The ability to differentiate stimuli predicting positive or negative outcomes is critical for survival, and perturbations of emotional processing underlie many psychiatric disease states. Synaptic plasticity in the basolateral amygdala complex (BLA) mediates the acquisition of associative memories, both positive1,2 and negative3,4,5,6,7. Different populations of BLA neurons may encode fearful or rewarding associations8,9,10, but the identifying features of these populations and the synaptic mechanisms of differentiating positive and negative emotional valence have remained unknown. Here we show that BLA neurons projecting to the nucleus accumbens (NAc projectors) or the centromedial amygdala (CeM projectors) undergo opposing synaptic changes following fear or reward conditioning. We find that photostimulation of NAc projectors supports positive reinforcement while photostimulation of CeM projectors mediates negative reinforcement. Photoinhibition of CeM projectors impairs fear conditioning and enhances reward conditioning. We characterize these functionally distinct neuronal populations by comparing their electrophysiological, morphological and genetic features. Overall, we provide a mechanistic explanation for the representation of positive and negative associations within the amygdala.
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We thank C. Wildes, G. Glober, R. Luck and G. F. Conyers for technical assistance. We thank the entire Tye laboratory, B. Okaty, U. Eser, J. di Iulio and A. Pfenning for helpful discussion. We thank E. J. Kremer for providing the CAV2-Cre virus, R. Neve for the HSV virus carrying Cre recombinase and mCherry, and the UNC vector core for the AAV viruses carrying double floxed constructs. K.M.T. is a New York Stem Cell Foundation–Robertson Investigator and holds a Whitehead Career Development Chair. This work was supported by funds from NIMH (R01-MH102441-01), NIDDK (DP2-DK-102256-01), the JPB Foundation (PIIF and PNDRF), NARSAD, Klingenstein, Whitehall and Sloan Foundations (K.M.T.). Additionally, S.Y. and J.G. were supported by NIMH (R01-MH101528-01), P.N. was supported by Singleton, Leventhal and Whitaker fellowships, and A.B. was supported by a fellowship from the Swiss National Science Foundation. R.W. was supported by post-doctoral fellowships from the Simons Center for the Social Brain and the Netherlands Organization for Scientific Research (NWO) RUBICON fellowship program. I.R.W. was supported by seed grants from the McGovern Institute for Brain Research, the Picower Institute for Learning and Memory, the MIT Department of Brain and Cognitive Sciences, and the Simons Center for the Social Brain, as well as by BRAIN Initiative awards from NIMH, NEI, and NINDS (U01-MH106018 and U01-NS090473) and NSF (IOS-1451202).
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Histological verification of retrobead injection sites and behavioural quantification of fear and reward conditioning for mice used in Fig. 1.
a, Representative differential interference contrast (DIC) image of a 300-µm thick coronal slice containing the centre of the retrobead injection in NAc . The white circle indicates the most ventral point at which fluorescence is brightest and corresponds to the filled green circle in b. b, Location of all retrobead injection sites (green circles) in the NAc for all mice used in Fig. 1. Each atlas schematic represents a 1.5 mm × 1.5 mm region of the atlas and the corresponding anteroposterior stereotaxic coordinate relative to Bregma is indicated below. c, Representative DIC image of a 300-µm thick coronal slice containing the centre of the retrobead injection in CeM as indicated by the white dot. d, Retrobead injection sites in CeM (red circles) for all mice used in Fig. 1, with the example from c indicated by the filled red circle. The corresponding anteroposterior stereotaxic coordinate relative to Bregma is indicated below. e, Experimental design for AMPAR/NMDAR ratios from Fig. 1. Either red or green retrobeads were injected in the NAc and the other colour in the contralateral CeM. Two weeks after injection, the retrobeads had travelled back to the cell bodies of the BLA neurons projecting to NAc or CeM. Animals were conditioned 1 day before ex vivo whole-cell patch-clamp recordings. Each mouse received one of six conditioning protocols, three protocols categorized under ‘fear conditioning’ and three protocols categorized under ‘reward conditioning’. Fear conditioning protocols: (i) naive, animals were naive to the operant chamber. (ii) Unpaired, animals were exposed to the conditioning chamber in two sessions. Animals received six tones in the first session and they received six foot shocks in the second session. Animals were returned to their home cage for ∼20 min between the two sessions. (iii) Paired, animals were exposed to the operant chamber in two sessions. Animals did not receive any tone or shock stimuli in the first session, and received tones co-terminating with shocks in the second session. Animals were returned to their home cage for 20 min between the two sessions. Protocols for unpaired and paired fear groups were adapted from ref. 18. Reward conditioning protocols: (i) naive food restricted (FR), animals naive to the operant chamber were food restricted two days before ex vivo experiments and had free access to food for 1 day before ex vivo experiments. We used this group to control for changes in synaptic strength caused by food restriction which was necessary in reward conditioning groups to expedite task acquisition, adapted from rats as in refs 1 and 2. (ii) Unpaired, animals received tones in the operant chamber, were returned to their home cage for ∼20 min after which they had free access to 1.8 ml of sucrose, followed by free access to food until ex vivo experiments. (iii) Paired, sucrose was delivered into a port 1 s after the onset of a tone, and the tone was terminated 400 ms after the animal entered the port to claim sucrose. The tone lasted for a maximum length of 30 s. If there was sucrose in the port during the onset of a tone (indicated by the absence of a port entry after the previous tone), then no sucrose was delivered in that trial. Mice could receive up to 120 sucrose deliveries and the conditioning session lasted about 4 h after which they had free access to food until ex vivo experiments. Behavioural performance from the second half of the conditioning session was used to assess performance and mice that met learning criterion (see Methods) were categorized in the learner group and the rest of the mice were categorized in the non-learner group. One day after conditioning, BLA neurons identified as either NAc or CeM projectors (retrobead positive) were recorded with whole-cell patch-clamp in ex vivo brain slices. Ex vivo data from both NAc and CeM projectors were collected from the following 7 groups: (1) naive (n = 12 mice, 9 for NAc pr., 7 for CeM pr.); (2) unpaired fear (n = 13 mice, 7 for NAc pr., 9 for CeM pr.); (3) paired fear (n = 10 mice, 7 for NAc pr., 7 for CeM pr.); (4) naive food restricted (n = 11 mice, 7 for NAc pr., n = 8 for CeM pr.); (5) unpaired (n = 11 mice, 7 for NAc pr., 5 for CeM pr.); (6) reward paired non-learner (n = 9 mice, 5 for NAc pr., 6 for CeM pr.); and (7) reward paired learner (n = 10 mice, 8 for NAc pr., 7 for CeM pr.) groups. The n values indicated here are the number of mice used in Fig. 1. Data from groups 1–5 and 7 are shown in Fig. 1 and Extended Data Figs 1, 2 and 3. Data from group 6 is shown only in Extended Data Fig. 3. f, Time course of percentage freezing for the paired fear group. Percentage freezing was estimated during the shock-predictive tone (excluding the final 2 s, where the foot shock was delivered). g, Average normalized histogram of port entries relative to the onset of the tone predicting sucrose delivery for mice that learned the conditioned–unconditioned stimulus association (learners, n = 11) and mice that did not (non-learners, n = 17; see Extended Data Fig. 3). Mice in the paired reward conditioning group were deemed learners if the number of port entries in the post-conditioned-stimulus period (1 to 8 s relative to conditioned stimulus onset, black line) were determined as significantly higher than the number of port entries in the pre-conditioned stimulus period (−8 to −1 s relative to conditioned stimulus onset, grey line) using a one-sided Wilcoxon rank sum test (P < 0.001).
Extended Data Figure 2 Location of BLA projectors recorded and analysed for each experimental group in Fig. 1.
Top, representative DIC image showing the location of the stimulation electrode around a bundle of fibres of the internal capsule and a neuron recorded in the BLA (at the tip of the micropipette). The location of the recorded cell is indicated by an orange open circle. Scale bar, 200 µm. Bottom, atlas schematics (1.5 mm × 1.5 mm) showing BLA at various anteroposterior positions relative to Bregma. Each circle represents the location of a neuron from which the AMPAR/NMDAR ratio was acquired (Fig. 1). NAc projector locations are summarized in rows 1 and 2 and CeM projector locations are summarized in rows 3 and 4. Colour of the circle represents the conditioning group of the animal from which the AMPAR/NMDAR ratio was acquired.
Extended Data Figure 3 Paired-pulse ratio and AMPAR/NMDAR ratio in non-learners and food-restricted naive animals.
a, Confocal image of a representative retrobead-positive neuron recorded in BLA after injection of retrobeads into NAc. This cell was recorded in an ex vivo slice, filled with biocytin and stained with streptavidin-CF405, pseudo-coloured white. b, In NAc projectors, the ratio of EPSC amplitude in response to paired-pulse stimulation (50 ms inter-pulse interval) of internal capsule inputs to the BLA was not related to experimental conditions of fear (one-way ANOVA, F2,44 = 0.5209, P = 0.5978). c, Paired-pulse ratio of EPSC amplitude was not related to experimental conditions of reward (one-way ANOVA, F3,61 = 0.5868, P = 0.6261). d, AMPAR/NMDAR ratio of internal capsule inputs onto NAc projectors in mice with unpaired tone and sucrose presentations (unpaired) and mice that did not learn the cue-reward association (non-learner) were not different (unpaired t-test t16 = 0.180, P = 0.8595). Both groups of mice received the same amount of total sucrose. e, AMPAR/NMDAR ratio on NAc projectors is significantly decreased by food restriction in naive mice (unpaired t-test, t20 = 2.626, P = 0.0162). f, Confocal image of a representative retrobead-positive neuron recorded in BLA after retrobead injection in CeM. g, Paired-pulse ratio of EPSC amplitude onto CeM projectors is not related to experimental conditions of fear (one-way ANOVA, F2,29 = 0.9040, P = 0.4169). h, Paired-pulse ratio of EPSC amplitude is not related to experimental conditions of reward (one-way ANOVA, F3,44 = 0.9770, P = 0.4129). i, AMPAR/NMDAR ratio on CeM projectors is similar in unpaired reward and paired reward non-learner mice (unpaired t-test t14 = 0.381, P = 0.7090). j, AMPAR/NMDAR ratio of internal capsule inputs onto CeM projectors is significantly increased by food restriction in naive mice (unpaired t-test t20 = 2.526, P = 0.0201). Results show mean and s.e.m.
Extended Data Figure 4 Histological verification of viral injection site and fibre placement for photostimulation experiments used in Fig. 2.
a, Center of the rabies virus injection in NAc for the animals tested in intra-cranial self-stimulation (ICSS) and real-time place avoidance (RTPA) paradigms (Fig. 2a–e). Rabies virus (RV)-ChR2–Venus injections are denoted with green circles, and RV-Venus injections are indicated with grey squares. b, Representative confocal image of viral expression in a mouse 6 days after RV-ChR2–Venus injection in NAc. Right panel, enlarged view of the brightest fluorescence point (white circle), corresponding to the filled green circle in a. c, Center of RV-ChR2–Venus (red diamonds) and RV-Venus (grey squares) injections in CeM of animals analysed in Fig. 2. d, Example of viral expression 6 days after RV-ChR2–Venus injection in CeM. Right panel, enlarged view of the brightest fluorescence point (white circle), corresponding to the filled red diamond in c. e, Optical fibre tip placements over BLA of animals with RV-ChR2–Venus injected in NAc (green circles), CeM (red diamonds) or RV-Venus in NAc or CeM (grey squares). Horizontal lines represent the thickness of the implanted fibre (300 µm). f, Representative confocal image showing optical fibre tip from a RV-ChR2–Venus injection in NAc, corresponding to the filled green circle in e. Region in the white rectangle is magnified in the right panel and shows rabies-virus-expressing NAc projectors. g, Representative optic fibre placement for RV-ChR2-Venus injection in CeM, corresponding to the filled diamond in (e). Right panel: enlarged image of the BLA, containing rabies-virus-expressing CeM projectors. Atlas schematic in a, c and e represent 1.5 mm × 1.5 mm of the brain and the corresponding anteroposterior coordinates relative to Bregma are specified below. Scale bars in b, d, f and g are 500 µm.
Extended Data Figure 5 Histological verification of viral injection site and fibre placement for photoinhibition experiments used in Fig. 3.
a, Centre of canine adenovirus (CAV)-Cre injection into bilateral NAc of mice with AAV5-EF1α-DIO-NpHR-eYFP (green circles) or AAV5-EF1α-DIO-eYFP (grey squares) injected bilaterally into the BLA. This approach allows for selective expression of NpHR–eYFP/eYFP, in NAc-projecting BLA neurons. b, Representative confocal image of the CAV-Cre injection site in NAc. c, Center of CAV-Cre injection into CeM from both hemispheres of mice with AAV5-EF1α-DIO-NpHR-eYFP (red diamonds) or AAV5-EF1α-DIO-eYFP (grey squares) injected bilaterally into BLA. In these animals, CeM-projecting BLA neurons express NpHR–eYFP or eYFP, respectively. d, Confocal image of a representative CeM injection and NpHR–eYFP-expressing cells bodies in the BLA. e, Optical fibre tip placements over BLA from both hemispheres in animals injected with AAV5-EF1α-DIO-NpHR-eYFP in BLA and CAV-Cre in NAc (green circles) or CeM (red diamonds), or AAV5-EF1α-DIO-eYFP in BLA and CAV-Cre in NAc/CeM (grey squares). Horizontal lines represent thickness of the implanted fibre (300 µm). f, Representative confocal images of optic fibre placements over BLA from both hemispheres of an animal injected with CAV-Cre in NAc and AAV5-EF1α-DIO-NpHR-eYFP in BLA. Note NpHR–eYFP-expressing NAc projectors in the BLA. Each atlas diagram and confocal image in a–f represents an area of 1.5 mm × 1.5 mm; anteroposterior stereotaxic coordinates relative to Bregma are specified to the left of each image.
Extended Data Figure 6 Tone-evoked freezing behaviour following inhibition of CeM or NAc projectors during auditory fear conditioning.
a, Experimental design. Mice were trained in an auditory fear conditioning paradigm, during which NAc or CeM projectors were selectively inhibited using a dual virus recombination approach (Fig. 3). On the day following conditioning, mice were exposed to eight presentations of the conditioned stimulus alone. They were tethered to a patch cable but no light was delivered. b, Time course of percentage freezing in mice expressing NpHR in NAc projectors (green circles), CeM projectors (red diamonds), or expressing eYFP in NAc or CeM projectors (grey squares) was quantified for each trial. c, There was no significant difference in freezing behaviour in response to the conditioned stimulus among the three groups of mice on test day (one-way ANOVA, F2,38 = 2.010, P = 0.1488). Results show mean and s.e.m.
Extended Data Figure 7 Membrane properties of retrobead-positive NAc/CeM-projecting BLA neurons and rabies-virus-expressing BLA neurons.
a, Access resistance, membrane resistance, and membrane capacitance were estimated from the current response of the cell to a 4 mV square voltage pulse using the Q-method35. Access and membrane resistance as well as the membrane capacitance and membrane potential were not significantly different between the two populations (unpaired t-tests: t20 = 0.788, P = 0.4400; t20 = 1.599, P = 0.1256; t20 = 1.847, P = 0.0796; and t18 = 0.2521, P = 0.8038, respectively). The holding current corresponds to the current injected to clamp the cell at −70 mV. This value was not significantly different between NAc and CeM projectors (unpaired t-test, t20 = 1.046, P = 0.3079). b, Confocal image of a BLA–NAc projectors expressing ChR2–eYFP transduced by rabies virus (RV) and recorded ex vivo in whole-cell patch-clamp. The cell was filled with biocytin during recording and stained with streptavidin-CF405 (in grey). c, Current response to a 1-s blue light pulse in a cell expressing rabies virus, 5 days after injection. d, Five days after viral injection, rabies-virus-expressing cells were able to respond with an action potential to every pulse of a 20 Hz light stimulation (5 ms pulses, top trace, blue line shows onset of light pulse). They also responded with an action potential to 250 pA, 5 ms current pulses injected at 20 Hz (middle trace). Rabies-virus-expressing cells also showed spontaneous post-synaptic excitatory and inhibitory currents (EPSCs and IPSCs, respectively) when clamped at −70 mV (bottom trace, 0 pA holding for this cell). e, Current/voltage curves are similar in retrobead (RB, grey circles, n = 5 cells) and rabies-virus-expressing cells (black circles, n = 3 cells). f, Average action potential for 11 retrobead-positive BLA–NAc projectors (grey) and six BLA–NAc projectors expressing rabies virus. g, Membrane properties of retrobead-positive versus rabies-virus-expressing neurons. None of the properties investigated were significantly altered in rabies-virus-expressing neurons (unpaired t-tests: access resistance, t15 = 1.299, P = 0.2135; membrane resistance, t15 = 2.057, P = 0.0575; membrane capacitance, t15 = 1.215, P = 0.2430; action potential threshold, t15 = 0.0756, P = 0.9407; holding current, t16 = 1.002, P = 0.3314). Results show mean and s.e.m.
Extended Data Figure 8 Morphological reconstructions of individual BLA neurons projecting to NAc or CeM.
Morphological reconstructions of all neurons used for Sholl analysis performed by Imaris software (Fig. 4i). Classification of each neuron as pyramidal or stellate is indicated in the top left corner of each reconstructed neuron (triangle or star, respectively). Each atlas schematic represents 1.5 mm × 1.5 mm area and the corresponding anteroposterior stereotaxic coordinates (relative to Bregma) are shown below.
Extended Data Figure 9 RNA-seq identification of candidate genes differentially expressed in NAc- and CeM-projecting BLA neurons.
a, Candidate differentially expressed genes were required to be enriched in only one group (either CeM or NAc projectors) in two independent experiments (NAc projectors collected from n = 8 mice; CeM projectors collected from n = 9 mice, total) at the indicated quantile fold-change threshold (light-blue column). One of the chance estimates (‘flip-flopped’, see Methods) is taken from genes that passed the quantile thresholds but were enriched in the opposite groups in the two experiments. Another chance estimate (‘permuted’, see Methods) is determined based on an analysis in which fold differences for each gene were permuted across genes within each of the two experiments before determining differential expression. A 0.02 quantile threshold was chosen to identify differentially expressed candidate genes in order to balance specificity and sensitivity, resulting in an estimated false discovery rate of 41.5%, calculated as the number expected by chance (flip-flopped) divided by the number of differentially expressed genes (see Extended Data Fig. 9c for candidate gene list). In Fig. 4k, a 0.01 quantile threshold was chosen to identify a more conservative list of differentially expressed candidate genes at a lower false discovery rate of 26.2%. b, Distribution of differentially expressed genes between NAc and CeM projectors from RNA-seq experiments 1 and 2 (see Methods). Light-blue shaded areas represent the 2nd and 98th percentiles of the distributions. c, RNA-seq heat map showing normalized expression levels of differentially expressed genes in NAc- and CeM-projecting BLA neurons. Differentially expressed genes were required to be enriched in either NAc or CeM projectors in two independent experiments (samples used in experiment 1 are indicated in black text below the heat map; experiment 2 samples are indicated in blue text) at a 0.02 quantile threshold (Extended Data Fig. 9a). Each RNA-seq library was prepared from 35–60 manually sorted retrobead-labelled cells taken from the BLA.
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Namburi, P., Beyeler, A., Yorozu, S. et al. A circuit mechanism for differentiating positive and negative associations. Nature 520, 675–678 (2015). https://doi.org/10.1038/nature14366
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