# An inhibitory hippocampal-thalamic pathway modulates remote memory retrieval

Gisella Vetere, Frances Xia, Adam I Ramsaran, Lina M Tran, Sheena A Josselyn, Paul W Frankland

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Gisella Vetere, Frances Xia, Adam I Ramsaran, Lina M Tran, Sheena A Josselyn, et al. An inhibitory hippocampal-thalamic pathway modulates remote memory retrieval. Nature Neuroscience, 2021, 24, pp.685 - 693. 10.1038/s41593-021-00819-3. hal-04053841

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1 **An inhibitory hippocampal-thalamic pathway modulates remote

2 memory retrieval

3 Gisella Vetere¹,²,*, Frances Xia¹,³,*, Adam I. Ramsaran¹,⁴, Lina M. Tran¹,³, Sheena A. Josselyn¹,³-

4,⁶ and Paul W. Frankland¹,³⁻⁵,⁷

6 * Co-first authors

7 ¹Program in Neurosciences & Mental Health, Hospital for Sick Children, 555 University Avenue,

9 Toronto, ON M5G 1X8, Canada

10 ²Team Cerebral Codes and Circuits Connectivity (C4), Plasticité du cerveau, ESPCI Paris,

11 CNRS, PSL University, 75005 Paris, France.

12 ³Department of Physiology, University of Toronto, Toronto, ON M5G 1X8, Canada

13 ⁴Department of Psychology, University of Toronto, Toronto, ON M5S 3G3, Canada

14 ⁵Institute of Medical Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada

15 ⁶Brain, Mind & Consciousness Program, Canadian Institute for Advanced Research, Toronto,

16 Ontario M5G 1M1, Canada

17 ⁷Child & Brain Development Program, Canadian Institute for Advanced Research, Toronto,

18 Ontario M5G 1M1, Canada

20

21

22

23

24

25

26
27 ABSTRACT

Memories are supported by distributed hippocampal-thalamic-cortical networks, but the brain

regions that contribute to network activity may vary with memory age. This process of

reorganization is referred to as systems consolidation, and previous studies have examined the

relationship between the activation of different hippocampal, thalamic, and cortical brain regions

and memory age at the time of recall. While the activation of some brain regions increases with

memory age, other regions become less active. Here we asked whether the active

disengagement of one such brain region, the anterodorsal thalamic nucleus (ADn), is necessary

for recall at remote time points, and in addition, which projection(s) mediate such inhibition.

Specifically, we identified a sparse inhibitory projection from CA3 to the ADn that becomes more

active during systems consolidation, such that it is necessary for contextual fear memory

retrieval at remote, but not recent, time-points post-learning.

39

40

41

42
43 INTRODUCTION

44
45 While memories for events may initially depend upon the hippocampus, with time these
46 memories become increasingly dependent upon the cortex for their expression¹⁻⁴. Post-learning
47 communication between the hippocampus and cortex, via the thalamus, is thought to drive this
48 time-dependent reorganization process called systems consolidation²,⁵⁻¹². While the
49 contributions of hippocampal and cortical regions have been studied in detail, much less is
50 known about how thalamic regions contribute to this memory reorganization process at different
51 post-encoding delays.

52
53 Using brainwide mapping of immediate early gene induction, we previously identified networks
54 of regions activated by recall of either recent or remote contextual fear memory¹¹,¹³. Thalamic
55 regions played a central role within these networks, with their retrieval-induced activation
56 generally correlated with activity in cortical and hippocampal regions at both retention delays.
57 However, one region, the anterodorsal thalamic nucleus (ADn), exhibited a distinct pattern.
58 Following recall of recent contextual fear memory, ADn activity was positively correlated with
59 activity in other regions, including cortical and hippocampal subregions (Supplementary Figure
60 1a). In contrast, at the remote time point, this pattern switched. Following recall of remote
61 contextual fear memory, ADn activity was negatively correlated with activity in other regions,
62 including cortical and hippocampal subregions (Supplementary Figure 1b). The ADn has
63 extensive connections with both the hippocampus and cortex¹⁴⁻¹⁶, and lesioning or inhibiting
64 ADn impairs performance in hippocampus-dependent memory tasks¹⁷⁻²². Furthermore, ADn
65 activity is tightly coupled with hippocampal oscillations that play important roles in memory
66 processing²³. In the network analysis¹¹,¹³, the inverse correlation between ADn and other brain
67 regions during remote memory recall raises an intriguing possibility. As other brain regions

68 become more actively engaged during remote memory recall, ADn activity is actively inhibited

69 by one or more brain regions, and such inhibition may be necessary for successful remote
70 memory recall.

71
72 To explore the role of the ADn, and its connections, at recent and remote time points here we
73 used contextual fear conditioning. We found that the ADn is necessary for the retrieval of recent,
74 but not remote, contextual fear memory. Consistent with this, retrieval-induced activity of ADn
75 decreases as a function of memory age. We then identified a novel long-range inhibitory
76 pathway from the CA3 subfield of the hippocampus to the ADn. This CA3-ADn inhibitory
77 pathway is recruited during memory consolidation, and becomes necessary for the retrieval of
78 remote fear memory. Previous studies have shown that consolidation of event memories
79 involves gradual recruitment of brain regions over time. Our current results suggest that active
80 disengagement of at least the ADn is additionally necessary for successful memory retrieval at
81 remote time points.

82
83 RESULTS

84 ADn activity is necessary for recall of recently acquired memory

85 We first assessed retrieval-induced activation of ADn as a function of memory age. To do this
86 we trained wild-type (WT) mice in contextual fear conditioning, and tested their memory at either
87 1 or 28 days post-training (Fig. 1a). Ninety minutes following testing, mice were perfused and
88 we analyzed the expression of the activity-regulated gene, *Fos*, in the ADn. c-Fos expression
89 was additionally assessed in 3 control groups of mice: (1) home cage mice; (2) mice trained
90 without a footshock and tested 1 or 28 days later, and (3) mice trained with an immediate shock
and tested 1 or 28 days later. For all mice that were tested 1 or 28 days later, c-Fos levels were
normalized to those levels in home cage controls (Fig. 1b-c). In comparison to control mice,
memory retrieval at 1 day post-training significantly increased the expression of c-Fos in the
ADn. In contrast, c-Fos levels were not increased in mice tested at the 28 day memory test
point, and were equivalent to those in control mice. Consistent with a previous observation in
rats¹⁹, these results suggest that the ADn is actively engaged during retrieval of a recently-, but
not remotely-acquired fear memory. At remote time-points, the ADn may either be not engaged
or, alternatively, actively disengaged or inhibited.

To test whether the activity of ADn is necessary for retrieval of a recent memory, we next
inhibited the ADn cells optogenetically during retrieval of a contextual fear memory. As ADn
contains excitatory neurons²⁴,²⁵ (Supplementary Fig. 2), we micro-infused an adeno-associated
virus (AAV) expressing the inhibitory opsin iC++ with a fluorescent reporter EYFP under the
CaMKII promoter (AAV-CaMKII-iC++-EYFP) bilaterally in the ADn in WT mice (Fig. 1d). The
iC++ opsin is a light-activated chloride channel that has improved chloride selectivity and
conductivity compared to previous generations of inhibitory opsins using chloride pumps²⁶, and
therefore allows efficient inhibition of targeted cells. Control mice received micro-infusion of a
virus expressing only the fluorescent reporter (AAV-GFP). Four weeks following surgery, after
allowing sufficient time for the virus to be expressed, mice were implanted with an optical fibre in
ADn, and trained in contextual fear conditioning. Contextual fear memory was tested either 1 or
28 days post-training. During the test, photo-stimulation was delivered during the last 3 minutes
of a 6 minute test session (Fig. 1e-f), allowing a within-animal comparison of the effect of
inhibiting ADn cells during memory recall.
113 Inhibition of ADn cells during 1 day test impaired memory retrieval. Mice infused with the iC++
114 virus froze less during the light-ON period than OFF, and also less than the control mice micro-
115 infused with the GFP virus (Fig. 1e). In contrast, inhibition of ADn cells during the 28 day test
116 had no effect on memory recall (Fig. 1f). In this case, mice infused with the iC++ virus froze at
117 similar levels during the light-ON and light-OFF periods, and at equivalent levels to those in
118 mice micro-infused with the control virus.

119 These results show that ADn is actively engaged and necessary for recent, but not remote,
120 memory retrieval. These results extend previous experiments in which pharmacological
121 inhibition of the anterior thalamus (including ADn) in rats impaired recall of recent, but not
122 remote, contextual fear memory¹⁹.

123 **ADn receives inhibitory projections from CA3.**

124 ADn may play no role in the remote recall of contextual fear conditioning. Alternatively,
125 successful remote memory recall may involve active inhibition of ADn by one or more brain
126 regions, and such inhibition may be necessary for successful remote memory retrieval. To
127 explore this latter possibility, we next examined afferent input to the ADn. Retrobeads were
128 unilaterally infused into the ADn, and retrogradely-labeled neurons were identified (Fig. 2a).
129 Consistent with previous findings¹⁵,¹⁶,²⁷⁻²⁹, we found labeled neurons in layer VI of anterior
130 cingulate cortex (ACC), retrosplenial cortex (RSP), pre-subiculum (PRE), indicating that these
131 regions send projections to the ADn (Supplementary Fig. 3a-b). In addition, we found
132 retrogradely labeled neurons in the ipsilateral stratum pyramidale of CA3 subfield of
133 hippocampus (Fig. 2a-b).
To our knowledge, this CA3 to ADn projection has not been previously described. We therefore next sought to verify this connection using complementary tracing methods. We first micro-infused the retrograde tracer cholera toxin subunit B (CTB; Fig. 2c-d, Supplementary Fig. 3c-d), unilaterally in the ADn. Consistent with our Retrobeads analysis, retrogradely-labeled neurons were found in the same regions, and specifically in the ipsilateral CA3 region of the hippocampus (Fig. 2d, Supplementary Fig. 3d). We next micro-infused the anterograde tracer Phytohaemagglutinin-L (PHA-L) unilaterally into the CA3 in a separate cohort of mice. We identified anterogradely labeled axons in the ipsilateral ADn, and also in ipsilateral CA1, and contralateral CA3 and CA1 (Fig. 2e-f, Supplementary Fig. 3e-f), as observed previously³⁰,³¹.

Because the ADn might be actively inhibited during remote memory recall, we next used three complementary approaches in order to assess whether CA3 sends inhibitory projections to the ADn. First, we micro-infused the retrograde tracer CTB unilaterally into the ADn, and performed immunohistochemical staining of CA3 sections using the inhibitory cell marker, GAD67 (Fig. 3a-b). We found sparse labeling of CTB+ cells in ipsilateral CA3 (2.5±0.7 CTB+ neurons / 10⁵ mm² in CA3), with the majority of the CTB-labeled CA3 cells co-localized with GAD67.

Second, we used a viral tracing approach to label only the CA3-ADn projections and performed immunohistochemistry to investigate whether the projections are inhibitory. Specifically, we micro-infused AAVrg-Cre-EGFP unilaterally into ADn and AAV-DIO-mCherry bilaterally into CA3 (Fig. 2g-h). Using this approach, Cre is retrogradely transported from axon terminals in ADn to neuronal cell bodies, where Cre allows recombination and expression of mCherry exclusively in the CA3-ADn pathway. Consistent with our other tracing methods, we observed mCherry-labeled somas and axons in CA3 and ADn, respectively, specifically labeling the CA3-ADn projection neurons. The majority of the mCherry-labeled cells were observed in CA3 ipsilateral
157 to the ADn infusion site -1.5 to -2.5 mm posterior to bregma, consistent with our other methods
158 (Supplementary Fig. 3g-h). We further co-stained CA3 and ADn sections with GAD67 and
159 VGAT, respectively, and found that many retrogradely-labeled (mCherry+) neurons in CA3 are
160 inhibitory (GAD67+), and many (mCherry+) axons in ADn are inhibitory (VGAT+) (Fig. 3e-g).

161 Third, we used an anterograde approach to examine inhibitory projections to the ADn.

162 Specifically, we micro-infused an AAV that expresses the fluorescent reporter (EYFP) in a Cre-
163 recombinase-dependent manner (AAV-DIO-EYFP) in mice expressing Cre recombinase only in
164 inhibitory (i.e., VGAT+) neurons (VGAT-Cre mice). We targeted cortical and hippocampal
165 regions sending projections to the ADn, as identified by the analyses above (Fig. 2a-f,
166 Supplementary Fig. 3a-d). These regions included CA3, retrosplenial cortex, pre-subiculum, and
167 anterior cingulate cortex (Fig. 3c-d, Supplementary Fig. 3i-n). Dense terminal projection labeling
168 in ADn was only observed following viral micro-infusions into CA3 (Fig. 3c-d). Together, these
169 converging lines of evidence indicate that CA3 sends long-range, inhibitory projections to the
170 ADn.

171 CA3 inhibitory cells projecting to the ADn become more active as memory ages

172 One possibility is that this CA3-ADn inhibitory projection is active during remote memory recall,
173 and the resulting suppression of ADn activity is necessary for successful memory retrieval. To
174 test this hypothesis, we analyzed the expression of c-Fos in these CA3 inhibitory cells that
175 project to the ADn during memory retrieval at 1 or 28 days post-training. We micro-infused a
176 retrograde AAV (AAV-Ef1a-DO_DIO-TdTdTdTdT_EGFP-WPRE-pA) in the ADn of VGAT-Cre
177 mice (Fig. 4a). This virus is retrogradely transported from the axon terminals to neuronal cell
178 bodies, where the double-floxed orientation (DO) allows the expression of the fluorescent tag,
179 TdTomato, only in the cells lacking Cre (Cre-Off). At the same time, the double-floxed inverted
180 open reading frame (DIO) allows the expression of another fluorescent tag, EGFP, only in
181 VGAT+ cells (Cre-On)$^{32}$. Therefore, using this approach, in CA3 we were able to simultaneously
182 visualize both excitatory (TdTomato+) and inhibitory (EGFP+) ADn-projection neurons.

183 Following micro-infusion in the ADn, mice were fear conditioned and then tested 1 or 28 days
184 later (Fig. 4a). Ninety minutes after the test, mice were perfused, and we analyzed the
185 expression of c-Fos in the excitatory (TdTomato+) and inhibitory (EGFP+) projection neurons in
186 the CA3. We found that the retrieval-induced c-Fos expression was elevated in inhibitory CA3
187 neurons at the 28 day time-point in comparison to the 1 day time-point (Fig. 4b-c). There was no
188 significant change in the activity of excitatory CA3 cells that project to the ADn across retrieval
189 delays (Fig. 4d). Nor was there any change in the overall CA3 c-Fos expression following recent
190 vs. remote contextual fear memory recall (Fig. 4e), most likely reflecting the fact that neurons
191 that project from CA3 to the ADn only represent a small fraction of all neurons in CA3. This
192 result supports the hypothesis that the activity of CA3 inhibitory cells projecting to the ADn
193 increases during remote memory recall.

194 The inhibitory pathway from CA3 to ADn is necessary for remote, but not recent, memory
195 retrieval.

196 Since CA3 inhibitory neurons projecting to ADn become more active 28 days after training in
197 comparison to 1 day, we hypothesized that CA3 may be inactivating the ADn during remote
198 memory recall. To investigate this, we micro-infused the ADn with an AAV that expresses the
199 fluorescent reporter (EYFP) with the inhibitory opsin iC++ in a Cre-recombinase-dependent
200 manner (DIO) (AAV-DIO-iC++-EYFP) in VGAT-Cre mice. A different group of mice were micro-
201 infused with a control virus that only contained the fluorescent tag (AAV-DIO-EYFP). We then
202 implanted an optical fiber above the ADn (Fig. 5a-b). We trained the mice in contextual fear
203 conditioning and then tested their memory 1 or 28 d later. During the memory test, the laser was
204 OFF during the first 3 minutes, then turned on during the subsequent 3 minutes to inactivate the
205 projection terminals of the CA3-ADn inhibitory cells.

206 At the 1-d delay, photo-stimulation did not alter freezing levels in either control or iC++ groups,
207 suggesting that the CA3-ADn inhibitory pathway is not necessary for memory recall at this time
208 point (Fig. 5c). At the 28-d delay, however, while both control and iC++-infused mice froze
209 equally during the OFF period, photo-stimulation reduced freezing in the iC++ group (Fig. 5d),
210 suggesting that selective silencing of this inhibitory CA3-ADn projection impairs recall of remote
211 contextual fear memory. We observed similar results in an independent replication of this
212 experiment (Supplementary Fig. 5a-c). Inhibition of the CA3-ADn inhibitory pathway does not
213 alter locomotor activity in either novel or familiar environments (tested either in the open field or
214 in the conditioning context; Supplementary Fig. 4a-f). These data indicate that the photo-
215 stimulation-induced reductions in freezing observed during the remote contextual memory test
216 were not confounded by non-specific locomotor effects.

217 We note that overall freezing levels were much lower in the 1 day test compared to the 28 day
218 test. Reduced freezing at the recent delay was additionally observed in the independent
219 replication of this experiment (Supplementary Fig. 5a-c). Because of reduced freezing levels in
220 the 1 day test, it remains possible that potential effects of manipulating this inhibitory projection
221 on recent memory are obscured by a floor effect. However, additional analyses suggest that
222 freezing in the VGAT-Cre mice in the recent test reflects successful memory retrieval and that
223 floor levels of freezing are much lower. First, freezing levels during the 1 day test were
224 significantly higher than those during the pre-shock period during training (Supplementary Fig.
225 5e-f). Second, when a separate cohort of VGAT-Cre mice were trained without shocks they
226 exhibited negligible freezing when tested 1 day later (Supplementary Fig. 5g), suggesting that
227 'floor' freezing is 0-1% in this strain. Third, behavioural (latent inhibition³³,³⁴, Supplementary Fig.
228 5h) and optogenetic (Fig. 5c-d, Supplementary Fig. 5b-e) interventions reduce freezing, even
229 when control levels are 20-40%. Nonetheless, future experiments should verify that selective
230 silencing of the inhibitory CA3-ADn projections impair remote memory in other mouse lines.

231 We also assessed how the activity of ADn changes when the CA3-ADn inhibitory projection was
232 silenced following recent vs. remote memory recall. Consistent with our previous analyses (Fig.
233 1b), retrieval-induced c-Fos expression was reduced at the remote, compared to recent, time-
234 point in EYFP-infused mice. Importantly, retrieval-induced c-Fos expression was elevated in
235 iC++-infused mice, only at the remote time point (Fig. 5e, Supplementary Fig. 5d). This result is
236 consistent with our hypothesis that the CA3-ADn inhibitory projection becomes more active over
237 time, such that it suppresses the activity of ADn during remote memory recall.

238 Together, our data show that the ADn becomes disengaged during memory consolidation. This
239 disengagement is a result of recruitment of a novel inhibitory pathway from CA3 to ADn.
240 Importantly, activation of this CA3-ADn inhibitory pathway is necessary for successful remote
241 fear memory recall.

## DISCUSSION

242
243
244
245 Event memories are supported by distributed hippocampal-thalamic-cortical networks¹¹, but the
246 specific brain regions that contribute to memory expression may vary with memory age. While
247 previous studies have examined the relationship between activation of brain regions and
248 memory age at the time of recall¹⁹,³⁵⁻³⁸, here we asked whether the active disengagement of one
249 such brain region, the anterodorsal thalamic nucleus (ADn), is necessary for recall, and in
250 addition, which projection(s) mediate such inhibition. Using a mouse contextual fear conditioning
251 paradigm, we find that retrieval-induced activity of ADn is reduced at remote (28 days)
252 compared to recent (1 day) time points following training. We identified a sparse, long-range
253 projection from inhibitory CA3 neurons to the ADn, and find that retrieval-induced activity of
254 these ADn projecting inhibitory neurons is increased from recent to remote memory recall. More
255 importantly, inhibiting the CA3-ADn inhibitory pathway impairs memory recall, only at the remote
256 time point.

257 As far as we know, this is the first time that this inhibitory anatomical projection has been
258 identified, likely due to its sparsity. Here, using seven different tracing methods (including single-
259 step, transneuronal, enzymatic, and viral anterograde and retrograde tracers), we provide
260 convergent evidence for an inhibitory CA3-ADn projection. Despite its sparsity, we additionally
261 show that this inhibitory CA3-ADn projection plays a critical role in the retrieval of remote
262 contextual fear memory. While most of the GABAergic cells in the brain target local neurons,
263 some examples of long-range inhibitory connections have also been previously reported. For
264 example, there are bidirectional long-range GABAergic projections between the hippocampus
265 (CA1, CA3, and DG) and entorhinal cortex³⁹, as well as between hippocampus (CA1 and CA3)
266 and medial septum⁴⁰,⁴¹. In addition, CA1 GABAergic cells also project to the subiculum and
267 retrosplenial cortex⁴². Functionally, these long-range inhibitory connections can modulate
268 memory function in a number of different ways, for example, by modulating theta oscillations in
269 the hippocampus that is critical for spatial memory³⁹, and facilitating interregional temporal
270
coordination of activity<sup>42,43</sup>. The CA3-ADn GABAergic connection that we identified here adds to
the functional complexity of these long-range inhibitory connections.

Our initial motivation to focus on the ADn was based on our previous study which examined c-Fos expression induced in 84 different brain regions by recall of recent and remote contextual fear in mice<sup>11</sup>. By examining how c-Fos expression co-varied across regions, this analysis identified collections of brain regions that were co-activated during recent and remote memory recall. One striking feature of these analyses was that whereas activity in the ADn was positively correlated with activity in other brain regions following recent memory recall, this pattern switched for remote memory recall, where activity in the ADn was negatively correlated with activity in other brain regions. Moreover, at the remote time point, the region with the strongest negative correlation with the ADn was CA3. It is interesting to note that at the recent time point, the correlation between CA3 and ADn was positive (r = 0.64, P = 0.085) in this earlier analysis, although not statistically significant<sup>11</sup>. This is consistent with our observation that the inhibitory CA3-ADn pathway is not active nor required for recent memory retrieval.

Our discovery of an inhibitory pathway from CA3 to the ADn may account for the differential role
of ADn in recent versus remote memory retrieval (Supplementary Fig. 1). The activity of this
projection is dynamically modulated by fear retrieval, with the CA3-ADn GABAergic projection
cells being most strongly activated by remote memory recall. This increase in retrieval-induced
activity coincides with a decrease in retrieval-induced ADn activation with memory age. More
importantly, we provide causal evidence that the inhibition of ADn activity via the CA3-ADn
pathway is necessary for remote memory recall. Consistent with previous studies, we observed
that CA3 is equivalently active at recent (day-old) and remote contextual fear memory recall
295 (month-old)$^{44}$. However, our results additionally suggest that there is time-dependent
296 recruitment of inhibitory CA3 neurons, and, in particular those that project to ADn
297 (Supplementary Fig. 6). Although we did not observe a statistically significant change in the
298 activation level of the excitatory CA3-ADn projections over time, our data do not exclude the
299 possibility that the excitatory projections also play a role in memory consolidation and retrieval.
300
301 Analysis of activity-dependent gene expression indicates that recall of recent and remote
302 contextual fear memory engages different networks, with remote memory in particular engaging
303 broad hippocampal-thalamic-cortical networks$^{11}$. The emerging importance of the CA3-ADn
304 inhibitory pathway in recall of contextual fear memory may reflect the switch from the recent to
305 the remote network state. One possible mechanism for this switch is via decrease in
306 feedforward inhibition from DG to CA3 over time as memory ages$^{45,46}$. Specifically, feedforward
307 inhibition from DG to CA3 is initially increased after fear learning, but is reduced at the remote
308 time point. Reduced inhibition of CA3 could lead to enhanced activity in specific subpopulations
309 of CA3 neurons, including the inhibitory CA3-ADn projection neurons that we studied here. Such
310 a contribution of CA3 to remote memory expression is inconsistent with the idea that the
311 hippocampus plays a time-limited role in expression of these types of memories$^{47}$. In fact, more
312 recent optogenetic$^{48,49}$ and imaging$^{44}$ studies suggest that the hippocampus contributes to
313 remote memory recall. These studies, together with our current data, are consistent with models
314 of systems consolidation that emphasize a prolonged role for the hippocampus in memory
315 retrieval, even at remote time-points$^{50}$.

316

317

318
319 **MATERIALS AND METHODS**

320 **Mice**

321 All procedures were approved by the Canadian Council for Animal Care (CCAC) and the Animal
322 Care Committees at the Hospital for Sick Children and the University of Toronto. Experiments
323 were conducted on 8-12 week old male and female F1 hybrid (C57BL/6NTac x 129S6/SvEvTac)
324 wild-type (WT) mice, or VGAT-Cre knock-in transgenic mice where Cre-recombinase was
325 targeted to the VGAT locus, without disrupting endogenous VGAT expression. The VGAT-Cre
326 mice were obtained from Jackson Lab. Heterozygous VGAT-Cre mice were maintained on a
327 C57BL/6NTac x 129S6/SvEvTac genetic background.

328
329 All mice were weaned at 21 days, and group housed with 2-5 mice per cage in a temperature-
330 controlled room with 12 h light/dark cycle (light on during the day). All experiments were
331 performed between 8 am and 12 pm. Mice were given *ad libitum* access to food and water. Mice
332 were randomly assigned to experimental groups. The experimenters were aware of the
333 experimental group assignment, as the same experimenters conducted the training and testing
334 of all mice, but were blinded during behavioural assessment and cell counting experiments.
335 Mice were excluded from analysis based on post-experimental histology: only mice with robust
336 expression of the viral vectors (as specified in each experiment) specifically in the targeted
337 region were included.

338
339 **Micro-infusion of viral vectors and tracers**
340
341 Various adeno-associated viral vectors (AAVs) and tracers were used, as specified in each
341 experiment. All viral vectors were infused bilaterally at 1.2 µl per side and 0.1 µl/min, unless
342 otherwise specified. Tracers were infused unilaterally at 0.5 µl volume and 0.1 µl/min
343 (Retrobeads), 0.1 µl volume and 0.01 µl/min (CTB), or 0.05 µl volume and 0.01 µl/min (PHA-L).

344 Specifically, in Figure 1, AAV8-CaMKII-iC++-EYFP virus was obtained from Stanford University
345 vector core, and micro-infused bilaterally in the ADn (-0.8 mm AP, ± 0.2 mm ML, -3 mm DV,
346 from bregma according to Paxinos and Franklin [2012]⁵¹) of WT mice to allow optogenetic
347 silencing of the ADn. The control group received ADn infusion of AAV(DJ)-CMV-GFP, where
348 GFP is expressed under the CMV promoter (made in house). Four weeks following surgery,
349 optical fibres were placed above ADn (-0.8 mm AP, ± 0 mm ML, -2.7 mm DV).

350 In Figure 2, Figure 3, and Supplementary Figure 3, the retrograde tracer Retrobeads (red) was
351 obtained from Lumafluor (Durham, NC) and micro-infused unilaterally in the ADn of WT mice to
352 retrogradely label regions that send monosynaptic projections to the ADn. To verify the results
353 obtained using retrobeads, in separate cohorts of mice, we micro-infused another retrograde
354 tracer cholera toxin subunit B (CTB, Thermo Fisher Scientific, Waltham, MA) into the ADn (Fig.
355 2, Supplementary Fig. 3). Mice were perfused 14 days following surgery inorder to visualize
356 projection neurons. To verify these results with an anterograde tracer, we micro-infused
357 Phytohaemagglutinin-L (PHA-L; Vector Laboratories, Burlingame, CA) into the CA3. Mice were
358 perfused 3 weeks following surgery inorder to visualize projection neurons. We additionally
359 verified the CA3-ADn projection by specifically labeling the projection using a dual-viral tracing
360 approach. We micro-infused 0.5 µl of pENN-AAVrg-hSyn-H-eGFP-Cre-WPRE-SV40 (#105540-
361 AAVrg, Addgene, Cambridge, MA) unilaterally into ADn and 0.6 µl of AAV(DJ)-hSyn-DIO-
362 mCherry bilaterally into CA3. Mice were perfused 4 weeks following surgery inorder to visualize
363 the CA3-ADn projection (mCherry+) neurons.
364 In Figure 4, AAVrg-EF1a-DO_DIO-TdTomato-EGFP was obtained from Addgene (Cambridge, MA), and micro-infused unilaterally in the ADn of VGAT-Cre mice to retrogradely label excitatory and inhibitory cells that project to the ADn. This construct allows Cre-dependent expression of EGFP, and expression of TdTomato in the absence of Cre³². The AAV is produced in a retrograde serotype to allow retrograde labeling or projection neurons to the site of injection.

369 In Figure 5, Supplementary Figure 4, and Supplementary Figure 5, AAV(DJ)-EF1a-DIO-iC++-EYFP was obtained from Stanford Neuroscience Gene Vector and Virus Core, and micro-infused bilaterally in CA3 (-2.2 mm AP, ± 2.7 mm ML, -2.4 mm DV, from bregma according to Paxinos and Franklin [2012]⁵¹) of VGAT-Cre mice. In this inhibitory opsin viral vector, the double-floxed inverted open reading frame of iC++ fused to EYFP can be expressed from the EF1a promoter after Cre-mediated recombination. Four weeks following surgery, optical fibres were placed above ADn to allow optogenetic silencing of CA3-ADn projections. The control group received infusion of AAV-hSyn-DIO-EGFP (Addgene) in CA3 of VGAT-Cre mice, where the double-floxed inverted open reading frame of the EGFP fluorescence tag can be expressed from the human synapsin (hSyn) promoter after Cre-mediated recombination.

379 The micro-infusion surgery protocol is similar to the previously described⁵². Specifically, mice were pretreated with atropine sulphate (0.1 mg/kg, intraperitoneal), then anesthetized with chloral hydrate (400 mg/kg, intraperitoneal). Mice were then placed on a stereotaxic frame, and holes were drilled in the skull at the targeted coordinates. Viral vector was micro-infused at 0.1 µl/min via glass pipettes connected to a Hamilton microsyringe with polyethylene tubing. After micro-infusion, the glass pipette was left in the brain for another 5 mins to allow sufficient time for the virus to diffuse. We have found that this infusion procedure produces high infection in the targeted region, without significant spread outside the region of interest⁵²,⁵³. For infusions of
387 CTB and PHA-L tracers we followed a similar procedure but performed injections using a
388 Nanoject III injector (Drummond Scientific, Broomall, PA) and left the pipette in the brain for 10-
389 15 minutes following infusions to minimize spread of tracers outside of regions of interest. Mice
390 were then treated with analgesic (ketoprofen, 5 mg/kg, subcutaneous) and 1 ml of 0.9% saline
391 (subcutaneous).

392 For the optogenetic experiments, mice received optical fibre implant 4 weeks following viral
393 infusion. Optical fibres were constructed in-house by attaching a 5 mm piece of 200-µm optical
394 fibre (with a 0.37 numerical-aperture, NA) to a 1.25-mm zirconia ferrule (final fibre extended 3
395 mm beyond ferrule). Fibres were attached with epoxy resin into ferrules, cut and polished.
396 Optical fibres were stabilized to the skull with screws and black dental cement to minimize light
397 leakage. For the 1 day groups, viral infusion was performed 5 weeks prior to training/testing. For
398 the 28 day groups, viral infusion was performed 1 week prior to contextual fear training, and 4
399 weeks prior to testing. Thus, for both 1 day and 28 day groups, the interval between viral
400 infusion and testing was 5 weeks.

401
402 **Behavioural Experiments**
403 Behavioural experiments for mice that did not undergo viral micro-infusion were carried out in 8
404 weeks old WT mice (Fig. 1a-b). Experiments for mice that were micro-infused with viral vectors
405 started one week following optical fibre implant (five weeks after micro-infusion).

406
407 *Contextual fear conditioning*
408 WT mice were trained in a standard contextual fear conditioning paradigm, as previously
409 described⁵⁴. Mice were first habituated to the conditioning chamber for 120 s, then given 3
shocks (0.5 mA each, 60 s apart), and remained in the chamber for another 60 s following the last shock. The amount of time mice spent freezing (% freezing, with minimum bout of 1 s) was monitored with overhead cameras, and calculated using automatic scoring software FreezeFrame (Actimetrics).

VGAT-Cre mice, when trained using the standard contextual fear conditioning protocol, froze less than the WT mice. Therefore, for the majority of experiments, VGAT-Cre mice were trained in an altered version of the contextual fear conditioning paradigm to ensure that they show successful fear memory retrieval (significantly higher freezing levels in comparison to pre-shock, Supplementary Fig. 5e-f). Specifically, VGAT-Cre mice were habituated to the conditioning chamber for 5 min one day prior to training. On the day of training, mice were habituated to the chamber for 120 s, then given 4 shocks (0.7 mA each, pseudo-random intervals), and remained in the context for another 30 s following the last shock (Supplementary Fig. 5a-d). For the independent replication, we extended the habituation period to 2 days (10 min, twice per day), as preliminary data showed that this extended habituation protocol slightly reduced variability in freezing levels. All other training and testing protocols were otherwise identical.

Either 1 or 28 days following training, mice were placed back into the training context for 5 mins, without shock. The amount of time mice spent freezing (% freezing) was monitored with overhead cameras, and manually scored by experiments blinded to the group assignment of mice. For the optogenetic experiments, the test session was 6 mins in duration, where the laser was OFF during the first 3 mins, then turned ON during the last 3 mins when continuous direct current blue laser photo-stimulation (473 nm, 10 mW power) was applied.
434 *c-Fos* analysis

435 To examine how the activity of ADn changes from recent (1 day) to remote (28 days) memory retrieval (Fig. 1a-b), we trained WT mice in contextual fear conditioning and tested them either 1 or 28 days later. Ninety minutes post-test, mice were perfused, and their brains used for c-Fos staining (see below). We analyzed c-Fos expression in the ADn in home cage control mice, immediate-shock controls (1 shock at 0.6mA upon exposure to fear conditioning chamber, tested at 1 or 28 days post-training), no-shock controls (5 min exploration in fear conditioning chamber, tested at 1 or 28 days post-training), and those that underwent contextual fear conditioning (tested at 1 or 28 days post-training). For quantification and visualizations, c-Fos levels in all groups were normalized to averaged c-Fos in home cage control mice.

444
445 To examine how the activity of the excitatory and inhibitory projection neurons from CA3 to ADn changes from recent to remote memory retrieval, VGAT-Cre mice were infused with the retrograde tracer AAVrg-EF1a-DO_DIO-TdTomato-EGFP. Five weeks following infusion, mice were trained in contextual fear conditioning paradigm, and tested 1 day later. For the 28 day group, mice were trained in contextual fear conditioning 1 week following viral infusion, and tested 4 weeks later. Ninety minutes post-test, all mice were perfused, and their brains used for c-Fos staining. We analyzed c-Fos expression in the CA3-ADn projection neurons (excitatory and inhibitory), as well as total c-Fos in CA3, in home cage control mice, and those that underwent recent or remote memory test (Fig. 4).

454
455 *Open field*

456
To control for the possibility that optogenetic manipulation of the CA3-ADn pathway alters locomotion in a novel environment, VGAT-Cre mice were micro-infused with AAV-DIO-iC++-EYFP or control virus (AAV-DIO-EYFP) in the CA3, then implanted with optical fibres in the ADn four weeks later. One week after optical fibre implant, mice were placed in the centre of an open square arena (45 cm x 45 cm x 20 cm height) and allowed to explore for 6 mins⁵⁵, where the laser was OFF during the first 3 mins, then turned ON during the last 3 mins (Supplementary Fig. 4a-b). To control for the possibility that optogenetic manipulation of the CA3-ADn pathway alters locomotion in a familiar environment, VGAT-Cre mice were micro-infused with AAV-DIO-iC++-EYFP or control virus (AAV-DIO-EYFP) in the CA3, then implanted with optical fibres in the ADn four weeks later. One week after optical fibre implant, mice were placed in the centre of an open square arena (45 cm x 45 cm x 20 cm height) and allowed to explore for 6 mins (open field pre-exposure)⁵⁵ on day 1. Twenty-four hours later, mice were placed back into the same open field arena and allowed to explore for 6 mins (open field test) where the laser was OFF during the first 3 mins, then turned ON during the last 3 mins (Supplementary Fig. 4c-e). We additionally assessed the effect of optogenetic inhibition of the CA3-ADn pathway on locomotion in a second familiar environment, the fear conditioning chamber (without shocks). VGAT-Cre mice were habituated to the fear conditioning chamber for 6 min without shocks, 1 day after open field test (fear chamber pre-exposure). Twenty-four hours later, mice were placed back into the same conditioning chamber to explore for 6 mins (fear test) where the laser was OFF during the first 3 mins, then turned ON during the last 3 mins (Supplementary Fig. 4c, f). The location of the mouse was tracked using an overhead camera. The total distance traveled was assessed using ImageJ and freezing levels were assessed using FreezeFrame.

## Immunohistochemistry
481 Immunofluorescence staining was conducted as previously described⁵⁶. Specifically, at the end of behaviour experiments, mice were transcardially perfused with 1x PBS followed by 4% paraformaldehyde. For the c-Fos experiment, mice were perfused 90 mins after behaviour test or training. Brains were fixed overnight at 4°C, and transferred to 30% sucrose solution for 48 hours. Brains were sectioned coronally using a cryostat (Leica CM1850), and 50 µm sections were obtained for the ADn or hippocampus.
482
483
484
485
486
487
488
489 For αCaMKII and GAD67 immunostaining, free-floating sections were blocked with PBS containing 2.5% bovine serum albumin and 0.3% Triton-X for 30 mins. Afterwards, sections were incubated in PBS containing mouse anti-αCaMKII primary antibody (1:200 dilution; Sigma Cat# C265 RRID:AB_258808) or mouse anti-GAD67 (1:500 dilution; EMD Millipore Cat# MAB5406 RRID: AB_2278725) primary antibody for 24 hours at 4 °C. Sections were washed with PBS (3 times), then incubated with PBS containing goat anti-mouse ALEXA Fluor 488 (1:500 dilution, Thermo Fisher Scientific Cat# A-11001 RRID:AB_2534069) secondary antibody for 24 hours at 4 °C. Sections were washed with PBS, mounted on gel-coated slides, and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories). Images were obtained using a confocal laser scanning microscope (LSM 710; Zeiss) with a 20X objective.
489
490
491
492
493
494
495
496
497
498
500 For c-Fos immunostaining, free-floating sections were blocked with PBS containing 2.5% bovine serum albumin and 0.3% Triton-X for 30 mins. Afterwards, sections were incubated in PBS containing rabbit polyclonal anti-c-Fos primary antibody (1:1000 dilution; Santa Cruz Biotechnology Cat# sc-52 RRID:AB_2106783) for 48 hours at 4°C. Sections were washed with PBS (3 times), then incubated with PBS containing goat anti-rabbit ALEXA Fluor 633 (for c-Fos, 1:500 dilution, Thermo Fisher Scientific Cat# A-21070 RRID:AB_2535731) secondary antibody
501
502
503
504
528 microscope (LSM 710; Zeiss) with 20X and 40X objectives. To visualize PHA-L anterogradely-
529 labeled cells, brain sections were co-stained with PHA-L antibody. Specifically, brain sections
530 were blocked as described above and incubated with rabbit anti-PHA (E+L) primary antibody
531 (1:1000 dilution, Vector Laboratories Cat# AS-2300-1 RRID: AB_2313686) for 24 hours at 4 °C.
532 Sections were washed with PBS (3 times), then incubated with PBS containing goat anti-rabbit
533 ALEXA Fluor 488 (1:500 dilution, Thermo Fisher Scientific Cat# A-11008 RRID:AB_143165)
534 secondary antibody for 24 hours at 4 °C. Sections were washed with PBS, mounted on gel-
535 coated slides, and coverslipped with Vectashield fluorescent mounting medium (Vector
536 Laboratories). Images were obtained using a confocal laser scanning microscope (LSM 710;
537 Zeiss) with 20X and 40X objectives.

538 To specifically visualize CA3-ADn projection neurons, brain sections containing CA3 from mice
539 infused with AAVrg-Cre-EGFP and AAV(DJ)-hSyn-DIO-mCherry (unilaterally in ADn and
540 bilaterally in CA3, respectively) were blocked as described above and then incubated with rabbit
541 anti-RFP (for mCherry, 1:1000 dilution, Rockland Cat# 600-401-379 RRID: AB_2209751) and
542 mouse anti-GAD67 (1:500 dilution; EMD Millipore Cat# MAB5406 RRID: AB_2278725) for 48
543 hours at 4 °C. Sections were washed with PBS (3 times), then incubated with PBS containing
544 goat anti-rabbit ALEXA Fluor 568 (for mCherry, 1:500 dilution, Thermo Fisher Scientific Cat# A-
545 11011 RRID:AB_143157) and goat anti-mouse ALEXA Fluor 647 (for GAD67, 1:500 dilution,
546 Thermo Fisher Scientific Cat# A-21235 RRID: AB_2535804) secondary antibodies for 24 hours
547 at 4 °C. To visualize the projection terminals of the same neurons, sections containing the ADn
548 from the same brains were blocked and then incubated with mouse anti-RFP (1:1000 dilution,
549 Rockland Cat# 200-301-379 RRID: AB_2611063) and rabbit anti-VGAT (1:500 dilution, EMD
550 Millipore Cat# AB2257 RRID: AB_1587623) primary antibodies for 48 hours at 4 °C. Sections
551 were washed with PBS (3 times), then incubated with PBS containing goat anti-mouse ALEXA

552 Fluor 568 (for mCherry, 1:500 dilution, Thermo Fisher Scientific Cat# A-11004

553 RRID:AB_2534072) and goat anti-rabbit ALEXA Fluor 633 (for VGAT, 1:500 dilution, Thermo

554 Fisher Scientific Cat# A-21070 RRID:AB_2535731) secondary antibodies for 24 hours at 4 °C.

555 Sections were washed with PBS, mounted on gel-coated slides, and coverslipped with

556 Vectashield fluorescent mounting medium (Vector Laboratories). Images were obtained using a

557 confocal laser scanning microscope (LSM 710; Zeiss) with 20X, 40X, and 100X objectives.

558

559 **Data availability**

560

561 The data generated during and/or analyzed during the current study are available from the

562 corresponding author on reasonable request.

563

564 **Statistical analysis**

565 No statistical tests were used to pre-determine sample size, but the sample sizes used are

566 similar to those generally used within the field. Data were analyzed using parametric two-way

567 repeated measures ANOVA, or two-sample Student's unpaired t-test. For comparisons to a

568 hypothetical mean of 1, one-sample t-test was used. Where appropriate, ANOVA was followed

569 by *post hoc* pairwise comparisons with Bonferroni correction. If data were significantly non-

570 normal (with α = 0.05), Kruskal-Wallis test or Mann-Whitney test (between-group comparisons)

571 and Wilcoxon signed-rank test (within-group comparisons) were used accordingly. All tests were

572 two-sided. Statistical analyses were performed using Graphpad Prism V8.

573

574 **ACKNOWLEDGMENTS**
This work was supported by Canadian Institutes of Health Research (CIHR) grants to PWF (FDN143227) and SAJ (MOP74650). FX was supported by fellowships from NSERC and Biological Sleep and Rhythms (CIHR) scholarships. GV was supported by the Biological Sleep and Rhythms (CIHR) fellowship. AIR was supported by fellowships from NSERC and NIMH (1 F31 MH120920-01). LMT was supported by fellowships from The Hospital for Sick Children RestraComp and NSERC scholarships. PWF and SAJ are senior fellows in the Child Brain & Development Program and the Brain, Mind & Consciousness programs, respectively, at the Canadian Institute for Advanced Research (CIFAR).

AUTHOR CONTRIBUTIONS
GV, FX, AIR, LMT, SAJ and PWF designed the experiments. GV, FX, AIR and LMT conducted all of the experiments and data analysis. PWF, FX, GV, AIR and LMT wrote the paper.

COMPETING INTERESTS
The authors declare no competing financial interests.

FIGURES AND FIGURE LEGENDS
<figure><img src="image_1.png" /><figcaption>Figure 1</figcaption></figure>
**Fig. 1 | ADn activity is necessary for recall of recently acquired contextual fear memory.**

**a**, Mice were trained in contextual fear conditioning and tested 1 or 28 days later. Ninety minutes after the test, mice were sacrificed and their brains processed for c-Fos expression in ADn. **b**, Expression of c-Fos in ADn (normalized to mean expression level in home cage control mice n = 16) in no shock (1 day n = 8; 28 days n = 7; Mann-Whitney test, *P* = 0.62; 1 day versus one one-sample Wilcoxon signed rank test, *P* = 0.55; 28 day versus one one-sample Wilcoxon signed rank test, *P* > 0.99) or immediate shock (Imm shock; 1 day n = 8; 28 days n = 8; Mann-Whitney test, *P* = 0.62; 1 day versus one one-sample Wilcoxon signed rank test, *P* > 0.99; 28 day versus one one-sample Wilcoxon signed rank test, *P* = 0.38) control groups were similar at 1 and 28 days, and not different from home cage controls. However, expression of c-Fos in ADn in mice trained in contextual fear conditioning (CFC) was higher in mice tested at 1 day, in comparison to home cage controls, but not in mice tested 28 days post-training (1 day n = 10; 28 days n = 10; Mann-Whitney test, *P* = 0.0003; 1 day versus one one-sample Wilcoxon signed rank test, *P* = 0.002; 28 day versus one one-sample Wilcoxon signed rank test, *P* = 0.70). **c**, Representative images showing expression of c-Fos in ADn in mice following no shock, immediate shock, or contextual fear conditioning and testing at 1 and 28 day time points (scale bar: 100 µm). **d**, Wild-type (WT) mice were micro-infused with AAV-iC++-EYFP or the control virus AAV-GFP in the ADn, and optical fibres were implanted above the ADn. During fear memory testing at 1 or 28 days post-training, ADn was optogenetically inactivated. **e**, At 1 day test, iC++- and GFP-infused mice froze equally during light-OFF epoch (3 minutes duration), but iC++-infused mice showed reduced freezing in comparison to GFP-infused mice during light-ON epoch (3 minute duration) (iC++ n = 6; GFP n = 6; Mann-Whitney test iC++ versus GFP, *P* = 0.004; OFF: Wilcoxon signed rank test iC++ versus GFP, *P* = 0.18; ON:
618 Wilcoxon signed rank test iC++ versus GFP, *P* = 0.03). *f*, At 28 day test, iC++- and GFP-infused

619 mice froze equally during light-OFF and light-ON epochs (iC++ *n* = 5; GFP *n* = 7; Mann-

620 Whitney test iC++ versus GFP, *P* = 0.47; OFF: Wilcoxon signed rank test iC++ versus GFP, *P* =

621 0.43; ON: Wilcoxon signed rank test iC++ versus GFP, *P* = 0.88). Data are individual mouse, or

622 mean ± s.e.m. (* *P* < 0.05, *** *P* < 0.001).

623
<figure><img src="image_2.png" /><figcaption>Figure 2</figcaption></figure>
**Fig. 2 | Identification of anatomical projections to the ADn.** WT mice were micro-infused with retrograde tracers **a-b**, Retrobeads ($n = 3$) or **c-d**, cholera toxin subunit B (CTB, $n = 3$) in the ADn and retrograde labeled neurons in CA3 were visualized. **e-f**, For anterograde labeling, WT mice were micro-infused with anterograde tracer Phytohaemagglutinin-L (PHA-L, $n = 4$) in the CA3 and anterograde labeling was visualized in ADn. **g-h**, To visualize specifically CA3-ADn projections, WT mice were micro-infused with AAV-DIO-mCherry bilaterally in CA3 and AAVrg-Cre-EGFP unilaterally in ADn. CA3-ADn (mCherry+) projections were visualized in ADn (blue scale bars: 100 µm, white scale bars: 50 µm).

634
<figure><img src="image_3.png" /><figcaption>Figure 3</figcaption></figure>
**Fig. 3** | Identification of inhibitory projections to the ADn. a-b, WT mice were micro-infused with cholera toxin subunit B (CTB, *n* = 3) in the ADn and retrograde labeled neurons in CA3 were visualized. Sections were co-stained with the inhibitory cell marker GAD67. Numerous CTB+ and GAD67+ co-localized cells were visualized in CA3 (white arrows). c-d, To identify inhibitory projections to the ADn, VGAT-Cre mice were micro-infused with AAV-DIO-EYFP in CA3 (*n* = 3). Strong terminal projections were visualized in the ADn. e-g, To visualize specifically CA3-ADn projections and examine whether they are inhibitory, WT mice were micro-infused with AAV-DIO-mCherry bilaterally in CA3 and AAVrg-Cre-EGFP unilaterally in ADn, and sections were co-stained with GAD67 (CA3) or VGAT (ADn) (*n* = 4). Numerous inhibitory CA3-ADn projection neurons in CA3 (mCherry+ and GAD67+, white arrows) and projections in ADn (mCherry+ and VGAT+, white arrows) were visualized (blue scale bars: 100 µm, white scale bars: 50 µm).
<figure><img src="image_4.png" /><figcaption>Figure 4</figcaption></figure>
**Fig. 4** | **CA3 inhibitory cells projecting to the ADn become more active at remote time point post-training.** a, VGAT-Cre mice were micro-infused AAV-Ef1a-DO_DIO-TdTomato_EGFP in ADn to retrogradely label inhibitory (EGFP+) and excitatory (TdTomato+) cells that project to the ADn. After viral infusion, mice were then trained in contextual fear conditioning and tested 1 or 28 days post-training. Ninety minutes after the test, mice were sacrificed and their brains processed to analyze c-Fos expression in CA3. b, Representative images showing viral infusion site in the ADn, retrogradely labelled inhibitory cells in CA3, and expression of c-Fos in both regions, at 1 and 28 day time points (scale bar: 100 µm). c, Mice tested at 28 days post-training showed higher co-localization of EYFP+ and c-Fos+ cells in CA3, in comparison to mice tested at 1 day post-training (co-localization of EYFP+ and c-Fos+ cells, normalized to mean co-localization level in home cage control mice (n = 4), adjusted for multiple-comparisons with Bonferroni-Dunn method; 1 day n = 6; 28 day n = 7; t-tests t₁₁ = 2.98, *P* = 0.038). d, Mice tested at 1 or 28 days post-training showed similar level of co-localization of TdTomato+ and c-Fos+ cells in CA3 (co-localization of EYFP+ and c-Fos+ cells, normalized to mean co-localization level in home cage control mice (n = 3), adjusted for multiple-comparisons with Bonferroni-Dunn method; 1 day n = 6; 28 day n = 9; t-tests t₁₃ = 1.62, *P* = 0.39). e, Mice tested at 1 or 28 days post-training showed similar level of total c-Fos+ cells in CA3 (total c-Fos+ cells normalized to mean c-Fos levels in home cage control mice (n = 7), adjusted for multiple-comparisons with Bonferroni-Dunn method; 1 day n = 9; 28 day n = 8; t-tests t₁₅ = 1.77, *P* = 0.29). Data are or mean ± s.e.m. (n.s.: not significant; \* *P* < 0.05).
<figure><img src="image_5.png" /><figcaption>Figure 5</figcaption></figure>
**Fig. 5** | CA3 inhibitory cells projecting to the ADn are necessary for the recall of remote memory. **a**, VGAT-Cre mice were micro-infused with AAV-DIO-iC++-EYFP or AAV-DIO-EYFP virus in CA3 and optical fibres were implanted in the ADn, to optogenetically inhibit the CA3-ADn projection during fear memory test, at 1 or 28 days post-training. Ninety minutes after the test, mice were sacrificed and their brains processed to analyze c-Fos expression in ADn. **b**, Representative images showing viral infection in CA3 and projections in ADn (scale bar = 100 µm). **c**, At 1 day test, iC++- and EYFP-infused mice froze equally during light-OFF and light-ON epochs (iC++ *n* = 14; EYFP *n* = 11; two-way repeated measures ANOVA iC++ versus EYFP x light-OFF versus light-ON; interaction, *F*<sub>1,46</sub> = 0.13, *P* = 0.72; light-OFF versus light-ON, *F*<sub>1,46</sub> = 5.71, *P* = 0.02; iC++ versus EYFP, *F*<sub>1,46</sub> = 1.35, *P* = 0.25). **d**, At 28 day test, iC++- and EYFP-infused mice froze equally during light-OFF epoch (3 minutes duration), but iC++-infused mice showed reduced freezing in comparison to EYFP-infused mice during light-ON epoch (3 minute duration) (iC++ *n* = 18; EYFP *n* = 17; Mann-Whitney test iC++ versus EYFP, *P* = 0.03; OFF: Wilcoxon signed rank test iC++ versus EYFP, *P* = 0.68; ON: Wilcoxon signed rank test iC++ versus EYFP, *P* = 0.014). **e**, At 1 day, iC++-infused mice showed equivalent level of c-Fos expression in the ADn (normalized to mean expression level in home cage control mice (*n* = 16)), in comparison to EYFP-infused control mice. But at 28 day, iC++-infused mice showed elevated level of c-Fos expression in the ADn, in comparison to controls. (1 day: iC++ *n* = 6; EYFP *n* = 5; 28 day: iC++ *n* = 9; EYFP *n* = 9; two-way ANOVA iC++ versus EYFP x 1 day versus 28 day; interaction, *F*<sub>1,25</sub> = 4.77, *P* = 0.039; 1 day versus 28 day, *F*<sub>1,25</sub> = 3.27, *P* = 0.083; iC++ versus EYFP, *F*<sub>1,25</sub> = 12.62, *P* = 0.0015; *post hoc* Bonferroni's test, 1 day iC++ versus EYFP *P* = 0.79, 28 day iC++ versus EYFP *P* = 0.0002). Data are individual mouse, or mean ± s.e.m. (* *P* < 0.05, *** *P* < 0.001).

698
<figure><img src="image_6.png" /><figcaption>Supplementary Figure 1</figcaption></figure>

<figure><img src="image_7.png" /></figure>
700
701 **Supplementary Figure 1 | Analysis of co-activation of ADn with 83 other brain regions following recent vs. remote fear memory retrieval.** a, Following recent fear memory recall, 702 ADn activity was positively correlated with activity in the majority of the other 83 brain regions. 703 (bars: correlation between ADn and a given region; green line and shaded region: mean 704 correlation between other 83 non-ADn brain regions and a given region ± s.e.m.). b, At the 705 remote time point, ADn activity became strongly inversely correlated with activity in the majority 706 (78/83) of other 83 brain regions. (bars: correlation between ADn and a given region; blue line 707 and shaded region: mean correlation between other 83 brain regions combined and a given 708 region ± s.e.m.). Data were obtained from Wheeler et al., 2013¹¹. 709
710
<figure><img src="image_8.png" /><figcaption>Supplementary Figure 2</figcaption></figure>
712

713

**Supplementary Figure 2 | Characterization of ADn cell type.** Representative images

714

showing immunohistochemical staining in ADn of WT home cage mice with **a**, inhibitory cell

715

marker GAD67, or **b**, excitatory cell marker aCamKII (scale bar: 100 µm).

716
<figure><img src="image_9.png" /><figcaption>Supplementary Figure 3</figcaption></figure>
**Supplementary Figure 3** | Identification of anatomical projections to the ADn. **a**, WT mice were micro-infused with Retrobeads in the ADn, **b**, and neurons in anterior cingulate cortex (ACC), retrosplenial cortex (RSP), and pre-subiculum (PRE) were retrogradely labeled (n = 3). **b**, WT mice were micro-infused with the retrograde tracer cholera toxin subunit B (CTB) in the ADn, and **d**, neurons in retrosplenial cortex (RSP), and pre-subiculum (PRE) were retrogradely labeled (n = 3). Additional examples of ADn infusion site and retrogradely labeled CA3 are also shown. **e**, WT mice were micro-infused with the anterograde tracer PHA-L in the CA3, and **f**, in addition to ipsilateral ADn (Fig. 2f), ipsilateral CA1, contralateral CA1 and CA3 were anterogradely labeled, while much less labeling was observed in contralateral ADn (n = 4). An additional example of anterograde labeling in ADn is shown. **g-h**, To visualize specifically CA3-ADn projections, WT mice were micro-infused with AAV-DIO-mCherry bilaterally in CA3 and AAVrg-Cre-EGFP unilaterally in ADn. Majority of the CA3-ADn (mCherry+) cells were visualized in CA3 ipsilateral to the site of ADn infusion. To see whether anatomical projections from other regions to CA3 are inhibitory, different VGAT-Cre mice were micro-infused with AAV-DIO-EYFP in **i-j**, retrosplenial cortex (RSP), **k-l**, anterior cingulate cortex (ACC), or **m-n**, pre-subiculum (PRE) (n = 3 for each region). Unlike the CA3 (Fig. 3c-d), none of these regions showed terminal projections in the ADn (blue scale bars: 100 µm, white scale bars: 50 µm).

737
<figure><img src="image_10.png" /><figcaption>Supplementary Figure 4</figcaption></figure>
**Supplementary Figure 4 | Inhibition of CA3-ADn inhibitory pathway does not alter locomotory activity in open field test.** a, VGAT-Cre mice were micro-infused with AAV-DIO-iC++-EYFP or AAV-DIO-EYFP virus in CA3 and optical fibres were implanted in the ADn, to optogenetically inhibit the CA3-ADn projection during test in a novel open field. b, iC++- and EYFP-infused mice showed similar levels of distance travelled during the light-OFF and light-ON epochs (3 minute duration) (iC++ *n* = 4; EYFP *n* = 4; two-way repeated measures ANOVA iC++ versus EYFP x light-OFF versus light-ON; interaction, *F*<sub>1,6</sub> = 0.92, *P* = 0.38; light-OFF versus light-ON, *F*<sub>1,6</sub> = 2.31, *P* = 0.179; iC++ versus EYFP, *F*<sub>1,6</sub> = 0.002, *P* = 0.97). c, VGAT-Cre mice were micro-infused with AAV-DIO-iC++-EYFP or AAV-DIO-EYFP virus in CA3 and optical fibres were implanted in the ADn. On day 1, mice were pre-exposed to open field chamber without light (6 min), then on day 2, the CA3-ADn projection was optogenetically inhibited by turning on the laser during the last 3 min of the open field test, to assess the effect of inhibition on locomotory activity in a familiar environment. To assess the effect of inhibition on locomotory activity in a second familiar environment, 1 day after open field test, mice were pre-exposed to the fear conditioning chamber without shocks and without light (6 min), and then 24 hours later, the CA3-ADn projection was optogenetically inhibited by turning on the laser during the last 3 min of the fear test. d, iC++- and EYFP-infused mice showed similar levels of distance travelled during the first and last 3 minutes of open field pre-exposure on day 1 without light (iC++ *n* = 6; EYFP *n* = 6; two-way repeated measures ANOVA iC++ versus EYFP x first-3-min versus last-3-min; interaction, *F*<sub>1,10</sub> = 2.05, *P* = 0.18; first-3-min versus last-3-min, *F*<sub>1,10</sub> = 48.40, *P* < 0.0001; iC++ versus EYFP, *F*<sub>1,10</sub> = 1.08, *P* = 0.32). e, iC++- and EYFP-infused mice showed similar levels of distance travelled during light-OFF and light-ON periods of open field test on day 2 (iC++ *n* = 6; EYFP *n* = 6; two-way repeated measures ANOVA iC++ versus EYFP x light-OFF versus light-ON; interaction, *F*<sub>1,10</sub> = 0.54, *P* = 0.48; light-OFF versus light-ON, *F*<sub>1,10</sub> = 0.15, *P* =
763 0.70; iC++ versus EYFP, $F_{1,10} = 0.11, P = 0.74$). f, iC++- and EYFP-infused mice showed similar

764 levels of freezing during light-OFF and light-ON periods of fear test 1 day after fear chamber

765 pre-exposure without shocks (iC++ *n* = 6; EYFP *n* = 6; two-way repeated measures ANOVA

766 iC++ versus EYFP x light-OFF versus light-ON; interaction, $F_{1,10} = 0.00, P = 0.77$; light-OFF

767 versus light-ON, $F_{1,10} = 3.40, P = 0.09$; iC++ versus EYFP, $F_{1,10} = 0.00, P = 0.76$). Data are

768 individual mouse, or mean ± s.e.m.

769
<figure><img src="image_11.png" /><figcaption>Supplementary Figure 5</figcaption></figure>
**Supplementary Figure 5 |** Independent replication experiment showing that CA3 inhibitory cells projecting to the ADn are necessary for the recall of remote memory. **a,** VGAT-Cre mice were micro-infused with AAV-DIO-iC++-EYFP or AAV-DIO-EYFP virus in CA3 and optical fibres were implanted in the ADn, to optogenetically inhibit the CA3-ADn projection during fear memory test, at 1 or 28 days post-training. **b,** At 1 day test, iC++- and EYFP-infused mice froze equally during light-OFF and light-ON epochs (iC++ *n* = 9; EYFP *n* = 8; Mann-Whitney test iC++ versus EYFP, *P* = 0.40; OFF: Wilcoxon signed rank test iC++ versus EYFP, *P* = 0.82; ON: Wilcoxon signed rank test iC++ versus EYFP, *P* = 0.28). **c,** At 28 day test, iC++- and EYFP-infused mice froze equally during light-OFF epoch (3 minutes duration), but iC++-infused mice showed reduced freezing in comparison to EYFP-infused mice during light-ON epoch (3 minute duration) (iC++ *n* = 12; EYFP *n* = 10; two-way repeated measures ANOVA iC++ versus EYFP x light-OFF versus light-ON; interaction, *F*<sub>1,20</sub> = 5.35, *P* = 0.031; light-OFF versus light-ON, *F*<sub>1,20</sub> = 0.43, *P* = 0.52; iC++ versus EYFP, *F*<sub>1,20</sub> = 3.45, *P* = 0.078; *post hoc* Bonferroni's test, light-OFF iC++ versus EYFP *P* > 0.99, light-ON iC++ versus EYFP *P* = 0.015). **d,** At 1 day, iC++-infused mice showed equivalent level of c-Fos expression in the ADn (normalized to mean expression level in home cage control mice (*n* = 16)), in comparison to EYFP-infused control mice. But at 28 day, iC++-infused mice showed elevated level of c-Fos expression in the ADn, in comparison to controls. (1 day: iC++ *n* = 7; EYFP *n* = 12; 28 day: iC++ *n* = 5; EYFP *n* = 7; two-way ANOVA iC++ versus EYFP x 1 day versus 28 day; interaction, *F*<sub>1,27</sub> = 18.39, *P* = 0.0002; 1 day versus 28 day, *F*<sub>1,27</sub> = 2.26, *P* = 0.14; iC++ versus EYFP, *F*<sub>1,27</sub> = 23.34, *P* < 0.0001; *post hoc* Bonferroni's test, 1 day iC++ versus EYFP *P* > 0.99, 28 day iC++ versus EYFP *P* < 0.0001). **e,** Both iC++ and EYFP groups froze significantly more during test (first 3 minutes) at 1 day than pre-shock period during training (iC++ *n* = 14; EYFP *n*
795 = 11; iC++: Wilcoxon matched-pair signed rank test pre-shock versus 1 day test, *P* = 0.0001;
796 EYFP: Wilcoxon matched-pair signed rank test pre-shock versus 1 day test, *P* = 0.001). f, Both
797 iC++ and EYFP groups froze significantly higher during test (last 3 minutes) at 28 day than pre-
798 shock period during training (iC++ *n* = 18; EYFP *n* = 17; iC++: Wilcoxon matched-pair signed
799 rank test pre-shock versus 28 day test, *P* < 0.0001; EYFP: Wilcoxon matched-pair signed rank
test pre-shock versus 28 day test, *P* < 0.0001). g. VGAT-Cre mice trained without shocks and
800 tested the following day showed baseline (floor) level freezing (*n* = 10; paired *t*-tests *t*<sub>9</sub> = 1.56, *P*
801 = 0.15). h. VGAT-Cre mice were habituated to the training context for 5 days (twice a day, 10
802 min each) before contextual fear conditioning. Mice showed significantly lower levels of freezing
803 following this latent inhibition protocol, in comparison to the 1 day habituation protocol (1 day
804 habituation (1D Hab): *n* = 7; 5 day habituation (5D Hab): *n* = 10; *t*-tests *t*<sub>15</sub> = 2.38, *P* = 0.03).
805 Data are individual mouse, or mean ± s.e.m. (* *P* < 0.05, *** *P* < 0.001, **** *P* < 0.0001).
806
807
<figure><img src="image_12.png" /><figcaption>Supplementary Figure 6</figcaption></figure>
809
810 Supplementary Figure 6 | CA3-ADn pathway is recruited from recent to remote fear
811 memory recall. a, ADn is activated and necessary for recent memory retrieval. At this time, the
812 inhibitory cells that project from CA3 to the ADn are relatively inactive. b, At the remote memory
813 retrieval, inhibitory cells that project from CA3 to the ADn are actively engaged to suppress the
814 activity in ADn. The suppression of ADn activity is necessary for remote memory retrieval.

815
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    <td></td>
  </tr>
  <tr>
    <td>956</td>
    <td>51</td>
    <td>Paxinos, G. & Franklin, K. B. J. The mouse brain in stereotaxic coordinates. (Academic Press, 2012).</td>
  </tr>
  <tr>
    <td>957</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>958</td>
    <td>52</td>
    <td>Richards, B. A. et al. Patterns across multiple memories are identified over time. Nat Neurosci 17, 981-986, doi:10.1038/nn.3736 (2014).</td>
  </tr>
  <tr>
    <td>959</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>960</td>
    <td>53</td>
    <td>Rashid, A. J. et al. Competition between engrams influences fear memory formation and recall. Science 353, 383-387, doi:10.1126/science.aaf0594 (2016).</td>
  </tr>
  <tr>
    <td>961</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>962</td>
    <td>54</td>
    <td>Wang, S. H., Teixeira, C. M., Wheeler, A. L. & Frankland, P. W. The precision of remote context memories does not require the hippocampus. Nat Neurosci 12, 253-255, doi:10.1038/nn.2263 (2009).</td>
  </tr>
  <tr>
    <td>963</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>964</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>965</td>
    <td>55</td>
    <td>Arruda-Carvalho, M. et al. Conditional deletion of alpha-CaMKII impairs integration of adult-generated granule cells into dentate gyrus circuits and hippocampus-dependent learning. J Neurosci 34, 11919-11928, doi:10.1523/JNEUROSCI.0652-14.2014 (2014).</td>
  </tr>
  <tr>
    <td>966</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>967</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>968</td>
    <td>56</td>
    <td>Restivo, L., Niibori, Y., Mercaldo, V., Josselyn, S. A. & Frankland, P. W. Development of Adult-Generated Cell Connectivity with Excitatory and Inhibitory Cell Populations in the Hippocampus. J Neurosci 35, 10600-10612, doi:10.1523/JNEUROSCI.3238-14.2015 (2015).</td>
  </tr>
  <tr>
    <td>969</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>970</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>971</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>972</td>
    <td></td>
    <td></td>
  </tr>
</table>