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A circuit mechanism for differentiating positive and negative associations.

Namburi P, Beyeler A, Yorozu S, Calhoon GG, Halbert SA, Wichmann R, Holden SS, Mertens KL, Anahtar M, Felix-Ortiz AC, Wickersham IR, Gray JM, Tye KM - Nature (2015)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: 1] The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [2] Neuroscience Graduate Program, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
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 positive and negative. Different populations of BLA neurons may encode fearful or rewarding associations, 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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Related in: MedlinePlus

Histological verification of retrobead injection sites and behavioral quantification of fear and reward conditioning for mice used in Figure 1a, Representative differential interference contrast (DIC) image of a 300 μm thick coronal slice containing the center of the retrobead injection in NAc. The white circle is over 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 1.5 mm × 1.5 mm region of the atlas and the corresponding antero-posterior (AP) stereotaxic coordinate relative to Bregma is indicated below c, Representative DIC image of a 300 μm thick coronal slice containing the center of the retrobead injection in CeM.d, Retrobead injection sites in CeM (red circles) for all mice used in Fig. 1. e, Experimental design for AMPAR/NMDAR ratios from Figure 1. Either red or green retrobeads were injected in the NAc and the other color in the contralateral CeM. Two weeks after injection, the retrobeads had travelled back to the cell bodies of the BLA neurons projecting NAc and CeM. Animals were conditioned one day prior to ex-vivo whole cells patch clamp recordings. Each mouse received one of 6 conditioning protocols, 3 protocols categorized under Fear conditioning and 3 protocols categorized under Reward conditioning. Fear conditioning protocols: (i) Naïve: Animals were naïve to the operant chamber. (ii) Unpaired: Animals were exposed to the conditioning chamber in 2 sessions. Animals received 6 tones in the first session and they received 6 foot shocks in the second session. Animals were returned to their home cage for 20 minutes between the two sessions. (iii) Paired: Animals were exposed to operant chamber in two sessions. Animals did not receive any tone or shock stimuli in the first session, and received shocks co-terminating with tones in the second session. Animals were returned to their home cage for 20 minutes between the two sessions. Protocols for Unpaired and Paired fear groups were adapted from Clem and Huganir (2010)18. Reward conditioning protocols: (i) Naïve Food Restricted (FR): Animals naïve to the operant chamber were food restricted two days prior to ex vivo experiments and had free access to food for one 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 Tye et al., 2008 and 20101,2. (ii) Unpaired: Animals received tones in the operant chamber, returned to their home cage for 20 minutes 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 hours after which they had free access to food until ex vivo experiments. Behavioral 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 in 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) Naïve, (2) Unpaired and (3) Paired after fear conditioning, (4) Naïve FR, (5) Unpaired, (6) Non learner and (7) Learner groups after reward conditioning. Data from groups (1)–(5) and (7) are shown in Fig. 1 and Extended Data Fig. 1,2,3. 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 2 last seconds, 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 CS-US 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-CS period (1 to 8 s relative to CS onset, black line) were determined as significantly higher than the number of port entries in the pre-CS period (−8 to −1 s relative to CS onset, grey line) using a one-sided Wilcoxon Rank Sum Test (P < 0.001).
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Figure 1: Histological verification of retrobead injection sites and behavioral quantification of fear and reward conditioning for mice used in Figure 1a, Representative differential interference contrast (DIC) image of a 300 μm thick coronal slice containing the center of the retrobead injection in NAc. The white circle is over 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 1.5 mm × 1.5 mm region of the atlas and the corresponding antero-posterior (AP) stereotaxic coordinate relative to Bregma is indicated below c, Representative DIC image of a 300 μm thick coronal slice containing the center of the retrobead injection in CeM.d, Retrobead injection sites in CeM (red circles) for all mice used in Fig. 1. e, Experimental design for AMPAR/NMDAR ratios from Figure 1. Either red or green retrobeads were injected in the NAc and the other color in the contralateral CeM. Two weeks after injection, the retrobeads had travelled back to the cell bodies of the BLA neurons projecting NAc and CeM. Animals were conditioned one day prior to ex-vivo whole cells patch clamp recordings. Each mouse received one of 6 conditioning protocols, 3 protocols categorized under Fear conditioning and 3 protocols categorized under Reward conditioning. Fear conditioning protocols: (i) Naïve: Animals were naïve to the operant chamber. (ii) Unpaired: Animals were exposed to the conditioning chamber in 2 sessions. Animals received 6 tones in the first session and they received 6 foot shocks in the second session. Animals were returned to their home cage for 20 minutes between the two sessions. (iii) Paired: Animals were exposed to operant chamber in two sessions. Animals did not receive any tone or shock stimuli in the first session, and received shocks co-terminating with tones in the second session. Animals were returned to their home cage for 20 minutes between the two sessions. Protocols for Unpaired and Paired fear groups were adapted from Clem and Huganir (2010)18. Reward conditioning protocols: (i) Naïve Food Restricted (FR): Animals naïve to the operant chamber were food restricted two days prior to ex vivo experiments and had free access to food for one 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 Tye et al., 2008 and 20101,2. (ii) Unpaired: Animals received tones in the operant chamber, returned to their home cage for 20 minutes 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 hours after which they had free access to food until ex vivo experiments. Behavioral 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 in 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) Naïve, (2) Unpaired and (3) Paired after fear conditioning, (4) Naïve FR, (5) Unpaired, (6) Non learner and (7) Learner groups after reward conditioning. Data from groups (1)–(5) and (7) are shown in Fig. 1 and Extended Data Fig. 1,2,3. 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 2 last seconds, 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 CS-US 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-CS period (1 to 8 s relative to CS onset, black line) were determined as significantly higher than the number of port entries in the pre-CS period (−8 to −1 s relative to CS onset, grey line) using a one-sided Wilcoxon Rank Sum Test (P < 0.001).

Mentions: To test this, we selected the NAc and CeM as candidate target regions and examined the synaptic changes onto either NAc-projecting BLA neurons (NAc projectors) or CeM-projecting BLA neurons (CeM projectors) following fear conditioning or reward conditioning (Fig. 1). To identify the projection target of BLA neurons, we injected retrogradely-traveling fluorescent beads (retrobeads) into either the NAc or CeM to label BLA neurons sending axon terminals to these regions (Fig. 1a; Extended Data Fig. 1). After retrobead migration upstream to BLA cell bodies, we trained mice in fear or reward conditioning paradigms wherein a tone was paired with either a foot shock or sucrose delivery. Mice in reward conditioning groups were food restricted 1 day before the conditioning session to increase motivation to seek sucrose (Extended Data Fig. 1). AMPAR/NMDAR ratio, a proxy for glutamatergic synaptic strength, increases after either fear or reward conditioning in the BLA1,2,5,18. We used matched experimental parameters across groups in an acute slice preparation stimulating axons arriving via the internal capsule and performing whole-cell patch-clamp recordings in retrobead-identified NAc projectors and CeM projectors, which we observed to be topographically intermingled (Fig. 1b; Extended Data Fig. 2).


A circuit mechanism for differentiating positive and negative associations.

Namburi P, Beyeler A, Yorozu S, Calhoon GG, Halbert SA, Wichmann R, Holden SS, Mertens KL, Anahtar M, Felix-Ortiz AC, Wickersham IR, Gray JM, Tye KM - Nature (2015)

Histological verification of retrobead injection sites and behavioral quantification of fear and reward conditioning for mice used in Figure 1a, Representative differential interference contrast (DIC) image of a 300 μm thick coronal slice containing the center of the retrobead injection in NAc. The white circle is over 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 1.5 mm × 1.5 mm region of the atlas and the corresponding antero-posterior (AP) stereotaxic coordinate relative to Bregma is indicated below c, Representative DIC image of a 300 μm thick coronal slice containing the center of the retrobead injection in CeM.d, Retrobead injection sites in CeM (red circles) for all mice used in Fig. 1. e, Experimental design for AMPAR/NMDAR ratios from Figure 1. Either red or green retrobeads were injected in the NAc and the other color in the contralateral CeM. Two weeks after injection, the retrobeads had travelled back to the cell bodies of the BLA neurons projecting NAc and CeM. Animals were conditioned one day prior to ex-vivo whole cells patch clamp recordings. Each mouse received one of 6 conditioning protocols, 3 protocols categorized under Fear conditioning and 3 protocols categorized under Reward conditioning. Fear conditioning protocols: (i) Naïve: Animals were naïve to the operant chamber. (ii) Unpaired: Animals were exposed to the conditioning chamber in 2 sessions. Animals received 6 tones in the first session and they received 6 foot shocks in the second session. Animals were returned to their home cage for 20 minutes between the two sessions. (iii) Paired: Animals were exposed to operant chamber in two sessions. Animals did not receive any tone or shock stimuli in the first session, and received shocks co-terminating with tones in the second session. Animals were returned to their home cage for 20 minutes between the two sessions. Protocols for Unpaired and Paired fear groups were adapted from Clem and Huganir (2010)18. Reward conditioning protocols: (i) Naïve Food Restricted (FR): Animals naïve to the operant chamber were food restricted two days prior to ex vivo experiments and had free access to food for one 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 Tye et al., 2008 and 20101,2. (ii) Unpaired: Animals received tones in the operant chamber, returned to their home cage for 20 minutes 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 hours after which they had free access to food until ex vivo experiments. Behavioral 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 in 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) Naïve, (2) Unpaired and (3) Paired after fear conditioning, (4) Naïve FR, (5) Unpaired, (6) Non learner and (7) Learner groups after reward conditioning. Data from groups (1)–(5) and (7) are shown in Fig. 1 and Extended Data Fig. 1,2,3. 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 2 last seconds, 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 CS-US 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-CS period (1 to 8 s relative to CS onset, black line) were determined as significantly higher than the number of port entries in the pre-CS period (−8 to −1 s relative to CS onset, grey line) using a one-sided Wilcoxon Rank Sum Test (P < 0.001).
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Related In: Results  -  Collection

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Figure 1: Histological verification of retrobead injection sites and behavioral quantification of fear and reward conditioning for mice used in Figure 1a, Representative differential interference contrast (DIC) image of a 300 μm thick coronal slice containing the center of the retrobead injection in NAc. The white circle is over 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 1.5 mm × 1.5 mm region of the atlas and the corresponding antero-posterior (AP) stereotaxic coordinate relative to Bregma is indicated below c, Representative DIC image of a 300 μm thick coronal slice containing the center of the retrobead injection in CeM.d, Retrobead injection sites in CeM (red circles) for all mice used in Fig. 1. e, Experimental design for AMPAR/NMDAR ratios from Figure 1. Either red or green retrobeads were injected in the NAc and the other color in the contralateral CeM. Two weeks after injection, the retrobeads had travelled back to the cell bodies of the BLA neurons projecting NAc and CeM. Animals were conditioned one day prior to ex-vivo whole cells patch clamp recordings. Each mouse received one of 6 conditioning protocols, 3 protocols categorized under Fear conditioning and 3 protocols categorized under Reward conditioning. Fear conditioning protocols: (i) Naïve: Animals were naïve to the operant chamber. (ii) Unpaired: Animals were exposed to the conditioning chamber in 2 sessions. Animals received 6 tones in the first session and they received 6 foot shocks in the second session. Animals were returned to their home cage for 20 minutes between the two sessions. (iii) Paired: Animals were exposed to operant chamber in two sessions. Animals did not receive any tone or shock stimuli in the first session, and received shocks co-terminating with tones in the second session. Animals were returned to their home cage for 20 minutes between the two sessions. Protocols for Unpaired and Paired fear groups were adapted from Clem and Huganir (2010)18. Reward conditioning protocols: (i) Naïve Food Restricted (FR): Animals naïve to the operant chamber were food restricted two days prior to ex vivo experiments and had free access to food for one 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 Tye et al., 2008 and 20101,2. (ii) Unpaired: Animals received tones in the operant chamber, returned to their home cage for 20 minutes 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 hours after which they had free access to food until ex vivo experiments. Behavioral 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 in 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) Naïve, (2) Unpaired and (3) Paired after fear conditioning, (4) Naïve FR, (5) Unpaired, (6) Non learner and (7) Learner groups after reward conditioning. Data from groups (1)–(5) and (7) are shown in Fig. 1 and Extended Data Fig. 1,2,3. 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 2 last seconds, 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 CS-US 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-CS period (1 to 8 s relative to CS onset, black line) were determined as significantly higher than the number of port entries in the pre-CS period (−8 to −1 s relative to CS onset, grey line) using a one-sided Wilcoxon Rank Sum Test (P < 0.001).
Mentions: To test this, we selected the NAc and CeM as candidate target regions and examined the synaptic changes onto either NAc-projecting BLA neurons (NAc projectors) or CeM-projecting BLA neurons (CeM projectors) following fear conditioning or reward conditioning (Fig. 1). To identify the projection target of BLA neurons, we injected retrogradely-traveling fluorescent beads (retrobeads) into either the NAc or CeM to label BLA neurons sending axon terminals to these regions (Fig. 1a; Extended Data Fig. 1). After retrobead migration upstream to BLA cell bodies, we trained mice in fear or reward conditioning paradigms wherein a tone was paired with either a foot shock or sucrose delivery. Mice in reward conditioning groups were food restricted 1 day before the conditioning session to increase motivation to seek sucrose (Extended Data Fig. 1). AMPAR/NMDAR ratio, a proxy for glutamatergic synaptic strength, increases after either fear or reward conditioning in the BLA1,2,5,18. We used matched experimental parameters across groups in an acute slice preparation stimulating axons arriving via the internal capsule and performing whole-cell patch-clamp recordings in retrobead-identified NAc projectors and CeM projectors, which we observed to be topographically intermingled (Fig. 1b; Extended Data Fig. 2).

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: 1] The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [2] Neuroscience Graduate Program, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
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 positive and negative. Different populations of BLA neurons may encode fearful or rewarding associations, 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.

Show MeSH
Related in: MedlinePlus