Valerio Crestan

A Neural Switch for Active and Passive Fear

The central nucleus of the amygdala (CeA) serves as a major output of this structure and plays a critical role in the expression of conditioned fear. By combining cell- and tissue-specific pharmacogenetic inhibition with functional magnetic resonance imaging (fMRI), we identified circuits downstream of CeA that control fear expression in mice. Selective inhibition of a subset of neurons in CeA led to decreased conditioned freezing behavior and increased cortical arousal as visualized by fMRI. Correlation analysis of fMRI signals identified functional connectivity between CeA, cholinergic forebrain nuclei, and activated cortical structures, and cortical arousal was blocked by cholinergic antagonists. Importantly, inhibition of these neurons switched behavioral responses to the fear stimulus from passive to active responses. Our findings identify a neural circuit in CeA that biases fear responses toward either passive or active coping strategies.

Differential Effects of Antipsychotic and Glutamatergic Agents on the phMRI Response to Phencyclidine

Acute administration of NMDA receptor (NMDAR) antagonists such as phencyclidine (PCP) or ketamine induces symptoms that closely resemble those of schizophrenia in humans, a finding that has led to the hypothesis that a decreased NMDAR function may be a predisposing or even causative factor in schizophrenia. However, the precise neuropharmacological mechanisms underlying these effects remain to be fully elucidated. Here, we applied pharmacological MRI (phMRI) to examine the brain circuitry underlying the psychotomimetic action of PCP in the anesthetized rat, and investigated how these functional changes are modulated by drugs that possess distinct pharmacological mechanisms. Acute administration of PCP (0.5u2009mg/kg i.v.) produced robust and sustained positive relative cerebral blood volume (rCBV) changes in discrete cortico-limbo-thalamic regions. Pretreatment with the selective D2 dopamine antagonist raclopride (0.3u2009mg/kg i.p.) did not significantly affect the rCBV response to PCP, while the atypical antipsychotic clozapine (5u2009mg/kg i.p.) produced region-dependent effects, with complete suppression of the rCBV response in the thalamus, and weaker attenuation of the response in cortical and hippocampal structures. The response to PCP was strongly suppressed in all regions by pretreatment with two drugs that can inhibit aberrant glutamatergic activity: the anticonvulsant lamotrigine (10u2009mg/kg i.p.) and the mGluR2/3 agonist LY354740 (10u2009mg/kg i.p.). Taken together, our findings corroborate the pivotal role of dysfunctional glutamatergic neurotransmission in the functional response elicited by PCP, while the lack of effect of raclopride argues against a primary role of dopamine D2 receptor activation in this process. Finally, the thalamic effect of clozapine could be key to elucidating the functional basis of its pharmacological action.

Concurrent pharmacological MRI and in situ microdialysis of cocaine reveal a complex relationship between the central hemodynamic response and local dopamine concentration.

The mechanisms underlying the signal changes observed with pharmacological magnetic resonance imaging (phMRI) remain to be fully elucidated. In this study, we obtained microdialysis samples in situ at 5-min intervals during phMRI experiments using a blood pool contrast agent to correlate relative cerebral blood volume (rCBV) changes with changes in dopamine and cocaine concentrations following acute cocaine challenge (0.5 mg/kg iv) in the rat over a duration of 30 min. Three brain areas were investigated: the dorsal striatum (n = 8), the medial prefrontal cortex (mPFC; n = 5), and the primary motor cortex (n = 8). In the striatum and mPFC groups, cocaine and dopamine temporal profiles were tightly correlated, peaking during the first 5-min period postinjection, then rapidly decreasing. However, the local rCBV changes were uncorrelated and exhibited broader temporal profiles than those of cocaine and dopamine, attaining maximal response 5-10 min later. This demonstrates that direct vasoactivity of dopamine is not the dominant component of the hemodynamic response in these regions. In the motor cortex group, microdialysis revealed no local change in dopamine in any of the animals, despite large local cocaine increase and strong rCBV response, indicating that the central hemodynamic response following acute iv cocaine challenge is not driven directly by local dopamine changes in the motor cortex. The combination of phMRI and in situ microdialysis promises to be of great value in elucidating the relationship between the phMRI response to psychoactive drugs and underlying neurochemical changes.

Region-specific effects of nicotine on brain activity: a pharmacological MRI study in the drug-naïve rat.

We have applied pharmacological magnetic resonance imaging (phMRI) methods to map the functional response to nicotine in drug-naïve rats. Nicotine (0.35u2009mg/kg intravenous (i.v.)) increased relative cerebral blood volume (rCBV) in cortical (including medial prefrontal, cingulate orbitofrontal, insular) and subcortical (including amygdala and dorsomedial hippocampus) structures. The pharmacological specificity of the effect was demonstrated by acute pretreatment with the nicotinic acetylcholine receptor (nAChR) ion-channel-blocking agent mecamylamine, which suppressed the rCBV response to nicotine. Control experiments with norepinephrine, a potent non-brain-penetrant vasopressor, at a dose that mimics the cardiovascular response induced by nicotine were performed to assess the potential confounding effects of peripheral blood pressure changes induced by nicotine. In an attempt to highlight the relative contribution of different nAChR subtypes to the observed activation pattern of nicotine, we also investigated the central phMRI response to an acute challenge with (R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide) (cpdA, at 5, 10, 20, and 30u2009mg/kg i.v.) and 5-iodo-A-85380 (5IA, 5u2009mg/kg i.v.). CpdA is a selective agonist at homomeric α7 nAChRs, while 5IA features high in vivo affinity for the α4β2* and other less-abundant β2-containing nicotinic receptors. CpdA did not produce significant rCBV changes at any of the doses tested, whereas 5IA induced a pattern of activation very similar to that induced by nicotine. The lack of phMRI response to cpdA together with the high spatial overlap between the activation profile of nicotine and 5IA, suggest that the acute functional response to nicotine in drug-naïve rats is mediated by β2-containing nAChR isoforms, presumably belonging to the α4β2* subtype.

Pharmacological stimulation of NMDA receptors via co-agonist site suppresses fMRI response to phencyclidine in the rat

RationaleIncreasing experimental evidence suggests that impaired N-methyl-d-aspartic acid (NMDA) receptor (NMDAr) function could be a key pathophysiological determinant of schizophrenia. Agonists at the allosteric glycine (Gly) binding site of the NMDA complex can promote NMDAr activity, a strategy that could provide therapeutic efficacy for the disorder. NMDAr antagonists like phencyclidine (PCP) can induce psychotic and dissociative symptoms similar to those observed in schizophrenia and are therefore widely used experimentally to impair NMDA neurotransmission in vivo.ObjectivesIn the present study, we used pharmacological magnetic resonance imaging (phMRI) to investigate the modulatory effects of endogenous and exogenous agonists at the NMDAr Gly site on the spatiotemporal patterns of brain activation induced by acute PCP challenge in the rat. The drugs investigated were d-serine, an endogenous agonist of the NMDAr Gly site, and SSR504734, a potent Gly transporter type 1 (GlyT-1) inhibitor that can potentiate NMDAr function by increasing synaptic levels of Gly.ResultsAcute administration of PCP induced robust and sustained activation of discrete cortico-limbo-thalamic circuits. Pretreatment with d-serine (1xa0g/kg) or SSR504734 (10xa0mg/kg) completely inhibited PCP-induced functional activation. This effect was accompanied by weak but sustained deactivation particularly in cortical areas.ConclusionsThese findings suggest that agents that stimulate NMDAr via Gly co-agonist site can potentiate NMDAr activity in the living brain and corroborate the potential for this class of drugs to provide selective enhancement of NMDAr neurotransmission in schizophrenia.

Drug–anaesthetic interaction in phMRI: the case of the psychotomimetic agent phencyclidine

Pharmacological magnetic resonance imaging (phMRI) provides a powerful means to map the effects of drugs on brain activity, with important applications in pharmacological research. However, phMRI studies in preclinical species are often conducted under general anaesthesia as a means to avoid head motion and to minimise the stress induced by the procedure. Under these conditions, the phMRI response to the drug of interest may be affected by interactions with the anaesthetic agent, with consequences for the interpretation of the data. Here, we have investigated the phMRI response to phencyclidine (PCP), an NMDA receptor blocker, in the halothane-anaesthetised rat for varying levels of anaesthesia and different PCP challenge doses. PCP induces psychotic-like symptoms in humans and laboratory animals and is widely applied as a pharmacological model of schizophrenia. However, PCP possesses anaesthetic properties per se, and its interactions with halothane might result in significant effects on the phMRI activation patterns. We observed two qualitatively different patterns of phMRI response. At 0.5 mg/kg iv PCP and 0.8% halothane maintenance anaesthesia, the lowest doses explored, an activation of discrete cortico-limbo-thalamic structures was observed, consistent with neuroimaging studies in humans and 2-deoxyglucose functional mapping in conscious animal models. However, higher anaesthetic concentrations or higher PCP challenge doses resulted in complete abolition of the positive response and in a widespread cortical deactivation (negative response). In the intermediate regime, we observed a dichotomic behaviour, with individual subjects showing one pattern or the other. These findings indicate a dose-dependent drug-anaesthetic interaction, with a complete reversal of the effects of PCP at higher challenge doses or HT concentrations.

A robust experimental protocol for pharmacological fMRI in rats and mice.

Pharmacological Magnetic Resonance Imaging (phMRI) methods have significantly expanded the stimulation repertoire available to preclinical fMRI research, by allowing to selectively probe the activity of specific brain circuitries and neurotransmitter systems. However, the application of phMRI to animal models is constrained by a number of experimental factors. Firstly, in order to prevent motion artefacts and reduce restraint-induced stress, phMRI studies are typically performed under anaesthesia. Moreover, several psychoactive drugs produce blood pressure changes and alterations in respiratory frequency that may perturb central haemodynamic readouts of brain function. Hence, the quality and outcome of phMRI studies is critically dependent on the ability to monitor and control peripheral physiological parameters (i.e. blood pressure, arterial blood gases) that could alter phMRI readouts. Here we provide a thorough methodological description of a robust protocol to measure drug-induced cerebral blood volume changes in anaesthetised rats and mice. We show that the protocol ensures stable physiological parameters and robust phMRI response to the psychostimulant drug d-amphetamine in three different rat strains. We also document the successful application of the protocol to map the central effects produced by d-amphetamine in C57Bl/6J mice, a strain commonly used as background for the generation of transgenic lines, thus paving the way to the implementation of phMRI in genetically engineered animals.

Antagonism at serotonin 5-HT2A receptors modulates functional activity of frontohippocampal circuit

RationaleSeveral second-generation antipsychotics are characterised by a significant antagonistic effect at serotonin 5-HT2A receptors (5-HT2AR), a feature that has been associated with lower incidence of extra-pyramidal symptoms and a putative amelioration of positive and negative symptoms experienced by schizophrenic patients. However, the neurofunctional substrate of 5-HT2A antagonism and its exact contribution to the complex pharmacological profile of these drugs remain to be elucidated.ObjectivesHere, we used pharmacological magnetic resonance imaging to map the modulatory effects of the selective 5-HT2AR antagonist Ml00907 on the spatiotemporal patterns of brain activity elicited by acute phencyclidine (PCP) challenge in the rat. PCP is a non-competitive NMDA receptor antagonist that induces dysregulation of corticolimbic glutamatergic neurotransmission and produces cognitive impairment and psychotic-like symptoms reminiscent of those observed in schizophrenia.ResultsPre-administration of M100907 produced focal and region-dependent attenuation of PCP-induced response in frontoseptohippocampal areas. As early studies highlighted a permissive role of 5-HT2AR on frontal dopamine release, the role of post-synaptic dopamine D1 receptors on PCP-induced response was examined by using the potent antagonist SCH23390. Interestingly, SCH23390 did not affect PCP’s response in any of the regions examined. This finding rules out a significant contribution of dopamine in the functional changes mapped and, indirectly, the inhibitory effect of M100907, in favour of a glutamatergic origin.ConclusionsOur data expand recent evidence suggesting a key role of 5-HT2AR in modulating glutamate-mediated cognitive performance in the prefrontal cortex and highlight the whole frontoseptohippocampal circuit as a key functional substrate of 5-HT2AR antagonism in normal and disease states.

Functional magnetic resonance mapping of intracerebroventricular infusion of a neuroactive peptide in the anaesthetised rat

Pharmacological magnetic resonance imaging (phMRI) methods map the cerebral haemodynamic response to challenge with psychotropic agents as a surrogate for drug-induced changes in brain activity. However, many neuroactive compounds present low blood-brain barrier penetration and thus systemic administration may result in insufficient brain concentration. Intracerebroventricular (ICV) administration has been long used as an effective way of bypassing the blood-brain barrier in studies with non-brain-penetrant compounds, such as neuropeptides. In order to extend the range of pharmacological substances accessible to phMRI, we have developed methods to map relative cerebral blood volume (rCBV) changes induced by in situ ICV administration of neuroactive agents in the anaesthetised rat. We have applied this method to study for the first time the phMRI response to central administration of a neuropeptide, the metabolically stable and potent NK1 receptor agonist GR-73632. ICV administration of 4.2 pmol of GR-73632 produced a rapid onset and sustained rCBV increase in several brain structures, such as the amygdala, the caudate putamen and the cortex. These results demonstrate the feasibility of phMRI as a tool to study the functional correlates of brain activity induced by central administration of neuroactive agents.

Brain penetration of local anaesthetics in the rat: Implications for experimental neuroscience.

Multiple experimental neuroscience techniques rely on the use of general anaesthesia to minimize the discomfort associated to animal restraint and to achieve a more effective control of relevant physiological parameters. In order to minimise potential interference on brain neuronal activity, such studies are typically conducted at low anaesthetic doses. This practice is often coupled to peripheral infiltration of local anaesthetics to provide supplementary analgesia and prevent sub-threshold activation of pain pathways that may confound central measurements of brain function. However, little is known of the effect of peripheral anaesthesia on central measurements of brain activity in small laboratory animal species. In order to begin to address this question, we measured total and free brain exposure of five different local anaesthetics following subcutaneous infiltration of analgesic doses in a surgical protocol widely used in rodent neuroimaging and electrophysiology studies. Notably, all the anaesthetics exhibited detectable total and free brain concentrations at all the time points examined. Lidocaine and mepivacaine showed the highest free brain exposures (>525 ng/g), followed by bupivacaine and ropivacaine (>70 ng/g). The ester-type local anaesthetic tetracaine produced the lowest free brain exposure (<8.6 ng/g). Our data suggest that peripheral administration of local anaesthetics in small laboratory animals could result in pharmacologically active brain exposures that might influence and confound central measurements of brain function. The use of the ester-type anaesthetic tetracaine produced considerably lower brain exposure, and may represent a viable experimental option when local anaesthesia is required.