Triclocarban Disrupts the Epigenetic Status of Neuronal Cells and Induces AHR/CAR-Mediated Apoptosis.

Triclocarban is a phenyl ether that has recently been classified as a contaminant of emerging concern. Evidence shows that triclocarban is present in human tissues, but little is known about the impact of triclocarban on the nervous system, particularly at early developmental stages. This study demonstrated that triclocarban that was used at environmentally relevant concentrations induced apoptosis in mouse embryonic neurons, inhibited sumoylation, and changed the epigenetic status, as evidenced by impaired activities of HDAC, sirtuins, and DNMT, global DNA hypomethylation, and alterations of methylation levels of bax, bcl2, Ahr, and Car genes. The use of selective antagonists and specific siRNAs, which was followed by the co-localization of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) in mouse neurons, points to the involvement of AHR and CAR in triclocarban-induced neurotoxicity. A 24-h treatment with triclocarban enhanced protein levels of the receptors which was paralleled by Car hypomethylation and Ahr hypermethylation. Car hypomethylation is in line with global DNA hypomethylation and explains the increased mRNA and protein levels of CAR in response to triclocarban. Ahr hypermethylation could reflect reduced Ahr mRNA expression and corresponds to lowered protein levels after 3- and 6-h exposures to triclocarban that is likely related to proteasomal degradation of activated AHR. We hypothesize that the triclocarban-induced apoptosis in mouse neurons and the disruption of epigenetic status involve both AHR- and CAR-mediated effects, which may substantiate a fetal basis of the adult onset of neurological diseases; however, the expression of the receptors is regulated in different ways.

Cocaine enhances ST8SiaII mRNA expression and neural cell adhesion molecule polysialylation in the rat medial prefrontal cortex.

The present study investigated whether cocaine (COC) administration evokes changes in the mRNA and protein levels of neural cell adhesion molecule (NCAM) and polysialylated neural cell adhesion molecule (PSA-NCAM) in the medial prefrontal cortex (mPFC) of rats. NCAM/PSA-NCAM is required for neuronal structural plasticity and is constitutively expressed in the mPFC. Rats were treated with a single dose of COC (15 mg/kg, i.p.), and mRNA levels of NCAM and the polysialyltransferases ST8SiaII and ST8SiaIV, enzymes involved in polysialylation of NCAM, were measured at 3, 6 and 24 h after COC treatment. At the same time points, the protein levels of NCAM and PSA-NCAM were measured via western blotting. Acute COC injection did not affect mRNA levels of NCAM and ST8SiaIV, but it increased the mRNA level of ST8SiaII 3 h after injection. At the same time point, an increase in PSA-NCAM, but not in NCAM, protein was observed. Morphological studies of PSA-NCAM protein expression patterns (immunocytochemistry/stereology) performed 3 h after COC administration revealed an enhancement of PSA-NCAM immunostaining in perisomatic-like sites and in the length density of PSA-NCAM-positive neuropil. Double immunofluorescence staining showed that PSA-NCAM perisomatic-like sites surround excitatory neurons. We also observed that a single injection of raclopride (0.4 mg/kg) or SCH 23390 (0.5 mg/kg), D2/D3 and D1 dopamine receptors antagonists, respectively, which were ineffective when given alone, abolished the effects of COC administration on mRNA and protein expression. The data in the present study indicate that COC administration may modify constitutive synaptic plasticity in the mPFC by increasing the NCAM polysialylation in perisomatic innervations of pyramidal neurons via activation of dopamine D1 and D2/D3 receptors.

The impact of maternal separation on the number of tyrosine hydroxylase-expressing midbrain neurons during different stages of ontogenesis.

Early life stressors have life-long functional and anatomical consequences. Though many neurotransmitters are involved in the functional impact of early life stress, dopamine seems to be important because of its roles in motor control, adaptation to stressful conditions, mood, cognition, attention and reward. Thus, in the present study, we investigated the way that early life stress, in the form of maternal separation (MS), affects the populations of tyrosine hydroxylase-immunoreactive (TH-IR) dopaminergic neurons in rat midbrain structures during ontogenesis. We included in the study the sub-regions of the substantia nigra (SN) and the ventral tegmental area (VTA). In both the control and MS rats, we found that the estimated total number of TH-expressing neurons fluctuated during ontogenesis. Moreover, MS influenced the number of TH-IR cells, especially in the SN pars reticulata (SNr) and VTA. Shortly after the termination of MS, on postnatal day (PND) 15, a decrease in the estimated total number of TH-IR neurons was observed in the SNr and VTA (in both males and females). On PND 35, MS caused a transient increase in the number of TH-IR cells only in the SNr of female rats. On PND 70, MS affected the number of TH-IR neurons in the VTA of females; specifically, an increase in the number of these cells was observed. Additionally, MS did not alter TH-IR cell sizes or the total levels of TH (measured by Western blot analysis) in the SN and VTA for all stages of ontogenesis in both males and females. The results from the study herein indicate that early life stress has enduring effects on the populations of midbrain TH-expressing dopaminergic neurons (especially in female rats), which are critically important for dopamine-regulated brain function throughout ontogenesis.

Maternal separation affects the number, proliferation and apoptosis of glia cells in the substantia nigra and ventral tegmental area of juvenile rats.

Adverse early life experiences can increase the risk of psychiatric and neurological disorders as the result of interference with brain development and maturation. In the present study, we tested whether early life stress, that is, maternal separation (MS), affects cell number, cell proliferation and constitutive apoptotic processes in the substantia nigra (SN) and the ventral tegmental area (VTA) of juvenile male and female rats. It was found that MS decreased the total number of glia but not neuronal cells in the SN pars compacta (SNc) and VTA of males. Moreover, MS reduced the number of S-100ß-immunoreactive (IR) glial cells in the SN of females and in the VTA of males. It was also observed that MS decreased the rate of proliferation (as measured by Ki-67-immunoreactivity) in the SN pars reticulata (SNr) and VTA of both males and females. Additionally, MS reduced the number of TUNEL-positive cells in the SNc of both males and females and in the SNr and VTA of males only. Moreover, MS decreased the number of cleaved caspase-9-IR cells in the SN and VTA of male rats. Cleaved caspase-9 was present in microglia cells, which exhibited the morphological hallmarks of apoptosis, but not in neuronal, astrocytic or oligodendrocytic cells. On the contrary, MS increased the number of cleaved caspase-3-IR cells in the SN and VTA of both male and female rats. Cleaved caspase-3-IR cells did not display signs of apoptosis and had an astrocytic phenotype (S-100ß-IR). In males exposed to MS, a decrease in caspase-3 enzymatic activity in the SN was also observed. In summary, the results of the present study revealed that early life stress affects the number, proliferation and naturally occurring apoptosis of glia cells in the SN and VTA in a sex-dependent manner and consequently may impair brain functions that are regulated by these structures.

5-HT1A receptors mediate detrimental effects of cocaine on long-term potentiation and expression of polysialylated neural cell adhesion molecule protein in rat dentate gyrus.

The present study investigated the involvement of 5-HT(1A) receptors in the inhibitory effect of single administration of cocaine (COC, 15 mg/kg i.p.) on the induction of long-term potentiation (LTP) in slices of rat dentate gyrus (DG), prepared 30 min and 2 days after COC administration. These effects of COC were blocked by an antagonist of 5-HT(1A) receptors, WAY 100635 (0.4 mg/kg i.p.), which had been administered 20 min before COC. The detrimental effect of COC on LTP in slices prepared 30 min after COC administration could be prevented by blocking glucocorticoid receptors (GRs) using mifepristone (RU 38486, 10 mg/kg s.c. given 1 h before COC), similar as in slices obtained 2 days after COC as reported previously [Mackowiak et al. (2008) Eur J Neurosci 27:2928-2937]. After a single administration of an agonist of 5-HT(1A) receptors, 8-OH-DPAT, (0.5 mg/kg i.p.), the level of LTP in slices prepared 2 days later was significantly decreased resembling the effect of COC. This effect of 8-OH-DPAT was antagonized by WAY 100635 (0.4 mg/kg i.p.), administered 20 min before 8-OH-DPAT and by RU 38486, given 1 h before 8-OH-DPAT. COC-induced inhibition of LTP could be blocked by the inhibitor of mitogen-activated protein kinase kinase 1/2 (MEK1/2), SL 327 (50 mg/kg i.p.), administered 1 h before COC, but not by the inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), LY 294002 (80 mg/kg i.p.). COC-induced reduction in the number of polysialylated neural cell adhesion molecule (PSA-NCAM)-positive neurons in rat dentate gyrus could also be prevented by WAY 100635, given 20 min before COC. These data indicate that the indirect 5-HT(1A) receptor activation by a single COC administration and subsequent stimulation of extracellular signal-regulated kinases (ERK 1/2) signaling pathway result in a decrease of the potential for long-term increase in synaptic efficacy in rat DG lasting at least two but less than 7 days, most likely via activation of the hypothalamic-pituitary-adrenal (HPA) axis.

Aryl hydrocarbon receptor-mediated apoptosis of neuronal cells: a possible interaction with estrogen receptor signaling.

Activation of aryl hydrocarbon receptors (AhRs) induces neuronal damage, but the mechanism by which this occurs is largely unknown. This study evaluated the effects of an AhR agonist, beta-naphthoflavone, on apoptotic pathways in mouse primary neuronal cell cultures. beta-Naphthoflavone (0.1-100 micronhanced caspase-3 activity and lactate dehydrogenase (LDH) release in neocortical and hippocampal cells. These data were supported at the cellular level with Hoechst 33342 and calcein AM staining. alpha-Naphthoflavone inhibited the action of beta-naphthoflavone, thus confirming specific activation of AhRs. A high-affinity estrogen receptor (ER) antagonist, ICI 182,780, and a selective estrogen receptor modulator (SERM), tamoxifen, enhanced beta-naphthoflavone-mediated apoptosis. Another SERM, raloxifene, and an ERalpha antagonist, methyl-piperidino-pyrazole, did not affect beta-naphthoflavone-induced caspase-3 activity. However, they inhibited beta-naphthoflavone-induced LDH release at a late hour of treatment, thus suggesting delayed control of AhR-mediated neuronal cell death. The apoptotic effects of beta-naphthoflavone were accompanied by increased levels of AhRs, and these receptors colocalized with ERbeta as demonstrated by confocal microscopy. These data strongly support apoptotic effects of AhR activation in neocortical and hippocampal tissues. Moreover, this study provides evidence for direct interaction of the AhR-mediated apoptotic pathway with estrogen receptor signaling, which provides insight into new strategies to treat or prevent AhR-mediated neurotoxicity.

Activation of CB1 cannabinoid receptors impairs memory consolidation and hippocampal polysialylated neural cell adhesion molecule expression in contextual fear conditioning.

We investigated the role of CB1 receptors in hippocampal-dependent memory consolidation mediated by polysialylated neural cell adhesion molecule (PSA-NCAM) during contextual fear conditioning (CFC). The CB1 receptor agonist 3-(1,1-dimethylheptyl)-(-)-11-hydroxy-Delta(8)-tetrahydrocannabinol (HU-210) (0.1 mg/kg) was given immediately after training during the memory consolidation phase, and freezing behavior was measured 24 h after conditioning. Administration of HU-210 attenuated freezing behavior measured in CFC. Western blot analysis showed that CFC induced a decrease in the expression of NCAM-180, but did not change the level of NCAM-140 and increased PSA-NCAM expression measured 24 h after training in the rat hippocampus. HU-210 (0.1 mg/kg) injection did not affect the reduction in NCAM-180 levels induced by CFC, but it blocked the increase in PSA-NCAM expression. Since the dentate gyrus (DG) of the hippocampus is known to be involved in memory consolidation and expresses a high level of PSA-NCAM protein, we measured the effects of CFC and HU-210 administration on PSA-NCAM-immunoreactive (IR) cells in the DG. CFC caused an increase in the number of PSA-NCAM-IR cells in the DG, but not K(i)-67- or doublecortin (DCX)-IR cells. This increase in PSA-NCAM-IR cells was abolished by HU-210 injection. Administration of the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251) (3 mg/kg immediately before HU-210) inhibited the effects of HU-210 on freezing behavior and PSA-NCAM expression in the DG. These results indicate that activation of CB1 receptors disturbs consolidation of fear memory in CFC, likely by affecting PSA-NCAM expression in the DG, which plays an important role in synaptic rearrangement during the formation of memory traces.

A search for colocalization of serotonin 5-HT2A and 5-HT1A receptors in the rat medial prefrontal and entorhinal cortices--immunohistochemical studies.

Recently developed antipsychotic drugs ameliorating the negative symptoms of schizophrenia act not only on dopamine D2 receptors but also on serotonin 2A (5-HT2A) and 1A (5-HT1A) receptors in specific regions of the cerebral cortex. Since it is not yet known whether serotonin 5-HT1A and 5-HT2A receptors coexist in the same population of neurons in the cortex, the present study investigated their colocalization in the rat medial prefrontal (MPC) and entorhinal (EC) cortices. Using antibodies that recognize epitopes specific to the serotonin 5-HT2A or 5-HT1A receptors, studies employing confocal microscopy have shown that in the MPC 5-HT2A receptors are preferentially, if not exclusively, present on the pyramidal neurons and that 5-HT1A-immunopositive material is present in the axonal hillocks and, to lower extend, in cytoplasm of presumably pyramidal cell bodies. With the regard of labeling of active receptors (i.e. present in shafts and axonal hillocks) we found that about 38% of neurons positive for the presence of serotonin 5-HT2A receptors, are also positive for serotonin 5-HT1A receptors in the MPC. In the EC, only 22% of serotonin 5-HT2A-positive neurons were positive for serotonin 5-HT1A receptor-immunoreactivity. In the respect of cytoplasmatic serotonin 5-HT1A receptor-immunoreactivity (possibly inactive receptors), 65% and 73% of serotonin 5-HT2A receptor-positive neurons were colocalized with serotonin 5-HT1A receptors in the MPC and EC, respectively. Data obtained on serotonin 5-HT2A and 5-HT1A receptor localization provide anatomical grounds for at least three distinct populations of pyramidal neurons, one governed only by 5-HT2A, one only by 5-HT1A and one by both types of serotonin receptors.

Impact of postnatal blockade of N-methyl-D-aspartate receptors on rat behavior: a search for a new developmental model of schizophrenia.

The malfunction of glutamatergic neurotransmission in the neonatal or postnatal periods may be a risk factor for the appearance of neuroanatomical, neurochemical or functional changes that are characteristic of schizophrenia. Thus, the present study was undertaken to investigate whether blockade of N-methyl-d-aspartate (NMDA) receptors in the postnatal period influences rat behavior in tests characterizing schizophrenia-like deficits such as psychomotor agitation, impairments of sensorimotor gating, working memory, and intensity of social interactions. (E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 40116), a competitive antagonist of NMDA receptors, was given postnatally (1.25 mg/kg on days 1, 3, 6, 9; 2.5 mg/kg on days 12, 15, 18; and finally 5 mg/kg on day 21, all injections s.c.), and rats were tested at 60 days old. We found that blockade of NMDA receptors in the postnatal period led to an enhancement of exploration, mimicking psychomotor agitation, impairments in sensorimotor gating as measured by a prepulse-evoked inhibition of acoustic startle response, and an impaired working memory, as measured by an increase in the latency to achieve accurate rate of response in the delayed alternation task. Decreases in non-aggressive social interactions and increases in aggressive interactions were also observed. In addition to cognitive deficits typical of schizophrenia, rats treated postnatally with NMDA receptor antagonists also showed higher level of fear exhibited in the elevated plus maze. Thus, the blockade of NMDA receptors in the postnatal period may model deficits that are characteristic of schizophrenia.

Determination of the electric field gradient asymmetry from 2D nutation NQR spectra of 75As nuclei in oriented samples of As2Se3 semiconductor.

The method of two-dimensional nutation nuclear quadrupole resonance in time domain is used to study the chalcogenide semiconductor As(2)Se(3). In this system, the nuclear quadrupole resonance (NQR) resonance line width is as large as 10 MHz; therefore, the radiofrequency field produced by a pulsed NQR spectrometer can excite only a portion of the nuclear spins. The proposed method relies on polarizing the melted glass specimen in a strong magnetic field so that orientational disorder is partially removed. After hardening the sample is placed in a spectrometer r.f. coil in such a way that that the axes of sample polarization and r.f. coil coincide. We demonstrate the application of this method to determine eta in glassy As(2)Se(3).

Glutamatergic neurons of rat medial prefrontal cortex innervating the ventral tegmental area are positive for serotonin 5-HT1A receptor protein.

The present study was designed to investigate whether serotonin 5-HT1A receptor protein (5-HT1A receptor-immunoreactivity), is present on cortical pyramidal neurons of the rat medial prefrontal cortex (MPC) innervating the ventral tegmental area (VTA). Recent data stress the role of serotonin 5-HT1A receptors in the pathology of schizophrenia, and in the mechanism of action of novel antipsychotic drugs. It was found that approximately 52% of cells in layers II/III of the MPC whose axons initial segments were immunoreactive for serotonin 5HT1A receptor were also labeled with Fluoro-Gold (FG), a retrograde tracer injected into the VTA, indicating that certain portion of neurons forming glutamatergic innervations of the VTA may be controlled by serotonin 5-HT1A receptors. In deep cortical layers (V/VI) retrogradely labeled neurons never colocalized with serotonin 5-HT1A receptormmunoreactivity. These anatomical data indicate that serotonin 5-HT1A receptors might potentially control the excitability and propagation of information transmitted by the pyramidal cells to the VTA. Moreover, our results indicate that the drugs operating via serotonin 5-HT1A receptors in the MPC, might control from this level the release of glutamate in the VTA and restore function of glutamate neurotransmission, whose dysfunction is observed for example in schizophrenia.

Brain MRI in late-onset multiple sclerosis.

Multiple sclerosis (MS) with clinical onset after 50 years of age is unusual (between 1 and 6%) and is frequently misdiagnosed. Furthermore, brain magnetic resonance imaging (MRI) abnormalities are frequently observed in subjects over 50 years of age. The aim of this study was to describe brain MRI in late-onset MS to evaluate the sensitivity and specificity of radiological MS criteria in patients aged over 50 years. We evaluated the brain MRI of 20 patients with onset of MS after 50 years of age. We compared these MRI with 26 controls matched for age, sex and vascular risk factors. MRI were blindly analysed by two neuroradiologists according to Paty et al.'s [Neurology38 (1988) 180] criteria, Fazekas et al.'s [Neurology38 (1988) 1822] criteria and Barkhof et al.'s [Brain120 (1997) 2059] criteria. The mean age at MRI scanning was 58 years. Sensitivity was 90% for Paty et al.'s criteria, 80% for Fazekas et al.'s criteria and 85% for Barkhof et al.'s criteria. Specificity was 54% for Paty et al.'s criteria, 69% for Fazekas et al.'s criteria and 65% for Barkhof et al.'s criteria. Barkhof et al.'s criteria are less specific in older patients than in young patients. We suggest that spinal cord MRI and cerebrospinal fluid analysis should be systematically performed in suspected late-onset MS in order to increase the specificity of the diagnosis.

DOI, an agonist of 5-HT2A/2C serotonin receptor, alters the expression of cyclooxygenase-2 in the rat parietal cortex.

The hallucinogenic effect of DOI, serotonin 5-HT2A/2C receptor agonist, is known to be associated with the activation of cortical 5-HT2 receptors. However, the effect of DOI on excitability of cortical neurons and their subsequent function is still not quite understood. Previous immunohistochemical studies using Fos proteins expression as a marker of neuronal activity showed the involvement of arachidonic acid cascade, particularly cyclooxygenase metabolic pathway, in DOI-induced Fos proteins expression in the rat parietal cortex. DOI increases arachidonic acid release which is transformed itself via acceleration of cyclooxygenase metabolic pathway to biologically active metabolites, such as prostaglandins and thromboxanes. Since cyclooxygenase-2 (COX-2) expression correlates with neuronal activity, it was of interest to investigate whether DOI is capable of influencing the level of COX-2 protein and mRNA expression in the rat parietal cortex. It was observed that neurons which were positive for 5-HT2A receptors showed constitutive COX-2 immunoreactivity. It was found further, that COX-2 protein level was increased at 1 h, and returned to the control level at 3 and 6 h after DOI (5 mg/kg) administration. In contrast, DOI decreased the COX-2 mRNA expression at all tested time points (1 h, 3h and 6h after DOI treatment). The obtained results further support the suggestion that COX-2 activation and possibly arachidonic acid metabolites generated by COX-2 may be considered as important mediators of functional responses generated by activation of cortical 5-HT2A/2C receptors.

Kainate receptor agonists, antagonists and allosteric modulators.

Interest in kainate receptors has increased over the past few years. Our understanding of their physiology and pharmacology has improved markedly since their original cloning and expression in the early 1990s. For example, agonist profiles at recombinant kainate receptors have been used to identify and distinguish kainate receptors in neurons. Furthermore, the development of selective antagonists for kainate receptor subtypes has increased our understanding of the functional roles of kainate receptors in neurons and synaptic transmission. In this review we described the activity of agonists and antagonists at kainate receptors and their selectivity profiles at NMDA and non-NMDA receptors.

[The involvement of 5-HT1a serotonin receptors in the pathophysiology and pharmacotherapy of schizophrenia]. / Udzial receptorów serotoninowych 5-HT1A w patofizjologii i farmakoterapii schizofrenii.

Many studies of schizophrenic brains indicate the dysfunction of dopamine and glutamate systems in the prefrontal and frontal cortex. It seems that better understanding of mechanisms regulating functions of these neuronal cortical systems could contribute to creation of new drugs acting in the cortex selectively. This might be profitable in cognition of dysfunction and negative symptoms in schizophrenia. This article presents preclinical data concerning the role of 5-HT1A serotonin receptors in the modulation of cortical dopamine system and in psychotomimetic effects of non-competitive NMDA receptor antagonists. Neurochemical studies have shown that 5-HT1A receptor agonists increase dopamine release in the rat prefrontal cortex on the one hand, and they inhibit the augmentation of dopamine release induced by stress or amphetamine, on the other. However, the increase of dopamine release induced by non-competitive NMDA receptor antagonists is blocked by 5-HT1A receptor antagonists. Blockade of 5-HT1A receptors seems to be important also in reduction of most psychotomimetic effects induced by non-competitive NMDA antagonists both involving (locomotor hyperactivity, working memory impair) and not involving (sensorimotor gating deficits) dopamine mechanism. Thus, binding with 5-HT1A receptors can be an important site for the regulation of cortical dopamine system, both in physiological conditions and in disregulation of the system induced by stress, psychostimulants or psychotomimetics. On the other hand, 5-HT1A receptors modulate most of psychotomimetic effects of non-competitive NMDA receptor antagonists. The above results of preclinical investigations indicate that 5-HT1A receptor can be involved in the pathology of schizophrenia, what is partly confirmed by clinical postmortem studies of schizophrenic brains. These studies showed the increase of 5-HT1A receptor density in prefrontal and frontal cortex in schizophrenic brains. It also seems that 5-HT1A receptors might be a good target for the antipsychotic drugs. Although the clinical studies have demonstrated controversial data, maybe further studies using substances with selectivity to 5-HT1A receptors would help to determine more precisely the role of these receptors in pathology and pharmacotherapy of schizophrenia.

WAY 100135, an antagonist of 5-HT1A serotonin receptors, attenuates psychotomimetic effects of MK-801.

In the present study, we investigated whether the antagonist of 5-HT1A receptors, WAY 100135, was capable of modifying the psychostimulant and psychotomimetic effects of MK-801, a non-competitive antagonist of NMDA receptors. It was found that: 1) WAY 100135 (10 and 20 mg/kg, but not 1.25, 2.5, and 5 mg/kg) transiently, in a dose dependent manner, attenuated the locomotor stimulant effects of MK-801 (0.4 mg/kg). Given alone, WAY 100135 had no effect on the locomotor activity of rats; 2) WAY 100135 (1.25 and 2.5 mg/kg, but not 10 or 20 mg/kg), attenuated or abolished the disruptive effects of MK-801 on the sensorimotor gating measured in a prepulse-induced inhibition of the acoustic startle response paradigm. WAY 100135 in all tested doses had no effect on the sensorimotor gating or amplitude of the acoustic startle response; 3) WAY 100135 (1.25, 2.5 mg/kg, but not 5 mg/kg) attenuated the detrimental effects of MK-801 on working memory and selective attention, measured in a delayed alternation task. Again, given alone, WAY 100135 did not influence the behavior of rats in that experimental paradigm; and 4) MK-801 (0.4 mg/kg) had no effect on the 5-HT1A receptor mRNA level in rat hippocampus, measured 2 and 24 hours after MK-801 administration. These data indicate that 5-HT1A receptors might be involved in the psychotomimetic effects of non-competitive NMDA receptor antagonists. In addition, 5-HT1A serotonin receptor antagonists and partial agonists may have potential antipsychotic properties.

Metyrapone, an inhibitor of corticosterone synthesis, blocks the kainic acid-induced expression of HSP 70.

It is shown in the present study that metyrapone (100 mg/kg), an inhibitor of corticosterone synthesis, given twice, 30 min before and 6 h after kainic acid (10 mg/kg) administration, blocks the kainic acid-evoked induction of heat shock proteins 72 kDa (HSP 70). Specifically, it was observed that metyrapone completely prevented kainic acid-induced appearance of HSP 70 in the rat amygdala, habenula, parietal cortex, and significantly decreased the number of HSP 70-positive neurons in the CA1, CA3, and CA4 subregions of hippocampus. The reduction in HSP 70 induction was paralleled by a complete prevention of the kainic acid-induced rise in the circulating corticosterone level by metyrapone; however, in applied doses metyrapone evoked slight enhancement of blood corticosterone. Despite the fact that metyrapone blocked/attenuated the kainic acid-evoked induction of HSP 70, its administration did not affect the behavioral effects of kainic acid, regarded as "limbic status epilepticus." It is concluded that the blockade of corticosterone synthesis might have neuroprotective effects in the pathological states associated with the overstimulation of glutamatergic receptors.

Cortical localization of dopamine D4 receptors in the rat brain--immunocytochemical study.

Using polyclonal antibody against dopamine D4 receptor we investigated cortical distribution of D4 receptors, with the special emphasis on regions of the prefrontal cortex. Prefrontal cortex is regarded as a target for neuroleptic drugs, and engaged in the regulation of the psychotic effects of various substances used in the experimental modeling of schizophrenia. Western blot analysis performed on samples from the rat cingulate, parietal, piriform cortices and also striatum revealed that antibody recognized one main band of approximately 40 kD, which corresponds to the predicted molecular weight of D4 receptor protein. In immunocytochemical studies we found D4 receptor-positive neurons in all regions of prefrontal cortex (cingulate, agranular/insular and orbital cortices) and all cortical regions adjacent to prefrontal cortex, such as frontal, parietal and piriform cortex. Substantial number of D4 receptor-positive neurons has also been observed within the striatum and nucleus accumbens. In general, a clear stratification of the D4 receptor-positive neurons was observed in the cortex with the highest density seen in layers II/III and V/VI. D4 immunopositive material was also found in the dendritic processes, particularly clearly visible in the layer II/III. At the cellular level D4 receptor immunoreactivity was seen predominantly on the periphery of the cell body, but a certain population of neurons with clear cytoplasmatic localization was also identified. In addition to cortical distribution of D4 receptor-positive neurons we tried also to define types of neurons expressing D4 receptor protein. In double-labeling experiments, D4 receptor protein was found in nonphosphorylated neurofilament H-positive, calbindin-D28k-positive, as well as parvalbumin-positive cells. Since, used proteins are markers of certain populations of pyramidal neurons and GABA-ergic interneurons, respectively, our data indicate that D4 receptors are located on cortical pyramidal output neurons and their dendritic processes as well as on interneurons. Above localization indicates that D4 receptors are not only directly influencing excitability of cortical inter- and output neurons but also might be engaged in dendritic spatial and temporal integration, required for the generation of axonal messages. Additionally, our data show that D4 receptors are widely distributed throughout the cortex of rat brain, and that their cortical localization exceeds the localization of dopaminergic terminals.

Distribution of dopamine D1 receptors in the nucleus paraventricularis of the hypothalamus in rats: an immunohistochemical study.

The present study investigated the distribution of dopamine D1 receptor protein in the nucleus paraventricularis of the hypothalamus. It was found that the nucleus paraventricularis of the hypothalamus contains a relatively large number of cells which are positive for presence of dopamine D1 receptor protein. The vast majority of dopamine D1 receptor-positive neurons was found in the magnocellular part, but they were also present in considerable quantity in the parvocellular part of this subregion of the hypothalamus. When measured by the Western blot technique, the quantity of D1 receptor protein found in the paraventricular nucleus of the hypothalamus was at the level found in the prefrontal cortex. It was also found that dopamine D1 receptor protein was present in neurons constitutively displaying phosphorylated CREB protein, i.e. neurons which are, as might be speculated, under the tonic influence of neurotransmitters whose receptors operate via cAMP and pCREB as second or third messengers. The presence of dopamine D1 receptors in the nucleus paraventricularis of the hypothalamus may suggest, at an anatomical level, that these receptors are involved in controlling the release of hormones, as well as their synthesis at the level of transcription, which is regulated by phosphorylation of CREB protein. Finally, the present immunocytochemical findings offer an anatomical substrate for the role of dopamine and its receptors of D1 subtype in the regulation of the activity of paraventricular neurons seen in the functional studies.