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Post-mortem studies suggest that schizophrenia is associated with abnormal expression of specific GABAA receptor (GABAAR) α subunits, including α5GABAAR. Positron emission tomography (PET) measures of GABAAR availability in schizophrenia, however, have not revealed consistent alterations in vivo. Animal studies using the GABAAR agonist [3H]-muscimol provide evidence that antipsychotic drugs influence GABAAR availability, in a region-specific manner, suggesting a potential confounding effect of these drugs. No such data, however, are available for more recently developed subunit-selective GABAAR radioligands. To address this, we therefore combined a rat model of clinically relevant antipsychotic drug exposure with quantitative receptor autoradiography. Haloperidol (0.5 and 2 mg/kg/day) or drug vehicle were administered continuously to adult male Sprague-Dawley rats via osmotic mini-pumps for 28 days. Quantitative receptor autoradiography was then performed post-mortem using the GABAAR subunit-selective radioligand [3H]-Ro15-4513 and the non-subunit selective radioligand [3H]-flumazenil. Chronic haloperidol exposure increased [3H]-Ro15-4513 binding in the CA1 sub-field of the rat dorsal hippocampus (pd = +1.3), which was not dose-dependent. [3H]-flumazenil binding also increased in most rat brain regions (pAR radioligands and is the first to demonstrate a potential effect of haloperidol on the binding of a α1/5GABAAR-selective radioligand. Although caution should be exerted when extrapolating results from animals to patients, our data support a view that exposure to antipsychotics may be a confounding factor in PET studies of GABAAR in the context of schizophrenia.

The pathophysiology of the majority of neuropsychiatric disorders, including schizophrenia and mood disorders, involves synaptic dysfunction and/or loss, manifesting as lower levels of several presynaptic and postsynaptic marker proteins. Whether chronic exposure to antipsychotic drugs may contribute to this pattern of synaptic loss remains controversial. In contrast, the mood stabiliser lithium has shown to exhibit neurotrophic actions and is thought to enhance synapse formation. Whilst these data are not unequivocal, they suggest that antipsychotic drugs and lithium have contrasting effects on synapse density. We therefore investigated the effect of chronic exposure to lithium and to two different antipsychotics, haloperidol and olanzapine, on presynaptic Synaptic Vesicle glycoprotein 2A (SV2A) and postsynaptic Neuroligin (NLGN) clusters in the rat frontal cortex. Chronic exposure (28 days) to haloperidol (0.5 mg/kg/d) or olanzapine (7.5 mg/kg/d) had no effect on either SV2A or NLGN clusters and no overall effect on synaptic clusters. In contrast, chronic lithium exposure (2 mmol/L eq./d) significantly increased NLGN cluster density as compared to vehicle, but did not affect either SV2A or total synaptic clusters. These data are consistent with and extend our prior work, confirming no effect of either antipsychotics or lithium on SV2A clustering, but suggest contrasting effects of these drugs on the post-synapse. Although caution needs to be exerted when extrapolating results from animals to patients, these data provide clarity with regard to the effect of antipsychotics and lithium on synaptic markers, thus facilitating discrimination of drug from illness effects in human studies of synaptic pathology in psychiatric disorders.

Postmortem studies suggest that schizophrenia is associated with abnormal expression of specific GABAA receptor (GABAAR) α subunits, including α5GABAAR. Positron emission tomography (PET) measures of GABAAR availability in schizophrenia, however, have not revealed consistent alterations in vivo. Animal studies using the GABAAR agonist [3H]-muscimol provide evidence that antipsychotic drugs influence GABAAR availability, in a region-specific manner, suggesting a potential confounding effect of these drugs. No such data, however, are available for more recently developed subunit-selective GABAAR radioligands. To address this, we combined a rat model of clinically relevant antipsychotic drug exposure with quantitative receptor autoradiography. Haloperidol (0.5 and 2 mg/kg/day) or drug vehicle were administered continuously to adult male Sprague-Dawley rats via osmotic mini-pumps for 28 days. Quantitative receptor autoradiography was then performed postmortem using the GABAAR subunit-selective radioligand [3H]-Ro15-4513 and the non-subunit selective radioligand [3H]-flumazenil. Chronic haloperidol exposure increased [3H]-Ro15-4513 binding in the CA1 sub-field of the rat dorsal hippocampus (p

The precise effect of antipsychotic drugs on either central or peripheral inflammation remains unclear. An important issue in this debate is to what extent the known peripheral metabolic effects of antipsychotics, including increased adiposity, may contribute to increased inflammation. Adipose tissue is known to contribute to the development of systemic inflammation, which can eventually lead to insulin resistance and metabolic dysregulation. As a first step to address this question, we evaluated whether chronic exposure to clinically comparable doses of haloperidol or olanzapine resulted in the immune activation of rat adipose tissue. Samples of visceral adipose tissue were sampled from male Sprague–Dawley rats exposed to, haloperidol, olanzapine or vehicle (all n = 8), for 8 weeks. From these we measured a cytokine profile, protein expression of F4/80 (a phenotypic macrophage marker) and translocator protein (TSPO), a target for radiotracers putatively indicating microgliosis in clinical neuroimaging studies. Chronic olanzapine exposure resulted in significantly higher adipose IL-6 levels compared with vehicle-controls (ANOVA p = 0.008, Bonferroni post-hoc test p = 0.006); in parallel, animals exposed to olanzapine had significantly higher F4/80 expression when compared with vehicle-controls (Mann Whitney Test, p = 0.014), whereas there was no difference between haloperidol and vehicle groups (Mann Whitney test, p = 0.1). There were no significant effects of either drug on adipose TSPO protein levels. Nevertheless, we found a positive correlation between F4/80 and TSPO adipose protein levels in the olanzapine-exposed rats (Spearman’s rho = 0.76, p = 0.037). Our data suggest that chronic exposure to olanzapine, but not haloperidol, increases production of the pro-inflammatory cytokine IL-6 in adipose tissue and increased macrophages expression (F4/80), in the absence of measurable changes in TSPO with respect to vehicle. This may have potentially important consequences in terms of metabolic dysregulation associated with long-term antipsychotic treatment.

Positron emission tomography studies using the synaptic vesicle glycoprotein 2A (SV2A) radioligand [11C]-UCB-J provide in vivo evidence for synaptic dysfunction and/or loss in the cingulate and frontal cortex of patients with schizophrenia. In exploring potential confounding effects of antipsychotic medication, we previously demonstrated that chronic (28-day) exposure to clinically relevant doses of haloperidol does not affect [3H]-UCB-J radioligand binding in the cingulate and frontal cortex of male rats. Furthermore, neither chronic haloperidol nor olanzapine exposure had any effect on SV2A protein levels in these brain regions. These data do not exclude the possibility, however, that more subtle changes in SV2A may occur at pre-synaptic terminals, or the post-synaptic density, following chronic antipsychotic drug exposure. Moreover, relatively little is known about the potential effects of psychotropic drugs other than antipsychotics on SV2A. To address these questions directly, we herein used immunostaining and confocal microscopy to explore the effect of chronic (28-day) exposure to clinically relevant doses of haloperidol, olanzapine or the mood stabilizer lithium on presynaptic SV2A, postsynaptic Neuroligin (NLGN) puncta and their overlap as a measure of total synaptic density in the rat prefrontal and anterior cingulate cortex. We found that, under the conditions tested here, exposure to antipsychotics had no effect on SV2A, NLGN, or overall synaptic puncta count. In contrast, chronic lithium exposure significantly increased NLGN puncta density relative to vehicle, with no effect on either SV2A or total synaptic puncta. Future studies are required to understand the functional consequences of these changes.

Background The synaptic dysfunction hypothesis of schizophrenia is supported by multiple lines of evidence. However, in vivo evidence is lacking. Moreover, it is not known if antipsychotics alter synaptic protein levels. We addressed this in a combined clinical study using [11C]UCB-J, a positron emission tomography (PET) radioligand specific for synaptic vesicle glycoprotein 2A (SV2A), and rodent study of clinically relevant antipsychotic drug exposure. Methods We scanned 18 subjects with schizophrenia (SCZ) and 18 healthy volunteers (HV) with [11C]UCB-J PET and T1-weighted MRI, estimating grey matter volumes of distribution (VT) and corrected grey matter volumes (GMV) for the frontal cortex (FC), anterior cingulate cortex (ACC) and hippocampus. In addition, we estimated the [11C]UCB-J distribution volume ratio (DVR) in these regions, using the centrum semiovale (CS) as a pseudoreference region. We collected clinical data including PANSS score, chlorpromazine-equivalent antipsychotic dose (CPZ) and duration of illness (DOI). In parallel, we investigated the effects of olanzapine and haloperidol administration on SV2A levels in the Sprague-Dawley rat frontal cortex using western blotting, [3H]UCB-J autoradiography and immunostaining with confocal microscopy. We used two-way analysis of variance (ANOVA) to test effects of group, ROI and group-by-ROI interaction on VT and DVR. We used planned post hoc t-tests to test group effects at each ROI, with false discovery-rate adjustment for multiple comparisons. We used two-way ANOVA with Bonferroni’s post-hoc test to analyse autoradiography and SV2A immunostaining data, and the Kruskal-Wallis test, with alpha=0.05, to analyse western blot data. Results Clinical study: We found significant group-by-region interaction (F2, 68=7.472, p=0.001), group (F1, 34=6.170, p=0.02) and ROI (F2, 68=426.0, p0.05) between VT and GMV, PANSS score, DOI and CPZ. Preclinical study: Chronic olanzapine or haloperidol exposure did not significantly affect total SV2A immunostaining intensity (p=0.97 and p=0.71 respectively) in the rat frontal cortex as compared to vehicle-exposed controls. Moreover, chronic haloperidol exposure did not significantly affect [3H]UCB-J specific binding (p=0.27) or SV2A protein levels (relative to GAPDH, p=0.71). Discussion These findings provide evidence for the first time that presynaptic marker protein levels are reduced in schizophrenia in vivo and that antipsychotic drug exposure is unlikely to account for this, consistent with the synaptic dysfunction hypothesis.

Background Post-mortem studies in schizophrenia (SZ) have reported abnormalities in the GABAergic system, more specifically, on αGABA-A receptors (αGABA-A R) (Benes, Kwok, Vincent, & Todtenkopf, 1998; Beneyto, Abbott, Hashimoto, & Lewis, 2011). Consistently, animal models of SZ have also reported changes in these receptor subtypes (Lodge, Behrens, & Grace, 2009; Richetto, Calabrese, Riva, & Meyer, 2014). Preclinical work in the methylazoxymethanol acetate (MAM) animal model of SZ has shown that administration of an α5GABA-A modulating drug rescues the psychosis-like phenotype (hyperdopaminergia) (Gill, Lodge, Cook, Aras, & Grace, 2011). However, this rescue does not occur if the animals are previously administered the first-generation antipsychotic haloperidol (Gill, Cook, Poe, & Grace, 2014). A recent study in patients with first-episode psychosis found increased baseline GABA levels as measured by magnetic resonance spectroscopy (de la Fuente-Sandoval et al., 2017). After 4 weeks of treatment with the antipsychotic risperidone, these GABA levels normalized in the medial prefrontal cortex and decreased at a trend level in the dorsal caudate (de la Fuente-Sandoval et al., 2017). All in all these studies suggest that antipsychotic medication besides directly regulating the dopaminergic system may also be acting on the GABAergic system, but the underlying mechanisms remain unknown. The aim of our study was thus to investigate, for the first time, the effects of clinically-relevant dosages of haloperidol on α5GABA-A R and benzodiazepine-sensitive α1–3;5GABA-A R in healthy rats. Methods A total of 36 male Sprague-Dawley rats were treated with vehicle, haloperidol 0.5 mg/kg/day or 2 mg/kg/day with an osmotic minipump for 28 days, representing clinically-relevant dosages of antipsychotic administration (Kapur, Vanderspek, Brownlee, & Nobrega, 2003). Quantitative autoradiography was conducted with [3H]Ro15-4513 and [3H]flumazenil, to assess the effects of haloperidol on α5GABA-A R and overall α1–3;5GABA-A R. Optical density measurements from cortical and subcortical regions were obtained from the autoradiographs using the MCID Software and converted into radioactivity values using a robust linear regression on GraphPad Prism. All data were analyzed using a mixed-effects model ANOVA on the same software and considered significant at p Results Administration of chronic haloperidol showed a dose-specific increase in [3H]Ro15-4513 in the dorsal CA1 area of the hippocampus, showing an increase in binding in the 0.5 mg/kg/day vs. vehicle (p Discussion This is the first study to investigate, in healthy rats, the effects of clinically-relevant doses of haloperidol on GABA-A R binding in a receptor-subtype specific manner. Our results show that exposure to haloperidol causes changes in α5GABA-A R predominantly in the dorsal CA1 and the nucleus accumbens. An overall change in benzodiazepine-sensitive α1–3;5GABA-A R is also observed, irrespective of region. These results suggest that exposure to antipsychotic medication impacts the GABAergic system and should be considered when testing new pharmacological strategies in humans that have been exposed to antipsychotic medication.

Genome-wide significant variants associated with combined risk for major psychiatric disorders on chromosome 10q24 affect the expression of the cytosolic 5’-nucleotidase II (NT5C2, cN-II) in population controls, implicating it as a psychiatric susceptibility gene. Risk alleles are associated with reduced expression of this gene in the developing and adult brain, but the resulting neurobiological risk mechanisms remain elusive. In this study, we provide further evidence for the association ofNT5C2with psychiatric disorders, and use a functional genetics approach to gain a deeper understanding of the function of this risk gene in the nervous system.NT5C2expression was significantly reduced in thepost-mortembrain of schizophrenia and bipolar disorder patients, and its protein predominately expressed in neurons within the adult brain. Using human neural progenitor cells (hNPCs), we found thatNT5C2expression peaked at the neural progenitor state, where the encoded protein was ubiquitously distributed through the cell.NT5C2knockdown in hNPCs elicited transcriptomic changes associated with protein translation, that were accompanied by regulation of adenosine monophosphate-activated protein kinase (AMPK) signalling and ribosomal protein S6 (rpS6) activity. To identify the effect of reduced neuronalNT5C2expression at a systems level, we knockdown its homologue,CG32549, inDrosophila melanogasterCNS. This elicited impaired climbing behaviour in the model organism. Collectively, our data implicateNT5C2expression in risk for psychiatric disorders and inDrosophila melanogastermotility, and further suggest that risk is mediated via regulation of AMPK signalling and protein translation during early neurodevelopment.

Neuroinflammation is increasingly implicated in the pathogenesis of Schizophrenia (SCZ). In addition, there is increasing evidence for a relationship between the dose and duration of antipsychotic drug (APD) treatment and reductions in grey matter volume. The potential contribution of microglia to these phenomena is however not yet defined. Adult rats were treated with a common vehicle, haloperidol (HAL, 2 mg/kg/day) or olanzapine (OLZ, 10 mg/kg/day) for 8 weeks via an osmotic mini-pump implanted subcutaneously. Microglial cells, identified by their Iba-1 immunoreactivity, were quantified in four regions of interest chosen based on previous neuroimaging data: the hippocampus, anterior cingulate cortex, corpus striatum, and secondary somatosensory cortex. Those cells were also analysed according to their morphology, providing an index of their activation state. Chronic APD treatment resulted in increased density of total microglia in the hippocampus, striatum, and somatosensory cortex, but not in the ACC. Importantly, in all brain regions studied, both APD tested led to a dramatic shift towards an amoeboid, reactive, microglial morphology after chronic treatment compared to vehicle-treated controls. These data provide the first in vivo evidence that chronic APD treatment at clinically relevant doses leads to microglial proliferation and morphological changes indicative of activated microglia in the naïve rat brain. Although caution needs to be exerted when extrapolating results from animals to patients, these data suggest a potential contribution of antipsychotic medication to markers of brain inflammation. Further investigation of the links between antipsychotic treatment and the immune system are warranted.

Neuroanatomical, electrophysiological and behavioural abnormalities following timed prenatal methylazoxymethanol acetate (MAM) treatment in rats model changes observed in schizophrenia. In particular, MAM treatment on gestational day 17 (E17) preferentially disrupts limbic–cortical circuits, and is a promising animal model of schizophrenia. The hypersensitivity of this model to the NMDA receptor antagonist-induced hyperactivity has been proposed to mimic the increase in sensitivity observed in schizophrenia patients following PCP and Ketamine administration. However, how this increase in sensitivity in both patients and animals translates to differences in EEG oscillatory activity is unknown. In this study we have shown that MAM-E17 treated animals have an increased response to the hyperlocomotor and wake promoting effects of Ketamine, PCP, and MK801 but not to the competitive antagonist SDZ 220,581. These behavioural changes were accompanied by altered EEG responses to the NMDAR antagonists, most evident in the gamma and high frequency (HFO) ranges; altered sensitivity of these neuronal network oscillations in MAM-exposed rats is regionally selective, and reflects altered interneuronal function in this neurodevelopmental model. This article is part of a Special Issue entitled ‘Schizophrenia’.

Background Evidence-based medicine suggests that a subset of schizophrenia is associated with an inflammatory syndrome. To fully harness the potential of novel immunomodulatory therapeutics, it is critical to first determine the impact of antipsychotics on microglial function in vivo. Evidence suggests antipsychotics are anti-inflammatory; however, this is based on in vitro models and non-clinical doses of antipsychotics in vivo. It therefore remains unknown if antipsychotics promote detrimental neuroinflammation or beneficial, homeostatic changes in the brain. To address this question, we explored the effects of chronic haloperidol treatment on microglia in a rat maternal immune activation (MIA) model, representative of schizophrenia pathology. Methods Pregnant SD rat dams were exposed to poly (I:C) on GD15 (4 mg/kg, i.v.; n=5; POL) to induce MIA, or saline (n=5; CON) as a control. At 4 months of age, male offspring from CON and POL (n=2 per litter), were randomly allocated to treatment with either haloperidol (0.5 mg/kg/d s.c.) or vehicle for 28 days by osmotic minipumps, giving four groups: CON/vehicle; CON/haloperidol; POL/vehicle and POL/haloperidol (all n=10). After 28d treatment, animals were culled and perfused transcardially with 4% PFA. Fixed brain tissues were dissected, cryoprotected and microtome sectioned (1 in 12 series, 40 µm thick). Serial sections were stained for Iba1 as a marker of microglia using an immunoperoxidase protocol. The density and morphology (soma size) of Iba1+ microglia were then assessed in the corpus striatum (CS) and anterior cingulate cortex (ACC) using unbiased stereology. Data were analysed using 2x2 ANOVA in SPSS with main effects of prenatal, postnatal and pre x post-natal interactions. Results There were significant main effects of prenatal exposure to POL on Iba1+ microglia density in the CS (F(1,32)=18.09; p

Background Positron emission tomography (PET) using radiolabeled ligands selective for the 18 kDa translocator protein (TSPO) is the most widely used technique to assess putative neuroimmune abnormalities in vivo. However, the results of TSPO PET in schizophrenia patients are ambiguous. In particular, the influence of antipsychotic medication exposure has not been definitively tested. We have previously established that chronic exposure to two different antipsychotics resulted in increased density and amoeboid morphology of brain microglia, particularly in the cortex (ACC) and corpus striatum (STR) (Cotel et al., 2015). Here we explore the consequences of such treatment on TSPO, in relation to changes in microglia. We hypothesize that chronic haloperidol treatment would increase the expression of TSPO in microglia and correlate with increased levels of circulating proinflammatory cytokines. Methods Adult male Sprague-Dawley rats (12 week-old) were implanted with subcutaneous osmotic mini-pumps (ALZET® 2ML4) delivering a continuous dose of either common vehicle (20% β-cyclodextrin + 5% ascorbic acid) or haloperidol (2mg/kg/d) for 28 days (Kapur et al., 2003). Plasma was collected before perfusing the animals with 4% buffered PFA. Brains were carefully removed and processed for immunostaining against Iba1 and TSPO. Levels of plasma cytokines were measured using the pro-inflammatory 9-plex panel from Meso Scale Discovery (MSD®). TSPO expression was measured by its area fraction using FIJI software (10 snaps per animal per brain region, 20x magnification) in the ACC, STR, hippocampus (HPC), secondary somatosensory cortex (S2), primary motor cortex (M1). Numbers of resting and amoeboid microglia were determined by unbiased stereology (Gundersen, 1987) in the ACC, STR, and HPC. The Cavalieri estimator (StereoInvestigator 12.0,MBF Bioscience) was used to determine the volume of these regions. Data were analyzed by t-tests corrected for multiple comparisons, using GraphPad Prism 6.0. Results Only 5 cytokines from the panel were above the limit of detection. Of these, IL-6 is significantly increased in haloperidol-treated animals (+186%, p

Based on the glutamatergic hypothesis of schizophrenia we assessed the effects of a novel mGlu5 positive allosteric modulator, LSN2463359 [N-(1-methylethyl)-5-(pyridin-4-ylethynyl)pyridine-2-carboxamide] on deficits in cognitive flexibility in two distinct rodent models of schizophrenia, the neurodevelopmental MAM E17 model and the acute PCP model. Cognitive flexibility was measured with the intra-dimensional and extra-dimensional set-shifting and reversal learning digging paradigm. Regional effects of MAM on the expression of parvalbumin-positive cells (PV) and mGlu5 receptors were also examined, to further characterize the model. Results showed that LSN2463359 selectively attenuated reversal learning deficits in the MAM but not acute PCP model. Whilst both models led to deficits in reversal learning and extra-dimensional set-shifting, the reversal impairments were qualitatively distinct, with MAM increasing perseverative responding, whereas the PCP deficit was mainly due to the inability of rats to maintain reinforced choice behavior. Reduction of PV and mGlu5 expression was found in the MAM model in several regions of importance in schizophrenia, such as the orbitofrontal and medial prefrontal cortex, which also mediate reversal learning and extra-dimensional set-shifting. The present findings confirm that the positive modulation of mGlu5 receptors may have beneficial effects in the treatment of certain aspects of cognitive impairment associated with schizophrenia. This study also illustrates the importance of studying putative cognitive enhancing drug effects in a number of models which may have implications for the future development of the compound.

Environmental enrichment has been used in a variety of transgenic mouse models of Alzheimer's disease (AD), however, with conflicting results. Here we studied the influence of environmental enrichment in a severely affected AD mouse model, showing a multiplicity of pathological alterations including hippocampal neuron loss. APP/PS1KI and wild type (WT) control mice were housed under standard conditions or in enriched cages equipped with various objects and running wheels. Amyloid plaque load, motor and working memory performance, axonopathy, as well as CA1 neuron number and hippocampal neurogenesis were assessed. Although a partial improvement in motor performance was observed, 4 months of enriched housing showed no beneficial effects in terms of working memory, Aβ plaque pathology, or neuron loss in APP/PS1KI mice. In addition, no changes in hippocampal neurogenesis and even an aggravation of the axonal phenotype were detected with a tendency toward a premature death. The APP/PS1KI model represents a model for mild to severe AD showing early behavioral deficits starting at 2 months of age with fast deterioration. Therefore our data might suggest that physical activity and enriched environment might be more beneficial in patients with mild cognitive impairment than in patients with incipient AD.

Loss of neurons in the hippocampus and other brain regions is, besides the occurrence of plaques and tangles, a neuropathological feature of Alzheimer's disease (AD). In recent years a plethora of transgenic mouse models overexpressing mutant amyloid precursor protein (APP) has been developed, which represent valuable research tools. Whereas extracellular plaque pathology is a common feature of these models, neuronal loss is a rather rare characteristic. In the present study, we quantified the number of neurons in the dentate gyrus granule layer (GCL) in 2- and 12-month-old APP/PS1KI mice, a mouse model that has been previously shown to have significant loss of neurons in the CA1 layer of the hippocampus. Stereological analysis revealed a strongly significant decrease of GCLs in aged APP/PS1KI mice, compared to age-matched PS1KI control animals (− 44%), however, the volume of the GCL was not different.