Basal Forebrain Transient Cholinergic Signal in Prefrontal

Cortex and its Role in Associative Learning

by

Gaqi Tu

A thesis submitted in conformity with the requirements

for the degree of Master of Arts

Department of Psychology

University of Toronto

© Copyright by Gaqi Tu 2018

ii

Basal Forebrain Transient Cholinergic Signal in Prefrontal

Cortex and its Role in Associative Learning

Gaqi Tu

Master of Arts

Department of Psychology

University of Toronto

2018

Abstract

Basal forebrain (BF) cholinergic neurons constitute a main neuromodulatory system in the brain

and densely innervate to neocortex and hippocampus. Traditionally, acetylcholine (ACh) released

from BF cholinergic neurons are thought to slowly influence neuronal network to regulate arousal,

attention, and learning and memory. However, accumulating studies report phasic ACh release

upon events with innate or learned salience. Thus, the present study investigated its role in

acquisition of trace eyeblink conditioning where a preceding tone is paired with an eyelid shock.

Here, it was shown that suppressing cholinergic activity in medial prefrontal cortex (mPFC) during

eyelid shock facilitated the formation of the tone-shock association. The enhanced learning was

accompanied by elevated mPFC network activities indicated by neuronal activity marker, c-Fos.

These findings suggest that phasic ACh release does not serve as a reinforcement signal in mPFC.

Rather, it may regulate cortical network state by differentially activating local excitatory and

inhibitory neurons.

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Acknowledgment

I want to thank my supervisor Dr. Kaori Takehara for her supports, guidance and

encouragement throughout the research project.

I am indebted to Dr. Laura corbit and Dr. John Yeomans for their active participation and

valuable comments on this thesis.

I would like to thank our lab members and volunteer for their helps over the past year, Sam

Gillman, Xiaotian Yu, Justin Jarovi, Maryna Pilkiw and Bohan Xing.

At last, I express my deepest gratitude to my parents for their enduring love and supports.

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Table of Contents

Acknowledgment ............................................................................................................................... iii

Table of Contents .............................................................................................................................. iv

List of Figures .................................................................................................................................... vi

List of Appendices ............................................................................................................................ vii

Introduction ........................................................................................................................................ 1

1.1 Central cholinergic system ............................................................................................... 1

1.2 Basal forebrain anatomy................................................................................................... 1

1.3 Firing activity of BF cholinergic neuron .......................................................................... 2

1.4 The BF cholinergic neuron and associative learning ....................................................... 4

1.5 Trace eyeblink conditioning ............................................................................................. 6

1.6 Hypothesis ........................................................................................................................ 8

2 Material and methods ........................................................................................................... 9

2.1 Subject .............................................................................................................................. 9

2.2 Surgery ............................................................................................................................. 9

2.3 Behavioral paradigm ...................................................................................................... 11

2.4 Histology ........................................................................................................................ 12

2.5 Data analysis .................................................................................................................. 13

3 Results ................................................................................................................................ 15

3.1 Optogenetic inhibition of BF cholinergic neurons ......................................................... 15

3.2 Inactivation of BF cholinergic neurons during the US facilitated acquisition in trace

eyeblink conditioning ................................................................................................................ 17

3.3 c-Fos expression in medial prefrontal cortex was increased in ArchT group following

the photoinhibition of BF cholinergic neuronal activity ........................................................... 19

v

3.4 Retrograde tracing results............................................................................................... 24

3.5 Photoinhibition of BF cholinergic terminals in mPFC during US facilitated acquisition

of trace eyeblink conditioning ................................................................................................... 25

3.6 c-Fos expression was increased in PrL in ArchT group mice following the photo-

inhibition of BF cholinergic terminals in mPFC ....................................................................... 26

4 Discussion .......................................................................................................................... 28

5 Reference: .......................................................................................................................... 37

vi

List of Figures

Figure 1 Optogenetic inhibition of BF cholinergic neurons ............................................................ 16

Figure 2 Inactivation of basal forebrain (BF) cholinergic neurons during the US facilitated

acquisition in trace eyeblink conditioning ........................................................................................ 18

Figure 3 BF cholinergic projection in targeted cortex ..................................................................... 19

Figure 4 Neuronal activity level was increased in both superficial and deep layers of prelimbic

cortex (PrL), but not in motor cortex ................................................................................................ 20

Figure 5 Cell-type specific activation in PrL ................................................................................... 21

Figure 6 The expression of c-Fos in excitatory neurons. ................................................................. 22

Figure 7 The expression of c-Fos in different cell type of interneurons. ......................................... 23

Figure 8 BF cholinergic projection to the PrL. ................................................................................ 25

Figure 9 Photoinhibition of BF cholinergic terminals in PrL during US facilitated acquisition in

trace eyeblink conditioning. ............................................................................................................. 26

Figure 10 The expression of c-Fos in different cell type. ................................................................ 27

vii

List of Appendices

Appendix A Genotype of the ChAT (IRES)-Cre mice………………………………………....34

Appendix B Schema of viral spread and optic fiber placements………………………………35

Appendix C Optic fiber placements in mPFC………………………………………………….36

1

Introduction

1.1 Central cholinergic system

Acetylcholine (ACh) is the first neurotransmitter identified in the nervous system. In the

brain, ACh is synthesized in neurons expressing the enzyme choline acetyltransferase (ChAT).

After released from the axon terminals of these cholinergic neurons, ACh binds to nicotinic

(nAChRs) or muscarinic receptors (mAChRs) and modulates the activity of other neurons

(Wonnacott, 1997). Cholinergic neurons are found in nuclei such as pedunculopontine and lateral

tegmental areas, medial habenula (Ren et al., 2011; Sutherland, 1982), and basal forebrain (BF) (J.

Woolf, 1991). In striatum and nucleus accumbens, some local interneurons are also capable of

releasing ACh (Butcher, 1981). Among these cholinergic nuclei, BF is of particular interest

because its cholinergic neurons are the main source of ACh in the neocortex (L. Zaborszky et al.,

2008; Laszlo Zaborszky et al., 2015) and hippocampus (Mesulam, Mufson, Wainer, & Levey,

1983), and degeneration of BF cholinergic neurons is associated with age-related cognitive decline

and neurodegenerative disorders like Alzheimer’s disease (Bartus, Dean, Beer, & Lippa, 1982).

1.2 Basal forebrain anatomy

BF is a highly heterogenous structure located in the middle and ventral part of the forebrain

and is composed of several subregions. The cholinergic and non-cholinergic populations are

intermingled and distributed throughout these subregions (Gritton et al., 2016; Laszlo Zaborszky &

Duque, 2000). Previous anatomical studies have divided BF cholinergic neurons to four different

clusters (CH1-CH4), and described their different projection pathways: cholinergic neurons in the

medial septum (MS) (CH1) and vertical diagonal band of Broca (VDB) (CH2) mainly innervate to

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the hippocampal and parahippocmapus region; the lateral part of the horizontal diagonal band of

Broca (HDB)(CH3) projects to the olfactory bulb; cholinergic neurons in the nucleus basalis of

Mynert (NBM) and parts of HDB form CH4 and project widely to the cortex and amygdala

(Mesulam et al., 1983).

1.3 Firing activity of BF cholinergic neuron

Previously, electrophysiological studies recorded BF neuronal activity mostly from acute

brain slice and restrained or anesthetized animals. In vitro studies showed that BF cholinergic

neurons discharge slowly followed by large and long-lasting afterhyperpolarization (Hedrick &

Waters, 2010; Unal, Golowasch, & Zaborszky, 2012). In vivo studies found that BF cholinergic

neurons discharge differentially throughout cortical states: approximately at 8 Hz during walking

and much lower during slow wave sleep (0.84 ± 0.42 Hz) in rodents (Lee, Hassani, Alonso, &

Jones, 2005; Simon, Poindessous-Jazat, Dutar, Epelbaum, & Bassant, 2006). In addition,

microdialysis studies revealed increased ACh release in several brain regions including frontal

cortex, hippocampus and striatum during walking (Day, Damsma, & Fibiger, 1991; Lee et al.,

2005; Marrosu et al., 1995). In parallel, early electron microscope studies reported that the vast

majority of cholinergic terminals projecting into hippocampus and cortex do not form classical

synapses. For instance, in rat (Umbriaco, Garcia, Beaulieu, & Descarries, 1995) and mouse

(Yamasaki, Matsui, & Watanabe, 2010) hippocampus only less than 20% of the entire cholinergic

terminals form typical chemical synapses with local neurons, whereas in macaque prefrontal cortex

(Mrzljak, Pappy, Leranth, & Goldman-Rakic, 1995) and human temporal lobe (Smiley, Morrell, &

Mesulam, 1997) the percentage is approximately 50%. Based on these findings, it is generally

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believed that the BF cholinergic system is functionally diffuse and slowly regulates its targets (N. J.

Woolf, 1991).

However, recent studies applied advanced techniques and challenged the traditional view.

Using single-unit recording in mice, Hangya et al. have uncovered precise and short-latency firing

responses (18 ± 2 ms) of BF cholinergic neurons upon the delivery of water reward and air-puff

punishment in an auditory detection task (Hangya, Ranade, Lorenc, & Kepecs, 2015). In the same

study, phasic firing activity of cholinergic neurons was also reported after the presentation of a foot

shock. Similarly, using in vivo microendoscopic calcium imaging, Harrison et al. demonstrated that

BF cholinergic neurons can be activated by both reward and punishment stimuli in an auditory

discrimination task also at the subsecond level (Harrison, Pinto, Brock, & Dan, 2016). These

cholinergic neuronal firings that are time-locked to innately salient stimuli suggest that BF

cholinergic neurons respond much faster than previously proposed. One potential function of this

cholinergic transient can be to broadcast occurrence of the salient event which inherently adds to

elicit responses and reinforce the formation of memory. Consistent with this view, optogenetic

stimulation of BF cholinergic terminals in the mouse primary visual cortex can be used as a

reinforcement signal to shape the firing activities of pyramidal neurons time-locked to the

presentation of visual stimulus (Liu, Coleman, Davoudi, Zhang, & Hussain Shuler, 2015); whereas

selective lesion of the BF cholinergic terminals in the same region would abolish such effect

(Chubykin, Roach, Bear, & Shuler, 2013).

In addition to the time-locked firing of cholinergic neurons to the innately salient stimuli,

other studies also reported phasic ACh release in medial prefrontal cortex (mPFC) upon events

with learned salience. Using choline sensitive microelectrodes for measuring the synaptic release of

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ACh, Parikh, et al., (Parikh, Kozak, Martinez, & Sarter, 2007) found phasic release of ACh in

mPFC only when rats detected a visual cue which predicts the reward. Furthermore, Gritton et al.,

(Gritton et al., 2016) demonstrated that enhancing the cholinergic transient during the cue

presentation period using optogenetic stimulation can improve cue detection, while stimulating

cholinergic terminals during a non-cued trial led to an orienting behavior, presumably due to a

mistaken “detection” of a cue. Conversely, optogenetic inhibition of BF cholinergic terminals

during the cue caused the rats to fail to approach the reward (Gritton et al., 2016). Together these

findings indicate that phasic ACh release from BF cholinergic terminals in mPFC is important for

detection of stimuli with learned salience.

The investigations reviewed above suggest how cholinergic signaling during innate and

learned salient events are related to the formation of event memories. However, the role of transient

cholinergic activity in learning and memory remains unclear.

1.4 The BF cholinergic neuron and associative learning

In 1982, Bartus first proposed “cholinergic hypothesis of geriatric memory dysfunction"

(Bartus et al., 1982). Since then, the role of ACh in learning and memory has been intensively

investigated using lesions and pharmacological treatments. In the past, using excitotoxic lesion to

the BF by treating with ibotenic acid or quisqualic acid researcher proposed its involvement in

learning and memory (Casamenti, Milan, & Pepeu, 1990; Wenk, 1990). However, the non-

cholinergic population damage was also reported in these treatments, and it was premature to

conclude that the learning and memory deficits in these studies were due specifically to the BF

cholinergic neuronal loss (Fibiger, 1991). This problem was solved with the application of

excitotoxins 192 IgG-saporin that induces irreversible damage specifically to the cholinergic

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neurons (Wiley, Oeltmann, & Lappi, 1991). Infusion of 192 IgG-saporin impaired the acquisition

and expression of associative memory tested in various paradigm, including inhibitory avoidance

task (Power, Thal, & McGaugh, 2002), trace eyeblink conditioning (Fontán-Lozano, Troncoso,

Múnera, Carrión, & Delgado-García, 2005) and feature-binding task (Botly & Rosa, 2009).

Consistently, similar memory deficits were also reported in pharmacological studies. Infusion

of mAChRs antagonism scopolamine was shown to impair the contextual fear conditioning (Gale,

Anagnostaras, & Fanselow, 2001; Wallenstein & Vago, 2001), trace fear conditioning (Baysinger,

Kent, & Brown, 2012), social transmission of food preference task (Boix-Trelis, Vale-Martínez,

Guillazo-Blanch, & Martí-Nicolovius, 2007; Carballo-Márquez, Vale-Martínez, Guillazo-Blanch,

& Martí-Nicolovius, 2009), odor-reward task (Carballo-Márquez et al., 2007), object-in-place

associative memory (Barker & Warburton, 2009), conditioned flavor preference (Rotella et al.,

2015) and odor-fear conditioning (Pavesi, Gooch, Lee, & Fletcher, 2013). In parallel, nAChRs

antagonists can also retard acquisition of trace eyeblink conditioning (Diana S. Woodruff-Pak,

2003; Diana S. Woodruff-Pak, Li, Kazmi, & Kem, 1994), contextual fear conditioning (André,

Leach, & Gould, 2011) and conditioned flavor avoidance (Rotella et al., 2015). Comparable

learning and memory deficits in trace eyeblink conditioning are also reported in genetically-

modified mice lacking subunits of nAChRs (Brown, Comalli, De Biasi, & Woodruff-Pak, 2010).

Parallel studies showed that infusion of ACh and ACh receptor agonist can enhance the

performance of these task (Disterhoft & Matthew Oh, 2003; Gould & Wehner, 1999; Kenney &

Gould, 2008).

In addition to the lesion and drug treatment, several microdialysis studies measured

endogenous ACh release in these tasks and found an elevated level of ACh influx in mice mPFC

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and hippocampus after learning an appetitive trace conditioning (Flesher, Butt, & Kinney-Hurd,

2011) and after classical conditioning in rat auditory cortex (Butt et al., 2009). However, such ACh

influx was not observed in animals which did not form the associative memory or in those in the

presentation of random sensory stimulus, suggesting that ACh is indispensable for the formation of

associative memory.

Taken together, these studies provide support for the essential role of ACh in the various

forms of associative learning. These studies, however, have several major limitations: firstly, the

temporal resolution is low. Learning and memory is a time-dependent dynamic process. Thus,

permanent damage induced by the lesion approach prevents researchers from attributing a specific

role in memory to BF cholinergic neurons. Second, a compensatory effect can exist in both

pharmacological and lesion studies. Due to lack of function in a given lesion region, the brain can

reorganize its functional connectivity leading to the recovery of function. Third, muscarinic

antagonism also binds to presynaptic M2-type mAChRs and induces increased extracellular ACh

level and overactivation of nAChRs. Therefore, the studies listed above failed to uncover the role

of transient cholinergic activity in associative learning in a temporally specific manner.

1.5 Trace eyeblink conditioning

One paradigm suitable for testing the role of transient cholinergic activity in the formation of

associative memory is trace eyeblink conditioning. It is a well-characterized behavioral paradigm,

in which the neutral conditioned stimulus (CS, e.g., tone) is paired with the unconditioned stimulus

(US, e.g., eyelid shock). A temporal gap called trace interval (TI) inserts between CS and US. One

main advantage of this paradigm is that the timing of CS, TI and US are precisely controlled. The

separated CS and US enable researchers to manipulate neuronal activity at a high temporal

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resolution and to dissect the underlying neuronal circuits mechanism. In addition, the task

performance can be monitored easily. For example, prior to the conditioning, the US induces a

specific reflex response (unconditioned response, UR), whereas the CS does not. Repeated pairings

of the CS and US build an association between them, resulting in the transfer of the reflex behavior

from the US to the CS, i.e., conditioned response (CR). Furthermore, the neuronal circuits

underlying this task have been investigated. The brainstem and cerebellum circuits have been

shown to be important for the timing of CR and association of CS and US in this simple form of

pavlovian conditioning (Anderson et al., 1999; Bracha, 2004; Kalmbach et al., 2010; Kalmbach,

Ohyama, Kreider, Riusech, & Mauk, 2009). However, the insertion of the stimulus free trace

interval makes the acquisition of this task dependent on many different brain areas. Hippocampal

lesion not only disrupts the formation of CS-US association, also severely impair the expression of

already acquired CS-US association when lesion was produced shortly after acquisition (Kim,

Clark, & Thompson, 1995; Takehara, Kawahara, & Kirino, 2003). Furthermore, greater

impairment induced by hippocampal lesion is correlated with longer trace interval (Tseng, Guan,

Disterhoft, & Weiss, 2004; Walker & Steinmetz, 2008). Thus, it is generally believed that

hippocampus plays an important role in binding temporally discontinuous stimulus. In addition to

hippocampus, several forebrain regions, including sensory cortex (Galvez, Weible, & Disterhoft,

2007), thalamus (Powell & Churchwell, 2002) and striatum (Flores & Disterhoft, 2013) are

involved in memory acquisition and expression. Accumulating evidence suggested that the mPFC

is also essential for the formation of CS-US association as damage to the mPFC impairs the

acquisition and later expression of CR (Kronforst-Collins & Disterhoft, 1998; Weible, McEchron,

& Disterhoft, 2000). In parallel, electrophysiological studies shows that mPFC neurons respond to

the CS and maintain persistent activity across trace interval (Hattori, Yoon, Disterhoft, & Weiss,

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2014; Siegel, Kalmbach, Chitwood, & Mauk, 2011; Takehara-Nishiuchi & McNaughton, 2008).

Such persistent activity lasts till the US onset and becomes stronger over the acquisition of CR,

presumably to bridge the interval between the CS and US (Hattori et al., 2014; Siegel et al., 2011;

Takehara-Nishiuchi & McNaughton, 2008). Finally, this paradigm is also applicable to the human.

For example, older adults and Alzheimer’s disease patient with degeneration of BF fail to acquire

this task (Cheng, Faulkner, Disterhoft, & Desmond, 2010; D. S. Woodruff-Pak & Papka, 1996).

Considering the evidence reviewed above, the trace eyeblink conditioning task is a suitable

behavioral paradigm to study the role of transient cholinergic activity in the associative learning.

1.6 Hypothesis

I hypothesize that the time-locked, transient cholinergic influx may provide signals on a

behaviorally salient outcome, thereby serving as a reinforcement signal for animals to associate

neutral stimuli to reinforcer. To specifically manipulate cholinergic neuronal activity, I expressed

inhibitory opsin-archaerhodopsin (ArchT) in BF cholinergic neurons in a Cre-recombinase

dependent way. With optic fibers implanted in the BF, delivery of green light induces outward

currents of proton in cholinergic neurons, and thereby inhibits cholinergic neuronal activity at

millisecond time scale.

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2 Material and methods

2.1 Subject

Adult male (2-4 months old) ChAT (IRES)-Cre mice were used in the behavioral experiments

and eight additional C57BL/6J wild-type mice (JAX #000664) were used in the retrograde tracing

study. ChAT (IRES)-Cre mice express Cre recombinase under the promotor of ChAT, and its

endogenous ChAT expression is not disrupted. Six homozygotes ChAT (IRES)-Cre mice were

purchased from the Jackson Laboratory (JAX #028861). The genotype of the six breeding mice had

been confirmed by The Center of Applied Genomics at the Hospital for SickKids (Appendix A).

One male and two female ChAT (IRES)-Cre mice were housed together as a harem to generates the

experimental cohorts. All the mice were maintained under a 12:12 light-dark cycle, with free

access to food and water. All the procedures have been approved by the University of Toronto

Institutional Animal Care Committee.

2.2 Surgery

ChAT (IRES)-Cre mice underwent two surgery protocols: they first received the viral vector

(2.2.1) and then were given optic fiber and electromyogram (EMG) electrodes implantation (2.2.2).

The C57BL/6J wild-type mice underwent retrobeads infusion surgery (2.2.3).

2.2.1 Viral vector infusion

The ChAT-Cre mice were anesthetized with 4% isoflurane and were administrated

Ketoprofen subcutaneously (10 mg/Kg) right after the induction. One and half to two and half

percent isoflurane was applied for surgical maintenance. The mice were mounted on a stereotaxic

frame and were provided with ophthalmic lubricant to their eyes. A heat pad was placed under the

mice to maintain their body temperature. After making an incision, skin was retracted. The

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anterior-posterior axis of the skull was adjusted, and two craniectomy windows were opened

symmetrically above BF (AP: +0.14 mm; ML: 1.40 mm). A 400~600 nl of recombinant adeno

associated virus (rAAV8) which either expresses archaerhodopsin (ArchT) with green fluorescence

protein (GFP) (rAAV8-CAG-Flex-ArchT-GFP) or only GFP (rAAV8-CAG-Flex-GFP) was

injected in each side at a depth of 5.2 mm in ten mins. After three-min delay, the pipette was raised

to 5.1 mm for a second infusion of another 200 nl volume of virus. Following a ten-min delay, the

pipette was slowly withdrawn. Skin was sutured, and 1 ml saline was given subcutaneously.

2.2.2 Optic fiber and EMG electrodes implantation

After 4-6 weeks of viral incubation period, mice were anesthetized following the same

procedure. Two craniectomies were made above BF (AP: +0.14 mm; ML: 3.0 mm) or mPFC (AP:

+2.0 mm; ML: 1.8 mm), and one craniectomy was made above the partial lobe for placing a ground

screw. Home-made optic fibers (200 µm; NA=0.39; efficiency output> 60%) were lowered to 5.1

mm beneath the cortical surface into BF with 15° lateral angle or 1.8 mm into mPFC with 35°

lateral angle and secured to the skull with 3M resin (Kdental). Four teflon-coated stainless-steel

wires (No. 791000; Carlsborg) were implanted subcutaneously in the upper left orbicularis oculi

(eyelid muscle) with two of them for recording EMG activity and the other two for delivering a

periorbital shock. All electrodes were connected to a connecting cap (PlasticOne) and secured with

stainless steel screws and dental acrylic resin. All the mice were given five to seven days to recover

from surgery before beginning daily conditioning session.

2.2.3 Retrobeads Infusion

The C57BL/6J wild-type mice were anesthetized and placed in the stereotaxic frame as

described above. After making an incision, a craniectomy was drilled above the right mPFC (AP:

+2.0 mm; ML: 0.4 mm). A 300 nl of Red RetrobeadsTM (LumaFlour) was injected with a 10 µl

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Hamilton Syringe into the right prelimbic region of the mPFC at a depth of 2.2 mm beneath the

cortical surface in ten mins. After a five-min delay, the pipette was slowly withdrawn. After the

surgery, 1ml of 0.9% saline was given subcutaneously. Retrobeads taken up by axon terminals in

mPFC were actively transported back to the cell bodies of mPFC-projecting neurons. Three weeks

after the surgery, mice were deeply anesthetized by urethane before cardiacally perfused with 0.9%

saline and 4% paraformaldehyde (PFA).

2.3 Behavioral paradigm

Mice were trained in trace eyeblink conditioning in light and sound-attenuating chamber.

For the first session, mice were individually placed in the conditioning chamber for fifty minutes

without any stimulus to habituate them to the new surroundings and to acquire the baseline

blinking rates (adaptation session 1). One the next day, mice were placed in the same chambers for

fifty minutes with only laser on (adaptation session 2). Starting from the third day, mice underwent

a ten-day acquisition session. Conditioned stimulus (CS), a 100-ms pure tone (85 dB, 2.5 kHz),

was delivered through a speaker mounted inside the chamber, and unconditioned stimulus (US), a

100-ms mild electrical shock to the eyelid (100 Hz square pulse), was given with a stimulus

isolator (ISO-Flex, A.M.P.I) through an RZ-5 recording system (Tucker-Davis Technologies) and

a microcontroller (Arduino). With an initial shock level of 0.2 mA, shock intensity was adjusted

individually and daily for each mouse to induce optimal head-turn responses which were

monitored using infrared cameras mounted inside the chamber. Each daily conditioning session

(around 50 mins) is comprised of 10 blocks, each of which consisted of 9 CS-US pairs and 1 CS-

only trial. The inter-trial intervals were pseudorandomized between 20 s and 40 s with a mean of

30 s. During conditioning, eyeblink responses were monitored by recording EMG activity from the

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left upper orbicularis oculi muscle through two surgically implanted stainless-steel wires. EMG

activity was band-pass filtered between 0.3 and 3.0 kHz, digitized at 6,102 Hz, and stored using an

RZ-5 recording system (Tucker-Davis Technologies).

During conditioning, a green (532 nm wavelength) laser from the DPSS laser system

(Laserglow Technologies) was delivered to the target region via a patch cord and optic fibers

connected to the animals. The laser illumination time was controlled by a MATLAB software: for

inhibition during US, the laser was turned on from the US onset and continuously shined up to 100

ms after the US delivery. The laser power was 20 mW for cell body inhibition and 15 mW for axon

terminal inhibition.

2.4 Histology

Ninety minutes after conditioning, all the mice were deeply euthanized by urethane and

cardiacally perfused by 0.9% saline and 4% PFA. After harvesting the brains, all tissues were

stored in 4% PFA overnight, transferred into 30% sucrose PBS and maintained at 4°C for 2-4 days.

The brains were sectioned into 35~45-µm coronal slices continuously using a cryostat. For

immunostaining, the brain sections were washed three times, five mins each, in PBS solution and

incubated in 10% donkey or horse serum (Sigma-Aldrich) PBS for two hours at room temperature.

After washing three times with PBS, five mins each, the sections were incubated with primary

antibodies diluted in 0.1% Triton-PBS for 48-72 hours at 4°C. Following PBS wash, secondary

antibodies were applied for two hours at room temperature. All slices were mounted on slides using

anti-fade mountant (ThermoFisher).

Series 1: For visualizing viral expression, the primary antibodies of Goat-anti-ChAT

(Millipore, 1:200) and Rabbit-anti-GFP (Cell signaling, 1:800) were used. Secondary antibodies

13

were Rhodamine Donkey-anti-Goat (Jackson Immuno Research, 1:200) and Alex Flour 488

Donkey-anti-Rabbit (Jackson Immuno Research, 1:200).

Series 2: For visualizing c-Fos expression in excitatory neurons, the primary antibodies of

Mouse-anti-CamkII α+ (Santa Cruz Biotechnology, 1:50) and Rabbit-anti-c-Fos (Cell signaling,

1:400) were used. Secondary antibodies were Alex Flour 594 Goat-anti-mouse (Jackson Immuno

Research, 1:200) and Alex Flour 488 Donkey-anti-Rabbit (Jackson Immuno Research, 1:200). This

series of slices were mounted with anti-fade mountant with DAPI (ThermoFisher).

Series 3: For visualizing c-Fos expression in interneurons, the primary antibodies of Rabbit-

anti-c-Fos (Cell signaling, 1:400), Rat anti-somatostatin (SST) (Millipore, 1:200) and Mouse-anti-

parvalbumin (PV) (Sigma-Aldrich, 1:800) were used. Secondary antibodies were Alex Flour 405

Donkey-anti-mouse (Jackson Immuno Research, 1:100), Alex Flour 488 Donkey-anti-Rat (Jackson

Immuno Research, 1:200) and Rhodamine Donkey-anti-rabbit (Jackson Immuno Research, 1:200).

2.5 Data analysis

2.5.1 EMG analysis

EMG activities were analyzed using a custom code written in MATLAB (Mathworks). The

EMG amplitudes were averaged during a 300-ms window before CS in each trial, and its median

plus one standard deviation was set as the baseline. A trial was defined as a CR trial if the EMG

amplitude during 200 ms before US onset (CR value) was at least five times of that during the 300

ms pre-CS period (pre-value). A “hyperactive” trial was defined as the pre-value exceeding ten

times the threshold because the mice engaged in grooming and teeth grinding immediately before

CS onset. These “hyperactive” trials were discarded. Performance of each mouse in each session

was evaluated by calculating the percentage of trials with CR in the total number of trials.

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2.5.2 Cell counting

All images were taken under a confocal microscope with a subjective of 20X. Approximately,

3-6 sections were counted and averaged per mouse, with 3–6 mice in each group. To examine the

viral infection efficiency and specificity, co-expression of ChAT+ cholinergic neurons and GFP+

neurons was calculated. To evaluate the activities of different cell types during learning, c-Fos co-

localization in CamKII+/SST+/PV+ cells (e.g., total numbers of c-Fos+ and SST+ co-localized

cells/total numbers of SST+ cells) was calculated. All the images were processed by ImageJ

software.

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3 Results

3.1 Optogenetic inhibition of BF cholinergic neurons

To silence the BF cholinergic neurons with inhibitory opsin, ArchT, I infused an adeno-

associated viral vector which encodes either ArchT with a fluorescence reporter (GFP) or only GFP

in a cre-recombinase manner (rAAV-CAG-Flex-ArchT-GFP or rAAV-CAG-Flex-GFP) into the

BF of mice expressing Cre recombinase under the ChAT promoter (Jackson Laboratory, Stock#:

006410; Figure 1a). After four to eight weeks of incubation period, the GFP+/ChAT+ neurons were

found in several subregions of BF, including HDB, SI, and NBM (Figure 1e; 1f; Appendix B). The

GFP+/ChAT+ neurons were not observed in MS/VDB which projects to the hippocampus

formation. Over 80% of the ChAT+ neurons were GFP+ (Figure 1c; 1f), suggesting effective viral

infection. Over 95% of the GFP+ neurons in the BF were ChAT+ (Figure 1d; 1f), indicating viral

expression highly specific to the cholinergic neurons. Four to six weeks after viral infusion, optic

fibers were bilaterally implanted into the BF (Figure 1b, Appendix B). The laser power was

adjusted to reach minimum 1 mW/mm2 in the most ventral part of the BF. Two mice with

misplaced optic fibers were excluded from the study.

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Figure 1 Optogenetic inhibition of BF cholinergic neurons.

(a) ChAT-Cre mice were bilaterally injected with rAAV8-CAG-Flex-ArchT-GFP (ArchT group) or rAAV8-CAG-

Felx-GFP (GFP group). (b) An example of optic fiber track, with white arrow indicating optic fiber placement above

the BF. (c, d) A high overlap of ChAT+ cells with those that are GFP+ (c, 81.11±7.25%; n = 10) and a high overlap of

GFP+ cells with those that are ChAT+ (d, 96.73±4.33%; n = 10) in the BF. (e, f) Representative images showing

cholinergic neurons in the BF that were transduced, including horizontal diagonal band of broca (HDB), substantia

innominata (SI) and ventral pallidum VP). Cholinergic neurons (ChAT+) are in red, virus transfected neurons (GFP+)

are in green in the BF. In merged microphotograph (f) white arrows point ChAT+/GFP+ neurons, i.e. transduced

cholinergic neurons; whereas red arrows show ChAT+/GFP- cells, i.e. cholinergic neurons that were not transduced.

17

3.2 Inactivation of BF cholinergic neurons during the US facilitated

acquisition in trace eyeblink conditioning

To determine the role of US-locked transient cholinergic activity in associative memory

formation, I trained the mice in trace eyeblink conditioning task and optogenetically inhibited

cholinergic neuronal activities during the US. To achieve an effective manipulation, the laser was

presented from the 100-ms US onset till 100 ms after the US offset (Figure 2a, 2b). Prior to the

training, I first tested whether delivering laser would alter reflex response. On this adaptation

session (adap 2), same 200 ms laser was presented every 20-40 s for fifty mins, but no difference in

spontaneous blinking rate was observed between the two groups. The spontaneous blinking rate

was also comparable to the first adaptation session (adap1) where no laser was presented.

Over the 10 days of acquisition sessions, both ArchT group (n=7) and control group mice

(n=5) learned to express anticipatory blinking responses peaking near the expected onset of the US

(CR) as is evident in the increased frequency of CR expression (CR%; Fig 2c). Surprisingly, the

ArchT group mice showed a significantly higher CR% than control group (Figure 2c) (Two-way

repeated ANOVA; the main effect of group F (1, 9) =5.049, p=0.048; the main effect of sessions F

(9. 90) =14.457, p<0.001; the group x session interaction F (9. 90) =0.72, p=0.689).

18

Figure 2 Inactivation of basal forebrain (BF) cholinergic neurons during the US facilitated acquisition

in trace eyeblink conditioning.

(a) A schematic of training with photoinhibition of BF cholinergic neurons which express inhibitory opsin ArchT. (b)

A daily acquisition session consists of 100 trials: 90 pairings of 100-ms tone (CS) and 100-ms periorbital shock (US)

separated by 500 ms-trace interval (TI) and 10 CS-only trials. Each trial was separated by 20-40 ms intertrial interval.

The laser illumination starts from US onset and ends 100 ms after the US offset during acquisition session. (c) Over the

10-day acquisition sessions, both ArchT group and control group showed increased CR%. However, CR% of ArchT

group is significantly higher than control group (Two-way repeated ANOVA; the main effect of group F (1, 9) =5.049,

p=0.048; the main effect of sessions F (9. 90) =14.457, p<0.001; the group x session interaction F (9. 90) =0.72,

p=0.689). Prior to the training, there was no difference between groups during the adaptation sessions. Adap 1: first

adaptation session without the CS, US and laser presentation. Adap 2: second adaptation session with only laser

delivered.

19

3.3 c-Fos expression in medial prefrontal cortex was increased in

ArchT group following the photoinhibition of BF cholinergic

neuronal activity

Next, I examined how the transient photoinhibition of cholinergic neurons affects neural

activity in the cortex during memory acquisition. Using immunohistochemistry approach, I found

that ArchT-GFP and GFP-expressing BF cholinergic terminals were primarily present in the

prefrontal cortex, motor cortex and somatosensory cortex (Figure 3), though in some mice sparse

GFP+ cholinergic terminals were also found in the basal lateral amygdala and auditory cortex (not

shown).

Figure 3 BF cholinergic projection in targeted cortex.

(a-d) The ArchT-GFP or GFP-expressing BF cholinergic terminals were detected largely in the midline frontal regions

including prelimbic cortex (PrL), primary motor cortex (M1), secondary motor cortex (M2) and anterior cingulate

cortex (ACC).

Further, I assessed the expression of an immediate early gene, c-Fos, a marker for neuronal

activity in these regions. Interestingly, c-Fos expression in both superficial and deep layers of the

20

prelimbic region (PrL) of medial prefrontal cortex (mPFC) was significantly higher in ArchT group

than controls (t test, p<0.001, p<0.001) (Figure 4a, 4b). Although, GFP+ cholinergic terminals

were found in motor cortex (Mo), the numbers of c-Fos expressing cells was comparable between

two groups in superficial and deep layers (t test, p=0.148, p=0.452) (Figure 4a, 4b). Together, these

results suggest that neuronal activities in PrL are specifically modulated by BF cholinergic

neurons.

Figure 4 Neuronal activity level was increased in both superficial and deep layers of prelimbic cortex

(PrL), but not in motor cortex.

21

(a) The representative images showing c-Fos+ neurons in superficial and deep layer of the PrL and Mo. Scale bar 50

µm. (b) The number of c-Fos+ neurons in ArchT group was significantly higher than controls in the superficial layer of

PrL (34.884±2.614 vs. 24.583 ±3.289, p<0.001, n=8) and deep layer of PrL (39.070±3.628 vs. 27.300 ±1.128, p<0.001,

n=8). (c) There was no significant difference between two groups in superficial layer (1.100±0.945 VS 1.500 ±0.353,

p=0.452, n=8) of Mo or in deep layer of Mo (1.700±1.124 VS 2.625 ± 0.176, p=0.148, n=8). *** p<0.001.

Next, to identify which types of neurons in PrL showed increased c-Fos expression after

photoinhibition, I double-stained sections with c-Fos and CamKII α for excitatory neurons,

parvalbumin (PV) for PV+ interneurons, or somatostatin (SST) for SST+ interneurons. In deep

layer of PrL (Figure 5a, 6a, 7a), the percentage of CamKII α+, PV+ and SST+ neurons that co-

express c-Fos was higher in ArchT group than controls (t test, p=0.012; p=0.013,p=0.030), whereas

in superficial layer of PrL (Figure 5b, 6b, 7b) only the percentage of PV+ neurons co-expressing c-

Fos was higher than controls, and there was no difference between SST+ and CamKII α+ neurons

(t test, p=0.064, p=0.17).

Figure 5 Cell-type specific activation in PrL.

(a) In deep layer of PrL, the overlap of c-Fos+ neurons were significantly higher in CamkIIα+ neurons (18.038±2.382

VS 13.073±2,402, p=0.012, n=6), PV+ neurons (24.762±2.02 VS 11.367±3.62, p=0.013, n=6), and SST+ neurons

(58.558±3.479 VS 34.635±9.693, p=0.030, n=6) in ArchT group than control. (b) In superficial layer of PrL, the

22

overlap of c-Fos+ neurons were significantly higher in PV+ neurons (4.167±7.216 VS 24.351±4.132, p=0.028, n=6).

The overlap of c-Fos+ neurons in SST+ neurons (53.571±5.051 VS 26.677±13.043, p=0.064, n=6) was comparable

between groups. There was no difference in the overlap of c-Fos+ neurons in CamkII α+ cells (17.803±3.218 VS

13.553±2.165, p=0.175, n=6). * p<0.05.

Figure 6 The expression of c-Fos in excitatory neurons.

23

Excitatory neurons were visualized by antibody against CamkII α. Representative double immunostainings for CamkII

α+(red) & c-Fos (green) & DAPI (blue) in deep layer (a) and superficial layer (b) of PrL of ArchT and control group.

In merged photograph, white arrows depict neurons which co-express CamkII α and c-Fos. Scale bar 14 µm.

Figure 7 The expression of c-Fos in different cell type of interneurons.

24

Representative double immunostainings for PV (blue)& c-Fos (red) and SST (green) & c-Fos in deep layer (a) and

superficial layer (b) of PrL of ArchT and control group. In merged photograph, white arrows depict the SST+ neurons

express c-Fos; red arrows depict PV+ neurons express c-Fos. Scale bar 14 µm.

3.4 Retrograde tracing results

In the above studies, virus injections were targeted only to the medial part of BF though it

spans along the anterior-posterior axis. Therefore, it is possible that PrL neurons are regulated by

larger amount of BF cholinergic neurons than the above experiments indicated. To answer this

question, I traced afferent projections from BF into PrL by micro-infusing a retrograde tracer,

retrobeads into PrL (Figure 8a). The majority of retrobeads were found in the PrL region, with a

minor spread to the ventral part of anterior cingulate cortex close to the injection site (Figure 8b).

Whereas in BF, the retrobeads cells were mainly distributed in the medial region of HDB and

ventralrostral parts of SI, along with a small population of red retrobeads labeled cells in the

MS/VDB. Notably, among all the retrobeads-labelled cells in the BF, only half of the retrobeads-

labelled cells expressed ChAT (Figure 8c). These data suggest that in the present study cholinergic

neurons located in the medial part of BF play the major role in modulating the activity of PrL

neurons.

25

Figure 8 BF cholinergic projection to the PrL.

(a) Rhodamine attached retrobeads were injected in the PrL. (b) The retro beads injection site in the PrL. (c) The

mPFC-projecting retrobeads+ neurons in the BF with immunohistochemistry staining for ChAT (green, cholinergic

neurons). The white arrows depict ChAT+ cholinergic neurons that projects to the mPFC. The yellow arrow depicts

non-cholinergic neuron that projects to the mPFC.

3.5 Photoinhibition of BF cholinergic terminals in mPFC during US

facilitated acquisition of trace eyeblink conditioning

To directly test the effect of cholinergic modulation of the PrL on memory acquisition, I

silenced axon terminals of cholinergic neurons in PrL by bilaterally implanting optic fibers into

PrL in virus-infected mice (Figure 9a, 9b; Appendix C). These group of mice followed the same

conditioning paradigm and laser manipulation: a 200 ms green laser illumination from the US

onset. Both ArchT group (n=8) and control group (n=11) mice increased their CR% over the ten-

day acquisition sessions. Consistent with previous cell body photo-manipulation data, ArchT group

mice had a significantly higher CR% (Figure 9c) (Two-way repeated ANOVA; the main effect of

26

group F (1, 16) =5.936, p=0.027; the main effect of sessions F (9, 144) =10.544, p<0.001; the

group x session interaction F (9, 144) =0.845, p=0.576).

Figure 9 Photoinhibition of BF cholinergic terminals in PrL during US facilitated acquisition in trace

eyeblink conditioning.

(a) Optic fibers were bilaterally implanted above the PrL. (b) White arrow indicates optic fiber track. (c) Over the 10-

day acquisition sessions, both ArchT group and control group increases the frequency of CR%. CR% of ArchT group

was significantly higher than control group (Two-way repeated ANOVA; the main effect of group F (1, 16) =5.936,

p=0.027; the main effect of sessions F (9, 144) =10.544, p<0.001; the group x session interaction F (9, 144) =0.845,

p=0.576). Prior to the training, there was no difference between groups on adaptation sessions.

3.6 c-Fos expression was increased in PrL in ArchT group mice

following the photo-inhibition of BF cholinergic terminals in

mPFC

To examine the neuronal activity level in different cell types, I double stained c-Fos with

different cell markers. Consistent with previous data, in deep layer of PrL (Figure 10a), the

percentage of CamKII α+ and SST+ neurons that co-express c-Fos were higher in ArchT group

than controls ( t test, p=0.006, p=0.001), but the percentage of PV+ neurons co-expressing c-Fos

27

was comparable (t test, p=0.088), whereas in superficial layer of PrL (Figure 10b) there was no

difference between groups among the activation level of CamKII α+, SST+ and PV+ neurons (t

test, p=0.196, p=0.093, p=0.305).

Figure 10 The expression of c-Fos in different cell type.

(a) In deep layer of PrL, the overlap of c-Fos+ neurons were significantly higher in CamkIIα+ neurons (12.286±1.135

VS 7.703±1.347, p=0.006, n=7), and SST+ neurons (46.528±3.182 VS 28.878±2.542, p=0.001, n=7) in ArchT group

than control. The percentage of PV+ co-express c-Fos was comparable between groups (16.203±6.197 VS 4.658±3.02,

p=0.088, n=7). (b) In superficial layer of PrL, the overlap of c-Fos+ neurons in PV+ neurons (12.255±12.5 VS

5.313±3.026, p=0.305, n=7), SST+ neurons (43.739±12.185 VS 25.259±5.775, p=0.093, n=7), and CamkII α+ cells

(14.532±4.898 VS 9.251±2.678, p=0.196, n=7) were comparable between groups. ** p<0.01.

28

4 Discussion

Recent studies showed that cholinergic neurons in the BF respond to innately salient events at

millisecond precision (Hangya, Ranade, Lorenc, & Kepecs, 2015; Harrison, Pinto, Brock, & Dan,

2016). By suppressing this transient cholinergic activity with optogenetic approaches, I have

investigated whether it serves as a reinforcement signal that facilitates the formation of temporal

event associations in trace eyeblink conditioning. Contrary to our hypothesis, inhibiting the

transient cholinergic activity during an aversive event facilitated the formation of its association

with a preceding neutral event. The enhanced learning was accompanied by the elevated expression

of a neuronal activity marker in the PrL region of mPFC, but not in the motor cortex. Within the

PrL, cholinergic inhibition increased the activity of excitatory neurons as well as inhibitory

neurons. We also confirmed that the PrL received strong monosynaptic projections from BF

neurons located in the medial region of HDB and ventralrostral parts of SI. Even though only half

of the PrL-projecting neurons are cholinergic neurons, inhibiting the cholinergic monosynaptic

projection reaching PrL was sufficient to replicate the enhanced learning with inhibition of

cholinergic cell bodies in BF. These findings suggest that transient cholinergic activity time-locked

to innately salient events does not serve as a reinforcement signal in temporal associative learning.

Rather, it may control the activation level of the PrL network during memory encoding through its

modulation on the activity of local excitatory and inhibitory neurons.

The observed enhanced learning with suppression of cholinergic activity sharply contrasts with

previous findings that disruption of transient cholinergic activity or its functional outcome impairs

learning in other memory tests. In auditory-cued fear conditioning, foot shock triggers

acetylcholine release which activates interneurons expressing vasoactive intestinal polypeptide

(VIP) in layer I of the primary auditory cortex (Letzkus et al., 2011). Optogenetic silencing of

29

VIP+ interneurons during the foot shock impairs fear learning (Letzkus et al., 2011). In the same

task, learning is impaired when cholinergic terminals in the basolateral amygdala were

optogenetically silenced during the CS-US pairing (Jiang et al., 2016). In the hippocampus, ACh

activates SST+ interneurons during foot shock, and inhibition of this shock-induced recruitment of

SST+ interneurons impairs contextual fear conditioning (Lovett-Barron et al., 2014). Collectively,

these results suggest that transient increase of acetylcholine during aversive events results in a

network state conducive to fear learning. This is opposite from what we observed in PrL in trace

eyeblink conditioning.

Although behavioral paradigms used in the studies mentioned above share some features,

cholinergic modulation may vary in a task-specific way. In trace eyeblink conditioning, the 500

ms-long trace interval between CS and US makes the acquisition of this task heavily depend on the

functional prefrontal and hippocampal network, and around 75% of mice are able to learn after

hundreds of repeated CS-US pairings. By contrast fear conditioning is an amygdala-dependent task

and can be acquired only with the single tone-shock pairing. Moreover, Jiang et al. (2016) inhibited

cholinergic terminals during the CS and US while our study limited photoinhibition to the US.

Thus, it is possible that the temporal pattern of cholinergic activity may be an important

determinant factor on the impact of cholinergic modulation on associative learning. In support,

researchers found that synaptic plasticity can be modified from long term potentiation to short term

depression by changing the temporal relationship between optogenetic manipulation of cholinergic

inputs and electrical stimulation of the CA3 to CA1 (Gu & Yakel, 2011). Future studies need to

examine the impact of photoinhibition during the CS or CS-US interval on acquisition in trace

eyeblink conditioning.

30

Another key difference between our study and the others is the brain region under

investigation. In the auditory cortex, VIP+ interneurons make inhibitory synapses on surrounding

interneurons of other types, such as PV+ and SST+. The acetylcholine-dependent activation of

VIP+ interneurons, therefore, disinhibits pyramidal neurons (Letzkus et al., 2011; Pi et al., 2013).

In fact, photostimulation of VIP+ neurons induces increased c-Fos expression, of which 92% are

expressed in pyramidal neurons and 8% are in VIP+ neurons (Pi et al., 2013). In line with this

disinhibition mechanism, our data suggested that inhibiting BF cholinergic neuronal activity during

eyelid shock augmented c-Fos expression in PV+ population in the superficial layer of the

prelimbic cortex, likely because of releasement from VIP+ neuronal inhibition. However, instead

of observing decreased pyramidal neuronal activation after photoinhibition, our data revealed an

enhanced neuronal network activity, with greater c-Fos expression in pyramidal neurons, PV+ and

SST+ interneurons located in deep layer of PrL. These findings suggest that the net effect of ACh

on the activity of local neurons greatly vary depending on brain regions likely due to differences in

microcircuit organization.

Photoinhibition of cell bodies or terminal fields of cholinergic neurons increased c-Fos

expression in excitatory and inhibitory neurons in the PrL during trace eyeblink conditioning,

suggesting that suppressing fast fluctuation of acetylcholine level augments the activation of the

PrL network during memory encoding. Accumulative studies have shown that the strong activation

of the prefrontal cortex is associated with encoding success (Volle et al., 2016). In humans, the

hippocampus and prefrontal cortex are co-activated during memory encoding, and the ability to

later remember events is predicted by the magnitude of prefrontal activation during encoding

(Kesteren, Rijpkema, Ruiter, & Fernández, 2010). Our lab recently found that artificially

generating this network state in rats is sufficient to enhance memory encoding in two variants of

31

trace eyeblink conditioning. Chemogenetically enhancing the activity of excitatory neurons in

mPFC enabled rats to learn CS-US associations over an extended temporal gap which was

prohibitively long for untreated rats to learn (Volle et al., 2016). In a follow-up study (Jarovi, in

prep), rats were exposed to two neutral stimuli in two environments whose contingency with an

aversive eyelid shock changed systematically across days. Chemogenetic mPFC activation

accelerated the initial acquisition of differential learning without affecting subsequent reversal

learning and set-shifting. Analyses of local field potentials (LFPs) in the mPFC of these rats

revealed that theta band activity differentiated the relevant CS (paired with the US) from the other

irrelevant CS (presented alone) and that the chemogenetic manipulation selectively augmented this

differential network response. This finding is consistent with the ability of the mPFC neurons to

respond to cues depending on their acquired behavioral relevance (i.e., biological significance or

value) (Euston, Gruber, & McNaughton, 2012; Levy & Glimcher, 2012). Specifically, single-unit

recording study showed that prefrontal neurons could differentially respond to the neutral stimuli

which predict reward or punishment even before rats show behavioral difference (Morrissey, Insel,

& Takehara-Nishiuchi, 2017; Takehara-Nishiuchi & McNaughton, 2008). Collectively, these

findings suggest that mPFC neurons rapidly detect the latent structure of events and compute the

value of those events on-the-fly. This internally-generated relevancy signal may provide a

relevance-signaling mechanism through which mPFC controls the mnemonic fate of event

information. Our finding supports this hypothesis and further extend it by demonstrating that the

transient cholinergic signal may serve as the key afferent system that controls this role of the mPFC

during memory encoding.

During memory encoding, neurons receive two types of information: 1) sensory inputs

perceived from an external environment, and 2) their interpretation based on an internal model of

32

the environment. Theories posit that ACh may play a key role in controlling the relative influence

of these competing inputs from external and internal sources (Hasselmo, 2006; Honey, Newman, &

Schapiro, 2017). In CA1 region of the hippocampus, ACh selectively suppresses excitatory

projections from CA3 region carrying internal inputs, while sparing projections from the entorhinal

cortex carrying external inputs (Lovett-Barron, 2014). Thus, the transient influx of ACh during

salient events will augment the relative impact of the external inputs on local neurons, leading to a

state appropriate for the formation of new representations of the external environment (Meeter,

Murre, & Talamini, 2004). In the mPFC, on the other hand, high ACh level makes local neurons

prioritize the external, afferent inputs over the internal, feedback inputs. This may impair the

mPFC’s ability to detect the latent structure of events without being distracted by their irrelevant

features, such as perceptual and incidental details. Consistent with this view, muscarinic M1

receptor agonist disrupts prefrontal neural activity selective for previously learned rules, a latent

task structure that defines a mapping of stimulus and adaptive behavior (Vijayraghavan, Major, &

Everling, 2018). Thus, inhibiting the transient cholinergic signal in present study likely increases

the relative influence of internal feedback inputs which contributes to generalization and

application of internal model, and in turn, facilitate CS-US association.

Finally, optogenetic inhibition can drive the cells outside their normal physiological range

(Kravitz & Bonci, 2013). Thus, it is possible that upon release of inhibition, potential rebound

excitation induces increased ACh release (Häusser, 2014). Importantly, optogenetic inhibition can

also synchronize populations of neurons from both optogenetic inhibition and potential rebound

spiking (Kravitz & Bonci, 2013). For example, recent study demonstrated that optogenetic

suppression of layer IV PV+ interneurons results in coordinated increase in both excitation and

33

inhibition (Moore, 2018). Future studies need to examine the efficacy of optogenetic inhibition and

potential rebound spiking it may induce.

The present finding begins to uncover millisecond-scale modulation that ACh exerts over

neural processing in the mPFC during memory encoding; however, a number of questions still

remains to be answered. Firstly, it is critical to determine how cholinergic neurons respond to the

US and how their responses change with learning. Secondly, even though, the cell-type specific

activation was reported above, it remains unknown how ACh and its suppression affect firing

responses of excitatory and inhibitory neurons in PrL during learning. To address these points,

future studies need to combine real-time activity recording like calcium imaging or single unit

recording and together with optogenetic manipulation.

In conclusion, the present study found that suppression of cholinergic activity during innately

salient events augments the activation of the mPFC network and in turn facilitates aversive

associative learning. This finding provides new insight into real-time cholinergic modulation of the

cortical network during encoding of event information.

34

Appendix A

Appendix A Genotype of the ChAT (IRES)-Cre mice

Confirmed genotype of the six ChAT (IRES)-Cre mice used to generate experimental cohorts in present study.

35

Appendix B

Appendix B Schema of viral spread and optic fiber placements

(a) Histological reconstruction of viral infection area. Transfected cholinergic neurons expressing the reporter GFP

were mainly located in several subregions of BF, including horizontal diagonal band of Broca (HDB), substantia

innominata (SI) and ventral pallidum (VP). (b) Schematic representations of the optic fiber location in the BF in

different coronal plates. (c) Example microphotographs with optic fiber track (red arrows).

36

Appendix C

Appendix C Optic fiber placements in mPFC

(a) Schematic representations of the optic fiber location in the PrL of mPFC in different coronal plates. (b) Example

microphotographs with optic fiber track (red arrows).

37

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