Amantadine and clozapine have proved to reduce abnormal involuntary movements (AIMs) in preclinical and clinical studies of L-DOPA-Induced Dyskinesias (LID). Even though both drugs decrease AIMs, they may have different action mechanisms by using different receptors and signaling profiles. Here we asked whether there are differences in how they modulate neuronal activity of multiple striatal neurons within the striatal microcircuit at histological level during the dose-peak of L-DOPA in ex-vivo brain slices obtained from dyskinetic mice. To answer this question, we used calcium imaging to record the activity of dozens of neurons of the dorsolateral striatum before and after drugs administration in vitro. We also developed an analysis framework to extract encoding insights from calcium imaging data by quantifying neuronal activity, identifying neuronal ensembles by linking neurons that coactivate using hierarchical cluster analysis and extracting network parameters using Graph Theory. The results show that while both drugs reduce LIDs scores behaviorally in a similar way, they have several different and specific actions on modulating the dyskinetic striatal microcircuit. The extracted features were highly accurate in separating amantadine and clozapine effects by means of principal components analysis (PCA) and support vector machine (SVM) algorithms. These results predict possible synergistic actions of amantadine and clozapine on the dyskinetic striatal microcircuit establishing a framework for a bioassay to test novel antidyskinetic drugs or treatments in vitro.

Chronic psychosocial stress modulates brain antioxidant systems and causes neuroinflammation that plays a role in the pathophysiology of depression. Although the antidepressant fluoxetine (FLX) represents the first-line treatment for depression and the atypical antipsychotic clozapine (CLZ) is considered as a second-line treatment for psychotic disorders, the downstream mechanisms of action of these treatments, beyond serotonergic or dopaminergic signaling, remain elusive. We examined behavioral changes, glutathione (GSH)-dependent defense and levels of proinflammatory mediators in the prefrontal cortex (PFC) of adult male Wistar rats exposed to 21days of chronic social isolation (CSIS). We also tested the ability of FLX (15mg/kg/day) or CLZ (20mg/kg/day), applied during CSIS, to prevent stress-induced changes. CSIS caused depressive- and anxiety-like behaviors, compromised GSH-dependent defense, and induced nuclear factor-kappa B (NF-κB) activation with a concomitant increase in cytosolic levels of proinflammatory mediators cyclooxigenase-2, interleukin-1beta and tumor necrosis factor-alpha in the PFC. NF-κB activation and proinflammatory response in the PFC were not found in CSIS rats treated with FLX or CLZ. In contrast, only FLX preserved GSH content in CSIS rats. CLZ not only failed to protect against CSIS-induced GSH depletion, but it diminished its levels when applied to non-stressed rats. In conclusion, prefrontal cortical GSH depletion and the proinflammatory response underlying depressive- and anxiety-like states induced by CSIS were prevented by FLX. The protective effect of CLZ, which was equally effective as FLX on the behavioral level, was limited to proinflammatory components. Hence, different mechanisms underlie the protective effects of these two drugs in CSIS rats.

The complex network of anatomical connections of the nucleus accumbens (NAc) makes it an interface responsible for the selection and integration of cognitive and affective information to modulate appetitive or aversively motivated behaviour. There is evidence for NAc dysfunction in schizophrenia. NAc also seems to be important for antipsychotic drug action, but the biochemical characteristics of drug-induced alterations within NAc remain incompletely characterized. In this study, a comprehensive proteomic analysis was performed to describe the differences in the mechanisms of action of clozapine (CLO) and risperidone (RIS) in the rat NAc. Both antipsychotics influenced the level of microtubule-regulating proteins, i.e., stathmin, and proteins of the collapsin response mediator protein family (CRMPs), and only CLO affected NAD-dependent protein deacetylase sirtuin-2 and septin 6. Both antipsychotics induced changes in levels of other cytoskeleton-related proteins. CLO exclusively up-regulated proteins involved in neuroprotection, such as glutathione synthetase, heat-shock 70-kDa protein 8 and mitochondrial heat-shock protein 75. RIS tuned cell function by changing the pattern of post-translational modifications of some proteins: it down-regulated the phosphorylated forms of stathmin and dopamine and the cyclic AMP-regulated phosphoprotein (DARPP-32) isoform but up-regulated cyclin-dependent kinase 5 (Cdk5). RIS modulated the level and phosphorylation state of synaptic proteins: synapsin-2, synaptotagmin-1 and adaptor-related protein-2 (AP-2) complex.

Dysregulation of GABAergic system is becoming increasingly associated with depression, psychiatric disorder that imposes severe clinical, social and economic burden. Special attention is paid to the fast-spiking parvalbumin-positive (PV+) interneurons, GABAergic neurons which are highly susceptible to redox dysregulation and oxidative stress and implicated in a variety of psychiatric diseases. Here we analyzed the number of PV+ and cleaved caspase-3-positive (CC3+) cells in the rat medial prefrontal cortical (mPFC) subregions following chronic social isolation (CSIS), an animal model of depression and schizophrenia. Also, we examined potential protective effects of antidepressant fluoxetine (FLX) and atypical antipsychotic clozapine (CLZ) on the number of these cells in mPFC subregions, when applied parallel with CSIS in doses that correspond to therapeutically effective ones in patients. Immunofluorescence analysis revealed decreased number of PV+ cells in cingulate cortex area 1, prelimbic area (PrL), infralimbic area (IL) and dorsal peduncular cortex of the mPFC in isolated rats, which coincided with depressive- and anxiety-like behaviors. In addition, CSIS-induced increase in the number of CC3+ cells was detected in aforementioned subregions of mPFC. Treatments with either FLX or CLZ prevented behavioral changes, decrease in PV+ and increase in CC3+ cell numbers in PrL and IL subregions in isolated rats. These results indicate the importance of intact GABAergic signaling in these areas for resistance against CSIS-induced behavioral changes, as well as subregion-specific protective effects of FLX and CLZ in mPFC of CSIS rats.

Anhedonia is a relevant symptom in depression and schizophrenia. Chronic stress exposure induces in rats escape deficit, disrupts the dopaminergic response to palatable food and the competence to acquire sucrose self-administration (SA), thus configuring a possible model of motivational anhedonia. Repeated lithium administration reverts stress effects and brings back to control values the breaking point (BP) score, a measure of reward motivation. In this study, we tested on this model two antidepressants, imipramine and fluoxetine, and two antipsychotics, haloperidol and clozapine. The dopaminergic response to sucrose consumption was studied in non food-deprived rats in terms of dopamine D1 receptor signaling in the nucleus accumbens shell (NAcS). More specifically, we studied the modifications in dopamine and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) phosphorylation pattern following sucrose consumption. Fluoxetine reverted the escape deficit and showed no effects on dopaminergic response and sucrose SA. Imipramine reverted sucrose SA and dopamine response deficit in half of the rats and the escape deficit in all animals. Haloperidol did not affect stress-induced deficits. Clozapine-treated rats recovered the dopaminergic response to sucrose consumption and the competence to acquire sucrose SA, although they still showed the escape deficit, thus confirming that motivation toward reward may be dissociated from that to punishment escape. These results indicate that imipramine or fluoxetine are not endowed with a rapid onset antianhedonic effect. On the other hand, clozapine treatment showed a motivational antianhedonic activity similar to that observed after lithium treatment.

Glutamate and dopamine disturbances are implicated in frontal cortical dysfunction in schizophrenia. Little, however, is known about the nature of dopamine D(1) and N-methyl-D-aspartate (NMDA) receptor interactions in the illness, nor of the extent of their co-involvement in antipsychotic drug response. It is well known that early life adversity may pre-date the development of schizophrenia. Using a neurodevelopmental model of schizophrenia, namely post weaning social isolation rearing (SIR), we studied the effect of SIR (post natal day 21-61) on frontal cortical NMDA and D(1) receptor binding characteristics with/without chronic haloperidol (0.1 mg/kg/day i.p.) or clozapine (5 mg/kg/day i.p.) treatment, undertaken from post-natal day 50-60. SIR increased frontal cortical NMDA-density, with decreased affinity (decreased pK(D)), but reduced D(1) receptor density (without effects on pK(D)). In socially reared animals, clozapine but not haloperidol increased NMDA receptor density without effects on pK(D.) Neither drug markedly affected D(1) receptor density, although clozapine increased D(1) affinity. Increased NMDA density in SIR animals was unaffected by haloperidol, but further increased by clozapine. However, SIR-associated decrease in NMDA affinity remained unaltered despite drug treatment. Reduced D(1) receptor density in SIR animals was exacerbated by haloperidol, but unaltered by clozapine, without changes in pK(D). SIR thus induces opposing effects on frontal cortical NMDA and D(1) radio-receptor binding characteristics, which has direct bearing on the mutual interplay of these receptors in schizophrenia. The ability of SIR to affect NMDA receptor affinity warrants deeper study. Furthermore, at the doses examined, in contrast to haloperidol, clozapine bolsters frontal cortical glutamatergic but stabilizes D(1) dopaminergic pathways in a neurodevelopmental animal model of schizophrenia, possibly explaining the atypical clinical characteristics of this drug.

The chemogenetic procedure DREADD (designer receptor exclusively activated by designer drugs) is an inventive way to selectively affect g-coupled protein receptors. In theory, DREADD receptors are only activated by administering inert compounds, primarily clozapine N-oxide (CNO). Research has shown that CNO does not cross the blood-brain barrier, and CNO is converted back to clozapine and N-desmethylclozapine (N-Des) in the brain. Clozapine and N-Des have many neurological effects including alterations in glutamate and dopamine (DA) levels in multiple brain regions. The current study examined the effects of peripheral administration of CNO on glutamate and DA levels in the medial prefrontal cortex (mPFC). Wistar rats were administered CNO, and microdialysis samples were collected from the mPFC. Administration of CNO significantly increased glutamate (31-87%) and DA (65-126%), CNO-induced increases in DA occurred for a longer duration than glutamate, and that for the two highest doses of CNO there was a significant correlation between the increase in glutamate and DA in the mPFC. In the mPFC, CNO-induced increases in DA occurred at 0.5 mg/kg, while increases in glutamate were observed at doses greater than 1.0 mg/kg. The source of the DA and glutamate could be caused by activation of projection neurons or local effects. The data replicate findings that CNO is not an inert compound and that interpretation of CNO-activated DREADD findings should be done with caution. The data indicate that low ('safe') doses of CNO still have neurochemical effects and that controlling for the actions of clozapine/N-Des in CNO-DREADD studies has many concerns.

We recently reported that serotonin excites a subpopulation of GABAergic neurons in the rat medial septum/diagonal band of Broca complex via multiple serotonin receptors, including the serotonin2A subtype. Since a subpopulation of medial septum/diagonal band GABAergic neurons projects to the hippocampus, in the present study we tested the effect of serotonin on antidromically-activated septohippocampal neurons using extracellular recordings. Bath-applied serotonin had an excitatory effect in a majority of septohippocampal neurons; serotonin-excited septohippocampal neurons had a mean conduction velocity -1.63 +/- 0.07 m/s (n=101). Pharmacologically, MDL 100,907, a selective serotonin2A antagonist blocked the excitatory effect of serotonin in 78% of septohippocampal neurons tested, with a mean pA2 of 8.51 +/- 0.12 (n=22). Additionally, the atypical antipsychotics risperidone and clozapine but not the typical antipsychotic haloperidol, blocked the excitatory effects of serotonin at clinically relevant concentrations. The pA2 values of 8.84 +/- 0.11, 6.57 +/- 0.13 and 5.94 +/- 0.27 for risperidone, clozapine and haloperidol, respectively, obtained in the present study, give a rank order of potency risperidone (1.6 nM) clozapine (269 nM) haloperidol (1.1 microM) which corresponds to that reported in binding studies. Additionally, in whole-cell patch-clamp recordings, risperidone (10 nM) blocked serotonin-induced increase in GABAergic synaptic currents. In conclusion, serotonin excites septohippocampal neurons primarily via the serotonin2A receptor and atypical antipsychotics block this excitation at clinically relevant concentrations.

Tardive dyskinesia (TD) is a potentially disabling condition encompassing all delayed, persistent, and often irreversible abnormal involuntary movements arising in a fraction of subjects during long-term exposure to centrally acting dopamine receptor-blocking agents such as antipsychotic drugs and metoclopramide. However, the pathogenesis of TD has proved complex and remains elusive. To investigate the mechanism underlying the development of TD, we have chronically exposed 17 Cebus apella monkeys to typical (11) or atypical (6) antipsychotic drugs. Six additional monkeys were used as controls. Using autoradiography, Western blot and in situ hybridization techniques, we compared neurochemical components of the dopamine, serotonin, and glutamate neurotransmitter systems modulating striatal activity in monkeys chronically exposed to haloperidol and clozapine. Five (5) out of 11 monkeys treated with haloperidol develop TD, whereas none of the monkeys treated with clozapine develop TD. Haloperidol treatment significantly upregulated the levels of serotonin 5-HT2A receptor, NR2A-containing NMDA receptors, and tyrosine hydroxylase contents in the monkey putamen, whereas clozapine regulated putamen NMDA receptor levels and tyrosine hydroxylase contents, and 5-HT2A and dopamine transporter outside the putamen. Comparisons of neurochemical alterations between dyskinetic and non dyskinetic animals within the haloperidol-treated group indicate that modulations of 5-HT2A, metabotropic glutamate type 5, NR2A- and NR2B-containing NMDA receptors, and vesicular monoamine transporter type 2 levels were restricted to the non dyskinetic group. The foregoing results suggest that TD is associated with complex deficient adaptation in aminergic and glutamatergic neurotransmission in the striatum of non-human primates chronically exposed to antipsychotic drugs.

NMDA receptor (NMDAr) hypofunction has been widely used as a schizophrenia model. Decreased activation of NMDAr is associated with a disrupted excitation/inhibition balance in the prefrontal cortex and with alterations in gamma synchronization. Our aim was to investigate whether this phenomenon could be reproduced in the spontaneous oscillatory activity generated by the local prefrontal network in vitro and, if so, to explore the effects of antipsychotics on the resulting activity. Extracellular recordings were obtained from prefrontal cortex slices bathed in in vivo-like ACSF solution. Slow (<1 Hz) oscillations consisting of interspersed Up (active) and Down (silent) states spontaneously emerged. Fast-frequency oscillations (15-90 Hz) occurred during Up states. We explored the effects of the NMDAr antagonist MK-801 on the spontaneously generated activity. Bath-applied MK-801 induced a dose-dependent decrease in Up-state duration and in the frequency of Up states. However, the beta/gamma power during Up states significantly increased; this increase was in turn prevented by the antipsychotic drug clozapine. The increased beta/gamma power with NMDAr blockade implies that NMDAr activation in physiological conditions prevents hypersynchronization in this frequency range. High-frequency hypersynchronization following NMDAr blockade occurring in cortical slices suggests that-at least part of-the underlying mechanisms of this schizophrenia feature persist in the local cortical circuit, even in the absence of long-range cortical or subcortical inputs. The observed action of clozapine decreasing hypersynchronization in the local circuit may be one of the mechanisms of action of clozapine in preventing schizophrenia symptoms derived from NMDA hypofunction.

Glycogen synthase kinase-3 (GSK-3) has been implicated in the action of antipsychotics, mood stabilizers, and antidepressants. Given that only antipsychotics are able to alleviate the positive symptoms of schizophrenia, the regulation of GSK-3 by antipsychotics would be expected to differ from other neuropsychiatric drugs if GSK-3 is involved in the alleviation of psychosis. Consequently, the current study examined the effects of antipsychotics (haloperidol and clozapine), mood stabilizers (lithium and valproic acid), and antidepressants (imipramine and fluoxetine) on GSK-3, as well as Akt and Wnt in the prefrontal cortex and striatum. Western blotting and co-immunoprecipitation experiments showed that only antipsychotic treatment increased Dvl-3, GSK-3, and β-catenin levels and enhanced the association of GSK-3 at the dopamine D2 receptor (D₂DR) complex in the rat prefrontal cortex. In the striatum, haloperidol had the same effect on Wnt signaling as observed in the prefrontal cortex, whereas clozapine did not affect Dvl-3, GSK-3 or β-catenin levels. All three classes of drugs were able to activate Akt signaling as shown by the increased phosphorylated Akt and phosphorylated GSK-3 protein levels in the prefrontal cortex and/or striatum. In conclusion, regulation of the Wnt pathway is specific to antipsychotics, whereas antipsychotics, mood stabilizers, and antidepressants all affect Akt.

We have recently shown that the expression of the nerve growth factor-inducible gene B (NGFI-B, or Nur77), a transcription factor belonging to the large ligand-activated nuclear receptor family, is modulated by antipsychotic drugs in the rat forebrain. In the present work, we have investigated the impact of antipsychotic drugs on a series of transcription factors also belonging to the nuclear receptor family. The receptors investigated include retinoid X receptor (RXR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), RAR-related orphan receptor (RZR) and Rev-erb receptor isoforms in addition to the NGFI-B transcript. We have used in situ hybridization to monitor their mRNA levels after acute and chronic antipsychotic drug administration. RZRbeta and NGFI-B mRNA levels are down-regulated after chronic haloperidol or clozapine treatment in the primary somatosensory cortex. The TRbeta1 isoform mainly expressed in the cingulate cortex is modulated only after chronic clozapine treatment, whereas TRalpha isoform mRNAs are modulated by both antipsychotics in the cingulate cortex and nucleus accumbens shell; two brain areas associated with limbic functions. The RXRgamma1 isoform, mostly expressed in the dorsolateral portion of the striatum is modestly affected by antipsychotics. Modulation of the expression of transcription factors belonging to the ligand-activated nuclear receptor family by antipsychotics represents an additional molecular event in the mechanism of action of these drugs. We suggest that modification of the pattern of transcription factor expression may play a role in long-term cellular responses to these drugs.

Specific neurons in the brain are the primary targets of the action of antipsychotic drugs. Identification and characterization of the nature of these neurons are important for understanding how antipsychotic drugs produce their effects. In previous studies GABAergic/dynorphinergic neurons were identified as a principal cell target of antipsychotic drugs in the shell of nucleus accumbens. In the present study, we further characterized which subpopulations of GABAergic neurons in this area respond after acute administration of antipsychotic drugs. Rats were treated with the typical antipsychotic haloperidol, or the prototype atypical antipsychotic clozapine and killed two hours after treatment. In appropriate sections of brain, double immunofluorescence labeling was performed with antibodies directed against markers specific to candidate cell types and Fos-like proteins (a marker to identify drug-induced cell activation). We reported here that haloperidol- and clozapine-activated neurons showed the following features: 1) approximately 54-57% of them express dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa (a marker for GABAergic medium spiny projection neurons), 2) they appear rarely to be GABAergic interneurons, marked by the calcium binding proteins, parvalbumin, calretinin or calbindin-D28K, 3) about 84-86% of them express the neuropeptide neurotensin (a neurotransmitter most often associated with projection neurons in the site tested). The results suggest that most of the antipsychotic drug-activated neurons in the shell of nucleus accumbens are likely to be neurotensin containing projection neurons.

Accumulating evidence suggests that glutamatergic signaling and synaptic plasticity underlie one of a number of ways psychiatric disorders appear. The present study reveals a possible mechanism by which this occurs, through highlighting the importance of LMTK3, in the brain. Behavioral analysis of Lmtk3-KO mice revealed a number of abnormalities that have been linked to psychiatric disease such as hyper-sociability, PPI deficits and cognitive dysfunction. Treatment with clozapine suppressed these behavioral changes in Lmtk3-KO mice. As synaptic dysfunction is implicated in human psychiatric disease, we analyzed the LTP of Lmtk3-KO mice and found that induction is severely impaired. Further investigation revealed abnormalities in GluA1 trafficking after AMPA stimulation in Lmtk3-KO neurons, along with a reduction in GluA1 expression in the post-synaptic density. Therefore, we hypothesize that LMTK3 is an important factor involved in the trafficking of GluA1 during LTP, and that disruption of this pathway contributes to the appearance of behavior associated with human psychiatric disease in mice.

Calcineurin is a calmodulin (CaM) dependent protein phosphatase recently found to be altered in the brains of patients suffering from schizophrenia and by repeated antipsychotic treatment in rats. Some data suggest, however, that antipsychotics and schizophrenia may have a more widespread effect on the CaM signaling axis than calcineurin alone. In the current study, the effects of selected psychoactive drugs were investigated using Western blotting, in situ hybridization and immunocytochemistry to determine if they target CaM, calmodulin-dependent protein kinases (CaMK) or calcineurin. Results indicated that repeated treatment with haloperidol, clozapine or risperidone increased CaM protein and CaMII mRNA levels but decreased calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) IV (CaMKIV), kinase alpha (CaMKKalpha), kinase beta (CaMKKbeta) and calcineurin protein levels in the striatum of Sprague-Dawley rats (Rattus Norvegicus). Closer examination of CaMKIV, CaMKKalpha and CaMKKbeta revealed that the observed decreases in protein levels were short-lived following antipsychotic treatment and reversed (i.e. upregulated) 24 h post-treatment similar to what was previously reported for calcineurin. The D(2)/D(3)dopamine receptor antagonist raclopride mimicked the decreases in CaMKIV, CaMKKalpha, CaMKKbeta and calcineurin observed following antipsychotic treatment whereas increases in these proteins were observed in an amphetamine model of the positive symptoms of schizophrenia. Mood stabilizers such as lithium and valproic acid or the antidepressant fluoxetine had no effect on CaMKIV, CaMKKalpha, CaMKKbeta and calcineurin with the exception of an increase in CaMKKbeta following lithium treatment. The results collectively suggest that antipsychotic specifically target several proteins associated with CaM signaling.

Pain is a common complication of herpes zoster (HZ) infection which results from reactivation of a latent varicella zoster virus (VZV). A third of HZ patients' progress to a chronic pain state known as post herpetic neuralgia (PHN), and about a quarter of these patients' have orofacial pain. The mechanisms controlling the pain responses are not understood. Studies suggest central pathways involving the thalamus could control pain related to HZ, and studies in our lab suggest (VGAT) in the lateral thalamus influences orofacial pain. We hypothesized that thalamic VGAT functions, in part, to reduce pain, particularly orofacial pain, associated with VZV. To address this hypothesis VZV was injected into the whisker pad. Affective and motivational aspects of pain were measured using the Place Escape/Avoidance Paradigm. Thalamic neuronal activity was modulated after injecting an adeno-associated virus (AAV) expressing an engineered acetylcholine Gi-protein-coupled receptor. This receptor inhibits neuronal firing when bound by clozapine-n-oxide (CNO). VGAT expression was attenuated in the thalamus by injecting an AAV construct that expressed a VGAT silencing shRNA. VZV-induced nociception was significantly decreased after administering CNO in male rats. Nociception significantly increased concomitant with increased thalamic c-fos expression after attenuating thalamic VGAT expression. These data establish that the lateral thalamus (posterior, ventral posteromedial, ventral posterolateral and/or reticular thalamic nucleus) controls VZV-induced nociception in the orofacial region, and that GABA in this region appears to reduce the response to VZV-induced nociception possibly by gating facial pain input.