Research Articles: Systems/Circuits Prefrontal α7nAChR signaling differentially modulates afferent drive and trace fear conditioning behavior in adolescent and adult rats https://doi.org/10.1523/JNEUROSCI.1941-20.2020
Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.1941-20.2020 Received: 27 July 2020 Revised: 29 November 2020 Accepted: 23 December 2020
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1 Prefrontal α7nAChR signaling differentially modulates afferent drive
2 and trace fear conditioning behavior in adolescent and adult rats 3 4 5 6 7 Running title: Prefrontal α7nAChR control of afferent drive 8 9 10 11 Anabel M. M. Miguelez Fernandez, Hanna M. Molla, Daniel R. Thomases, and Kuei Y. Tseng* 12 13 Department of Anatomy and Cell Biology, University of Illinois at Chicago, IL 14 15 16 17 *Corresponding Author: Kuei Y. Tseng, MD, PhD 18 Department of Anatomy and Cell Biology 19 University of Illinois at Chicago – College of Medicine 20 Chicago, IL 60612, USA 21 Email: [email protected] 22 23 24 Number of figures: 8 25 Number of tables: 0 26 Abstract: 250 27 Main text: 4,030 words (Introduction: 451; Methods: 1,205; Results: 979; Discussion: 1,395) 28 29 30 31 32 Acknowledgements 33 Supported by NIH Grants R01-MH086507 and R01-MH105488 to KYT, and UIC College of Medicine 34 funds to KYT. We thank Dr. Adriana Caballero for thoughtful comments on the manuscript. This study 35 was initiated at Rosalind Franklin University – Chicago Medical School. 36
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37 Abstract
38 Increased level of kynurenic acid is thought to contribute to the development of cognitive deficits in
39 schizophrenia through an α7nAChR-mediated mechanism in the prefrontal cortex (PFC). Yet, it
40 remains unclear to what extent disruption of PFC α7nAChR signaling impacts afferent transmission and
41 its modulation of behavior. Here we found that PFC infusion of methyllycaconitine (MLA, α7nAChR
42 antagonist) shifts ventral hippocampal-induced local field potential (LFP) suppression to LFP facilitation,
43 an effect only observed in adult male rats. Hippocampal stimulation can also elicit a GluN2B-mediated
44 LFP potentiation (when PFC GABAAR is blocked) that is insensitive to MLA. Conversely, PFC infusion
45 of MLA diminished the gain of amygdalar transmission, which is already enabled by postnatal day 30.
46 Behaviorally, the impact of prefrontal MLA on trace fear-conditioning and extinction was also age-
47 related. While freezing behavior during conditioning was reduced by MLA only in adults, it elicited
48 opposite effects in adolescent and adult rats during extinction as revealed by the level of reduced and
49 increased freezing response, respectively. We next asked if the late-adolescent onset of α7nAChR
50 modulation of hippocampal inputs contributes to the age-dependent effect of MLA during extinction.
51 Data revealed that the increased freezing behavior elicited by MLA in adult rats could be driven by a
52 dysregulation of the GluN2B transmission in the PFC. Collectively, these results indicate that distinct
53 neural circuits are recruited during the extinction of trace fear memory in adolescents and adults, likely
54 due to the late-adolescent maturation of the ventral hippocampal-PFC functional connectivity and its
55 modulation by α7nAChR signaling.
56
57
58
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59 Significance statement
60 Abnormal elevation of the astrocyte-derived metabolite kynurenic acid in the prefrontal cortex is thought
61 to impair cognitive functions in schizophrenia through an α7nAChR-mediated mechanism. Here we
62 found that prefrontal α7nAChR signaling is recruited to control the gain of hippocampal and amygdalar
63 afferent transmission in an input-specific, age-related manner during the adolescent transition to
64 adulthood. Behaviorally, prefrontal α7nAChR modulation of trace fear memory was also age-related,
65 likely due to the late-adolescent maturation of the ventral hippocampal pathway and its recruitment of
66 PFC GABAergic transmission enabled by local α7nAChR signaling. Collectively, these results reveal
67 that distinct α7nAChR-sensitive neural circuits contribute to regulate behavior responses in adolescents
68 and adults, particularly those requiring proper integration of hippocampal and amygdalar inputs by the
69 prefrontal cortex.
70
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71 Introduction
72 Several neural processes contributing to prefrontal cortex (PFC) maturation undergo major remodeling
73 during adolescence (Caballero et al., 2016; Caballero and Tseng, 2016) to enable the acquisition of
74 adult cognitive abilities (Casey et al., 2000; Best and Miller, 2010). As PFC processing and integration
75 of inputs matures through adolescence (Caballero et al., 2016), any disruption that compromises the
76 protracted trajectory of prefrontal development is expected to confer vulnerability to the onset of mental
77 disorders (Caballero and Tseng, 2016) that display cognitive deficits and associated dysregulation of
78 affect (Paus et al., 2008; Gogtay et al., 2011; Volk and Lewis, 2014). Thus, elucidating which signaling
79 mechanisms are recruited to strengthen the functional connectivity of PFC afferent transmission is key
80 to reveal how cognitive impairments could emerge in psychiatric disorders when such recruitment fails
81 to occur (Caballero et al., 2016; Caballero and Tseng, 2016).
82 Of particular interest is the increased level of kynurenic acid in the PFC and its potential link to
83 the onset of cognitive deficits in schizophrenia (Erhardt et al., 2007; Wonodi and Schwarcz, 2010;
84 Myint, 2012). In addition to disrupting NMDAR function (Kessler et al., 1989; Parsons et al., 1997),
85 nanomolar concentrations of kynurenic acid can elicit a state of excitatory-inhibitory imbalance in the
86 PFC through a presynaptic α7nAChR-mediated mechanism (Flores-Barrera et al., 2017). Certainly,
87 prefrontal regulation of cognitive behavior, such as working memory, behavioral flexibility and attention,
88 requires proper levels of α7nAChR and NMDAR function (Alexander et al., 2012; Alexander et al.,
89 2013; Phenis et al., 2020) and integration of hippocampal and amygdalar inputs by the PFC (Floresco
90 et al., 1997; Ishikawa and Nakamura, 2003; Tse et al., 2015). Since α7nAChRs are well-positioned to
91 regulate glutamate release in the PFC (Bortz et al., 2016), any disruption of local α7nAChR signaling is
92 expected to impact the gain of afferent glutamatergic transmission and the recruitment of postsynaptic
93 NMDAR-mediated plasticity by hippocampal and amygdalar inputs (Flores-Barrera et al., 2014).
94 The aim of the present study is to determine how disruption of α7nAChR signaling in the PFC
95 impacts afferent information processing and its control of behavioral responses in adolescent and adult
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 4
96 rats. To this end, local field potential recordings were combined with PFC infusions of antagonists to
97 reveal whether α7nAChR modulation of ventral hippocampal and basolateral amygdalar transmission is
98 input-specific and age-regulated between. Similar pharmacological manipulations were implemented to
99 assess the contribution of prefrontal α7nAChR signaling in modulating behavior using a trace fear
100 conditioning paradigm. Such a behavioral construct was preferred because proper processing of
101 hippocampal and amygdalar afferent information by the PFC is needed for the learning and extinction
102 of conditioned fear memories (Ishikawa and Nakamura, 2003; Sierra-Mercado et al., 2011; Gilmartin et
103 al., 2012; Sotres-Bayon et al., 2012; Gilmartin et al., 2014).
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104 Materials and Methods
105 All experimental procedures were approved by the University of Illinois at Chicago Institutional Animal
106 Care and Use Committee and met the National Institutes of Health guidelines for care and use of
107 laboratory animals. Male Sprague Dawley rats were purchased from Envigo. Upon arrival, rats were
108 allowed to habituate for at least 7 days before being subjected to any surgical procedures. They were
109 group housed (2-3 rats/cage), maintained under constant temperature (21-23°C) and light/dark cycle
110 (14/10hs) with food and water available ad libitum. All chemicals were obtained from Sigma, except for
111 methyllycaconitine (MLA) and Indiplon that were obtained from Tocris.
112
113 In vivo recordings of local field potential (LFP) responses in the prefrontal cortex (PFC). All recordings
114 and PFC infusions procedures were conducted as previously described (Cass et al., 2013; Thomases
115 et al., 2013; Caballero et al., 2014b; Thomases et al., 2014). Briefly, rats were anesthetized with 8%
116 chloral hydrate (400mg/kg, i.p.), placed in a stereotaxic frame, and maintained at 37-38°C with a steady
117 supplement of 300-400 μL/hour of 8% chloral hydrate. After exposing the skull, two burr holes were
118 drilled to place the recording electrode in the medial PFC and the stimulating electrode within the
119 ventral hippocampus or the basolateral amygdala. All LFP recordings were obtained using a concentric
120 bipolar electrode attached to a 28G cannula (PlasticsOne), amplified (Cygnus Technology), filtered (1–
121 100 Hz bandwidth), and digitized (Digidata 1440A, Molecular Devices) at a sampling rate of 10 kHz.
122 The intensity of stimulation was chosen from the 0.2–0.8 mA range (mean intensity: ~0.6mA) using 300
123 μs duration square pulses delivered every 15 s through a computer-controlled pulse generator (Master
124 8, A.M.P.I.). Typically, a 10 min LFP baseline recording was collected prior PFC infusions of 0.8 μL (0.1
125 μL/min) artificial cerebrospinal fluid (aCSF)-containing vehicle, picrotoxin (50μM in 0.1% DMSO), MLA
126 (300nM) or picrotoxin + MLA. The dose of MLA was chosen because it blocks α7nAChR function and
127 disrupts PFC synaptic transmission in vivo and ex vivo (Flores-Barrera et al., 2017). A protocol of high-
128 frequency stimulation (4 trains of 50 pulses each at 100 Hz every 15 s) was then delivered into the
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 6
129 ventral hippocampus or basolateral amygdala ~30 min post-PFC infusion, and changes in the slope of
130 LFP responses (from the onset to peak amplitude) were determined. The 30 min post-infusion period
131 was included to monitor the stability of the evoked LFP response. Only recordings with a reliable 10-12
132 min baseline of LFP responses (<15% variability in slope using a bin size of 2 min) prior to the delivery
133 of the high-frequency stimulation protocol were included.
134
135 Assessing the effects of PFC infusion of MLA on trace fear conditioning and extinction. Rats underwent
136 survival surgery for bilateral cannula placement targeting the medial PFC region at least 8 days (range:
137 8-13 days) prior to behavioral testing. All PFC infusions were performed 20 min prior to the start of
138 behavioral testing and consisted of simultaneous delivery of 0.8 μL aCSF alone or in combination with
139 MLA (300 nM), MLA+Ifenprodil (10 μM) or MLA+Indiplon (10 μM/0.04% DMSO), using a 33G infusion
140 cannula protruding 0.5 mm beyond the tip of the guide cannula. The doses of MLA, ifenprodil and
141 Indiplon were chosen from previous studies showing their preferential effects on α7nAChR, GluN2B
142 and GABA-AR transmission, respectively (Flores-Barrera et al., 2014; Flores-Barrera et al., 2017).
143 Survival surgery: Rats were deeply anesthetized in a chamber saturated with 5% isoflurane
144 (Somnosuite Unit, Kent Scientific). The level of anesthesia was monitored by assessing absence of the
145 withdrawal reflex (hindlimb compression reflex). Prior to mounting the rats in the stereotaxic apparatus
146 using non-rupture ear bars (Kopf), the head was shaved and the skin overlying the skull was then
147 infiltrated with 2% lidocaine hydrochloride. Throughout the surgical procedure, isoflurane anesthesia (3-
148 5%) was maintained using a Somnosuite Unit with the body temperature kept within 37-38°C (TCAT-
149 2LV heating pad, Physitemp). Burr holes were drilled in the skull to enable placement of a 26G guide
150 cannula (Plastics One) targeting the dorsal border of the medial PFC bilaterally at a 25° angle (2.7-3.2
151 mm anterior to bregma; 3.2 mm lateral; 3.5-4.0 mm below the brain surface). At least 2 skull screws
152 (Plastics One, VA) were used to anchor the acrylic cement head assembly. After securing the guide
153 cannula with acrylic cement (Stoelting), a 33G dummy cannula was screwed into the guide cannula to
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154 prevent clogging. All dummy cannulas were replaced by those protruding 0.5 mm beyond the tip of the
155 guide cannula 24 h prior to testing the impact of PFC infusions on behavior.
156 Behavioral testing: We adapted a fear conditioning paradigm used by Zhang and Rosenkranz
157 (2013). Briefly, all testing chambers (Ugo Basile) were housed in sound attenuating cabinets with white
158 noise (60-70 dB; Scientific Design). The conditioning phase begins with a 120 s habituation period
159 followed by the presentation of 5 trials of 220 s each using a pseudorandom inter-trial interval of 240-
160 280 s. In each trial, a neutral tone (10 s, 1500 Hz, 85 dB) was paired with a footshock (1 s, 0.4 mA) at a
161 delay of 20 s from the end of the tone (ANY-Maze, Stoelting). The extinction phase begins 24 h later in
162 a visually and tactilely distinct chamber. Following 120 s of habituation, rats were tested with 14 trials of
163 60 s each from which the conditioned tone was presented for 20 s without footshock (ANY-Maze,
164 Stoelting). The acquisition of fear extinction is typically revealed by the degree of conditioned freezing
165 to the tone that diminishes over repeated trials. All behavioral changes were recorded by an infrared
166 camera connected to a computer, and the time spent freezing (lack of non-respiratory movement >0.5
167 s) per trial (% freezing) was determined offline from trial to trial as previously described (Caballero et
168 al., 2020; Flores-Barrera et al., 2020).
169 Finally, a separate cohort of rats was included to assess the effect of MLA on contextual fear
170 memory. Briefly, rats were trained using the same trace fear conditioning protocol described above.
171 However, the shock intensity was increased to 0.5 mA to obtain sufficient levels of freezing response
172 during the contextual testing phase. The context retention test begins 24 h later in the same training
173 chamber in the absence of tone and shock. After 120 s of habituation, changes in freezing behavior
174 were recorded for 16 min, and the time spent freezing per epoch of 4 min (% freezing) was determined.
175
176 Histology. At the end of the experiments (electrophysiology and behavior), rats were euthanized, and
177 their brains quickly removed. Brains were then blocked, fixed in 10% formalin overnight, and stored in
178 30% sucrose before sectioning as previously described (Cass et al., 2013; Thomases et al., 2013). The
179 exact location of all recording, stimulating and infusion sites were determined by Nissl staining.
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180
181 Statistical analysis. Data were summarized as mean ± SEM and differences among experimental
182 conditions were considered statistically significant at p<0.05. More specifically, all electrophysiological
183 changes resulting from PFC infusion of MLA were compared to aCSF controls by student t-test
184 because they involve a single continuous dependent variable. On the other hand, changes in the level
185 of freezing behavior across treatment conditions or age groups were assessed by two-way and three-
186 way ANOVA (treatment or age x trials or epochs) for testing comparisons along 3 or more dependent
187 variables.
188
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189 Results
190 We first examined how blocking α7nAChR signaling in the PFC impacts afferent drive originated from
191 the ventral hippocampus by means of local field potential (LFP) recordings in vivo. To this end, the
192 α7nAChR antagonist MLA (300 nM) was locally infused into the PFC prior to high frequency stimulation
193 of the ventral hippocampus (4 trains of 50 pulses each delivered at 100 Hz every 15 s). This stimulation
194 protocol typically elicits a sustained suppression of LFP in the PFC that emerges after P45 (Caballero
195 et al., 2014b) (Fig. 1) through the recruitment of a developmentally regulated prefrontal GABAergic
196 transmission (Caballero et al., 2014a; Caballero et al., 2020). Relative to aCSF controls (n=6), PFC
197 infusion of MLA (n=8) shifted hippocampal-induced LFP suppression to LFP facilitation (Fig. 1a,b), an
198 effect that was not present in adolescent P30-44 rats (n=5 aCSF, n=7 MLA; Fig. 1c). Interestingly, such
199 an effect was no longer apparent in the presence of picrotoxin (n=6 picrotoxin, n=7 picrotoxin+MLA;
200 Fig. 2) indicating that MLA is disrupting the GABAergic component of the PFC response. In addition,
201 these results further revealed that the NMDAR-GluN2B component contributing to the potentiation of
202 LFP in the PFC when local GABAA receptors are blocked (Caballero et al., 2014a; Flores-Barrera et al.,
203 2014) is insensitive to MLA (Fig. 2). Together, these findings point to an age-dependent recruitment of
204 α7nAChR signaling by ventral hippocampal inputs that preferentially impacts the gain of prefrontal
205 GABAergic function.
206 We next asked whether PFC inputs originated from the basolateral amygdala are also
207 modulated by α7nAChR signaling. Contrary to the impact of hippocampal stimulation, basolateral
208 amygdala high frequency stimulation elicits a pattern of sustained LFP facilitation in the PFC that is
209 already enabled by P30 (Caballero et al., 2014b) (Fig. 3). Accordingly, the amplitude of the potentiated
210 amygdalar LFP response was markedly attenuated by PFC infusion of MLA in both P30-44 (n=5 aCSF,
211 n=5 MLA) and P60-90 (n=10 aCSF, n=7 MLA) age groups (Fig. 3). Thus, PFC α7nAChR signaling is
212 recruited as early as P30 to facilitate afferent transmission of basolateral amygdala inputs.
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213 At the behavioral level, we implemented a trace-fear conditioning paradigm paired with local
214 infusion of MLA to determine how PFC disruption of α7nAChR signaling impacts behavior in adolescent
215 (P38-44) and adult (P70-90) rats. Although the age ranges in the behavioral cohort are more narrowed
216 than that included in the electrophysiology groups due to the recovery period from the survival surgical
217 procedure for cannula placement (see Materials & Methods), they are still within P30-44 and P60-90.
218 Data obtained from all aCSF groups revealed that adolescent rats (n=15) show lower freezing than
219 adults (n=16) during acquisition (Fig. 4a), while both age groups display similar patterns of extinction
220 behavior (Fig. 4b). Of note, the impact of MLA during acquisition was also age-related, such that it
221 reduced the freezing response in adults (n=6 aCSF, n=8 MLA; Fig. 5a-c) without disrupting the pattern
222 of freezing behavior in adolescents (n=6 aCSF, n=8 MLA; Fig. 5d-f). Conversely, PFC infusion of MLA
223 elicited opposite effects in adolescent and adult rats during extinction (Fig. 6). Relative to aCSF
224 controls, MLA increased the level of freezing response to the conditioned tone in adults (n=6 aCSF,
225 n=8 MLA; Fig. 6a-c), while it reduced freezing in adolescents (n=7 aCSF, n=8 MLA; Fig. 6d-f). These
226 results suggest that adolescent and adult rats recruit distinct neural circuits during the extinction of
227 trace fear memory, a behavioral response known to require proper integration of ventral hippocampal
228 and amygdalar inputs by the PFC (Sierra-Mercado et al., 2011). Thus, it is possible that the delayed
229 maturation of the ventral hippocampal pathway (Caballero et al., 2014b) contributes to the opposite,
230 age-dependent effect of MLA as α7nAChR signaling in the PFC emerges after P45 to enable the
231 GABAA-component of the hippocampal-evoked response (Fig. 1).
232 If the late-adolescent onset of PFC α7nAChR modulation of hippocampal inputs (Fig. 1) sets the
233 age-dependent effect of MLA during extinction (Fig. 6), the increased freezing behavior observed in
234 adult rats could result from two concurrent events triggered by a disruption of the GABAA-mediated LFP
235 suppression (Fig. 1), while the GluN2B-mediated LFP potentiation component remains intact (Fig. 2).
236 To test this hypothesis, ifenprodil (10μM) was co-delivered with MLA into the PFC to block the GluN2B-
237 mediated potentiation of the hippocampal transmission (Flores-Barrera et al., 2014). Data revealed that
238 the inclusion of ifenprodil was sufficient to mitigate the enhanced freezing response elicited by MLA in
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 11
239 adult rats during extinction (n=5 aCSF, n=5 MLA+ifenprodil; Fig. 7a,b). However, this was not the case
240 when the GABAA receptor positive allosteric modulator indiplon (10μM) (Flores-Barrera et al., 2017)
241 was delivered along with MLA to partially compensate for the diminished GABAergic function (n=6
242 aCSF, n=6 MLA+indiplon; Fig. 7c). Together, the results indicate that an imbalanced potentiation of
243 PFC GluN2B transmission underlies the increased level of freezing response during extinction when
244 prefrontal disruption of α7nAChR signaling occurs in adults.
245 Finally, it is possible that α7nAChR signaling in the PFC is also recruited during contextual fear
246 association in trace conditioning (Gilmartin and Helmstetter, 2010; Orsini et al., 2011; Kim and Cho,
247 2017; Twining et al., 2020). Thus, another cohort of adolescent and adult rats was generated to
248 examine the impact of MLA during the context retention test 24 h later. Here, the shock intensity during
249 conditioning was increased to obtain sufficient levels of freezing behavior during the contextual testing
250 phase. As a result, the age effect obtained during conditioning with a lower shock intensity (Fig. 4a) is
251 no longer apparent (Fig. 8a). Remarkably, adolescent rats continue to show a pattern of lower freezing
252 than adults during re-exposure to the training context (Fig. 8b). However, PFC infusion of MLA did not
253 alter the distinct levels of freezing response observed in adolescent (P38-44: n=6 aCSF, n=7 MLA) and
254 adult (P70-90: n=6 aCSF, n=7 MLA) (Fig. 8b). Together, these results show that the level of contextual
255 freezing response is developmentally regulated, but independent of prefrontal α7nAChR signaling.
256
257
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258 Discussion
259 The present study reveals that prefrontal α7nAChR signaling is recruited by ventral hippocampal and
260 basolateral amygdalar inputs to modulate the gain of afferent transmission in an age-related manner.
261 While amygdalar inputs in the PFC are already enabled by α7nAChR signaling at P30, its modulation of
262 ventral hippocampal transmission does not emerge until late adolescence when the GABAA component
263 of the prefrontal response becomes online. Remarkably, a similar age-related modulation of trace fear
264 behavior by PFC α7nAChR was observed in tandem with the delayed maturation of the hippocampal
265 pathway. Thus, it is conceivable that distinct α7nAChR-sensitive neural circuits contribute to regulate
266 behavioral responses in adolescents and adults, particularly when proper integration of hippocampal
267 and amygdalar inputs by the PFC is required.
268 Despite its widespread expression, our data indicate that the recruitment α7nAChR signaling by
269 PFC afferent transmission is input- and synapse-specific. While the GluN2B-mediated potentiation of
270 hippocampal inputs (Flores-Barrera et al., 2014) is insensitive to MLA, PFC α7nAChR modulation of the
271 amygdalar pathway is already enabled by P30, likely through a mechanism that facilitates glutamate
272 release (Konradsson-Geuken et al., 2009; Bortz et al., 2016; Yarur et al., 2020). GABAergic synapses
273 in the PFC are also regulated by α7nAChR (Couey et al., 2007; Aracri et al., 2010; Flores-Barrera et
274 al., 2017) in a manner that enables ventral hippocampal inputs to enhance PFC inhibitory control of
275 afferent drive after P45 (Caballero et al., 2014b). In fact, the pattern of LFP potentiation observed in the
276 PFC following MLA infusion (Fig. 1) resembles those elicited by picrotoxin (Cass et al., 2013; Caballero
277 et al., 2014b; Thomases et al., 2014), which point to a GABAergic mechanism underlying the α7nAChR
278 control of hippocampal inputs. Thus, any disruption that compromises α7nAChR function in the PFC is
279 expected to limit its optimal computational capacity and the control of input selectivity by local inhibition
280 (Lew and Tseng, 2014).
281 Parallel to the gain of GABA function in the PFC during adolescence are the increased level of
282 parvalbumin (PV) expression and glutamatergic transmission onto PV-positive fast-spiking interneurons
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283 (FSI) (Caballero et al., 2014a). Although not all FSI express α7nAChR-sesitive currents (Porter et al.,
284 1999; Couey et al., 2007; Poorthuis et al., 2013), nAChR stimulation facilitates the transmission of
285 excitatory inputs onto these interneurons in the PFC, likely through a presynaptic mechanism (Couey et
286 al., 2007). Therefore, α7nAChR signaling could strengthen PFC inhibitory control of afferent drive by
287 increasing the gain of excitatory synapses onto PV-positive FSI during adolescence. In fact, genetic
288 deletion of α7nAChR markedly reduced the level of cortical PV and markers of GABA function (Lin et
289 al., 2014), resembling the GABAergic deficit observed in the PFC when local recruitment of PV-positive
290 FSI during adolescence is limited (Cass et al., 2013; Caballero et al., 2020; Flores-Barrera et al., 2020).
291 Collectively, these results suggest an α7nAChR mechanism underlying the maturation of PV-positive
292 FSI in the PFC. A deficient recruitment of prefrontal FSI function by α7nAChR in adulthood will likely
293 disrupt the inhibitory control of afferent drive and its impact on behavior, as seen when PV expression
294 in the PFC fails to reach adult levels (Caballero et al., 2020).
295 Prefrontal control of behavior requiring proper integration of hippocampal and amygdalar inputs
296 also undergoes developmental changes during adolescence. Of particular interest is the regulation of
297 conditioned fear memories by PFC nAChRs (Raybuck and Gould, 2010; Kutlu et al., 2018) and the
298 underlying glutamatergic mechanism driving freezing behavior (Gilmartin and Helmstetter, 2010;
299 Gilmartin et al., 2012; 2013a; Gilmartin et al., 2013b). In this regard, the age-dependent facilitation of
300 freezing response observed during trace fear conditioning following nanomolar infusion of MLA could
301 result from the gain of PFC GluN2B function to strengthen hippocampal inputs after P45 (Flores-
302 Barrera et al., 2014). However, it is unlikely that inhibition of such GluN2B transmission is driving the
303 reduced level of freezing observed following PFC α7nAChR blockade in adults since this glutamatergic
304 input is insensitive to MLA. Instead, the effect of MLA during conditioning is likely due to a disruption of
305 basolateral amygdala transmission to the PFC as similar behavioral deficit was observed following
306 functional disconnection of the amygdalar-prefrontal pathway (Gilmartin et al., 2012). Our data also
307 revealed that α7nAChR signaling is not recruited during the acquisition of trace fear conditioning in
308 adolescent rats, further indicating that different PFC dependent neural processes regulate this behavior
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 14
309 in an age-related manner. In addition to ventral hippocampal and amygdalar inputs, thalamic afferents
310 also continue to develop into early adulthood, which are likely to impact PFC maturation and its control
311 of behavior (Ferguson and Gao, 2014; Parnaudeau et al., 2018).
312 Associated with the strengthening of glutamatergic transmission is the functional maturation of
313 prefrontal GABAergic circuits during adolescence that enables ventral hippocampal inputs to enhance
314 PFC inhibitory control of afferent drive in adulthood (Caballero et al., 2016; Caballero and Tseng,
315 2016). Such gain of GABAergic function is critical to support the extinction of a trace fear memory that
316 requires intact hippocampal-PFC connectivity (Caballero et al., 2020; Flores-Barrera et al., 2020).
317 Remarkably, the enhanced freezing response elicited by MLA in adult rats during extinction resembles
318 the behavioral deficit observed following a developmental disruption that renders the PFC disinhibited
319 (Caballero et al., 2020; Flores-Barrera et al., 2020). While these results are consistent with the view
320 that PFC interneurons are recruited to facilitate the extinction of learned behaviors (Sotres-Bayon et al.,
321 2012; Courtin et al., 2014; Sparta et al., 2014), they also imply a disinhibitory mechanism underlying the
322 effect of MLA in adults. By limiting the gain of inhibitory synapses (Flores-Barrera et al., 2017), the
323 resulting impact of MLA during extinction becomes shifted due to an imbalanced facilitation of the PFC
324 GluN2B transmission (Flores-Barrera et al., 2014). Accordingly, the enhanced freezing response
325 elicited by MLA during extinction was mitigated by the GluN2B antagonist ifenprodil, which also blunted
326 the potentiation of hippocampal-driven LFP response in the PFC (Flores-Barrera et al., 2014). Thus, it
327 is possible that coordinated feedforward inhibitory control of glutamatergic inputs by α7nAChR in the
328 PFC dictates the level of conditioned freezing that diminishes over repeated trials during trace fear
329 extinction. The opposite freezing response elicited by MLA in adolescent rats further reveals that
330 distinct PFC mechanisms are recruited to regulate trace fear extinction behavior when the functional
331 connectivity of the hippocampal-prefrontal pathway is not fully matured.
332 The level of contextual freezing behavior is also developmentally regulated, but insensitive to
333 PFC infusion of MLA. Relative to adolescents, adult rats displayed an enhanced freezing response
334 during re-exposure to the training context. While several mechanisms (Gilmartin and Helmstetter, 2010;
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 15
335 Gilmartin et al., 2012; 2013a; Gilmartin et al., 2013b) could contribute to driving such age-dependent
336 freezing response, recent work has highlighted the role of ventral hippocampal inputs to the PFC in
337 modulating contextual fear association in trace conditioning (Twining et al., 2020). In this regard, the
338 enhanced contextual freezing response observed in adults could result from the gain of PFC GluN2B
339 transmission that potentiates ventral hippocampal inputs after P45 (Flores-Barrera et al., 2014) that is
340 also insensitive to MLA. Although the gain of ventral hippocampal inputs after P45 also strengthens
341 PFC inhibitory control of afferent drive (Caballero et al., 2014b), it is unlikely that this component is
342 recruited during the contextual retention testing because of its sensitivity to MLA, whereas the
343 behavioral response is not. Collectively, our data suggest that contextual and cue-mediated association
344 in trace fear conditioning can be dissociated at the level of circuitry and PFC α7nAChR signaling.
345 Whether the contextual control of extinction observed in adolescents and adults involve hippocampal-
346 PFC connections directly or indirectly via midline thalamic inputs (Orsini et al., 2011; Xu and Sudhof,
347 2013; Kim and Cho, 2017) remains to be determined.
348 In sum, any disruption of prefrontal α7nAChR function during development (e.g. by elevation of
349 brain kynurenic acid levels) is expected to limit the gain of afferent transmission and negatively impact
350 the functional connectivity between the PFC, ventral hippocampus and basolateral amygdala in an age-
351 dependent manner. Our data also reveal that behavioral responses requiring PFC integration of
352 hippocampal and amygdalar inputs mature through adolescence, which can be compromised by a
353 deficient prefrontal α7nAChR function as seen in psychiatric disorders exhibiting deficits in cognitive
354 and affective domains (Young and Geyer, 2013; Parikh et al., 2016; Notarangelo and Pocivavsek,
355 2017). Future studies are warranted to identify the cholinergic origin underlying the input-specific, age-
356 related recruitment of α7nAChR transmission in the PFC, and its contribution to the maturation of
357 prefrontal cognitive functions during adolescence.
358
359
360
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 16
361 Figure Legends
362 Figure 1. PFC infusion of MLA shifts ventral hippocampal-induced LFP suppression to LFP
363 facilitation. (a) Summary diagram and coronal sections (inset images) showing the anatomical location
364 (mm relative to bregma) of LFP recordings within the medial PFC and the stimulation sites in the ventral
365 hippocampus shown in b. (b) Ventral hippocampal HFS typically elicits a pattern of sustained LFP
366 suppression in the PFC of adult rats (P60-90) that remained unaltered following aCSF infusion (n=6).
367 However, a potentiation of LFP responses emerged in the PFC following local infusion of MLA (n=8).
368 Bar graph summarizing the mean normalized LFP response obtained from the last 10 min post-HFS
369 (***p<0.0001, unpaired t-test). Inset traces are examples of hippocampal-evoked LFP taken from 5 min
370 pre-HFS (-5) and 35 min post-HFS (+35) illustrating the effect of MLA (calibration: 2mV/20ms). (c) No
371 apparent changes in prefrontal LFP responses were observed following hippocampal HFS in P30-44
372 rats. Relative to aCSF (n=5), PFC infusion of MLA (n=7) failed to disrupt the pattern of hippocampal-
373 evoked LFP.
374
375 Figure 2. PFC infusion of MLA does not disrupt the LFP facilitation elicited from the ventral
376 hippocampus. (a) Summary of the recording and stimulating electrodes placement (mm relative to
377 bregma). (b) Ventral hippocampal HFS typically elicits a pattern of sustained LFP potentiation in the
378 PFC of adult rats (P60-90) when local GABAA receptors are blocked with picrotoxin (n=6). Infusion of
379 MLA along with picrotoxin (n=7) failed to disrupt this facilitation. Inset bar graph summarizing the mean
380 LFP response obtained from the last 10 min post-HFS. Examples traces of hippocampal-evoked LFP
381 taken from 5 min pre-HFS (-5) and 35 min post-HFS (+35) illustrating the lack of effect of MLA
382 (calibration: 2mV/ 20ms).
383
384 Figure 3. PFC infusion of MLA attenuates basolateral amygdalar-induced facilitation of LFP. (a)
385 Summary diagram and coronal sections (inset images) showing the anatomical location (mm relative to
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 17
386 bregma) of LFP recordings within the medial PFC and the stimulation sites in the basolateral amygdala
387 shown in b. (b) Relative to aCSF (n=10), PFC infusion of MLA in adult rats (P60-90, n=7) markedly
388 diminished the amplitude of basolateral amygdalar-induced LFP facilitation as revealed by the mean
389 normalized LFP response obtained from the last 10 min post-HFS (bar graph, **p<0.001, unpaired t-
390 test). Inset traces are examples of amygdalar-evoked LFP taken from 5 min pre-HFS (-5) and 35 min
391 post-HFS (+35) illustrating the effect of MLA observed in adult rats (calibration: 2mV/ 20ms). (c) PFC
392 infusion of MLA (n=5) also reduced the amplitude of amygdalar-induced LFP facilitation observed in the
393 PFC of P30-44 rats (aCSF, n=5). Bar graph summarizing the mean normalized LFP response obtained
394 from the last 10 min post-HFS (**p<0.001, unpaired t-test). Inset traces are examples of amygdalar-
395 evoked LFP taken from 5 min pre-HFS (-5) and 35 min post-HFS (+35) illustrating the effect of MLA
396 (calibration: 2mV/ 20ms).
397
398 Figure 4. Adolescent and adult rats exhibited different levels of freezing response during trace
399 fear conditioning and extinction. (a) A progressive increase in freezing behavior was observed in
400 both adult (P70-90; n=16) and adolescent (P38-44; n=15) rats during conditioning (main effect of trials,
401 F4,145= 30.0, p<0.0001; two-way ANOVA). Yet, the adolescent group showed an overall lower freezing
402 pattern than adults (main effect of age, F1,145= 46.3, ***p<0.0001; two-way ANOVA). (b) Similarly, both
403 age group of rats display comparable patterns of conditioned freezing to the tone that diminishes over
404 repeated trials during extinction testing 24 hours later (main effect of trial F13,406= 27.1, p<0.0001; two-
405 way ANOVA). The two-way ANOVA also revealed a significant main effect of age (F1,406= 7.6, *p<0.01)
406 as a result of an overall lower freezing response in the adolescent group.
407
408 Figure 5. PFC infusion of MLA reduces the level of freezing response during the acquisition of
409 trace fear memory only in adult rats. (a) Summary of aCSF and MLA infusion sites within the PFC of
410 adult rats. (b) Relative to aCSF (n=6), infusion of MLA (n=8) into the PFC of adult rats (P70-90)
411 markedly diminished the freezing response during conditioning (main effect of treatment, F1,60=67.9,
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 18
412 ***p<0.0001; main effect of trials, F4,60=8.7, p<0.0001; two-way ANOVA). (c) Twenty-four hours later,
413 MLA-treated rats showed a lower level of conditioned freezing response during extinction testing (main
414 effect of treatment, F1,168=23.5, ***p<0.0001; main effect of trials, F13,168=10.3, p<0.0001; two-way
415 ANOVA). (d) Summary of aCSF and MLA infusion sites within the PFC of adolescent rats. (e) Relative
416 to aCSF (n=8), PFC infusion of MLA (n=9) did not alter the pattern of freezing response in P38-44 rats
417 during conditioning. (f) Both aCSF- and MLA-treated rats showed similar levels of conditioned freezing
418 behavior during extinction testing 24 hours later.
419
420 Figure 6. PFC infusion of MLA enhances the level of freezing response during extinction testing
421 only in adult rats. (a) Summary of aCSF and MLA infusion sites within the PFC of adult rats. (b) All
422 adult rats (P70-90) assigned to receive either aCSF or MLA during extinction testing (day 2) showed
423 similar level of freezing response during conditioning (day 1). (c) Relative of aCSF (n=6), PFC infusion
424 of MLA (n=8) markedly increased the level of conditioned freezing response during extinction testing
425 (main effect of treatment, F1,140=81.7, ***p<0.0001; main effect of trials, F13,140=13.4, p<0.0001; two-way
426 ANOVA). (d) Summary of aCSF and MLA infusion sites within the PFC of adolescent rats. (e) All P38-
427 44 rats assigned to receive either aCSF or MLA during extinction testing (day 2) showed similar level of
428 freezing response during conditioning (day 1). (f) Relative of aCSF (n=7), PFC infusion of MLA (n=8)
429 diminished the overall level of freezing response in P38-44 rats during extinction (main effect of
430 treatment, F1,182=12.4, **p<0.001; main effect of trials, F13,182=5.6, p<0.0001; two-way ANOVA).
431
432 Figure 7. PFC infusion of ifenprodil prevents the enhanced freezing response elicited by MLA in
433 adult rats during extinction. (a) All adult rats (P70-90) assigned to receive PFC infusions exhibited
434 similar patterns of increased freezing response during conditioning. (b) Relative of aCSF (n=5), the
435 typical heightened freezing behavior observed with MLA alone (Fig. 6c) is no longer apparent when the
436 GluN2B antagonist Ifenprodil (10μM, n=5) was co-administer into the PFC (main effect of treatment,
437 p=0.15; main effect of trials, F13,112=14.5, p<0.0001; two-way ANOVA). (c) In contrast, PFC co-infusion
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 19
438 of the GABAAα1-positive allosteric modulator Indiplon (10μM, n=6) along with MLA failed to reduce the
439 enhanced freezing response elicited by MLA alone (main effect of treatment, F1,126=19.1, ***p<0.0001;
440 main effect of trials, F13,126=15.4, p<0.0001; two-way ANOVA).
441
442 Figure 8. PFC infusion of MLA does not disrupt the pattern of freezing behavior during re-
443 exposure to the training context. (a) All adults (P70-90) and adolescents (P38-44) rats assigned to
444 receive either aCSF or MLA during the context retention test (day 2) showed similar level of freezing
445 response during conditioning (day 1). (b) Relative to aCSF (n=6, P70-90; n=6, P38-44), PFC infusion of
446 MLA in adult (n=7, P70-90) and adolescent (n=7, P38-44) rats failed to disrupt the level conditioned
447 freezing response that diminishes over time (4 min/epoch) during context retention testing (main effect
448 of epochs, F3,88=8.1, ***p<0.0001; three-way ANOVA). Notably, adolescents displayed an overall lower
449 freezing pattern than adults (main effect of age, F1,88=11.2, **p<0.002; three-way ANOVA).
450
Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 20
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Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 23 Figure 1_a7nAChR paper Miguelez Fernandez et al. 2020 a stimulating electrode recording electrode -6.3 -6.1 +3.2 PFC +2.7
ventral hippocampus b P60-90 aCSF MLA c P30-44 aCSF MLA 1.5 MLA 1.5 -5 +35 ***
1.0 1.0 aCSF -5 +35 normalized LFP normalized LFP
HFS HFS 0.5 0.5 -10 0 10 20 30 40 -100 10203040 min min Figure 2_a7nAChR paper Miguelez Fernandez et al. 2020 a b P60-90 ptx ptx+MLA PFC +3.2 1.5 ptx +2.7 -5 +35
1.0 -6.3 -6.1 1.4 ptx+MLA -5 +35 1.2 LFP normalized LFP
normalized 1.0 30-40’ post-HFS 0.5 HFS ventral hippocampus -10 0 10 20 30 40 min Figure 3_a7nAChR paper Miguelez Fernandez et al. 2020
a stimulating electrode recording electrode basolateral +3.2 PFC amygdala +2.7
-3.4 -3.0 b P60-90 aCSF MLA c P30-44 aCSF MLA 1.5 1.5
** ** 1.0 1.0 aCSF MLA aCSF MLA -5 +35 -5 +35 -5 +35 -5 +35 normalized LFP normalized LFP
0.5 HFS 0.5 HFS -10 0 10 20 30 40 min -10 0 10 20 30 40 min Figure 4_a7nAChR paper Miguelez Fernandez et al. 2020
a conditioning (day 1) b extinction (day 2) *** * 80 80
60 60
40 40
% freezing 20 % freezing 20 P70-90 0 P38-44 0 H12345 02468101214 trials trials Figure 5_a7nAChR paper Miguelez Fernandez et al. 2020
a P70-90 b conditioning (day 1) c extinction (day 2) +2.7 aCSF *** *** 80 aCSF 80 +3.2 MLA 60 60
40 40 +2.7 MLA % freezing 20 % freezing 20 P70-90 +3.2 0 0 aCSF H12345 0 2 4 6 8 10 12 14 or MLA trials trials d P38-44 e conditioning (day 1) f extinction (day 2) +2.7 aCSF 80 aCSF 80 +3.2 MLA 60 60
40 40 +2.7 MLA % freezing 20 % freezing 20 P38-44 +3.2 0 0 aCSF H12345 0 2 4 6 8 10 12 14 or MLA trials trials Figure 6_a7nAChR paper Miguelez Fernandez et al. 2020
a P70-90 b conditioning (day 1) c extinction (day 2) +2.7 aCSF *** 80 80 +3.2 60 60
40 40 +2.7 MLA % freezing 20 % freezing 20 aCSF P70-90 MLA +3.2 0 0 H12345 aCSF 02468101214 trials or MLA trials d P38-44 e conditioning (day 1) f extinction (day 2) aCSF +2.7 aCSF 80 80 MLA +3.2 ** 60 60
40 40 +2.7 MLA % freezing 20 % freezing 20 P38-44 +3.2 0 0 H12345 aCSF 02468101214 trials or MLA trials Figure 7_a7nAChR paper Miguelez Fernandez et al. 2020
a conditioning (day 1) b +2.7 aCSF 80 +3.2 60 +2.7 +2.7 40
% freezing 20 +3.2 +3.2 P70-90 0 H12345trials MLA+Infenprodil MLA+Indiplon
c extinction (day 2) d extinction (day 2) *** 80 80
60 60
40 40
% freezing 20 20 aCSF aCSF MLA+Ifenprodil MLA+Indiplon 0 0
PFC 02468101214 PFC 0 2 4 6 8 10 12 14 infusions trials infusions trials Figure 8_a7nAChR paper Miguelez Fernandez et al. 2020
a conditioning (day 1) b context test (day 2) 80 80 aCSF MLA 60 60 ***
40 40 **
% freezing 20 20 P70-90 0 P38-44 0 H12345 aCSF 1234 trials or MLA epochs