Molecular Profiles of Pyramidal Neurons in the Superior Temporal Cortex in Schizophrenia

Disrupted synchronized oscillatory firing of pyramidal neuronal networks in the cerebral cortex in the gamma frequency band (i.e., 30–100 Hz) mediates many of the cognitive deficits and symptoms of schizophrenia. In fact, the density of dendritic spines and the average somal area of pyramidal neurons in layer 3 of the cerebral cortex, which mediate both long-range (associational) and local (intrinsic) corticocortical connections, are decreased in subjects with this illness. To explore the molecular pathophysiology of pyramidal neuronal dysfunction, we extracted ribonucleic acid (RNA) from laser-captured pyramidal neurons from layer 3 of Brodmann’s area 42 of the superior temporal gyrus (STG) from postmortem brains from schizophrenia and normal control subjects. We then profiled the messenger RNA (mRNA) expression of these neurons, using microarray technology. We identified 1331 mRNAs that were differentially expressed in schizophrenia, including genes that belong to the transforming growth factor beta (TGF-β) and the bone morphogenetic proteins (BMPs) signaling pathways. Disturbances of these signaling mechanisms may in part contribute to the altered expression of other genes found to be differentially expressed in this study, such as those that regulate extracellular matrix (ECM), apoptosis, and cytoskeletal and synaptic plasticity. In addition, we identified 10 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of their predicted gene targets revealed signaling pathways and gene networks that were found by microarray to be dysregulated, raising an interesting possibility that dysfunction of pyramidal neurons in schizophrenia may in part be mediated by a concerted dysregulation of gene network functions as a result of the altered expression of a relatively small number of miRNAs. Taken together, findings of this study provide a neurobiological framework within which specific hypotheses about the molecular mechanisms of pyramidal cell dysfunction in schizophrenia can be formulated

An Animal Model of Emotional Blunting in Schizophrenia

Schizophrenia is often associated with emotional blunting—the diminished ability to respond to emotionally salient stimuli—particularly those stimuli representative of negative emotional states, such as fear. This disturbance may stem from dysfunction of the amygdala, a brain region involved in fear processing. The present article describes a novel animal model of emotional blunting in schizophrenia. This model involves interfering with normal fear processing (classical conditioning) in rats by means of acute ketamine administration. We confirm, in a series of experiments comprised of cFos staining, behavioral analysis and neurochemical determinations, that ketamine interferes with the behavioral expression of fear and with normal fear processing in the amygdala and related brain regions. We further show that the atypical antipsychotic drug clozapine, but not the typical antipsychotic haloperidol nor an experimental glutamate receptor 2/3 agonist, inhibits ketamine's effects and retains normal fear processing in the amygdala at a neurochemical level, despite the observation that fear-related behavior is still inhibited due to ketamine administration. Our results suggest that the relative resistance of emotional blunting to drug treatment may be partially due to an inability of conventional therapies to target the multiple anatomical and functional brain systems involved in emotional processing. A conceptual model reconciling our findings in terms of neurochemistry and behavior is postulated and discussed

The role of corticotropin-releasing factor in anxiety disorders

Thesis (MSc)--University of Stellenbosch, 2001.ENGLISH ABSTRACT: SEPARATION STUDY Traumatic experiences during childhood can have a negative impact on behaviour later in life. Kendier et al. (1992) found that the loss of a parent during childhood increased the risk to develop major anxiety disorders and could also lead to depressive-like behaviour (Furukawa et al., 1999). Methods: We subjected rat pups to maternal separation and determined the effects thereof on adult behaviour. We removed rat pups from their mothers for 3 hours daily from postnatal day 2 to 14. On day 60, the behaviours of the rats were tested using the elevated plus-maze and the open field test. Controls were reared normally. Behaviours: Amount of time spent and the number of entries into the arms of the maze were noted on the elevated plus-maze, while the total time spent in each zone (inner versus outer) and the number of zone crossings were noted for each rat on the open field arena. The latency to move from the initial placement in the outer zone to the inner zone as well as the number of quadrant crossings was also determined. Defecation, freezing, rearing and grooming behaviours were also noted. Neurotransmitter levels: Noradrenaline, serotonin and their metabolites were evaluated in maternally separated rats and compared to controls. Their concentrations at basal level, immediately after restraint stress and 15 minutes after restraint stress, were also determined. A HPLC method was followed in these determinations. ACTH Determinations: All rats were subjected to restraint stress for a lO-minute period. Trunk blood was collected for basal, as well as 15 and 60 minutes postrestraint stress for ACTH determinations. Results: Behaviours: The amount of entries was significantly reduced in the separated animals, indicating decreased locomotion. They spent significantly more time in the closed maze arms. A significant increase in defecation frequency and rearing behaviour was noted. These observations are typical of anxious behaviour. In the open field test, the behavioural results were less convincing. Only a significant increase in defecation frequency and a significant decrease in rearing behaviour in separated animals, were observed. Neurotransmitter levels: No significant differences were noted between separated animals and controls with respect to basal monoamine levels. However, noradrenaline levels were significantly decreased in the frontal cortex 15 minutes after restraint stress and immediately after restraint stress in the hypothalamus and hippocampus in separated animals. MHPG levels were significantly decreased in the frontal cortex immediately after restraint stress. No significant differences were found with respect to serotonin levels. However, significant increases were found in 5HIAA levels in the frontal cortex and hippocampus of separated rats, 15 minutes after restraint stress. The basal turnover ratios of serotonin (5HIAA/5HT) and noradrenaline (MHPGINA) did not yield significant results. However, immediately after restraint stress, a significant increase was found in serotonin turnover in the hypothalamus of separated rats when compared to controls. This turnover rate was also increased in separated rats, 15 minutes after restraint stress in the frontal cortex and hypothalamus. ACTH Determinations: Basal ACTH levels were significantly higher in separated animals. At 15 minutes post-restraint stress, the levels were significantly lower than controls, indicating a blunted stress response. Our results therefore showed that maternal separation could lead to anxious behaviours in adult life. These behavioural abnormalities were associated with alterations in the central nervous and neuroendocrinological systems, particularly in response to stressful situations. CRF STUDY The maternal separation study indicated that elevated CRF levels could possibly be causally related to abnormalities observed in the anxious animals. We therefore hypothesised that adverse development factors, such as maternal separation, predisposes individuals to develop psychopathologies later in life and that this process was driven by a presence of high CRF levels. Methods: Cannulas were implanted into the left lateral ventricles of normal rats, making use of stereotaxic procedures. CRF (3 flg/fll) was injected into the ventricles daily for 5 days. Saline controls were handled similarly, but only injected with saline for the same time period. Both groups of animals were then compared to naïve controls. Histology was performed to determine the correct placement of the cannulas. Behaviours: The Elevated Plus-maze was employed to determine whether their behaviours were anxious. The number of entries into the various arms of the maze as well as the amount of time spent in the open and closed arms was accumulated. Rearing, freezing, defecation and grooming were also noted. ACTH Determinations: The ACTH levels ofCRF-injected, saline-injected and naïve rats were determined 15 minutes after restraint stress. Results: Behaviours: A decrease in the number of entries into the closed arms of the maze was noted in the CRF-injected rats when compared to naïve controls. No significant differences were found between the groups with respect to the amount of time spent in the various arms and the behaviours noted during the experiment. ACTH Determinations: A decrease in ACTH levels was noted in CRF-injected rats 15 minutes after restraint stress when compared to naïve controls. Therefore, although the CRF injections did not alter the behaviour of the rat, they did exhibit a blunted stress response to the stressor. Conclusion: Our experiments led us to conclude that early adverse experiences, such as maternal separation, can lead to the development of psychopathologies later in life. CRF, however, is not pivotal in the development of these abnormalities; rather it seems that the neurochemical abnormalities (serotonin and noradrenaline) play a more important role in the development of these mental disturbances. Finally, we hypothesise that combination drug therapy that targets both the noradrenergic and serotonergic neurotransmitter systems could be preferred above those aimed at rectifying the individual neurotransmitter systems in the treatment of psychopathologies, such as anxiety disorders.AFRIKAANSE OPSOMMING: MOEDERLIKE SKEIDINGS STUDIE Traumatiese gebeurtenisse wat gedurende kinderjare ervaar word, kan 'n negatiewe impak op die gedrag van dieselfde individue hê, as hulle volwassenheid bereik het. Kendier et al. (1992) het waargeneem dat die verlies van 'n ouer tydens die kinderjare, die risiko om angssteumisse te ontwikkel, dramaties verhoog en kan ook lei tot 'n depressiewe gemoedtoestand (Furukawa et al., 1999). Metodes: Ons het neonatale rotte aan moederlike skeiding blootgestel en die effekte daarvan op gedrag tydens hul volwasse lewe beoordeel. Ons het daagliks die moeders vir 3 ure van die kleintjies afweggeneem, vanafpostnatale dag 2 tot 14. Op dag 60, het ons die gedrag van die diere op die "elevated plus-maze" en die" open field test" getoets. Kontrole rotte het onder normale omstandighede opgegroei. Gedrag parameters: Die hoeveelheid tyd en aantal kere wat die rotte in die verskillende arms van die "elevated plus-maze" gespandeer het, was waargeneem. Die totale tyd in die "open field" toets se binneste ofbuitenste sones, die hoeveelheid kruisings tussen die twee sones, die tyd wat dit neem om beweging in die binneste sone te inisiëer, sowel as die hoeveelheid kwadrante wat gekruis was, is genotuleer. Defekasie, botstilstande, steiering, en versorgingsgedragte was ook waargeneem terwyl die rotte in die doolhowe was. Neurochemiese oordragstowwe: Die hippokampus, hipotalamus en frontale korteks van moerderlik-geskeide rotte en kontroles, was uit hul brein gedissekteer om die vlakke van noradrenalien, serotonien en hul metaboliete daarin te bepaal. Basale vlakke sowel as hul konsentrasies onmiddelik na stres en 15 minute na stres, was gedetermineer. 'n HPLC metode was gebruik vir hierdie bepalings. ACTH bepalings: Rotte, moederlik-geskei en kontroles, was onderwerp aan beperkingstres vir 'n tydsduur van 10 minute. Bloed was op die volgende tydsintervalle gekollekteer vir die bepaling van ACTH vlakke, naamlik basaal, 15 minute en 60 minute na die einde van stresperiode. Resultate: Gedrag: Op die "elevated plus-maze" was moederlik-geskeide rotte minder beweeglik omdat hul aanmerklik minder die arms van die doolhowe binne gegaan het. Hulle het ook baie meer tyd in die geslote arms gespandeer. Verder het die eksperimentele rotte meer defekasie bolusse uitgeskei en was die aantal steieringe uitgevoer, ook aanmerklik verhoog. Hierdie patroon van gedrag is tipies die van angstigheid. Neurochemiese oordragstowwe: Daar was geen betekenisvolle verskil tussen die basale neurotransmitter vlakke van moederlik-geskeide rotte en hul kontroles. Daarenteen was die vlakke van noradrenalien in die frontale korteks dramaties verhoog by die 15 minute tydsinterval na die stres, asook onmiddelik na die stres in die hipotalamus en hippokampus. MHPG vlakke was egter aanmerklik verlaag in die frontale korteks onmiddelik na die stres. Terwyl daar geen noemenswaardige verskil in serotonien vlakke waargeneem is nie, was die vlakke van 5HlAA betekenisvol verhoog in die frontale korteks en hippokampus van moederlik-geskeide rotte, 15 minute na die beperkingstres. Geen verskil in die omsettingsverhoudinge van basale serotonien (5HlAA/5HT) ofnoradrenalien (MHPGINA) vlakke is gevind nie. Daar was egter 'n betekenisvolle verhoging in die serotonien omset in die hipotalamus van moerdlik-geskeide rotte, onmiddelik na beperkingstres. Hierdie verskil het ook voorgekom 15 minute na die stresperiode in die hipotalamus, sowel as in die frontale korteks. ACTH bepalings: Rotte wat onderwerp was aan moederlike skeiding het verhoogde basale konsentrasies van ACTH getoon. Die ACTH vlakke was egter aanmerklik laer 15 minute na stres toe dit met kontrole groepe vergelyk is. Ons resultate toon dus dat moerderlike-skeiding wel tot angstige gedrag tydens die volwasse lewe kan lei. Hierdie afwyking in gedrag was geassosieër met abnormaliteite in die sentrale senuwee sisteem sowel as die neuroendokrienologiese sisteem van die dier, veralonder toestande van stres. Na gelang van ons bevindinge in die moerderlike skeidingstudie, het dit geblyk dat CRF 'n belangrike rol speel tot daarstelling van angstige gedrag. Daarom het ons in die tweede deel van ons studie gaan kyk ofverhoogde vlakke van CRF in die brein moontlik die gedrag van die rot kon verander. CRF STUDIE Metodes: Kannules was in die linker ventrikel van die breine van normale rotte geïmplanteer deur gebruik te maak van stereotaktiese prosedures. CRF (3 Ilg/IlI) was daagliks vir 5 dae aan die rotte toegedien. Rotte wat presies dieselfde gehanteer was het 'n fisiologiese soutoplossing ontvang. Hierdie rotte was met naïewe rotte vergelyk. Die korrekte plasing van kannules was met histologiese metodes bevestig. Gedrag: Die "elevated plus-maze" was gebruik om te bepaal of angstige gedragte by behandelde rotte ontlok was. Die aantal kere wat 'n rot die verskillende arms van die doolhofbinne gaan, sowel as die tyd wat die dier op elke arm deurbring was genotuleer. Die aantal steierings, botstilstande, defekasies en versorgingsbewegings was weereens waargeneem. ACTH bepalings: Die vlakke van ACTH was bepaal in al die rotgroepe, 15 minute nadat hulle aan 10 minute beperkingstres onderwerp was. Resultate: Gedrag: Rotte wat met CRF toegedien was, het op minder geleenthede die toe arms van die "elevated plus-maze" binne gegaan toe hulle met die naïewe groep rotte vergelyk was. Hierdie verskil was betekenisvol. Daar was geen ander noemenswaardige verskille ten opsigte van die ander gedragsparameter nie. ACTH bepalings: Daar was 'n afname in die ACTH vlakke, 15 minute na die stres toegedien was in rotte wat CRF ontvang het, in vergelyking tot die naïewe kontrole groep. Hierdie resultate dui daarop dat die toediening van CRF in die brein nie die rot se gedrag, maar wel die dier se respons op stres, beïnvloed het. Gevolgtrekking: In die lig van die voorafgaande resultate verky, blyk dit dat moederlike-skeiding tydens die vroeë kinderjare wel kan aanleiding gee tot angstige gedrag tydens volwassenheid. Ons studies dui ook aan dat CRF nie die primêre bron van hierdie gedrags afwykings is nie, maar dat abnormaliteite in die neurochemiese oordragstowwe (serotonien en noradrenalien) eerder die bepalende faktore is. Ten slotte, ons beveel aan dat geneesmiddels wat geskoei is om die serotonerge sowel as die noradrenerge sisteme aan te spreek, voordeel moet geniet in die behandeling van gedragstoomisse, soos angs

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons

Ketamine administration disturbs behavioural and distributed neural correlates of fear conditioning in the rat

The neurotransmitter glutamate and its associated receptors perform an important role in the brain circuitry underlying normal fear processing. The glutamate NMDA receptor, in particular, is necessary for the acquisition and recollection of conditioned-fear responses. Here the authors examine how acute blockage of the NMDA receptor with sub-anaesthetic doses of ketamine affects behavioural assays of fear-conditioned stress (e.g. freezing) and cFos expression in a network of brain areas that have previously been implicated in fear processing. Fear-conditioned rats displayed significantly more freezing behaviour than non-conditioned controls. In fear-conditioned rats that also received ketamine, this conditioning effect was largely neutralised. Fear conditioning also led to increased cFos expression in various areas central to fear processing, including the basolateral nucleus of the amygdala, the paraventricular nucleus of the hypothalamus and the anterior cingulate. Ketamine abolished such increases in cFos expression in most brain areas investigated. The present study therefore demonstrates that systemic ketamine administration in rats interferes with fear conditioning on a behavioural level and in a network of brain regions associated with fear and anxiety. The combination of ketamine and fear conditioning may therefore provide a useful model of abnormal fear processing, as observed in certain psychiatric conditions. (c) 2006 Elsevier Inc. All rights reserved

Comparison of brain and blood gene expression in an animal model of negative symptoms in schizophrenia

Objectives: To investigate the potential of white blood cells as probes for central processes we have measured gene expression in both the anterior cingulate cortex and white blood cells using a putative animal model of negative symptoms in schizophrenia. Methods: The model is based on the capability of ketamine to induce psychotic symptoms in healthy volunteers and to worsen such symptoms in schizophrenic patients. Classical fear conditioning is used to assess emotional processing and cognitive function in animals exposed to sub-chronic ketamine vs. controls. Gene expression was measured using a commercially sourced whole genome rat gene array. Data analyses were performed using ANOVA (Systat 11). Results: In both anterior cingulate cortex and white blood cells a significant interaction between ketamine and fear conditioning could be observed. The outcome is largely supported by our subsequent metagene analysis. Moreover, the correlation between gene expression in brain and blood is about constant when no ketamine is present (r-0.4). With ketamine, however, the correlation becomes very low (r similar to 02) when there is no fear, but it increases to -0.6 when fear and ketamine are both present. Our results show that under normal conditions ketamine lowers gene expression in the brain, but this effect is completely reversed in combination with fear conditioning, indicating a stimulatory action. Conclusion: This paradoxical outcome indicates that extreme care must be taken when using gene expression data from white blood cells as marker for psychiatric disorders, especially when pharmacological and environmental interactions are at play. Crown Copyright (c) 2012 Published by Elsevier Inc. All rights reserved

Molecular Profiles of Parvalbumin-Immunoreactive Neurons in the Superior Temporal Cortex in Schizophrenia

Dysregulation of pyramidal cell network function by the soma- and axon-targeting inhibitory neurons that contain the calcium-binding protein parvalbumin (PV) represents a core pathophysiological feature of schizophrenia. In order to gain insight into the molecular basis of their functional impairment, we used laser capture microdissection (LCM) to isolate PVimmunolabeled neurons from layer 3 of Brodmann’s area 42 of the superior temporal gyrus (STG) from postmortem schizophrenia and normal control brains. We then extracted ribonucleic acid (RNA) from these neurons and determined their messenger RNA (mRNA) expression profile using the Affymetrix platform of microarray technology. Seven hundred thirty-nine mRNA transcripts were found to be differentially expressed in PV neurons in subjects with schizophrenia, including genes associated with WNT (wingless-type), NOTCH, and PGE2 (prostaglandin E2) signaling, in addition to genes that regulate cell cycle and apoptosis. Of these 739 genes, only 89 (12%) were also differentially expressed in pyramidal neurons, as described in the accompanying paper, suggesting that the molecular pathophysiology of schizophrenia appears to be predominantly neuronal type specific. In addition, we identified 15 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of the predicted targets of these miRNAs included the signaling pathways found by microarray to be dysregulated in schizophrenia. Taken together, findings of this study provide a neurobiological framework within which hypotheses of the molecular mechanisms that underlie the dysfunction of PV neurons in schizophrenia can be generated and experimentally explored and, as such, may ultimately inform the conceptualization of rational targeted molecular intervention for this debilitating disorder