Gastrin-Releasing Peptide Signaling Plays a Limited and Subtle Role in Amygdala Physiology and Aversive Memory

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-Phe6, Leu-NHEt13, des-Met14)-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

The rostral anterior cingulate cortex modulates depression but not anxiety-related behaviour in the rat.

A growing body of functional imaging studies suggests that human depression and anxiety symptoms are associated with functional abnormalities in the circuitry formed by the rostral anterior cingulate cortex (rACC) and its direct limbic and paralimbic connections. In rodents however, the role of the rACC (rCG1/rCG2) remains unknown in depression-related behaviours and elusive in acute anxiety. In order to address this, we specifically lesioned the rat rCG1/rCG2, and assessed the behavioural outcome using a modified forced swim test (FST) and the elevated plus maze (EPM), tests for depression and anxiety related behaviours respectively. Lesions of the rostral anterior cingulate cortex significantly increased the time spent immobile in the FST without affecting climbing or swimming performances, suggesting a pro-depressant effect. On the contrary, none of the parameters measured in the EPM was affected by the lesion. These data point to an involvement of the rCG1/rCG2 in depression-related coping behaviours

The rostral anterior cingulate cortex modulates the efficiency of amygdala-dependent fear learning.

BACKGROUND: The rostral anterior cingulate cortex (rACC) and the amygdala consistently emerge from neuroimaging studies as brain regions crucially involved in normal and abnormal fear processing. To date, however, the role of the rACC specifically during the acquisition of auditory fear conditioning still remains unknown. The aim of this study is to investigate a possible top-down control of a specific rACC sub-region over amygdala activation during pavlovian fear acquisition. METHODS: We performed excitotoxic lesions, temporal inactivation, and activation of a specific sub-region of the rACC that we identified by tracing studies as supporting most of the connectivity with the basolateral amygdala (r(Amy)-ACC). The effects of these manipulations over amygdala function were investigated with a classical tone-shock associative fear conditioning paradigm in the rat. RESULTS: Excitotoxic lesions and transient inactivation of the r(Amy)-ACC pre-training selectively produced deficits in the acquisition of the tone-shock associative learning (but not context). This effect was specific for the acquisition phase. However, the deficit was found to be transient and could be overcome by overtraining. Conversely, pre-training transient activation of the r(Amy)-ACC facilitated associative learning and increased fear expression. CONCLUSIONS: Our results suggest that a subregion of the rACC is key to gating the efficiency of amygdala-dependent auditory fear conditioning learning. Because r(Amy)-ACC inputs were confirmed to be glutamatergic, we propose that recruitment of this brain area might modulate overall basolateral amygdala excitatory tone during conditioned stimulus-unconditioned stimulus concomitant processing. In the light of clinical research, our results provide new insight on the effect of inappropriate rACC recruitment during emotional events

Differential roles of mGlu7 and mGlu8 in amygdala physiology and in amygdala-dependent behavior

Glutamate transmission within the amygdala is crucial for amygdaloid plasticity and the learning and expression of conditioned fear. Glutamate activates both ionotropic glutamate receptors and eight subtypes of metabotropic glutamate receptors (mGlu1-8). In the present study, the roles of mGlu7 and mGlu8 in different in vitro and in vivo paradigms of amygdaloid plasticity and behavior were investigated. We show that mGlu7-deficient but not mGlu8-deficient mice have attenuated long-term potention (LTP) within the amygdala. mGlu7-deficient mice express a general deficit in conditioned fear wheras in mGlu8-deficient mice, only contextual fear is strongly reduced . The mGlu7 agonist AMN082 reduces amygdaloid LTP and blocks the learning of conditioned fear after intra-amygdala injections. In contrast, the mGlu8 agonist DCPG decreased synaptic transmission within the amygdala but not LTP. Intra-amygdala injections of DCPG do only affect expression of contextual fear but not the learning and expression of cued fear. Taken together, these data revealed very different roles for amygdaloid mGlu7 and mGlu8 in the learning and expression of conditioned fear. Both receptors may be promising targets for the treatment of anxiety disorders; mGlu7 for anxiety disorders with pathological fear learning and mGlu8 for anxiety disorders with exaggerated contextual fear

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

<p><b>A</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034963#pone-0034963-g002" target="_blank">Fig. 2C</a>, 10 min prior to testing freezing in the conditioning context 24 h later. <b>B</b>) Effect of intra-amygdala infusion of 600 ng GRP 10 min prior to testing freezing in response to the CS. <b>C</b>) Location of the bilateral injection sites determined from post-hoc histological analysis.</p

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

<p><b>A</b>) 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>B</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.</p

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

<p><b>A</b>) 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>B</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. <b>C,D</b>) As in A,B except that cortical afferents were stimulated to evoke fEPSPs in the LA. <b>E,F</b>) 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. <b>G,H</b>) 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. <b>I,J</b>) 1 µM GRP also did not significantly affect cortico-LA LTP.</p