The paraventricular thalamus controls a central amygdala fear circuit

Appropriate responses to an imminent threat brace us for adversities. The ability to sense and predict threatening or stressful events is essential for such adaptive behavior. In the mammalian brain, one putative stress sensor is the paraventricular nucleus of the thalamus (PVT), an area that is readily activated by both physical and psychological stressors1-3. However, the role of PVT in the establishment of adaptive behavioral responses remains unclear. Here we show in mice that PVT regulates fear processing in the lateral division of the central amygdala (CeL), a structure that orchestrates fear learning and expression4,5. Selective inactivation of CeL-projecting PVT neurons prevented fear conditioning, an effect that can be accounted for by an impairment in fear conditioning-induced synaptic potentiation onto somatostatin-expressing (SOM+) CeL neurons, which has previously been shown to store fear memory6. Consistently, we found that PVT neurons preferentially innervate SOM+ neurons in the CeL, and stimulation of PVT afferents facilitated SOM+ neuron activity and promoted intra-CeL inhibition, two processes that are critical for fear learning and expression5,6. Notably, PVT modulation of SOM+ CeL neurons was mediated by activation of the brain-derived neurotrophic factor (BDNF) receptor tropomysin-related kinase B (TrkB). As a result, selective deletion of either Bdnf in PVT or Trkb in SOM+ CeL neurons impaired fear conditioning, while infusion of BDNF into CeL enhanced fear learning and elicited unconditioned fear responses. Our results demonstrate that the PVT–CeL pathway constitutes a novel circuit essential for both the establishment of fear memory and the expression of fear responses, and uncover mechanisms linking stress detection in PVT with the emergence of adaptive behavior.

conditioning-induced synaptic potentiation onto somatostatin-expressing (SOM + ) CeL neurons, which has previously been shown to store fear memory 6 . Consistently, we found that PVT neurons preferentially innervate SOM + neurons in the CeL, and stimulation of PVT afferents facilitated SOM + neuron activity and promoted intra-CeL inhibition, two processes that are critical for fear learning and expression 5,6 . Notably, PVT modulation of SOM + CeL neurons was mediated by activation of the brain-derived neurotrophic factor (BDNF) receptor tropomysin-related kinase B (TrkB). As a result, selective deletion of either Bdnf in PVT or Trkb in SOM + CeL neurons impaired fear conditioning, while infusion of BDNF into CeL enhanced fear learning and elicited unconditioned fear responses. Our results demonstrate that the PVT-CeL pathway constitutes a novel circuit essential for both the establishment of fear memory and the expression of fear responses, and uncover mechanisms linking stress detection in PVT with the emergence of adaptive behavior.
To probe the sensitivity of PVT to threatening events, we examined the expression of c-Fos, a marker of recent neuronal excitation, both following fear conditioning and after fear memory retrieval test. Fear conditioning markedly increased the number of neurons expressing c-Fos in the posterior PVT (pPVT) (Extended Data Fig. 1), consistent with the finding that pPVT receives direct inputs from the nociceptive parabrachial nucleus and the periaqueductal grey 7,8 . Interestingly, fear memory retrieval induced a similar increase in pPVT c-Fos expression (Extended Data Fig. 1). These results demonstrate that pPVT is recruited by both the US (unconditioned stimulus) and the threat-predicting CS (conditioned stimulus), and raise the possibility that it might be instrumental in fear conditioning.
Indeed, pPVT strongly projects to CeL 9,10 , with weaker projections to other amygdala nuclei, such as the basolateral amygdala (BLA) 10 . To examine the distribution patterns of pPVT neurons innervating different amygdala subregions, we injected CeL and BLA, respectively, with the retrograde tracer cholera toxin subunit B conjugated to Alexa Fluor-488 (CTB-488) or Alexa Fluor-555 (CTB-555). This approach resulted in dense labeling throughout the ipsilateral pPVT, indicating prominent pPVT-amygdala projections (Extended Data Fig. 2). Notably, pPVT neurons projecting to CeL and BLA were largely non-overlapping (Extended Data Fig. 2).
To determine whether the pPVT-CeL pathway, the most prominent projection originating from pPVT 9,10 , is involved in fear conditioning, we sought to selectively inhibit CeLprojecting neurons in pPVT with a chemogenetic method 11 . We bilaterally injected CeL with a retrograde canine adenovirus expressing the Cre recombinase (CAV2-Cre) 12 , followed by injection into pPVT of the AAV-DIO-hM4Di-mCherry, an adeno-associated virus harboring a double floxed inverted open reading frame (AAV-DIO) that expresses, in a Cre-dependent manner, an engineered G i -coupled receptor hM4Di tagged with a fluorescent protein mCherry (hM4Di-mCherry). This intersectional strategy effectively targeted CeLprojecting pPVT neurons (Fig. 1a, b), which can subsequently be suppressed by treating mice with CNO, the agonist of hM4Di 11 . Remarkably, selective suppression of the CeLprojecting pPVT neurons during either conditioning or 24 h memory retrieval test significantly impaired fear responses measured as freezing in the retrieval test ( Fig. 1c; Extended Data Fig. 3). Importantly, these behavioral effects significantly correlated with the number of hM4Di-expressing neurons in pPVT (Fig. 1d), showing the specificity and potency of our manipulation. Altogether, these results indicate that pPVT is crucial for both the establishment and expression of fear memory.
We next determined the mechanisms by which the pPVT-CeL circuit contributes to fear regulation. Fear conditioning induces a potentiation of excitatory synapses onto SOM + CeL neurons that stores fear memory 6 . To investigate whether pPVT is required for this plasticity, we labeled SOM + CeL neurons with eYFP by injecting CeL with AAV-DIO-eYFP in Som-Cre mice, in which the Cre recombinase is expressed under the endogenous Som promoter 6 . In addition, we injected pPVT in the same mice with a mixture of AAV-GFP-Cre and AAV-DIO-hM4Di-mCherry (Fig. 2a-c). This strategy allowed us to inhibit pPVT neurons using the chemogenetic method during fear conditioning, and subsequently determine the effect on SOM + (eYFP + ) CeL neurons ( Fig. 2; Extended Data Fig. 3).
As previously reported 6 , fear conditioning significantly enhanced excitatory synaptic transmission -measured as an increase in both the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) -onto SOM + CeL neurons (Fig. 2d, e). This synaptic potentiation can be detected at both 3 h and 24 h following conditioning 6 . Interestingly, inhibition of pPVT neurons during fear conditioning did not affect this synaptic potentiation when examined 3 h after conditioning (Fig. 2d, e). In contrast, the same manipulation completely abolished synaptic potentiation measured 24 h after conditioning (Fig. 2d, e). These results indicate that pPVT is required for the maintenance or consolidation, but not the initial induction of CeL plasticity, and are consistent with findings in an accompanying study that long-term (≥ 24 h), but not short-term (0.5 & 6 h) fear memories are susceptible to PVT manipulations (Do-Monte et al., this issue).
Because mEPSCs do not allow distinguishing between different synaptic pathways, we next examined whether pPVT inactivation impairs plasticity at the lateral amygdala (LA)-CeL synapses, a pathway that presumably conveys CS information to the CeL. For this purpose we used the Som-Cre;Rosa26-stop flox -H2b-GFP (Som-Cre;H2b-GFP) mice 13 in which all SOM + CeL neurons are readily identified based on the nucleus-localized GFP signal (Extended Data Fig. 4a). We inhibited pPVT neurons during fear conditioning using the same method as described above (Fig. 2a, b). We then simultaneously recorded pairs of adjacent SOM + (green-fluorescent) and SOM -(non-fluorescent) CeL neurons in acute brain slices, while EPSCs were evoked by electrical stimulation in LA (Extended Data Fig. 4a). As previously reported 6 , in naïve control mice AMPA receptor (AMPAR)-mediated EPSCs were significantly larger in SOMneurons than in SOM + neurons (Extended Data Fig. 4b, c), indicating a clear distinction between these two cell types in their intrinsic functional connectivity. However, fear conditioning reversed this relationship such that AMPARmediated transmission was significantly stronger in SOM + neurons than in SOMneurons (Extended Data Fig. 4b, c).
Fear conditioning also induced a decrease in the paired-pulse ratio (PPR) of EPSCs -an indicator of increased presynaptic release probability -onto SOM + CeL neurons (Extended Data Fig. 4d). This result, when considered together with data obtained from the paired recording (Extended Data Fig. 4a-c) and mEPSC recording (Fig. 2d, e) experiments (also see Ref 6 ), demonstrates that fear conditioning strengthens excitatory synaptic transmission onto SOM + CeL neurons, and that an increase in presynaptic release probability is likely the major underlying mechanism. Importantly, inhibition of pPVT during conditioning largely blocked these synaptic changes in the LA-CeL pathway (Extended Data Fig. 4b-d).
Altogether, these results indicate that pPVT participates in fear memory formation by regulating the maintenance of fear conditioning-induced plasticity at the LA-CeL synapses. SOM + and SOMneurons -the latter being predominantly protein kinase C-δ-expressing (PKC-δ + ) 6 -constitute two major CeL populations that are mutually inhibitory 6,14 . We reasoned that pPVT might control CeL synaptic plasticity by regulating either one or both of these populations. To distinguish between these possibilities, we used a modified rabies virus system that can trace the monosynaptic inputs onto genetically identified neurons 15 (see Methods). This approach revealed that pPVT projects to both populations (Fig. 3a-c), with the distinction that pPVT neurons innervating SOM + neurons were twice as many as those innervating PKC-δ + neurons (the connectivity indices, calculated as the ratio of the number of rabies virus-labeled cells in pPVT to that of starter cells in CeL, for the two cell types are: SOM + , 0.99 ± 0.08, n = 3 mice; PKC-δ + , 0.49 ± 0.03, n = 3 mice; P < 0.01, t test), suggesting that pPVT afferents in CeL preferentially innervate SOM + neurons.
To assess the functional connectivity between pPVT and CeL, we used the Som-Cre;Ai14 mice, in which SOM + cells can be identified by their red fluorescence, and injected pPVT with an AAV expressing the channelrodhopsin-2 (AAV-ChR2-YFP) that allows photostimulation of axonal projections 16 (Fig. 3d, e). Bright ChR2-YFP-labeled fibers were readily observed throughout the CeL in acute slices prepared from these mice, confirming a strong pPVT-CeL projection. To our surprise, brief light pulses, which reliably evoked excitatory synaptic transmission in BLA neurons (Extended Data Fig. 5a-c), failed to evoke any detectable fast synaptic transmission in all the recorded SOM + or SOM -CeL neurons (Fig. 3e, f). Interestingly, high frequency photo-stimulation of pPVT afferents in CeL induced a slow inward current exclusively in SOM + neurons ( Given that stimulation of pPVT afferents in CeL evoked slow inward currents, rather than canonical fast synaptic responses in SOM + neurons, it is likely that the pPVT-CeL SOM transmission is mediated by a neuromodulator. One notable candidate for this is BDNF, a known modulator of synaptic function 17 , whose mRNA expression in pPVT is the highest within the dorsal thalamus 18 . In fact, we found that the majority (~70%; n = 5 mice) of CeLprojecting pPVT neurons expressed BDNF (Extended Data Fig. 6a, b). In contrast, only 29% (n = 2 mice) of BLA-projecting PVT neurons expressed BDNF, a value that is likely an overestimation owing to contamination by CeL-projecting pPVT neurons caused by tracer diffusion into CeL (Extended Data Fig. 6c, d). In addition, CeL expression of the highaffinity BDNF receptor TrkB was largely restricted to SOM + neurons (76% colocalization, n = 4 mice; Extended Data Fig. 7a), consistent with the preferential targeting of this particular cell-type by inputs from pPVT (Fig. 3). These results suggest that BDNF might be a critical factor mediating pPVT to CeL communication.
To examine the functional role of BDNF in pPVT-CeL SOM interaction, we bred the Trkb lox/lox ;Som-Flp mice, which carry the Trkb lox/lox conditional alleles 19 and in which the Flp recombinase is expressed under the endogenous Som promoter 20 . In these mice, Trkb can be deleted in CeL by infection with the AAV-GFP-Cre, while SOM + neurons can be tagged by infection with AAV-fDIO-mCherry -an AAV harboring a double FRT-flanked inverted open reading frame (fDIO) that expresses mCherry in an Flp-dependent mannerthereby allowing their identification in acute slices for electrophysiological recording (Extended Data Fig. 7b, c). Based on this strategy we found that the pPVT-driven slow excitatory inward currents were selectively abolished in SOM + neurons in which Trkb was deleted (mCherry and GFP double-positive neurons; Extended Data Fig. 7c-f). Consistent with this result, bath application of the BDNF scavenger TrkB-Fc in acute slices abolished the pPVT-driven increase in inhibition onto SOM -CeL neurons (Extended Data Fig. 7g, h), whereas exogenous application of BDNF mimicked this increase in inhibition (Extended Data Fig. 7i, j). These results indicate that BDNF/TrkB signaling is a mediator of pPVT-CeL SOM communication.
Next, to determine whether this BDNF/TrkB-mediated pPVT-CeL SOM communication is important for fear processing, we selectively deleted either Bdnf from pPVT, or Trkb in SOM + CeL neurons. To achieve the former, we employed a mouse line carrying the Bdnf lox/lox conditional alleles 21 and injected PVT with the AAV-GFP-Cre ( Fig. 4a-c). To achieve the latter, we designed an AAV-fDIO-CreGFP that expresses CreGFP (a Cre and GFP fusion protein) under the control of Flp (Extended Data Fig. 8). This virus, when injected into CeL of the Trkb lox/lox ;Som-Flp mice, expresses Cre -and hence leads to Trkb deletion -specifically in SOM + neurons. This Intersectional Recombinases-mediated Arealand-cell-Specific gene Excision (IRASE) method can be generally applied for gene deletion with spatial and cell-type specificity.
Deletion of Bdnf in pPVT depleted BDNF from CeL (BDNF + cells in CeL: GFP group, 156.36 ± 39.40 cells/mm 2 , n = 8 mice; GFP-Cre group, 41.20 ± 12.00 cells/mm 2 , n = 7 mice; P < 0.05, t test) (Fig. 4a These results indicate that PVT is a major source of BDNF for CeL, and BDNF/TrkB-mediated pPVT-CeL SOM interaction has an important role in fear processing. Of note, Bdnf deletion in pPVT or Trkb deletion in SOM + CeL neurons not only impaired CS-evoked freezing, a measurement of tone-associated memory, but also reduced pre-CS freezing, which presumably represents contextual memory (Fig. 4c, e). Alternatively, or additionally, these behavioral effects could reflect a general reduction in fear responses, such as that caused by altered arousal or negative behavioral states 22 .
To disentangle these issues, we first determined whether disruption of BDNF signaling affects the synaptic potentiation onto SOM + CeL neurons that, as mentioned above, can serve as a fear memory trace 6 . Deletion of Trkb in SOM + CeL neurons by IRASE prevented the fear conditioning-induced increase in frequency, but not amplitude, of mEPSCs (Extended Data Fig. 9a-d), suggesting that the presynaptic potentiation -the major component of fear conditioning-driven CeL plasticity 6 -depends on BDNF signaling. We next examined the effects of enhancing BDNF signaling. Bath application of BDNF on slices was sufficient to induce LTP (long-term potentiation) of excitatory synaptic transmission onto SOM + CeL neurons, which was accompanied by a reduction in PPR (Extended Data Fig. 9e-g), indicating a presynaptic mechanism. These presynaptic effects in CeL induced by BDNF/TrkB manipulations are consistent with the presynaptic action of BDNF in other brain areas 17 . Notably, infusion of BDNF into CeL facilitated conditioning to a mild foot shock (Fig. 4g, h). These results, together with the above finding regarding the role of the pPVT-CeL pathway in fear learning (Fig. 1, Fig. 2, Extended Data Fig. 4), indicate that BDNF is a critical regulator of fear memory in CeL.
In light of our previously finding that activation of SOM + CeL neurons is sufficient to drive unconditioned fear responses and is required for the expression of conditioned fear 6  . These results, together with the above finding that the pPVT-CeL pathway is required for the expression of conditioned fear (Fig. 1), suggest that BDNF signaling in the pPVT-CeL SOM pathway may facilitate the expression of fear by promoting SOM + CeL neuron activation.
On the basis of our collective results, we propose that BDNF/TrkB-mediated pPVT-CeL communication promotes both synaptic plasticity and excitability of SOM + CeL neurons, thereby facilitating not only the formation of stable fear memories but also the expression of fear responses.
Recent studies suggest a role of PVT in anxiety-related behaviors 23 . In addition, altered BDNF signaling has been implicated in anxiety disorders 24,25 . Our findings that pPVT recruits BDNF/TrkB signaling to control CeL function define a functional framework for pPVT that can subserve its role in the coordination of behavioral responses to stress.

Mice
All procedures were approved by the Institutional Animal Care and Use Committees of Cold Spring Harbor Laboratory and conducted in accordance to the US National Institute of Health guidelines. Mice were housed under a 12-h light-dark cycle (9 a.m. to 9 p.m. light), with food and water available ad libitum. Som-Cre 6 , Prkcd-Cre 14 , Som-Flp 20 , Bdnf lox/lox21 , Trkb lox/lox19 , Ai14 6 , H2b-GFP 13 , and Rosa26-stop flox -tTA 26 mice have all been described elsewhere. All mice were bred onto C57BL/6J genetic background. Male and female mice 6-9 weeks of age were used for all the experiments. All subjects were randomly allocated to the different experimental conditions used in this study.

Viral vectors
AAV-DIO-hM4Di-mCherry, AAV-DIO-eYFP, AAV-GFP-Cre, AAV-ChR2-YFP, and AAV-TRE-hGFP-TVA-G were produced by the University of North Carolina (UNC) Vector Core Facilities (Chapel Hill, NC) and have been previously described 6,15 . For the generation of the AAV-fDIO-CreGFP and AAV-fDIO-mCherry, standard cloning procedures were used to subclone the CreGFP or mCherry cassettes into the backbone of a Flippase-dependent AAV-Ef1a-fDIOYFP expression plasmid 20 (gift from K. Deisseroth). Following DNA sequencing screening, the AAV plasmid was packaged into AAV serotype 8 virus from UNC Vector Core, with titers of 8.3 × 10 12 virus particles/ml. The EnvApseudotyped, protein G-deleted rabies-EnvA-SAD-ΔG-mCherry virus 15 was produced by the Viral Vector Core Facility at Salk Institute. CAV2-Cre was prepared using E1transcomplementing dog kidney (DK) cells and purified by sucrose and CsCl gradient centrifugation, and was re-suspended in 1× Hanks Balanced Saline Solution (HBSS) 12 . All viral vectors were stored in aliquots at −80°C until use.

Stereotaxic surgery
Viral injections were performed using previously described procedures 6 at the following stereotaxic coordinates: pPVT, -1.34 mm from Bregma, 0.05 mm lateral from midline, and 3.03 mm vertical from cortical surface; CeL, −1.22 mm from Bregma, 2.9 mm lateral from midline, and 4.6 mm vertical from cortical surface; BLA, -1.80 mm from Bregma, 3.4 mm lateral from midline, and 5.4 mm vertical from cortical surface. For pPVT injections we used a 6.5° angle to avoid damage of the superior sagittal sinus. Animals were kept on a heating pad throughout the entire surgical procedures and were brought back to their home cages after 24 h post-surgery recovery and monitoring. Postoperative care included intraperitoneal injection with 0.3-0.5 ml of lactated Ringers solution and metacam (meloxicam, 1-2 mg/kg) for analgesia and anti-inflammatory purposes. All AAVs and the CAV2-Cre were injected at a total volume of approximately 1 μl (except for the monosynaptic rabies viral tracing, see below), and were allowed at least two weeks for maximal expression. For retrograde tracing of amygdala-projecting pPVT cells, CTB-555 or CTB-488 (0.1-0.3 μl, 0.5% in PBS) (Invitrogen) was injected into CeL and BLA and allowed 3-5 days for sufficient retrograde transport.

Monosynaptic tracing with pseudotyped rabies virus
Retrograde tracing of monosynaptic inputs onto genetically-defined cell populations of CeL was accomplished using a previously described method 15 . Briefly, the Som-Cre;Rosa26stop flox -tTA mice and the Prkcd-Cre;Rosa26-stop flox -tTA mice, which express tTA in SOM + cells and PKC-δ + cells, respectively, were injected in CeL with the AAV-TRE-hGFP-TVA-G (0.2-0.3 μl) that expresses the following components in a tTA-dependent manner: a fluorescent reporter histone-GFP (hGFP), TVA (which is a receptor for the avian virus envelope protein EnvA), and the rabies envelope glycoprotein (G). Two weeks later mice were injected in the same location with the rabies-EnvA-SAD-ΔG-mCherry (1.2 μl), a rabies virus that is pseudotyped with EnvA, lacks G, and expresses mCherry. This method ensures that the rabies virus exclusively infects cells expressing TVA. Furthermore, complementation of the modified rabies virus with G in the TVA-expressing cells allows the generation of infectious particles, which then can trans-synaptically infect presynaptic neurons.

Histology
Animals were deeply anesthetized and transcardially perfused with PBS, followed by perfusion with 4% paraformaldehyde (PFA) in PBS. Brains were extracted and postfixed in 4% PFA at 4 °C for two hours for BDNF and TrkB immunohistochemistry and overnight for all other experiments. This was followed by cryoprotection in a 30% PBS-buffered sucrose solution until brains were saturated (~36 h). Coronal brain sections (40 μm) were cut using a freezing microtome (SM 2010R, Leica). Brain sections were first washed in PBS (3 × 5 min) and then incubated in PBST (0.1% Triton X-100 in PBS) for 15 min at room temperature (RT). Next, sections were blocked in 10% normal goat serum (NGS) in PBST for 30 min at RT, followed by incubation with primary antibodies overnight at 4 °C. Sections were then washed with PBST (5 × 15 min) and incubated with fluorescent secondary antibodies at RT for 1 h. After washing with PBS (5 × 15 min), sections were mounted onto glass slides with Fluoromount-G (Beckman Coulter). Images were taken using a LSM 780 laser-scanning confocal microscope (Carl Zeiss).

Fear conditioning
Mice were initially handled and habituated to the conditioning cage, a Mouse Test Cage (18 cm × 18 cm × 30 cm) with an electrifiable floor connected to a H13-15 shock generator (Coulbourn Instruments, Whitehall, PA). The Test Cage was located inside a sound attenuated cabinet (H10-24A; Coulbourn Instruments). Before each conditioning session the Test Cage was wiped clean with 70% ethanol. During conditioning the cabinet was illuminated and the behavior was captured with a monochrome CCD-camera (Panasonic WV-BP334) at 3.7 Hz and stored on a personal computer. The FreezeFrame software (Coulbourn Instruments) was used to control the delivery of both tones and foot shocks. For habituation, five 4-kHz 75-dB tones (conditioned stimulus; CS), each of which was 30 s in duration, were delivered at variable intervals. During conditioning, mice received five presentations of the CS, each of which co-terminated with a 2-s 1-mA foot shock (unconditioned stimulus; US). In the experiment to determine the effect of BDNF infusion on fear learning (Fig. 4g, h), weaker (2-s 0.5-mA) foot shocks were used. The test for fear memory was performed 24 h following conditioning in a novel illuminated context, where mice were exposed to two presentations of unreinforced CS (120 s inter-CS interval). The novel context was a cage with a different shape (22 cm × 22 cm × 21 cm) and floor texture compared with the conditioning cage. Prior to each use the floor and walls of the cage were wiped clean with 0.5% acetic acid to make the scent distinct from that of the conditioning cage. Behavioral responses to the CS were recorded. Freezing behavior was analyzed with FreezeFrame (Coulbourn Instruments).
For plasticity experiments, recordings were always performed on interleaved naïve and fearconditioned animals. Simultaneous whole-cell patch-clamp recordings from SOM + /SOMneuronal pairs in CeL were obtained with Multiclamp 700B amplifiers (Molecular Devices). Recordings were under visual guidance using an Olympus BX51 microscope with transmitted light illumination, and SOM + cells were identified based on their fluorescence (hGFP or tdTomato). Synaptic responses were evoked with a bipolar stimulating electrode placed in BLA approximately 0.2 mm away from the recorded cell bodies in CeL. Electrical stimulation was delivered every 10 seconds and synaptic responses were low-pass filtered at 1 KHz and recorded at holding potentials of −70 mV (for AMPA-receptor-mediated responses), +40 -mV (for -NMDA-receptor-mediated responses), or 0 mV (for GABA-Areceptor-mediated responses). NMDA-receptor-mediated responses were quantified as the mean current amplitude between 50 ms and 60 ms after stimulation. Recordings were made in the ACSF. The internal solution for voltage-clamp experiments contained 115 mM cesium methanesulphonate, 20 mM CsCl, 10 mM HEPES, 2.5 mM MgCl 2 , 4 mM Na 2 -ATP, 0.4 mM Na 3 GTP, 10 mM Na-phosphocreatine and 0.6 mM EGTA (pH 7.2). Evoked EPSCs were recorded with picrotoxin (100 μM) added to the ACSF. mEPSCs were recorded in the presence of tetrodotoxin (TTX; 1 μM) and picrotoxin (100 μM) and analyzed using Mini Analysis software (Synaptosoft). To assess presynaptic function, a paired-pulse stimulation protocol (50 ms inter-stimulus interval) was used to evoke double-EPSCs, and paired-pulse ratio (PPR) was quantified as the ratio of the peak amplitude of the second EPSC to that of the first EPSC. While most of the above electrophysiology experiments were carried out by M.A.P., plasticity results were replicated by V.R. who performed these experiments blind.
To evoke pPVT-driven synaptic transmission onto CeL neurons, the AAV-ChR2-YFP was injected into the pPVT of Som-Cre;Ai14 mice and allowed to express for 3 weeks. Acute brain slices were prepared and a blue light was used to stimulate ChR2-expressing axons. The light source was a single-wavelength LED system (λ = 470 nm; CoolLED.com) There was no significant difference in performance among groups (F (2,155) = 0.51, P > 0.05, two-way ANOVA). b. There was no significant difference in performance between saline-treated mice and CNO-treated mice (F (1,70) = 0.43, P > 0.05, two-way ANOVA). c. There was no significant difference in performance between the two groups (F (1,85) = 0.73, P > 0.05, two-way ANOVA). d. There was no significant difference in performance between the two groups (F (1,75) = 0.45, P > 0.05, twoway ANOVA). Data are presented as mean ± s.e.m. Figure 4. pPVT is required for fear conditioning-induced synaptic plasticity in the LA-CeL SOM pathway a. Top: a schematic of the whole-cell paired-recording configuration. A pair of SOM + and SOM -CeL neurons was simultaneously recorded, and EPSCs were evoked by stimulation of the LA. We used the Som-Cre;H2b-GFP mice, in which the SOM + neurons were tagged with H2b-GFP and the pPVT neurons were infected with hM4Di as in Figure 2a &

Extended Data
Bottom: a representative image of a slice used for recording, in which a SOM + (arrow) and an adjacent SOM -(arrowhead) neuron were recorded. b. Sample EPSC traces obtained from the simultaneous paired-recording experiment. Naïve control mice (left) and fear conditioned mice treated with either saline (middle) or CNO (right) were used. Saline or CNO were administered 40 min prior to, and recording were performed 24 hours following conditioning. Upper and lower traces represent EPSCs recorded at +40 mV and -70 mV holding potentials, respectively. c. Quantification of AMPA (left) and NMDA (right) currents (Control, n = 9 pairs (2 mice); "Fear, saline", n = 8 pairs (3 mice); "Fear, CNO", n = 14 pairs (3 mice); *P < 0.05, **P < 0.001, n.s., non-significant, paired t test). EPSC values are normalized to the average EPSC value of SOMcells for each group. d. Quantification of paired-pulse ratio (PPR) (see Methods) of EPSCs measured at -70 mV (comparing "control", "Fear, saline", and "Fear, CNO" groups for SOM + neurons: *P < 0.05, **P < 0.01, n.s., non-significant; one-way ANOVA followed by Tukey's test). Control mice for all experiments were injected with the same viral vectors as the experimental groups. Data are presented as mean ± s.e.m. effect of BDNF on EPSC peak amplitude, for which the first peak in the paired-pulse was measured and normalized to the baseline (that is, the average pre-BDNF amplitude). BDNF significantly enhanced EPSC amplitude (pre-BDNF, 98 ± 1.74%, post-BDNF, 146 ± 17.6%, n = 6 neurons (3 mice), P < 0.05, paired t test). g. BDNF application decreased paired-pulse ratio (PPR) (see Methods) of the EPSCs (pre-BDNF, 1.17 ± 0.18; post-BDNF, 0.80 ± 0.09; n = 6 neurons (3 mice), *P < 0.05, paired t test). Data are presented as mean ± s.e.m. Extended Data Figure 10. Exogenous application of BDNF in CeL increases the excitability of SOM + neurons and elicits unconditioned freezing response (a-d) BDNF increases the excitability of SOM + CeL neurons. a. A schematic of the experimental approach, in which photostimulation was used to assess the excitability of SOM + CeL neurons expressing ChR2. b. Sample traces of photostimulation-evoked spikes recorded in cell-attached mode, before (baseline; left) and after (right) bath application of BDNF (100 ng/ml). Light intensity was adjusted to evoke spikes with ~50% probability at baseline. c. A sample recording, in which the spike probability of a SOM + CeL neuron was followed before, during, and after BDNF application. d. Quantification of the effect of BDNF on spike probability (baseline, 0.50 ± 0.02; BDNF, 0.74 ± 0.08; n = 8 neurons (4 mice), *P < 0.05, paired t test). (e, f) Infusion of BDNF into CeL elicits unconditioned freezing response. e. Drawing of the cannula sites. Each dot denotes where the tip of the injection cannula was located in each mouse. f. Quantification of freezing levels following CeL infusion of saline and BDNF (saline, 2.93 ± 1.84 %; BDNF, 32.22 ± 9.19 %; n = 6 mice, *P<0.05, paired t test). Data are presented as mean ± s.e.m.