Charmaine Pietersen - Academia.edu (original) (raw)

Papers by Charmaine Pietersen

Stellenbosch University http://scholar.sun.ac.za IV ACTH Determinations: All rats were subjected ... more Stellenbosch University http://scholar.sun.ac.za IV 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 Stellenbosch University http://scholar.sun.ac.za v 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 Stellenbosch University http://scholar.sun.ac.za VI 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.

Hormones and Behavior, Jun 1, 2004

Abstract: Disrupted synchronized oscillatory fi ring of pyramidal neuronal networks in the cerebr... more Abstract: Disrupted synchronized oscillatory fi ring of pyramidal neuronal networks in the cerebral cortex in the gamma frequency band (i.e., 30 – 100 Hz) mediates many of the cognitive defi cits 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 profi led the messenger RNA (mRNA) expression of these neurons, using microarray technology. We identifi ed 1331 mRNAs that were differentially expressed in schizophrenia, including genes that belong t...

Journal of Psychiatric Research, 2018

Schizophrenia is a neurodevelopmental disorder with the typical age of onset of overt symptoms an... more Schizophrenia is a neurodevelopmental disorder with the typical age of onset of overt symptoms and deficits occurring during late adolescence or early adulthood, coinciding with the final maturation of the cortical network involving the prefrontal cortex. These observations have led to the hypothesis that disturbances of the developmental events that take place in the prefrontal cortex during this period, specifically the remodeling of synaptic connectivities between pyramidal neurons, may contribute to the onset of illness. In this context, we investigated the gene expression changes of pyramidal neurons in the human prefrontal cortex during normal periadolescent development in order to gain insight into the possible molecular mechanisms involved in synaptic remodeling of pyramidal neuronal circuitry. Our data suggest that genes associated with the ubiquitination system, which has been implicated in the biology of synaptic plasticity, may play a major role. Among these genes, UBE3B, which encodes the ubiquitin ligase E3, was found to undergo periadolescent increase and was validated at the protein level to be upregulated during periadolescent development. Furthermore, we found that the density of UBE3B-immunoreactive pyramidal neurons was decreased in schizophrenia subjects, consistent with the result of a previous study of decreased UBE3B mRNA expression in pyramidal neurons in this illness. Altogether these findings point to the novel hypothesis that this specific ligase may play a role in

Schizophrenia research, 2015

The pathophysiology of schizophrenia involves disturbances of information processing across brain... more The pathophysiology of schizophrenia involves disturbances of information processing across brain regions, possibly reflecting, at least in part, a disruption in the underlying axonal connectivity. This disruption is thought to be a consequence of the pathology of myelin ensheathment, the integrity of which is tightly regulated by oligodendrocytes. In order to gain insight into the possible neurobiological mechanisms of myelin deficit, we determined the messenger RNA (mRNA) expression profile of laser captured cells that were immunoreactive for 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a marker for oligodendrocyte progenitor cells (OPCs) in addition to differentiating and myelinating oligodendrocytes, in the white matter of the prefrontal cortex in schizophrenia subjects. Our findings pointed to the hypothesis that OPC differentiation might be impaired in schizophrenia. To address this hypothesis, we quantified cells that were immunoreactive for neural/glia...

Journal of Visualized Experiments, 2009

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schiz... more 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. Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51μg, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

Metabolic Brain Disease, 2000

Methods in Molecular Biology, 2011

The human brain is an exceptionally heterogeneous structure. In order to gain insight into the ne... more The human brain is an exceptionally heterogeneous structure. In order to gain insight into the neurobiological basis of neural circuit disturbances in various neurologic or psychiatric diseases, it is often important to define the molecular cascades that are associated with these disturbances in a neuronal type-specific manner. This can be achieved by the use of laser microdissection, in combination with molecular techniques such as gene expression profiling. To identify neurons in human postmortem brain tissue, one can use the inherent properties of the neuron, such as pigmentation and morphology or its structural composition through immunohistochemistry (IHC). Here, we describe the isolation of homogeneous neuronal cells and high-quality RNA from human postmortem brain material using a combination of rapid IHC, Nissl staining, or simple morphology with Laser-Capture Microdissection (LCM) or Laser Microdissection (LMD).

Schizophrenia Research, 2010

affected vs. unaffected co-twins). The concentration of this sphingomyelin was significantly asso... more affected vs. unaffected co-twins). The concentration of this sphingomyelin was significantly associated with the reductions in gray matter density including regions of the temporal and occipital lobes as well as corpus callosum, as confirmed by permutation testing and separately for each hemisphere (P's < 0.05). The significant association remained if corpus callosum was excluded from the analysis, for the right hemisphere only. Discussion: Our findings support the view that lipid abnormalities are intrinsic to schizophrenia. Sphingomyelin (SM) is an abundant brain lipid which is also a precursor of lipotoxic ceramide. Elevated ceramide levels have been identified in white matter of schizophrenic subjects in a recent study (Schwartz et al., 2008). SM is also an abundant lipid in circulation, found particularly in LDL and HDL particles (Kotronen et al., 2009). The twin design allowed us to link abnormal serum SM changes to schizophrenia independent of the genetic Background. Associations of SM to specific brain regions of potential pathogenic relevance suggest SM may play a role in schizophrenia. Our findings emphasize the importance of further studies aimed to establish a mechanistic link between the alterations in circulating SM and related changes in brain lipids and structure. References

Schizophrenia Research, 2014

Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2012

Meta gene analysis White blood cells Objectives: To investigate the potential of white blood cell... more Meta gene analysis White blood cells 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~0.2) 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.

Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2006

The neurotransmitter glutamate and its associated receptors perform an important role in the brai... more 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.

PLoS ONE, 2007

Schizophrenia is often associated with emotional blunting-the diminished ability to respond to em... more Schizophrenia is often associated with emotional blunting-the diminished ability to respond to emotionally salient stimuliparticularly 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.

Neurochemistry International, 2010

We have investigated effects of continuous SSRI administration and abrupt discontinuation on bioc... more We have investigated effects of continuous SSRI administration and abrupt discontinuation on biochemical and behavioral indices of rat brain serotonin function, and attempted to identify underlying mechanisms. Biochemistry of serotonin was assessed with brain tissue assays and microdialysis; behavior was assessed as the acoustic startle reflex. Long-term SSRI administration to rats reduced the content of 5-HT and its main metabolite shortly after inhibition of 5-HT synthesis in many brain areas with more than 50%. Turnover was not appreciably decreased, but significantly increased within 48h of drug discontinuation. The microdialysis experiments indicate that neuronal release of 5-HT depends strongly on new synthesis and emphasize the role of 5-HT(1B) receptors in the regulation of these processes. Discontinuation of the SSRI rapidly increased behavioral reactivity to the external stimulus. Additional startle experiments suggest that the increased reactivity is more likely related to the reduced extracellular 5-HT levels than to impaired synthesis. The combination of the marked reduction of serotonin content and limited synthesis may destabilize brain serotonin transmission during long-term SSRI treatment. These combined effects may compromise the efficacy of an SSRI therapy and facilitate behavioral changes following non-compliance.

Metabolic Brain Disease, 2004

Adverse early life experiences can have a negative impact on behavior later in life. We subjected... more Adverse early life experiences can have a negative impact on behavior later in life. We subjected rat pups to maternal separation and determined the effects thereof on adult behavior. We removed rat pups from their mothers for 3 h daily from postnatal days 2 to 14. While controls were reared normally on day 60, the behaviors of the rats were tested using the elevated plus-maze. Some rats were subsequently subjected to restraint stress for a 10-min period. Trunk blood was collected for basal, as well as 15-and 60-min postrestraint stress ACTH determinations. Neurotransmitter levels (noradrenaline (NA), serotonin (5HT), and their metabolites, MHPG and 5HIAA, respectively) were also determined at basal, immediately and 15-min post-restraint stress in the hypothalamus, hippocampus, and frontal cortex in another group of animals. The amount of entries into the arms of the elevated plus-maze was significantly reduced in the separated animals, indicating decreased locomotion. They spent significantly more time in the closed arms of the maze. A significant increase in defecation frequency was noted. These observations suggested anxious behavior. Basal ACTH levels were significantly higher in separated animals. At 15-min post-restraint stress, the ACTH levels were significantly lower than controls, indicating a blunted stress response. A decrease in noradrenaline was noted first in limbic regions and an increase in 5HIAA levels was found in the frontal cortex and hippocampus. We conclude that maternal separation induced abnormal behaviors and stress responses that were associated with altered neurotransmitter levels.

Laboratory Animals, 2006

Many fear conditioning studies use electric shock as the aversive stimulus. The intensity of shoc... more Many fear conditioning studies use electric shock as the aversive stimulus. The intensity of shocks varies throughout the literature. In this study, shock intensities ranging from 0 to 1.5 mA were used, and the effects on the rats assessed by both behavioural and biochemical stress parameters. Results indicated a significant difference with respect to defaecation and freezing behaviour between controls and those animals that received a shock. Significant differences in corticosterone levels were also noted between controls and those groups that received a shock. No significant differences were found between the shock groups with regards to the stress parameters measured in our fear conditioning paradigm, indicating that the two shock groups were similarly stressed. Increased significance levels were noted for freezing behaviour as well as a lower standard error of means was found in the highest shock intensity group. We therefore recommend using the higher shock intensity (1.5 mA) a...

Journal of Neurogenetics, 2014

Dysregulation of pyramidal cell network function by the soma-and axon-targeting inhibitory neuron... more 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 PGE 2 (prostaglandin E 2) 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

Journal of Neurogenetics, 2014

Disrupted synchronized oscillatory fi ring of pyramidal neuronal networks in the cerebral cortex ... more Disrupted synchronized oscillatory fi ring of pyramidal neuronal networks in the cerebral cortex in the gamma frequency band (i.e., 30-100 Hz) mediates many of the cognitive defi cits 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 profi led the messenger RNA (mRNA) expression of these neurons, using microarray technology. We identifi ed 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 identifi ed 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, fi ndings of this study provide a neurobiological framework within which specifi c hypotheses about the molecular mechanisms of pyramidal cell dysfunction in schizophrenia can be formulated.

Annals of the New York Academy of Sciences, 1994

CORTICOTROPIN-RELEASING FACTOR IN ANXIETY DISORDERS Corticotropin-releasing factor (CRF), a 41-re... more CORTICOTROPIN-RELEASING FACTOR IN ANXIETY DISORDERS Corticotropin-releasing factor (CRF), a 41-residue hypothalamic peptide, is the major physiological regulator of adrenocorticotropin (ACTH) and P-endorphin release from the anterior pituitary gland during stress.lS2 However, like other previously identified hypothalamic regulatory peptides, CRF is not confined to the hypothalamus. Indeed, immunohistochemical and radioimmunoassay studies have revealed that CRF-like immunoreactivity is distributed throughout the mammalian central nervous system (CNS).3-4 Notably, CRF-stained cells and fibers can be found in the paraventricular nucleus of the hypothalamus (PVN), the central nucleus of the amygdala, the bed nucleus of the stria terminalis, the substantia innominata, the region of the locus ceruleus, and parabrachialis nucleus. Similarly, biochemical and autoradiographic studies have identified specific CRF receptors in the CNS in particular in the cerebral cortex and the limbic ~y s t e m .~ When administered directly into the CNS, CRF produces a variety of behav-ioraI6s7 and physiological effects6 independent of the hypothalamo-pituitary-adrenal axis (HPA). These effects are similar to an animal's response to a stressor as well as to many of the symptoms of both affective and anxiety disorders in humans. These findings suggest that CRF, acting as a neurotransmitter, may be responsible for integrating the endocrine as well as the autonomic and behavioral responses to a stressor. More compelling evidence for a role of endogenous CRF in behavioral responses to stress is the demonstration that an antagonist to the CRF receptor, a-helical CRF-(9-41) blocks the behavioral and physiological responses to a stressor when administered into the CNS. In an example from a recent study, intracerebroventricular (i.c.v.) and intra-amygdala administration of CRF antagonist reversed the decrease in exploration of the open arms of an elevated plus-maze caused by exposure to a social s t r e~s o r .~ Furthermore, i.c.v. administration of this CRF antagonist attenuates the behavioral reactivity to a wide range of stressors including physical restraint and electric Taken together, these data provide compelling evidence that endogenous CRF mediates a number of responses associated with the encountering of a stressor.

Stellenbosch University http://scholar.sun.ac.za IV ACTH Determinations: All rats were subjected ... more Stellenbosch University http://scholar.sun.ac.za IV 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 Stellenbosch University http://scholar.sun.ac.za v 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 Stellenbosch University http://scholar.sun.ac.za VI 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.

Hormones and Behavior, Jun 1, 2004

Abstract: Disrupted synchronized oscillatory fi ring of pyramidal neuronal networks in the cerebr... more Abstract: Disrupted synchronized oscillatory fi ring of pyramidal neuronal networks in the cerebral cortex in the gamma frequency band (i.e., 30 – 100 Hz) mediates many of the cognitive defi cits 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 profi led the messenger RNA (mRNA) expression of these neurons, using microarray technology. We identifi ed 1331 mRNAs that were differentially expressed in schizophrenia, including genes that belong t...

Journal of Psychiatric Research, 2018

Schizophrenia is a neurodevelopmental disorder with the typical age of onset of overt symptoms an... more Schizophrenia is a neurodevelopmental disorder with the typical age of onset of overt symptoms and deficits occurring during late adolescence or early adulthood, coinciding with the final maturation of the cortical network involving the prefrontal cortex. These observations have led to the hypothesis that disturbances of the developmental events that take place in the prefrontal cortex during this period, specifically the remodeling of synaptic connectivities between pyramidal neurons, may contribute to the onset of illness. In this context, we investigated the gene expression changes of pyramidal neurons in the human prefrontal cortex during normal periadolescent development in order to gain insight into the possible molecular mechanisms involved in synaptic remodeling of pyramidal neuronal circuitry. Our data suggest that genes associated with the ubiquitination system, which has been implicated in the biology of synaptic plasticity, may play a major role. Among these genes, UBE3B, which encodes the ubiquitin ligase E3, was found to undergo periadolescent increase and was validated at the protein level to be upregulated during periadolescent development. Furthermore, we found that the density of UBE3B-immunoreactive pyramidal neurons was decreased in schizophrenia subjects, consistent with the result of a previous study of decreased UBE3B mRNA expression in pyramidal neurons in this illness. Altogether these findings point to the novel hypothesis that this specific ligase may play a role in

Schizophrenia research, 2015

The pathophysiology of schizophrenia involves disturbances of information processing across brain... more The pathophysiology of schizophrenia involves disturbances of information processing across brain regions, possibly reflecting, at least in part, a disruption in the underlying axonal connectivity. This disruption is thought to be a consequence of the pathology of myelin ensheathment, the integrity of which is tightly regulated by oligodendrocytes. In order to gain insight into the possible neurobiological mechanisms of myelin deficit, we determined the messenger RNA (mRNA) expression profile of laser captured cells that were immunoreactive for 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a marker for oligodendrocyte progenitor cells (OPCs) in addition to differentiating and myelinating oligodendrocytes, in the white matter of the prefrontal cortex in schizophrenia subjects. Our findings pointed to the hypothesis that OPC differentiation might be impaired in schizophrenia. To address this hypothesis, we quantified cells that were immunoreactive for neural/glia...

Journal of Visualized Experiments, 2009

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schiz... more 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. Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51μg, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

Metabolic Brain Disease, 2000

Methods in Molecular Biology, 2011

The human brain is an exceptionally heterogeneous structure. In order to gain insight into the ne... more The human brain is an exceptionally heterogeneous structure. In order to gain insight into the neurobiological basis of neural circuit disturbances in various neurologic or psychiatric diseases, it is often important to define the molecular cascades that are associated with these disturbances in a neuronal type-specific manner. This can be achieved by the use of laser microdissection, in combination with molecular techniques such as gene expression profiling. To identify neurons in human postmortem brain tissue, one can use the inherent properties of the neuron, such as pigmentation and morphology or its structural composition through immunohistochemistry (IHC). Here, we describe the isolation of homogeneous neuronal cells and high-quality RNA from human postmortem brain material using a combination of rapid IHC, Nissl staining, or simple morphology with Laser-Capture Microdissection (LCM) or Laser Microdissection (LMD).

Schizophrenia Research, 2010

affected vs. unaffected co-twins). The concentration of this sphingomyelin was significantly asso... more affected vs. unaffected co-twins). The concentration of this sphingomyelin was significantly associated with the reductions in gray matter density including regions of the temporal and occipital lobes as well as corpus callosum, as confirmed by permutation testing and separately for each hemisphere (P's < 0.05). The significant association remained if corpus callosum was excluded from the analysis, for the right hemisphere only. Discussion: Our findings support the view that lipid abnormalities are intrinsic to schizophrenia. Sphingomyelin (SM) is an abundant brain lipid which is also a precursor of lipotoxic ceramide. Elevated ceramide levels have been identified in white matter of schizophrenic subjects in a recent study (Schwartz et al., 2008). SM is also an abundant lipid in circulation, found particularly in LDL and HDL particles (Kotronen et al., 2009). The twin design allowed us to link abnormal serum SM changes to schizophrenia independent of the genetic Background. Associations of SM to specific brain regions of potential pathogenic relevance suggest SM may play a role in schizophrenia. Our findings emphasize the importance of further studies aimed to establish a mechanistic link between the alterations in circulating SM and related changes in brain lipids and structure. References

Schizophrenia Research, 2014

Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2012

Meta gene analysis White blood cells Objectives: To investigate the potential of white blood cell... more Meta gene analysis White blood cells 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~0.2) 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.

Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2006

The neurotransmitter glutamate and its associated receptors perform an important role in the brai... more 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.

PLoS ONE, 2007

Schizophrenia is often associated with emotional blunting-the diminished ability to respond to em... more Schizophrenia is often associated with emotional blunting-the diminished ability to respond to emotionally salient stimuliparticularly 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.

Neurochemistry International, 2010

We have investigated effects of continuous SSRI administration and abrupt discontinuation on bioc... more We have investigated effects of continuous SSRI administration and abrupt discontinuation on biochemical and behavioral indices of rat brain serotonin function, and attempted to identify underlying mechanisms. Biochemistry of serotonin was assessed with brain tissue assays and microdialysis; behavior was assessed as the acoustic startle reflex. Long-term SSRI administration to rats reduced the content of 5-HT and its main metabolite shortly after inhibition of 5-HT synthesis in many brain areas with more than 50%. Turnover was not appreciably decreased, but significantly increased within 48h of drug discontinuation. The microdialysis experiments indicate that neuronal release of 5-HT depends strongly on new synthesis and emphasize the role of 5-HT(1B) receptors in the regulation of these processes. Discontinuation of the SSRI rapidly increased behavioral reactivity to the external stimulus. Additional startle experiments suggest that the increased reactivity is more likely related to the reduced extracellular 5-HT levels than to impaired synthesis. The combination of the marked reduction of serotonin content and limited synthesis may destabilize brain serotonin transmission during long-term SSRI treatment. These combined effects may compromise the efficacy of an SSRI therapy and facilitate behavioral changes following non-compliance.

Metabolic Brain Disease, 2004

Adverse early life experiences can have a negative impact on behavior later in life. We subjected... more Adverse early life experiences can have a negative impact on behavior later in life. We subjected rat pups to maternal separation and determined the effects thereof on adult behavior. We removed rat pups from their mothers for 3 h daily from postnatal days 2 to 14. While controls were reared normally on day 60, the behaviors of the rats were tested using the elevated plus-maze. Some rats were subsequently subjected to restraint stress for a 10-min period. Trunk blood was collected for basal, as well as 15-and 60-min postrestraint stress ACTH determinations. Neurotransmitter levels (noradrenaline (NA), serotonin (5HT), and their metabolites, MHPG and 5HIAA, respectively) were also determined at basal, immediately and 15-min post-restraint stress in the hypothalamus, hippocampus, and frontal cortex in another group of animals. The amount of entries into the arms of the elevated plus-maze was significantly reduced in the separated animals, indicating decreased locomotion. They spent significantly more time in the closed arms of the maze. A significant increase in defecation frequency was noted. These observations suggested anxious behavior. Basal ACTH levels were significantly higher in separated animals. At 15-min post-restraint stress, the ACTH levels were significantly lower than controls, indicating a blunted stress response. A decrease in noradrenaline was noted first in limbic regions and an increase in 5HIAA levels was found in the frontal cortex and hippocampus. We conclude that maternal separation induced abnormal behaviors and stress responses that were associated with altered neurotransmitter levels.

Laboratory Animals, 2006

Many fear conditioning studies use electric shock as the aversive stimulus. The intensity of shoc... more Many fear conditioning studies use electric shock as the aversive stimulus. The intensity of shocks varies throughout the literature. In this study, shock intensities ranging from 0 to 1.5 mA were used, and the effects on the rats assessed by both behavioural and biochemical stress parameters. Results indicated a significant difference with respect to defaecation and freezing behaviour between controls and those animals that received a shock. Significant differences in corticosterone levels were also noted between controls and those groups that received a shock. No significant differences were found between the shock groups with regards to the stress parameters measured in our fear conditioning paradigm, indicating that the two shock groups were similarly stressed. Increased significance levels were noted for freezing behaviour as well as a lower standard error of means was found in the highest shock intensity group. We therefore recommend using the higher shock intensity (1.5 mA) a...

Journal of Neurogenetics, 2014

Dysregulation of pyramidal cell network function by the soma-and axon-targeting inhibitory neuron... more 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 PGE 2 (prostaglandin E 2) 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

Journal of Neurogenetics, 2014

Disrupted synchronized oscillatory fi ring of pyramidal neuronal networks in the cerebral cortex ... more Disrupted synchronized oscillatory fi ring of pyramidal neuronal networks in the cerebral cortex in the gamma frequency band (i.e., 30-100 Hz) mediates many of the cognitive defi cits 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 profi led the messenger RNA (mRNA) expression of these neurons, using microarray technology. We identifi ed 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 identifi ed 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, fi ndings of this study provide a neurobiological framework within which specifi c hypotheses about the molecular mechanisms of pyramidal cell dysfunction in schizophrenia can be formulated.

Annals of the New York Academy of Sciences, 1994

CORTICOTROPIN-RELEASING FACTOR IN ANXIETY DISORDERS Corticotropin-releasing factor (CRF), a 41-re... more CORTICOTROPIN-RELEASING FACTOR IN ANXIETY DISORDERS Corticotropin-releasing factor (CRF), a 41-residue hypothalamic peptide, is the major physiological regulator of adrenocorticotropin (ACTH) and P-endorphin release from the anterior pituitary gland during stress.lS2 However, like other previously identified hypothalamic regulatory peptides, CRF is not confined to the hypothalamus. Indeed, immunohistochemical and radioimmunoassay studies have revealed that CRF-like immunoreactivity is distributed throughout the mammalian central nervous system (CNS).3-4 Notably, CRF-stained cells and fibers can be found in the paraventricular nucleus of the hypothalamus (PVN), the central nucleus of the amygdala, the bed nucleus of the stria terminalis, the substantia innominata, the region of the locus ceruleus, and parabrachialis nucleus. Similarly, biochemical and autoradiographic studies have identified specific CRF receptors in the CNS in particular in the cerebral cortex and the limbic ~y s t e m .~ When administered directly into the CNS, CRF produces a variety of behav-ioraI6s7 and physiological effects6 independent of the hypothalamo-pituitary-adrenal axis (HPA). These effects are similar to an animal's response to a stressor as well as to many of the symptoms of both affective and anxiety disorders in humans. These findings suggest that CRF, acting as a neurotransmitter, may be responsible for integrating the endocrine as well as the autonomic and behavioral responses to a stressor. More compelling evidence for a role of endogenous CRF in behavioral responses to stress is the demonstration that an antagonist to the CRF receptor, a-helical CRF-(9-41) blocks the behavioral and physiological responses to a stressor when administered into the CNS. In an example from a recent study, intracerebroventricular (i.c.v.) and intra-amygdala administration of CRF antagonist reversed the decrease in exploration of the open arms of an elevated plus-maze caused by exposure to a social s t r e~s o r .~ Furthermore, i.c.v. administration of this CRF antagonist attenuates the behavioral reactivity to a wide range of stressors including physical restraint and electric Taken together, these data provide compelling evidence that endogenous CRF mediates a number of responses associated with the encountering of a stressor.