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Neuronal encoding of the switch from specific to generalized fear

Nature Neuroscience volume 18, pages 112–120 (2015)Cite this article

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Abstract

Fear memories are crucial for survival. However, excessive generalization of such memories, characterized by a failure to discriminate dangerous from safe stimuli, is common in anxiety disorders. Neuronal encoding of the transition from cue-specific to generalized fear is poorly understood. We identified distinct neuronal populations in the lateral amygdala (LA) of rats that signaled generalized versus cue-specific associations and determined how their distributions switched during fear generalization. Notably, the same LA neurons that were cue specific before the behavioral shift to generalized fear lost their specificity afterwards, thereby tilting the balance of activity toward a greater proportion of generalizing neurons. Neuronal activity in the LA, but not the auditory cortex, was necessary for fear generalization. Furthermore, targeted activation of cAMP–PKA signaling in the LA increased neuronal excitability of LA neurons and led to generalized fear. These results provide a cellular basis in the amygdala for the alteration of emotional states from normal to pathological fear.

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Figure 1: Discriminative conditioning with weak US causes increase in CS+-evoked, but not CS−-evoked, spiking in LA neurons.
Figure 2: A strong US causes generalization of conditioned fear and changes the population distribution of LA neurons encoding cue specificity versus generalization.
Figure 3: Neuronal activity in the LA, but not ACx, is necessary for generalization of conditioned fear.
Figure 4: Distinct neuronal transitions when the same animal switched from low to high fear generalization.
Figure 5: Occlusion of further shifts in behavioral and neuronal generalization in animals exhibiting intrinsically higher generalization after weak US conditioning.
Figure 6: Activation of cAMP–PKA signaling in the LA during weak US conditioning is sufficient to cause fear generalization.
Figure 7: Increased spiking at shorter latencies in response to tone and shock after switch to higher generalization.

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Acknowledgements

We are grateful to A. Lüthi and M. Thattai for discussions and advice. This work was supported by the National Centre for Biological Sciences and Department of Biotechnology, India, and an International Senior Research Fellowship to S.C. (GR 0701339 MA) from The Wellcome Trust, UK.

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  1. Supriya Ghosh

    Present address: Present address: Department of Neurobiology, University of Chicago, Chicago, Illinois, USA.,

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  1. National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India

    Supriya Ghosh & Sumantra Chattarji

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Contributions

S.G. and S.C. designed the experiments. S.G. performed the experiments and analyzed the data. S.G. and S.C. interpreted the results and wrote the manuscript.

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Correspondence to Sumantra Chattarji.

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Integrated supplementary information

Supplementary Figure 1 Cluster sorting for isolation of single units in chronic extracellular recordings from the LA

(a-c) Sorted clusters from a single electrode on consecutive days based upon principal component scores. (d) Cluster quality based upon J3 and Davies-Bouldin index (DB) statistics. (e) Cluster stability across recording sessions was estimated by Mahalanobis-distance between the waveforms.

Supplementary Figure 2 Classification of cue-specific and generalizing neurons using a bootstrap resampling method

(a, c) Scatter plots showing re-sampled (n = 1,000) z-scores averaged over 5 trials over a duration of 200 ms after tone-onset during the habituation (gray) and testing (blue) sessions for a representative Cue-specific (a) and Generalizing (c) neuron. In each re-sampling, five trials were picked up randomly with replacement. Line plots on top and bottom right depict the distribution of the z-scores for CS+ and CS– respectively. (b, d) Re-sampled normalized z-scores (bottom left) for the representative neurons shown in (a) and (c). Distributions of responses evoked by CS+ (top) and CS– (bottom right) showing a CS+-specific (b) and non-specific (d) increase (>1) after conditioning.

Supplementary Figure 3 Placement of infusion cannulae into the LA

(a) Representative coronal brain section stained with cresyl violet (0.2%), indicating bilateral cannulae tacks, implanted chronically for drug infusion (muscimol or forskolin) into the LA. (b) Schematic representations of coronal sections showing cannulae placements (blue lines) in the LA for all experimental animals (N = 27).

Supplementary Figure 4 Infusion of muscimol into the auditory cortex (ACx) prior to conditioning reversibly blocks neuronal spiking

(a) Representative coronal sections showing the infusion site and spread of a fluorescently labeled muscimol (MW, 607 g) across most of the ACx. Dose: 1.0 μl/side, 10 mM. (b) Simultaneous in vivo recording of neuronal spiking and muscimol infusion in the ACx, indicating inactivation of spontaneous firing 20 min after the infusion (1.0 μl/side, 1 μg/μl), which recovered 24 h later [at a time point when fear memory was tested in conditioned animals (Fig. 3)].

Supplementary Figure 5 Placement of infusion cannulae into the ACx for muscimol experiments

Schematic representations of coronal sections showing cannulae placements (blue lines) in the ACx for all experimental animals (N = 6).

Supplementary Figure 6 Transitions in the responses of LA neurons to CS+ and CS– over the course of enhanced generalization in the same animal.

Raster plots (top) and peristimulus time histograms (bottom) of a LA neuron during the behavioral switch from low (testing, day 2) to high (testing, day 3) generalized fear. A neuron that did not change its response after weak-US conditioning (i.e. Non-conditioned cell, day 2), fired more strongly to the CS+ than CS– after strong-US conditioning (i.e. Cue-specific, day 3). Brown bar indicates presentation of the auditory tone (bin size, 20 ms). Insets show superimposed spike waveforms.

Supplementary Figure 7 Reconditioning with the same weak US does not cause generalization at the behavioral and neuronal levels.

(a) Experimental protocol. The same animals were conditioned twice with the same intensity of weak-US (0.5 mA). (b) Mean freezing levels (N = 6) in the same rats. During habituation, CS+ and CS– elicited equally low levels of freezing (grey square). During testing, 1 day after weak-US conditioning, only the CS+, evoked significantly higher freezing compared to habituation (solid blue square; p < 0.01) and CS– (p < 0.05). Reconditioning with the same weak-US failed to cause any further significant increase in freezing values to either tone (open blue square; p > 0.05). Average IBG values (inset) were low after weak-US conditioning and additional session of weak-US conditioning failed to cause any further change (p > 0.05). (c) Scatter plots illustrating population distribution of all LA neurons (n = 51, N = 6) based on their normalized responses to the CS+ and CS–, after 1st weak and 2nd weak-US conditioning. (d) Pie-plots illustrating non-significant shifts in population distribution of neuronal responses in the LA during the 1st weak-US to 2nd weak-US reconditioning [Generalizing: 8% (4/51) → 6% (3/51), Cue-specific: 42% (21/51) → 46% (23/51); n = 51, χ2(2) = 0.25, p = 0.88]. (e) Population responses of all cue-specific and generalizing neurons during habituation and testing trials (n = 26/51). Weak-US conditioning induced a significant increase in the response to the CS+ (p < 0.01) and but not CS–. The two responses were also significantly different, and this was unchanged even after 2nd weak-US conditioning (p < 0.01). Brown horizontal bar, tone-presentation. Bin size, 20 ms. Error bars, ±s.e.m.

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Supplementary Figure 8 Spontaneous firing in LA neurons was greater in rats exhibiting high (N = 6; Fig. 5) levels of fear generalization compared to those showing low (N = 8; Fig. 4) fear generalization after weak US conditioning.

Cumulative probability distribution of spontaneous firing rates of all recorded LA neurons (solid lines) and only those showing conditioning-induced increase in firing (dotted lines) from rats exhibiting low (grey, n = 131, N = 8) and high (black, n = 114, N = 6) fear generalization. A subset of all the recorded LA neurons exhibited tone-evoked increased spiking after weak-US conditioning from low (grey, n = 37/131) and high (black, n = 43/114) generalizing rats. The frequency of spontaneous firing in all the recorded neurons (k = 0.28; p < 0.001, Kolmogorov-Smirnov test), as well as the smaller subset of conditioned neurons (k = 0.33; p = 0.02, Kolmogorov-Smirnov test), are significantly greater in the rats exhibiting high fear generalization compared to those showing low fear generalization. Spontaneous firing rate is estimated over 100 s pre-tone period (10 s, 10 trials) during habituation session. ***p < 0.001; *p< 0.05.

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Ghosh, S., Chattarji, S. Neuronal encoding of the switch from specific to generalized fear. Nat Neurosci 18, 112–120 (2015). https://doi.org/10.1038/nn.3888

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