CITY UNIVERSITY OF HONG KONG
香港城市大學
Cholecystokinin Dependent Long-Term
Potentiation in Auditory Thalamocortical
Pathway
聽覺丘腦皮層通路中膽囊收縮素依賴的
長時程增強
Submitted to
Department of Biomedical Sciences
生物醫學科學系
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
哲學博士學位
by
FENG Jingyu
馮經宇
March 2018
二零一八年三月
Abstract
Previous studies have demonstrated that thalamocortical synapses in young adult
rodents regain plasticity after the critical period. However, the mechanism(s)
underlying this recurrence remains elusive. Cholecystokinin (CCK) is the most
commonly found neuropeptide in the brain and is widely distributed throughout the
central nervous system including the medial geniculate body (MGB) and the auditory
cortex (ACx). Our recent findings suggest that CCK plays a crucial role in synaptic
plasticity in rodents. In this study, we investigated whether the age-dependent
plasticity in the adult auditory thalamocortical pathway is modulated by CCK.
Our first objective was to confirm the auditory thalamocortical plasticity in the young
adult rodents. Theta burst stimulation (TBS) in the ventral MGB (MGv) was used to
induce long-term potentiation (LTP) in the thalamocortical pathway. Robust LTP was
achieved after TBS in MGv. Next, we investigated whether this thalamocortical LTP
is also CCK dependent. Immunostaining results revealed that nearly all the
thalamocortical projecting neurons in the MGv expressed CCK. Furthermore, TBS
failed to induce the thalamocortical LTP in CCK-/- mice, and the CCK-B receptor
antagonist, L365, 260 completely blocked the thalamocortical LTP. These results
suggest that TBS-induced thalamocortical LTP is CCK dependent. To further test the
sufficiency of CCK positive MGv neurons activation in the thalamocortical LTP
induction, AAV virus was injected into CCK-Cre mice to express hChR2 in CCK
2
positive neurons of MGv, and high-frequency laser stimulation of these neurons in
MGv was applied. This successfully induced LTP in the thalamocortical pathway.
To investigate the mechanism of CCK release in the auditory thalamocortical pathway,
N-methyl-D-aspartate (NMDA) receptor, metabotropic glutamate receptor 1 and 5
(mGluR1/5) antagonists were utilized, respectively. The results suggest that the
thalamocortical LTP is not NMDA receptor but mGluR1/5 dependent.
Immunostaining results also showed the existence of mGluR1/5 on the CCK positive
thalamocortical presynaptic terminals. These results may suggest that CCK release in
the auditory thalamocortical pathway is probably controlled by mGluR1/5.
We further hypothesized that the recurrence of plasticity in the thalamocortical
pathway of young adult rodents is regulated by the emergence of CCK expression in
the MGB, during development. To test this hypothesis, both LTP induction and CCK
expression levels were examined at different postnatal days (P10, P14, P21, P28, and
adult). We could not observe LTP in rats at P10 and P14 in vivo. Immunostaining
results also demonstrated that CCK was barely detectable in MGB in first two
postnatal weeks. At the end of the third postnatal week, a weak CCK expression was
detected in MGB, and minor LTP could be induced by TBS. Both CCK expression
level and LTP continued to increase after P21 until adulthood. There was only a sparse
CCK expression in MGB of old rats (>18 months), and the auditory thalamocortical
pathway lost the plasticity. CCK-8S injection in the auditory cortex of the old rats
could partially restore the plasticity of the auditory thalamocortical pathway. Another
3
interesting finding was that the peak latency of the thalamocortical activation became
shorter (31.55±0.75ms ~ 7.40±0.16ms) during the developmental stage (P10-P60),
which is a sign of a maturation process of the thalamocortical synapses. This finding
also supports the subplate neuron model of the thalamocortical development.
Finally, to examine the plasticity induction ability of CCK in behaving condition,
mice were injected with CCK-8S in the auditory cortex and were exposed to the tone
stimulation. Prepulse inhibition level of modified gap startle reflex was measured.
The animals showed a significant increase in prepulse inhibition compared to the
control group. This evidence suggests that CCK could significantly enhance the
frequency differentiation ability of mice.
This study is consistent with previous findings that the auditory thalamocortical
pathway develops plasticity during the young adulthood and shows for the first time
how CCK regulates the thalamocortical plasticity at different ages. Further, these
findings may be translated to develop a therapeutic intervention for treating hearing
loss and tinnitus.
4
Relevant Publications
Journal publications
Xiao Li, Kai Yu, Zicong Zhang, Wenjian Sun, Zhou Yang, Jingyu Feng, Xi Chen,
Chun-Hua Liu, Haitao Wang, Yi Ping Guo & Jufang He. Cholecystokinin from the
entorhinal cortex enables neural plasticity in the auditory cortex. Cell Research
volume 24, pages 307–330 (2014)
Xiao Li, Xi Chen, Yin Ting Wong, Haitao Wang, Hemin Feng, Xuejiao Zheng,
Jingyu Feng, Joewel T. Baibado, Robert Jesky, Yujie Peng, Zhedi Wang, Hui Xie,
Wenjian Sun, Zicong Zhang, Xu Zhang, Ling He, Nan Zhang, Zijian Zhang, Peng
Tang, Jun-Feng Su, Ling-Li Hu, Qing Liu, Xiao-Bin He, Ailian Tan, Xia Sun, Min
Li, Kelvin Wong, Xiaoyu Wang, Yi Ping Guo, Fuqiang Xu, Jufang He.
Cholecystokinin release triggered by presynaptic NMDA receptors produces LTP and
sound-sound associative memory formation. bioRxiv 188839
Jingyu Feng, Xuejiao Zheng, Peter Jendrichovsky, Joewel T. Baibado, Xi Chen,
Jufang He. Cholecystokinin dependent long-term potentiation in the auditory
thalamocortical pathway. (In preparation)
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Acknowledgments
I would like to express my deep gratitude to my supervisor, Prof. Jufang He, whose
worthy guidance, inspirations, motivations and generous support and encouragements
enabled me to accomplish this project.
It is my great pleasure to thank all my dear colleagues who accompanied me these
years and made this thesis possible, especially to mention Dr. Xi Chen, Dr. Xiao Li,
Dr. Kai Yu, Ms. Xuejiao Zheng, Mr. Peter Jendrichovsky, Mr. Joewel T. Baibado,
and other members in the lab.
Especially, I would like to express my sincere appreciation to my dear wife. She is
always showing patience and unreserved support to my study and research work.
I would like to thank my parents for giving me life and supporting me to pursue my
academic career.
Finally, I also appreciate the lovely people studying and working in CityU and PolyU.
They made these places warm and beautiful.
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Table of Contents
Abstract ..................................................................................................................... 2
Relevant Publications ............................................................................................... 5
Acknowledgments ..................................................................................................... 6
1. Introduction ........................................................................................................ 10
1.1 Ascending auditory pathway ....................................................................... 10
1.2 Long-term potentiation and thalamocortical LTP .................................... 19
1.2.1 Long-term potentiation .......................................................................... 19
1.2.2 Thalamocortical LTP ............................................................................. 20
1.3 Cholecystokinin and its function ................................................................. 22
1.4 Aims of this study .......................................................................................... 25
2. Material and Methods ........................................................................................ 27
2.1 Animals .......................................................................................................... 27
2.2 Chemicals and Antibodies ............................................................................ 27
2.3 Surgery ........................................................................................................... 28
2.3.1 Acute electrophysiological experiments ............................................... 28
2.3.2 Virus and retrograde tracer injection .................................................. 29
2.3.3 Cannula implantation ............................................................................ 29
2. 4 Auditory, electrical and laser stimuli ......................................................... 30
7
2.5 In vivo acute electrophysiological recording .............................................. 31
2.6 Immunohistochemistry and imaging .......................................................... 34
2.7 Frequency discrimination test ..................................................................... 35
2.8 Data analysis .................................................................................................. 36
3. Results .................................................................................................................. 38
3.1 Theta burst stimulation (TBS) induced thalamocortical LTP in the
auditory cortex in vivo ........................................................................................ 38
3.2 TBS enhanced noise responses of auditory cortical neurons .................... 45
3.3 Auditory thalamocortical projecting neurons were Cholecystokinin
(CCK) expressing neurons ................................................................................. 49
3.4 The loss of the auditory thalamocortical LTP in CCK-/- mice................. 51
3.5 Thalamocortical LTP was blocked by CCK-B receptor antagonist ........ 53
4.6 High-frequency (HF) optical activation of CCK positive neurons in the
MGv induced thalamocortical LTP .................................................................. 55
3.7 TBS-induced thalamocortical LTP was group I mGluR dependent ........ 63
3.8 The ontogeny of CCK-dependent thalamocortical LTP during
development ......................................................................................................... 69
3.9 Artificial CCK-8S injection partially restored thalamocortical LTP in old
rats ........................................................................................................................ 75
3.10 CCK enhanced the frequency discrimination ability in adult mice ....... 81
8
4. Discussion ........................................................................................................... 89
References: .............................................................................................................. 96
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1. Introduction
1.1 Ascending auditory pathway
Hearing is one of the most important sensory modalities in animals. It plays a crucial
role in localizing and identifying sound resources, which could help the animals sense
the unseen dangers or potential preys in the wild. For some species, the hearing also
guides the learning of vocal behavior, like understanding and production of speech in
human (Jeffery A. Winer & Schreiner, 2011). To have a deep understanding of the
function of the auditory system, it is very crucial to have a brief review of the anatomy
of the auditory pathway.
It is well-established that there are two major pathways in the central nervous system
for auditory information processing. The ascending auditory pathway, which from the
cochlea to the auditory cortex, is responsible for the perception of sound. The
descending pathway, on the other hand, sending signals from the auditory cortex
down to the cochlea, plays a vital role in filtering the sound information, which will
not be explicated in this study. The ascending auditory pathway contains six major
components, cochlear nucleus (CN), superior olivary complex (SOC), lateral
lemniscus (LL), inferior colliculus (IC), medial geniculate body (MGB), and finally
the auditory cortex, Figure I.
10
Figure I. Diagram of the ascending auditory pathway in human (CIBA Collection of
Medical Illustrations, Volume 1)
11
Sound waves travel through the ear canal and vibrate the eardrum. The eardrum, then,
transmits the vibration to the cochlea via the ossicles. In the cochlea, the mechanical
sound vibration is transformed into an electrical signal by the electro-mechanical
channels of the hair cells. The hair cells lay inside of the organ of Corti on the basilar
membrane of the cochlea. Because of the characteristics of the basilar membrane, the
hair cells show the significant frequency specificity, as the hair cells in the basal end
of the cochlea respond to high frequencies and those located in the apical end detect
low frequencies (Kros & Evans, 2006). While the electro-mechanical transduction
happens in the hair cells, cochlear ganglion cells digitize the signals received from
the hair cells through one to one connection and send the information to the first stop
in the central nervous system, the cochlear nuclei, via cochlear nerve. The cochlear
nuclei, which located in the brainstem, possess two essential features. One is the
tonotopic organization. The cochlear nerve terminals arborize in the cochlear nuclei
tonotopically. The fibers, which convey the high-frequency sound information,
terminate in the dorsal part of the dorsal and ventral cochlear nuclei and those carrying
low-frequency sound information terminate ventrally (Bourk, Mielcarz, & Norris,
1981). The other feature is that each cochlear nerve fiber connects to different types
of neurons in different areas of the cochlear nuclei. In this manner, the ascending
auditory pathway is separated into more than four parallel pathways, which convey
different features of the sound (Cant, 1992). Once they leave CN, most of the fibers
of the cochlear nucleus neurons cross over to the contralateral side of the brain, which
is different from that found in the visual system. Each hemisphere processes the
12
acoustic information mostly comes from the contralateral ear. The next stop in the
brainstem is SOC, which is the first nuclei that receive information from both ears.
Because of this characteristic, SOC nuclei is very important for sound location in
many vertebrates. They have different circuits for detecting the interaural time
difference and the interaural intensity differences, as the lateral superior olive (LSO)
is responsible for the interaural intensity differences detection and the medial superior
olive (MSO) is assigned to analyze the interaural time differences (Park, Grothe,
Pollak, Schuller, & Koch, 1996; Pecka, Brand, Behrend, & Grothe, 2008). After
passing through or around LL, all the auditory information from the brainstem level
(CN, SOC) converge into IC and is, then, sent to the auditory cortex via MGB.
MGB is the principal thalamic nucleus related to the auditory perception. It receives
inputs from IC, thalamic reticular nucleus (TRN), the auditory cortex, and other areas
and project primarily to the auditory cortex, and other subcortical regions, like the
amygdala and TRN (LeDoux, Farb, & Ruggiero, 1990; Yu, Xu, He, & He, 2009).
There are three subdivisions of MGB, the medial (MGm), dorsal (MGd) and ventral
(MGv), which are distinguished by the afferent and efferent connections, the cell
morphology, physiology, and being conservative across many species (J. a Winer,
1984; J A Winer & Larue, 1996). Among the three subdivisions, the MGv receives
the projection from the central nucleus of IC, TRN, and the corticothalamic projection
sending from the auditory cortex. The output of MGv mainly target on the primary
auditory cortex (AI) and is defined as the lemniscal auditory thalamocortical pathway
because of the tonotopical organization of MGv (He, Yu, Xiong, Hashikawa, & Chan,
13
2002). It was thought that the primary function of the lemniscal auditory
thalamocortical pathway was merely relaying the frequency and intensity information
to the cortex. However, more and more evidence suggests that the plasticity of this
pathway is highly involved in the development and refinement of the cortical
tonotopic map and experience-dependent reorganization of the auditory cortex
(Steriade & Timofeev, 2003), which will be further introduced in the next section.
MGd and MGm, on the other hand, are categorized as non-lemniscal nuclei of MGB
since these two subdivisions show no clear tonotopic map. They receive inputs from
the non-tonotopic area of IC and send projections to the secondary auditory cortex
(AII), limbic area and amygdala (C. C. Lee, 2015), Figure II.
14
Figure II. The schematic summary of thalamocortical projections from different
subdivisions of MGB (C. C. Lee, 2015)
15
The final station on the ascending auditory pathway is the auditory cortex, which
located in the temporal lobe of the cortex. The auditory cortex was first described by
Sir. David Ferrier, who used stimulation and ablation of various gyri and surface
regions of the monkeys` brains to identify the function of the brain, in 1875 and has
been intensively studied since then (Jeffery A. Winer & Schreiner, 2011). Like all the
sensory cortices in the brain, the auditory cortex shares the six-layer structure. Layer
I, which also called the molecular layer, contains the thalamocortical axons originated
from MGm and the apical dendrites of pyramidal neurons in deep layers (Huang &
Winer, 2000). Only a few of neurons are found in layer I, and they are mainly
GABAergic neurons. They have diverse lateral connections with the apical dendrites
from other layers (J. A. Winer & Larue, 1989). Layer II is relatively rich in pyramidal
neurons, also some unique neurons, and it primarily projects to other cortical areas (J
A Winer, 1985). Layer III consists of two sub-layers, layer IIIa, a pyramidal layer,
and layer IIIb, a non-pyramidal layer. It contains corticocortical feedforward
connections and is the main origin of the commissural projections (Thomas & López,
2003). Layer IIIb and layer IV are the main input layers where the lemniscal
thalamocortical projections target on (J. a Winer, 1984), and the output of layer IV is
local (layer II, III) (Mitani et al., 1984). Layer V, the internal pyramidal layer, consists
of corticocortical, corticothalamic and corticocollicular projecting neurons (Jeffery A
Winer, 2001). Layer VI contains various shapes of neurons, mostly excitatory, and
the major target of layer VI neurons is MGB.
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The auditory cortex is also divided into many fields based on the different cortical
functions, and the functional organization of the auditory fields varies within different
species. There are three auditory fields in the rat (Shi & Cassell, 1997), thirteen in the
cat (C. C. Lee & Winer, 2005), and twelve in the monkey (Hackett, 1999). Among
these auditory fields, the primary auditory cortex is the largest part, which receives
the lemniscal projections from MGv (Sally & Kelly, 1988). It has a short latency of
the responses to the sound and represents the frequencies tonotopically. In rodents,
the anterior part of AI responds to the high frequencies and the posterior part respond
to the low frequencies (Recanzone, Schreiner, & Merzenich, 1993). The function of
the primary auditory cortex was studied for a long time. It was thought that AI could
only represent the auditory information from the outside world and viewed as neither
critical site of learning, nor memory storage location. In 1956, Galambos et al.
established the first electrophysiological study of the auditory cortex during learning
(G Sheatz, 1956). They found that fear conditioning could cause a significant increase
in the amplitude of evoked potentials to the conditioned stimulus (CS) in the auditory
cortex. Since then, lots of works have been done to study the plasticity of the auditory
cortex (Blake, Heiser, Caywood, & Merzenich, 2006; Polley, Heiser, Blake, Schreiner,
& Merzenich, 2004; Weinberger, 2007). Nearly all the experiments demonstrated
associative neural plasticity in the primary auditory cortex after a sound was paired
with another meaningful event (reward or punishment), the responses of the cortical
neurons changed. In 1986, Gonzalez-Lima and Scheich reported that tone-shock
pairing produced a specific increase in uptake of the metabolic marker in an area of
17
the primary auditory cortex of the rat that processed the tone stimulus (GonzalezLima & Scheich, 1986)，which was the first demonstration revealing that learning
specifically modified auditory coding in the primary auditory cortex. From then,
electrophysiological experiments on tuning curve were introduced to the primary
auditory cortex. Weinberger and colleagues found that when a tone was followed by
a shock in the guinea pig, tuning curves of the auditory cortical neurons would shift
toward or even to the frequency of the tone (Bakin & Weinberger, 1990). Later studies
found that there are various experimental manipulations that can change the neuronal
representations of sounds, such as changing the statistics of an acoustic environment
(de Villers-Sidani, Chang, Bao, & Merzenich, 2007; Norẽa, Gourevich, Aizawa, &
Eggermont, 2006; Zhang, Bao, & Merzenich, 2001), pairing acoustic stimulation with
neuromodulatory signals (Froemke, Merzenich, & Schreiner, 2007; Kilgard &
Merzenich, 1998; Swanson & Köhler, 1986), and manipulating the behavioral
significance of a sound (Blake et al., 2006; Fritz, Shamma, Elhilali, & Klein, 2003;
Polley, Steinberg, & Merzenich, 2006; Recanzone et al., 1993; Schnupp, Hall,
Kokelaar, & Ahmed, 2006).
As the studies above elucidated that numerous neuronal connectivity changes
contribute to the plasticity of AI, in the present study, we focused on the plasticity of
the lemniscal auditory thalamocortical pathway, which is from MGv to AI.
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1.2 Long-term potentiation and thalamocortical LTP
1.2.1 Long-term potentiation
Long-term potentiation has been studied for more than forty years and is considered
as the major candidate cellular mechanism of learning and memory (Roberts &
Glanzman, 2003). It was first discovered in the hippocampus of a rabbit by Bliss and
Lomo in 1973 (Bliss & Gardner-Medwin, 1973). They stimulated perforant pathway
which projecting from the entorhinal cortex and recorded the evoked response in the
dentate gyrus of the hippocampus. As a result, high-frequency electrical stimulation
(100Hz) would significantly increase the synaptic strength of this neuronal pathway
and an increased firing possibility in response to the constant synaptic input. These
two findings together were defined as LTP. After the original finding of LTP in
hippocampus, lots of experiments have been done to investigate LTP in different areas
of the brain, such as cerebral cortex (Cooke & Bear, 2010), amygdala (Clugnet &
LeDoux, 1990) and thalamocortical pathway (Crair & Malenka, 1995; Hogsden &
Dringenberg, 2009; Isaac, Crair, Nicoll, & Malenka, 1997). Different forms of LTP
are observed in different neuronal structures. Based on the signaling pathways that
recruited for the formation of LTP, LTP is categorized N-methyl-D-aspartate (NMDA)
receptor-dependent (Lüscher & Malenka, 2012) and NMDA receptor-independent
(Futatsugi et al., 1999), in which some of NMDA independent LTP are mGluR
dependent (H. Wang et al., 2016). The pre-post synaptic activation which required to
induce LTP is another factor for LTP classification. According to the induction
19
protocol, LTP is also divided into Hebbian and non-Hebbian (Lechner & Byrne,
1998). As Hebbian`s theory postulated that cells that fire together, wire together,
Hebbian LTP induction requires pre- and post-synaptic activation at the same time.
Parallelly, Non-Hebbian LTP does not require the simultaneous activation of pre- and
post-synapses.
The function of LTP has been discussed for over forty years in different circuits of
the brain. It has been intensively interpreted as the underlying mechanism for learning
and memory in the hippocampal system (Bliss & Collingridge, 1993), amygdala
(Clugnet & LeDoux, 1990), and most of the cerebral cortex (Rioult-Pedotti, 2000).
During the development, LTP has also been considered as the mechanism for the
maturation and refinement of the topographical map in the sensory cortices of
neonatal rodents (Daw, Scott, & Isaac, 2007; Feldman, Nicoll, & Malenka, 1999;
Martini, Moreno-Juan, Filipchuk, Valdeolmillos, & López-Bendito, 2018).
1.2.2 Thalamocortical LTP
As mentioned in the previous section, LTP of the thalamocortical pathway plays a
key role in the formation and refinement of the topographical map in the sensory
cortices during development. In the somatosensory system, LTP could be induced by
pairing the pre-and postsynaptic activation within the first postnatal week in vitro
(Crair & Malenka, 1995). Furthermore, by applying NMDA receptor antagonist to
the neonatal rats, the formation of the topographical map of barrel cortex was
disturbed (Schlaggar, FOX, & O`Leary, 1993). Also, by exposing the neonatal mice
20
with the tone presentation during the auditory critical period, the tonotopical map of
the auditory cortex was significantly impaired (Zhang et al., 2001). At the end of the
critical period of each sensory modality, LTP in the thalamocortical pathway vanished
because of the switching of NMDA receptor subunits during the development (Barkat,
Polley, & Hensch, 2011; Malenka & Bear, 2004). Many methods have been tried to
reverse the critical period to make the sensory cortex more plastic in the adult brain.
By bringing the adult or juvenile rats back to a moderate noise environment or
reducing the adenosine receptor 1 (A1R) signaling in MGB, the primary auditory
cortex became easier to be manipulated by passive tone exposure (Blundon et al.,
2017; Zhou, Panizzutti, de Villers-Sidani, Madeira, & Merzenich, 2011). Also,
peripheral sensory deprivation could restore the critical period of the thalamocortical
input in the somatosensory cortex (Seungsoo Chung et al., 2017). Inspired by these
studies, new treatment plans could be established to help patients with the
developmental disorder (Zhu et al., 2014). The thalamocortical LTP has also been
found in the adult brain, which serves as the potential mechanism of sensory
experience-dependent plasticity in the sensory cortex. In 2001, Bear and colleagues
found that, by applying theta burst stimulation to the dLGN, LTP could be induced in
the visual cortex in vivo (Heynen & Bear, 2001). Also, the result was successfully
repeated in the auditory thalamocortical pathway in vitro with the existence of
picrotoxin (Chun, Bayazitov, Blundon, & Zakharenko, 2013). However, the
underlying mechanism of thalamocortical LTP in the adult brain remains elusive.
21
1.3 Cholecystokinin and its function
Cholecystokinin (CCK) was first discovered as a molecule that causes gallbladder
contraction. In 1975, van der Haagen and colleagues found that CCK is one of the
gastrointestinal peptides in mammalian brains (van der Haagen, 1975). CCK family
has several biologically active fragments. Among the family, CCK-8S is the most
dominant member in the brain tissue and shows a wide distribution in both the
peripheral and central nervous system. It has been found in the neurons of olfactory
tubercle, the substantia nigra, the ventromedial thalamus, the septum, the nucleus
accumben, the ventral tegmental area, the interpeduncular nucleus, the hypothalamus,
the posterior lobe of the pituitary and the spinal cord (Beinfeld, Meyer, Eskay, Jensen,
& Brownstein, 1981). This neuropeptide has also been detected in cortical areas and
limbic structures such as the hippocampus and the amygdala (Lotstra &
Vanderhaeghen, 1987), Figure III. In the auditory pathway, Senatorov found CCK
mRNA expression in reciprocally-connected MGB and AC, which implies the
modulatory function of CCK in the thalamo-cortico-thalamic circuit (Senatorov,
Trudeau, & Hu, 1997).
22
Figure III. Distribution of CCK neurons and their major projections in the rat
brain (Beinfeld, 2013)
For CCK receptors, there are two subtypes: CCK-A and CCK-B. CCK-A receptors
are constitutively expressed in the alimentary tract, which are closely involved in the
endocrine regulation of digestion and feeding behavior, whereas CCK-B receptors are
widely distributed in the central nervous system, such as the hippocampus, the
amygdala, and cortices (Saito, Sankaran, Goldfine, & Williams, 1980; Zarbin, Innis,
Wamsley, Snyder, & Kuhar, 1983). Moreover, one recent preliminary study from our
lab also indicated that there might be a third unidentified CCK receptor in the cortical
area, which plays a crucial role in the plasticity of GABAergic synapses (Unpublished
data).
It has been found that CCK is involved in satiety (Gibbs et al. 1973, Zhang et al. 1986),
nociception (De Araujo et al. 1998), fear (Fekete et al. 1984), and most importantly,
23
learning and memory (Harro et al. 1993, Hadjiivanova et al. 2003, Li 2014, 2017).
Fekete reported that infusion of CCK-8 into nucleus accumben facilitated the
extinction of active avoidance behavior and attenuated the retention of passive
avoidance behavior, and applying CCK-8 to the central amygdala caused opposite
effects. An in vitro study suggested that LTP, but it substantially prolonged LTP of
both field excitatory postsynaptic potential (fEPSP) and population spikes (PS) from
about 1 to at least 3h. The CCK antagonist PD 135158 did not influence the induction
of potentiation of the fEPSP slope but decreased the potentiation of PS amplitude.
The results support the view that CCK-8S may facilitate long-term changes of
glutamatergic synaptic transmission in the hippocampal LTP (Balschun & Reymann,
1994). Behavioral test of an appetitive task also provided the evidence that
administration of CCK-B agonist improves the performance, and CCK-B antagonist
can abolish the improvement if it is injected into the hippocampus (Matto, Harro, &
Allikmets, 1997). Moreover, CCK knockout mice exhibited attenuated performance
in a passive avoidance task and impaired spatial memory in the Morris Water Maze
Test (Lo et al., 2008). In 2014, our lab found that local CCK-8S injection into the
auditory cortex could significantly enhance the neuronal response to the sound
stimulation and synaptic strength was potentiated after pairing the presynaptic and
postsynaptic activity in the presence of CCK (Li et al., 2013). Another recent
experiment showed that CCK, which released from the entorhinal cortical projecting
terminals in the auditory cortex, could induce the cortico-cortical LTP and soundsound associative memory (Li, Chen, Wong, Wang, & Feng, 2017).
24
All the evidence above suggests that CCK is an important neuromodulator that
participates in the neural plasticity throughout the brain.
1.4 Aims of this study
It has been shown that the thalamocortical plasticity still exists in many sensory
modalities of the adult brain. However, the underlying mechanism of the
thalamocortical LTP and its emergence after the critical period remain elusive. Based
on the previous findings of our lab, we hypothesized that CCK, which expressed in
MGB, plays an essential role in the formation of thalamocortical LTP and the
expression level of CCK is correlated with the emergence of thalamocortical LTP
during development. To test this hypothesis, CCK-/- mouse line was used to test the
existence of the thalamocortical LTP and CCK-B receptor antagonist was
administrated to block the thalamocortical LTP which induced by TBS. To further
confirm the sufficiency of CCK in the thalamocortical LTP induction, CCK positive
neurons in MGv of CCK-Cre mice were labeled by an optogenetic virus that
expressing hChR2, and high-frequency laser activation of CCK positive neurons in
MGv was then used to test whether activating the CCK positive neurons is sufficient
to induce the thalamocortical LTP. Next, to test the hypothesis raised in our previous
work that CCK release is mediated by presynaptic NMDA receptor activation during
TBS, NMDA receptor antagonist and group I mGluR (mGluR 1/5) antagonists were
applied to block the thalamocortical LTP. Furthermore, the ontogeny of CCK
expression in MGB and the thalamocortical LTP were also examined by
immunostaining and electrophysiological recording in the neonatal rats, respectively.
25
Finally, to explore the potential therapeutic function of CCK in the loss of neural
plasticity in adult and aged brains, CCK was infused into the auditory cortex, and the
plasticity was measured on both electrophysiology and behavioral level.
26
2. Material and Methods
2.1 Animals
In the present study, rats and mice of both sexes were used to investigate the
thalamocortical LTP. For rats, Sprague-Dawley rats in different ages (neonatal: P10P28, young adult: 6-8weeks, old: older than 18 months), with clean external ear canals,
were used in the electrophysiological and immune-histochemical experiments. For
the mouse lines, C57BL/6 wild-type mouse, CCK-ires-Cre (Jax#012706), CckCreERT2 (Jax#012710) were used for the electrophysiological, optogenetic, immunehistochemical and C57 mice were also used in the behavioral experiments. All
experimental procedures were approved by the Animal Subjects Ethics SubCommittees of The City University of Hong Kong.
2.2 Chemicals and Antibodies
For in vivo electrophysiological experiments, CCK-8S (Cat.No. 1166), selective
CCK-B receptor antagonist, L-365, 260 (Cat. No. 2767), DMSO (Cat. No. 3176),
NMDA receptor antagonist, DL-APV sodium salt (Cat. No. 3693), selective noncompetitive mGluR5 antagonist MPEP hydrochloride (Cat. No. 1212), selective noncompetitive mGluR1 antagonist YM 298198 hydrochloride (Cat. No. 2448) were
purchased from Tocris Bioscience (Hong Kong SAR). Artificial cerebrospinal fluid
(item# 59-7316) was used as a solvent for the antagonists above and was purchased
from Harvard Apparatus (U.S.). For the immunohistochemistry, the primary
antibodies used are rabbit polyclonal CCK-8S antibody (given by Prof. Tomas
27
Hokfelt, 1: 500), anti-GFP antibody (Ab13970, 1:500), anti-CCK B receptor
(Ab137495, 1:500). Anti-mGluR1 antibody (Ab82211, 1:200), anti-mGluR5
antibody (Ab53090, 1:200), purchased from Abcam (U.S.). Secondary antibodies,
including Alexa Fluor 488-conjugated goat anti-chicken, CY5-conjugated goat antirabbit, and Alexa Fluor 405-conjugated goat anti-mouse, were purchased from
Invitrogen.
2.3 Surgery
2.3.1 Acute electrophysiological experiments
Rats or mice were anesthetized with urethane sodium (1.6g /kg IP; Sigma., US).
Anesthesia was maintained throughout surgery. Atropine sulfate (0.05 mg/kg SC;
Sigma., US) was administered 15 min before the induction of anesthesia to inhibit
tracheal secretion. The animal was mounted in a stereotaxic frame (Narishige, Japan)
and a midline incision was made in the scalp after a liberal application of a local
anesthetic (Xylocaine, 2%). Cranial windows were prepared, and the dura mater was
minimally opened followed by the silicon oil application to the surface of the brain to
prevent drying. The animal’s body temperature was maintained at 37-38°C with a
feedback-controlled heating pad (RWD, China). After the recording, those animals
were sacrificed, and the brains were harvested for histological confirmation or
immunostaining.
28
2.3.2 Virus and retrograde tracer injection
CCK-Cre mice or SD rats were anesthetized with Pentobarbital (5mg/kg IP, France)
and kept under anesthetic status by supplying one-third of the initial dosage once per
hour. For the optogenetic virus injection, two small holes were drilled bilaterally in
the skull of CCK-Cre mice according to the coordination of MGv (AP: -3.16mm, ML:
1.9mm, DV: 3.2mm). 100nl, 2.13x10^12 titer, rAAV5-EF1a-DIO-hChR2
(E123T/T159C)-EYFP (UNC vector core, U.S.) was injected into MGv. For the
retrograde tracer injection, True Blue (5%, 1 µl, Invitrogen) in 0.1 M PBS was infused
into the auditory cortex of rats. After injection, animals were sutured and sent back to
the home cage for recovery. Antiseptic and analgesic balm was applied on the surface
of the wound during first three days after the surgery.
2.3.3 Cannula implantation
C57 mice were anesthetized, and the scalp was opened as mentioned above. Two
small holes were prepared bilaterally based on the location of the primary auditory
cortex (AP: -3.0mm, ML: 4mm, DV: 2.2mm). Drug injection cannulas with metallic
caps and dummies (Length: 6mm, Diameter: 0.6mm., RWD Science., China) were
inserted into the primary auditory cortex bilaterally and then fixed with C&BMetaBond adhesive luting cement (Parkell, U.S.) and dental cement (Mega Press,
German). After the implantation, the animals were sent back to the home cages for
recovery. Antiseptic and analgesic balm was applied on the surface of the wound on
first three days after the surgery.
29
2. 4 Auditory, electrical and laser stimuli
Auditory stimuli, including pure tones and noise bursts, were generated by TuckerDavis Technologies (TDT, U.S.) workstation and delivered through an electrostatic
speaker (ED1, TDT). Auditory stimuli were played via the speaker placed at 20 cm
away from the behavioral animals or directly to the ear contralateral to the implanted
electrodes via a hollow ear bar for the anesthetized animals. The sound pressure level
of the speaker was calibrated with a condenser microphone (B&K, Denmark).
The electrical stimulation was generated by an ISO-Flex isolator (A.M.P.I., Israel),
which was controlled by a multifunction processor (RX6, TDT). The electrical current
pulses for baseline test were 0.5ms, 50-150µA and presented every 10s. The theta
burst stimulation (TBS) contained four trails of 10 bursts at 5 Hz with the interval of
10s between the trails, and each burst consisted of 5 pulses at 100 Hz.
The laser stimulation was produced by a laser generator (Wavelength=473nm, Ike
Cool, China) controlled by RX6 and delivered to the brain by an optic fiber (Thorlabs,
U.S.) which was connected to the generator. The output power of the fiber was
measured and calibrated by an optical power meter (Item# PM120A, Thorlabs, U.S.)
before inserted into the brain. The laser pulse width was 5ms, and the interval for
baseline testing was 10s. For the high-frequency (HF) laser stimulation, the laser
pulse train was comprised of four trails of 10 bursts at 5 Hz with the interval of 10s
between the trails, and each burst consisted of five pulses at 80 Hz.
30
2.5 In vivo acute electrophysiological recording
In TBS-induced thalamocortical LTP experiments, two customized microelectrode
arrays with four tungsten electrodes each, impedance: 0.5-1 MΩ (recording), and 200500kΩ (ES) (FHC, U.S.), were used for the auditory cortical neuronal activity
recording and MGv electrical stimulation and monitoring, respectively. The two
electrode arrays were driven by two micro-manipulators separately. 70 dB, 100ms,
noise bursts were presented every 10s during the insertion of the electrodes arrays,
and neuronal activities were monitored as the electrode arrays were lowered until
robust noise responses were achieved on both arrays. The final stimulating and
recording positions were determined by maximizing the cortical field excitatory
postsynaptic potential (fEPSP) amplitude triggered by the electrical stimulation in
MGv. Histological confirmation showed that the stimulating electrode tip was placed
in MGv (Figure 1). In neonatal rat experiments, the coordinates of MGB and ACx
were readjusted according to ATLAS OF THE DEVELOPING RAT BRAIN IN
STEREOTAXIC COORDINATES (Khazipov et al., 2015).
The fEPSPs, which were elicited by 0.5ms electrical current pulses, were amplified
(×1000) and filtered (1Hz -5kHz), with the 25kHz sampling rate, stored in PC by
OpenEx software (TDT). In the young adult animals, fEPSPs with less than 10ms
peak latency were considered as monosynaptic transmission. However, the peak
latencies of evoked fEPSPs in neonatal rats were significantly longer than the adult
rats because of the immaturity of the thalamocortical pathway. Before the recording,
an input-output function was measured. A stimulation current, which elicited a fEPSP
31
amplitude 50% of maximum, was chosen for the baseline and after TBS recording.
The fEPSPs were collected for 15min and 1 hour, before and after TBS, respectively.
For TBS, 10 negative electrical current bursts were delivered in 5Hz and repeated 4
trials. The inter-trial interval was 10s. Each burst contained 5, 0.5ms pulse, in 100Hz.
The current amplitude was selected 75% of the maximal response from the inputoutput relationship. The slopes of the evoked fEPSPs were calculated and normalized
by customized MATLAB script, and the group data was plotted as mean±SEM. Also,
50 noise bursts (Intensity=70dB, Duration=100ms, Inter-stimulus-interval=10s) were
presented before and after LTP induction session in rat experiments, and multiunit
noise responses of the auditory cortex were recorded. A threshold of 3 standard
deviations (SDs) above baseline was set to identify spikes online. Z-scores was then
used to compare the noise responses before and after TBS in MGv. The Z-score of
the neuronal response to the tone stimulus was calculated against the mean
spontaneous firing rate before the stimulus onset; bin width was 5ms. It represented
the difference between stimulus-evoked neuronal responses and average spontaneous
firing in units of SD. Neuronal responses larger than 3.5 SD above baseline were
considered as noise responses.
In the optogenetic experiments, two to three tungsten electrodes were placed into the
deep layer III and layer IV (350-500μm) of the auditory cortex in CCK-Cre mice 5-6
weeks after virus injection. The optic fiber was then inserted into either the auditory
cortex, right next to the electrodes, or MGv, and the laser-evoked responses in the
auditory cortex for both stimulating sites were tested. As the hChR2 expressing
32
thalamocortical projecting terminals could not follow high-frequency stimulation
(Figure 14), fEPSPs evoked by laser stimulation in MGv were recorded and analyzed
in this experiment. The procedures for the HF laser-induced LTP was similar as
previously described in the TBS-induced LTP experiment, except the high-frequency
burst containing laser pulses in 80Hz rather than 100 Hz.
In the antagonist infusion experiments, L-365,260 (250nM in 10% DMSO, 1μl) was
injected into the rat auditory cortex, and MPEP (43μM, 1μl), YM298198 (250nM,
1μl) and DL-APV (40μM, 1μl) were injected into the mouse auditory cortex by microinjector, before TBS, within 10min. ACSF (10% DMSO) or ACSF was injected in all
the drug infusion experiments as a control.
In the old rat thalamocortical LTP induction experiments, CCK-8S (1μΜ, 1μl) or
ACSF was injected into the auditory cortex of old rats. Low-frequency electrical
stimulation, which contains 200, 0.5ms, 75% saturated current pulses in 0.1Hz were
delivered into MGv after the injection.
In the tuning curve test, tones spanning 6 octaves (750-48kHz, 0.2-0.3 octave spacing)
and 60dB (10-70 dB, 5-10 dB spacing) lasting 100ms were presented every 500ms in
a pseudo-random sequence before and after the pairing to measure the receptive field
of cortical neurons. For the pairing protocol, CCK-8S (1μΜ, 1μl) or ACSF was
injected locally near the recording site, and non-Characteristic Frequency (non-CF)
pure tone stimuli, one octave within the CF, were presented once per 2s for 200 trails,
5mins after the injection. We limited all analysis to action potentials that occurred
33
less than 30ms after the onset of the tone during the test. The threshold was
determined as the lowest intensity bin in the receptive field, and CF was the middle
frequency bin at that intensity. To quantify the changes of the tuning curve, frequency
responding threshold was calculated as an indicator of the change. The data were
plotted as mean ±SEM.
After the recording, animals were sacrificed, and the brains were harvested for
histological confirmation or immunostaining.
2.6 Immunohistochemistry and imaging
Rats and mice were euthanized and then perfused with ice-cold saline followed by 4%
paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The brains were then
harvested and soaked in 4% PFA for post-fixation. The brains were dehydrated by
30% sucrose and sectioned with a cryostat (thickness 50μm), and the brain slices were
washed with PBS followed by incubated in a blocking buffer, which contains 10%
goat serum, 0.2% Triton-100 for 2h at room temperature. After the blocking, brain
slices were incubated with primary antibodies (24-48h) at 4°C and washed three times
for 10 min with PBS before incubation with secondary antibodies (1:500) for 2h.
After being intensively washed with PBS, brain slices were mounted with mounting
medium with or without DAPI for imaging and analysis. Slices were imaged with
Zeiss Laser Scanning Microscope LSM 880 NLO with Airyscan, or Nikon Eclipse
Ni-E upright fluorescence microscope.
34
2.7 Frequency discrimination test
Modified prepulse inhibition of the acoustic startle response test was adopted to
examine the frequency discrimination ability of mice in CCK-8S injection group and
ACSF control group. Three customized soundproof chambers equipped with
vibration sensors on the bottoms for the startle reflex detection, MF-1 multi-field
magnetic speakers for tone presentation and high-power tweeter speakers for startle
white noise presentation. 3 days after the cannula implantation surgery, mice were
placed into the self-designed plastic tubes individually with opening slots on both
sides and front for habituation, 5-10min every day for 3 days. The mice were, then,
randomly separated into two groups, one CCK-8S injection group, and one ACSF
injection control group. CCK-8S (10nM, 1μl) or ACSF (1μl) was infused by
microinjector at the speed of 0.2 μl/min bilaterally into the auditory cortices. 5min
after the injection, mice were placed in the tubes and exposed to pure tone stimulation
for 0.5h in the soundproof chamber. For the tone exposure protocol, a pure tone of
9.8kHz or 16.4kHz (Intensity=70dB, duration=100ms) was used. The tone exposure
consisted of 900 trains of tones, and the trains were presented every 2s. Each train
contained 5 tones presented at 5Hz. 24hrs after the exposure, mice were restrained
into the tubes again for the startle test. The tubes were stabilized on the vibration
sensors in the soundproof box during the experiment. The startle reflexes were
detected by the sensors and then amplified and recorded by TDT. The whole
experiment was divided into 4 blocks; a background tone (9.8 kHz or 16.4 kHz) was
continuously presented at 70 dB throughout the experiment. Block 1 was a 5min
35
acclimation period in which only the background tone was presented. Block 2
contained 9-startle only trials in which a 120dB, 20ms white noise (WN) burst was
presented. Block 3 consisted of prepulse inhibition trials in a pseudorandom order.
Each prepulse trial consisted of a 70dB 80ms prepulse (pure-tone frequency was 0%,
2%, 4%, 8%, 16%, or 32% lower than the background tone, Δf), followed by a 120dB,
20ms white noise startle pulse, and then return to the background tone after the startle.
Every trial in Block 3 was presented 15 times. Block 4 was identical with Block 2,
and it was used to detect any habituation within the experiment. The inter-trial interval
was 10-20s. The startle reflex was measured as peak-to-peak amplitude of the raw
waveform detected by the sensor. Prepulse inhibition percentage was calculated from
Block 3 data as follows: [1 – (prepulse trial/Δf=0% prepulse trial)] ×100 and the data
was plotted as mean±SEM. Startle reflex amplitude in Block 2 and Block 4 were
compared with each other as an internal control for startle attenuation over the whole
experiment. Mice in which there were statistically significant differences between
both trials were removed from the analysis.
2.8 Data analysis
All data are presented as mean ±SEM, and n represents the number of recording sites
or animals in each experiment. We used one-way ANOVA for three or more groups.
Two-way ANOVA was used to compare mean values under different interventions
with in two groups. If the data fails to pass the normality test, ANOVA on rank was
used. Tukey test was used for the post-hoc test. P values < 0.05 were considered
36
statistically significant. These statistical analyses were performed using SigmaPlot
12.5
37
3. Results
3.1 Theta burst stimulation (TBS) induced thalamocortical LTP in the
auditory cortex in vivo
To confirm the existence of the thalamocortical LTP in the auditory cortex (ACx) of
the adult rats, ES array was inserted into the medial geniculate body (MGB), and the
recording electrodes were placed into the primary auditory cortex (according to the
vasculature). Unlike Bear`s study, they recorded the evoked potential in the
superficial layer of the visual cortex (Heynen & Bear, 2001). In the present study, we
focused on the plasticity of the lemniscal auditory thalamocortical pathway, which is
from the ventral medial geniculate body (MGv) to the deep layer III and layer IV of
the auditory cortex (ACx). To confirm the location, after recording, the lesion was
made to the MGv by driving 10mA current. Figure 1 shows the ES array inserted into
MGv, and the recording electrodes were into the thalamorecipient layers of ACx (500700µm deep from the surface).
38
Figure 1. Experimental setup for thalamocortical LTP induction and histological
confirmation of the position of ES array in MGv A) Schematic diagram represents
the experimental setup, ES array was placed in the MGv, and the recording electrodes
array was inserted into the auditory cortex. B) Nissl stained brain slice shows the
position of the ES electrode tips in the MGv, as indicated by the asterisk. Scale bar:
500µm. LTP: long-term potentiation, MGd: dorsal medial geniculate body,
MGm, medial medial medial geniculate body, MGv: ventral medial geniculate
body, ACx: auditory cortex.
39
During the insertion, sound responses were monitored to determine the precision of
the electrode positions at the MGv and the ACx. 100 ms noise bursts in 70 dB were
played every ten seconds, and the sharp responses with the shortest latency indicated
that the electrodes were in the desired locations (Figure 2).
Figure 2. Raw signal of the extracellular multiunit recording in MGv and ACx.
A) 100ms, 70dB noise bursts were presented during the electrodes insertion. Robust
sound responses were achieved when the electrodes reached the layer IV of the ACx
and the MGv, respectively. B) Detected spike waveforms of neuronal firing in ACx.
40
Subsequently, to test the connectivity between the stimulating and recording sites, 0.5
ms electrical pulses were delivered through one of the ES electrodes in the MGv every
10s. The position of the recording electrodes was then optimized to achieve the
maximal fEPSPs with shortest peak latency (less than 10ms) triggered by the
stimulation. The short peak latency of the fEPSP reflects the monosynaptic
transmission of the thalamocortical pathway (Chun et al., 2013).
Before the LTP induction, the relationship between the stimulation intensity in the
MGv and the evoked fEPSPs in the ACx was tested. The stimulation current was
gradually increased by the step of 50 µA, and the evoked potentials were recorded
and averaged every 6 trials. Such input-output relationship is shown in Figure 3. As
seen from the figure, the fEPSPs elicited by the ES is a function of stimulation
intensity and reached the saturated level when the current went up to 300µA. The
baseline probing current for the LTP induction was selected as the current elicited 50%
of the saturated fEPSP, and 75% was used for TBS stimulation.
41
Figure 3. The input-output relationship between stimulation intensity in MGv
and evoked fEPSP in ACx. Negative current pulses, started from 50µA and
increased by 50µA per step, were presented in MGv and the evoked fEPSPs were
recorded from ACx to calculate the input-output relationship between the stimulation
intensity and the level of the response. The baseline stimulation current was set at a
value that could evoke 50% of the maximum response, while the TBS current was set
at 75%. fEPSP: field excitatory postsynaptic potential
42
Figure 4. TBS protocol for LTP induction. The schematic diagram illustrates the
stimulation protocol. 10 negative electrical current bursts were delivered in 5Hz and
repeated 4 trials. The inter-trial interval was 10s. Each burst contained 5, 0.5ms pulse,
in 100Hz. The current amplitude was selected 75% of the maximal response from the
input-output relationship as shown in Figure 3.
43
During the LTP induction, a stable baseline of the evoked fEPSP was recorded for
15min, TBS (Figure 4) was then applied to the MGv, and fEPSPs were recording of
for another 1h following the stimulation. Consistent with the previous findings in
other sensory modalities (Heynen & Bear, 2001; K. K. Y. Lee, Soutar, & Dringenberg,
2018), TBS successfully induced the auditory thalamocortical LTP in the adult rats.
The slopes of fEPSPs were measured 1h after TBS increased to 156 ± 7.2% of
baseline (Figure 5) (n=11, one-way ANOVA, p<0.001).
Figure 5. TBS-induced LTP in the auditory thalamocortical pathway. Population
data of normalized fEPSP slopes before and after TBS. (n=11, One-way ANOVA,
p<0.001). Sample fEPSP waveforms before and after TBS (1, 2). Scale bar: 5ms,
0.2mV.
44
3.2 TBS enhanced noise responses of auditory cortical neurons
As mentioned in the definition of LTP, the postsynaptic firing possibility to a constant
synaptic input should also increase when LTP happens (Bliss & Gardner-Medwin,
1973). To test this, 50 trials of noise bursts with low sound intensities were presented
before and after the LTP induction, and subsequently, the noise responses of the
auditory cortical neurons were recorded (Figure 6). As we expected, the firing rate
of the noise responses significantly increased after TBS and is shown by PSTH and
raster plots in Figure 7. The latency of the responses also became shorter.
Furthermore, the spontaneous activity and the rebound oscillation in the auditory
cortex were also increased (data not shown), which may suggest that the overall of
the auditory cortex became more active after TBS in the MGv. We repeated the
experiments on 6 rats, and the mean Z-score of the noise responses showed a
significant increase (Figure 8, n=12, two-way ANOVA，p<0.001; post-hoc: Tukey
test, before vs after TBS, **, p< 0.001, *, p<0.05). These results suggest that the
thalamocortical plasticity does still exist in the auditory system of adult rats and could
be induced by TBS.
45
Figure 6. Schematic diagram of the experimental setup and the experimental
protocol. 50 trials of noise responses in the auditory cortex were tested and compared
before and after the thalamocortical LTP induction.
46
Figure 7. Noise responses in ACx before and after TBS in MGv. A) PSTH and
raster plot (grey) show weak neuronal responses to the noise burst stimulation before
TBS. B) PSTH and raster plot (red) show the increased neuronal responses to the
noise burst increased after TBS.
47
Figure 8. Mean Z-score of noise responses before and after TBS. Z-score (mean
±SEM) of the cortical neuronal responses to the noise bursts before (grey) and after 。
(crimson) TBS in the MGv. Bin width: 5ms. The blue dash line represents the
threshold of the sound response, 3.5 standard-deviation. The noise responses were
significantly increased after TBS (n=12, two-way ANOVA, p<0.001; post-hoc:
Tukey test, before vs after TBS, **, p<0.001, *, p<0.05)
48
3.3 Auditory thalamocortical projecting neurons were Cholecystokinin
(CCK) expressing neurons
After confirming the existence of the thalamocortical LTP in the auditory cortex of
adult rats, we wanted to investigate the mechanism of the adult thalamocortical LTP.
Cholecystokinin (CCK) is a neuropeptide, which is widely distributed in the central
nervous system. In 1997, Senatorov and colleagues reported that CCK is expressed
in both MGB and the deep layer of the auditory cortex (Senatorov et al., 1997). Recent
experimental results of our laboratory demonstrated that local CCK infusion into the
auditory cortex potentiates the cortical neuronal responses to the natural sound (ref).
Furthermore, CCK, sent from the entorhinal cortex, can enable the cortical neural
plasticity ( Li, Chen, Wong, Wang, & Feng, 2017; X. Li et al., 2013). Based on these
evidence above, we hypothesized that CCK is involved in the auditory
thalamocortical LTP. To test this hypothesis, first, we investigated whether CCKexpressing neurons in the MGv send projections to the auditory cortex. True Blue, a
retrograde tracer, was injected into the auditory cortex, and CCK expression in the
retrogradely labeled neurons was then detected by the CCK antibody. In the MGv,
the thalamocortical projecting neurons were labeled as blue, and the CCK expression
was shown as red (Figure 9). The high level of True blue and CCK colocalization
indicated that most of the thalamocortical projecting neurons in the MGv expressed
CCK.
49
Figure 9. Auditory thalamocortical projecting neurons expressed CCK. The
schematic shows the injection site of the True Blue tracer. A) True Blue retrogradely
labeled neurons were found in the MGv. B) The immunostaining shows that CCK
was expressed in MGv neurons. C) The merged picture indicates that nearly all the
projecting neurons contained CCK. Scale bar: 50µm
50
3.4 The loss of the auditory thalamocortical LTP in CCK-/- mice
As the immunostaining result showed that a clear majority of the thalamocortical
neurons express CCK; if CCK works as a key molecule that controls the
thalamocortical LTP, deficits of the thalamocortical plasticity were expected in the
CCK-/- animals. In the next experiment, CCK-/- mice were used to examine the
thalamocortical LTP. As we predicted, TBS could not induce LTP in the
thalamocortical pathway of CCK-/- mice, whereas significant LTP was successfully
induced in C57 wild-type mice (Figure 10. CCK-/-, n=9; C57, n=8, two-way
ANOVA, p<0.001; post-hoc: Tukey test, CCK-/-, p>0.05; C57, p<0.001). The result
indicates that CCK is necessary for the thalamocortical LTP. However, we could not
rule out the possibility that there might be some congenital deficit besides CCK
expression that had contributed to the loss of plasticity in the auditory thalamocortical
pathway of CCK-/- mice. Although, the cortical neuronal responses to the auditory
stimuli in these mice were similar comparing with C57, suggesting the normal
function of the auditory pathway (Li et al., 2017).
51
Figure 10. TBS failed to induce the thalamocortical LTP in CCK-/- mice. A)
Population data of normalized slopes of fEPSPs before and after TBS in CCK-/- mice
(blue open circle). Sample fEPSP waveforms before (1) and after (2) TBS in CCK-/mice. B) Population data of normalized slopes of fEPSP before and after TBS in C57
mice (blue filled circle). Sample fEPSP waveform before (1) and after (2) TBS in C57
mice. Scale bar: 5ms, 0.3mV. (CCK-/-, n=9; C57, n=8, two-way ANOVA, p<0.001,
post-hoc: Tukey test, CCK-/-, p>0.05; C57, p<0.001).
52
3.5 Thalamocortical LTP was blocked by CCK-B receptor antagonist
To confirm our hypothesis that CCK plays a key role in the thalamocortical LTP
induction, L365, 260, a CCK-B receptor antagonist, was infused into the auditory
cortex of rats before TBS. Another group of rats (control) received artificial cerebralspinal fluid (ACSF). We found that L365, 260 infusion totally blocked the
thalamocortical LTP induction after TBS, whereas the LTP induction in the control
group remained intact (Figure 11, L365, 260, n=9; ACSF, n=8, two-way ANOVA,
p<0.001, post-hoc: Tukey test, L365, 260, p>0.05; ACSF, p<0.001), suggesting that
CCK-B receptor activation is also necessary for the thalamocortical LTP induction.
53
Figure 11. CCK-B receptor antagonist blocked the thalamocortical LTP. A)
Population data of normalized slopes of fEPSPs before and after L355, 260 infusion
and TBS (Red filled circle). Sample fEPSP waveforms before (1) and after (2)
L365,260 infusion and TBS. B) Population data of normalized slopes of fEPSPs
before and after ACSF injection and TBS (Gray filled circle). Sample fEPSP
waveforms before (1) and after (2) ACSF injection and TBS. Scale Bar: 5ms, 0.3mV.
(L365, 260, n=9; ACSF, n=8, two-way ANOVA, p<0.001, post-hoc: Tukey test, L365,
260, p>0.05; ACSF, p<0.001).
54
4.6 High-frequency (HF) optical activation of CCK positive neurons in the
MGv induced thalamocortical LTP
Previous studies suggest that electrical stimulation in the MGB, the thalamic
radiations or the white matter beneath the sensory cortices may activate not only the
thalamocortical projections but also antidromically activate the corticothalamic
projections, which also contain CCK (Beierlein, 2003; Senatorov et al., 1997). To
activate the thalamocortical pathway exclusively and to test the sufficiency of
activation of CCK positive MGB neurons in the thalamocortical LTP induction, we
used the optogenetic method. We injected a Cre-dependent AAV virus, AAV-EFIaDIO-hChR2 (E123T/T159C)-EYFP, which specifically infected CCK-positive
neurons and expressed hChR2 into the MGv of CCK-Cre mice. As shown in Figure
12, nearly all the neurons in the MGv were labeled with EYFP, indicating that most
of the thalamocortical projecting neurons were CCK positive. Also, EYPF labeled
thalamocortical projection fibers mainly targeted on the Layer IV of the primary
auditory cortex. The previous study also mentioned that there is a high density of
CCK binding sites in the layer IV of the auditory cortex (Zarbin et al., 1983),
suggesting the active site of the thalamocortical CCK. To examine this hypothesis,
CCK B receptors were stained on the brain slides, which contained the labeled
thalamocortical terminals in the auditory cortex. We found that the CCK B receptors
(red) and the CCK-positive thalamocortical terminals (green) were next to each other,
resembling a synaptic structure. This result implies that CCK may release from the
55
thalamocortical projection terminals and has effects on the postsynaptic neurons in
the layer IV, which express CCK-B receptors.
56
Figure 12. Cre-dependent virus infected CCK-positive thalamocortical
projecting neurons in CCK-Cre mice. The schematic showing the virus injection
site in the MGB and thalamocortical projection in the ACx. A-B) Histological
confirmation of the virus expression in both ACx and MGB in CCK-/- mice (Green:
EYFP, Blue: DAPI. Scale bar: ACx=100µm, MGB=50µm). C) CCK B receptors
were visualized as red by immunostaining (white triangles). D) CCK positive
thalamocortical fibers and terminals were labeled as green by EYFP (white triangles).
E) CCK positive terminals and CCK B receptors were next to each other (white
triangles). Scale Bar: 5 µm.
57
In our previous studies, we found that CCK dependent plasticity requires the pre- and
post-synaptic activation and the presence of CCK (Li et al., 2013). Also, CCK release
from the presynaptic terminals required high-frequency (HF) stimulation (Li et al.,
2017). Therefore, in the current experiment, HF laser stimulation was utilized to
trigger the CCK release, which in turn, induced LTP. However, as a key feature of
the thalamocortical connections, the thalamocortical responses show strong shortterm depression because of the decreased glutamate release during the repeated
thalamic radiation or white matter activation (Z Gil, Connors, & Amitai, 1997; Ziv
Gil, Connors, & Amitai, 1999). Therefore, before the HF laser LTP induction
experiment, we tested the efficacy of the laser-induced thalamocortical activation in
both MGv and ACx. The fEPSPs evoked by laser in the auditory cortex were
significantly smaller than those evoked by the identical power of the laser in the MGv,
whereas the peak latency was longer for those fEPSPs evoked by the laser in the MGv
(Figure 13). This result suggests that laser activating thalamocortical fibers was less
efficient to elicit the thalamocortical responses in the ACx than the MGv. The shortterm depression of the thalamocortical response was also compared between two laser
activation sites by using HF laser stimulation for the LTP induction (Figure 14). In
agreement with the previous studies (Sooyoung Chung, Li, & Nelson, 2002; Ziv Gil
et al., 1999), stimulating the thalamocortical fibers in the ACx, the thalamocortical
responses in the ACx showed clear depression. As the HF laser bursts were delivered,
the fEPSPs decreased and stopped to follow the stimulation after the third laser burst.
On the contrary, flashing the laser to the MGv could induce reliable fEPSPs in the
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ACx, which could follow the HF activation. Although the fEPSPs also showed a
depression within one laser burst and partially recovered when the next laser burst
arrived, this method could reliably activate the pre- and post-synaptic components.
For the LTP induction of thalamocortical laser-response, one optic fiber was inserted
into the MGv, and the recording electrodes were placed in the layer IV (350-500µm)
of the ACx. Laser evoked thalamocortical fEPSPs were recorded. After a stable
baseline achieved, HF laser stimulation was then delivered into the MGv. The result
was similar to what we found while using ES. A robust LTP was achieved after the
HF laser stimulation (Figure 15; n=8, one-way ANOVA, p<0.001). This result
suggests that HF activation of CCK-positive thalamocortical projecting neurons may
trigger CCK release at the thalamocortical synapses, which in turn enables the
thalamocortical LTP.
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Figure 13. Auditory cortical responses evoked by activation of CCK positive
thalamocortical fibers and cell bodies. The schematics show the laser fiber
positions in ACx (left) and in MGv (right). Laser evoked fEPSPs were recorded in
both conditions. For the identical laser power, stimulating in the MGv elicited larger
responses in ACx than stimulating the thalamocortical fibers in ACx
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Figure 14. Responses evoked by HF laser stimulation in ACx and MGv. A) HF
laser stimulation protocol. 4 trials of HF laser stimulation were delivered with intertrial-interval was 10s. Each trial contained 10 bursts, and each burst consisted 5, 5ms
pulses in 80Hz (40% duty cycle). B) Raw waveforms of fEPSPs evoked by HF laser
stimulation shows that fEPSPs in the ACx could follow the laser stimulation in the
MGv, but not ACx. HF: high frequency
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Figure 15. HF laser stimulation of CCK positive thalamocortical neurons in the
MGv induced LTP in the thalamocortical pathway. Normalized slopes of laserevoked fEPSPs 15mins before and 1h after HF laser stimulation in the MGv (n=8,
one-way ANOVA, p<0.01). Sample laser evoked fEPSP waveforms before (1) and
after (2) HF laser stimulation. Scale bar: 5ms, 0.1mV.
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3.7 TBS-induced thalamocortical LTP was group I mGluR dependent
The results above suggest that TBS-induced thalamocortical LTP depends on CCK
release from the thalamocortical terminals; however, the underlying mechanism for
CCK release remains unclear. Based on our previous findings in the intracortical LTP
(Li et al., 2017), CCK release is gated by presynaptic NMDA receptor activation. To
test whether the same mechanism also works in the thalamocortical pathway, NMDA
receptor antagonist, DL-APV, was injected into the auditory cortex before the LTP
induction. We found that DL-APV did not block the thalamocortical LTP (Figure
16A, n=8, one-way ANOVA, p<0.001). However, group I metabotropic glutamate
receptors, mGluR1/5, were involved in the thalamocortical LTP induction, as the
results showed that MPEP, mGluR5 antagonist, and LY298198, mGluR1 antagonist
blocked TBS-induced thalamocortical LTP (Figure 16B-C, MPEP, n=7, one-way
ANOVA, p>0.05, LY298198, n=9, one-way ANOVA, p>0.05). Previous studies have
shown that mGluR1/5 mainly work in a postsynaptic manner and are involved in
different types of LTP, which are induced by different protocols and in different areas
(Huemmeke, Eysel, & Mittmann, 2002; Stiefel, Tennigkeit, & Singer, 2005; Vickery,
Morris, & Bindman, 1997; X. F. Wang & Daw, 2003). Furthermore, group I mGluR
agonists could depolarize hippocampal neurons and elevate intracellular Ca2+
(Bianchi, Young, & Wong, 1999). However, in some special case, they are expressed
in the presynaptic area to regulate the neurotransmitter release (Xie, Chen, & Pan,
2017). In the present study, we hypothesized that group I mGluR regulate the release
of CCK in the auditory thalamocortical pathway presynaptically. mGluR1/5 were
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stained on the brain slices with a presynaptic biomarker, synaptotagmine, and EYPF
labeled CCK-positive thalamocortical projecting fibers. We found that mGluR1 and
mGluR5 were colocalized with CCK-positive thalamocortical terminals in layer IV
of the auditory cortex, which may suggest that mGluR1/5 control CCK release in a
presynaptic manner. However, further studies are necessary to confirm the role of
mGluR1/5 in the thalamocortical LTP.
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Figure 16. TBS-induced thalamocortical LTP was group I mGluRs dependent,
not NMDAR dependent. A) Normalized fEPSP slopes before and after the injection
of D-APV in the ACx and TBS in the MGv (DL-APV, n= 7, one-way ANOVA,
p<0.01). Sample fEPSP waveforms before (1) and after (2) DL-APV injection and
TBS. Scale bar: 10ms, 0.3mV. B) Normalized fEPSP slopes before and after the
injection of in the ACx and TBS in the MGv (LY298198, n=9, one-way ANOVA,
p>0.05). Sample fEPSP waveforms before (1) and after (2) LY298198 injection and
TBS. Scale bar: 10ms, 0.3mV. C) Normalized fEPSP slopes before and after the
injection of in the ACx and TBS in the MGv (MPEP, n=9, one-way ANOVA, p>0.05).
Sample fEPSP waveforms before (1) and after (2) MPEP injection and TBS. Scale
bar: 10ms, 0.3mV.
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Figure 17 Presynaptic mGluR1/5 on the CCK-positive thalamocortical
projecting terminals. A) Co-labeling of EYPF (green), mGluR1 (red), and
synaptotagmin (blue) in the auditory cortex of CCK-Cre mice, which was infected
with AAV-EF1α-DIO-hChR2 (E123T/T159C)-EYFP in the MGv. The white
triangles indicate the colocalization of mGluR1 and the presynaptic terminals of
CCK-positive thalamocortical projecting fibers in the layer IV of the auditory cortex.
Scale bar: 10µm. B) Co-labeling of EYPF (green), mGluR5 (red), and synaptotagmin
(blue) in the auditory cortex of CCK-Cre mice, which was infected with AAV-EF1αDIO-hChR2(E123T/T159C)-EYFP in the MGv. The white triangles indicate the
colocalization of mGluR5 and the presynaptic terminals of CCK-positive
thalamocortical projecting fibers in the layer IV of the auditory cortex. Scale bar:
10µm.
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3.8 The ontogeny of CCK-dependent thalamocortical LTP during development
Since we found that the auditory thalamocortical plasticity in the adult rodents is
CCK-dependent, which is different from the mechanism of LTP in the neonatal brains
(Barkat et al., 2011; Crair & Malenka, 1995), the second question we wanted to
address was when TBS-induced CCK-dependent plasticity starts to emerge during the
thalamocortical development. To answer this question, the thalamocortical plasticity
was examined in the rats at different postnatal days (P10, P14, P21, P28) in vivo. In
agreement with in vitro experimental results (Crair & Malenka, 1995), the
thalamocortical LTP did not exist in the neonatal rats at P10 and P14 (Figure 18, P10,
n=9, one-way ANOVA, p>0.05, P14, n=11, one-way ANOVA, p>0.05). However, a
weak potentiation (113±4.8%) after TBS was observed at the end of the third postnatal
week (Figure 18, P21, n=13, one-way ANOVA, p<0.05), and the potentiation kept
increasing to the adult level (143±11.3%) around P28 (Figure 18, n=7, one-way
ANOVA, p<0.001). There are two critical factors may account for this CCKdependent thalamocortical plasticity developmental pattern. One factor is CCK
expression level in the MGB during the development. By staining the CCK expression
in the brain slices of rats at different postnatal days, we found that CCK was barely
detectable in P10, and weak signals were detected in P14 rats (Figure 19). The
expression level started to increase around P21 and reached the adult level until P28.
The higher CCK expression level indicates that the thalamocortical pathway would
be more plastic after the critical period. The other factor of CCK-dependent
thalamocortical plasticity developmental pattern is the maturation of the
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thalamocortical connection. The peak latencies of evoked fEPSPs during
development were measured (Figure 20). The peak latency was 31.55±0.75ms in P10.
The peak latency kept shortening as the rats grew and finally reached the adulthood
in P28 (8.82±0.25ms) (P10, n=9, P14, n=9, P21, n=9, P28, n=9, Adult, n=9, one-way
ANOVA, p<0.001, post-hoc: Tukey test, *, p<0.05). These results reflected the
progress of the thalamocortical projection maturation during the development. In the
auditory system, MGB projections enter the subplate area and activate the subplate
neurons as early as P2 (Viswanathan, Bandyopadhyay, Kao, & Kanold, 2012; Zhao,
Kao, & Kanold, 2009). These neurons, which have direct connections with the layer
IV neurons in the auditory cortex, guide the thalamocortical projections to the layer
IV neurons during the first two postnatal weeks. During these two postnatal weeks,
two important events happen concomitantly: the opening of the ears and the auditory
critical period. As the thalamocortical connections getting mature, the subplate
neurons eventually die out around the fourth postnatal week. Also, the myelination
change of the thalamocortical projection during development affects the conduction
velocity, which in turns results in the peak latency (Salami, Itami, Tsumoto, & Kimura,
2003). Also, the myelin staining revealed that, before the second postnatal week, the
whole brain was unmyelinated. As the animals grew and the myelin sheath started to
cover the thalamocortical projection except for the section that entering the cortex.
Thus, the increased conduction velocity caused by the growing myelination of the
thalamocortical projection also contributes to the shortened peak latency that we
observed. These three findings together suggest that the CCK-dependent plasticity
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started to emerge in the auditory thalamocortical pathway when CCK started to
express in the MGB and the thalamocortical projections were mature enough to
follow the HF stimulation, which triggered the CCK release from the synapses.
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Figure 18. TBS-induced thalamcortical plasticity of the rats at different
postnatal days Normalized fEPSP slopes before and after TBS in the MGv at
different postnatal days, P10 (open triangle), P14 (pink spade), P21 (orange circle)
and P28 (red square). In P10 and P14, no LTP was induced by TBS in the MGv (P10,
n=9, one-way ANOVA, p>0.05; P14, n=11, one-way ANOVA, p>0.05). A minor
increase was observed after TBS at P21 (n=13, one-way ANOVA, p<0.05) and P28
rats showed significant thalamocortical LTP induced by TBS (n=7, one-way ANOVA,
p<0.001).
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Figure 19. Peak latencies of the evoked fEPSPs in the thalamocortical pathway
of the rats at different postnatal days. Left: sample fEPSP waveforms at different
postnatal days (P10, P14, P21, P28, and Adult). The blue dash lines indicate the peak
latencies of the representative fEPSPs. Right: population data of the peak latencies.
(P10, n=9, P14, n=9, P21, n=9, P28, n=9, Adult, n=9, one-way ANOVA, p<0.001
post-hoc: Tukey test, *, p<0.05)
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Figure 20. CCK expression level in the MGv neurons of the rats at different
postnatal days. Immunostained CCK expression of the MGv neurons at P10, P14,
P21, P28. CCK: red; DAPI: blue. Scale bar: 20 µm.
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3.9 Artificial CCK-8S injection partially restored thalamocortical LTP in old
rats
LTP, working as a possible mechanism of learning and memory, is also subject to
aging (Barnes, 2003). Previous studies revealed that age-related impairment of
hippocampal-dependent memory is partially caused by altered synaptic plasticity,
including LTP (Deupree, Bradley, & Turner, 1993). During aging, the synapses also
become less plastic in the thalamocortical pathway (Hogsden & Dringenberg, 2009).
To investigate the thalamocortical plasticity in the aged brains, older than 18 months
rats were examined in the current study. We tried to induce the thalamocortical LTP
by TBS in these rats. As we inserted the recording electrodes into the auditory cortex,
low spontaneous activity was observed, and neurons were less responsive to the sound
stimulation throughout the insertion process (data not shown). However, the fEPSP
could still be elicited by the electrical stimulation in MGv, except that the evoked
fEPSP was easily saturated by the electrical current less than 200µA. After baseline
recording, TBS failed to induce LTP in these rats, and minor decreases of fEPSPs
were also observed in some cases (Figure 21, n=9, one-way ANOVA, p>0.05). This
result suggests that the thalamocortical synapses lose the plasticity in the old age.
According to our hypothesis, we speculated that the loss of the thalamocortical LTP
must be accompanied by the decreased expression of CCK in the MGB. As we
expected, the CCK expression level in the MGB decreased in the old rats (Figure 22).
The results of CCK downregulation in the MGB and thalamocortical LTP loss is
likely a causal relationship. If the loss of thalamocortical LTP is caused by the
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decreased CCK expression, an introduction of artificial CCK should rescue this agerelated impairment. To test this, CCK-8S was infused into the auditory cortex, and
low-frequency stimulation (LFS), 200 trials of electrical stimulation (75% of the
saturated current pulse in 0.1Hz) were applied to the MGv after the infusion. The
slopes of evoked fEPSPs increased to 126±5.1% of the baseline in CCK-8S infused
rats (n=8, two-way ANOVA, p<0.001, post-hoc: Tukey test, p<0.001). ACSF control
rats had no such effect (n=8, two-way ANOVA, post-hoc: Tukey test, p>0.05), Figure
23. The reason that CCK-8S infusion did not have the similar effects as TBS may be
due to the decreased CCK-B receptor expression in the auditory cortex, or the loss of
neurons in the thalamocortical pathway. These results indicate that CCK could
partially restore thalamocortical LTP.
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Figure 21. TBS failed to induce the thalamocortical LTP in old rats. Normalized
fEPSP slopes before and after TBS in the MGv of the old rats (n=9, one-way ANOVA,
p>0.05). Sample fEPSP waveforms before (1) and after (2) TBS in the MGv of the
old rats. Scale bar: 5ms, 0.2mV.
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Figure 22. CCK expression was downregulated in the MGv neurons of the old
rats. CCK expression level in the MGv neurons of the old rats (2 years old) was
detected by the immunostaining. CCK: Red; DAPI: Blue. Scale bar: 50 µm
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Figure 23. CCK-8S injection in the auditory cortex induced thalamocortical LTP
in the old rats. A) Normalized fEPSP slopes before and after CCK-8S injection in
the ACx and LFS in the MGv of the old rats (n=8, two-way ANOVA, p<0.001, posthoc: Tukey test, p<0.001). Sample fEPSP waveforms before (1) and after (2) the
CCK-8S injection and LFS. Scale bar: 5ms, 0.1mV. B) Normalized fEPSP slopes
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before and after ACSF injection in the ACx and LFS in the MGv of the old rats (n=8,
two-way ANOVA, post-hoc: Tukey test, p>0.05). Sample fEPSP waveforms before
(1) and after (2) the ACSF injection and LFS. Scale bar: 5ms, 0.1mV.
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3.10 CCK enhanced the frequency discrimination ability in adult mice
Based on our above findings, CCK is involved in the thalamocortical plasticity in both
juvenile and adult animals. Also, it could partially restore the thalamocortical LTP in
older rodent brains, indicating a therapeutic potential of CCK in the developmental
impairments or age-related diseases. Since the auditory cortical plasticity is less
susceptible to the tone exposure after the critical period unless it is linked to
meaningful events such as punishments or rewards (M. Weinberger, 2012), we
wanted to investigate whether artificial CCK administration could enable the
thalamocortical plasticity triggered by passive tone exposure, which in turn changes
the frequency discrimination ability of adult mice. CCK-8S was injected into the
auditory cortex of anesthetized rats, and supra-threshold tone stimulation was
delivered for 200 trails after the CCK-8S injection. The tuning curves of the auditory
cortical neurons were measured before and after the intervention. We found CCK-8S
did not entirely shift the characteristic frequency (CF) of the tuning curve to the
pairing tone frequency. Around the exposed frequencies, it only enhanced the
neuronal responses and lowered the responding threshold to the frequencies in the
range of one octave (Figure 24, CCK, n=15; ACSF n=12, two-way ANOVA,
p<0.001, post hoc: Tukey test, CCK-8S vs ACSF, *p<0.05, **p<0.001), suggesting
that CCK-8S potentiated thalamocortical activation during the exposure.
In this study, we also conducted the prepulse inhibition acoustic startle test. After
recovery from cannula implantation surgery and habituation of the testing tube, CCK8S and ACSF control injected mice were exposed to the tone stimulations (9.8 or
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16.4kHz) in soundproof testing chambers immediately after the injections (Figure
25). 24h after the exposure, the frequency discrimination ability was tested by
comparing the prepulse inhibition level between the two groups. The prepulse
inhibition level (PPI) indicates how well the animals perceive the frequency
differences. The higher PPI means the animal can differentiate the two frequencies
better. We found that, in both ACSF control groups (Figure 26C, 9.8kHz; Figure 26,
Figure 26; 16.4kHz, black lines), the animals showed a gradual increase of prepulse
inhibition as the differences between the background tone frequency and the prepulse
tone frequency became larger. The increased prepulse inhibition level indicates the
normal frequency discrimination ability of mice (Figure 26B). However, in the CCK8S infusion groups, the prepulse inhibition levels were significantly increased in
detecting the 2% difference between the background tone and the prepulse tone
(Figure 26C, 9.8kHz; Figure 27, Figure 27; 16.4kHz, red lines) (9.8kHz; CCK-8S
group n=7, ACSF group n=7, two-way ANOVA, p<0.05, post-hoc: Tukey test, CCK8S vs ACSF, **, p<0.001; 16.4kHz; CCK group n=7, ACSF group n=7, two-way
ANOVA, p<0.05, post-hoc: Tukey test, CCK-8S vs ACSF, **, p<0.001). This result
indicates that CCK enhanced the frequency discrimination ability of mice by
potentiating the thalamocortical responses triggered by passive tone exposure.
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Figure 24. CCK-8S injection lowered the responding threshold of exposed tone.
A) Sample tuning curves before and after CCK-8S or ACSF infusion and a
suprathreshold non-CF tone exposure. Red arrow above the horizontal axis indicates
the exposed frequency (EF), 20 kHz in this case. B) Population data of the responding
threshold changes after CCK-8S (filled circle line) or ACSF (open circle line),
followed by tone exposure. (CCK-8S, n=15; ACSF n=12, two-way ANOVA, p<0.001,
post-hoc: Tukey test, CCK-8S vs ACSF, *p<0.05, **p<0.001). EF: exposed
frequency
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Figure 25. Experimental setup of prepulse inhibition acoustic startle test and
passive tone exposure with CCK/ACSF injection procedures. A) Schematic
diagram of the experimental setup B) The experimental procedures of passive tone
exposure with CCK/ACSF injection.
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Figure 26. The frequency discrimination ability of mice was enhanced after
CCK-8S infusion and passive tone exposure. A) Prepulse inhibition acoustic startle
test protocol: 70dB, background pure tone (9.8kHz or 16.4kHz) was played
continuously test, except when the prepulse tones and the startle noises were delivered.
Prepulse inhibition trials were presented in a pseudorandom order. Each prepulse trial
consisted of a 70dB 80ms prepulse (Δf, pure-tone frequency was 0%, 1%, 2%, 4%,
8%, 16%, or 32% lower than the background tone), followed by a 120dB, 20ms white
noise startle pulse, and then return to the background tone after the startle. Every trial
was presented 15 times; Inlet: example averaged startle waveforms of one intact
mouse. B) Two individual example waveform series of prepulse inhibition startle
from CCK-8S injection group (red) and ACSF injection group (black). C) Mean
prepulse inhibition of the startle responses in C57 mice exposed to 9.8kHz tone after
CCK-8S (red line) or ACSF (black line) injection into the auditory cortex (CCK-8S
n=7, ACSF n=7, two-way ANOVA, p<0.05, post-hoc: Tukey test, CCK-8S vs ACSF,
**, p<0.001). Prepulse frequencies were 0, 2, 4, 8, 16, or 32% lower than background
tone, 9.8 kHz. PPI: prepulse inhibition. WN: white noise.
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Figure 27. Mean prepulse inhibition of the startle responses in C57 mice exposed to
16.4 kHz tone after CCK-8S (red line) or ACSF (black line) injection into the auditory
cortex (CCK-8S n=8, ACSF n=7, two-way ANOVA, p<0.05, post-hoc: Tukey test,
CCK-8S vs ACSF , **, p<0.001). Prepulse frequencies were 0, 2, 4, 8, 16, or 32%
lower than background tone, 16.4 kHz.
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4. Discussion
In the present study, we have shown that TBS in the MGv successfully induced LTP
in the auditory thalamocortical pathway of adult rodents, which was CCK dependent
and mediated by the presynaptic group I mGluRs. Also, the recurrence of the
thalamocortical LTP after the critical period was due to the maturation of the
thalamocortical projection and increasing expression of CCK in the MGB during the
development. However, in the older animals, the auditory thalamocortical pathway
lost LTP again because of the downregulation of CCK expression in the MGB. But,
an artificial CCK injection in the auditory cortex partially restored the thalamocortical
LTP. We found that artificial CCK introduction in the auditory cortex enabled the
plasticity triggered by passive tone exposure, which in turns enhanced the frequency
discrimination ability in adult behaving mice. These findings imply a great
therapeutic potential of CCK in treating developmental impairments and age-related
degenerations.
Previous in vitro study done on the somatosensory thalamocortical pathway showed
that there is a critical period for NMDA dependent LTP at the thalamocortical
synapses (Crair & Malenka, 1995). By pairing the presynaptic stimulation and
postsynaptic depolarization, thalamocortical LTP could be significantly induced in
the neonatal brain slices from P3 to P7. However, this plasticity disappeared after the
end of the first postnatal week. The reason behind the loss of the neonatal
thalamocortical LTP was that the switch of the subunits of NMDA receptors (NR2B
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to NR2A) on the thalamocortical synapses increased the difficulty in the
thalamocortical LTP induction during the development. Later, they found that the
silent synapses were activated by the LTP induction and contributed to the neonatal
thalamocortical LTP, and the loss of those silent synapses during the development
could be another possible mechanism for the critical period of the neonatal
thalamocortical plasticity (Isaac et al., 1997). In 2011, Hensch and colleagues also
found a similar critical period for the auditory thalamocortical connectivity by
detecting the tonotopic map reorganization caused by passive tone exposure during
the development (Barkat et al., 2011). Hence, it was thought that the thalamocortical
inputs become less plastic after the critical period. However, some recent studies have
demonstrated that the thalamocortical plasticity does still exist in the adult brain and
could be induced by different protocols (Bear 2001, 2010, Hogsden, 2009, Hirata,
2005, Petrus, 2014, Blundon, 2011). To investigate the ontogeny of the
thalamocortical LTP in the adult brain and its underlying mechanism, standard LTP
induction protocol was used in our study. In support of the results from Heynen and
Bear (Heynen & Bear, 2001), the thalamocortical LTP could be successfully induced
by tetanic stimulation of MGv in our experiments, indicating that the thalamocortical
synapses are still plastic after the critical period. One major difference between their
study and ours is that they did not focus on the thalamorecipient layers. Instead, they
mainly recorded the evoked potentials from the superficial layers of the visual cortex.
However, the increased evoked potentials in these layers cannot reflect the real
changes of the thalamocortical inputs directly. Furthermore, an in vitro study showed
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that TBS could not induce LTP in the layer IV of the visual cortex without GABA
antagonist (X. F. Wang & Daw, 2003). In the current study, we have shown for the
first time that TBS induces the auditory thalamocortical LTP in the adult rodents in
vivo.
Our previous studies suggest that CCK plays an important role in the plasticity of the
neocortex. Local CCK infusion potentiated the sound response of the auditory cortex
and enabled intercortical plasticity in the adult animals. This result was confirmed by
which was confirmed by cross-modal association on behavioral rodents (Li et al.,
2013, 2017). Senatorov and his colleagues have reported that CCK mRNA was found
in the reciprocally connected areas of MGB and auditory cortex, and high density of
CCK B receptors was also found in layer IV of the auditory cortex (Senatorov et al.,
1997; Zarbin et al., 1983). These results imply a potential function of CCK in the
thalamocortical plasticity. In the present study, we hypothesized that the auditory
thalamocortical LTP in the adult rodents is CCK dependent. By using retrograde
labeling and immunostaining techniques, we confirmed that nearly all the
thalamocortical projecting neurons expressed CCK. Also, the electrophysiological
results showed that thalamocortical LTP could not be induced by TBS in the CCK-/mice, and the CCK-B receptor antagonist, L365, 260 blocked the thalamocortical LTP,
suggesting that CCK is required for the formation of thalamocortical LTP in the adult
rodent brain. However, we could not exclude the possibility that there might be some
congenital deficits account for the loss of the thalamocortical plasticity in CCK-/mice. In a future study, CCK conditional knockout or RNAi knockdown methods may
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be utilized to rule out these possibilities. We further tested the sufficiency of CCKpositive MGv neurons activation in the thalamocortical LTP induction. By using the
optogenetic method, we could specifically target the CCK-positive neurons with the
Cre-dependent virus. We could also prevent the activation of the corticothalamic
pathway or the cholinergic fibers from the nucleus basalis caused by electrical
stimulation in the thalamic radiation (Chun et al., 2013). According to our previous
result, HF electrical stimulation of CCK containing neurons triggered CCK release
(Li et al., 2017). In the current study, HF laser stimulation was utilized to activate the
CCK-positive neurons in MGv exclusively. To prevent short-term depression caused
by repeated activation of the thalamocortical fibers, the optic fiber was inserted into
the MGv instead of the ACx. The HF laser stimulation of the CCK-positive neurons
in MGv resulted in successful induction of thalamocortical LTP, supporting our
hypothesis that the thalamocortical LTP is CCK dependent.
Previous findings from Wang and Daw illustrated that there are different types of LTP
in the visual cortex such as NMDA-dependent, mGluR dependent, and voltagesensitive calcium channel dependent (X. F. Wang & Daw, 2003). Also, our previous
study (Li et al., 2017) had shown that CCK release from the entorhinal projecting
neurons was controlled by presynaptic NMDA receptors. To test whether this CCKdependent thalamocortical LTP is also mediated by NMDA receptors, NMDA
receptor antagonist, DL-APV and group I mGluR (mGluR1/5) antagonists, MPEP
and LY298198 were applied to the auditory cortex, respectively. As a result, the
thalamocortical LTP was blocked by group I mGluR antagonists and DL-APV
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showed no such effect to this thalamocortical LTP. To further investigate the function
of mGluR1/5 in the CCK-dependent thalamocortical LTP, our immunostaining study
revealed that there were many mGluR1/5 colocalized with the CCK-positive
thalamocortical terminals. These results above may imply the potential presynaptic
function that is triggering the CCK release from the thalamocortical terminals, since
group I mGluRs could depolarize the neurons and elevate the intracellular Ca2+
concentration (Anwyl, 2009). More whole-cell recording and immunoelectron
microscopic studies may be done to confirm this result.
In vivo thalamocortical LTP induction in the neonatal rats at different postnatal days
revealed for the first time that the TBS-induced thalamocortical LTP appeared around
P21 as the CCK expression level started to rise and the peak latency of the evoked
potential became shorter and shorter. In agreement with the theory of the subplate
neurons during the thalamocortical development (Zhao et al., 2009), our results
indicate that the CCK-dependent thalamocortical LTP starts to appear when the
synapses are getting matured and ready to follow the HF stimulation, which triggers
the CCK release. Also, the mechanism of the thalamocortical LTP in the adult animal
is different from that during the critical period (Isaac et al., 1997).
As Hogsden and colleagues reported that the thalamocortical plasticity is also subject
to aging (Hogsden & Dringenberg, 2009). In the present study, thalamocortical LTP
in the old rodent brains was also examined. We found that TBS failed to induce the
thalamocortical LTP in the rats older than 18 months of age and the CCK expression
93
in the MGB was also downregulated. According to our experimental results, CCK
injection into the auditory cortex partially induced the thalamocortical LTP. This
limited restoration may be due to a decrease of CCK-B receptors in the auditory cortex
and the loss of the thalamocortical connectivity caused by aging. More immunehistochemical studies needed be done to further confirm this result.
In the current study, we have demonstrated that CCK plays a crucial role in the
thalamocortical plasticity of both young and old rodents. These results suggest the
potential of CCK as a therapeutic drug to promote plasticity. We have shown that
CCK significantly increased the frequency discrimination ability via facilitating the
plasticity induced by the passive tone exposure. The thalamocortical plasticity in the
adult brain has been reported to be induced by reversing the critical period of the
sensory cortices in different ways (Blundon et al., 2017; Seungsoo Chung et al., 2017;
Zhu et al., 2014). Zhu and colleagues reported that exposing the juvenile or adult rats
to a moderate-level noise could reinstate the critical period plasticity (Zhu et al., 2014).
Also, critical-period-like thalamocortical plasticity in the adult somatosensory cortex
was induced by peripheral sensory deprivation (Seungsoo Chung et al., 2017). In a
recent study, Blundon and colleagues demonstrated that the thalamocortical plasticity
could be restored by restricting the thalamic adenosine signaling (Blundon et al.,
2017). In parallel, our current study proposed another potential method to trigger the
thalamocortical plasticity in the adult brain. Furthermore, CCK induced plasticity
may provide a possible treatment plan which can be applied to enhance the function
of the cochlear implant in the prelingually deaf children ( Lee, 2001). Also, CCK is
94
found within the entire thalamic area rather than MGB only (Burgunder & Young,
1988). The thalamocortical plasticity does not only participate in the refinement of
the sensory cortices but also the learning process (Biane et al., 2016). Thus, our
findings may be generalized in different sensory modalities not only in the perception
but also in the perceptual learning and memory.
95
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