Annarosa Ugolini

Chronic stress-induced alterations in amygdala responsiveness and behavior – modulation by trait anxiety and corticotropin-releasing factor systems

The basolateral nucleus of the amygdala (BLA) plays a key role in emotional arousal and anxiety, and expresses high levels of corticotropin‐releasing factor receptor (CRFR)1. In rat brain slices, we have recently shown that afferent activation of the BLA is increased following application of exogenous corticotropin‐releasing factor (CRF). Here we examined the impact of chronic unpredictable stress (CUS) on this effect of CRF and whether blockade of CRFR1 could prevent stress‐induced changes in the electrophysiological response, the animal’s behavior and in cell proliferation in the hippocampus. The behavior of the rats was monitored via a series of tests that formed part of the CUS. Electrophysiological measures of the BLA response to CRF, cell proliferation in the dentate gyrus and the expression of CRF and CRFR1 mRNA in amygdaloid nuclei were determined ex vivo after completion of the CUS. CRF‐induced potentiation of afferent activation of the BLA was reduced in rats exposed to CUS, an effect that was inhibited by chronic antagonism of CRFR1. Furthermore, the reduction in BLA response to CRF was correlated with the anxiety trait of the animals, determined prior to initiation of the CUS. These results implicate CRFR1 in chronic stress‐induced alterations in amygdala function and behavior. Furthermore, they show that CRFR1 antagonists can prevent changes induced by chronic stress, in particular in those animals that are highly anxious.

Synthesis and pharmacological characterisation of 2,4-Dicarboxy-pyrroles as selective non-Competitive mGluR1 antagonists

Metabotropic glutamate receptors (mGluRs) are an unusual family of G-protein coupled receptor (GPCR), and are characterised by a large extracellular N-terminal domain that contains the glutamate binding site. We have identified a new class of non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonists, 2,4-dicarboxy-pyrroles which are endowed with nanomolar potency. They interact within the 7 transmembrane (7TM) domain of the receptor and show antinociceptive properties when tested in a number of different animal models.

CRF1 receptor activation increases the response of neurons in the basolateral nucleus of the amygdala to afferent stimulation

The basolateral nucleus (BLA) of the amygdala contributes to the consolidation of memories for emotional or stressful events. The nucleus contains a high density of CRF1 receptors that are activated by corticotropin-releasing factor (CRF). Modulation of the excitability of neurons in the BLA by CRF may regulate the immediate response to stressful events and the formation of associated memories. In the present study, CRF was found to increase the amplitude of field potentials recorded in the BLA following excitatory afferent stimulation, in vitro. The increase was mediated by CRF1 receptors, since it could be blocked by the selective, non-peptide antagonists, NBI30775 and NBI35583, but not by the CRF2-selective antagonist, astressin 2B. Furthermore, the CRF2-selective agonist, urocortin II had no effect on field potential amplitude. The increase induced by CRF was long-lasting, could not be reversed by subsequent administration of NBI35583, and required the activation of protein kinase C. This effect of CRF in the BLA may be important for increasing the salience of aversive stimuli under stressful conditions, and for enhancing the consolidation of associated memories. The results provide further justification for studying the efficacy of selective antagonists of the CRF1 receptor to reduce memory formation linked to emotional or traumatic events, and suggest that these compounds might be useful as prophylactic treatments for stress-related illnesses such as post-traumatic stress disorder.

AMN082, an allosteric mGluR7 agonist that inhibits afferent glutamatergic transmission in rat basolateral amygdala.

Glutamatergic neurotransmission has been implicated in the pathophysiology of psychiatric disorders, such as anxiety and depression. The possible contribution of group III metabotropic glutamate receptors has been poorly investigated, due to the lack of selective pharmacological tools. However, a selective agonist of mGLUR7, AMN082 has been identified recently, and shown to act through an allosteric mechanism in recombinant cells expressing the receptor. Thus, using AMN082, we examined the role of mGLUR7 in modulating synaptic transmission in the rat basolateral amygdala (BLA), a brain region known to be important for the genesis of anxious states. We found that bath application of AMN082 (1-10microM) produced a concentration-dependent inhibition of synaptic transmission evoked at 2Hz, but had no effect on transmission evoked at 0.05Hz. However, at this lower frequency, AMN082 (10microM) significantly increased the synaptic inhibition produced by a group III mGLUR agonist, L-AP4 (100microM). This effect was blocked by pre-application of CPPG (500microM), a group III mGLUR-preferring antagonist, consistent with the involvement of mGLUR7. Thus, we have shown that AMN082 can modulate high frequency synaptic transmission in the BLA, in vitro, and appears to act on the receptor via an allosteric mechanism. These results suggest that mGLUR7 has a unique role in regulating neuronal activity in the BLA and may be a target for novel drugs for the treatment of anxiety.

Transient forebrain over-expression of CRF induces plasma corticosterone and mild behavioural changes in adult conditional CRF transgenic mice.

BACKGROUND Converging findings support a role for extra-hypothalamic CRF in the mediation of the stress response. The influence of CRF in the amygdala is well established, while less is known of its role in other areas of the forebrain where CRF and CRF(1) receptors are also expressed. In the present study CRF was genetically induced to allow forebrain-restricted expression in a temporally-defined manner at any time during the mouse lifespan. This mouse model may offer the possibility to establish a model of the pathogenesis of recurrent episodes of depression. METHODS Mice were engineered to carry both the rtTA transcription factor driven by the CamKII alpha promoter and the doxycycline-regulated operator (tetO) upstream of the CRF coding sequence. Molecular, biochemical and behavioural characterisation of this mouse is described. RESULTS Following a three-week period of transcriptional induction, double transgenic mice showed approximately 2-fold increased expression of CRF mRNA in the hippocampus and cortex, but not hypothalamus. These changes were associated with 2-fold increase in morning corticosterone levels, although responses to the dexamethasone suppression test or acute stress were unaffected. In contrast, induced mice displayed modestly altered behaviour in the Light and Dark test and Forced Swim test. CONCLUSIONS Transient induction of CRF expression in mouse forebrain was associated with endocrine and mild anxiety-like behavioural changes consistent with enhanced central CRF neurotransmission. This mouse allows the implementation of regimens with longer or repeated periods of induction which may model the initial stages of the pathology underlying recurrent depressive disorders.

The identification of structurally novel, selective, orally bioavailable positive modulators of mGluR2

The optimisation of an HTS hit series (1) leading to the identification of structurally novel, selective, orally bioavailable mGluR2 positive modulators GSK1331258 and GSK1331268 is described. Structure-activity relationships, attenuation of dopaminergic activity, and potentiation of mGluR2 responses in rat hippocampal MPP-DG synapses are also reported.

Combined treatment with a selective PDE10A inhibitor TAK‐063 and either haloperidol or olanzapine at subeffective doses produces potent antipsychotic‐like effects without affecting plasma prolactin levels and cataleptic responses in rodents

Activation of indirect pathway medium spiny neurons (MSNs) via promotion of cAMP production is the principal mechanism of action of current antipsychotics with dopamine D2 receptor antagonism. TAK‐063 [1‐[2‐fluoro‐4‐(1H‐pyrazol‐1‐yl)phenyl]‐5‐methoxy‐3‐(1‐phenyl‐1H‐pyrazol‐5‐yl)pyridazin‐4(1H)‐one] is a novel phosphodiesterase 10A inhibitor that activates both direct and indirect pathway MSNs through increasing both cAMP and cGMP levels by inhibition of their degradation. The activation of indirect pathway MSNs through the distinct mechanism of action of these drugs raises the possibility of augmented pharmacological effects by combination therapy. In this study, we evaluated the potential of combination therapy with TAK‐063 and current antipsychotics, such as haloperidol or olanzapine after oral administration. Combined treatment with TAK‐063 and either haloperidol or olanzapine produced a significant increase in phosphorylation of glutamate receptor subunit 1 in the rat striatum. An electrophysiological study using rat corticostriatal slices showed that TAK‐063 enhanced N‐methyl‐D‐aspartic acid receptor‐mediated synaptic responses in both direct and indirect pathway MSNs to a similar extent. Further evaluation using pathway‐specific markers revealed that coadministration of TAK‐063 with haloperidol or olanzapine additively activated the indirect pathway, but not the direct pathway. Combined treatment with TAK‐063 and either haloperidol or olanzapine at subeffective doses produced significant effects on methamphetamine‐ or MK‐801‐induced hyperactivity in rats and MK‐801‐induced deficits in prepulse inhibition in mice. TAK‐063 at 0.1 mg/kg did not affect plasma prolactin levels and cataleptic response from antipsychotics in rats. Thus, TAK‐063 may produce augmented antipsychotic‐like activities in combination with antipsychotics without effects on plasma prolactin levels and cataleptic responses in rodents.

S223. COMBINED TREATMENT WITH A SELECTIVE PDE10A INHIBITOR TAK-063 AND ANTIPSYCHOTICS AT SUBEFFECTIVE DOSES PRODUCES POTENT ANTIPSYCHOTIC-LIKE EFFECTS WITHOUT EXACERBATING SIDE EFFECTS PROFILE IN RODENTS

Abstract Background Activation of indirect pathway medium spiny neurons (MSNs) via promotion of cAMP production is the principal mechanism of action (MOA) of current antipsychotics with dopamine D2 receptor antagonism. Phosphodiesterase 10A (PDE10A) inhibitors activate both direct and indirect pathway MSNs by increasing both cAMP and cGMP levels by inhibiting their degradation, which might be expected to promote activation of intracellular signaling similar to that of D2 antagonists in the indirect pathway MSNs. Thus, the activation of the indirect MSN pathway through the distinct MOA of these compounds raises the possibility of augmented pharmacologic effects with combined treatment. In this study, we compared gene-regulation patterns in the indirect pathway MSNs induced by the PDE10A inhibitors T-773 and T-609, and the D2 antagonist haloperidol, using a cell-type-specific comprehensive gene expression analysis in Drd2-bacTRAP (translating ribosome affinity purification) mice. The pharmacologic effects of combined treatment with another PDE10A inhibitor, TAK-063, and clinically used antipsychotics, haloperidol (HAL) and olanzapine (OLA), were evaluated in multiple rodent models. Methods Male ICR mice, Drd2-bacTRAP mice, and Sprague-Dawley rats were used. The indirect pathway MSN-specific gene expression changes by T-773, T-609, and HAL were investigated using RNA sequencing of striatal samples of Drd2-bacTRAP mice. The activation of MSNs in rats was evaluated by measuring glutamate receptor subunit 1 phosphorylation (pGluR1) levels. An in vitro electrophysiological study on the corticostrial pathway in rats was conducted in a slice preparation. The activation of each MSN pathway was assessed by inducing genes as pathway-specific markers: enkephalin for the indirect pathway and substance P for the direct pathway. Suppression of MK-801- or methamphetamine (METH)-induced hyperactivity was assessed by measuring locomotor activity for 2 hours after administration of these stimulants to rats. Improvement of prepulse inhibition (PPI) was investigated in a MK-801-induced PPI deficit mouse model. Results Translational profiling in Drd2-bacTRAP mice treated with the PDE10A selective inhibitors, T-773 and T-609, and with HAL suggested regulatory of a largely overlapping signaling pathway by these compounds in the indirect pathway MSNs: 87% of the genes regulated by HAL were also regulated by both T-773 and T-609. Combined treatment with TAK-063 and either HAL or OLA produced an augmented effect on pGluR1 in the rat striatum. An electrophysiological study in rat brain slices indicated that TAK-063 enhanced synaptic responses to a similar extent in both direct and indirect pathway MSNs. Additional evaluation using MSN pathway-specific markers revealed that coadministration of TAK-063 with HAL or OLA additively activated the indirect pathway, but not the direct pathway. Combined treatment with TAK-063 (0.1 mg/kg p.o.) and either HAL (0.3 mg/kg p.o.) or OLA (3 mg/kg p.o.) at subeffective doses produced augmented effects on METH- or MK-801–induced hyperactivity in rats and MK-801–induced PPI deficits in mice. TAK-063 at 0.1 mg/kg did not affect plasma prolactin levels and cataleptic response induced by HAL or OLA in rats. Discussion PDE10A inhibitors and HAL showed similar patterns of gene regulation in indirect pathway MSNs in mice. Combined treatment with TAK-063 and either HAL or OLA at subeffective doses produced significant antipsychotic-like effects but no augmentation of the plasma prolactin level and cataleptic response. Although further preclinical and clinical studies will be needed, TAK-063 may provide a novel mechanism as a PDE10A inhibitor for use as combination therapy in schizophrenia.