Mini review of molecules involved in altered postnatal neurogenesis in autism.

The neurobiology of autism is complex, but emerging research points to potential abnormalities and alterations in neurogenesis. The aim of the present review is to describe the advances in the understanding of the role of selected neurotrophins, neuropeptides, and other compounds secreted by neuronal cells in the processes of postnatal neurogenesis in conjunction with autism. We characterize the fundamental mechanisms of neuronal cell proliferation, generation of major neuronal cell types with special emphasis on neurogenic niches - the subventricular zone and hippocampal areas. We also discuss changes in intracellular calcium levels and calcium-dependent transcription factors in the context of the regulation of neurogenesis and cell fate determination. To sum up, this review provides specific insight into the known association between alterations in the function of the entire spectrum of molecules involved in neurogenesis and the etiology of autism pathogenesis.

Effects of antipsychotics, haloperidol and olanzapine, on the expression of apoptosis-related genes in mouse mHippoE-2 cells and rat hippocampus.

Objective. Modified levels of pro- (caspase3, Bax) and anti-apoptotic (Bcl-2) regulatory proteins have been detected in certain brain areas of schizophrenic patients indicating a possible dysregulation of apoptosis. In the present study, effects of antipsychotics, haloperidol (HAL) and olanzapine (OLA), on the gene expression of caspase3 (casp3), Bax and Bcl-2 were studied in vitro in mouse hippocampal mHippoE-2 cell line and in vivo in the hippocampus of MK-801 animal schizophrenia model with the aim to provide evidence that antipsychotics may affect the activity of apoptosis-related markers. Methods. mHippoE-2 cells were incubated with MK-801 (20 µM), HAL (10 µM), and OLA (10 µM) alone or combined, MK-801+HAL/OLA, for 24, 48, and 72 h. Male Sprague Dawley rats were injected with saline or MK-801 (0.5 mg/kg) for 6 days and since the 7th day, they were treated with vehicle (VEH), HAL (1 mg/kg) or OLA (2 mg/kg) for the next 7 days. The casp3, Bax and Bcl-2 gene expression in mHippoE-2 cells and rat hippocampus was measured by RT-PCR. Results. In mHippoE-2 cells, casp3 gene expression was increased by MK-801 and OLA treatments alone for 48 h, HAL treatment alone for 24 and 72 h, and co-treatment with MK-801+OLA for 24 and 72 h compared to controls. HAL and OLA suppressed the stimulatory effect of MK-801 on casp3 mRNA levels in cells after 48 h of incubation. Bax mRNA levels in mHippoE-2 cells were decreased after HAL treatment for 24 and 48 h, and also after co-treatment with MK-801+HAL for 72 h. In vivo, MK-801 decreased mRNA levels of both pro-apoptotic markers, casp3 and Bax, in hippocampus of VEH-treated rats and Bax mRNA levels in hippocampus of HAL-treated animals. OLA reversed the inhibitory effect of MK-801 on casp3 expression in the VEH-treated animals. Neither MK-801 nor antipsychotics induced changes in the gene expression of anti-apoptotic marker Bcl-2 in mHippoE-2 cells as well as hippocampus of rats. Conclusions. The results of the present study demonstrate that antipsychotics, HAL and OLA, may affect mRNA levels of pro-apoptotic markers in hippocampal cells in vitro, but not in vivo. The obtained data do not clearly support the assumed potentiating role of MK-801 in inducing apoptosis in specific brain areas and a possible protective role of antipsychotics against induction of apoptosis. The obtained data may contribute to a deeper insight into the neurodevelopmental changes connected with schizophrenia.

Effect of Haloperidol and Olanzapine on Hippocampal Cells' Proliferation in Animal Model of Schizophrenia.

Aberrant neurogenesis in the subventricular zone (SVZ) and hippocampus (HIP) contributes to schizophrenia pathogenesis. Haloperidol (HAL) and olanzapine (OLA), commonly prescribed antipsychotics for schizophrenia treatment, affect neurogenesis too. The effect of HAL and OLA on an mHippoE-2 cell line was studied in vitro where we measured the cell number and projection length. In vivo, we studied the gene expression of DCX, Sox2, BDNF, and NeuN in the SVZ and HIP in an MK-801-induced animal schizophrenia model. Cells were incubated with HAL, OLA, and MK-801 for 24, 48, and 72 h. Animals were injected for 6 days with saline or MK801 (0.5 mg/kg), and from the 7th day with either vehicle HAL (1 mg/kg) or OLA (2 mg/kg), for the next 7 days. In vitro, HAL and OLA dose/time-dependently suppressed cells' proliferation and shortened their projection length. HAL/OLA co-treatment with MK-801 for 24 h reversed HAL's/OLA's inhibitory effect. In vivo, HAL and OLA suppressed DCX and NeuN genes' expression in the HIP and SVZ. MK-801 decreased DCX and NeuN genes' expression in the HIP and OLA prevented this effect. The data suggest that subchronic HAL/OLA treatment can inhibit DCX and NeuN expression. In an MK-801 schizophrenia model, OLA reversed the MK-801 inhibitory effect on DCX and NeuN and HAL reversed the effect on DCX expression; however, only in the HIP.

Possible involvement of apoptosis in the antipsychotics side effects: A mini-review.

Antipsychotics are used in the treatment of schizophrenia and other psychiatric disorders. Generally, they are divided into typical and atypical ones, according to the fact that atypical antipsychotics induce fewer side effects and are more effective in terms of social and cognitive improvements. Their pharmacological effects are mediated via broad range of receptors that consequently influence different cellular signalling pathways. Antipsychotics produce undesirable side effects that range from relatively minor to life threatening. In vitro and in vivo studies have pointed to neurotoxic effect exerted by some antipsychotics and have shown that apoptosis might play role in some side effects induced by antipsychotics, including tardive dyskinesia, weight gain, agranulocytosis, osteoporosis, myocarditis, etc. Although cumulative data have suggested safety of atypical antipsychotics use during pregnancy, some of them have been shown to induce apoptotic neurodegenerative and structural changes in fetal brains with long-lasting impact on cognitive impairment of offspring. Typical antipsychotics seem to be more cytotoxic than atypical ones. Recently, epidemiological studies have shown lower incidence of cancer in schizophrenic patients that suggest the ability of antipsychotics to suppress risk of cancer development. Some antipsychotics have been reported to inhibit cancer cell proliferation and induce their apoptosis. Therefore, antipsychotics apoptotic effect may be used as a tool in the treatment of some types of cancer, especially in combinatorial therapies. In this mini-review, we focused on pro- and antiapoptotic or 'Dr. Jekyll and Mr. Hyde' effects of antipsychotics, which can be involved in their side effects, as well as their promising therapeutic indications.

Haloperidol and aripiprazole affects CRH system and behaviour of animals exposed to chronic mild stress.

CRH system integrates responses to stress challenges, whereas antipsychotics may impinge on this process. Effect of haloperidol (HAL) and aripiprazole (ARI) on chronic mild stress (CMS) induced neurobehavioral and CRH/CRHR1 system changes was studied in functionally interconnected rat brain areas including prefrontal cortex (PFC), bed nucleus of the stria terminalis (BNST), hypothalamic paraventricular nucleus (PVN), hippocampus (HIP), and amygdala (AMY). Animals were exposed to CMS for 3-weeks and since the 7th day of CMS injected with vehicle (VEH), HAL (1 mg/kg) or ARI (10 mg/kg) for 4-weeks. Expression levels of CRH, CRHR1, and c-fos genes and anxiety-like and anhedonia behavioural patterns were evaluated. CMS in VEH animals suppressed CRH gene expression in the PFC and BNST, c-fos expression in all areas, except HIP, and increased CRHR1 gene expression in the HIP. Antipsychotics decreased CRH gene expression in all areas, except HIP and by CMS elevated CRHR1 expression in the HIP (ARI also in AMY). CMS and antipsychotics decreased the sucrose preference. Aripiprazole prevented CRH expression decrease in the BNST and sucrose preference induced by CMS. Haloperidol increased time spent in the EPM open arms. These data indicate that HAL and ARI selectively influenced behavioural parameters and CRH/CRHR1 gene expression levels in CMS animals.

Combined In Silico, Ex Vivo, and In Vivo Assessment of L-17, a Thiadiazine Derivative with Putative Neuro- and Cardioprotective and Antidepressant Effects.

Depression associated with poor general medical condition, such as post-stroke (PSD) or post-myocardial infarction (PMID) depression, is characterized by resistance to classical antidepressants. Special treatment strategies should thus be developed for these conditions. Our study aims to investigate the mechanism of action of 2-morpholino-5-phenyl-6H-1,3,4-thiadiazine, hydrobromide (L-17), a recently designed thiadiazine derivative with putative neuro- and cardioprotective and antidepressant-like effects, using combined in silico (for prediction of the molecular binding mechanisms), ex vivo (for assessment of the neural excitability using c-Fos immunocytochemistry), and in vivo (for direct examination of the neuronal excitability) methodological approaches. We found that the predicted binding affinities of L-17 to serotonin (5-HT) transporter (SERT) and 5-HT3 and 5-HT1A receptors are compatible with selective 5-HT serotonin reuptake inhibitors (SSRIs) and antagonists of 5-HT3 and 5-HT1A receptors, respectively. L-17 robustly increased c-Fos immunoreactivity in the amygdala and decreased it in the hippocampus. L-17 dose-dependently inhibited 5-HT neurons of the dorsal raphe nucleus; this inhibition was partially reversed by the 5-HT1A antagonist WAY100135. We suggest that L-17 is a potent 5-HT reuptake inhibitor and partial antagonist of 5-HT3 and 5-HT1A receptors; the effects of L-17 on amygdaloid and hippocampal excitability might be mediated via 5-HT, and putatively mediate the antidepressant-like effects of this drug. Since L-17 also possesses neuro- and cardioprotective properties, it can be beneficial in PSD and PMID. Combined in silico predictions with ex vivo neurochemical and in vivo electrophysiological assessments might be a useful strategy for early assessment of the efficacy and neural mechanism of action of novel CNS drugs.

Haloperidol and aripiprazole impact on the BDNF and glucocorticoid receptor levels in the rat hippocampus and prefrontal cortex: effect of the chronic mild stress.

Objective. Changes in the brain derived neurotrophic factor (BDNF) and glucocorticoid receptor (GR) expression in the prefrontal cortex (PFC) and hippocampus (HIP) are associated with psychiatric diseases and stress response. Chronic mild stress (CMS) may alter BDNF as well as GR levels in both the PFC and the HIP. The aim of the present study was to find out whether chronic treatment with a typical antipsychotic haloperidol (HAL) and an atypical antipsychotic aripiprazole (ARI) may modify the CMS effect on the BDNF and GR expression in the above-mentioned structures. Methods. The rats were exposed to CMS for 3 weeks and from the 7th day of CMS injected with vehicle (VEH), HAL (1 mg/kg) or ARI (10 mg/kg) for 4 weeks. BDNF and GR mRNA levels were established in the PFC and the HIP by Real Time PCR, whereas, PFC and HIP samples were obtained by punching them from 500 µm thick frozen sections. C-Fos immunoreactivity was analyzed in the PFC and the HIP on 30 µm thick paraformaldehyde fixed sections. Weight gain and corticosterone (CORT) levels were also measured. Results. The CMS and HAL suppressed the BDNF and GR mRNA levels in the PFC. In the HIP, CMS elevated BDNF mRNA levels that were suppressed by HAL and ARI treatments. The CMS decreased the c-Fos immunoreactivity in the PFC in both HAL- and ARI-treated animals. In the HIP, HAL increased the c-Fos immunoreactivity that was again diminished in animals exposed to CMS. Stressed animals gained markedly less weight until the 7th day of CMS, however, later their weight gain did not differ from the unstressed ones or was even higher in CMS+HAL group. Un-stressed HAL and ARI animals gained less weight than the VEH ones. Neither CMS nor HAL/ARI affected the plasma CORT levels. Conclusion. The present data indicate that HAL and ARI in the doses 1 mg/kg or 10 mg/kg, respectively, does not modify the effect of the CMS preconditioning on the BDNF and GR mRNA levels in the PFC or the HIP. However, HAL seems to modify the CMS effect on the HIP activation.

Extra-forebrain impact of antipsychotics indicated by c-Fos or FosB/ΔFosB expression: A minireview.

It is apparent that the c-Fos and FosB/ΔFosB immunohistochemistry has generally become a useful tool for determining the different antipsychotic (AP) drugs activities in the brain. It is also noteworthy that there are no spatial limits, while to the extent of their identification over the whole brain axis. In addition, they can be in a parallel manner utilized in the unmasking of the brain cell phenotype character activated by APs and by this way also to identify the possible brain circuits underwent to the APs action. However, up to date, the number of APs involved in the extra-striatal studies is still limited, what prevents the possibility to fully understand their extra-striatal effects as a complex as well as differentiate their extra-striatal impact in qualitative and quantitative dimensions. Actually, it is very believable that more and more anatomical/functional knowledge might bring new insights into the APs extra-striatal actions by identifying new region-specific activities of APs as well as novel cellular targets affected by APs, which might reveal more details of their possible side effects of the extra-striatal origin.

The effect of amisulpride, olanzapine, quetiapine, and aripiprazole single administration on c-Fos expression in vasopressinergic and oxytocinergic neurons of the rat hypothalamic paraventricular nucleus.

Antipsychotics, including amisulpride (AMI), quetiapine (QUE), aripiprazole (ARI), and olanzapine (OLA), are used to treat mental illnesses associated with psychotic symptoms. The effect of these drugs on c-Fos expression in vasopressinergic (AVP) and oxytocinergic (OXY) neurons was studied in the hypothalamic paraventricular nucleus (PVN) of rats. The presence of c-Fos in AVP and OXY perikarya was investigated in seven PVN cells segregations: the anterior (Ant), dorsal cup (Dc), wing-shaped (Wi), periventricular zone (Pe), circle-shaped core (Co) and shell of core (Sh), and the posterior (pPVN) after an acute treatment with AMI-20 mg/kg, QUE-15 mg/kg, ARI-10 mg/kg, and OLA-5 mg/kg/bw in rats. Ninety min after treatments, the animals were sacrificed by transcardial perfusion with fixative and the PVN area sliced into 35 µm thick coronal sections for immunohistochemistry. The c-Fos was processed by avidin-biotin-peroxidase complex intensified with nickel-enhanced 3,3'-diaminobenzidine tetrahydrochloride. Visualization of AVP- and OXY-synthesizing neurons was achieved by a fluorescent marker Alexa Flour 568. The c-Fos-AVP and c-Fos-OXY colocalizations were evaluated from c-Fos stained sections merged with AVP or OXY ones. AMI, QUE, ARI, and OLA, single administration distinctly increased the c-Fos expression in each of the PVN cells segregations. QUE induced the highest magnitude of activation of AVP and OXY neurons, while OLA and AMI had only moderate effects. Incontestable variabilities detected in c-Fos expression in PVN AVP and OXY neurons extend the knowledge of selected antipsychotics extra-striatal actions and may also be helpful in a presumption of their possible functional impact.

c-Fos and FosB/ΔFosB colocalizations in selected forebrain structures after olanzapine, amisulpride, aripiprazole, and quetiapine single administration in rats preconditioned by two different mild stressors sequences.

OBJECTIVE: Olanzapine (OLA), amisulpride (AMI), aripiprazole (ARI), and quetiapine (QUE) belong to antipsychotics, which administration represents still most reliable way for the treatment of schizophrenic and bipolar disorders. The intention of the present study was to explore whether the acute administration of a particular antipsychotic, indicated by the presence of c-Fos, will: a) stimulate neurons already activated by a long lasting homogeneous or heterogeneous stress preconditioning, indicated by the FosB/ΔFosB (ΔFosB) expression, or b) have a stimulatory effect only on a not activated, so called silent neurons. The pattern of ΔFosB and c-Fos spatial relationship was investigated in three forebrain structures, including the septal ventrolateral nucleus (seVL), the striatal dorsolateral area (stDL), and the shell of the nucleus accumbens (shell). METHODS: The rats were divided into 10 groups and exposed to two types of stressors. Half of them was exposed to a sequence of homogeneous stressor - handling (HDL) and the other half to a heterogeneous stressor (CMS) daily for 20 days. CMS consisted of five types of stressors: crowding, air-puff, wet bedding, predator stress, and forced swimming applied in an unexpected order. On the 21st day of the experiment, the rats were free of the stress exposure and on the 22nd day, both groups of animals receive a single intraperitoneal injection of vehicle (4% DMSO in saline, 0.1 ml/100 g) or OLA (5 mg/kg), AMI (20 mg/kg), ARI (10 mg/kg), and QUE (15 mg/kg). 90 min after the drugs administration the animals were transcardially perfused, brains removed, cut into 30 µm thick coronal sections, and double stained: first with ΔFosB antibody linked with Alexa488 fluorescent dye and second with c-Fos antibody linked to Alexa596 one. Quantitative evaluation of ΔFosB and c-Fos colocalizations was performed on fluorescence photomicrographs transformed into a final picture containing only yellow, green, and red colored circles. RESULTS: The data of this investigation demonstrate that ΔFosB and c-Fos colocalizations occurred in each of the three areas investigated, i.e. seVL, stDL, and shell ones, in both HDL as well as CMS preconditioned rats. The levels of ΔFosB and c-Fos colocalizations varied in the individual forebrain areas studied. From the total 22 areas measured, level of c-Fos colocalization prevailed over ΔFosB in 18 ones. However, neither c-Fos nor ΔFosB reached 100% level of colocalization in any of the forebrain areas investigated. CONCLUSION: The present findings indicate that ΔFosB and c-Fos colocalizations occurred in each of the three areas investigated, i.e. seVL, stDL, and shell, in both HDL and CMS preconditioned rats, whereas the parallel occurrence of free c-Fos as well as c-Fos colocalized with ΔFosB might speak out for a possible involvement of the c-Fos activated by antipsychotics applied in dual, i.e. short- and long-lasting, functions.

Effect of amisulpride, olanzapine, quetiapine, and aripiprazole single administration on c-Fos expression in vasopressinergic and oxytocinergic neurons of the rat hypothalamic supraoptic nucleus.

OBJECTIVE: The goal of this study was to reveal the impact of four types of atypical antipsychotics including amisulpride (AMI), olanzapine (OLA), quetiapine (QUE), and aripiprazole (ARI), with different receptor-affinity profile and dissociation constant, on the activity of hypothalamic supraoptic nucleus (SON) vasopressinergic and oxytocinergic neurons. METHODS: Male Sprague Dawley rats received a single injection of vehicle (VEH) (0.1 ml/100g), AMI (20 mg/kg), OLA (5 mg/kg), QUE (15 mg/kg/) or ARI (10 mg/kg). Ninety min after treatment, the animals were fixed by transcardial perfusion, the brains removed, and cryocut into serial coronal sections of 35 µm thickness. The sections were processed for c-Fos staining using an avidin-biotin-peroxidase complex and visualized by nickel intensified diaminobenzidine to reach black end product. Afterwards, the sections were exposed to vasopressin (AVP) and oxytocin (OXY) antibodies and the reaction product visualized by biotin-labeled fluorescent Alexa Fluor 568 dye. The data were evaluated from c-Fos and AVP or OXY merged sections. RESULTS: The present study shows that all four antipsychotics applied induced c-Fos expression in the SON. With respect to the stimulation efficacy of the individual antipsychotics, estimated based on the quantity of c-Fos-labeled AVP and OXY neurons, could be a preferential action assigned to QUE over moderate effect of ARI and lower effect to OLA and reduced effect of AMI (VEH < AMI < OLA < ARI < QUE). CONCLUSION: The present data for the first time provide an insight into the quantitative pattern of brain activity within the clusters of SON AVP and OXY cells in response to different atypical antipsychotics single treatment.

Clozapine impact on FosB/ΔFosB expression in stress preconditioned rats: response to a novel stressor.

OBJECTIVE: Prolonged treatment with neuroleptics has been shown to induce FosB/ΔFosB expression in several brain regions including the medial prefrontal cortex, dorsomedial and dorsolateral striatum, ventrolateral and dorsolateral septum, nucleus accumbens shell and core, and the hypothalamic paraventricular nucleus (PVN). Some of these regions are known to be also stress responsive. This study was designed to determine whether repeated clozapine (CLZ) administration for 7 consecutive days to Wistar rats may modify FosB/ΔFosB expression in the above-mentioned brain areas induced by acute stress or novel stressor that followed 13-day chronic mild stress preconditioning. METHODS: Following experimental groups were used: unstressed animals treated with vehicle/ CLZ for 7 days; 7-day vehicle/CLZ-treated animals on the last day exposed to acute stress - forced swimming (FSW); and animals preconditioned with stress for 13 days treated from the 8th day with vehicle/CLZ and on the 14th day exposed to novel stress - FSW. RESULTS: In the unstressed animals CLZ markedly increased FosB/ΔFosB immunoreactivity in the ventrolateral septum and PVN. FSW elevated FosB/ΔFosB expression in the medial prefrontal cortex, striatum, and septum. CLZ markedly potentiated the effect of the FSW on FosB/ΔFosB expression in the PVN, but suppressed it in the dorsomedial striatum. Novel stress with stress preconditioning increased FosB/ΔFosB immunoreactivity in the prefrontal cortex, striatum, ventrolateral septum, and the PVN. In the nucleus accumbens the effect of the novel stressor was potentiated by CLZ. CONCLUSION: Our data indicate that CLZ may modulate the acute as well as novel stress effects on FosB/ΔFosB expression but its effect differs within the individual brain regions.

Spatial relationship between the c-Fos distribution and enkephalinergic, substance P, and tyrosine hydroxylase innervation fields after acute treatment with neuroleptics olanzapine, amisulpride, quetiapine, and aripiprazole in the rat septum.

OBJECTIVE: The aim of the present study was to demonstrate the spatial relationship between the c-Fos immunoreactive cells elicited by an acute treatment with neuroleptics including amisulpride (AMI), olanzapine (OLA), quetiapine (QUE), and aripiprazole (ARI) and enkephalinergic (ENK), substance P (SP), and tyrosine hydroxylase (TH) innervation fields in the rat septum. METHODS: Male Sprague Dawley rats received a single injection of OLA (5 mg), ARI (10 mg), AMI (20 mg), QUE (15 mg/kg/b.w.). Ninety min after antipsychotics administration, the animals were transcardially perfused with a fixative and the brains cryocut into serial coronal sections of 35 µm thickness. The sections were processed for c-Fos staining using an avidin-biotin-peroxidase complex and visualized by nickel intensified diaminobenzidine to reach black endproduct. Afterwards, the sections were exposed to ENK, SP, and TH antibodies and the reaction product visualized by biotin-labeled fluorescent AlexaFluor 564 dye. The data were evaluated from the sections either simultaneously illuminated with fluorescent and transmission microscope beams or after merging the separately illuminated sections in the Adobe Photoshop 7.0 software. RESULTS: ENK, SP, and TH displayed characteristic spatial images formed by a dense accumulation of immunoreactive fibers and terminals on the both sides of the septum. A dense plexus of axons formed by ENK and SP immunopositive terminals were situated predominantly in the lateral, while TH ones more medial portion of the septum. QUE and AMI activated distinct amount of c-Fos expression in cells located within the SP-immunoreactive principal innervation field. The OLA effect on the c-Fos expression was very pronounced in the ventral TH-labeled principal innervation field including the space between the ENK field ventral portion and the dorsal margin of the accumbens nucleus shell. Generally, the occurrence of c-Fos cells in the ENK-immunoreactive principal innervation field, in comparison with the surrounding septal area, was less abundant after all of the four antipsychotics treatments. CONCLUSION: The data of the present study indicate that ENK, SP, and TH innervation fields may influence separate populations of septal cells activated by AMI, OLA, QUE, and ARI and that each of these region-differently innervated cells may be associated with the functional heterogeneity of the individual lateral septal nuclei.

c-Fos expression response to olanzapine, amisulpride, aripiprazole, and quetiapine single administration in the rat forebrain: Effect of a mild stress preconditioning.

Antipsychotics have been shown to stimulate different forebrain areas, whereas some of them are sensitive to stress. In the present study, effect of a single administration of olanzapine (OLA), amisulpride (AMI), aripiprazole (ARI), and quetiapine (QUE) on the activity of cells in the striatal dorsolateral (stDL) area, the periventricular zone (peVZ), the septal ventrolateral (seVL) nucleus, and the accumbens nucleus shell (shACC) and core (coACC) was investigated in male rats preconditioned with a mild stress complex (CMS) for 20 days. The objective of the study was to extend the anatomical-functional knowledge on the mechanism of selected antipsychotics with the goals: 1) to analyze the ability of the selected antipsychotics to induce c-Fos protein expression in the above mentioned forebrain structures and to map the pattern of their topography and 2) to find out whether longer-lasting mild stress preconditioning may modify the impact of the selected antipsychotics on the activity of cells in the forebrain areas in adult rats. Ten groups of rats were used. CMS complex contained five stressors: cage crowding, air-puff noising, wet bedding, predator stress, and forced swimming. AMI (20 mg/kg), OLA (5 mg/kg), QUE (15 mg/kg), and ARI (10 mg/kg/b.w.) were administered intraperitoneally and 90 min later the animals transcardially perfused by fixative. c-Fos was visualized by ABC complex. In unstressed animals, OLA and ARI elevated c-Fos expression in all areas studied, AMI and QUE in all areas except stDL, seVL and coACC, shACC FL-2 (shACC posterior level), respectively. CMS potentiated the effect of AMI in coACC, and QUE in shACC FL-2 and suppressed the effect of AMI in peVZ, and ARI in peVZ and seVL. The present data provide new insights into activity of cells in response to CMS challenge, which might be helpful in understanding the diverse clinical effects of atypical antipsychotics.

Clozapine impact on c-Fos expression in mild stress preconditioned male rats exposed to a novelty stressor.

Clozapine (CLZ) stimulates several brain areas some of them being sensitive to stress. Aim of the present study was to reveal whether 7-day CLZ administration may: (1) activate the selected forebrain areas; (2) modulate response of these structures to a single forced swimming episode (FSW); (3) modulate response of these structures to FSW after 13-day preconditioning with mild unpredictable stress complex (CMS). Used groups of male Wistar rats: (a) vehicle or CLZ treated for 7 days; (b) vehicle or CLZ treated for 7 days and on the 7th day exposed to FSW; (c) CMS exposed for 13 days, from the 8th day injected with vehicle or CLZ and on the 14th day exposed to FSW. Vehicle or CLZ (10 mg kg-1  day-1 in 0.1% acetic acid) were administered intraperitoneally. c-Fos quantification was performed 90 min after FSW in the medial prefrontal cortex (mPFC), dorsolateral (dLS) and ventrolateral (vLS) septum, dorsolateral (DLStr) and dorsomedial (DMStr) striatum, nucleus accumbens shell (NAc shell) and core (NAc core), and hypothalamic paraventricular nucleus (PVN). In unstressed animals CLZ increased c-Fos expression in the mPFC, vLS, and PVN. After a single FSW, CLZ decreased the number of c-Fos immunoreactive cells in the vLS, DMStr, NAc shell, and NAc core. In CMS rats, CLZ suppressed c-Fos immunoreactivity in response to FSW in the PVN. Our data indicate that CLZ elicits different impact on neuronal activities in the brain areas studied and modifies the response of these structures to stress. CLZ effect seems to be affected by stress duration.

Acute Impact of Selected Pyridoindole Derivatives on Fos Expression in Different Structures of the Rat Brain.

The impacts of three pyridoindole derivatives (PDs), designated as PD144, PD143, and PD104, which have previously been shown to have antidepressant (PD144) and anxiolytic (PD143, PD104) properties, were investigated on the Fos expressions in 11 different rat brain areas, including the medial prefrontal cortex, striatum, septum, accumbens nucleus (shell, core), bed nucleus of the stria terminalis, hypothalamic paraventricular nucleus, central amygdala, locus coeruleus, dorsal raphe nucleus, and the solitary tract nucleus. Control rats received vehicle, while the other three groups the PDs in a dose of 25 mg/kg/b.w. The animals were transcardially perfused with a fixative 90 min after the treatments. Coronal sections of 40-µm thickness were processed for Fos-immunostaining by avidin-biotin-peroxidase complex and visualized by nickel-intensified diaminobenzidine complex. Fos-labeled sections were counterstained with neuropeptides including corticoliberine (CRH), oxytocin (OXY), vasopressin (AVP), and vasoactive intestinal polypeptide (VIP) and processed for immunofluorescence staining using Alexa Fluor 555 dye. In all the three groups of animals, the upregulation of PDs-induced Fos expression only in 2 of 11 brain areas was investigated, namely, in the hypothalamic paraventricular nucleus (PVN) and the central amygdaloid nucleus (CeA). The other brain structures studied were devoid of Fos expression. Counterstaining of the Fos-labeled CeA-containing sections with VIP antibody revealed that the Fos expression stimulated by the PDs was upregulated in all the CeA subdivisions (lateral, ventral, capsular), except the medial one. Dual immunoprocessings showed Fos/CRH-labeling in both the PVN and the amygdala and Fos/OXY in the PVN. No Fos/AVP colocalizations were seen in the PVN. The obtained data provide the first view on the intracerebral effects of three new PDs derivatives, which effects were restricted only to the PVN and CeA areas. The present data may help to improve our understanding of the impact of the selected PDs on the brain and to anticipate possible behavioral and neuroendocrine consequences.

Impact of repeated asenapine treatment on FosB/ΔFosB expression in the forebrain structures under normal conditions and mild stress preconditioning in the rat.

Long-term effect of asenapine (ASE), an atypical antipsychotic drug, on FosB/ΔFosB quantitative variations in the striatum, septum, nucleus accumbens, and prefrontal cortex, was light microscopically evaluated in normal rats and rats preconditioned with chronic unpredictable mild stress (CMS). CMS included restraint, social isolation, crowding, swimming, and cold. The rats were exposed to CMS for 21 days. From the 7th day of CMS, the rats were injected subcutaneously with saline (300µl/rat) or ASE (0.3mg/kg b.w.), twice a day for 14 days. On the 22nd day, i.e. 16-18h after the last treatment, the animals were perfused with fixative and the brains cut into 30µm thick coronal sections. FosB/ΔFosB protein was immunohistochemically visualized by avidin-biotin peroxidase complex (ABC). Four groups of animals were investigated: control+vehicle, control+ASE, CMS+vehicle, and CMS+ASE. Repeated ASE treatment significantly increased the amount of FosB/ΔFosB immunostained cell nuclei in the dorsolateral and dorsomedial striatum and the shell of the nucleus accumbens, followed by strVM and coACC, as assessed by numerical analysis in both total (different size for each structure) and unified (equal size for each structure) brain sectors. The effect of ASE was significantly lowered by CMS preconditioning only in the dorsolateral striatum, dorsomedial striatum, and the shell of the nucleus accumbens, indicated by both total and unified calculations. Although, highest FosB/ΔFosB expression was seen in the prefrontal cortex and lowest in the dorsolateral and ventrolateral septum, no differences between the groups occurred. CMS itself did not affect FosB/ΔFosB expression level. These findings demonstrate for the first time that repeated administration of ASE may result in eliciting of long-lasting FosB/ΔFosB-like transcription factors that could mediate some of the persistent and region-specific changes in brain function, interconnected with chronic drug exposure. However, it cannot be excluded that the impact of repeated ASE exposure might be influenced by an ambient stressogen leverage.

Sympathectomy reduces tumor weight and affects expression of tumor-related genes in melanoma tissue in the mouse.

Accumulated evidence indicates that sympathetic nerves may potentiate tumor growth, including melanoma. To elucidate possible mechanisms for this effect, we performed chemical sympathectomy by intraperitoneal (i.p.) injection of the neurotoxin 6-hydroxydopamine hydrobromide (100 mg/kg of body weight); in nine adult male C57BL/6J mice; nine control mice received i.p. vehicle (VEH). Seven days later, all mice were injected subcutaneously with 3 × 10(3) B16-F10 melanoma cells. Mice were euthanized 20 d after injection of melanoma cells, for measurement of tumor weight and expression of genes related to sympathetic signaling, apoptosis, hypoxia and angiogenesis in tumor tissue. To assess potential involvement of the hypothalamo-pituitary-adrenocortical axis in the effect of sympathectomy on melanoma growth, concentrations of plasma corticosterone and level of glucocorticoid receptor mRNA in tumor tissue were determined. We found that sympathectomy significantly attenuated melanoma growth (tumor weight 0.29 ± 0.16 g versus 1.02 ± 0.30 g in controls; p < 0.05). In tumor tissue from sympathectomized mice, we found significantly increased gene expression (measured by real-time PCR), relative to VEH-injected controls, of tyrosine hydroxylase, neuropeptide Y and glucocorticoid receptor (all p < 0.05), and alpha1, beta1 and beta3 adrenergic receptors (all p < 0.025), and factors related to apoptosis (Bcl-2 and caspase-3; p < 0.05) and hypoxia (hypoxia inducible factor 1 alpha) (p = 0.005). Plasma corticosterone concentrations were significantly elevated (p < 0.05) in these mice. Our findings indicate that sympathectomy induces complex changes in the tumor microenvironment reducing melanoma growth. Such complex changes should be considered in the prediction of responses of cancer patients to interventions affecting sympathetic signaling in tumor tissue and its environment.

Chronic liquid nutrition feeding affects blood pressure, heart and kidney morphology, and serum lipid profile in Wistar rats.

We determined the effect of chronic liquid nutrition (Fresubin) intake in different developmental stages on the cardiovascular and renal system of male Wistar rats. Body weight, water intake and blood pressure were periodically measured. Selected serum and urine biochemical parameters reflecting metabolic and homeostatic changes after Fresubin intake were investigated as well. Heart and kidney weight, diameter of cardiomyocytes, diameter and length of cardiomyocyte nuclei, wall thickness of thoracic aorta, the diameter and the area of renal corpuscles and serum and urine biochemical parameters were assessed at the end of experiment. We showed that Fresubin intake differently affects the investigated morphological and biochemical parameters in rats and this effect was dependent on the developmental stage when Fresubin was provided. Importantly, we have shown that Fresubin-induced elevation of blood pressure is a reversible phenomenon and it is independent of weight gain and subsequent development of obesity.

Role of nucleus of the solitary tract noradrenergic neurons in post-stress cardiovascular and hormonal control in male rats.

Chronic stress causes hypothalamo-pituitary-adrenal (HPA) axis hyperactivity and cardiovascular dyshomeostasis. Noradrenergic (NA) neurons in the nucleus of the solitary tract (NTS) are considered to play a role in these changes. In this study, we tested the hypothesis that NTS NA A2 neurons are required for cardiovascular and HPA axis responses to both acute and chronic stress. Adult male rats received bilateral microinjection into the NTS of 6-hydroxydopamine (6-OHDA) to lesion A2 neurons [cardiovascular study, n = 5; HPA study, n = 5] or vehicle [cardiovascular study, n = 6; HPA study, n = 4]. Rats were exposed to acute restraint stress followed by 14 d of chronic variable stress (CVS). On the last day of testing, rats were placed in a novel elevated plus maze (EPM) to test post-CVS stress responses. Lesions of NTS A2 neurons reduced the tachycardic response to acute restraint, confirming that A2 neurons promote sympathetic activation following acute stress. In addition, CVS increased the ratio of low-frequency to high-frequency power for heart rate variability, indicative of sympathovagal imbalance, and this effect was significantly attenuated by 6-OHDA lesion. Lesions of NTS A2 neurons reduced acute restraint-induced corticosterone secretion, but did not affect the corticosterone response to the EPM, indicating that A2 neurons promote acute HPA axis responses, but are not involved in CVS-mediated HPA axis sensitization. Collectively, these data indicate that A2 neurons promote both cardiovascular and HPA axis responses to acute stress. Moreover, A2 catecholaminergic neurons may contribute to the potentially deleterious enhancement of sympathetic drive following chronic stress.