Panic results in unique molecular and network changes in the amygdala that facilitate fear responses

Abstract

Recurrent panic attacks (PAs) are a common feature of panic disorder (PD) and post-traumatic stress disorder (PTSD). Several distinct brain regions are involved in the regulation of panic responses, such as perifornical hypothalamus (PeF), periaqueductal gray, amygdala and frontal cortex. We have previously shown that inhibition of GABA synthesis in the PeF produces panic-vulnerable rats. Here, we investigate the mechanisms by which a panic-vulnerable state could lead to persistent fear. We first show that optogenetic activation of glutamatergic terminals from the PeF to the basolateral amygdala (BLA) enhanced the acquisition, delayed the extinction and induced the persistence of fear responses 3 weeks later, confirming a functional PeF-amygdala pathway involved in fear learning. Similar to optogenetic activation of PeF, panic-prone rats also exhibited delayed extinction. Next, we demonstrate that panic-prone rats had altered inhibitory and enhanced excitatory synaptic transmission of the principal neurons, and reduced protein levels of metabotropic glutamate type 2 receptor (mGluR2) in the BLA. Application of an mGluR2-positive allosteric modulator (PAM) reduced glutamate neurotransmission in the BLA slices from panic-prone rats. Treating panic-prone rats with mGluR2 PAM blocked sodium lactate (NaLac)-induced panic responses and normalized fear extinction deficits. Finally, in a subset of patients with comorbid PD, treatment with mGluR2 PAM resulted in complete remission of panic symptoms. These data demonstrate that a panic-prone state leads to specific reduction in mGluR2 function within the amygdala network and facilitates fear, and mGluR2 PAMs could be a targeted treatment for panic symptoms in PD and PTSD patients.

Figure 1.. Optogenetic activation in the amygdala of terminals from the panic-inducing PeF results in enhanced acquisition, consolidation, delayed extinction, and the persistence of conditioned fear.

a) Schematic representation of the experimental setup. b-d) Optogenetic stimulation of PeF glutamatergic inputs in the BLA enhanced acquisition (b) as well as, consolidation (c), and delayed extinction (d) of fear memories compared to animals injected with control virus (n = 8–9/group). e) Three weeks after optical stimulation ChR2-expressing animals demonstrated significantly higher freezing during the spontaneous memory recovery test in the absence of additional optogenetic stimulation. f) Representative coronal hypothalamus containing sections from control (left) or ChR2 (right) animals showing GFP/eYFP (green) and DAPI (blue) double immunostaining. Scale bars, 500 μm. Bregma −2.92. g) Representative high magnification images showing regions in white boxes from (f) with dual GFP/DAPI immunostaining in PeF cells (arrows). green – GFP, blue – DAPI. Scale bars, 10 μm. Bregma −2.92. h) Representative fluorescent images showing GFP- and eYFP-positive PeF terminals (green) and c-fos (red) immunoreactivity (ir) in the BLA. Arrows represent c-fos-ir BLA neurons. Scale bars, 50 μm. Bregma −2.52. i) Group data showing an increased number of c-fos-ir neurons after stimulation of ChR2 PeF terminals in the BLA compared to control animals. *p < 0.05, unpaired t-test. j) Representative BLA traces depicting oEPSPs evoked by light pulses in the presence of aCSF and 10 min after perfusion with AMPA and NMDA antagonists DNQX (20 μm) and APV (25 μm). k) and l) Consistent with optogenetic results, rats with pharmacological disinhibition of PeF neurons, i.e., panic-prone rats (chronic l-AG infusions into the PeF to inhibit GABA synthesis) compared to control rats (chronic inactive d-AG infusions into the PeF), displayed delayed extinction of fear. Except where otherwise specified, *p < 0.05, ANOVA, Sidak’s within subject posthoc analysis, ^#p < 0.05, ANOVA, Sidak’s between subjects posthoc analysis. All data reported as mean + S.E.M.

Figure 2.. Disinhibition of panic network (with l-AG) was associated with an increase in the excitability of BLA pyramidal neurons.

a,e) BLA neurons from I-AG animals (n = 14) displayed higher input resistance compared to d-AG controls (n = 19). a) Representative whole cell voltage responses to 600 pA current 700 ms pulses from the BLA of l-AG and d-AG animals. e) Group data indicating significantly higher input resistance in BLA neurons recorded from l-AG animals (l-AG, n = 9) compared to d-AG controls (d-AG, n = 14). b,f) BLA neurons from l-AG animals (n = 17) have smaller inward rectification (sag) compared to d-AG controls (n = 15). b) Whole cell voltage responses to 1200 pA current 700 ms pulses from the BLA neurons of l-AG and d-AG animals. f) Summary of the sag amplitude in d-AG and l-AG animals. c,d,g) I_h was suppressed in BLA neurons from l-AG animals (n = 14) compared to d-AG controls (n = 12). c) Representative current traces of I_h induced by applying hyperpolarizing voltage steps from −60 to 120 mV (step = −10 mV) in d-AG (top) and l-AG animals (middle). Representative examples of raw current traces (bottom) in response to voltage step to −110 mV from −60 mV. d) Plots of instantaneous current (I_ins, ● and ●, as shown in (c) and steady state currents (I_ss, ■ and ■, as shown in (c) against the membrane potential in the BLA neurons from l-AG (n = 14) and d-AG animals (n = 12). g) Plots of I_h against the membrane potential in the BLA neurons from l-AG (n = 14) and d-AG animals (n = 12). Note that the difference between I_ss and I_ins corresponds to I_h. *p < 0.05, ANOVA, Sidak’s between subjects posthoc analysis. h) and o) Depolarizing currents induced significantly more APs in neurons recorded from l-AG animals (n = 19), compared to d-AG animals (n = 14). *p < 0.05, ANOVA, Sidak’s between subjects posthoc analysis. i) Example traces showing the voltage response to current step used to induce a mAHP (APs are truncated). j) Group data indicating significant effect of l-AG treatment (l-AG, n = 9) on the amplitude of mAHP (d-AG, n = 14). l) Half-width and m) decay of APs of BLA neurons from l-AG animals (n = 16) were significantly different compared to d-AG rats (n = 16). k) Example traces of ePSPs in the BLA neurons from l-AG and d-AG animals. n) Group data showing that l-AG treatment significantly reduced amplitude of eIPSPs without affecting amplitude of eEPSPs (n = 18). p) Representative recordings of spontaneous activity in l-AG and d-AG animals. r) Group data showing that amplitude and frequency of sIPSPs were significantly reduced in l-AG animals (n = 16) compared to d-AG rats (n = 18) at holding potential of −50 mV. s,q) Group data showing that frequency, but not amplitude of sEPSPs were significantly increased in l-AG animals (n = 16) compared to d-AG control (n = 18) at holding potential of −50 mV (s) and −70 mV (q). Except where otherwise specified, *p < 0.05, t-test. All data reported as mean + S.E.M.

Figure 3.. Increased BLA excitability seen in panic-prone animals was associated with decreased mGluR2 protein and gene expression in the BLA and CeA and mGluR2 PAM reduced enhanced glutamatergic neurotransmission in the BLA.

Pre-treatment with l-AG induced significant changes of a,d) GABA related genes in the BLA, and b,e) GABA/Glutamate related genes in the CeA. c) Group data and representative Western blots, normalized to β-actin, illustrating reduced mGluR2 protein levels in the BLA and CeA of l-AG rats (n = 8–9) compared to d-AG controls (n = 7). f,i) Bath application of JNJ-42153605 significantly reduced the amplitude of oEPSPs evoked by light stimulation of PeF→BLA terminals in ChR2-expressing animals (n = 8) compared to time control group (n = 4). Representative traces of oEPSPs evoked by light pulses before (black trace) and during (gray trace) bath application of JNJ-42153605 (i). *p ≤ 0.05, ANOVA. g,j) Bath application of JNJ-42153605 significantly reduced the frequency (j), but not the amplitude (g) of sEPSPs in recordings from the BLA of l-AG (n = 9) and d-AG animals (n = 9). h,k) Pre-incubation with JNJ-42153605 did not affect the amplitude (h) and the frequency (k) of sEPSPs in recordings from the CeA of l-AG (n = 7) and d-AG rats (n = 7). l) Bar graph indicating significant reduction of paired-pulse ratio (oEPSP₂/oEPSP₁) recorded 5 min before and 10 min after bath application of JNJ-42153605 (n = 8). Except where otherwise specified, *p < 0.05, t-test. All data represented as mean + S.E.M.

Figure 4.. Pretreating panic-prone rats (chronic l-AG) with mGluR2 PAM attenuated NaLac-induced panic symptoms and facilitated extinction of fear, without affecting general locomotor activity.

a) Anxiety-like behavior (as measured by reduced SI times) displayed by animals treated with 5 and 20 mg/kg i.p. of JNJ-40411813. b,c) JNJ-4041183 did not affect locomotor activity of panic-prone rats as measured by distance travelled (b) and average speed (c) during open field test. d) Bar graph demonstrating that panic-prone animals showed significant increase of social avoidance episodes. Pretreatment with 5 mg/kg or 20 mg/kg of JNJ-40411813 significantly reduced number of social avoidance episodes. *p < 0.05, compared to pre-l-AG group, ^#p < 0.05, compared to l-AG group. e) Heart rate response displayed by animals treated with 5 and 20 mg/kg doses of JNJ-40411813. n = 6–7 per group. Gray shading in line graphs indicates onset and duration of intravenous 0.5M NaLac infusions. ^#, ^a and ^bp < 0.05. f,g) Pretreating panic-prone rats with mGluR2 PAM JNJ-42153605 (20 mg/kg), did not alter acquisition of fear-induced freezing (f), but did attenuate the resistant extinction of fear-induced freezing on the recall/extinction (g). n = 5 per group. *p < 0.05, ANOVA. All data represented as mean + S.E.M.

Figure 5.. Data demonstrating clinical effects of the mGluR2 PAM JNJ-40411813 on panic symptoms.

The changes in Panic Disorder Severity Scale (PDSS) show that all five subjects exhibited remission of their panic symptoms following 2 – 4 weeks of mGluR2 PAM therapy. NOTE: PDSS range = 0 – 28, Scores ≥ 8 consistent with DSM-IV Panic Disorder.