Gastrin-releasing peptide signaling plays a limited and subtle role in amygdala physiology and aversive memory

Abstract

Links between synaptic plasticity in the lateral amygdala (LA) and Pavlovian fear learning are well established. Neuropeptides including gastrin-releasing peptide (GRP) can modulate LA function. GRP increases inhibition in the LA and mice lacking the GRP receptor (GRPR KO) show more pronounced and persistent fear after single-trial associative learning. Here, we confirmed these initial findings and examined whether they extrapolate to more aspects of amygdala physiology and to other forms of aversive associative learning. GRP application in brain slices from wildtype but not GRPR KO mice increased spontaneous inhibitory activity in LA pyramidal neurons. In amygdala slices from GRPR KO mice, GRP did not increase inhibitory activity. In comparison to wildtype, short- but not long-term plasticity was increased in the cortico-lateral amygdala (LA) pathway of GRPR KO amygdala slices, whereas no changes were detected in the thalamo-LA pathway. In addition, GRPR KO mice showed enhanced fear evoked by single-trial conditioning and reduced spontaneous firing of neurons in the central nucleus of the amygdala (CeA). Altogether, these results are consistent with a potentially important modulatory role of GRP/GRPR signaling in the amygdala. However, administration of GRP or the GRPR antagonist (D-Phe(6), Leu-NHEt(13), des-Met(14))-Bombesin (6-14) did not affect amygdala LTP in brain slices, nor did they affect the expression of conditioned fear following intra-amygdala administration. GRPR KO mice also failed to show differences in fear expression and extinction after multiple-trial fear conditioning, and there were no differences in conditioned taste aversion or gustatory neophobia. Collectively, our data indicate that GRP/GRPR signaling modulates amygdala physiology in a paradigm-specific fashion that likely is insufficient to generate therapeutic effects across amygdala-dependent disorders.

Figure 1. The gastrin-releasing peptide receptor is expressed in interneurons in the lateral amygdala and affects amygdala physiology.

A) In situ hybridization of the GRPR in the amygdala. B) Binary version of A) that more clearly distinguishes the ISH signal (white dots), mainly in the lateral (LA) and basolateral (BLA) with only very few labeled cells in the central lateral (CeL) and central medial (CeM) nuclei. C) Higher power image showing ISH signal in neurons that were D) co-immunolabeled for eGFP being expressed under control of the GAD67 promoter. E) Sample recordings of spontaneous inhibitory postsynaptic currents recorded from LA pyramidal neurons in control conditions, in the presence of GRP and after addition of picrotoxin in slices made from WT and GRPR KO mice. The patch pipette contained high Cl⁻ therefore IPSCs were inward at the holding potential of −70 mV. CNQX (20 µM) was present to block fast excitatory activity. Picrotoxin (100 µM) blocked all the inward currents confirming their inhibitory nature. F) Quantification of the results from 6 slices from WT and 4 slices from GRPR KO mice. G) Typical example of the most rostral and ventral in vivo recording position in the central medial nucleus of the amygdala. H) Cumulative frequency plot of CeM single unit activity from 12 WT and 12 GRPR KO mice. Inset shows a sample record.

Figure 2. Expression of conditioned fear is altered in GRPR KO mice after single-pairing but not multiple-pairing conditioning.

A,B) Fear conditioning was induced by a single CS-US (tone-shock) pairing in context 1. 24 h and 2 weeks later the freezing response in the same context was tested A and response to the cue alone was tested in a new context B (WT n = 12, GRPR KO n = 11). C,D) To test for extinction of conditioned fear GRPR KO (n = 12) and WT mice (n = 11) were subjected to multiple CS-US (tone-shock) pairing in context 1. Freezing levels during acquisition are shown in the inset in C. C) At the start of extinction training, baseline (pre-cue) and cue-related freezing responses were tested in a new context. D) At the end of 4 days of extinction training, the freezing response to the cue was tested in mice that were handled but not given the training (no Extinction; n = 12) and mice subjected to extinction training (Extinction; 10 presentations of CS alone each day).

Figure 3. Long-term potentiation in the LA is not changed in GRPR KO mice or by agonist/antagonist application.

A) Thalamic afferents were stimulated to evoke field excitatory postsynaptic potentials (fEPSPs) in the LA. Inset shows sample averaged traces (10 sweeps) from the 10 min baseline period (black) immediately before applying the tetanus (5×100 Hz/1 s trains, 20 s inter-train interval) and 40 min after the tetanus (grey). B) Mean ± s.e.m. of the change in fEPSP slope in the first 2 minutes after the tetanus (PTP) and 30–40 min after the tetanus. C,D) As in A,B except that cortical afferents were stimulated to evoke fEPSPs in the LA. E,F) Cortical afferents were stimulated at 30 s intervals to evoke EPSCs recorded from LA pyramidal neurons at −70 mV with the whole-cell voltage clamp technique. After a 10 min baseline 80 stimuli at 2 Hz were paired with depolarization to 30 mV. G,H) Long-term potentiation of cortico-LA fEPSPs induced by 5×100 Hz/1 s trains was not affected by bath application of 1 µM (D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴)-Bombesin(6–14). Reducing inhibitory inputs by addition of 5 µM picrotoxin increased LTP. I,J) 1 µM GRP also did not significantly affect cortico-LA LTP.

Figure 4. Exogenous GRP or GRPR antagonist did not affect expression of conditioned fear.

A) 600 ng GRP or 3000 ng GRPR antagonist (D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴)-Bombesin(6–14) was infused into the amygdala of C57BL/6 mice, that were conditioned with 6 CS-US pairings as in Fig. 2C, 10 min prior to testing freezing in the conditioning context 24 h later. B) Effect of intra-amygdala infusion of 600 ng GRP 10 min prior to testing freezing in response to the CS. C) Location of the bilateral injection sites determined from post-hoc histological analysis.

Figure 5. GRPR KO animals showed no differences in conditioned tast aversion (CTA) or neophobia.

A) CTA was evoked by pairing a novel taste, saccharin, with a LiCl injection to induce illness the day before testing (LiCl; n = 12 mice per group). Control animals were offered the novel taste saccharin but injected with NaCl (NaCl groups; n = 11 mice per group) or given only water to drink and injected with NaCl the previous day (saccharin naive groups; n = 12 mice per group). B) Attenuation of neophobia and neophobia were assessed by comparing the aversion to saccharin on first exposure (saccharin naive) with the aversion shown by mice that were exposed to saccharin the previous day (NaCl). On successive days the neophobia was attenuated by repeatedly being given the chance to drink saccharin flavored water.