Origins of Axons in the Cat's Acoustic Striae Determined
by Injection of Horseradish Peroxidase into
Severed Tracts'
JOE C . A D A M S 2
AND W . BRUCE WARR
Eaton-Peabody Laboratory of Auditory Physiology, Massachusetts Eye
and Ear Infirmary, 2 4 3 Charles Street, Boston, Massachusetts
02114 a n d Department of A n a t o m y , Boston University
School of Medicine, Boston, Massachusetts 021 18
ABSTRACT
Origins and terminations of fibers of the dorsal and intermediate
acoustic striae were studied by surgically severing these tracts and injecting
HRP into the incision. This procedure results i n filling the severed axons with
HRP. Filled axons were traced to cell groups of origin and to some terminations
of the acoustic striae. HRP-labeled terminals were found i n the cochlear nuclei
as well as in periolivary cell groups. Filling of cells with HRP ranged from being
complete, resulting i n Golgi-like images, to being barely detectable. Labeled
cells were abundant in the dorsal and posteroventral cochlear nucleus adjacent
to the injection as well as scattered throughout the periolivary cell groups of
both sides, being highest i n concentration around the ipsilateral lateral superior
olive. On the side contralateral to the injection, labeled cells were found along the
medial border of the dorsal cochlear nucleus, i n the interstitial nucleus of the
stria of Held, and sparsely throughout the ventral cochlear nucleus. The distribution of labeled cells was similar following HRP injections of the dorsal
cochlear nucleus, except that these injections revealed additional descending projections from the inferior colliculi and from the ventral nucleus of the trapezoid
body of both sides. These additional projections were interpreted as entering
the CN by a ventral route. Findings of this study are i n accord with physiological recordings made from fibers of the acoustic striae.
The cochlear nucleus (CN) of the cat
is divided into anterior and posterior portions by the auditory nerve fibers entering
from the cochlea (Cajal, '09). The posterior
portion consists of the posteroventral
nucleus (PVCN) and caudal to this the dorsal cochlear nucleus (DCN). Further partitions of the CN have been made by Lorente de NO ('33) and more recently by
Brawer et al. ('74) Fibers connecting posterior portions of the CN to more central
structures course both ventrally and dorsally. Ventrally, ascending and descending
fibers constitute major portions of the
trapezoid body. Dorsally, fibers leave the
CN via the stria of Held, or intermediate
acoustic stria (IAS) and the dorsal acoustic stria (DAS). Cells of the DCN and
PVCN that send fibers centrally via the
IAS and DAS have been studied using retrograde chromatolysis (Osen, '69; Warr,
'69, '72). In addition to ascending projections, there are also descending projections
J.
COMP. NEW%170: 107-122.
that enter the CN via the DAS (Rasmussen, '60; Van Noort, '69). No evidence is
available regarding which cells give rise to
these descending projections. Physiological
recordings made from fibers of the DAS
and IAS raised questions regarding the
cells of origins of these fibers (Adams, '76).
To investigate the origins of strial fibers
a variation on the method of retrograde
marking of cells with horseradish peroxidase (HRP) (Kristensson and Olsson, '71;
LaVail and LaVail, '72) was employed.
Since the first report of tracing central
pathways using HRP as a retrograde
marker (LaVail and LaVail, '72) there
have been a growing number of studies
using this technique. The procedure usually employed consists of injecting a small
1 Preliminary results of the paper were presented at
the 28th Annual Meeting of the Cajal Club, March, 1974.
2 Present address: Laboratory of Neuro-otolaryngology,
National Institute of Neurological and Communicative
Disorders and Stroke, National Institutes of Health,
Building 36, Room 5D32, Bethesda, Maryland 20014.
107
108
JOE C. ADAMS A N D W . BRUCE WARR
volume of HRP solution into gray matter
and later identifying cells in other regions
which have been labeled by virtue of HRP
accumulations in their somata. It is generally believed that mechanisms underlying
this labeling involve uptake of injected
HRP by axonal endings and accumulation of this HRP i n neural somata due
to retrograde axonal transport. One drawback of this procedure is that fiber pathways involved in the HRP transport are
usually not labeled, so that the route taken
by axons of labeled cells can remain unknown. To overcome this drawback and to
identify cells of origin of fibers that enter
and leave the C N dorsally, the IAS and
DAS were transected and HRP injected
into the incision. This procedure resulted
in intense labeling of axons and nearby
cells known to send axons through the
region of the incision. Labeling of other
cells indicated that there are many sources
of DAS and IAS fibers which would be
difficult to identify using traditional pathway tracing techniques.
er-stained with cresyl violet and in some
cases the presence of acetylcholinesterase
(AChE) was demonstrated according to the
method of El-Badawi and Schenk (‘66).
In these cases the incubation for HRP
reaction product followed that for the
AChE product and 4-Chloro-1-naphtholreplaced 0-dianisidine in the HRP incubation medium. This replacement resulted in
a black HRP reaction product (Nakane,
’68) and made it possible to distinguish
the brown AChE reaction product from
that of the HRP.
In order to map the location of labeled
cells, enlarged drawings of the principal
features of representative sections were
traced using a macrophotography device.
The locations of labeled cells as seen
through a microscope were projected onto
these drawings by means of a spot of light
from a laser which was attached to the
microscope stage and moved as the tissue
section was scanned (Patterson et al., ’76).
When plotting results in this manner (fig.
1), labeled cells present only in each individual
section were depicted. Not all
METHODS
labeled cells of the CN were plotted beKittens (10-30 days old) were anesthe- cause of space limitations. In addition to
tized with Dial (75 mglkg) and the cere- injections of the striae, HRP was injected
bellum aspirated to expose the dorsal tip in a variety of other places as control exof the DCN. The DAS and IAS were tran- periments. A total of 40 animals were insected at the dorso-medial tip of the DCN cluded in the present study. The nomenwith a No. 11 scapel blade. A glass pipette clature of cell groups and nuclei used to
with a tip diameter of about 100 micra was describe results is that of Taber (‘61) and
inserted into the incision and approximate- Morest (‘68).
ly one microliter of 10% Sigma Type VI
RESULTS
horseradish peroxidase (HRP) in saline
General observations
was injected over a period of 1.5 hours.
When the injection was completed the
The general appearance of the tissue
wound was closed and the animal main- following HRP injections into cut tracts
tained at normal body temperature. After resembled that reported by earlier invesa post-injection period of 24 hours in the tigators after injections into central nuclei
animal was perfused through the aorta with (LaVail and LaVail, ’72). The injection
0.1 M cacodylate buffer ( p n 7.2) followed site was surrounded by a pale brown difby a mixture of 4 % paraformaldehyde and fusion spot of HRP reaction product.
5 % glutaraldehyde in buffer. The brain Axons labeled with the reaction product
was removed and placed in a solution of radiated from the diffusion spot. In some
30% sucrose (in fixative) until it sank. cases fine processes of axons in the form
Frozen sections were cut at 40 pm thick- of collaterals or terminal plexuses were
ness and stored in buffer until incubated. labeled. Nerve cells colored by the reacSections were incubated in a saturated tion product took one of several forms.
solution of 0-dianisidine for the demon- First, within the diffusion spot many cells
stration of the HRP reaction product ac- were a homogeneous pale brown of apcording to the method of Graham and proximately the same density as the backKarnovsky (‘65). Some sections were count- ground. This is considered to be an arte-
ORIGINS OF ACOUSTIC STRIAE
fact. In a second type of labeling, seen i n
approximately 10% of the animals, cells
were so filled with opaque reaction product
that fine details of their soma, axons, and
dendritic arborizations were clearly visible
(figs. 2 , 3). Another form of labeling was
in the form of granular accumulations of
the reaction product within somata (figs. 36, 8-10). Often diffuse and granular labeling occurred in the same cells (figs. 3 ,
4). The opaque, completely filled cells
were found only within approximately 3
mm of the injection site. Cells containing
both diffuse and granular reaction product
were found close to the injection site among
the completely filled cells, and those containing only granular reaction product
were found as far as one centimeter away
from the injection site. In general, the
amount of the granular reaction product i n
individual cells appeared to decrease with
increasing distance from the injection site.
A. Auditory projections
Labeled axons of the DAS and IAS could
be traced from the injection site coursing
both laterally into the CN and medially
across the brain stem. Labeled cells were
prominent in the caudal portion of the
CN and in the superior olivary complex
(SOC) ipsilateral to the injection. In both
these regions labeled axonal terminals
were found. To facilitate description of
labeled structures, findings i n one animal
will be given i n detail. Results found i n this
animal are representative and are corroborated and extended (where noted by those
of the remaining cases.
Origin of ascending components of
t h e DAS and IAS
Figure 1 shows the location of labelled
cells following injection of HRP into a n
incision which transected the DAS and
IAS. The greatest number of labeled cells
is i n the CN ipsilateral to the injection. A
low magnification micrograph of the posterior CN ipsilateral to the injectionis shown
in figure 2. Just beneath the DCN surface
the layer of labeled fusiform cells stands
out. Labeled fusiform cells at higher
magnification are shown i n figure 3. This
figure shows that details of dendritic arborizations, including dendritic spines, are
revealed i n favorable preparations. The level
109
of detail observable i n such preparations
compares favorably with that of Golgi
preparations of the same type cells (Kane,
'74). Note that some cells i n figure 3 contain both diffuse and granular reaction
product. Further removed from the injection site, unlabeled fusiform cells were
found adjacent to fusiform cells containing only granular reaction product.
A number of labeled cells were also
found superficial to the layer of fusiform
cells. One instance of such a cell, oriented
parallel to the ependymal surface of the
DCN, is shown i n figure 3 (arrow) enmeshed
i n the dendrites of a fusiform cell.
Below the fusiform layer of the DCN,
cells of the polymorph layer were also
labeled (figs. 1, 2 ) . Somata, but not the
dendritic processes, were often heavily
labeled i n this layer of the DCN, a s shown
in figure 4.
The wedge-shaped region lying beneath
the polymorph layer of the DCN is the posterior portion of the PVCN (fig. 1: PV).
Figure 1 indicates that many neurons located i n this region were labeled following the injection of HRP into the DAS and
IAS. Labeled cells and axons in the PVCN
are shown i n figure 5. In this region, as
i n the polymorph layer of the DCN, somata,
but not dendritic trees, were often filled
with reaction product. Most labeled cells
were located caudally, in regions corresponding to the dorsal, ventral and central
divisions of this nucleus as described by
Brawer et al. ('74). A few were found scattered as far rostra1 as the entrance of the
auditory nerve into the CN (fig. 1E).
The principal cell type labeled i n the
PVCN was the octopus cell (fig. 5), but
smaller, elongate cells located along the
medial and ventral margins of the caudal
PVCN also were labeled (fig. 6). In addition, labeled cells were located i n the interstitial nucleus of the stria of Held (INSH),
which lies at the dorsal peak of the PVCN
within the intermediate stria (fig. 1B).
Origins of descending projections to the C N
The principal sources of descending auditory projections entering the CN dorsally
were found to be the periolivary cell
groups of the ipsilateral SOC (fig. 1).
Periolivary groups are clusters of cells
that surround the medial and lateral sup-
110
JOE C. ADAMS A N D W . BRUCE WARR
perior olives and the medial nucleus of the
trapezoid body. Rostrally there is no clear
demarcation between periolivary groups
and the ventral nucleus of the lateral lemniscus (Taber, '61).
Labeled cells were found in virtually
all the periolivary cell groups. Most of these
cells were located in the ipsilateral dorsolateral periolivary group and lateral nucleus
of the trapezoid body. Some labeled cells
A
Figure 1
ORIGINS OF ACOUSTIC STRIAE
were also found i n the posterior periolivary group, dorsomedial periolivary group,
medial nucleus of the trapezoid body,
ventral nucleus of the trapezoid body, and
the ventral nucleus of the lateral lemniscus. In the case shown in figure 1 approximately 70% of all labeled periolivary cells
were located in cell groups adjacent to
the ipsilateral lateral superior olive. Another
10% were in other ipsilateral periolivary
groups. The remaining 20% were located
in cell groups of the SOC contralateral to
the injection site, without any apparent
concentration in a particular cell group
(fig. 1). Cell counts in three other animals
with a profusion of labeled cells revealed
similar distributions of labeling in periolivary groups.
Other projections into the CN via the
dorsal tracts were revealed by the presence
of labeled cells located along the medial
margin of the DCN contralateral to the
injection (fig. 1). These cells were spindleAbbreviations
AV, anteroventral cochlear nucleus
D, descending vestibular nucleus
DAS, dorsal acoustic stria
DC, dorsal cochlear nucleus
FG, facial genu
IAS, intermediate acoustic stria
IC, inferior colliculus
ICP, inferior cerebellar peduncle
INSH, interstitial nucleus of the stria of Held
1 0 , inferior olive
L, lateral vestibular nucleus
LC, locus coeruleus
LSO, lateral superior olivary nucleus
LT, lateral nucleus of the trapezoid body
M, medial vestibular nucleus
MCP, middle cerebellar peduncle
MSO, medial superior olivary nucleus
MT, medial nucleus of the trapezoid body
NA, nucleus annularis
NLL, nucleus of the lateral lemniscus
P, pyramidal tract
PGL, nucleus paragigiantocellularis lateralis
PH, nucleus prepositus hypoglossi
Pop, posterior periolivary nucleus
RD, dorsal raphe nucleus
RTP, nucleus reticularis tegmenti ponti
S, superior vestibular nucleus
SCP, superior cerebellar peduncle
ST, spinal tract of trigeminal nerve
TB, trapezoid body
VN, vestibular nerve
~
Fig. 1 Labeled neurons and HRP-filled axons i n
a series of transverse sections of the brain stem
in a 20-day-old kitten. Each dot represents one
labeled neuron, except in the ipsilateral (right)
cochlear nucleus where there were more labeled
neurons t h a n could be indicated. Section A is
most caudal.
111
shaped with their long axis oriented parallel to the fibers of the striae. In most
cases they were sparsely distributed from
the dorsal tip of the DCN down to and including the INSH (or in some cases, of
which figure 1 is a n example, only i n
the INSH). A few labeled cells were scattered throughout the ventral CN as far
rostrally as the AVCN (fig. 1). These cells
had no readily characterizable morphological traits nor were their exact locations consistent from animal to animal.
Nerve fibers and terminals
Individual fibers were labeled following
injection of HRP into the cut striae. ,In
favorable cases the DAS and IAS could
be traced from the injection site as far as
the lateral lemniscus of the opposite side
(fig. 1). Some fibers coursed from the injection site almost directly toward the ipsilateral SOC, while other followed the route
previously described as being that of the
DAS (Fernandez and Karapas, '67; Warr,
'69) and ran more horizontally across the
brainstem above the SOC (fig. 1D-G).
Above the contralateral SOC collaterals
coursing ventrally toward the SOC were
found leaving the horizontal fibers of the
DAS.
Medial to the injection the IAS could
be easily identified because its fibers take
a characteristic course medial to the restiform body and descend ventrally through
the trigeminal spinal tract and nucleus.
At the ventral limit of the spinal trigeminal tract the IAS turns to cross the brain
stem. At this point (fig. 1C: arrow) pericellular arborizations were sometimes seen
around cells clustered just lateral to the
facial nucleus. The fibers of the IAS traverse the rostra1 portion of the facial nucleus and pass among the periolivary cell
groups caudal and dorsal to the lateral superior olive (Fernandez and Karapas, '67;
Warr, '69). In several cases a dense afferent plexus was found in the ipsilateral
posterior and dorsolateral periolivary cell
groups (figs. 7, 8). Collaterals leaving IAS
fibers were common and terminals upon
periolivary cells could be identified. Figure
7 shows a n elaborate terminal plexus which
originated from a single axon contacting
a n unlabeled cell located in the posterior
periolivary cell group. Figure 8 shows a
labeled cell of the same cell group receiv-
112
J O E C. A D A M S A N D W . B R U C E WARR
Fig. 2 Extensive labeling of neurons of the dorsal a n d posteroventral cochlear nuclei i n a
20-day-old kitten. Note labeled axons ( a ) of t h e dorsal acoustic stria deep within the dorsal
cochlear nucleus a n d t h e faintly labeled afferent plexus (p) i n the postero-ventral cochlear
nucleus. Pericytes a n d pia-arachnoid macrophages are also heavily labeled.
ing several terminal branches from one
of several nearby HRP filled axons.
Lateral to the injection site, labeled
fibers were prominent as they entered the
C N (figs. 1 , 2). Numerous labeled fibers
were present in all but the ependymal
layer of the DCN. In the molecular layer
fine fibers ( < 1 pm in diameter) ran
parallel to the surface through the region
of the apical dendrites of the fusiform
ORIGINS OF ACOUSTIC STRIAE
cells (fig. 3, upper left). In addition to fine
axons within the fusiform cell layer, larger fibers ( 'y.1pm), probably axons of
fusiform cells, were in abundance. In
those cases where fusiform cells were completely filled with reaction product, axons
could often be seen leaving the soma or a
proximal dendrite and joining other fibers
coursing through the region. Evidence
was sought for descending fibers which
were reported to enter the DCN dorsally
and terminate in the fusiform cell layer
(Held, '93). Figure 9 shows a fine fiber
descending from the molecular layer and
terminating on the soma of a fusiform
cell. However, terminals such as this were
rare, The polymorph layer of the DCN,
which contains relatively few cells, was
nearly filled with labeled fibers. Although
axonal arborizations were readily observed
in this layer, terminals ending on cells were
never observed.
Numerous HRP filled fibers were also
observed in the PVCN. In addition to large
fibers ( > 1 pm) which are probably the
axons of large cells of the PVCN, many
fine fibers entered the PVCN from the
dorsal aspect and formed plexuses resembling those found in the SOC (fig. 5),
but terminals ending on cells were seldom
observed. Figure 10 shows one of the rare
instances where a delicate HRP-filled fiber
supplied a thick dendrite of a n octopus cell
with several bulblike terminals. A profusion
of axonal terminals was also observed among
the cells of the interstitial nucleus of the
stria of Held (fig. 1B).
B. Non-auditory projections a n d controls
In injecting the DAS and IAS, non-auditory structures were exposed to the HRP.
Accordingly, many non-auditory cells were
found to be labeled, particularly in cell
groups which project to or from the cerebellum and vestibular nuclei (fig. 1). These
included Purkinje cells and cells of the
fastigial nuclei, and inferior olive, pontine
nuclei, as well as the nucleus reticularis
tegmenti pontis. Nuclei of the reticular
formation also contained labeled cells.
These nuclei included gigiantocellularis,
parvocellularis, paragigiantocellularis dorsalis, pontis centralis caudalis, paramedian, reticularis dorsalis, lateralis reticularis
magnocellularis, and lateralis reticularis
subtrigeminalis. In addition, cells located
113
in the region of paragigiantocellularis
lateralis (fig. 1 , PGL) of both sides were
labeled. These cells were situated approximately half way between the pyramidal
tract and the rostra1 portion of the facial
nucleus. Small cells located peripheral to
(within approximately 200 p ) and in the
margins of the facial nucleus were labeled.
A few labeled cells were located i n the
medial and lateral parabrachial nuclei,
in the lateral cuneate nuclei, and in the
nucleus tractus spinal trigemini oralis
and nucleus nervi trigemini sensibilis principalis, subnucleus ventralis, bilaterally.
Following injection of the DAS and IAS,
HRP spread extensively into the lateral,
medial, and portions of the inferior vestibular nuclei. Ipsilateral to the injection
the diffusion spot tended to obscure cells
of these nuclei. Vestibular commissural
fibers were labeled, and on the side contralateral to the injection labeled cells were
located in the four principal vestibular
nuclei, as well as in nucleus Y, the interstitial nucleus of the vestibular nerve, and
the nucleus prepositus hypoglossi bilaterally.
Labeled cells were also found in nuclei
not usually thought of as having connections with the auditory system, the cerebellum, or the vestibular system. These
include the locus coeruleus of both sides
as well as several raphe nuclei. The latter
included the dorsal raphe, pontis oralis,
median raphe, and raphe obscurus.
In order to identify cells with cerebellar
connections which were labeled due to
stria1 injections, HRP injections were made
into the cerebellum. These injections produced no labeled cells in auditory nuclei,
but a number were present in the pontine
nuclei, the inferior olive, the raphe nuclei,
the locus coeruleus, the lateral cuneate,
the prepositus hypoglossi, the parabrachial
nuclei, the superior, medial, and inferior
vestibular, the above mentioned trigeminal
nuclei, small cells peripheral to the facial
nucleus, and all nuclei of the reticular
formation mentioned above except paragigiantocellularis lateralis.
Injections were also made directly into the DCN in order to label inputs to
this nucleus and minimize the spread of
HRP into vestibular and cerebellar structures. In these cases, the distribution of
labeled cells in non-auditory cell groups
114
JOE C . ADAMS AND W . BRUCE WARR
115
ORIGINS OF ACOUSTIC STRIAE
was similar to that which resulted from
cerebellar injections because part of the
cerebellum was aspirated in the DCN
exposures and the damaged cerebellar
surfaces were contracted by HRP spread
by cerebrospinal fluid. However, the incidence of labeled cells in vestibular structures was limited to a few cells in the medial
and superior nuclei. Results of the cerebellar and DCN injections suggest that nonauditory cells which were labeled following
strial injections were labeled due to involvement of cerebellar and vestibular
structures. An exception to this account
were cells of nucleus paragigiantocellularis
lateralis.
Following HRP injections of the DCN,
there were labeled cells in the same superior olivary nuclei as there were following strial injections but the number of
labeled cells in the ventral nucleus of the
trapezoid body of both sides was much increased, with the contralateral nucleus
showing more labeled cells than the ipsilateral nucleus. In addition, following injections of the DCN, cells of the inferior colliculus of both sides were labeled.
The distribution of labeled cells in the
SOC following DAS/IAS injections was
similar to the distribution of cells labeled
when HRP was injected into the cochlea
(Warr, ’75). Warr found that periolivary
cells which were labeled with HRP following cochlear injections also showed a positive reaction when stained for AChE. Because of the possibility of a spread of HRP
injected into the DAS/IAS through the
cochlear aqueduct into the cochlea, a test
for the presence of both enzymes in single
SOC cells was made in several animals
of the present study. In no instance could
Fig. 3 Fusiform cell (fc) in the dorsal cochlear
nucleus is completely filled with HRP reaction
product. Note spines on superficial dendrites at
top of picture. A horizontally oriented neuron
nestled among the dendrites of the fusiform cell
is also labeled (arrow).
Fig. 4 Labeled neuronal perikarya and axons
in deep (polymorph) layer of the dorsal cochlear
nucleus.
Fig. 5 Afferent plexus among labeled neurons
of the posteroventral cochlear nucleus. Thick axons
joining the striae from below belong to octopus
cells which contribute to the intermediate acoustic
stria.
Fig. 6 Three slender elongate cells (arrows)
located along the medial edge of the posteroventral
cochlear nucleus contain HRP reaction product,
both enzymes be identified in a single cell.
Figure 11 shows a summary of origins
of ascending and descending fibers which
make up the DAS and IAS. Cells of origin
plotted i n periolivary cell groups are meant
to show relative numbers of cells i n different groups and not how many an “average” section might contain. Figure 11
shows that cells sending descending fibers
from the SOC are found over the entire
rostro-caudal extent of the olivary complex of both sides and that 80% of these
cells are ipsilateral to the CN to which they
project. Projections also originate from the
borders of the contralateral CN, including
the medial border of the DCN, the INSH,
and scattered locations within the PVCN.
In contrast to the scattered origins of
descending fibers, ascending fibers which
leave the CN dorsally originate in relatively restricted regions, primarily the
DCN and caudal PVCN, with only a few
arising from the more rostra1 PVCN.
DISCUSSION
Results of the present study show that
introducing HRP where axons have been
severed can result in filling of axons (figs.
2-10) and in maximal labeling of somata
(fig. 3 ) located well outside the diffusion
spot. Many authors have observed soma
and axon filling near injection sites and
have assumed such filling to be due to cell
damage. The present results support that
interpretation. Such labeling opens possibilities for studying cytological details as
well as tracing pathways. Besides labeling
cells and axons in the retrograde direction from the injection site, anterograde
axonal labeling also occurs. This is evidenced by the fact that the routes taken
by the labeled fibers of the DAS and IAS
(fig. 1) match those described for these
tracts in anterograde silver degeneration
studies (Fernandez and Karapas, ’67;
Warr, ’69, ’72; Van Noort, ’69). In addition, the presence of afferent plexuses and
terminals (figs. 7-10) indicate that anterograde labeling of axons occurred. It seems
likely that axonal and complete cell filling
are a consequence of HRP being introduced to intracellular space. This interpretation is supported by results of experiments done under conditions where it was
possible to control cell damage accompanying injections of HRP (LaVail and LaVail,
116
J O E C . A D A M S AND W . B R U C E WARR
ORIGINS OF ACOUSTIC STRIAE
’74). In that study, when intravitreal HRP
injections were made, filled axons were
seen only following mechanical disruption
of the retina. These findings, along with
those of Kristensson and Olsson (‘74) and
Scalia and Coleman (‘74) indicate that
optimal labeling can be achieved by introducing HRP to cut or damaged axons.
In the present study the procedure of
severing axons and injecting HRP into the
incision made possible the discovery of
details of projections which would have
been difficult to detect by conventional
HRP methods. For example, the locations
of cells in the SOC and contralateral CN
which were found to project to the posterior CN were demonstrated by injecting
HRP directly into the DCN, but the fact
that axons of these fibers enter the CN
via dorsal pathways came to light because
injections were made directly into the
tracts, and was not shown by CN injections. Another example comes from labeling of axons which demonstrated terminals on periolivary cells (figs. 7, 8).
Projections to periolivary cells from the
PVCN had been previously demonstrated
by silver degeneration studies (Fernandez
and Karapas, ’67; Warr, ’69). However, results of the present study indicate that
some cells receiving inputs from the CN
also send projections back to the CN via a
dorsal pathway (fig. 8). This indication of
reciprocity of connections was found only
because HRP was placed on severed axons.
The present findings confirm and extend
those of previous reports on the cell types
of the CN which give rise to the DAS and
IAS. Held (‘93), using the Golgi method,
Fig. 7 A single labeled axon of the intermediate
acoustic stria branches repeatedly before terminating upon a neuron located in the posterior periolivary cell group ipsilateral to the injection. The
first bifurcation of the parent axon is obscured
by a labeled pericyte at top right of picture.
Fig. 8 Coarse axons of the intermediate acoustic stria traverse the posterior periolivary nucleus
and provide collateral arborizations, one of which
terminates (arrow) on a dendrite of a labeled
neuron.
Fig. 9 Labeled fusiform cells of the dorsal
cochlear nucleus is contacted by axonal terminal
(arrow) near the base of its superficial dendrite.
Axon (ax) of fusiform cell is visible.
Fig. 10 Axo-dendritic contacts made by a thin
HRP-labeled axon (arrow) in relation to a faintly
labeled dendrite of an octopus cell of the posteroventral cochlear nucleus.
117
described cells of the DCN and PVCN sending axons centrally via these tracts. More
recently Osen (‘72), using retrograde
chromatolysis, found that “pyramidal”
cells of the DCN (fusiform cells of the
present nomenclature) project to the contralateral inferior colliculus via the DAS,
and that large cells of the polymorph layer
of the DCN send projections centrally via
the DAS. Osen also reported that octopus
cells of the PVCN were the cells of origin
of the IAS. Warr’s results (‘69, ’72) support Osen’s interpretation of the origin of
the IAS. Results of the present investigation confirm reports that fusiform cells and
polymorph cells of the DCN project from
the nucleus dorsally (figs. 3, 4), as do octopus cells of the posterior PVCN (fig. 5).
In addition, present findings indicate that
axons of cells of the INSH (fig. l), small,
elongate PVCN cells (fig. 6), and scattered
cells located as far rostral as the entrance
of the eighth nerve (fig. 1) also leave the
CN dorsally. These additional ascending
projections, together with projections which
were found to enter the CN dorsally from
the SOC and the contralateral CN, indicate
that the fiber composition of the dorsal
tracts is much more complex than had
previously been suspected. The newly described sources of dorsal tract fibers help
to account for the finding in the previous
paper (Adams, ’76) that the number of
fibers in the combined IASIDAS far exceeded
the total number of CN cells previously
reported to contribute fibers to the striae.
Results of the present study indicate that
additional sources of stria1 fibers include
ascending, descending, and crossed projections.
One source of fibers which enters the
CN via the DAS was not studied in the present experiments. Gacek (‘73) found a projection from the posterior vermis to the
PVCN that enters the CN in the DAS.
Evidence regarding the origin of this
projection was not obtained in present experiments because the posterior vermis
and lateral cerebellar hemisphere were removed to expose the injection site. It may
be that some of the fine fibers seen in the
present material entering the PVCN from
above (figs. 2, 5) are those described by
Gacek, but the origins of these fibers could
not be determined in the present material.
118
JOE C. ADAMS AND W. BRUCE WARR
Fig. 1 1 Summary of results. The locations and relative numbers of labeled neurons, based
on quantitative study of four animals, are indicated by dots. The loci containing labeled cells
in control idections of the cerebellum are excluded. Section A is most caudal. Abbreviations
as in figure 1 .
ORIGINS OF ACOUSTIC STRIAE
Descending projections from periolivary
groups may also be a source of these fine
fibers.
Rasmussen ('60) reported a projection
arising from the ventral nucleus of the
lateral lernniscus sending fibers to the
contralateral DCN via the DAS. Van Noort
('69) was unable to confirm Rasmussen's
finding but described degeneration in the
DAS following a large lesion of the ipsilateral SOC. The present results provide
little support for the projection described
by Rasmussen. Labeled cells in the ventral nucleus of the lateral lemniscus were
sparse in all animals. The significance of
negative or marginal findings utilizing the
present methods is not yet clear but the
apparent discrepancy could be accounted
for if Rasmussen's results were due to
lesions of as yet undescribed fibers of
passage. On the other hand, the present
results confirm Van Noort's reported projection from the ipsilateral olivary complex through the DAS. The scattered distribution of periolivary cells (fig. 1) makes
it difficult to study their projections using
methods that require discrete lesions to
demonstrate degenerating fibers by an anterograde technique. A retrograde marking
technique, such as the one employed in
the present study, offers an especial advantage for demonstrating sources of inputs to a given region when the sources
are diffusely distributed.
In addition to descending projections
from periolivary cell groups, the present
study indicated the presence of descending inputs to the CN that originate in
regions not traditionally considered as
part of the auditory system. Labeled neurons were located in the nucleus reticularis paragigiantocellularis lateralis. It
seems reasonable to consider labeled cells
of this group as a caudal extension of
the SOC periolovary groups since they
are continuous with them (Taber, '61 ;
Warr, '75). This view is supported by reports that this region receives projections
from the ventral CN (Van Noort, '69;
Warr, '72) and may be a source of axons
which innervate the peripheral auditory
apparatus (Warr, '75).
The distribution of labeled cells in the
SOC following HRP injections of the DAS/
IAS was similar to that found when HRP
was injected into the cochlea (Warr, '75).
Because HRP that is injected into the sub-
119
arachnoid space of guinea pigs arrives
quickly in scale tympani (Duvall and
Sutherland, '72), the possibility must be
entertained that the labeled periolivary
cells of the present study could be olivocochlear, and were labeled because their
axonal endings were exposed to HRP
which had spread to the cochlea following injection of the striae. There are several arguments against this interpretation.
First, the distributions of labeled cells
following cochlear and acoustic striae
injections were not identical. One class of
labeled olivary cells which characterized
cochlear injections was a group of small
cells located bilaterally along the margins
of the lateral superior olive which were
especially prominent in the dorsal hilus.
Cells of this group were never found to be
labeled when HRP was injected into the
DAS/IAS. However, in one animal which
had HRP injected into the severed olivocochlear bundle, these marginal cells of
the lateral superior olive were labeled.
Secondly, in those cases where AChE stains
were done on tissue following HRP injections of DAS/IAS, no instances of cells
containing reaction products were found.
This finding contrasts with that of Warr
('75), who found many cells containing
both reaction products following cochlear
injections of HRP. These results suggest
that there are two classes of periolivary
cells which are origins of descending projections. One class shows positive staining
for AChE and projects to the cochlea. The
other class does not show staining for AChE
and projects to the CN.
Following HRP injections into the DCN,
labeled cells were found in the same locations as when the DAS and IAS were injected. In addition, labeled cells were found
in the inferior colliculi and the ventral
nuclei of the trapezoid body. Projections
from these additional regions apparently
enter the DCN ventrally. This interpretation is supported by previous reports of
projections from the colliculi to the DCN
which course through the trapezoid body
(Rasmussen, '60, '64, '67; Van Noort, '69)
and of projections from the ventral nuclei
of the trapezoid body to the DCN which
also travel in the trapezoid body (Van
Noort, '69). Van Noort, '69 assumed the
projections from the ventral nuclei of the
trapezoid body to the collaterals of the
olivocochlear bundle. Results of the present
120
JOE C. ADAMS AND W. BRUCE WARR
study and of previous studies do not support this assumption. Following HRP injections into the DCN there were dense accumulations of labeled cells in the ventral
nuclei of the trapezoid body, particularly
on the side contralateral to the injection.
Similar accumulations of labeled cells were
not seen by Warr ('75) following cochlear
injections of HRP. Furthermore, in this
region there is no similar accumulation of
cells that stain heavily for AChE (Rasmussen, '64; Osen and Roth, '67; Warr, '75).
These findings indicate that, like periolivary
projections that enter the DCN dorsally,
fibers that enter the DCN ventrally are
distinct from the olivocochlear bundle.
It has been known for some time that
acoustic stimulation of one ear can affect
single unit activity in the CN of the opposite side (Pfalz, '62; Mast, '71; Hochfeld, '73). The neural pathways underlying
such contralateral effects have not been
demonstrated. Activity underlying these effects could be transmitted by projections
to the CN from either the SOC or the inferior colliculi, or both (Rasmussen, '60,
'64, '67; Van Noort, '69). The presence of
direct C N to CN projection (fig. 1) adds
another possible route by which neural
activity of one CN could be affected by
sound delivered to the opposite ear. This
finding may not be taken as evidence for
a connection of second order neurons.
First of all, the cells that receive the
crossed CN projections have not been identified. Secondly, for the case of the one
identifiable group of cells giving rise to
crossed projection, the INSH, it appears
that they are at least third order neurons.
Warr ('69) found no projections from the
cochlea to this nucleus, but inputs from
the PVCN were described. Until more detailed investigations of crossed CN projections have been completed, the lowest level
from which projections arise to innervate
structures of the opposite side must be
assumed to be at least third order.
Results of the present study have significance for physiological recordings from
fibers in the DAS and IAS (Kiang et al.,
'73; Adams, '76). Response patterns of
single units recorded in the striae resemble
those of cells in the posterior CN (Godfrey et al., '75a,b). The locations of CN
cells giving rise to the DAS and IAS fibers
match the locations of cells with discharge
patterns similar to those recorded in the
striae. The presence of fibers entering the
CN via the DAS was shown physiologically by recordings made in the DAS at a
point medial to where the DAS had been
completely severed (Adams, '76). Results
of the present study verified the existence
of such projections anatomically and suggest some origins of these descending fibers.
Recordings have also been made from strial
fibers which showed discharge properties
similar to cells located in the contralateral
CN and the ipsilateral medial nucleus of
the trapezoid body (Adams, '76: fig. 4).
The present findings of labeled cells in
the ipsilateral nucleus of the trapezoid
body and the contralateral C N following
HRP injections of the striae (fig. 1) can
account for these physiological findings.
With the present anatomical findings in
hand, it is possible to plan further physiological experiments to demonstrate the
sources of activity recorded in the dorsal
striae.
ACKNOWLEDGMENTS
This work was supported by PHS Grants
5 R 0 1 NS 01344-16, 1 R01 NS 11000-01,
5 PO1 GM 14940, 1-F02-NS 53172-01,
and 5 R01 NS 07720-07. The technical
assistance of S. Katherine Stanton and
Heidi Van Arsdell is gratefully acknowledged. The support of the staff of EatonPeabody Laboratory of Auditory Physiology
contributed greatly to successful completion of the work. In particular, special
thanks go to Dr. Nelson Y-S. Kiang for his
continued criticism, guidance, and support.
Thanks also go to Dr, Jorgen Fex for a
critical reading of the manuscript.
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