Differential roles of mGlu(7) and mGlu(8) in amygdala-dependent behavior and physiology.

Glutamate transmission and synaptic plasticity in the amygdala are essential for 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, we investigated the roles of mGlu7 and mGlu8 in amygdala-dependent behavior and synaptic plasticity. We show that ablation of mGlu7 but not mGlu8 attenuates long-term potentiation (LTP) at thalamo-lateral amygdala (LA) synapses where a strong association between LTP and learning has been demonstrated. mGlu7-deficient mice express a general deficit in conditioned fear whereas mGlu8-deficient mice show a dramatic reduction in contextual fear. The mGlu7 agonist AMN082 reduced thalamo-LA LTP and intra-amygdala administration blocked conditioned fear learning. In contrast, the mGlu8 agonist DCPG decreased synaptic transmission but not LTP at thalamo-LA synapses. Intra-amygdala DCPG selectively reduced the expression of contextual fear but did not affect the acquisition and expression of cued fear. Taken together, these data revealed very different roles for mGlu7 and mGlu8 in amygdala synaptic transmission, fear learning and its expression. These receptors seem promising targets for treating anxiety disorders with different underlying pathologies with exaggerated fear learning (mGlu7) or contextual fear (mGlu8).

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-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.

Endocrine and behavioural responses to acute central CRF challenge are antagonized in the periphery and CNS, respectively, in C57BL/6 mice.

Corticotropin releasing factor (CRF) is a major mediator of central and peripheral responses to environmental stressors, and antagonism of its receptors (CRF-R1, -R2) is an active area of pharmacotherapeutic research for stress-related disorders. Stress responses include CRF activation of the hypothalamus-pituitary-adrenal axis and behavioural inhibition. Valid in vivo models for the study of these neuro-endocrine and -behavioural CRF pathways and their central-peripheral antagonism are important. The aims of this study in C57BL/6 mice were to describe the acute effects of intracerebroventricular (ICV) CRF using plasma ACTH-CORT titres and locomotor activity as readouts, and to study the impact on these readouts of central versus peripheral pre-treatment with the CRF-R1/2 antagonist, astressin. The following experiments were performed: Effects of (i) serial blood sampling (SBS) per se, (ii) physical confinement+SBS, (iii) ICV saline infusion+SBS, on plasma titres of ACTH-CORT. (iv) Effects of ICV or IP CRF infusion on plasma ACTH-CORT. (v) Effects of ICV CRF on plasma CRF. (vi) Effects of ICV or IP astressin on ICV or IP CRF-stimulated plasma CORT. (vii) Effects of ICV or IP astressin on ICV CRF-induced locomotor inactivity. Main findings were: (i)-(ii) Serial blood sampling per se and physical confinement+SBS led to similar, mild increases in plasma ACTH-CORT. (iii) ICV saline infusion led to a marked increase in plasma ACTH, possibly due to assay crossreactivity with "washed out" pituitary peptides, and a mild increase in plasma CORT. (iv) ICV CRF (0.001-1µg) induced no further increase in plasma ACTH versus vehicle, and induced dose-dependent increased plasma CORT. 1µg ICV CRF also reduced locomotor activity. (v) ICV CRF-induced dose-dependent increased plasma CRF. (vi) ICV astressin failed to block ICV CRF-induced increased plasma CORT, whereas IP astressin did do so. (vii) ICV CRF-induced locomotor inactivity was blocked by ICV astressin, but not by IP astressin. Therefore, ICV CRF-induced a dose-dependent increase in plasma CORT via a peripheral pathway and a reduction in locomotion via a central pathway, indicated by the double dissociation in the ability of astressin to antagonize these effects relative to its route of administration, IP or ICV, respectively. The preparation described here could be readily used to provide initial indications on the central and peripheral activity of CRF-R antagonists, including pharmacokinetics following peripheral administration.

Intra-amygdala injections of neuropeptide S block fear-potentiated startle.

Injections of neuropeptide S (NPS) into the lateral ventricle induce a strong hyperactivity. Since most behavioral paradigms are dependent of spontaneous locomotor activity, this makes it difficult to interpret the role of NPS in such paradigms. The aim of the present experiment was to investigate the effects of NPS in fear-potentiated startle, a behavioral fear paradigm which we believe is less sensitive to general changes in locomotor activity. Furthermore, NPS was directly injected into the amygdala, the central site of the neural fear circuitry. Our data shows that intra-amygdala NPS injections dose-dependently block the expression of conditioned fear and that this effect is independent of NPS effects on locomotor activity. This strongly supports a crucial role of amygdaloid NPS in conditioned fear.

Fear-reducing effects of intra-amygdala neuropeptide Y infusion in animal models of conditioned fear: an NPY Y1 receptor independent effect.

RATIONALE: Neuropeptide Y (NPY) and its receptors are densely localized in brain regions involved in the mediation and modulation of fear, including the amygdala. Several studies showed that central NPY is involved in the modulation of fear and anxiety. OBJECTIVES: In the present study, we investigated (1) whether intra-amygdala injections of NPY affect the expression of conditioned fear and (2) whether NPY Y1 receptors (Y1R) mediates the effects of these intra-amygdaloid NPY injections. RESULTS: Intra-amygdala NPY injections robustly decreased the expression of conditioned fear measured by conditioned freezing and fear-potentiated startle. These NPY effects were not mimicked by intra-amygdala injections of the Y1R agonists Y-28 or Y-36, and co-infusion of the Y1R antagonist BIBO 3304 did not block the NPY effects. Furthermore, we tested Y1R-deficient mice in conditioned freezing and found no differences between wild type and mutant littermates. Finally, we injected NPY into the amygdala of Y1R-deficient mice. Y1R deficiency had no effect on the fear-reducing effects of intra-amygdala NPY. CONCLUSIONS: These data show an important role of the transmitter NPY within the amygdala for the expression of conditioned fear. Y1R do not appear to be involved in the mediation of the observed intra-amygdala NPY effects suggesting that these effects are mediated via other NPY receptors.

Translocator protein (18 kD) as target for anxiolytics without benzodiazepine-like side effects.

Most antianxiety drugs (anxiolytics) work by modulating neurotransmitters in the brain. Benzodiazepines are fast and effective anxiolytic drugs; however, their long-term use is limited by the development of tolerance and withdrawal symptoms. Ligands of the translocator protein [18 kilodaltons (kD)] may promote the synthesis of endogenous neurosteroids, which also exert anxiolytic effects in animal models. Here, we found that the translocator protein (18 kD) ligand XBD173 enhanced gamma-aminobutyric acid-mediated neurotransmission and counteracted induced panic attacks in rodents in the absence of sedation and tolerance development. XBD173 also exerted antipanic activity in humans and, in contrast to benzodiazepines, did not cause sedation or withdrawal symptoms. Thus, translocator protein (18 kD) ligands are promising candidates for fast-acting anxiolytic drugs with less severe side effects than benzodiazepines.

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.

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.

A selective metabotropic glutamate receptor 7 agonist: activation of receptor signaling via an allosteric site modulates stress parameters in vivo.

Metabotropic glutamate receptor (mGluR) subtypes (mGluR1 to mGluR8) act as important pre- and postsynaptic regulators of neurotransmission in the CNS. These receptors consist of two domains, an extracellular region containing the orthosteric agonist site and a transmembrane heptahelical domain involved in G protein activation and recognition of several recently synthesized pharmacological modulators. The presynaptic receptor mGluR7 shows the highest evolutionary conservation within the family, but no selective pharmacological tool was known. Here we characterize an mGluR7-selective agonist, N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), which directly activates receptor signaling via an allosteric site in the transmembrane domain. At transfected mammalian cells expressing mGluR7, AMN082 potently inhibits cAMP accumulation and stimulates GTPgammaS binding (EC50-values, 64-290 nM) with agonist efficacies comparable with those of L-2-amino-4-phosphonobutyrate (L-AP4) and superior to those of L-glutamate. AMN082 (< or = 10 microM) failed to show appreciable activating or inhibitory effects at other mGluR subtypes and selected ionotropic GluRs. Chimeric receptor studies position the binding site of AMN082 in the transmembrane region of mGluR7, and we demonstrate that this allosteric agonist has little, if any, effect on the potency of orthosteric ligands. Here we provide evidence for full agonist activity mediated by the heptahelical domain of family 3 G protein-coupled receptors (which have mGluR-like structure) that may lead to drug development opportunities. Further, AMN082 is orally active, penetrates the blood-brain barrier, and elevates the plasma stress hormones corticosterone and corticotropin in an mGluR7-dependent fashion. Therefore, AMN082 is a valuable tool for unraveling the role of mGluR7 in stress-related CNS disorders.

Differential effects of iloperidone, clozapine, and haloperidol on working memory of rats in the delayed non-matching-to-position paradigm.

RATIONALE: Because cognitive function, particularly working memory (WM), is severely impaired in schizophrenia, evaluation of neuroleptic medication should include investigation of possible effects on cognition. Iloperidone is a promising, novel atypical neuroleptic drug (NL), for which no cognitive data is presently available. OBJECTIVE: To investigate whether the novel atypical NL iloperidone would affect performance of rats on a WM test, using a delayed non-matching-to-position (DNMTP) paradigm, and compare its effects with those of the atypical NL clozapine and the typical NL haloperidol. METHODS: Male Lister Hooded rats trained to criterion in an operant DNMTP task (0-64 s delay intervals) were administered vehicle, iloperidone (0.03, 0.1 mg/kg, i.p.), clozapine (0.1, 0.3 mg/kg, s.c.), haloperidol (0.003, 0.01, 0.03 mg/kg, s.c.), or scopolamine (0.05 mg/kg, s.c.). Together with choice accuracy, the motor performance of the task was measured. RESULTS: It was found that: (1) iloperidone significantly improved choice accuracy delay-dependently while impairing task performance; (2) the atypical NL clozapine had no effect on choice accuracy and parameters related to motor function, but significantly increased the number of uncompleted trials; (3) haloperidol did not affect choice accuracy except at the longest delay with the highest dose, but in contrast to clozapine it significantly impaired task performance. CONCLUSION: In accordance with their different pharmacological profiles, the three NLs iloperidone, clozapine, and haloperidol have different effects in this preclinical cognitive task. These results might provide important information for the development of NLs with beneficial effects on cognition.