by
B.S. Communication Disorders
University of Massachusetts, Amherst, 1998
M.S. Audiology
Brooklyn College, 2000
Submitted to the Harvard-M.I.T. Division of Health Sciences and Technology in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
August 2006
© 2006 Keith N. Darrow. All rights reserved.
The author hereby grants to M.I.T. permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part.
Signature of Author: ........
August 23, 2006
Certified By:............. ...................... ..... M Charles Liberm
Harvard-M.I.T. Division of Health Sciences and Technology
August 23, 2006
A ccepted B y: .............................................................................
Martha L. Gray, Ph.D.
OF TECHNOLOGY
Edward Hood Taplin Profe sor of Medical and Electrical Engineering
Director, Harvard-M.I.T. Di ision of Health Sciences and Technology
August 23, 2006
NOV 15 2006
-1•7

by
Keith N. Darrow
Submitted to the Harvard-M.I.T Division of Health Sciences and Technology on
August 1, 2005 in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
ABSTRACT
Sensory cells and afferent auditory neurons in the cochlea receive efferent feedback via olivocochlear (OC) neurons originating in the brainstem's olivary complex. The OC system comprises 1) medial (M)OC neurons that decrease electromotility in outer hair cells, and 2) lateral (L)OC neurons that elicit slow excitation or inhibition of auditory nerve dendrites that contact inner hair cells. We investigated the organization and function of the LOC system by immunohistochemical and physiological studies in mice with unilateral stereotaxic destruction of
LOC cell bodies. Double immunostaining in control cochleas and brainstems revealed two cytochemical subgroups of LOC neurons: a majority cholinergic population and a minority dopaminergic population. The observation of two LOC subgroups is consistent with reports that
LOC activation can either excite or inhibit auditory nerve activity. In lesioned mice, we observed two physiological abnormalities. First, ipsilateral ears were more vulnerable to noise-induced auditory nerve dysfunction, consistent with speculation that dopaminergic transmission controls glutamate excitotoxicity of auditory nerve dendrites after acoustic overexposure. Second, ipsilateral auditory nerve responses were increased while contralateral responses were decreased, and the normal tight correlation of neural excitability between the two ears was disrupted. A neural circuit is proposed to explain bilateral effects from unilateral LOC innervation. We suggest that a key LOC function is to bilaterally balance ascending inputs to olivary complex neurons, which are responsible for computing sound location based on the interaural level differences coded in the response rates of auditory nerve fibers.
Thesis Supervisor: M. Charles Liberman, Ph.D.
Title: Professor of Otology and Laryngology, Harvard Medical School

The path leading me to this thesis has been a great journey. As I set out many years ago as an undergraduate student in Amherst, I don't think I could have imagined it ending here, in
Boston. While so many people mean so much to me, Mom and Dad, this thesis is dedicated to you. You have enabled me to reach as high as I possibly can, and even further. Your dedications to life and to your family have inspired me to overcome every personal and educational obstacle put before me. You have made everything possible.
This thesis has required a great deal of time and dedication from so many people. Charlie
Liberman, my advisor and friend, you have dedicated your time, effort, and knowledge to my goals, thank you for helping me develop into a scientist. I would also like to thank my committee members, John Guinan, Chris Brown, and Doug Vetter, your guidance and thoughtful contributions to my thesis and education are greatly appreciated. Stephane (aka Steve) Maison, my Honorary Advisor, thank you for always helping me "lighten the load." I will miss not working with you everyday (well, most days), but I know we will remain friends, always. Mary
Andrianopolous, your continued guidance and support of my career goals are so greatly appreciated. Richard Freyman, Rochelle Cherry, Adrianne Rubenstein and Shlomo Silman, your help along the way have helped me get this far; thank you. My success at MIT, and in my lab,
EPL, has been made possible by so many people including Leslie Liberman, Melissa. Wood,
Brad Buran, Erik Larsen, Connie Miller, and Dianna Sands; thanks guys for all your help.
In addition to the educational support and guidance of so many people at so many different institutions, my closest family and friends have stood by me from start to finish. To my brothers, Mark and Rob, thanks for being my best men. Kerry, my dearest friend and sister, words can not being to explain the love and admiration I have for you and your family; thank you for always being by my side whenever and wherever I've needed you. Dillon, my nephew, I want you to know (once you are able to read this) that your smile, laughter, and sheer excitement for life make me the proudest uncle, ever. My (soon to be) in-laws, the Byrons and Lovetts, you are great addition to my family. Jeff, Egg and Drew, my other best-men, ROCK ON! Leo and
Ally, Joey, Darren, Hector, Mario and the rest of the HST Softball crew, thanks for such a
GREAT TIME all these years.
Finnigan, my little girl, you have seen me through every personal milestone in the last 5 years of my life. You have seen my ups and my downs, my good moods and my bad moods, my laughter and my tears, and all without a hint of hesitation. You are the greatest 401bs Chihuahua, ever.
Now to the future, this October
8 th
I will begin the newest chapter of my life; I am marrying my best-friend, Laura Byron. Laura, you are the most special, most beautiful, most supportive, most loving and most perfect person for me. Finding you makes me the most lucky person, ever. Over the past 3 years we have done everything together, and loved every minute of it. So now I've asked you to do everything with me for the rest of my life. I will love you unconditionally, forever. I can't wait to start our life together; me, you and Finny have so much to look forward to.

LIST OF FIG URES............................................................................. .................................
7-8
Chapter 1: BACKGROUND AND INTRODUCTION TO THE THESIS.......................9-15
1.1 Anatomy of the Olivocochlear Efferent System.............................................................9.......
1.2 Physiology of the Medial OC Efferent System.................................... ........
10
1.3 Physiology of the Lateral OC Efferent System...................................................................11
1.4 Role of the OC Efferent System in Protection from Acoustic Injury...........................18
1.5 Summary of Experimental Procedures, Results and Significance............................ 14
Chapter 2: DOPAMINERGIC INNERVATION OF THE MOUSE INNER EAR:
EVIDENCE FOR A SEPARATE CYTOCHEMICAL GROUP OF COCHLEAR
EFFERENT FIBERS .............................................................................................................
2.1 Abstract ........................ ...........................................................
16-31
.....................................
16
2.2 Introduction................................................................................ .......................................
16
2.3 M aterials and M ethods ...................................................... .............................................. 17
2.3.1 Animals, Surgery & Histological Processing... ................................. .... 17
2.3.2 Im m unostaining .......................... .............................................................................. 18
2.3.3 M orphometric Analysis.............. ........... ......................................... 19
2.4 Results........................................................................................ ........................................
20
2.4.1. Analysis of Cochlear Projections...................................................................... 20
2.4.1.1 Single-Staining R esults......... ........ ....................................................... ...20
2.4.1.2 Double-Staining results..... ...................... ................................ 21
2.4.2. A nalysis of Brainstem Origins........................................
2.4.2.1 B rainstem Lesions...............................................
................................... 25
........................................ 25
2.4.2.2 Brainstem Im m unostaining........................................................................... 26
2.5 D iscussion................................................................................... ......................................
28
2.5.1 Cochlear terminations of dopaminergic neurons................... ...............
28
2.5.2 Central origins of dopaminergic neurons .................................. ................... 29
2.5.3 Functional significance of a cochlear dopaminergic innervation ........................ 31
Chapter 3: COCHLEAR EFFERENT FEEDBACK: EVIDENCE FOR A ROLE IN
BALANCING INERAURAL SENSITIVITY .................................. ........
32-42

3.1 Abstract ....................................................................................
3.2 Introduction................... ..................................
.................................... 32
32
3.3 Materials and Methods .......................................................................................................
34
3.3.1 Stereotaxic Surgery..................................................................... ......................... 34
3.3.2 Histological analysis & Immunocytochemistry................................................... 34
3.3.3 In vivo functional assays.............................................................34
3.4 R esults.....................................................................................................................................35
3.4.1 Assessment of Lesion Success.............................................35
3.4.2 Effects of LOC De-efferentation on Cochlear Function............ ..........................36
3.5 D iscussion........................................................................... ............................................... 39
3.5.1 The OC System and its Influence on Cochlear Physiology.............................. 39
3.5.2 The OC System and its Role in Hearing.............................................................41
Chapter 4: SELECTIVE REMOVAL OF LATERAL OLIVOCOCHEAR EFFERENTS
INCREASES VUNERABILITY TO ACUTE ACOUSTIC INJURYo ..........................
43-61
4.1 Abstract ....................................................................................................................... 43
4.2 Introduction................................................................................................43
4.3 Materials and Methods......................................................................................................45
4.3.1 Stere otaxic Surgery................................................... ......................................... ..45
4.3.2 ABR and DPOAE Measurements.........................................45
4.3.3 A coustic O verexposure ................................................................. ....................... 46
4.3.4 Medial Olivocochlear (MOC) Assay... .......................................................
46
4.3.5 Histological Preparations................................46
4.3.6 Morphometric Analyses.................. ...................................... .. 46
4.3.6.1 B rainstem s........................................................................................ .....46
4.3.6.2 Cochleas....................... ....................................... . ............... .......46
4.4 R esults..................................................................................................................................47
4.4.1 Histological assessment of completeness and selectivity of the LOC lesion...........47
4 .4 .1.1 B rainstem s.............................................................................................................47
4 .4 .1.2 C ochleas.................................................................................................... 4 9
4.4.1.3 Correlating Cochleas and Brainstems....................................49
4.4.2 Functional Integrity of the MOC System...................................................... 53
4.4.3 Effect of LOC lesion on cochlear threshold and supra-threshold response............. 55
4.4.4 Effect of LOC lesion on vulnerability to acoustic injury......................................... 56
4.5 Discussion............................................................................................................................... 58
4.5.1 Peripheral effects of the LOC system in modulating neural excitability ........... 58

4.5.2 Acoustic injury and olivocochlear feedback.........................................................59
BIBLIOGRAPHY..........................................................................................................62-68

Chapter 1
Figure 1.1: Anatomical schematic of central and peripheral projection patterns of the OC system in m o u se ....................................................................................................................................... 9
Chapter 2
Figure 2.1: TH immunostaining of cochlear sections and whole mounts reveals a TH-positive innervation of the modiolus, osseous spiral lamina and organ of Corti....................................21
Figure 2.2: Double-immunostaining for TH (DAergic) and VAT (cholinergic) in a confocal zseries shows lack of co-localization of these two transmitter markers................................ 22
Figure 2.3: Comparison of xy projection and a single slice from a double-immunostained confocal z-series shows that apparent co-localization in the projection view often arises from superposition of signal from different focal levels and thus different terminals........................23
Figure 2.4: Semi-quantitative analysis of the density of VAT-positive and TH-positive terminals in the inner hair cell area shows that TH immunoreactivity in control ears is uniformly distributed along the cochlear spiral and that, after LOC lesion, both types of immunoreactivity decline to a sim ilar degree....................................................... ................................................ 24
Figure 2.5: Confocal image merging the TH signal and the DIC image shows that TH immunoreactivity in the mouse cochlea is spotty: regions of the inner spiral bundle with high density of labeled terminals can be flanked by regions of low density, and in turn by regions with no im m unostaining. .................................................................................... .............................. 24
Figure 2.6: Double-immunostaining for TH and VAT in a control cochlea and the opposite cochlea after either section of the olivocochlear bundle, or injection of neurotoxin into the LSO.
..........................................................................................................
26
Figure 2.7: TH-positive cell bodies located within and around the LSO................................ 27
Figure 2.8: Double-staining of brainstem sections through the superior olivary complex suggests that the TH-positive innervation of the cochlea arises from a separate population of noncholinergic neurons, in the 'shell' around the LSO............................... .............
27
Chapter 3
Figure 3.1: Anatomical schematic of central and peripheral projection patterns of the OC system in m ouse......................................................................................... ........................................... 33
Figure 3.2: Histol[ogical verification of LOC lesions, as seen in AChE-stained brainstem sections or in cochlear whole mounts immunostained for a cholinergic marker. ....................................
36

Figure 3.3: Selective disruption of the LOC system does not alter baseline cochlear thresholds, as measured by ABR or DPOAE; however, suprathreshold auditory nerve responses, as measured by Wave 1 amplitude, are increased in the ipsilateral ears of LOC Hit cases..........37
Figure 3.4: Changes in ABR amplitudes as a function of test frequency for all injected mice.
Comparisons of each ear from LOC Hit and Miss cases are made to control mice; as well as comparisons of right and left ears within both surgical groups............................... ..... 38
Figure 3.5: Anatomical schematic illustrating the LOC feedback circuit.............................. 41
Chapter 4
Figure 4.1: Anatomical schematic of central and peripheral projection patterns of the OC system in m ou se ................................... ........................................ 4 4
Figure 4.2: Histological verification of LOC lesions, as seen in AChE-stained brainstem sections or in cochlear whole mounts double-immunostained for a cholinergic and dopaminergic m arker ............................. 48
Figure 4.3: Lesion locations within the brainstems of all LOC Hit cases. In each case, the lesion is outlined and superimposed on "atlas" sections................................50
Figure 4.4: Lesion locations within the brainstems of all LOC Miss cases. In each case, the lesion is outlined and superimposed on "atlas" sections............................ ...........
51
Figure 4.5: Semi-quantitative analysis of cholinergic and dopaminergic markers in the IHC and
OHC areas of injected and control mice........................................................52
Figure 4.6: Analysis of lesion success based on the fractional survival of the LSO, as seen in
AChE-stained brainstem sections, and the fractional survival of cholinergic terminals in the IHC area, as seen in immunostained cochlear whole mounts.................................. ......
52
Figure 4.7: Bilateral suppression of DPOAEs, elicited via midline electrical stimulation of MOC fibers, suggests that MOC function is minimally affected by the lesions.............................. 54
Figure 4.8: Mean cochlear thresholds, as measured by ABR and DPOAE were not affected by a successful LOC le:sion, nor were interaural threshold differences for both measures................54
Figure 4.9: ABR amplitudes are enhanced in the ipsilateral LOC Hit ears and not in LOC Miss ears, whereas DPOAE amplitudes are unaffected in all groups................................ ..... 56
Figure 4.10: Mean ABR threshold shifts in LOC Hit mice measured at 6 hrs and 1 week after acoustic overexposure............................................................................ .................................. 57

1.1 Anatomy of the Olivocochlear Efferent System
The pioneering studies of Rasmussen (1946, 1953), and later work of Warr (1975), provide anatomical evidence of an efferent pathway that originates in the superior olivary complex
(SOC) and projects to the cochlea; thus termed the olivocochlear system. After injection of retrograde neuronal tracers in the cochlea (Warr and Guinan, 1979), two distinct efferent systems were defined based on the location of their cell bodies: a population of large cells with myelinated axons in the medial peri-olivary nuclei (termed the medial (M)OC system), and a population of small cells with unmyelinated axons located in and around the lateral superior olivary (LSO) complex (termed the lateral (L)OC system). Guinan et al. (1984), using anterograde neuronal tracers injected into the MOC vs. LOC regions of the brainstem, observed distinct patterns of cochlear projections for the two systems: MOC efferents projected mainly to the contralateral cochlea and innervated the outer hair cells (OHC), whereas LOC efferents projected mainly to the ipsilateral cochlea and terminated in the inner hair cell (IHC) area (see
Figures la,b). While similar studies have been performed in other mammalian species (guinea pigs, rats, mouse, chinchillas, monkeys; for review see Warr et al. 1986), the same general pattern holds.
Figure 1.1: Anatomical schematic of central (A) and peripheral projection patterns of the OC system in mouse. A) Almost all LOC cell bodies originate in the ipsilateral LSO; whereas MOC cell bodies are located in the contralateral (75%) and ipsilateral
(25%) peri-olivary nuclei. B) The main targets of the
MOC are the OHC soma; whereas the LOC's primary target is the afferent terminal of the Type I auditory nerve fiber, with sparse innervation of the IHC soma. Bold arrows indicate the direction of action potentials.
A number of later morphological studies further clarified the projection patterns of MOC and LOC fibers (Liberman and Brown, 1986; Brown, 1989). Convincing evidence for the projection patterns of MOC neurons comes from intracellular labeling experiments in which single MOC fibers were recorded from and then labeled via HRP. These labeled fibers were always myelinated, always originated from the MOC cell group, always projected to OHCs and never sent branches to the IHC area (Liberman and Brown, 1986; Brown, 1989). Morphological studies of LOC neurons suggest two subsets of LOC cells: small cells located "intrinsic" to the

LSO and large cells that surround the "shell" of the LSO (Vetter and Mugnaini, 1992).
Interestingly, these two subsets have subsequently been shown to have distinct cochlear projections (for review see Warr et el. 1997): 1) intrinsic neurons enter the cochlea and send unidirectional, either basally or apically, processes to a small select region of the cochlea (in guinea pigs these fibers have terminal regions that span < 1% the entire distance of the cochlea
(Brown, 1987) and 2) shell neurons enter the cochlea and diverge in a bi-directional manner, both basally and apically, and span large sections of the cochlea (in guinea pig, single fiber reconstruction revealed the terminal region of a bi-directional fiber spanned > 10% the distance of the cochlea). LOC fibers primary target is the dendrites of spiral ganglion fibers in the IHC area; however, there is also a sparse (-15%) innervation of IHC somata as well (see Figure 2;
Brown, 1985; Liberman, 1980; Liberman and Brown, 1985). In the mouse, the LOC system: 1) appears to have a 99% ipsilateral projection pattern (Campbell and Henson, 1988), and 2) does not appear to have any projections to the OHC area (Maison et al. (2003a).
Immunocytochemical studies in different species are in general agreement that OC terminals in the cochlea contain a variety of neurotransmitters including acetylcholine (ACh), yamino butyric acid (GABA) and dopamine (DA), as well as a several peptidergic transmitters including CGRP and enkephalins (for review see Eybalin,'93). In mouse, OC terminals in the both the IHC and OHC areas contain, and co-localize, ACh, CGRP, and GABA (Maison et al.
2003a). Other transmitters, such as DA, opioid peptides, and urocortin have also been localized to IHC area terminals, but no extensive studies in mice have been done to determine what, if any, subgroups may exist.
1.2 Physiology of the Medial OC Efferent System
Many studies aimed at understanding the in vivo physiology of the OC system have exploited the fact that MOC fibers decussate close to the floor of the IV ventricle. In many experiments, the crossing fibers of the olivocochlear bundle have been electrically stimulated while simultaneously monitoring different measures of cochlear function. These studies have revealed what are now termed the 'classic OC effects': 1) suppression of the compound action potential, a measure of summed cochlear auditory nerve activity (Galambos, 1956), 2) suppression of IHC DC and AC potentials in response to sound stimulation (Brown and Nuttall,
1984), 3) reduction of basilar membrane (BM) motion in response to sound (Murugasu and
Russell, 1996), 4) reduction of distortion product otoacoustic emissions amplitude (Mountain,
1980), and 5) an increase in cochlear microphonic (CM) that results from an increased conductance of the OHCs (Bonfils et al. 1987; Gifford and Guinan, 1987). By comparing cochlear effects of midline stimulation to cochlear effects seen when directly stimulating MOC vs. LOC cell bodies, it has been concluded that all "classic" effects arise solely from MOC stimulation (Gifford and Guinan 1987). Recent work with mouse knockout lines, in particular the mice with deletion of the o9 nAChR located on the OHCs, corroborates this conclusion: all classic effects are mediated via the cholinergic actions of the MOC neurons (Vetter et al. 1999).
Evidence from both in vitro and in vivo studies suggests that the MOC's "fast" suppressive effects arise by modifying the contribution of OHC electromotility to cochlear amplification
(Evans et al. 1997; Murugasu and Russell, 1996).
1.3 Physiology of the Lateral OC Efferent System
In contrast to the MOC system, it has proven difficult to study the LOC system by either electrically stimulating or recording from these unmyelinated fibers. Recent insight to LOC

peripheral effects comes from 1) intra-cochlear perfusion of putative LOC neurotransmitters, 2) genetic manipulation of putative LOC neurotransmitters or their respective receptors, 3) indirect electrical stimulation of the LOC, and 4) LOC lesioning experiments.
Numerous studies have applied putative LOC neurotransmitters, or their agonists and antagonists, to the guinea pig cochlea or Xenopus lateral line. In the guinea pig, application of
ACh increases both spontaneous and glutamate-induced auditory nerve activity (meant to mimic sound-induced activity; Felix and Ehrenberger 1992). Perfusion of GABA does not affect spontaneous activity; but decreases glutamate-induced and ACh-induced activity (Felix and
Ehrenberger, 1992; Arnold et al. 1998). In the lateral line, CGRP has been shown to increase spontaneous activity and decrease mechanically driven rates (Bailey and Sewell, 2000a,b). DA appears to have a primarily inhibitory effect on auditory nerve function. In guinea pigs, cochlear perfusion of DA results in a decrease of both spontaneous and sound-driven activity (d'Aldin et al. 1995; Oestreicher 1997). In addition, removing DA, via cochlear perfusion of DA antagonists, results in vacuolization of auditory nerve dendrites under the IHCs (Ruel et al. 2001); thus suggesting a tonic inhibitory function that may serve to protect auditory nerve dendrites from excitotoxic damage.
Maison et all. (2003b, 2005) have complemented these pharmacological lines of work by describing the auditory phenotype of the oa-CGRP null mice and several GABA-A receptor subunit knockout mice. In the a-CGRP null mice, OHC function appeared normal: DPOAE amplitudes and the magnitude of electrically-evoked MOC suppression of DPOAEs were identical to wild-type littermates. However, a decrease in neural activity, as shown by a reduction of the ABR wave 1 at suprathreshold levels, was demonstrated. Several of the studied
GABA-A receptor subunit mice (al, ox2, cx6, 6) revealed no auditory phenotype. However, three subunit knockouts, P3, P2, and x5 showed differences in cochlear function re: wildtype littermates. The P33 phenotype suggested anomalies in cochlear development; whereas the 32 and o5 knockout mice had late-onset cochlear dysfunction suggesting a degenerative neuropathy
(ABR threshold shifts > DPOAE threshold shifts).
In addition to pharmacological and genetic manipulation of OC function, LOC peripheral effects have been studied in chronically de-efferented animals (Liberman 1990; Walsh et al.
1998; Zheng et al. 1999). Two approaches have classically been used: 1) a midline cut that interrupts 2/3 of the MOC innervation and spares virtually all LOC innervation; and 2) a lateral cut which interrupts the entire ipsilateral OC system (both MOC and LOC). In combination, these studies have suggested that the LOC system is capable of modulating spontaneous and sound-evoked discharge rates in the auditory nerve. Removing OC innervation in adult animals causes little change in thresholds or tuning (Q10 values); however, there is a substantial compression of spontaneous rates towards lower levels (Liberman, 1990; Zheng et al. 1999). The lack of effect on thresholds and tuning suggests that an MOC innervation is not required for normal OHC function. The selective effects on discharge rate are consistent with a post-synaptic effect of the LOC innervation on auditory nerve dendrites. OCB section in neonates also results in decrease in spontaneous rates. These changes in spontaneous rate are likely a result of LOC removal, since a midline section, which removes 2/3 of the MOC innervation without affecting the LOC system, did not affect spontaneous rates. However, in contrast to the studies in adults, neonatal de-efferentation increased thresholds and decreased tuning (Walsh et al. 1998), suggesting an important role for the OC system in cochlear development (Pujol and Carlier,
1982; Walsh et al. 1998).

A third lesioning technique that aims to selectively remove the LOC system via unilateral stereotaxic lesioning of the LOC cell bodies in the LSO was recently demonstrated by Le Prell et al. (2003). In using this technique, Le Prell and colleagues (2003) have described cochlear function in adult guinea pigs lacking unilateral LOC innervation. They report a reduction of suprathreshold CAP amplitudes in the de-efferented ears, similar to the effects described in the a-CGRP null mouse (Maison et al. 2003b). However, interpreting their results is complicated by a number of factors, including the high variability of cochlear sensitivity as measured by CAP, even in control ears: an effect likely attributed to a middle-ear reaction that resulted from the chronically implanted round window electrodes.
Further insight to LOC function comes from recent attempts to indirectly activate the LOC system by shocking the inferior colliculus. Using this approach, Groff and Liberman (2003) provide evidence that LOC efferents can elicit either enhancing or suppressive effects on auditory nerve (AN) activity (as assayed via CAPs) without affecting OHC function (as assayed via DPOAEs and cochlear microphonics). These observed effects were slow-onset and slowoffset, as would be expected from an unmyelinated modulating system.
Taken together, these studies of immunohistochemistry, surgical lesions, electrical stimulation and pharmacological or genetic manipulation of receptors suggest that LOC subsystems may exist, and that they may have complementary effects of excitation or inhibition on auditory nerve output for long periods of time (t = 10 min; Liberman, 1990; Felix and
Ehrenberger 1992; d'Aldin et al. 1995; Oestreicher 1997; Arnold et al. 1998; Walsh et al. 1998;
Zheng et al. 1999; Bailey and Sewell, 2000a,b; Ruel et al. 2001; Maison et al. 2003b; Le Prell et al. 2003; Groff and Liberman 2003; Maison et al. 2005).
In the present experimental series we will address the question of the LOC subgroup existence by immunohistochemical means of staining for cholinergic and dopaminergic markers in both the brainstem and in cochlear whole-mounts. To determine the origin of terminals in the
IHC area of the mouse cochlea, we will perform stereotaxic destruction of LOC cell bodies on one side of the brainstem and determine the pattern of terminal loss within both cochleas. In addition, by using the same unilateral stereotaxic technique of disrupting the LOC cell bodies, we will revisit the question of ensemble LOC effects on cochlear function in unilaterally LOC de-efferented mice. By monitoring the relatively noninvasive measures of ABR and DPOAE, we will avoid the complications experienced by Le Prell et al. (2003) in which their guinea pigs had changing thresholds that likely resulted from the chronic implanting of cochlear round window electrodes. Also, by studying the mouse, we can directly compare the results of removing the entire LOC system with existing and future studies of mutant mouse lines lacking LOC system subcomponents (e.g. the oc-CGRP knockout study, Maison et al. 2002).
1.4 Role of the OC efferent system in Protection from Acoustic Injury
The OC efferent system has been implicated in protecting the cochlea from noise-induced injury. Acoustic overexposure can cause both temporary threshold shift (TTS) and permanent threshold shift (PTS). The mechanisms underlying these two forms of acoustic trauma are very different. While severe PTS can be destructive enough to breakdown the entire organ of corti, loss of OHCs and damage to the stereocilia are common observations in cochleas with mild to moderate levels of PTS (Liberman and Kiang, 1978, Robertson, 1983; Liberman and Dodds,
1984; Liberman and Mulroy, 1984). It appears that the MOC system's cholinergic action on
OHC motion is capable of protecting the OHCs from acoustic injury. In guinea pigs, simultaneous MOC stimulation and acoustic overexposure reduce the amount of overall

threshold shift (Rajan, 1988; Reiter and Liberman, 1995). Recently, more definitive evidence that the MOC system is directly involved in protection was demonstrated in mice with overexpression of the a9 nAChR in OHCs: "over-expresser" mice had an enhanced resistance to acoustic injury (Maison et al. 2002).
A common acute pathology in ears < 24 hrs after exposure to a traumatic stimulus is swelling of the auditory nerve dendrites in the IHC area (Liberman and Mulroy 1982; Robertson
1983; Puel et al. 1998; Le Prell et al. 2005). These vacuolizations appear to result from excessive glutamate exposure: 1) dendritic swellings can be mimicked in cochleas perfused with glutamate receptor agonists and 2) when noise is presented with simultaneous intra-cochlear perfusion of a glutamate antagonist, there are fewer vacuoles and there is less threshold shift (Puel et al. 1998).
These observations suggest that dendritic swelling is a type of excitotoxicity brought on by excessive release of glutamate from the IHC. In cochleas perfused with dopamine antagonist, without exposure to intense noise, an increase in dendritic vacuolization has also been observed, suggesting that the LOC's dopaminergic component might counteract this glutamate-induced excitotoxicity (Ruel et al. 2001).
Histological and pharmacological evidence have indirectly implicated the LOC system in protecting auditory nerve dendrites from glutamate-induced excitotoxicity. Dendritic vacuolization, similar to that seen in cochleas with acute acoustic injury, has been reported after acute section of the OC bundle (Bodian and Gucer 1980); thus bringing about the hypothesis that the LOC system may protect from acoustic injury, as well as possibly provide protection from sound levels that are lower then those considered hazardous. This, in combination with the observation that blocking dopaminergic transmission in the cochlea results in dendritic vacuolization (Ruel et al. 2001), provide support for an LOC role in protecting auditory nerve dendrites from the damaging effects of TTS.
In the present experimental series, we will attempt to directly assess the contribution of the
LOC to protection. We will stereotaxically lesion the LOC system (Le Prell et al., 2003) in adult mice and subsequently compare threshold shifts in the ears ipsilateral and contralateral to the lesion. We chose the mouse CBA/CaJ because of the reduced variability in response to acoustic trauma, compared to out-bred experimental animals such as the guinea pig (Wang et al., 2003).
To rule out any effects of the lesion on the MOC system, we will assess the integrity of the MOC pathway both morphologically (in the brainstem and cochlea) and functionally (with bilateral measurement of MOC-mediated suppression of cochlear responses).
In the following three chapters we use a combination of immunohistochemical and surgical-lesion techniques to study the LOC system in mice. More specifically, the goals of this study are to investigate: 1) the existence of cytochemical subgroups within the LOC system, 2) the effect of chronic unilateral LOC lesion on cochlear output in both ears, and c) the involvement of LOC system in the protection from acoustic injury. We study the LOC system in mice to complement the ongoing effort in our laboratory that exploits mouse lines with targeted deletion of putative LOC-system receptors or neurotransmitters in the inner ear.
In chapter 2, we analyzed cholinergic and dopaminergic transmitter co-localization of the
LOC system in both the brainstem and in cochlear whole mounts. By immunostaining mouse cochleas for tyrosine hydroxylase (TH) and dopamine P-hydroxylase, we observed a rich adrenergic innervation throughout the auditory nerve trunk, and a small dopaminergic innervation in the IHC area. Double-immunostaining of cholinergic and dopaminergic markers

suggests little, if any, co-localization of these terminals in the IHC area; quantification of terminal density suggest TH-positive fibers constitute only 10-20% of the efferent innervation of the IHC area. Correspondingly, double-staining in the brainstem revealed two distinct populations: a small population of dopaminergic cells surrounding the LSO and a large population of cholinergic cells inside the LSO. Subsequent stereotaxic lesion of the LSO confirmed that both populations of efferent terminals in the IHC region of the cochlea are of
LOC origin. The presence of two LOC cytochemical subgroups in mice is consistent with earlier reports in that LOC somata have two distinct .brainstem locations and two distinct cochlear innervation patterns. Specifically, the two LOC subgroups in mice appear to represent the intrinsic and shell populations of LOC neurons described by Warr et al. (1997).
In chapter 3, we examined cochlear physiology in mice with unilateral disruption of the
LOC feedback. Successful LOC lesions did not alter baseline cochlear thresholds (ABR and
DPOAE) in either ear; this was expected based on earlier experiments that showed OC innervation is not necessary to maintain thresholds in adult animals. In lesioned mice, however, there were abnormalities in ABR growth functions of both cochleas: the neural output was increased in the ipsilateral ear and decreased in the contralateral ear. In control and sham surgery mice there was a strong tendency for ABR to be bilaterally symmetric. The observation of abnormal ABR growth functions in lesioned mice, contrasted with normal growth functions for
DPOAEs, is consistent with a selective postsynaptic effect of the LOC system on auditory nerve output. A neural circuit is proposed to explain the both the normal tight correlation of cochlear output, as well as the bilateral abnormal growth functions in mice with unilateral disruption of the LOC system. We then suggest that a major LOC function in vivo is to bilaterally balance ascending inputs to olivary complex neurons, which are responsible for computing sound location based on the interaural level differences coded in the response rates of auditory nerve fibers.
In chapter 4, a subset of the surgical cases was entered into an acoustic overexposure paradigm to evaluate the influence of the LOC system on protecting the cochlea from acoustic injury. ABR threshold shifts in ipsilateral ears of LOC lesioned cases were 10-15 dB higher then the contralateral ears. This asymmetry was not present in DPOAE threshold shifts, nor was it present in the ABR or DPOAE thresholds shifts of sham-surgery mice. This is the first direct evidence of a key role of the LOC system in protecting the neural elements within the cochlea, namely the IHC -- auditory nerve synapse, from acoustic injury. Thus, both the LOC and the
MOC (Maison et al. 2002) appear directly involved in protecting their respective target cells from noise-induced damage. The mechanisms involved in these separate, yet complementary, protective effects may be different (e.g. over-expression of the o9 nAChR in mice results in increased resistance to acoustic injury, but the 09 nAChR is only present in the OHCs and not in the IHC area of adult mice). The observation that ipsilateral ears in LOC lesioned mice were more susceptible to noise-induced auditory nerve dysfunction is consistent with speculation that
LOC dopaminergic transmission decreases glutamate excitotoxicity of auditory nerve dendrites after acoustic over-exposure.
The notion that the LOC system is heterogeneous is not new. The observation of abnormal ABR growth functions in LOC lesioned mice is compatible with the existence of two
LOC subgroups capable of enhancing and inhibiting auditory nerve activity. Abnormal neural growth functions are a common finding in experiments with OC- and LOC-lesion animals
(Liberman 1990; Zheng et al. 1999; Le Prell et al. 2003), as well as in transgenic mice with targeted deletion of individual LOC components (Maison et al. 2003b). Although the results of

all these experiments can be qualitatively different, these differences may arise from a different resting balance of resting activation levels of the LOC subgroups in different species. The LOC system's dopaminergic innervation has been suggested to protect noise-induced glutamate excitotoxicity in auditory nerve dendrites; we show that LOC-deefferented ears are more susceptible to acoustic injury. In combination, this report provides the first direct evidence that cytochemical heterogeneity exists within the LOC system, and that these LOC subgroups may have different effects on cochlear function.

Immunostaining mouse cochleas for tyrosine hydroxylase (TH) and dopamine
hydroxylase suggests there is a rich adrenergic innervation throughout the auditory nerve trunk, and a small dopaminergic innervation of the sensory cell areas. Surgical cuts in the brainstem confirm these dopaminergic fibers as part of the olivocochlear efferent bundle. Within the sensory epithelium, TH-positive terminals are seen only in the inner hair cell area, where they intermingle with other olivocochlear terminals expressing cholinergic markers (vesicular acetylcholine transporter -VAT). Double-immunostaining suggest little co-localization of TH and VAT; quantification of terminal volumes suggest TH-positive fibers constitute only 10-20% of the efferent innervation of the inner hair cell area. Immunostaining of mouse brainstem revealed a small population of TH-positive cells in and around the lateral superior olive.
Consistent with cochlear projections, double-staining for the cholinergic marker acetylcholinesterase suggested that TH-positive somata are not cholinergic and vice versa. All observations are consistent with the view that a small dopaminergic subgroup of lateral olivocochlear neurons 1) projects to the inner hair cell area, 2) is distinct from the larger cholinergic group projecting there, and 3) may correspond to lateral olivocochlear "shell" neurons described by others (Warr et al., 1997).
2.2 Introduction
The lateral olivocochlear (LOC) system forms a neuronal feedback system from the lateral superior olivary complex in the brainstem to the organ of Corti (Guinan et al., 1983). The main peripheral targets of these unmyelinated fibers include the peripheral unmyelinated terminals of auditory nerve afferents in the region under the inner hair cells (Liberman, 1980); however a smaller population of synapses on the inner hair cells themselves may also arise from the LOC system (Liberman et al., 1990; Sobkowicz and Slapnick, 1994; Sobkowicz et al., 1997).
The functional role of the LOC system has remained less clear than that of the medial olivocochlear (MOC) system to the outer hair cells, in large part, because it is only the myelinated axons of the MOC system that are activated when the olivocochlear bundle is electrically stimulated (Gifford and Guinan, 1987), and only the myelinated axons of the MOC system are large enough to record single-fiber responses to sound with glass micropipets (Fex,
1962; Liberman and Brown, 1986).
A recent electrophysiological study suggested that the LOC system could be activated indirectly by electrical stimulation of the inferior colliculus (Groff and Liberman, 2003). This study reported that, depending on electrode placement in the colliculus, the shocks could cause either a slow enhancement or a slow depression of cochlear neural potentials, without changing outer hair cell based responses such as the cochlear microphonic and distortion product

otoacoustic emissions. These slow changes in afferent excitability, with onset time constants greater than 60 seconds, were consistent with expected effects of LOC terminals acting postsynaptically on auditory nerve dendrites. However, the presence of both enhancing and suppressing effects suggested the existence of at least two functional subgroups of LOC fibers.
Such a suggestion for multiple peripheral effects of the LOC system is not unexpected given the apparent plethora of transmitter and neuromodulatory systems it comprises. Over the last 25 years, immunohistochemical evidence has accumulated for the presence of cholinergic,
GABAergic, catecholaminergic and peptidergic transmission in the peripheral terminals of this fiber system (for review, see (Eybalin, 1993)). Although it is clear that the LOC terminals, as a group, are heterogeneous with respect to their transmitter content, the key question of cytochemical subgroups remains unanswered: are all transmitters co-localized in one class of terminals, or might each transmitter be released by a separate class of fibers? Only a handful of explicit co-localization studies have been performed on the LOC system; and all to date have concluded that all transmitter systems are present in all terminals, i.e. that there is no histological evidence for functional subgroups (Safieddine and Eybalin, 1992; Satake and Liberman, 1996;
Maison et al., 2003a), when the transmitters acetylcholine, GABA and CGRP are considered.
The purpose of the present study was to extend the analysis of transmitter co-localization and cytochemical subgroups in the LOC system to include the cochlea's dopaminergic innervation.
We use mouse as an experimental animal in this study, because of an ongoing effort in our laboratory to exploit the availability of mouse lines with targeted deletion of putative LOCsystem receptors or neurotransmitters in the inner ear (e.g. Maison et al. 2003b) as a useful tool in the analysis of LOC peripheral effects and the functional significance of this neuronal feedback pathway.
2.3.1 Animals, Surgery and Histological Processing
CBA/CaJ mice between 4 and 9 weeks of age were used in this study. All procedures were approved by the IACUC of the Massachusetts Eye and Ear Infirmary. For histological processing, animals were anesthetized with ketamine and xylazine, and intravascularly perfused with 4% paraformaldehyde in phosphate buffer saline (PBS). In some cases, the fixative also included 0.1 or 0.05% glutaraldehyde. Vascular perfusion was followed by cochlear dissection and perfusion of the cochlear scalae and overnight post-fixation in the same fixative. Fixation was followed by decalcification in ethylenediaminetetraacetic acid (EDTA). Some ears were then embedded and sectioned, whereas others were dissected and evaluated as whole mounts.
For cases to be sectioned, decalcified cochleas were dehydrated in alcohol and embedded in polyester wax, cut at 25 gLm and mounted on gel-subbed slides. Prior to staining, the polyester wax sections were treated with Histoclear@. Whole-mount ears were carefully dissected into 5 half-turns with the tecctorial membrane and spiral ligament are removed prior to immunostaining.
A subset of animals (n=6) underwent surgery designed to sever the entire OCB to one ear.
After anesthetization with ketamine and xylazine, a posterior craniotomy was performed and the cerebellum was elevated. Using surface landmarks visible on the floor of the IVth ventricle, a microknife was inserted and an incision was made at the sulcus limitans, positioned at the rostro-

caudal position of the facial nerve genu and angled in the sagittal plane so as to sever the olivocochlear bundle to left ear only; the opposite ear served as control.
A larger subset of animals (n=17) underwent surgery and a stereotaxic injection of neurotoxin designed to lesion the LOC system unilaterally (Le Prell et al., 2003). After anesthetization with ketamine and xylazine, the mouse was fixed in a Kopf stereotaxic apparatus via snout clamp and ear bars. The skin overlaying the skull was slit and retracted to reveal bregma and lambdoidal sutures. A hole was made over the left lambdoidal suture, and a micropipette filled with a solution of mellitin in saline was inserted into the brainstem using stereotaxic coordinates derived originally from an atlas of the mouse brain (Franklin and
Paxinos, 1997), and modified by trial and error. After the pipette was lowered into the lateral superior olive (LSO) on the right side, an injection of 2 gtl was made with a 10 gl syringe
(Hamilton) attached to the micropipette. The opposite ear served as control. Immediately after injection, the scalp was sutured, and the animal placed in a padded cage with heat lights for ~ 1 hr post surgery. Four weeks later, the brainstem and both cochleas were harvested from each animal.
A final group of (control) animals underwent no surgical procedures before tissue harvest and subsequent immunohistochemistry. Some of these controls (n=3 animals, 6 olivary complexes) were used to study the brainstem distribution of cholinergic and dopaminergic neurons in the LSO. Others (n=14 animals, 16 cochleas) were used to study the cochlear distributions. Of the cochleas, 9 were double-immunostained for vesicular acetylcholine transporter (VAT) and tyrosine hydroxylase (TH), while the remaining 7 were immunostained for TH only.
For all surgical cases, the brainstems were fixed, sectioned and stained for acetylcholinesterase (AChE) as described elsewhere (Osen and Roth, 1969). The cochleas were fixed, dissected and double-immunostained for VAT and TH (see below). After analysis of the
AChE-stained brainstem sections, each surgical case was classified as a hit or a miss: 1/6 OCB sections was fully successful; however, 10/17 of the LSO lesions were at least partially successful in removing the LOC system unilaterally. All cochleas from the both the "hit" and
"miss" cases were analyzed (see below).
2.3.2 Immunostaining
Primary antibodies were as follows: sheep anti-TH from Calbiochem (657014, lot#
D19854), rabbit anti-VAT from Sigma (V5387, lot# 049H4884) and mouse anti-dopamine beta hydroxylase (DBH) from Alpha Diagnostics (DBHl -M, lot# Xcc0103). The anti-DBH antibody was raised against bovine DBH protein. The anti-TH antibody was raised against native rat pheochromocytoma tyrosine hydroxylase. The specificity of these antibody was assessed by documentation of the locations of immunopositive cells throughout serial sections of the mouse brainstem, compared to that described in the literature and illustrated in an atlas of the mouse brain (Franklin and Paxinos, 1997). The anti-VAT antibody was raised against a synthetic peptide corresponding to the C-terminal of rat vesicular acetylcholine transporter (amino acids
512-530). The specificity of the VAT antibody was tested by an adsorption control with a custom peptide from Sigma Genosys, according to the manufacturer's specifications: 1.3 mg peptide was mixed with 1.3 ml water and 15 ul of this solution was added to 500 ul of a 1:100 dilution of antibody, vortexed, then put on shaker at room temp for 4 hrs. This pre-adsorbed solution was diluted (1:1000 antibody concentration) and staining of alternate cochlear pieces (with non-pre-

adsorbed antibody at the same dilution) was compared: preadsorption eliminated all specific staining.
Cochlear material was immunostained as sections or as dissected whole mounts. Sectioned material (n=8 cochleas) and some wholemounts (n=3 cochleas) were singly immunostained for
TH (1:1000) or DBH (1:2000). After first blocking in 5% normal horse serum (with Triton), sections or whole mounts were incubated in the primary antibody followed by an appropriate biotinylated secondary antibody (at 1:200). Antibody was visualized in these singly immunostained sections and whole mounts via an ABC kit (Vector Laboratories) followed by chromogen reactions with diaminobenzidine (Adams, 1977). Negative controls were included in each staining run, consisting of sections treated identically except for the lack of the primary antibody incubation.
Double-immunostaining for TH and VAT was carried out in cochlear whole mounts only.
This approach was applied to 9 cochleas from control animals (no surgery), as well as to both cochleas from each of the animals undergoing unilateral OC lesions (see above: n=23 animals).
After the blocking step, cochlear pieces were incubated in the sheep anti-TH (1:500) and the rabbit anti-VAT (1:1000) overnight, followed by a 1 hr incubation in the VAT secondary (1:400 biotinylated donkey anti-rabbit) and, finally, an overnight incubation in the TH secondary
(1:1000 chicken anti-sheep coupled to AlexaFluor488) and the VAT tertiary (1:1000 streptavidin-coupled AlexaFluor 568). Tissue pieces were analyzed and photographed either on a Nikon E-800 microscope equipped with epifluorescence or a Leica confocal microscope.
Immunostaining of brainstem sections for TH was carried out in 5 cases using the sheep anti-TH (1:500) followed by two AlexaFluor488-coupled secondary antibodies: chicken antigoat followed by goat anti-chicken. For these sections, the immunostaining was followed by histochemical processing for AChE (Osen and Roth, 1969).
2.3.3 Morphometric Analysis
For cochlear whole mount preparations, cochlear lengths were measured along the pillar heads by computerized planimetry. Cochlear location was converted to frequency according to published maps for mouse (Muller et al., 2005). Cochlear locations in each case corresponding to 10 roughly log-spaced frequency correlates were identified.
Two types of systematic analyses were carried out. In an exhaustive "semi-quantitative" analysis, an observer blinded to the surgical histories, separately rated the innervation densities for VAT- and TH-positive terminals at each of the 10 points along the cochlear spiral in a set of
36 cochleas including: 10 cochleas ipsilateral to a successful LOC lesion (see above) and the 10 opposite-ear controls; 7 cochleas ipsilateral to a brainstem injection that missed the LOC and the
7 opposite-ear controls; and 2 cochleas from animals without any brainstem surgery. Separate rating scales were used in inner hair cell and outer hair cell areas. A 3-point scale was used for
VAT-positive terminals in the OHC area: the observer's task was to estimate the fraction OHCs with at least one VAT-positive terminal: 3 = 100-66%, 2 = 66-33%, 1 =33-0%. VAT- and THpositive terminals in the IHC area were evaluated with a 4-point scale: 3 = profuse , 2 = moderate, 1= sparse, and 0= none. Each immunostain was separately referenced to its own maximum values: i.e. TH-positive terminals are much rarer than VAT terminals, but maximum density for each would receive a rating "3".
In a second, more quantitative analysis of 4 control cochleas (no brainstem surgery), quantification of the relative areas of TH-positive and VAT-positive terminals in the doubleimmunostained material was carried out using confocal z-stacks obtained with z-spacing of 0.25

ýpm.. The z-stacks were ported to a 3-D visualization and morphometry software package
(Amira@), in which the 3-D volumes of each terminal type were computed: a criterion pixel intensity is selected and the software automatically 1) segments each slice to encircle areas within which the criterion intensity is exceeded, 2) assembles the slices to create 3-D surfaces, and 3) computes the surface area and enclosed volumes.
2.4.1. Analysis of Cochlear Projections
2.4.1.1 Single-Staining Results
To differentiate adrenergic from dopaminergic innervation, cochleas were immunostained with either tyrosine hydroxylase (TH) or dopamine P hydroxylase (DBH). Since TH catalyzes the first step in the biosynthetic pathway common to dopamine and norepinephrine, whereas
DBH catalyzes the conversion of dopamine to norepinephrine, adrenergic fibers are immunopositive for both TH and DBH; whereas dopaminergic fibers should be TH-positive and
DBH-negative.
TH immunoreactivity was seen in all cochleas examined as sectioned material (n=8).
Analysis of 70 immunostained sections revealed beaded TH-positive fibers throughout the modiolus and osseous spiral lamina (e.g. Figure 2.1A). A few of the TH-positive fibers were clearly associated with blood vessels, spiraling around their circumference; however the majority were not, taking an uncoiled trajectory through the neuropil of the modiolar region (e.g. filled arrow in Fig. 2.1A). In many sections (59/70), immunopositive puncta were also clearly seen in the inner spiral bundle, in the region under the inner hair cell (e.g. within the dashed circle in
Figure 2. 1B). Labeled swellings were never seen in the outer hair cell area.
Sections stained with anti-DBH also showed immunopositive beaded fibers in the modiolus and osseous spiral lamina; however, immunopositive puncta were never seen in the inner spiral bundle (50 immunostained sections were analyzed, with multiple views of the organ of Corti in each section). Together, these observations suggest there is a rich adrenegeric innervation of the modiolus, as previously reported (e.g. (Spoendlin and Lichtensteiger, 1966; Terayama et al.,
1966; Hozawa et al., 1989), and a sparse dopaminergic innervation of the cochlear epithelium.
To better assess the morphology of TH-positive fibers in the organ of Corti, several cochleas were dissected and immunoreacted as whole mounts. In this whole-mount material, immunopositive swellings were clearly visible within the inner spiral bundle beneath the inner hair cells (e.g. open arrows in Fig. 2.1 C). In some material, the thin axonal processes connecting the en passant swellings were also immunostained. In all cases, the total volume of TH-positive puncta in the inner spiral bundle was significantly less than that seen when similar material is immunostained for SNAP25, synaptophysin or other markers for all olivocochlear efferent terminals ((Maison et al., 2003a); and see below). However, individual TH-positive swellings were darkly labeled, suggesting that incomplete labeling was not the cause of the small numbers of immunopositive terminals.

I
Figure 2.1: TH immunostaining of cochlear sections (A) and whole mounts (B) reveals a TH-positive innervation of the modiolus, osseous spiral lamina and organ of Corti. A: Immunostained cochlear section from the upper basal turn shows TH-positive beaded fibers in the osseous spiral lamina (OSL) and modiolus (e.g. arrow). Inset (B) shows a higher magnification view of the immunostained puncta in the inner spiral bundle (ISB). The position of the inner hair cell nucleus is indicated by the arrowhead. This image is from the upper basal turn. C: Immunostained cochlear whole mount shows strings of THpositive en passant swellings (three are indicated by unfilled arrows) in the inner spiral bundle beneath the inner hair cells. Nuclei of three adjacent inner hair cells are indicated by the filled arrowheads.
2.4.1.2 Double-Staining results
Previous immunohistochemical analysis of the olivocochlear innervation of the mouse cochlea suggested that the vast majority of olivocochlear terminals in the inner spiral bundle express the cholinergic marker vesicular acetylcholine transporter (VAT), and also co-localize a
GABAergic marker as well as the neuropeptide CGRP (Maison et al. 2002). To address whether
TH-positive fibers in the organ of Corti represent a distinct fiber group or a subset of the cholinergic/GABAergic/CGRPergic population, we double-stained a number of cochlear wholemount preparations for TH and VAT.
The analysis of these double-stained cochleas suggests that TH-positive fibers in the organ of
Corti represent a small, separate population: i.e. the small number of TH-positive fibers in the inner spiral bundle do not generally express VAT, and the large number of VAT-positive fibers
in the inner spiral bundle do not generally express TH. The merged confocal image of Figure
2.2A illustrates the point: in the inner spiral bundle, VAT-positive (red) terminals greatly
outnumber the TH-positive (green) terminals, and the OHC area shows a robust VAT-positive
innervation, whereas no TH-immunostaining is visible.
The majority of TH-staining in the inner spiral bundle area appears complementary to the
VAT staining (e.g. arrow); however, the occasional yellow profile suggests some co-localization.
This apparent co-localization in merged projections arises from superposition of TH and VATimmunoreactivity from different z planes. This superposition is illustrated in Figure 2.3: for the two yellow terminals in the projection view (Fig. 2.3), examination of the single z plane from
which the TH signal arises (Fig. 2.3B) shows no co-localization in either terminal. The signal in
the VAT channel was from other terminals at a different focal level (not shown).

To assess whether there is any spatial segregation of TH-positive and VAT-positive terminals
in the inner hair cell area, we obtained confocal z-stacks and viewed the projections in all three orthogonal planes. As illustrated by the xy, xz and yz projections of one such stack (Figs. 2.2A,
2.2B and 2.2C, respectively), there is no clear evidence of any spatial segregation. Note that comparison of all three projections reveals that the cluster of apparent co-localization in the yz
projection (Fig. 2.2C) arises largely because of the superposition of green and red pixels from
different z-levels. Similar stacks were obtained and examined from all regions along the cochlear spiral in numerous cases: no clearcut evidence for spatial segregation was seen.
Figure 2.2: Double-immunostaining for TH
(green) and VAT (red) in a confocal z-series shows lack of co-localization of these two transmitter markers. A-C show xy, xz and yz projections, respectively. All projections suggest there is minimal co-localization of TH and VAT in efferent terminals (co-localization of TH and VAT would appear as yellow terminals). None shows evidence for spatial segregation of TH- and VAT positive terminals.
D-F show isosurfaces viewed in the xy plane as generated by Amira 3-D visualization software: isosurfaces generated in 3-D enclose all voxels with signal intensity > 90 in 8-bit images. Red surfaces were generated from the
VAT signal; green from the TH signal. See text for further details. Scale bar in B applies to all panels. All images are from the same confocal z-stack (114 slices at 0.25 Lm per slice) from the lower second turn.

To quantify the ratio of TH-positive to VAT-positive profiles in the inner hair cell area, as well as the fraction of terminals expressing both markers, we performed 3-D morphometry on these confocal z-stacks using a software package (Amira) which allows rapid derivation and measurement of 3-D isosurfaces enclosing all voxels exceeding user-specified intensities (e.g.
see terminals in Figs. 2.2D-F). Based on a sample of three cochlear regions in one representative ear (at positions about 25%, 50% and 75% from the base), the volume fraction of all labeled terminals in the inner spiral bundle that were TH-positive was 23.4%, 9.1% and 14.2%, respectively. More than half of these TH-positive terminals did not co-localize VAT: the volume fraction of those TH-positive terminals that were not also VAT-positive was 77%, 94% and 58% respectively. Qualitative evaluation of dozens of other well-stained cochleas suggests that these values are representative.
A p * .
i
Figure 2.3: Comparison of xy projection (A) and a single slice (B) from a doubleimmunostained confocal z-series shows that apparent co-localization in the projection view (yellow arrows in A) often arises from superposition of signal from different focal levels and thus different terminals: the source of the green-channel (TH) signal in A is shown by the arrows in B at that focal level there is no red (VAT signal). All images are from the same confocal z-stack (114 slices at
0.25 gtm per slice) from the middle of the second turn. Scale bar in B applies to A.
Semi-quantitative evaluation of all cochlear regions of all double-stained cochleas suggested that there is no apical-basal gradient of TH-immunoreactivity along the cochlear spiral (Fig.
2.4B); the slight gradient for VAT staining (Fig 2.4A: broad mid-cochlear peak with slight dropoff towards the base and apex) is consistent with a prior quantitative analysis of LOC innervation density in mouse (Maison et al., 2003a). Note that the rating scale for each immunostain was normalized to its own maximum density, thus differences in absolute terminal densities between
VAT and TH immunostains are not captured by this analysis.

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Figure 2.4: Semi-quantitative analysis of the density of VAT-positive (A) and TH-positive (B) terminals in the inner hair cell area shows that TH immunoreactivity in control ears is uniformly distributed along the cochlear spiral and that, after LOC lesions, both types of immunoreactivity declined to a similar degree.
This analysis included 36 cochleas: the observer (blind to case history) separately rated the density of VATpositive or TH-positive puncta on a 4-point scale (see Methods) at 10 positions along the cochlear spiral in each case. Cochleas were from 17 animals with unilateral brainstem lesions: based on analysis of brainstem sections, the LOC cells were at least partially destroyed in 10/17 cases ("LOC lesion"). Control data are from the 17 opposite ears and 2 ears from animals without any brainstem surgery. Mean data (±SEM) are shown for each group, normalized for each immunostain by dividing by the maximum mean value for that stain.
An additional feature of the difference between the distributions of VAT- and TH-positive terminals is captured by the error bars in Figure 2.4. Whereas the distribution of VAT immunoreactive terminals appears continuous, and the density of VAT-positive terminals in one cochlea changes slowly with cochlear position; the TH innervation is discontinuous, such that a localized region of high terminal density can be flanked by regions with no immunoreactivity
(Fig. 2.5). It is possible that each "hot spot" of TH innervation corresponds to the terminal arbor of a single parent fiber.
Figure 2.5: Confocal image merging the
TH signal (green) and the DIC image
(gray) shows that TH immunoreactivity in the mouse cochlea is spotty: regions of the inner spiral bundle with high density of labeled terminals (filled green arrowhead) can be flanked by regions of low density
(unfilled green arrowheads), and in turn by regions with no immunostaining (white open arrowheads). This image is the xy projection of a z stack comprising 21 steps

2.4.2. Analysis of Brainstem Origins
2.4.2.1 Brainsteni Lesions
To investigate the brainstem origins of the TH-positive fibers in the modiolus and the organ of Corti, we lesioned the olivocochlear system in two different ways. In one set of experiments, we cut the OCB at the floor of the IVth ventricle near the sulcus limitans. At this lateral position, the cuts can interrupt all fibers of the LOC and MOC systems (Liberman and Gao, 1995). In a second, larger set of experiments, we selectively lesioned the LOC system unilaterally, by stereotaxic injection of a neurotoxin (Le Prell et al., 2003). Since the projections of the LOC are almost entirely ipsilateral, this lesion should selectively disrupt the LOC innervation to only one cochlea, leaving the other ear as a control. The success of the lesions was evaluated by examination of brainstem sections stained for acetylcholinesterase, which reveals the olivocochlear bundle as well as the cholinergic cells of the MOC and LOC systems in the superior olivary complex.
The most successful OC bundle cut is shown in Figure 2.6B: the VAT staining shows the marked reduction in efferent innervation of OHCs and the inner hair cell area; the green channel shows a complete loss of TH-positive terminals in the inner spiral bundle area, without obvious diminution of the profuse TH-positive innervation of the osseous spiral lamina by beaded fibers.
These observations are consistent with the view that the TH-positive innervation of the organ of
Corti arises from the olivocochlear system, whereas the beaded fibers in the lamina are part of the adrenergic innervation arising from the superior cervical ganglion (Spoendlin and
Lichtensteiger, 1966; Terayama et al., 1966; Hozawa et al., 1989).
Unilateral LOC lesions also caused loss of both VAT and TH immunoreactivity in the ipsilateral IHC area, but without affecting the VAT-positive terminals on OHCs (or the THpositive beaded fibers in the osseous spiral lamina) in either ear, consistent with the view that the
OHC innervation arises exclusively from the MOC system. Confocal projections of efferent terminals in the organ of Corti from a representative "LOC hit' case (Fig. 2.6D) can be compared with the place-matched cochlear region from the ear opposite the lesion (Fig. 2.6C). Results of the semi-quantitative analysis of all cochleas from the LOC lesion study, by an observer blind to the surgical history of each case, further confirm the view that both the VAT- and the THpositive terminals in the IHC area originate from the LOC cells located in the ipsilateral LSO; whereas the VAT-positive terminals in the OHC area arise from MOC cells located more medially in the brainstem. For both stains, the differences in density ratings in the IHC area for the lesioned vs control (opposite) ears was statistically significant by 2-way ANOVA (TH: p=0.001, F=12.755; VAT: p< 0.000, F=18.733), while there was no change in VAT-positive terminals in the OHC area (p=0.595, F=0.288; data not shown). For those cases in which the neurotoxin injection missed the LOC, differences in TH and VAT terminal densities between the injected and control sides were not significant by 2-way ANOVA (TH: p=0.206, F=1.683; VAT: p=0.245, F=1.398; data not shown).

Figure 2.6: Double-immunostaining for TH (green) and VAT (red) in a control cochlea (A,C) and the lesion-side cochlea (B,D) after either (B) section of the olivocochlear bundle , or (D) injection of neurotoxin into the LSO. A is from a control cochlea showing a few TH-positive (green) swellings (e.g.
green arrow) among the VAT-positive (red) terminals in the inner spiral bundle (ISB). B is the placematched cochlear region from the partially de-efferented opposite ear, showing loss of VAT-positive terminals in ISB and outer hair cell (OHC) areas, loss of TH-positive terminals in the ISB area, and no obvious loss of TH-positive beaded fibers in the osseous spiral lamina (OSL). Both images are from the lower second turn and comprise xy projections of confocal z series spanning 33 40 gm of focal depth in 0.5 gm steps. Scale bar in A applies to B. C shows TH and VAT immunostaining of the organ of
Corti a control ear; while D shows the place-matched cochlear region from the opposite ear, which was ipsilateral to a neurotoxin injection which hit the LSO. Both images are from the upper basal turn and comprise xy projections of confocal z series. Scale bar in D applies to C.
To confirm and clarify the origins of this putative dopaminergic innervation of the mouse cochlea, we examined immunostained sections of the olivary complex for presence or absence of
TH immunoreactivity. As illustrated by the micrographs in Figure 2.7, there were a small number of immunoreactive cell bodies located within or immediately around the lateral superior olive (LSO). In two cases, we evaluated serial sections through the LSO and counted the total number of TH-positive cell bodies on both sides: we found a total of only 62 TH-positive cells in the four LSOs combined. Of these, 47 were in the "shell" region on the outer perimeter of the
LSO (Warr et al., 1997) and only 15 were within the LSO itself. In one case, of the 7 "intrinsic" neurons found, 6/7 were in the (high-frequency) medial limb and only one was in the (lowfrequency) lateral limb. In the other case, they were more evenly distributed. As shown by the images in Figures 2.7 and 2.8, TH-positive shell neurons tended to be found lateral to the LSO.

Figure 2.7: TH-positive cell bodies (white arrows) located within and around the LSO.
An approximate outline of the LSO is indicated by the dashed lines, and the location of lateral and medial limbs are indicated. Image is an xy projection of a zstack through one 15 ýtm section acquired on a conventional light microscope (0.5 [tm
/slice).
To further confirm the general lack of co-localization of cholinergic and dopaminergic markers seen in the cochlea, we double-stained brainstem sections for acetylcholinesterase
(histochemically) and TH (immunohistochemically). As shown in Figure 9, there appeared to be little overlap: cell bodies and axons which were TH-positive were not strongly positive for the cholinergic marker and vice versa.
Figure 2.8: Double-staining (AChE red and TH green) of brainstem sections through the superior olivary complex suggests that the TH-positive innervation of the cochlea arises from a separate population of noncholinergic neurons, in the 'shell' around the LSO. Panel A shows an AChE-stained section of mouse brain: filled arrows point to three of many AChE-positive cholinergic neurons within the LSO. One of many AChEpositive axons encircling the LSO is indicated by the unfilled arrow. Panel B three TH-positive cells (filled arrows) and one TH-positive axon (unfilled arrow) are highlighted. Panel C merge of images in Panels A and
B shows the complementary distribution of AChE and TH signal in both cell bodies and axons. The AChE was visualized with a histochemical stain producing a black reaction product; the TH was visualized with a fluorescent antibody. The AChE image is shown as red to simplifying merging of the images.

2.5
2.5.1 Cochlear terminations of dopaminergic neurons
The two major catecholamine neurotransmitters, dopamine and noradrenaline, are both present in fiber :systems projecting to the inner ear. As will be described below, evidence suggests that the former is present in fibers originating in the superior olivary complex and comprising part of the olivocochlear bundle; whereas the latter is found in fibers originating in the superior cervical ganglion and comprising the sympathetic innervation of the inner ear.
Since the 19610's, histochemical techniques at both the light- and electron-microscopic level have been used to identify an adrenergic innervation of the inner ear in cat, guinea pig rabbit and squirrel monkey; this sympathetic innervation originates in the superior cervical ganglion, projects exclusively to the ipsilateral ear and terminates in a plexus of vessel-associated and vessel-independent fibers in both the modiolus and the osseous spiral lamina (Terayama et al.,
1966; Spoendlin and Lichtensteiger, 1967; Hozawa et al., 1989). There is widespread agreement among neuroanatomical studies that these adrenergic fibers do not enter the organ of Corti; rather, they appear to make synaptic contact with the fibers of the auditory nerve in the region near the habenula perforata (Densert and Flock, 1974) in all mammals, as well as within the spiral ganglion in monkey (Hozawa and Kimura, 1990). The adrenergic fibers presumably correspond in the present study to the rich innervation of the osseous spiral lamina and modiolus by beaded TH-positive fibers.
Since the 1980's, immunohistochemical evidence has accumulated for a population of dopaminergic neurons that originates in the superior olivary complex and, as part of the olivocochlear bundle, projects to the inner spiral bundle where the fibers send branches or en passant swellings to contact unmyelinated dendrites of the auditory nerve (d'Aldin et al., 1995).
Since dopamine is a precursor of noradrenaline, both fiber types are immunopositive for tyrosine hydroxylase (TH), whereas only adrenergic fibers are immunopositive for dopamine 0 hydroxylase (DBH). Thus, the presence of TH-positive, DBH- negative fibers in the organ of
Corti has provided anatomical evidence for a dopaminergic innervation in the guinea pig (e.g.
(Jones et al., 1987; Niu and Canlon, 2002; Mulders and Robertson, 2004) and now in the mouse
(present study).
In the present study, we showed that surgical interruption of the olivocochlear bundle at the floor of the fourth ventricle, or stereotaxic destruction of the lateral superior olivary complex, resulted in loss of TH-immunolabeling in the organ of Corti, without reducing the rich plexus of
TH-positive fibers in the osseous spiral lamina (Figs. 2.6 B,D). These observations are consistent with the idea that the adrenergic innervation of the modiolus does not extend into the organ of Corti, and that the TH-positive fibers in the organ of Corti are part of the olivocochlear efferent system, in particular part of the lateral olivocochlear system.
All studies of TH-positive fibers in the organ of Corti agree that such fibers are restricted to the inner and tunnel spiral bundles, i.e. they do not appear to project to the outer hair cell region.
Two previous studies in the guinea pig address the question of an apex-to-base gradient in the density of the TH-positive innervation (Niu and Canlon, 2002; Mulders and Robertson, 2004).
Although neither previous study quantified the gradient, both suggest that TH-positive terminals are more numerous in the basal turn, and sparse in the apical turn. These results in guinea pig are nominally in contrast to those from the mouse, where the distribution of TH-positive terminals was quite uniform from base to apex (Fig. 2.4). However, the fact that the mouse is a highfrequency mammal and that the apicalmost point on its cochlear spiral is tuned to -4.0 kHz

(Muller et al., 2005), which in the guinea pig corresponds to the upper basal turn (Tsuji and
Liberman, 1997), suggests the hypothesis that a dopaminergic innervation is important for highfrequency regions of the mammalian cochlea in an absolute (> 3 kHz), rather than a relative
(apex vs. base) sense.
No previous study of the dopaminergic innervation of the organ of Corti has addressed the important issues of 1) colocalization of TH with other efferent-terminal markers or 2) the relative numbers of TH-positive terminals compared to the total numbers of olivocochlear terminals in the inner hair cell area. In the present study, we found the volume of TH-positive terminals to be only 10-25% of the volume of VAT-positive terminals. We used VAT, a cholinergic marker, because a previous study in mouse (Maison et al., 2003a) found 1) that cholinergic, GABAergic and peptidergic markers were all co-localized in terminals of the inner spiral bundle, and 2) that VAT-positive terminal areas were similar to terminal areas immunostained by SNAP25, a marker of synaptic vesicles expected to label all efferent terminals. However, no double-staining studies were done to determine if a small VAT-negative
/ SNAP25-positive subset existed.
Combining the results of the present study, with the previous co-localization study in mouse suggests that there may be two cytochemical subgroups within the LOC system: one large group of fibers that co-localizes ACh, GABA and CGRP, and a smaller group of fibers that may be solely or largely dopaminergic and that projects mainly to the inner hair cell area throughout the cochlear spiral. Such a view is not inconsistent with a previous co-localization study in guinea pig and rat which also concluded that, in the cochlear terminals of OC fibers, ACh, CGRP and opioid peptides were also extensively co-localized (Saffiedine and Eybalin, 1992).
The present study showed that a small fraction of TH-positive terminals also showed some
VAT immunoreactivity. suggesting there is not an absolute distinction between dopaminergic and cholinergic neurons, rather there might be a continuum of expression levels. Nevertheless the degree of separation between the dopaminergic innervation and the cholinergic innervation is much more robust than that between the cholinergic and GABAergic, for example, for which the expression patterns in cochlear terminals overlapped completely (Maison et al, 2002).
The data on TH-immunoreactivity in the lateral superior olive are largely consistent with the view afforded by evaluation of the immunostained cochlea in both mouse (the present study) and guinea pig (Mulders and Robertson, 2004).
In mouse, we found a small number of TH-positive neurons in and around LSO: an average of only 16 per side. The estimated total number of LOC cells in mouse (n=310), derived from a study of retrograde cochlear transport of HRP (Campbell and Henson, 1988), suggests that the
TH-positive cell bodies comprise ~ 5% of the LOC system. Such an estimate is not inconsistent with the estimates derived from comparison of the cochlear innervation densities, although they suggest that the TH-positive cells may tend to be more highly branched than the cholinergic neurons (an inference that is also consistent with other lines of argument presented below).
In guinea pig, the numbers of TH-positive LSO cells are greater, in both absolute and relative terms; however, the idea that they constitute a small fraction of the total LOC system is supported by two studies. One group (Mulders and Robertson, 2004) counted TH-positive cells in alternate sections of two LSOs from guinea pig after labeling LOC neurons with a retrograde tracer injected into the cochlea. They report that only a "small number" of LOC cells (i.e. tracerpositive cells in the LSO region) were TH positive, but that almost all of the TH-positive cells

were also tracer positive (i.e. part of the olivocochlear system) and that they tended to be found
"at the edge of the LSO". In all, 92 TH-positive cells were counted in one animal, and 111 in another, in every second section. If we multiply by 2 (ignoring the double-count correction which must be significant here), we get at most -200 TH-positive neurons. According to another retrograde label study, the total LOC population in one guinea pig LSO is ~ 1300 (Aschoff and
Ostwald, 1987); thus, the TH-positive subset constitutes, at most, just over 15%. In another study of LOC cells identified by retrograde transport from the cochlea, the total number of THpositive LOC cells was found to be -155 per side and the fraction of total LOC neurons that this number represented was estimated at 35% (Niu et al., 2004).
These observations from single-antigen immunolabeling coupled with retrograde transport to identify LOC neurons are hard to square with data on triple-immunostaining of LSO neurons for cholinergic (anti-ChAT), GABAergic (anti-GAD) and catecholaminergic (anti-TH) markers by
Saffeidine and colleagues (Safieddine et al., 1997) studying rats and guinea pigs. These studies, which did not use retrograde tracing techniques to identify OC neurons directly, concluded that
94% of neurons in the LSO regions that were immunopositive for ChAT were also immunopositive foir GAD and TH. One way to resolve the two sets of studies is to hypothesize that large numbers of cholinergic LSO cells are not part of the OC system. However, that hypothesis is not consistent with reports of Vetter and colleagues in the rat (Vetter et al., 1991).
The majority of TH-positive neurons in the present study were located near the edges, or just outside the LSO borders, and thus may correspond to LOC "shell" neurons. As extensively studied by Warr and colleagues (Warr et al., 1997; Sanchez-Gonzalez et al., 2003; Warr and
Boche, 2003), LOC cochlear projections appear to be of two major subtypes: 1) a large population of small intrinsic neurons, located within the LSO proper, and 2) a smaller population of shell neurons, located around the LSO border, which tend to be larger in size. In rat, intrinsic shells outnumber shell neurons by ~10:1; data are not available for mouse (Sanchez-Gonzalez et al., 2003). Anterograde tracer studies further suggest that shell neurons give rise to highly branched peripheral projections which may spiral to innervate more than half of the cochlea's length, in contrast to intrinsic LOC neurons, which give rise to projections with more restricted trajectories within the inner spiral bundle (Brown, 1987).
Thus, both by cell location and cell proportions, the present data are consistent with the idea that dopaminergic cochlear innervation arises largely from LOC shell neurons. However, it does not appear likely that all shell neurons are dopaminergic. Vetter and colleagues report that about half of the shell LOC neurons, identified by retrograde HRP transport from the cochlea, immunostain for cholinergic or GABAergic markers; whereas about half stain for neither (Vetter et al., 1991). If half of shell neurons in rat are dopaminergic, and if shell neurons comprise about
10% of the total number of LOC cells in rat (Sanchez-Gonzalez et al., 2003), then dopaminergic
LOC cells would constitute about 5% of the total LOC somata, closely corresponding to the estimates derived above for mouse. If, furthermore, these relatively rare dopaminergic shell neurons are particularly highly branched relative to the more common intrinsic (cholinergic) neurons, the observed ratios of immunolabeled dopaminergic to cholinergic terminals in the inner spiral bundle should be significantly amplified, as indeed they were (to values of 9-24%).
2.5.3 Functional significance of a cochlear dopaminergic innervation
The evidence that the lateral olivocochlear efferent system contains a functional dopaminergic innervation now derives from many sources. Beyond the histochemical and immunohistochemical evidence described in the previous section, there is biochemical evidence

showing electrically evoked release of dopamine from the cochlea which can be blocked by tetrodotoxin (Gaborjan et al., 1999); there is pharmacological evidence showing that cochlear dopamine perfusion decreases spontaneous and sound-evoked activity in the auditory nerve
(Ruel et al., 2001); and there is RT-PCR data showing cochlear expression of several dopamine receptors in the cochlea (Karadaghy et al., 1997), although there are currently no data on the tissue localization of such receptors.
A recent electrophysiological study suggested that electrical activation of the LOC system by shocking the inferior colliculus can either decrease or increase the amplitude of the cochlear neural potentials, depending on where the stimulus was applied within the colliculus (Groff and
Liberman, 2003). Since these neural effects were seen without changes in outer hair cell function
(i.e. DPOAE magnitude), it was suggested that they arise from interactions between the LOC terminals and their peripheral targets the auditory nerve fibers and/or inner hair cells. It was further suggested that there might be two functional subgroups of neurons within the LOC system. Data from the present study suggest the obvious possibility that the suppressive effects arise by selective activation of the dopaminergic system, whereas the excitatory effects arise by activation of the system which colocalizes ACh, GABA and CGRP.
One interesting hypotheses concerning the functional significance of this dopaminergic innervation is that it acts to minimize glutamate-like excitotoxicity at the inner hair cell/ afferent synapse after acoustic overexposure (Pujol and Puel, 1999). This excitotoxic effect of acoustic overstimulation is seen as a marked swelling of afferent dendrites during the acute phase of the response to the insult (Liberman and Mulroy, 1982; Robertson, 1983). It has been shown that cochlear perfusion of dopamine-receptor blocker eticlopride can result in a similar type of dendritic vacuolization under the inner hair cells (Ruel et al., 2001) and that acoustic overstimulation can lead to depletion of TH immunoreactivity in the cochlea (Niu and Canlon,
2002). It has further been shown that selective destruction of the LOC system in mouse by the same types of stereotaxic injections used in the present study leads to an enhanced vulnerability to the acute effects of acoustic overexposure (see chapter 4) : incorporating results from the present study would suggest that this protective effect of the LOC system arises from the small cytochemically distinct population of dopaminergic fibers.

The synapse between the auditory nerve and cochlear inner hair cells (IHCs) is responsible for relaying afferent information from the periphery to the central nervous system. The principal cells of the lateral superior olivary (LSO) complex, located within the mammalian brainstem, receive this ascending information from both ears and in-turn calculate sound location in the azimuthal plane. Interestingly, the LSO complex is also where lateral olivocochlear (LOC) neurons originate and project to the ipsilateral cochlea to innervate both the auditory nerve and
IHC soma. Immunohistochemical, pharmacological and physiological studies suggest that this
LOC feedback consists of (at least) two subgroups capable of enhancing or inhibiting auditory nerve output. Here, we explore LOC function in vivo via selective stereotaxic removal of LOC cell bodies in mouse. LOC lesions were verified histologically by analyzing the brainstem and cochlear whole-mounts in each surgical case. In both control and sham surgery mice (when the injection missed the LOC cell bodies) there was a strong tendency for the neural output of the two ears to be bilaterally symmetric at all cochlear frequencies. In cases where the LOC system was disrupted, this interaural correlation was lost. These lesion-induced neural asymmetries were not observed in OHC-based measurements. It appears that the data in this report, in combination with studies that have demonstrated a) that LSO principal cells compute sound level differences at the two ears (Boudreau and Tsuchitani 1968) and b) that the LOC system can modulate auditory nerve activity (Groff and Liberman 2004), suggests that a function of the LOC system is to maintain bilateral symmetry and ensure that LSO principal cells receive a balanced representation of sound from each ear.
The lateral superior olivary (LSO) complex of the mammalian brainstem has two distinct features: 1) it is a major point of converging afferent information from both ears and 2) neurons of the lateral olivocochlear (LOC) efferent system originate from the LSO. This feedback loop is a means by which the brain can regulate cochlear function. The LSO is a tonotopically organized nucleus whose principal cells receive inputs from the two ears. These inputs relay information about the differences in sound level at the two ears; interaural level differences (ILD) are a principal cue for sound localization of high-frequency stimuli in the azimuthal plane. This cue is processed by LSO principal cells that receive inhibitory inputs from the contralateral ear and excitatory from the ipsilateral ear.
The unmyelinated LOC efferent system innervates almost exclusively the ipsilateral cochlea: its primary target is the afferent dendrites of auditory nerve fibers located at the base of the inner hair cell (IHC, see figure lb), however sparse innervation of IHC soma has also been observed
(-15%, Liberman 1980). At least two cytochemical subgroups of LOC terminals have been localized in the IHC area of mice; and while it is not clear if these two groups differentially

target the auditory nerve dendrites vs. the IHC soma (Darrow et al. 2006b), they have been proposed to represent the two LOC-based effects which appear capable of enhancing or inhibiting auditory nerve activity (Groff and Liberman 2004). In addition to the LOC system, there is a separate medial (M)OC system. The myelinated MOC component controls the gain of the cochlear amplifier by affecting the function of its terminal targets, the outer hair cells (OHC).
In contrast to the LOC, the MOC has a bilateral innervation pattern and primarily innervates the contralateral cochlea (see figure 3.1A,B).
The neurons within the LSO complex, both the LSO principal cells and the LOC cells, appear positioned to compare inputs from the two ears and subsequently regulate auditory nerve output. This feedback loop may play a crucial role in the ability of the LSO principal cells to compute sound location. If LSO principal cells are expected to correctly compare their bilateral inputs and relay information about sound location, then there must be a means by which the ascending inputs are regulated. The mediator of this bilateral symmetry may be the LOC system.
We propose that LOC cells are capable of integrating information from the two ears over long time periods, thus allowing the LOC system to make slow adjustments to the output of each ear to keep them bilaterally balanced.
To test the hypothesis that the LOC system is important for regulating auditory nerve output in the two ears, we used a stereotaxic approach to create a unilateral LSO lesion (Le Prell et al.
2003) in adult mice and subsequently compared auditory nerve activity in both ears of LOC deefferented mice, as well as in both ears of sham-surgery and control mice.
I
A i
Figure 3.1: Anatomical schematic of central (A) and peripheral projection patterns of the OC system in mouse. A) Almost all LOC cell bodies originate in the ipsilateral LSO; whereas MOC cell bodies are located in the contralateral (75%) and ipsilateral (25%) peri-olivary nuclei. B) The main targets of the
MOC are the OHC soma; whereas the LOC's primary target is the afferent terminal of the Type I auditory nerve fiber, with sparse innervation of the IHC soma. Bold arrows indicate the direction of action potentials.

3.3.1 Stereotaxic Surgery: Experimental animals were CBA/CaJ mice aged 6-8 weeks, weighing between 25-30g, and of either sex. Following anesthesia (xylazine 20 mg/kg i.p. and ketamine 100 mg/kg i.p.) the mouse was held in a Kopf small-animal stereotaxic apparatus by snout clamp and ear bars. Care was taken to ensure the position of the skull was flat in both the vertical and horizontal planes. The skin overlaying the skull was slit and retracted to reveal bregma and lambdoidal sutures. Rongeurs were used to make a hole in the skull located .488mm
caudal and .12mm lateral re: the bregma suture. A micropipette filled with a solution of melittin in saline was lowered -0.688mm re: the height of the bregma suture. Once the pipette reached the appropriate penetration depth, an injection of 2gl was made with a 10tl syringe (Hamilton) attached to the mrnicropipette. Immediately after injection the scalp was sutured and the animal placed in a padded cage with heat lights for ~ hour post surgery.
3.3.2 Histological analysis and immunocytochemistry: After final testing, all animals were perfused intracardially with 10% formalin. The brainstems were extracted, post-fixed overnight, cryoprotected in sucrose, frozen and cut on a sliding microtome at 80 jim in the transverse plane.
Slide-mounted sections were stained for acetylcholinesterase (AChE) activity to allow for visualization of the cholinergic LOC cells in the LSO, as well as to verify that the microelectrode pipette did not sever the OC bundle. The success of the LSO lesion was quantified by using a drawing tube to trace the outline of surviving LSO cells in all sections through the LSO on both injected and control sides, from its caudal to rostral extreme. Tracings were digitized, and the areas of medial and lateral limbs were determined by computerized planimetry. Cochleas were extracted, post-fixed overnight, decalcified in EDTA for -48 hrs and dissected into half-turn segments. Sections were incubated in primary antibody overnight (rabbit anti-VAT: vesicular acetylcholine transporter) followed by a fluorescently labeled secondary antibody (Alexafluor
568). A cochlear frequency map was computed for each cochlea by measuring the individual cochlear segments from base to apex (Muller et al., 2005). Fractional survival of VAT-positive terminals was analyzed semi-quantitatively by an observer blinded to case history and true case number. Separate rating scales were used in IHC and OHC areas. A 3-point scale was used for
VAT-positive terminals in the OHC area: the observer's task was to estimate the fraction OHCs with at least one VAT-positive terminal: 3 = 100-66%, 2 = 66-33%, 1 =33-0%. VAT-positive terminals in the IHC area were evaluated with a 3-point scale: 3 = profuse, 2 = moderate and 1= sparse.
3.3.3 In vivo functional assays: For ABR and DPOAE measurements, mice were anaesthetized with xylazine and ketamine. For ABR, needle electrodes were inserted at vertex and pinna. ABR was evoked with 5-ms tone pips (0.5-ms rise-fall, with a cos2 envelope, at 35 per/s). The response was amplified (10,000x), filtered (0.1-3 kHz), and averaged with an A/D board in a
PC-based data-acquisition system (Labview). Sound level was raised in 5-dB steps from 0 to 80 dB SPL. At each level, 1,024 responses were averaged (with stimulus polarity alternated) after
'artifact rejection'. Threshold was determined by visual inspection. The DPOAE at 2fl-f2 was recorded in response to two primary tones: fl and f2, with f2>fl by 1:2 and the f2 level 10 dB lower than the fl level. Ear-canal sound pressure was amplified and digitally sampled at 4gts intervals (16 bit DAQ boards, NI 6052E National Instruments. Fast-Fourier transforms were computed from averaged waveforms of ear-canal sound pressure, and the DPOAE amplitude at

2fl-f2 and surrounding noise floor were extracted. Iso-response contours were interpolated from plots of amplitude versus sound level, performed in 5-dB steps of fl level. Threshold is defined as the fl level required to produce a DPOAE at 5 dB SPL. All data presented in this report are from the 4-week testing interval.
3.4 Results
3.4.1 Assessment of Lesion Success
Stereotaxic lesion of the LSO via injection of melittin (a neurotoxin), as previously performed in guinea pigs (Le Prell et al. 2003), was attempted in CBA/CaJ mice. Thirty-one mice survived stereotaxic surgery, and six mice served as uninjected controls. Since acetylcholine (ACh) is a common neurotransmitter of both the MOC and LOC systems, the success and selectivity of the lesion were measured by using cholinergic markers in both the brainstem and cochlea to determine: 1) if LOC cell bodies were destroyed and if there was a reduction of efferent terminals in the IHC area and 2) if MOC cell bodies were destroyed, and if there was a reduction of MOC terminals under the OHCs.
When the neurotoxin successfully targeted the LSO, there was a clear loss of cholinergic neurons from the LSO area and a clear disruption of the LSO neuropil (figure 3.2A-D). Based on the projections of the LOC system, a successful "Hit" should yield a reduction in the number of efferent terminals in the IHC area of the ipsilateral cochlea. Indeed, in each case where the injection successfully disrupted at least a portion of the LSO (n=17), there was a noticeable decrease in LOC terminals in the IHC area of the ipsilateral ear (figure 3.2C,D). The average loss of terminals in the ipsilateral IHC area of Hit cases was approximately 50% from the base to apex of the cochlea; thus termed a successful "LOC Hit" case. The correlation between LSO
"Hit" and "ISB Hit" was nearly perfect with exception to one case in which the LOC terminals were missing in the ipsilateral cochlea with no discernable effect of the injection on the LSO; perhaps due to damage of LOC fibers.
Since the integrity of the MOC system is important to the interpretation of the results, we measured MOC terminals in the OHC area from both cochleas in each surgical case. In most cases, the injection did not impinge upon MOC cell bodies; thus OHC efferent terminals were, in general, present in equivalent numbers in each ear (figure 3.2C,D). There were two exceptions: in one case that successfully targeted the LSO, as well as one that did not, there appeared to be damage to a small subset of MOC neurons; accordingly these two cases had a slight reduction of terminal density in the OHC area of the contralateral ear (data not shown).

(nntrl nide Iniected side
Figure 3.2: Histological verification of LOC lesions, as seen in AChE-stained brainstem sections (A,B) or in cochlear whole mounts immunostained for a cholinergic marker (VAT; C,D). A,B: Brainstems are from opposite sides of one LOC Hit case. Dashed lines indicate the outline of the surviving LSO in each section: on the control side, lateral and medial limbs are indicated. The AChE-positive fibers of the VIIth nerve, visible in each section, were used to identify comparable rostro-caudal locations on the two sides. Scale in B also applies to A. C,D: images are from the 22.6 kHz region of opposite sides of a LOC Hit case (different from the one shown in A and
B. Scale in D also applies to C.
3.4.2 Effect of LOC De-efferentation on Cochlear Function
Cochlear function was assessed at 2- and 4-weeks post surgery. Auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were recorded bilaterally and compared across surgical groups and across ears within each animal. The LOC and MOC neurons have different cochlear innervation patterns: the MOC system contacts the
OHCs while the LOC system terminates in the IHC area (see fig 3.1B), thus measuring both
DPOAE and ABR can give insight to the function of these two terminal targets. The ABR represents synchronous electrical activity generated by neurons in the ascending auditory system in response to short tone bursts. In particular, Wave 1 of the ABR represents the sum of auditory nerve potentials in response to sound. The distortion product otoacoustic emission (DPOAE) assay provides a measure of OHC amplification. Mechanical distortions are created in the inner ear when two primary tones (fl and f2) are presented. These distortions are amplified by OHCs and propagated back through the middle ear to the ear canal where they are measured in the sound pressure waveform. Both the ABR and DPOAE were assessed at various frequencies to test cochlear function across the range of mouse hearing.
Unilateral LSO lesion did not alter cochlear thresholds; however, ABR amplitudes were enhanced ipsilaterally at suprathreshold levels, without changes in suprathreshold DPOAEs

(Figure 3.3A-D). Threshold and level-series were measured in each ear of both surgical groups
(Figure 3.3A-D), as well as in uninjected age-matched mice. Disruption of the LOC innervation to one ear can lead to a complementary shift in auditory nerve output: a) auditory nerve activity increases in the ipsilateral ear (the LOC de-efferented ear) and b) auditory nerve activity appears to decrease in the contralateral ear (the ear with normal LOC innervation). These neural asymmetries in suprathreshold cochlear neural responses (ABR wave 1) were seen in the majority of LOC Hit cases; such inter-aural asymmetries in neural responses were not seen in the
LOC Miss or un-injected controls. Also, when the data are calculated as a percent difference at
50, 60, 70 and 80 dB (dashed circle in figure 3.3C), for each case, and then averaged across cases within each surgical group, there appears to be a level independent affect on cochlear function in
LOC Hit cases (insert Figure 3.3C).
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ABR (A) or DPOAE (B).
However, suprathreshold auditory nerve responses, as measured by Wave I amplitude, are increased in the ipsilateral ears of LOC Hit cases (C). The interaural percent differences at
50, 60, 70 and 80 dB for LOC
Hit ears (insert in Panel C) indicate that ipsilateral enhancement is level independent. Key in panel A also applies for B, C and D.
Mean data ±SEM from the combined 2 and 4 week postsurgery test times are shown in panels A-D.
Viewed differently, in figures 3.4A-D the data from both ears of both surgical groups are referenced to the mean non-surgical controls and the data from each ear, at each frequency, is collapsed across levels (mean value is calculated at 60-80 dB, see insert Fig. 3.3C). The complementary shifts of neural output from each LSO Hit case appear to be frequency independent in both the contralateral and ipsilateral ears. Although there is significant scatter in
ABR amplitudes of the LOC Miss ears (figure 3.4C,D), there is no consistent trend towards excitation or inhibition in either ear.
When the data from each ipsilateral ear is referred to its contralateral counterpart, the results reveal the robust bilateral symmetry seen in mice with an intact LOC system (figure 3.4F). On average, there was a 40% difference between ipsilateral and contralateral ears of LOC Hit cases; these differences were not observed in the DPOAE data (data not shown). Despite the variation in Wave 1 ABR amplitudes observed in the LOC Miss ears (figure 3.4C,D) an intact LOC system

maintains bilateral symmetry between the two ears (figure 3.4F); whereas disruption of the LOC system results in loss of this interaural correlation (figure 3.4E).
Aspects of the LOC-based effects described above have been seen before (Groff and
Liberman 2003): 1) frequency independence LOC innervation is relatively uniform from base to apex, 2) level independence LOC based effects on auditory nerve activity are a constant percentage at low and high sound pressure levels (see figure O1C insert) and 3) effects on neural responses only peripheral effects of the LOC system are only observed in the neural response and not in OHC-based measures (compare figures 3.3C,D). However, by studying the cochlear function in both ears of LOC lesioned mice, we have also demonstrated that a robust bilateral symmetry of neural output is present in animals with normal OC innervation, and that this interaural correlation is lost if LOC feedback is disrupted.
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10
6 8 10 30
Test Frequency (kHz)
50
Test Frequency (kHz)
-1 00 -
6 8 10
LOC Miss
30 50
Test Frequency (kHz)
Figure 3.4: Changes in ABR amplitudes as a function of test frequency for all injected mice: LOC Hit cases in the top row (A,C,E) and LOC Miss cases in the bottom row (B,D,F). In the first two columns (A,B,C,D) amplitudes are referred to average amplitudes in non-surgical controls; the first column (A,B) for contralateral ears, the second (C,D) for ears ipsi to injection. In the third column (E,F), data from the first two columns are re-plotted: within each animal,
Wave I amplitudes in the ipsilateral ears are compared to the contralateral ears. The single value plotted for each case, for each frequency, represents the mean fractional change in ABR amplitude averaged over the highest three levels presented (60, 70 and 80 dB SPL). Key in panels A and C also apply to B and D, respectively.

3.5
3.5.1 The OC System and its Influence on Cochlear Physiology
Since the discovery of the OC system (Rasmussen 1946) different techniques, including electrical stimulation and surgical lesions, have been used to study its role on cochlear physiology. While these studies have provided a wealth of information about the MOC system and its effects on cochlear physiology, it is only recently that any effects directly attributable to the LOC system have begun to emerge (Groff and Liberman 2003; Le Prell et al. 2003).
Many studies that have stimulated the crossing MOC fibers while making simultaneous measurements of cochlear physiology. In summary, these studies have demonstrated the ability of the MOC system to decrease OHC contribution to cochlear partition movement (Mountain,
1980; Murugasu and Russell, 1996), and that this reduction is reflected in "downstream" IHC and auditory nerve responses. Evidence from both in vitro and in vivo studies suggests that the
MOC's suppressive effects arise by modifying OHC electromotility (Murugasu and Russell
1996; Dallos et al. 1997; Cooper and Guinan 2003). Recent work with the MOC's a9 nAChR knockout mouse (Vetter et al. 1999; Maison et al. 2002) has confirmed the earlier conclusion of
Gifford and Guinan (1987): all the effects observed with midline stimulation are mediate via the
MOC system.
In contrast to the MOC system, it has proven difficult to study the unmyelinated LOC system by way of direct electrical stimulation (Gifford and Guinan 1987). However, a series of lesion experiments that section the OC innervation to one cochlea (both the MOC and LOC) have provided insight to the LOC's role in regulating auditory nerve activity. These studies suggest that the LOC can modulate both spontaneous and sound-driven discharge rates of auditory nerve fibers. Removing the OC bundle yields little change in thresholds or tuning (Q10 values); suggesting that LOC or MOC innervation is not required for normal OHC function. However, auditory nerve fibers do undergo a substantial compression of spontaneous activity towards lower rates (Liberman, 1990; Zheng et al. 1999). This overall decrease in spontaneous rates is consistent with a post-synaptic effect of the LOC innervation on auditory nerve dendrites rather then the MOC system's effect on the mechanical OHCs.
More recently, descending projections from the IC to the LSO complex (van Noort 1969) were exploited to indirectly stimulate the LOC system and study its effect on cochlear output
(Groff and Liberman 2004). In this study both OHC-based and neural-based summed potentials were recorded with vs. without electrical activation. Long-lasting effects on neural activity, which were not observed in OHC-based recordings (DPOAEs), were consistent with LOC activation, and not of the MOC system (Groff and Liberman 2004). It appears that stimulation of the LOC system is capable of increasing or decreasing auditory nerve output over long time periods (t ~ 10 min); whereas MOC effects only last for tens of seconds (,t - 25-50 sec). These
LOC-based effects on neural output were both level- and frequency-independent: changes in auditory nerve output were a constant percentage at low and high sound levels, and these changes were the same percent across all cochlear frequencies. In contrast, MOC effects are both level- and frequency-dependent: reduction of OHC-based activity is largest at low sound pressure levels and the degree of these effects are closely tied to the non-monotonic innervation of MOC terminals throughout the cochlea (Maison et al. 2003a).
In accordance with the dual effects of LOC stimulation on auditory nerve activity, the LOC system has (at least) two types of neurons (see Vetter and Mugnaini 1992 and Warr et al. 1997

for review): 1) small intrinsic neurons that each have an average of two dendritic arbors extend in opposite directions to span the LSO neuropil and 2) large shell neurons whose dendrites extend deep into the LSO. Although both groups of LOC cells primarily innervate the ipsilateral cochlea, the intrinsic neurons course unidiredctionally along the cochlear spiral to terminate on small sections of the IHC area whereas shell neurons course bidriectionally to terminate over large portions of the IHC area. These two LOC neurons also appear cytochemically distinct: intrinsic terminals co-localize ACh, GABA, and CGRP (Maison et al. 2003) and shell neurons contain DA (Darrow et al. 2006b).
The present study used a stereotaxic technique adapted from Le Prell et al. (2003) to examine
LOC function in mice with chronic destruction of LOC cell bodies on one side of the brainstem.
We assessed the functional consequence of this unilateral LOC lesion on cochlear output in both ears. In doing so, we have demonstrated two striking results: 1) the robust bilateral symmetry of auditory nerve output is lost in mice with unilateral disruption of the LOC system, and 2) removal of LOC innervation to one ear can result in complementary shifts of auditory nerve output from the two ears (figure 3.4a-b). In LOC de-efferented ears there is a clear increase in neural output at suprathreshold sound levels re the opposite ear. Although this net increase in auditory nerve activity contrasts with the results of Le Prell et al. (2003), which showed a net decrease of neural output in LOC de-efferented ears, they are similar to the increase in sounddriven discharge rates of auditory nerve fibers in OC sectioned chinchillas (Zheng et al. 1999).
Accordingly, the observations in this report, that Wave 1 ABR increases without any changes in
OHC-based activity, is indicative of an effect modulated by the LOC system. Although the results in this report are qualitatively different from that of Le Prell et al. (2003) these differences may arise from a different balance of resting activation levels of the LOC subgroups in different species, as well as; potentially different affects of different anesthesia regimens.
In order to understand the complementary shifts of auditory nerve output in LOC deefferented mice, it is useful to study the circuitry of the LSO (figure 3.5). The LSO is a tonotopically organized nucleus whose principal cells receive converging information from the two ears. These principal cells have similar dendritic morphology to LOC intrinsic cells: each having an average of two to three large dendritic arbors that extend in opposite directions to reach the borders of the LSO neuropil (see Sanes et al. 1990 figure 10). The most common response property of these principal cells to sound is an "IE" response: they are inhibited (I) by input to the contralateral ear and excited (E) by input to the ipsilateral ear. The inhibitory input to the LSO originates from the globular cells of the contralateral cochlear nucleus (CN) and innervates the LSO by way of inhibitory interneurons of the medial nucleus of the trapezoid body (MNTB). The excitatory inputs to LSO principal cells arise directly from the spherical and globular cells of the ipsilateral CN.

Figure 3.5: Anatomical schematic illustrates the LOC feedback circuit. Dashed red circle indicates the lesion area. AN Auditory Nerve; IC Inferior Colliculus; LOC Lateral Olivocochlear; MNTB
Medial Nucleus of the Trapezoid Body; PC Principal Cells; VCN Ventral Cochlear Nucleus.
The observation that disruption of the LOC system results in a breakdown of bilateral symmetry, suggests that a function of the LOC system is to correct interaural disparities: e.g.
slow increases in cochlear nerve excitability will increase negative feedback ipsilaterally and, via the sign change in the MNTB, subsequently decreases negative feedback to the contralateral ear.
While the only demonstration of a direct input to LOC cells is from the ipsilateral CN
(Thompson and Thompson 1991), they, like LSO principal cells, may also receive bilateral "IE"' inputs: their cell body locations and dendritic morphology are similar to that of LSO principal cells. Although this simple circuit can explain how the LOC can maintain bilateral symmetry, it does not, per se, explain why unilateral LOC disruption resulted in complementary shifts of bilateral auditory nerve output. However, the LSO lesion may interrupt the fibers that cross ventral to the LSO that connect the ipsilateral CN to the contralateral MNTB: disrupting this pathway would remove the inhibitory input to the contralateral LOC cells and therefore result in an increase of LOC inhibition in the contralateral cochlea. Alternatively the complementary coupling of the two ears could involve a more complex circuitry involving the known bilateral ascending inputs from the LSO to the IC (Nieuwenhuys 1984) and the bilateral descending projections from IC to LOC systems (van Noort 1969; Groff and Liberman 2004).
3.5.2 The OC System and its Role in Hearing
Based on the OC-mediated effects on cochlear physiology, described above, OC feedback is thought to play a crucial role in psychophysical tasks, e.g. OC-enhanced signal detection in noise. It has long been hypothesized that the MOC's ability to turn down the gain of the cochlear amplifier enables the cochlea to detect transient signals in background noise (Dewson 1967). In a series of experiments by Kawase and colleagues (1993a,b), auditory nerve fiber responses to

sound in the presence of background noise was shown to be enhanced by activation of the contralateral sound-evoked MOC reflex. Since MOC activation can decrease the level of adaptation in the auditory nerve, and since adapted auditory nerve fibers are less responsive then un-adapted fibers (Smith 1977; 1978), in theory, MOC-induced decreases in adaptation allows for auditory nerve fibers to better respond to such transient stimuli as speech. Unlike the MOC, the role of the LOC system in providing increased signal detection in noise has yet to be directly studied. However, it is plausible that the LOC's direct connection to the IHC soma (Liberman
1980), can also reduce adaptation and allow the auditory nerve to better respond to transient signals in background noise.
Many studies have aimed to determine the role of the OC system in human hearing.
These studies have been undertaken in humans with vestibular neurotomy (VN); along with sectioning the vestibular nerve (to relieve suffering from severe vertigo) the OC bundle is sectioned to the ipsilateral ear. These patients have been tested to see how they perform on different psychophysical tasks such as tone detection, intensity discrimination, frequency discrimination and speech detection in noise (Scharf et al. 1994; 1997, Giraud et al. 1996).
Although there are hints that the OC system does provide humans with increased signal detection in noise, the exact nature of the OC system in hearing remains unknown.
Considering that both the MOC and LOC system may each receive bilateral inputs, as well as both systems possibly being capable of altering cochlear output bilaterally, directly or indirectly, it is tempting to speculate that that the OC system might play a role in bilateral hearing tasks, e.g.
determining sound location. May et al. (2004) attempted to create bilateral MOC lesions in cats and subsequently measure sound localization ability. In the weeks after surgery, the deefferented cats showed a decreased ability to locate sounds in the azimuthal plane in background noise. This result provides insight as to the role of the OC system in bilateral hearing.
Unfortunately, the contributions of the MOC vs. LOC systems to this effect are not clear based on the fact that: a) surgical sections intended to only disrupt bilateral MOC projections likely affected the neighboring LOC neurons (see Figure 2A in May et al. 2004) and b) no cochlear histology was reported to verify the success, selectivity, and extent of the lesions.
Bilateral hearing tasks, such as those assessed in May et al. (2004), cannot be assessed in our
LOC lesioned mice: stereotaxic injection destroys both the descending LOC cells and the ascending LSO principal cells. However, if one considers that a) the OC system plays an integral role in maintaining sound localization ability (May et al. 2004) and b) that the LOC system is crucial for maintaining bilateral symmetry, then a plausible hypothesis of LOC function is to maintain cochlear output such that LSO principal cells are able to correctly compute sound location.
The notion that the LOC system can act as a bilateral balancer was first introduced by Guinan
(1996): "a possible function of the lateral efferent neurons is to adjust the overall output from
each cochlea so as to maintain a balance between the inputs (to the LSO) from each ear." The present study is the first to provide evidence that bilateral symmetry does indeed exist, and that loss of the LOC system disrupts this inherent interaural correlation.

Sensory cells and neurons in the mammalian cochlea receive efferent feedback from the olivocochlear (OC) system. The myelinated medial component of the OC system and its effects on the cochlear outer hair cells (OHC) have been implicated in protection from acoustic injury.
The unmyelinated lateral (L)OC fibers target the ipsilateral cochlear nerve dendrites in the inner hair cell (IHC) area, and pharmacological studies suggest the LOC's dopaminergic component may protect these dendrites from excitotoxic effects of acoustic overexposure. Here, we explore
LOC function in vivo via selective stereotaxic removal of LOC cell bodies in mouse. Lesion success in removing the LOC, and sparing the MOC, was assessed by histological analysis of brainstem sections and cochlear whole-mounts. In cases where the LOC was at least partially destroyed, there were interaural asymmetries in suprathreshold neural responses that were frequency- and level-independent, and not attributable to OHC-based effects. These interaural response asymmetries were not found in uninjected controls, or in cases where the lesion missed the LOC. In LOC-lesion cases over-exposed bilaterally to a traumatic stimulus, temporary threshold shifts were greater in the ipsilateral ear, but only when measured in the neural response: otoacoustic emission threshold shifts were always bilaterally symmetrical, suggesting
OHC vulnerability was unaffected. Interaural asymmetries in threshold shift were not found in either uninjected controls or lesion cases which missed the LOC. These findings suggest that: 1) the LOC modulates cochlear nerve excitability, and 2) the LOC system protects the cochlea from neural damage in acute acoustic injury.
The olivocochlear (OC) bundle, a feedback pathway that regulates inner-ear function, is conserved in virtually all vertebrate hair cell systems. In the mammal, this OC system comprises two separate components (Figure 4. 1A,B): 1) myelinated medial (M)OC neurons which originate in peri-olivary nuclei and primarily (75%) cross the midline to innervate outer hair cells (OHC), and 2) unmyelinated lateral (L)OC neurons which originate from the lateral superior olive (LSO) and course to the ipsilateral cochlea to innervate auditory nerve dendrites, as well as the inner hair cell (IHC) soma (-15% of total LOC innervation; Liberman 1980). While MOC terminals appear to co-localize acetylcholine (ACh), y-aminobutyratic acid (GABA), and calcitonin generelated peptide (CGRP; Maison et al. 2003a), all known effects of this system on cochlear physiology stem from its cholinergic actions (Vetter et al. 1999). In contrast, the LOC system is a more complex and heterogeneous system consisting of at least two morphological subgroups
(Vetter and Mugnaini 1992, Warr et al. 1997; Maison et al., 2003; Darrow et al., 2006b): 1) its
"intrinsic" neurons innervate small portions of the cochlear spiral and co-localize ACh, GABA, and CGRP, whereas 2) its "shell" neurons contain dopamine (DA) and spiral for long distances under the inner hair cells. These two groups may mediate the complementary effects of the LOC system seen on auditory nerve output: indirect activation of the LOC via electrical stimulation of the inferior colliculus can enhance or inhibit auditory nerve activity, depending on the site of

A
~___-~_~_.._~_;_
Figure 4.1: Anatomical schematic of central (A) and peripheral projection patterns of the OC system in mouse. A) Almost all LOC cell bodies originate in the ipsilateral LSO; whereas MOC cell bodies are located in the contralateral (75%) and ipsilateral (25%) peri-olivary nuclei. B) The main targets of the
MOC are the OHC soma; whereas the LOC's primary target is the afferent terminal of the Type I auditory nerve fiber, with sparse innervation of the IHC soma. Bold arrows indicate the direction of action potentials.
Many studies have implicated this OC feedback pathway in protecting the cochlea from acoustic injury: electrical stimulation of the OC bundle reduces temporary threshold shifts from acoustic overexposure (Rajan 1988, Reiter and Liberman 1995), and chronic section of the OC bundle renders the ear more vulnerable to permanent acoustic injury (Handrock and Zeisberg
1982; Kujawa and Liberman, 1997). Although OC contributions to protection are well documented, many experiments left some ambiguity as to the relative contributions of LOC vs.
MOC systems, given that: 1) LOC and MOC axons run together in the OC bundle at the floor of the IVth ventricle, thus both electrical stimulation and surgical section experiments may involve both systems, and 2) both LOC and MOC systems are cholinergic, thus pharmacological blockers presented in tandem with acoustic overexposure may not be selective. Recently, definitive evidence for a protective mechanism involving the cholinergic MOC system was demonstrated in mice with over-expression of the a9 ACh receptors in OHCs: over-expresser mice had enhanced resistance to acoustic injury (Maison et al. 2002).
Although no direct evidence exists for an LOC protective role, several lines of evidence have implicated the LOC system in controlling the excitotoxicity seen in the acute stages of acoustic injury. The morphological evidence for noise-induced excitotoxicity is the swelling of afferent dendrites in the IHC area within the first 24 hours after noise exposure (Liberman and Mulroy
1982; Robertson 1983; Puel et al. 1998; Le Prell et al. 2005). This vacuolization appears to result from excessive exposure to glutamate released by the inner hair cell: 1) application of glutamate receptor antagonists during acoustic overexposure decreases the degree of vacuolization and the amount of temporary threshold shift (Puel et al. 1998): 2) dendritic swelling can be mimicked
without acoustic overexposure by perfusing the cochlea with glutamate receptor agonists (Puel et al. 1997). Pharmacological evidence suggests that the dopaminergic component of the LOC is responsible for regulating the degree of vacuolization: cochleas perfused with dopamine antagonists show a marked increase in vacuolization (Ruel et al. 2001). Together, these studies support the hypothesis that the LOC, more specifically its dopaminergic component, protects the cochlea from acoustically driven, glutamate-induced excitotoxicity.

To directly assess the contribution of the LOC to protection from acoustic injury, we stereotaxically lesioned the LOC system (Le Prell et al., 2003) in adult mice and compared noise-induced threshold shifts following binaural noise exposure in the ears ipsilateral and contralateral to the lesion. The integrity of the MOC pathway was verified both morphologically
(in the brainstem and cochlea) and functionally (with bilateral measurement of MOC-mediated suppression of cochlear responses). In animals with unilateral LOC lesions, the ear ipsilateral to the lesion showed larger threshold shifts than the contralateral ears, but only when measured via the cochlear neural responses; threshold shifts measured by outer hair-cell based responses
(otoacoustic emissions) remained bilaterally symmetrical. The results are consistent with an
LOC-mediated protection of the cochlear nerve dendrites during acoustic overexposure.
4.3 Materials and Methods
4.3.1 Stereotaxic Surgery: All procedures were approved by the IACUC of the Massachusetts
Eye and Ear Infirmary. Experimental animals were CBA/CaJ mice aged 6-8 weeks, weighing between 25-30g, and of either sex. Following anesthesia with xylazine (20 mg/kg i.p.) and ketamine (100 mg/kg i.p.), the mouse was held in a Kopf small-animal stereotaxic apparatus by snout clamp and ear bars. The skin overlaying the skull was slit and retracted to reveal the bregma and lambdoidal sutures. Rongeurs were used to make an opening in the skull over the right lambdoidal suture. Using coordinates modified from Franklin and Paxinos (1997), a micropipette filled with 10mM melittin in saline was lowered into the brain at a position .49 mm caudal and .12 mm lateral to the bregma. When a depth of 0.69 mm was reached, 2 gl of solution was injected via a 10gl syringe (Hamilton) coupled to the micropipette. Immediately after injection, the scalp was sutured, and the animal placed in a padded cage with heat lights for -1 hour post surgery. A total of 18 mice underwent surgery. Animals in which the injection missed the target (see below) serve as surgical controls. In addition, 8 mice without surgery were included as an additional control.
4.3.2 ABR and DPOAE Measurements: For auditory brainstem responses (ABRs) and distortion product otoacoustic emission (DPOAE) recordings, mice were anesthetized with ketamine and xylazine, and needle electrodes were inserted at the vertex and pinna. The ABR was evoked with 5-ms tone pips (0.5-ms rise-fall, with a cos 2 envelope, at 35/sec). The response was amplified (10,000x), filtered (0.1-3 kHz), and averaged with a digital I-O board in a PCbased data-acquisition system. Sound level was raised in 5-dB steps from 0 to 80 dB SPL. At each level, 1,024 responses were averaged (with stimulus polarity alternated) after 'artifact rejection'. Threshold was determined by visual inspection. DPOAEs at 2f]-f response to primary tones: fl and f
2
, with f
2
/f = 1.2 and f
2
2 were recorded in level 10 dB < fl level. FFTs were computed and averaged over 5 waveform traces, and 2fl-f
2
DPOAE amplitude and surrounding noise floor were extracted. Iso-response contours were interpolated from plots of amplitude vs.
sound level, performed in 5 dB steps of primary level. "Threshold" is defined as the primary level required to produce a DPOAE at 0 dB SPL.
4.3.3 Acoustic Overexposure: Animals were exposed free-field, awake and unrestrained, in a small reverberant chamber. Acoustic trauma consisted of a 15-minute exposure to an 8-16 kHz octave band noise presented at 94 dB SPL. Exposure level was measured at four positions inside

the cage and varied by < 0.5 dB. In each animal, ABR and DPOAE measurements were made iweek prior to exposure, 6 hours post-exposure and again at I week post-exposure.
4.3.4 Medial Olivocochlear (MOC) Assay: MOC assays were performed on 6 control animals
(no surgery and no acoustic exposure) and a randomly chosen subset (n= 12) of the experimental animals (at least I wk after the exposure to noise). Animals were anesthetized as for ABR and
DPOAE testing and a posterior craniotomy and partial cerebellar aspiration exposed the floor of the IVth ventricle. Shocks (monophasic 150 gs pulses at 200/sec) were applied through silver wires at the midline. Shock threshold for facial twitch was determined, paralysis induced with cx-
D-tubocurarine (1.25 mg/kg i.p.), and the animal connected to a respirator. Shock levels were raised 6 dB above twitch threshold. The MOC suppression effects on DPOAEs were then assessed in both cochleae. f
2 level was set to produce a DPOAE -10 dB > noise floor. Repeated measures of baseline DPOAE amplitude (n=12) were made prior to a series of 17 contiguous periods in which ]DPOAE amplitudes were measured with shocks to the OC bundle. The shock epoch (-90 seconds) was followed by a series of DPOAE amplitude measurements (n=36) to observe the extinction of the MOC effect.
4.3.5 Histological Preparation: After final testing (-7 weeks post-surgery), animals were perfused intracardially with 10% formalin. The brainstems were extracted, post-fixed overnight, cryoprotected in sucrose, frozen and cut on a sliding microtome at 80 .tm in the transverse plane.
Slide-mounted sections were stained for acetylcholinesterase (AChE) activity to allow for visualization of the cholinergic OC cells in the brainstem (Osen and Roth 1969), as well as to verify that the microelectrode pipette did not sever the OC bundle. Cochleas on both sides were extracted, post-fixed overnight, decalcified in EDTA for -48 hrs, dissected into half-turn segments and double immunostained with rabbit anti-VAT (vesicular acetylcholine transporter) and sheep anti-TH (tyrosine hydroxylase), followed by a species-appropriate fluorophorecoupled secondary (VAT Alexafluor 568, TH Alexafluor 488).
4.3.6 Morphometric Analysis:
4.3.6.1 Brainstenms: The location, size and success of the brainstem lesion were quantified by comparing the area of the lateral superior olive (LSO) on both sides of the brainstem. A drawing tube was used to trace the outline of surviving LSO cells in all sections from its caudal to rostral extreme. Tracings were digitized and the areas of both medial and lateral limbs were determined by computerized planimetry.
4.3.6.1 Cochleas: Cochlear location was converted to frequency (Muller et al., 2005), and 10 log-spaced frequency loci were identified in each case. At each locus in each case, an observer blind to the physiology and brainstem analyses separately rated the innervation densities of
VAT- and TH-positive terminals in both the inner and outer hair cell areas. A 3-point scale was used for VAT-positive terminals in the OHC area: the observer's task was to estimate the fraction of OHCs with at least one VAT-positive terminal: 3 = 100-66%, 2 = 66-33%, and 1 =
33-0%. VAT- and TH-positive terminals in the IHC area were evaluated with a 4-point scale: 3 = profuse, 2 = moderate, 1= sparse and 0 = none. Each immunostain was separately referenced to its own maximum values: i.e. TH-positive terminals are much rarer than VAT terminals, but maximum density for each would receive a rating "3".

4.4.1 Histological assessment of completeness and selectivity of the LOC lesion
Brainstems and cochlear whole mounts were evaluated to assess the completeness and the selectivity of the stereotaxic lesions in this study, i.e. the extent to which they were successful in eliminating the LOC system and sparing the MOC system. Because the vast majority of OC neurons from both systems are cholinergic, brainstem sections and cochlear whole mounts from each case were stained with cholinergic markers: acetylcholinesterase histochemistry (AChE) was used to mark cell bodies and axons in the brainstem; and immunostaining for vesicular acetylcholine transporter (VAT) was used to locate cholinergic terminals in the IHC and OHC areas of the cochlea). Because of the suggested role of dopamine in cochlear anti-excitotoxicity
(Ruel et al. 2001), and because a separate subgroup of non-cholinergic LOC neurons in mouse are dopaminergic (Darrow et al. 2006), cochlear whole mounts were also immunostained for tyrosine hydroxylase [TH], an enzyme in the biosynthetic pathway that produces dopamine.
4.4.1.1 Brainstems
When the neurotoxin successfully targeted the LSO, there was a clear loss of cholinergic neurons from the LSO area and a clear disruption of the LSO neuropil: the paired micrographs in
Figures 4.2A and 4.2B compare opposite sides of the brainstem from one injected case. To quantify the extent of lesion in each case, we outlined the surviving portion of the LSO (dashed lines in Fig. 4.2B) throughout its rostro-caudal extent and compared the measured areas to those seen on the contralateral side of the brainstem (dashed lines in Fig. 4.2A) and to LSO areas in uninjected controls. Because the LSO is tonotopically organized (Kelley 1998), we separately assessed the areas of the lateral (low-frequency) and medial (high-frequency) limbs. The fractional survival of medial vs. lateral limbs (Fig. 4.6A) suggests a clear distinction between
LSO Hit and LSO Miss cases. The data also suggest that in a subset of cases (n=3), there was greater success in destroying the medial limb than the lateral limb (arrowheads in Fig. 4.6A).

( ntrnl qirip Inic&eted side
Figure 4.2: Histological verification of LOC lesions, as seen in AChE-stained brainstem sections (A,B) or in cochlear whole mounts double-immunostained for a cholinergic marker (VAT: red) and a dopaminergic marker
(TH: green). (C,D). A,B: Brainstems are from opposite sides of one LOC Hit case. Dashed lines indicate the outline of the surviving LSO in each section: on the control side, lateral and medial limbs are indicated. The AChEpositive fibers of the VIIth nerve, visible in each section, were used to identify comparable rostro-caudal locations on the two sides. Scale in B also applies to A. C,D: images are from the 22.6 kHz region of opposite sides of a LOC
Hit case (different from the one shown in A and B. Scale in D also applies to C.
When estimating the extent of lesion success on the LOC cell bodies, it is also important to assess, in both the Hit and Miss cases, which others structures were damaged. In particular the extent of MOC involvement is key to interpreting the results. As shown in Figure 4.2A,B, and as schematized in Figures 4.3 and 4.4, the cholinergic MOC cells form an elongate cluster, the caudal end of which is ventro-medial to the LSO, and the rostral end which extends well beyond the rostral tip of the LSO (Campbell and Henson 1988; Brown 1993). In each LSO Hit case (Fig.
4.3, n=10), the injection destroyed part of the LSO; in only one case (arrowhead in Sections 11 and 13) did the lesion appear to significantly impinge on a portion of the MOC cell cluster, near its rostral extent. In half of the LSO Miss cases, there was no sign of injection, suggesting that the pipet tip may have clogged. In the remaining cases (n=4), the lesion was always rostral to the
LSO (Fig. 4.4). In one LSO Miss case, the lesion impinged on MOC cell bodies, rostral to the
LSO (Fig. 4.4: arrowheads in Sections 11 and 13).
In addition to considering involvement of the MOC system, it is also important to rule-out any contribution of the stapedial motorneuron pathway when studying protection from acoustic overexposure: the sound-driven stapedius reflex has been demonstrated to protect the cochlea from acoustic injury (Henderson et al. 1994; Ryan et al. 1994). Previous retrograde labeling experiments of stapedius motorneurons in the rat (Shibayama et al. 1990) have demonstrated a

majority population of neurons that originate in the ipsilateral brainstem ventro-medial to the facial nucleus, and a minority population (-6%) of ipsilateral cell bodes that are scattered more rostrally at the level of the facial nerve exit from the brainstem. Assuming that stapedius motorneurons are similarly located in rat and mouse, we can qualitatively determine if the neurotoxin may have affected them in either the Hit or Miss cases. Analyzing figures 4.3 and 4.4, suggests that only in the Hit cases is it possible that the lesion may have included the small subset of stapedius motorneurons located near the facial nerve exit.
Based on the known cochlear projections and laterality of the LOC and MOC systems (Fig.
4.1A), a successful and selective LSO Hit should yield: 1) a reduction of OC terminals in the
IHC area, and not the OHC area, of the ipsilateral ear (See Figures 4.2C,D), and 2) no change in
OC terminal density of the IHC and OHC areas of the contralateral ear. Recent work in mouse suggests that the LOC innervation of the IHC area comprises two populations: a majority population of cholinergic terminals and a minority population of dopaminergic terminals, with virtually no co-localization (Darrow et al. 2006b). In contrast, there appears to be a single population of OC terminals in the OHC area, all of which are cholinergic (though they may colocalize GABAergic and other neurotransmitter markers). Accordingly, each cochlea was double-stained with a cholinergic (VAT) and dopaminergic (TH) antibody, and the innervation density in the IHC and OHC areas were semi-quantitatively evaluated by an observer blind to both brainstem histology and cochlear physiology. As shown in Figure 4.5, in the cases classified as LSO Hit based on the brainstem histology (Fig. 4.6A), there was, on average, a 50% reduction in the density of both cholinergic and dopaminergic terminals in the IHC area of the ipsilateral cochlea (Figs. 4.5A and B, respectively), without any changes in OC terminals in the OHC area
(Fig. 4.5C). There was no significant change of OC innervation in the ipsilateral ears of LSO
Miss cases; nor was there any mean loss of OC innervation in the IHC or OHC areas of contralateral ears in either surgical group (Figs. 4.5A-C).
Considered individually, each case classified as LSO Hit based on the brainstem sections
(Fig. 4.6A) showed a reduction of OC terminals in its ipsilateral cochlea (Fig. 4.6B); these 9 cases are unambiguously classified as "LOC Hits". In one injected case, the ipsilateral cochlea showed obvious loss of OC terminals, however the cholinergic cells in the LSO appeared intact.
It is possible that this exceptional case arose because the pipette or the neurotoxin destroyed OC axons without affecting the cell bodies. (It would be harder to explain a case with a clear central lesion and no obvious peripheral defect). This case is considered ambiguous and has been removed from further consideration. There was no obvious correlation between the apical-basal gradient of cochlear de-efferentation and the destruction of medial vs. lateral limb of the LSO: in the three brainstems with a "medial limb only" lesion (arrowheads in Fig 4.6A), there was a decrease in LOC terminals in both the apex and base of the cochlea (arrowheads in Fig. 13B indicate the same three cases). This discrepancy may also reflect the fact that some of the LOC cell bodies survived, though their peripheral projections had degenerated.

'
Figure 4.3: Lesion locations in all LOC Hit cases. In each case, the lesion is outlined (dashed lines, white centers) and superimposed on an "atlas" sections (derived from an AChE-stained control mouse). Alternate 80m sections are shown:
Section 1 is the most caudal, and Section 17 the most rostral to include the OC system. One case with partial damage to
MOC system is indicated by arrowheads in Sections 11 and 13. Abbreviations are: 4th V,4th ventricle; 5n, trigeminal nerve or nucleus; 7n, facial nerve or nucleus; 8n, cochlear nerve; AVCN, anteroventral cochlear Nucleus; DCN, dorsal cochlear nucleus; IC, inferior colliculus; LC, locus coeruleus; LSO, lateral superior olive; MiTg, Microcellular tegmental nucleus; MOC, medial olivocochlear cells; OCB, olivocochlear bundle; Pn, pontine nuclei; RTG, reticulotegmental nucleus; TB, trapazoid body.

Figure 4.4: Lesion locations in all LOC Miss cases. One case with partial damage to the MOC system is indicated by the arrowheads in Sections 11 and 13. See Figure 4 legend for further details.

IHC Area - VAT
1
IHC Area TH
4 "•f•
I LU
A
A
100-
80-
60-
40
20-
4•kA
0o
100-
80-
1
C
_. .
4 6 8110
-- LOC Miss - Ipsi
-0- LOC Hit- Ipsi
-- Hit & Miss Contra
-- Unhsected ews (n=2)
· · _ _
30 50 70
OHC Area - VAT
-- Hit & Miss - Contra
-X- Uninkcted ears (n=2)
· A
IZUL D
4 6 8 10
-*-t1 C Miss- Ip,
Figure 4.5: Semi-quantitative analysis of cholinergic (VAT:
A,C,D) and dopaminergic (TH:
B) markers in the IHC (A,B) and
OHC (C,D) areas. In each cochlea, innervation density was estimated at 10 locations along the cochlear spiral (apical and basal halves of each of 5 dissected
-4*-Hft
AMS-• tra pieces). In A-C, the mean (±SEM) innervation density in ipsilateral
30 50 70 ears from LOC Hit (open circles;
OHC Area -
--+--~-0--0-Z)
VAT
· , .· n = 10) and LOC Miss (dark-filled circles; n = 8) are compared to all contralateral ears and uninjected controls (grey-filled circles; n =
19). In D, individual contralateral ears from both surgical groups are shown to indicate the two cases,
(dashed lines) with decreased terminal density in the OHC area.
See Methods for details of semiquantitative analysis.
2
1
-- KND 429-Contra
-O-KND 429-Contra
-e-Other Contra
4 6 8 10 30 50 70
Cochlear Frequency (kHz)
4 6 8 10 30 50 70
Cochlear Frequency (kHz)
Brainstem - AChE Cochlea - VAT
A
M S140
120
() 100.
.0 80,
E
60-
>00
>0
00
;
A ,
40-
O 20-
0
0O
I e
SLSO Miss c
° n f I
20 40 60 80
LSO Medial Limb
100 120 140
(% Survival)
120
W
1 0 0 r 80-
0.
>o 0
>0
0, oO
9
I
I # l I I
0 20 40 60 80 100 120 140
Basal Innervation (% Survival)
Figure 4.6: Analysis of lesion success based on (A) the fractional survival of the LSO, as seen in AChE-stained brainstem sections, and (B) the fractional survival of cholinergic terminals in the IHC area, as seen in immunostained cochlear whole mounts. Filled arrowheads in B are the three cases in A (filled arrowheads) where the lesion affected the medial limb only. A) Fractional survival is the total surviving area (as depicted in
Fig. 2A,B) of each LSO limb from its rostral to caudal extent (the LSO spans -480 gm in the rostro-caudal plane), normalized to the mean area of control sides. B) Fractional survival of cholinergic (VAT-positive) terminals in the IHC area is calculated by dividing the cochlear spiral into two bins (apical vs. basal to the midpoint), and averaging the semi-quantitative estimates of fractional survival in each bin for each case.

On a case-by case-basis, there was a good correlation between the brainstem and cochlear data with respect to MOC involvement. As shown in Figure 4.5D, the cochlear analysis identified only two cases as having a decrease in efferent terminals in the OHC area of the contralateral ear; as would be expected based on the largely contralateral projection of the MOC system (Fig. 4.1A). Correspondingly, these two cases, one LOC Hit and one LOC Miss, are the only cases for which the brainstem analysis suggested that the lesion had impinged on MOC cell bodies (Figs. 4.3 and 4.4).
In addition to assessing the morphological integrity of the MOC system, we assessed MOC function by electrically stimulating the OC bundle at the midline and simultaneously recorded suppression of distortion product otoacoustic emissions (DPOAEs) in both ears. DPOAEs are distortions produced and amplified by normal healthy OHCs, which are propagated as mechanical vibrations back to through the middle ear to the ear cabal where they can be measured in the ear canal sound pressure. Because activation of the MOC feedback system essentially turns down the gain of the OHC electromechanical amplifier (Galambos 1956; Brown and Nuttall 1984; Mountain 1980; Bonfils et al. 1987; Gifford and Guinan 1987; Murugasu and
Russell 1996), the suppression of DPOAEs provide a rapid and reliable assay of MOC activation level. We measured the bilateral symmetry of MOC suppression in 6 control animals (no neurotoxin injection) and 12 injected animals (evaluated at a time when the success of the LSO lesion was not known). The maximum effect of MOC stimulation on DPOAE amplitude is for primary tones near 22.6 kHz (Maison et al. 2006), thus we show data only for 22.6 kHz (Fig.
4.7), however, results at other test frequencies were similar.
The bilateral symmetry of MOC effect size did not differ significantly in the control cases vs.
the injected cases (Fig. 4.7), suggesting that: 1) MOC function is relatively symmetric in animals with normal OC innervation, and 2) our lesions did not significantly perturb MOC function.
Interestingly, the two injected cases for which there was histological evidence of MOC involvement (see Figs. 4.3, 4.4, 4.5D) showed some asymmetry of the MOC effect: i.e. roughly half as large in the contralateral vs. the ipsilateral ear (arrowheads in Fig. 4.7B). The paired reduction of contralateral MOC effects and of OC terminals in the contralateral OHC area agrees with the known projection patterns of the MOC system (Fig. 4.1: 75% contralateral).

rr
L m cI-
~0
CO.
"o
-J
0 100 200
Time (sec)
300 -20 -15 -10 -0
MOC Effect Right/Ipsi (dB)
U
Figure 4.7: Bilateral suppression of DPOAEs, elicited via midline electrical stimulation that MOC function is minimally affected by the lesions. A: One run of of MOC fibers, suggests the MOC assay in a control case demonstrates symmetrical DPOAE suppression in right and left ears. "MOC effect" between mean DPOAE amplitude in the first three measures after shock onset, is defined as the dB difference compared to the pre-shock baseline.
B: MOC effects in right and left ears of control, LOC Hit and LOC Miss cases. cases with histological evidence of MOC lesions (Figures 4, 5, and 6D). For data
Large arrowheads indicate the shown here, f2 was 22.6 kHz.
Co
S-.
It w l
M
, 70-
0.
C) 60-
+-- LOC Mins- Col
-A LOC Miss -
"1 so-
40
.
-0- LOC Hit - I=
&LOC Hit -
30.
S20
10n
T~ t.I
, , , ,
,
6 8 10
I LOC Miss
-O-LOC Hit
FE
30
"
701
'l
,
0
60-
50a
40-
30-
20-
50
10-
O
U
6 8 10
DPOAEs
30 50
U
Test Frequency (kHz)
S6810 30 f2 Frequency (kHz)
50
Figure 4.8: Mean cochlear thresholds, as measured by ABR (A) and DPOAE (B) were not successful LOC lesion, nor were interaural threshold differences as measured by either affected by a
ABR (C) or DPOAE
(D). Keys in A and C also apply to B and D, respectively.

Given that chronic section of the entire OC bundle (including both MOC and LOC components) in adult animals does not affect cochlear thresholds (Liberman 1990; Zheng et al.
1999), an effect of unilateral LOC lesion on thresholds was not expected. Indeed, baseline cochlear thresholds, as measured by either auditory brainstem responses (ABRs) or DPOAEs, were unaffected by the LOC lesions (Fig. 4.8A-D). However, significant binaural asymmetries of suprathreshold neural responses (ABR; Figs. 4.9A,B) were observed in LOC Hit cases, without corresponding changes in DPOAE (Fig. 4.9C,D).
The ABR represents the summed activity of auditory neurons along the ascending afferent pathway, and the earliest wave, Wave 1, represents the activity of the cochlear nerve. The
DPOAEs reflect
<events
"upstream" of the ABR, in the sense that the OHCs' contribution to cochlear amplification is a necessary but not sufficient component of a normal ABR response.
The latter also relies on the integrity of synaptic transmission between the IHCs and the cochlear nerve, and of the cochlear nerve fibers themselves. Thus, selective changes in ABR amplitudes without accompanying shifts in DPOAE responses are consistent with expected LOC-based effects on neural activity only.
In LOC Hit cases, mean ABR amplitudes were enhanced in the ipsilateral ear (Fig. 4.9A): the difference between the two ears was statistically significant in the Hit cases (e.g. at 22.6 kHz: p =
0.019, F = 6.007, by two way ANOVA) and were not in the Miss cases (e.g. at 22.6 kHz: p =
0.387, F= 0.762, by two way ANOVA) .
This enhancement of ABR amplitude was roughly a constant percentage as tone level increased (data not shown). Thus, to summarize changes across frequency, the mean interaural amplitude difference across sound levels (50-80 dB SPL) was computed for each frequency, for each case, and then averaged across cases within each group.
When viewed in this way, ABR enhancement are seen across all test frequencies (Fig. 4.9B), and not in DPOAEs (Fig. 4.9D) In those cases where the brainstem lesion appeared to spare the lateral (high-frequency) limb (Fig. 4.6A arrowheads) there was no obvious difference in ABR enhancements between low and high frequency regions, thus mirroring the lack of a base-apex gradient in the cochlear efferent innervation in these same cases.
1 In a larger sample of cases, summarized in a separate study of the LOC role in balancing bilateral neural excitability (Chapter 3; Darrow et al. 2006a), a complementary reduction was seen in the ABR amplitudes in the contralateral ears of LOC Hit cases. We hypothesize that the lack of contralateral effects in the smaller subset of animals (see fig 4.9A) used in these acoustic injury experiments reflects (chance) differences in the degree to which the crossing ascending projections to the contralateral LSO were interrupted by the lesions (see Chapter 3; Darrow et al.
2006a).

22.6 kHz All Fron.annIiie
I·_
S10 20 30 40 50 60 70 80
Tone Pip Level (dB SPL) Test Frequency (kHz) oUn
-4lOC Mess
-t-lOC Hit
-.-Control
401
) 50
" 40.
30.
20
E 20.
LU
0
.
0
-IIf.
· · -· · · · ·
10 20 30 40 50 60 70 80
Primary Level (dB SPL)
UYY
· · · · ·
6 8 10
· ·
30 f2 Frequency (kHz)
· r
Figure 4.9: ABR amplitudes
(A,B) are enhanced in the ipsilateral LOC Hit ears and not in LOC Miss ears, whereas DPOAE amplitudes
(C,D) are unaffected in all groups. A,C: Mean (±SEM) amplitude vs. level functions for ABR and DPOAE, respectively, for responses at
22.6 kHz: key in panel C applies to A. B,D: mean interaural discrepancies in
ABR and DPOAE amplitudes, respectively. Key in D applies to B. See text for further details.
Dendritic vacuolization in the IHC area, the morphological signs of glutamate excitotoxicity, is only seen in the acute stages (<24 hours) of acoustic injury (Liberman and Mulroy, 1982;
Robertson 1983; Puel et al. 1998); thus, we designed an exposure stimulus (94 dB at 8-16 kHz for 15 min) to create a moderate (-35 dB) threshold shift when tested 6 hrs post-exposure (Fig.
4.10A,B), and to recover when tested at 1 wk post-exposure (Fig. 4.10E,F). Although not explicitly assessed here, a previous study of noise-exposed CBA/CaJ mice in our laboratory showed that a 40 dB threshold shift measured 12 hours after exposure to a similar noise band was associated with striking vacuolization under IHCs in the tonotopically appropriate region of the cochlea (Wang et al, 2002, Figure 13).
Following exposure, threshold shifts in the LOC Miss cases were bilaterally symmetrical at 6 hrs post exposure, whether measured by ABRs (Fig. 4.10A,C) or DPOAEs (Fig 4.10B,D).
Furthermore, threshold shifts in the LOC Miss cases are of similar magnitude when measured by
ABRs (Fig. 4.10A) or DPOAEs (Fig. 4.10B), suggesting that the functionally most important changes are occurring at, or "upstream" of, the OHCs. Similar results have been reported in other
TTS experiments on normal animals in which both ABR and DPOAE threshold shifts have been measured (Mills et al. 1997; Wang et al. 2002; Maison et al. 2003; Kujawa and Liberman 2006).

In contrast, in the LOC Hit cases, the ABR threshold shifts were significantly larger in the ipsilateral ear when compared to either the contralateral ear or either of the LOC Miss ears (Fig.
4.10A,C). Differences between the two ears of LOC Hit mice were highly significant (p =
0.001***, F = 17.385, by two way ANOVA). Although it appears that the mean threshold shifts of the contralateral ears in the LOC Hit group were slightly lower then those from the LOC Miss cases, the differences were not statistically significant (p < .525, F= 0.426, by two way
ANOVA). Importantly, the interaural asymmetries in ABR threshold shift were not mirrored in the DPOAE data (Fig. 4.10B,D), indicating the additional vulnerability arising from the loss of the LOC system involves additional damage to IHCs or neural elements, not to OHC function.
m
9-
Co r-
I--
21
ABRs 6 hrs Post-Exposure DPOAEs
40-
6 8 10 30 50
+:LOC Miss
Hit
-10
• ..
;i";"" ..........
6 8 10
Test Frequency (kHz)
1 wk Post-Exposure rr
,~
30 f2 Frequency (kHz)
-LOC Miss-
LOC Miss -" ntra
-0- LOC Hit - II
-LOC Hit - Contra
Mean ABR
Figure 4.10: mice
LOC Hit in d
6 hrs after acoustic overexposure, were 10-15 dB higher in the ipsilateral ear; this asymmetry was not present in LOC Miss cases (A,C) or in the mean
DPOAE data from either Hit or
Miss groups (B,D). A,B: threshold shift is defined as the difference from mean pre-exposure values
1or the same group. interaural
C.,D: threshold-shift difference defined as thresholds in ipsilateral minus contralateral ears of each case. At 1 wk post exposure, mean ABR (E) and
DPOAE (F) threshold shifts returned to pre-exposure levels.
Key in F, also applies to A,B and
E. Key in D also applies to C.
Error bars in all panels indicate
±+SEMs.
-c·i~ ·;
6 8 10 30
Test Frequency (kHz)
':a -.
6 8 10 30 f2 Frequency (kHz)

4.5
4.5.1 Peripheral effects of the LOC system in modulating neural excitability
In mammals, the OC efferent system has two major components: an MOC component that innervates the OHCs, and an LOC component that primarily innervates the auditory nerve dendrites under IHCs (for review see Warr et al. 1986). The MOC system represents a soundevoked negative-feedback reflex (Fex 1967; Cody and Johnstone 1982a,b; Liberman and Brown
1986), and activation by sound or electric shocks suppresses cochlear responses to sound by cholinergic actions on its terminal targets, the mechanically active OHCs. The proposed functional roles of this suppressive feedback include 1) improving signal detection in noise (antimasking), 2) mediating selective attention and 3) protection from acoustic injury (for a review see Guinan, 1996).
The LOC system's peripheral effects and functional role are more poorly understood, due, in large part, to the lack of myelination of its neurons, and the difficulty in electrically stimulating their axons when shocking at the floor of the IVth ventricle (Gifford and Guinan 1987).
Furthermore, it is still not clear if, or how, LOC neurons respond to sound (Adams 1995).
However, a recent study succeeded in indirectly activating the LOC pathway via electrical stimulation of the inferior colliculus: it demonstrated two functional subsystems capable of eliciting slow suppressive or excitatory effects on auditory nerve activity (Groff and Liberman
2003). Correspondingly, the LOC is a cytochemically heterogeneous system: co-localization studies in mice suggest that a majority population of cholinergic cells also contain GABA and
CGRP, while a second minority group of neurons are dopaminergic (Darrow et al. 2006b). There is also immunohistochemical evidence for urocortin and various opioids in LOC terminals: although evidence is fragmentary, these transmitters may also co-localize in the cholinergic terminals (see Eybalin 1993 for extensive review).
Pharmacological studies using cochlear perfusion of agonists and antagonists for putative
LOC neurotransmitters have suggested both excitatory and inhibitory effects on auditory nerve activity. In the guinea pig, application of ACh increases both spontaneous and glutamate-induced
(meant to mimic sound-induced) auditory nerve activity (Felix and Ehrenberger 1992). In contrast, perfusion of GABA did not affect spontaneous activity, but decreased glutamateinduced, and ACh-induced, activity (Felix and Ehrenberger, 1992; Arnold et al. 1998). Although
CGRP perfusion has not been described in mammals, in the lateral line organ, CGRP increases spontaneous rates, and decreases mechanically driven rates, of primary afferent neurons (Bailey and Sewell, 2000a,b). Dopamine is primarily inhibitory: in guinea pigs, cochlear perfusion of dopamine resulted in a decrease of both spontaneous and sound-driven activity (d'Aldin et al.
1995; Oestreicher 1997).
Studies of mice with targeted gene deletion have provided additional insight into effects of individual LOC transmitters. For example, Maison et al. (2003b) reported that oX-CGRP-null mice show an auditory phenotype consistent with a post-synaptic, LOC-based effect: i.e. leveland frequency-independent decreases in suprathreshold neural responses, whereas OHC-based activity (DPOAE amplitude and MOC-mediated suppression) was identical to wildtype controls.
Previous surgical lesion studies have cut the OC bundle and tried to infer those effects attributable to the LOC vs. MOC system (Liberman 1990; Walsh et al. 1998; Zheng et al. 1999).
Two approaches have been compared: 1) a midline cut that interrupts 2/3 of the MOC

innervation to both ears and spares virtually all LOC innervation; and 2) a lateral cut which interrupts the entire ipsilateral OC system (both MOC and LOC). These studies have suggested that the LOC can modulate spontaneous and sound-evoked discharge rates in the auditory nerve.
Removing the entire OC system in adults causes little change in thresholds or tuning; however, there is a substantial reduction of spontaneous rates, not seen after midline (MOC) section
(Liberman, 1990; Zheng et al. 1999), and an increase in sound-driven discharge rates in chinchillas (Zheng et al. 1999), but not in cats (Liberman, 1990). The selective effects on discharge rate are consistent with a post-synaptic effect of LOC innervation on auditory nerve dendrites. The chinchilla results further suggest that resting tone in the LOC pathway tends to suppress sound-evoked responses, consistent with the post-lesion enhancement of ABR amplitudes seen here (Fig. 49A,B)
In a previous study of selective LOC lesions in guinea pig via stereotaxic LSO ablation, neural response amplitudes were decreased in the successful LOC lesions (Le Prell et al 2003), suggesting, in contrast to the present results, that resting LOC tone tends enhance auditory nerve response. The fact that LOC destruction, either by itself or in conjunction with the MOC system, can in some experiments increase, and in others decrease, neural excitability, may simply reflect the existence of multiple LOC subgroups with both excitatory and inhibitory effects on cochlear nerve response. If the balance between the resting activation levels of these different subgroups is different in different species, or is differentially affected by different anesthesia regimens, it would be easy to produce such qualitatively different effects of LOC destruction.
4.5.2 Acoustic injury and olivocochlear feedback
In any acoustic injury study, involvement of the stapedius reflex must be considered since an intact stapedius reflex can attenuate the input stimulus as it passes through the middle ear (Pilz et al. 1997). Based on the location of the lesions in our surgical animals, it is possible, in the Hit cases, that a small subset of stapedius motorneurons was affected. However, the TTS results
(figure 4.10) suggest the functional integrity of the reflex was not compromised. If it were, the
OHC-based TTS in Hit cases should be greater than Miss cases: this was not the case.
OHC dysfunction plays a major role in the genesis of both temporary threshold shifts (TTSs) and permanent threshold shifts (PTSs): e.g. slow OHC depolarizations are well correlated with
TTS magnitude (Patuzzi 2002), and the loss of OHCs and/or damage to their stereocilia are well correlated with PTS magnitude (Liberman 1984). A longstanding theory of MOC function is that it protects the cochlea from both TTS and PTS via its actions on OHCs (Handrock and Zeisberg
1982; Rajan 1988; Reiter and Liberman 1995; Kujawa and Liberman 1997; Maison and
Liberman 2000). The idea has been supported by four types of experimental findings: 1) the degree of TTS is decreased when the OC bundle is electrically stimulated simultaneously with the acoustic overexposure (Rajan 1988), 2) in animals with chronic OC bundle section, including both MOC and LOC components, de-efferented ears are more vulnerable to both TTS and PTS
(Handrock and Zeisberg 1982; Kujawa and Liberman 1997); 3) the strength of the MOC reflex
(measured as a acoustically driven suppression of DPOAE amplitude) is strongly correlated to vulnerability (Maison and Liberman 2000)); and, most definitively, 4) transgenic mice with overexpression of the ACh receptor specifically in OHCs show enhanced MOC effects (via the same assay illustrated in Figure 17) coupled with enhanced resistance to both TTS and PTS (Maison et al., 2002).

Although MOC-mediated cholinergic effects on OHCs can clearly reduce acoustic vulnerability, there are hints in previous lesion studies that some of the increased vulnerability seen after complete de-efferentation is attributable to loss of the LOC system. For example, when acoustic vulnerability was assessed in totally de-efferented guinea pigs, neurally derived threshold shifts were 10-15 dB higher than OHC-derived shifts (a disparity not present in controls, exactly as observed in the present study with sham-surgery controls: Fig. 4.10); and vulnerability was not significantly affected by the midline lesion, which interrupts 2/3 of the
MOC while sparing the LOC (Kujawa and Liberman, 1997). However, this and other prior evidence linking the LOC system to protection from acoustic injury is indirect.
Neuronal damage is suggested to play an important role in the genesis of noise-induced TTSs
(Liberman and Mulroy 1982; Robertson 1983; Puel et al. 1998), and the targeting of cochlear neurons by the LOC system makes LOC-mediated protection from acoustic injury an attractive hypothesis. A common cochlear pathology seen in the first 24 hrs after acoustic overexposure is swelling of the auditory nerve dendrites in the IHC area (Liberman and Mulroy 1982; Robertson
1983; Puel et al. 1998). This type of dendritic swelling can also be observed in cochleas perfused with glutamate agonists (without noise exposure: Puel et al. 1994; d'Aldin et al. 1997).
Furthermore, when noise is presented with simultaneous intra-cochlear perfusion of a glutamate antagonist, there is less threshold shift (after washout of the glutamate antagonist) and fewer vacuoles (Puel et al. 1998). These observations suggest that dendritic swelling is a type of excitotoxicity brought on by excessive release of glutamate from the IHC. In cochleas perfused with dopamine antagonist, without exposure to intense noise, an increase in dendritic vacuolization has also been observed, suggesting that the LOC's dopaminergic component might counteract this glutamate-induced excitotoxicity (Ruel et al. 2001).
Previous work in our laboratory has documented the presence of IHC area vacuoles in mice with 40 dB of TTS, i.e. similar in magnitude to that produced in this study (Wang et al., 2002).
The dramatic nature of dendritic swelling suggests that it should be accompanied by a loss of synaptic transmission between the IHC and cochlear nerve: electron microscopic images show loss of intracellular components from, and membrane rupture of, the post-synaptic afferent terminal (Robertson 1983; Puel et al. 1998; Ruel et al. 2001; Le Prell et al. 2004). It follows that loss of synaptic transmission should contribute to the acute threshold shift, and that TTS should be larger in neural measures (e.g. ABRs) than in OHC-based measures (e.g. DPOAEs). In the present study, LOC lesions resulted in enhanced ABR threshold shifts re DPOAE threshold shifts, consistent with a significant component of "additional" TTS attributable to a dysfunction at the level of the inner hair cell or cochlear nerve. In contrast, mice with normal LOC innervation showed almost identical degrees of ABR and DPOAE shift (Fig. 4.10), suggesting that, with an intact LOC system, all the functionally important changes underlying the TTS were present at the level of OHC-dominated active cochlear mechanics.
If dendritic vacuolization is a functionally relevant contributor to TTS, why don't ears with an intact LOC system show larger ABR shifts than DPOAE shifts? In comparing ABR- and
DPOAE-based threshold shifts it is important to consider the relative insensitivity of neuralbased sound-evoked gross potentials such as ABRs to a distributed loss of neural elements. In adult chinchillas with selective IHC loss, which is functionally similar to primary neuronal degeneration as far as ABR generation is concerned, a distributed loss of 50% of the responding neurons in a particular cochlear region resulted in a neural-based gross potential shift of <6 dB
(Liberman et al. 1997). This can be understood by considering that a sound level increase of 6

dB can double the discharge rate of individual cochlear neurons near threshold (Liberman 1990) and thereby compensate for the loss of half the responsive neural elements. This line of argument resolves the apparent paradox that significant numbers of dysfunctional-looking cochlear nerve dendrites can be observed in normal animals with TTS, without a significant degree of
"additional" threshold shift in ABRs re DPOAEs.
The insensitivity of neural-based metrics to distributed IHC loss, and subsequently to neural degeneration, also implies that the 10-15 dB of "additional" ABR shift observed in LOClesioned ears (Fig. 4.10) must correspond to a very large number of dysfunctional auditory nerve fibers. Furthermore, considering that the LOC lesions in this report were incomplete, an average of -30-50% destruction from base to apex, the data also suggests a very strong anti-excitotoxic effect of an intact LOC system. Given the existing pharmacological data to implicate dopaminergic transmission in blocking the generation of dendritic swelling (Ruel et al. 2001), and given the recent report that the mouse LOC system consists of two functional subsystems
(one cholinergic and one dopaminergic, (Darrow et al. 2006b)), it is tempting to speculate that the loss of the dopaminergic component is responsible for the anti-excitotoxic effects described here. However, recent evidence implies that the LOC's GABAergic innervation may also protect cochlear afferent dendrites from acoustic injury: Maison et al. (2005) observed an "additional"
20 dB of ABR shift (re DPOAE shifts) in mutant mice lacking one of the GABA-A receptor subunits (05). Thus, one, or both, of the LOC subsystems may play a role in protecting the auditory nerve dendrites from glutamate induced excitotoxicity.
It has been suggested that OC-mediated protection is an epiphenomenon rather then a functional role that evolved because it confers selective advantage, given that the traumatic acoustic exposures used in "protection" experiments are well above levels that ever existed in the pre-industrial age (Kirk and Smith 2003). The present study uses sound pressures that are significantly lower than those used in previous studies of MOC protection (94 dB vs. > 105 dB
SPL) and thus makes it more plausible that anti-excitotoxicity is a true functional role of the
LOC system rather than an epiphenomenon. Furthermore, the incompleteness of the present lesions and the magnitude of the protective effect they reveal make it likely that significant protective effects may be present at even lower sound level exposures.

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