Agnieszka Chocyk

Serotonin 5-HT1A receptors might control the output of cortical glutamatergic neurons in rat cingulate cortex

The present study was designed to investigate the distribution of serotonin 5-HT1A receptor protein (5-HT1A-immunoreactivity) and its localization within cortical pyramidal neurons of the rat cingulate cortex. This experimental direction was inspired by recent data showing the role of 5-HT1A receptors in the pathology of schizophrenia, and in the mechanism of action of novel antipsychotic drugs as well as by the importance of the cingulate cortex in regulation of cognitive functions. It was found that 5-HT1A-immunoreactivity was densely distributed in neuronal eyelash-like elements, and their size, shape and spatial orientation may suggest concentration of 5-HT1A-immunopositive material in the proximal fragments of axons of cortical neurons. Moreover, it was observed that these 5-HT1A-immunopositive fragments were present predominately on proximal fragments of axons of pyramidal neurons, which was evidenced by double labeling experiments using glutamate and non-phosphorylated neurofilament H as markers of the cortical pyramidal cells. The 5-HT1A receptor immunoreactivity was localized distally to the inhibitory GABAergic terminals of chandelier and basket cells surrounding the pyramidal cell bodies and occasionally surrounding short initial segment of axonal hillock of pyramidal neurons. These anatomical data indicate that 5-HT1A receptors might control the excitability and propagation of information transmitted by the pyramidal cells. Moreover, our results indicate that drugs operating via 5-HT1A receptors in the cingulate cortex might control from this level the release of glutamate in the subcortical structures. Finally, the 5-HT1A receptors present in the cingulate cortex, as demonstrated in the present study, may constitute an important target for drugs used to repair dysfunction of glutamate neurotransmission, which is observed for example in 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.

Early-life stress affects the structural and functional plasticity of the medial prefrontal cortex in adolescent rats.

Early life experiences are crucial factors that shape brain development and function due to their ability to induce structural and functional plasticity. Among these experiences, early‐life stress (ELS) is known to interfere with brain development and maturation, increasing the risk of future psychopathologies, including depression, anxiety, and personality disorders. Moreover, ELS may contribute to the emergence of these psychopathologies during adolescence. In this present study, we investigated the effects of ELS, in the form of maternal separation (MS), on the structural and functional plasticity of the medial prefrontal cortex (mPFC) and anxiety‐like behavior in adolescent male rats. We found that the MS procedure resulted in disturbances in mother–pup interactions that lasted until weaning and were most strongly demonstrated by increases in nursing behavior. Moreover, MS caused atrophy of the basal dendritic tree and reduced spine density on both the apical and basal dendrites in layer II/III pyramidal neurons of the mPFC. The structural changes were accompanied by an impairment of long‐term potentiation processes and increased expression of key proteins, specifically glutamate receptor 1, glutamate receptor 2, postsynaptic density protein 95, αCa2+/calmodulin‐dependent protein kinase II and αCa2+/calmodulin‐dependent protein kinase II phosphorylated at residue Thr305, that are engaged in long‐term potentiation induction and maintenance in the mPFC. We also found that the MS animals were more anxious in the light/dark exploration test. The results of this study indicate that ELS has a significant impact on the structural and functional plasticity of the mPFC in adolescents. ELS‐induced adaptive plasticity may underlie the pathomechanisms of some early‐onset psychopathologies observed in adolescents.

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.

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.

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.

Regulation of α-Synuclein Expression in Limbic and Motor Brain Regions of Morphine-Treated Mice

Chronic exposure to opiates produces dependence and addiction, which may result from neuroadaptations in the dopaminergic reward pathway and its target brain regions. The neuronal protein α-synuclein has been implicated in neuronal plasticity and proposed to serve as a negative regulator of dopamine neurotransmission. Thus, α-synuclein could mediate some effects of opiates in the brain. The present study investigated the influence of acute and chronic morphine administration on α-synuclein mRNA and protein expression in the brains of mice. Downregulation of α-synuclein mRNA was observed in the basolateral amygdala, dorsal striatum, nucleus accumbens, and ventral tegmental area of mice withdrawn from chronic morphine treatment. The changes were the most pronounced after longer periods of withdrawal (48 h). In contrast, levels of α-synuclein protein, as assessed by Western blotting, were significantly increased in the amygdala and striatum/accumbens (but not in the mesencephalon) of morphine-withdrawn mice. In both brain regions, levels of α-synuclein were elevated for as long as 2 weeks after treatment cessation. Because α-synuclein is a presynaptic protein, the detected opposite changes in its mRNA and protein levels are likely to take place in different populations of projection neurons whose somata are in different brain areas. Axonal localization of α-synuclein was confirmed by immunofluorescent labeling. An attempt to identify postsynaptic neurons innervated by α-synuclein-containing axon terminals revealed their selective apposition to calbindin D28K-negative projection neurons in the basolateral amygdala. The observed changes in α-synuclein levels are discussed in connection with their putative role in mediating suppression of dopaminergic neurotransmission during opiate withdrawal.

Cannabinoid CB1 receptors in rat medial prefrontal cortex are colocalized with calbindin- but not parvalbumin- and calretinin-positive GABA-ergic neurons.

In the present study, we investigate putative localization of cannabinoid receptors 1 (CB1) protein on a population of cortical gamma-aminobutyric acid (GABA) - positive interneurons characterized by expression of calcium-binding proteins in rat medial prefrontal cortex (MPC). Parvalbumin (PARV)/calretinin (CALR)- and calbindin (CALB)-positive neurons form two distinct populations of GABA-ergic interneurons that comprise the axo-somatic/axo-axonic and axo-dendritic inhibitory systems of pyramidal cells. It has been found that CB1 receptor-positive cells are randomly distributed across the rat MPC. All spotted neurons that were positive for CB1 receptors were positive for GABA; however, the number of GABA-positive cells drastically exceeded the number of CB1 receptor-positive neurons. Subsequent experiments with double-labelling of CB1 receptors with PARV and CALR revealed no colocalization. CALB-positive neurons (e.g., double bouquet and bipolar cells) display colocalization: the degree of colocalization among CB1 receptor-positive cells reached 18%. The appearance of CB1 receptors in double bouquet and bipolar neurons indicates that CB1 receptors may control the activity of pyramidal neurons from presynaptic sites in axo-dendritic synapses formed on apical and basilar dendrites of pyramidal neurons, as is characteristic for CALB-positive cortical interneurons. The phenotype of GABA- and CB1 receptor-positive but CALB-negative neurons may represent a population of inhibitory neurons that allow axo-somatic control of information flow, governed by principal neurons of the MPC.

8-OHDPAT-Induced Disruption of Prepulse Inhibition in Rats is Attenuated by Prolonged Corticosterone Treatment

The present study investigated the impact of acute and repeated administrations of corticosterone (10 mg/kg, twice daily, for 7 days) on serotonin (5-HT)1A receptor function, density and expression. The effect on 5-HT1A receptor function was assayed in rats by assessing the corticosterone-induced modulation of disruption of prepulse inhibition (PPI) of acoustic startle response induced by 8-OHDPAT, a 5-HT1A receptor agonist. Our experiments revealed that repeated but not acute treatment with corticosterone attenuated the 8-OHDPAT-evoked disruption of PPI without having any effect on PPI or startle amplitude alone. Chronic corticosterone treatment modulated also the neuronal activity of serotonergic pathways in the brain decreasing the level of 5-HIAA in the raphe nuclei and increasing both 5-HT and 5-HIAA levels in the hippocampus. Nevertheless, the effects of 8-OHDPAT on 5-HT metabolism were not changed by corticosterone. However, 5-HT1A receptor binding in the ventral hippocampus and entorhinal cortex but not in the raphe nuclei was decreased after chronic corticosterone treatment. It is concluded that chronically elevated corticosterone level is capable of inducing functional desensitization of 5-HT1A receptors which is paralleled by decreases in the 5-HT1A receptor binding in the ventral hippocampus and entorhinal cortex, the brain structures shown to be engaged in the regulation of PPI. Alterations in 5-HT1A receptors may be one of important mechanisms by which glucocorticoids/stress influence various psychiatric conditions.

c-Fos proteins, induced by the serotonin receptor agonist DOI, are not expressed in 5-HT2A positive cortical neurons.

In the present study, we tried to find out whether the expression of c-Fos proteins induced by DOI, an agonist of 5-HT2A/2C receptor subtypes is colocalized with 5-HT2A receptor protein in cortical neurons. 5-HT2A receptor protein was found in two major neuronal elements: dendritic processes (seen in layers II/III-V) and less abundantly in cell bodies (layer V). In our experiment, DOI (8 mg/kg) induced a robust appearance of c-Fos proteins mainly in neuronal nuclei of the upper part of layer V/IV, and a moderate amount of sparsely distributed nuclei in deep cortical layers (V and VI). It was found that c-Fos proteins never occurred in cortical neurons, which were immunopositive for the presence of 5-HT2A receptor protein. It is concluded that the induction of c-Fos proteins expression by DOI though initiated by activation of 5-HT2A receptors, requires the involvement of intermediate neurotransmitter(s). Additionally, our study indicates that the appearance of DOI-induced c-Fos proteins cannot be used as a simple and direct marker of localization and site of activation of 5-HT2A receptors.