Antibiotic-induced microbiota perturbation causes gut endocannabinoidome changes, hippocampal neuroglial reorganization and depression in mice.

The microbiota-gut-brain axis (MGBA) regulates the reciprocal interaction between chronic inflammatory bowel and psychiatric disorders. This interaction involves multiple pathways that are highly debated. We examined the behavioural, biochemical and electrophysiological alterations, as well as gut microbiota composition in a model of antibiotic-induced experimental dysbiosis. Inflammation of the small intestine was also assessed. Mice were exposed to a mixture of antimicrobials for 2weeks. Afterwards, they received Lactobacillus casei DG (LCDG) or a vehicle for up to 7days via oral gavage. Perturbation of microbiota was accompanied by a general inflammatory state and alteration of some endocannabinoidome members in the gut. Behavioural changes, including increased immobility in the tail suspension test and reduced social recognition were observed, and were associated with altered BDNF/TrkB signalling, TRPV1 phosphorylation and neuronal firing in the hippocampus. Moreover, morphological rearrangements of non-neuronal cells in brain areas controlling emotional behaviour were detected. Subsequent probiotic administration, compared with vehicle, counteracted most of these gut inflammatory, behavioural, biochemical and functional alterations. Interestingly, levels of Lachnospiraceae were found to significantly correlate with the behavioural changes observed in dysbiotic mice. Our findings clarify some of the biomolecular and functional modifications leading to the development of affective disorders associated with gut microbiota alterations.

Palmitoylethanolamide reduces pain-related behaviors and restores glutamatergic synapses homeostasis in the medial prefrontal cortex of neuropathic mice.

BACKGROUND: Enhanced supraspinal glutamate levels following nerve injury are associated with pathophysiological mechanisms responsible for neuropathic pain. Chronic pain can interfere with specific brain areas involved in glutamate-dependent neuropsychological processes, such as cognition, memory, and decision-making. The medial prefrontal cortex (mPFC) is thought to play a critical role in pain-related depression and anxiety, which are frequent co-morbidities of chronic pain. Using an animal model of spared nerve injury (SNI) of the sciatic nerve, we assess bio-molecular modifications in glutamatergic synapses in the mPFC that underlie neuropathic pain-induced plastic changes at 30 days post-surgery. Moreover, we examine the effects of palmitoylethanolamide (PEA) administration on pain-related behaviours, as well as the cortical biochemical and morphological changes that occur in SNI animals. RESULTS: At 1 month, SNI was associated with mechanical and thermal hypersensitivity, as well as depression-like behaviour, cognitive impairments, and obsessive-compulsive activities. Moreover, we observed an overall glutamate synapse modification in the mPFC, characterized by changes in synaptic density proteins and amino acid levels. Finally, with regard to the resolution of pain and depressive-like syndrome in SNI mice, PEA restored the glutamatergic synapse proteins and changes in amino acid release. CONCLUSIONS: Given the potential role of the mPFC in pain mechanisms, our findings may provide novel insights into neuropathic pain forebrain processes and indicate PEA as a new pharmacological tool to treat neuropathic pain and the related negative affective states. Graphical Abstract Palmitoylethanolamide: a new pharmacological tool to treat neuropathic pain and the related negative affective states.

A role for D-aspartate oxidase in schizophrenia and in schizophrenia-related symptoms induced by phencyclidine in mice.

Increasing evidence points to a role for dysfunctional glutamate N-methyl-D-aspartate receptor (NMDAR) neurotransmission in schizophrenia. D-aspartate is an atypical amino acid that activates NMDARs through binding to the glutamate site on GluN2 subunits. D-aspartate is present in high amounts in the embryonic brain of mammals and rapidly decreases after birth, due to the activity of the enzyme D-aspartate oxidase (DDO). The agonistic activity exerted by D-aspartate on NMDARs and its neurodevelopmental occurrence make this D-amino acid a potential mediator for some of the NMDAR-related alterations observed in schizophrenia. Consistently, substantial reductions of D-aspartate and NMDA were recently observed in the postmortem prefrontal cortex of schizophrenic patients. Here we show that DDO mRNA expression is increased in prefrontal samples of schizophrenic patients, thus suggesting a plausible molecular event responsible for the D-aspartate imbalance previously described. To investigate whether altered D-aspartate levels can modulate schizophrenia-relevant circuits and behaviors, we also measured the psychotomimetic effects produced by the NMDAR antagonist, phencyclidine, in Ddo knockout mice (Ddo(-)(/-)), an animal model characterized by tonically increased D-aspartate levels since perinatal life. We show that Ddo(-/-) mice display a significant reduction in motor hyperactivity and prepulse inhibition deficit induced by phencyclidine, compared with controls. Furthermore, we reveal that increased levels of D-aspartate in Ddo(-/-) animals can significantly inhibit functional circuits activated by phencyclidine, and affect the development of cortico-hippocampal connectivity networks potentially involved in schizophrenia. Collectively, the present results suggest that altered D-aspartate levels can influence neurodevelopmental brain processes relevant to schizophrenia.

Dysfunctional dopaminergic neurotransmission in asocial BTBR mice.

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by pronounced social and communication deficits and stereotyped behaviours. Recent psychosocial and neuroimaging studies have highlighted reward-processing deficits and reduced dopamine (DA) mesolimbic circuit reactivity in ASD patients. However, the neurobiological and molecular determinants of these deficits remain undetermined. Mouse models recapitulating ASD-like phenotypes could help generate hypotheses about the origin and neurophysiological underpinnings of clinically relevant traits. Here we used functional magnetic resonance imaging (fMRI), behavioural and molecular readouts to probe dopamine neurotransmission responsivity in BTBR T(+) Itpr3(tf)/J mice (BTBR), an inbred mouse line widely used to model ASD-like symptoms owing to its robust social and communication deficits, and high level of repetitive stereotyped behaviours. C57BL/6J (B6) mice were used as normosocial reference comparators. DA reuptake inhibition with GBR 12909 produced significant striatal DA release in both strains, but failed to elicit fMRI activation in widespread forebrain areas of BTBR mice, including mesolimbic reward and striatal terminals. In addition, BTBR mice exhibited no appreciable motor responses to GBR 12909. DA D1 receptor-dependent behavioural and signalling responses were found to be unaltered in BTBR mice, whereas dramatic reductions in pre- and postsynaptic DA D2 and adenosine A2A receptor function was observed in these animals. Overall these results document profoundly compromised DA D2-mediated neurotransmission in BTBR mice, a finding that is likely to have a role in the distinctive social and behavioural deficits exhibited by these mice. Our results call for a deeper investigation of the role of dopaminergic dysfunction in mouse lines exhibiting ASD-like phenotypes, and possibly in ASD patient populations.

Free D-aspartate regulates neuronal dendritic morphology, synaptic plasticity, gray matter volume and brain activity in mammals.

D-aspartate (D-Asp) is an atypical amino acid, which is especially abundant in the developing mammalian brain, and can bind to and activate N-methyl-D-Aspartate receptors (NMDARs). In line with its pharmacological features, we find that mice chronically treated with D-Asp show enhanced NMDAR-mediated miniature excitatory postsynaptic currents and basal cerebral blood volume in fronto-hippocampal areas. In addition, we show that both chronic administration of D-Asp and deletion of the gene coding for the catabolic enzyme D-aspartate oxidase (DDO) trigger plastic modifications of neuronal cytoarchitecture in the prefrontal cortex and CA1 subfield of the hippocampus and promote a cytochalasin D-sensitive form of synaptic plasticity in adult mouse brains. To translate these findings in humans and consistent with the experiments using Ddo gene targeting in animals, we performed a hierarchical stepwise translational genetic approach. Specifically, we investigated the association of variation in the gene coding for DDO with complex human prefrontal phenotypes. We demonstrate that genetic variation predicting reduced expression of DDO in postmortem human prefrontal cortex is mapped on greater prefrontal gray matter and activity during working memory as measured with MRI. In conclusion our results identify novel NMDAR-dependent effects of D-Asp on plasticity and physiology in rodents, which also map to prefrontal phenotypes in humans.

The A1 adenosine receptor as a new player in microglia physiology.

The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2 X4,7 and P2 Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca(2+) influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive-specific neuron before and after application of resting or ATP-treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone.

Abnormal NMDA receptor function exacerbates experimental autoimmune encephalomyelitis.

BACKGROUND AND PURPOSE: Glutamate transmission is dysregulated in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the animal model of MS. A characteristic of EAE is increased glutamate transmission associated with up-regulation of AMPA receptors. However, little is known about the role of NMDA receptors in the synaptic modifications induced by EAE. EXPERIMENTAL APPROACH: The contribution of NMDA receptors to the alterations of glutamate transmission and disease severity in EAE mice was assessed by means of neurophysiological, morphological, Western blot, metabolic and clinical score assessments. KEY RESULTS: In our EAE mice, there was an NMDA receptor-dependent increase of glutamate release, associated with marked activation of the astroglia. Presynaptic NMDA receptors became overactive during EAE, increasing synaptic glutamate release by a mechanism dependent on voltage-gated sodium channels. By means of NAD(P)H autofluorescence analysis, we also found that EAE has a glutamate and NMDA receptor-dependent dysfunction of mitochondrial activity, which is known to contribute to the neurodegenerative damage of MS and EAE. Furthermore, pharmacological blockade of NMDA receptors in vivo ameliorated both synaptic transmission defects and of the clinical disease course of EAE mice, while EAE induced in mice with a genetically enhanced NMDA receptor signalling had opposite effects. CONCLUSIONS AND IMPLICATIONS: Our data, showing both sensitization of NMDA receptors and their involvement in the progression of the EAE disease, supggest that pharmacological impairment of NMDA receptor signalling would be a component of a neuroprotection strategy in MS.

Bimodal effect of D-aspartate on brain aging processes: insights from animal models.

Nowadays it is widely recognized that D-amino acids are present in bacteria as well as in eukaryotes, including mammals. In particular, free D-serine and D-aspartate are found in the brain of mammals. Notably, D-aspartate occurs at substantial levels in the embryo brain to then consistently decrease at post-natal phases. Temporal regulation of D-aspartate content depends on the post-natal onset of D-aspartate oxidase expression, the only known enzyme able to catabolize this D-amino acid. Pharmacological evidence indicates that D-aspartate binds and activates NMDA receptors (NMDARs). To decipher the physiological function of D-aspartate in mammals, in the last years, genetic and pharmacological mouse models with abnormally higher levels of this D-amino acid have been generated. Overall, these animal models have pointed out a significant neuromodulatory role for D-aspartate in the regulation of NMDAR-dependent functions. Indeed, increased content of D-aspartate are able to increase hippocampal NMDAR-dependent long-term potentiation (LTP) and spatial memory of adult mice. However, if exposure to elevated levels of D-Asp lasts for the entire lifetime of mice, enhancement of synaptic plasticity turns into a dramatic worsening, thus triggering an acceleration of the NMDAR-dependent aging processes in the hippocampus. Nonetheless, administration of D-Asp to old mice can restore the physiological age-related decay of hippocampal NMDA-related LTP. Besides its effect on hippocampus-dependent processes in mouse models, different points of evidence are indicating, today, a potential role for D-Asp in neurologic and psychiatric disorders associated with aberrant signalling of NMDARs.

Vitamin A deficiency induces motor impairments and striatal cholinergic dysfunction in rats.

Vitamin A and its derivatives, retinoids, are involved in the regulation of gene expression by binding two nuclear receptor families, retinoic acid receptors and retinoid X receptors. Retinoid receptors are highly expressed in the striatum, revealing an involvement of this system in the control of movement as demonstrated by previous observations in knockout mice. To further assess the role of retinoids in adult striatal function, the present study investigated the effect of vitamin A deprivation on rat motor activity and coordination, the rate of synthesis and release of dopamine, the functioning of D1 and D2 receptors and their expression in the striatum. Moreover, the content of acetylcholine in the striatum was measured. Results show that 24 weeks of postnatal vitamin A deprivation induced severe locomotor deficits and impaired motor coordination. Vitamin A deprivation rats showed a significant hyperactivity following D1 receptor stimulation by R(+)-6-chloro-7,8-dihydroxy-1-phenyil-2,3,4,5-tetrahydro-1H-3-benzazepine or amphetamine and reduced catalepsy in response to haloperidol treatment. This different response to the above drugs is not due to a change in striatal DA release or synthesis between vitamin A deprivation and control animals. In situ hybridization experiments showed identical level of expression for the D1 and D2 receptor transcripts. On the other hand, the striatal tissue content of acetylcholine was reduced significantly by about 30% starting from the initial manifestation of motor deficits. We suggest that the locomotor impairment could be imputable to the dysfunction in striatal cholinergic interneurons. Our results stress the basic role of vitamin A in the maintenance of basal ganglia motor function in the adult rat brain.

Pharmacological validation of a mouse model of l-DOPA-induced dyskinesia.

Dyskinesia (abnormal involuntary movements) is a common complication of l-DOPA pharmacotherapy in Parkinson's disease, and is thought to depend on abnormal cell signaling in the basal ganglia. Dopamine (DA) denervated mice can exhibit behavioral and cellular signs of dyskinesia when they are treated with l-DOPA, but the clinical relevance of this animal model remains to be established. In this study, we have examined the pharmacological profile of l-DOPA-induced abnormal involuntary movements (AIMs) in the mouse. C57BL/6 mice sustained unilateral injections of 6-hydroxydopamine (6-OHDA) in the striatum. The animals were treated chronically with daily doses of l-DOPA that were sufficient to ameliorate akinetic features without inducing overt signs of dyskinesia upon their first administration. In parallel, other groups of mice were treated with antiparkinsonian agents that do not induce dyskinesia when administered de novo, that is, the D2/D3 agonist ropinirole, and the adenosine A2a antagonist KW-6002. During 3 weeks of treatment, l-DOPA-treated mice developed AIMs affecting the head, trunk and forelimb on the side contralateral to the lesion. These movements were not expressed by animals treated with ropinirole or KW-6002 at doses that improved forelimb akinesia. The severity of l-DOPA-induced rodent AIMs was significantly reduced by the acute administration of compounds that have been shown to alleviate l-DOPA-induced dyskinesia both in parkinsonian patients and in rat and monkey models of Parkinson's disease (amantadine, -47%; buspirone, -46%; riluzole, -33%). The present data indicate that the mouse AIMs are indeed a functional equivalent of l-DOPA-induced dyskinesia.

Differential contribution of dopamine D2S and D2L receptors in the modulation of glutamate and GABA transmission in the striatum.

Compelling evidence indicates that the long (D2L) and the short (D2S) isoform of dopamine (DA) D2 receptors serve distinct physiological functions in vivo. To address the involvement of these isoforms in the control of synaptic transmission in the striatum, we measured the sensitivity to D2 receptor stimulation of glutamate- and GABA-mediated currents recorded from striatal neurons of three mutant mice, in which the expression of D2L and D2S receptors was either ablated or variably altered. Our data indicate that both isoforms participate in the presynaptic inhibition of GABA transmission in the striatum, while the D2-receptor-dependent modulation of glutamate release preferentially involves the D2S receptor. Accordingly, the inhibitory effects of the DA D2 receptor agonist quinpirole (10 microM) on GABA(A)-mediated spontaneous inhibitory postsynaptic currents (IPSCs)correlate with the total number of D2 receptor sites in the striatum, irrespective of the specific receptor isoform expressed. In contrast, glutamate-mediated spontaneous excitatory postsynaptic currents (EPSCs) were significantly inhibited by quinpirole only when the total number of D2 receptor sites, normally composed by both D2L and D2S receptors in a ratio favoring the D2L isoform, was modified to express only the D2S isoform at higher than normal levels. Understanding the physiological roles of DA D2 receptors in the striatum is essential for the treatment of several neuropsychiatric conditions, such as Parkinson's disease, Tourette's syndrome, schizophrenia, and drug addiction.

Caffeine as a psychomotor stimulant: mechanism of action.

The popularity of caffeine as a psychoactive drug is due to its stimulant properties, which depend on its ability to reduce adenosine transmission in the brain. Adenosine A(1) and A(2A) receptors are expressed in the basal ganglia, a group of structures involved in various aspects of motor control. Caffeine acts as an antagonist to both types of receptors. Increasing evidence indicates that the psychomotor stimulant effect of caffeine is generated by affecting a particular group of projection neurons located in the striatum, the main receiving area of the basal ganglia. These cells express high levels of adenosine A(2A) receptors, which are involved in various intracellular processes, including the expression of immediate early genes and regulation of the dopamine- and cyclic AMP-regulated 32-kDa phosphoprotein DARPP-32. The present review focuses on the effects of caffeine on striatal signal transduction and on their involvement in caffeine-mediated motor stimulation.

Distinct functions of the two isoforms of dopamine D2 receptors.

Signalling through dopamine D2 receptors governs physiological functions related to locomotion, hormone production and drug abuse. D2 receptors are also known targets of antipsychotic drugs that are used to treat neuropsychiatric disorders such as schizophrenia. By a mechanism of alternative splicing, the D2 receptor gene encodes two molecularly distinct isoforms, D2S and D2L, previously thought to have the same function. Here we show that these receptors have distinct functions in vivo; D2L acts mainly at postsynaptic sites and D2S serves presynaptic autoreceptor functions. The cataleptic effects of the widely used antipsychotic haloperidol are absent in D2L-deficient mice. This suggests that D2L is targeted by haloperidol, with implications for treatment of neuropsychiatric disorders. The absence of D2L reveals that D2S inhibits D1 receptor-mediated functions, uncovering a circuit of signalling interference between dopamine receptors.

Topographical evaluation of behavioural phenotype in a line of mice with targeted gene deletion of the D2 dopamine receptor.

The phenotype of spontaneous and dopamine D2-like agonist-induced behaviour was assessed topographically in a line of mice with targeted gene deletion of the D1 receptor. An ethologically-based, rapid time-sampling behavioural check-list technique was used to resolve and quantify all behaviours in the natural repertoire of the mouse. Relative to wildtypes [D2+/+], D2-null [D2-/-] mice evidenced over a 1 h period of initial exploration modest but significant reductions in locomotion, grooming, rearing free and rearing to wall; rearing seated, sniffing, sifting and stillness were not altered. Individual elements of behaviour habituated similarly over a 6 h period for both genotypes. The dose-dependent induction of stereotyped sniffing and ponderous locomotion by the D2-like agonist RU 24213 (0.1-12.5 mg/kg) in wildtypes was essentially absent in D2-null mice. The ethogram of spontaneous behaviour in D2-null mice was characterised by only modest reductions in, and topographical shifts between, certain individual elements of behaviour. Essential abolition of D2-like agonist responsivity in D2-null mice vis-à-vis considerable preservation of spontaneous behavioural topography suggests compensatory processes subsequent to developmental absence of the D2 receptor that are able to sustain function under naturalistic, tonic conditions but not during phasic challenge.

Neonatal 192 IgG-saporin lesions of basal forebrain cholinergic neurons selectively impair response to spatial novelty in adult rats.

The role of the developing cholinergic basal forebrain system on cognitive behaviors was examined in 7 day-old rats by giving lesions with intraventricular injections of 192 IgG-saporin or saline. Rats were subjected to passive avoidance on postnatal days (PND) 22-23, water maze testing on PND 50-60, and a open-field test (in which reactions to spatial and object novelty were measured) on PND 54. Behavioral effects of the lesions were evident only in the open-field test with 5 objects. Unlike controls, the lesioned rats did not detect a spatial change after a displacement of 2 of the 5 objects. Control and lesioned rats, however, showed comparable novelty responses to an unfamiliar object. Lesion effectiveness was confirmed by 75% and 84% decreases in choline acetyltransferase activity in cortex and hippocampus. These results suggest that the developing cholinergic system may be involved in spatial information processing or attention to spatial modifications.

N-methyl-D-aspartate and dopamine receptor involvement in the modulation of locomotor activity and memory processes.

In this study we report on the effects of N-methyl-D-aspartate (NMDA)- and dopamine (DA)-receptor manipulation on the modulation of one-trial inhibitory avoidance response and the encoding of spatial information, as assessed with a non-associative task. Further, a comparison with the well-known effects of the manipulation of these two receptor systems on locomotor activity is outlined. It is well assessed that NMDA-receptor blockage induces a stimulatory action on locomotor activity similar to that exerted by DA agonists. There is evidence showing that the nucleus accumbens is involved in the response induced by both NMDA antagonists and DA agonists. We show results indicating a functional interaction between these two neural systems in modulating locomotor activity, with D2 DA-receptor antagonists (sulpiride and haloperidol) being more effective than the D1 antagonist (SCH 23390) in blocking MK-801-induced locomotion. A different profile is shown in the effects of NMDA antagonists and DA agonists in the modulation of memory processes. In one-trial inhibitory avoidance response, NMDA antagonists (MK-801 and CPP) impair the response on test day, while DA agonists exert a facilitatory effect; furthermore, sub-effective doses of both D1 (SKF 23390) and D2 (quinpirole) are able to attenuate the impairing effect in a way similar to that induced by NMDA antagonists. The effects of NMDA- and DA-acting drugs on the response to spatial novelty, as assessed with a task designed to study the ability of animals to react to discrete spatial changes, are in good accord with the effects observed on passive avoidance. The results show that NMDA as well as DA antagonists, at low doses, selectively impair the reactivity of mice to spatial changes. In a last series of experiments, the possible role of NMDA receptors located in the nucleus accumbens was investigated regarding reactivity to spatial novelty. The experiments gave apparently contrasting results: while showing an impairing effect of focal administrations of NMDA antagonists in the nucleus accumbens on reactivity to spatial novelty, no effect of ibotenic acid lesions of the same structure was observed.

N-methyl-D-aspartate receptors in the nucleus accumbens are involved in detection of spatial novelty in mice.

The aim of this study was to investigate the role played by intra-accumbens N-methyl-D-aspartate (NMDA) receptors in spatial information encoding. For this purpose, the effect of local administration of both competitive (AP-5) and non-competitive (MK-801) NMDA antagonists was assessed in a task designed to estimate the ability of rodents to encode spatial relationships between discrete stimuli. The task consists of placing mice in an open field containing five objects and, after three sessions of habituation, examining their reactivity to object displacement (spatial novelty) and object substitution (object novelty). The results show that both doses of MK-801 (0.15 and 0.3 microg/side) induced a selective impairment in the capability of mice to detect spatial novelty. A similar effect was obtained by injecting the low dose of the competitive antagonist AP-5 (0.1 microg/side), whereas the high dose (0.15 microg/side) abolished detection of both spatial and object novelty. Taken together, these results show that intra-accumbens injections of low doses of competitive and non-competitive NMDA antagonists can produce selective deficits in processing spatial information resembling those observed after hippocampal damage. Moreover, the fact that pharmacological treatments spare memory processes involved in habituation suggests that NMDA antagonists may interfere with the formation of spatial representations rather than producing memory deficits per se.