Tineke Koch

Methylazoxymethanol acetate-induced abnormalities in the entorhinal cortex of the rat; parallels with morphological findings in schizophrenia.

It has been suggested repeatedly that the non-heritable factors in the pathogenesis of schizophrenia involve abnormalities of prenatal neurodevelopment. Furthermore, post-mortem studies show neuropathology of apparently developmental origin in the entorhinal cortex and other brain regions of schizophrenic subjects. In an attempt to model a developmental defect of the entorhinal region in the rat, cerebrocortical proliferation was briefly interrupted during its earliest stages, when the entorhinal area is thought to undergo major cell division. Specifically, the experimental set-up involved the administration of methylazoxymethanol acetate (MAM) on 1 of 4 consecutive days of embryonal development, from E9 to E12. Analysis of the forebrain in adult animals shows reduction of the entorhinal cortex in rats treated on each of these days. This effect shifts from lateral to medial divisions of the entorhinal cortex with later administration of MAM, following a known developmental gradient. Morphological consequences of MAM administration appear to be largely confined to the entorhinal cortex in the groups treated on E9 to E11, although slight reductions of the frontal and occipital neocortex were also observed in these animals. MAM treatment on E12 produces relatively more widespread damage, as reflected among other in a small reduction of brain weight. The described brain abnormalities are not accompanied by obvious phenotypical changes in any, but the E12-treated group. They, moreover, involve cortical thinning, disorganised cortical layering, and abnormal temporal asymmetries. These finding bare some similarity to observations in brains of schizophrenic subjects. The possible relevance of this approach in modeling neurodevelopmental aspects of schizophrenia is discussed.

DIFFERENTIAL FOS-PROTEIN INDUCTION IN RAT FOREBRAIN REGIONS AFTER ACUTE AND LONG-TERM HALOPERIDOL AND CLOZAPINE TREATMENT

Both acute and long-term effects of haloperidol and clozapine on Fos-like immunoreactive nuclei in several rat forebrain areas were quantified. Rats were treated with saline (1 ml/kg.day, control), haloperidol (1 mg/kg.day) and clozapine (20 mg/kg.day) i.p. for 21 days. Two hours before perfusion fixation a single (acute treatment) or last (long-term treatment) dose of the drug was given. Drug-induced catalepsy and gain in body weight were also measured. A single dose of haloperidol produced large increases in Fos-like immunoreactive nuclei in the striatum, the nucleus accumbens and central amygdala. Following long-term treatment these increases were reduced in all nuclei studied, except the lateral septum. Acute clozapine treatment had slight (if any) effects on the number of Fos-like immunoreactivity-expressing nuclei in the striatum, but the increases in the nucleus accumbens, the lateral septum, the paraventricular and supraoptic nuclei of the hypothalamus and the central amygdala were substantial. Long-term clozapine treatment reduced the acute response significantly in all the areas except the nucleus accumbens. Both haloperidol and clozapine treatment reduced the weight gain of the rats. Haloperidol, but not clozapine, induced catalepsy that remained maximal during the long-term haloperidol treatment. These results indicate that in most brain areas high Fos-protein levels are not necessary to maintain antipsychotic activity or side-effects. The persisting effect of clozapine in the nucleus accumbens may be of significance to the efficacy of this drug in treatment-refractory schizophrenia.

AN ENZYME-REACTOR FOR ELECTROCHEMICAL MONITORING OF CHOLINE AND ACETYLCHOLINE - APPLICATIONS IN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY, BRAIN-TISSUE, MICRODIALYSIS AND CEREBROSPINAL-FLUID

A sandwich-type enzyme reactor in which the enzymes are physically immobilized in a minimal dead space between two cellulose membranes, resulting in improved sensitivity, was developed for the electro-chemical detection of choline (Ch) and acetylcholine (ACh). The reactor contains the enzymes choline oxidase with or without acetylcholine esterase, for the detection of ACh and Ch, respectively. For the HPLC analysis of Ch and ACh the detection system was coupled post column. Levels of Ch and ACh of rat striatum tissue and human cerebrospinal fluid were found to be similar to those determined with published methods. Because of low back pressure--a further advantage of the reactor--the detection system could also be directly coupled to the outlet of a microdialysis device, allowing the on-line real-time measurement of extracellular brain Ch. The versatility of the enzyme reactor for the monitoring of analytes in HPLC eluates, flow injection analysis, with or without prepurification, is emphasized. The usefulness of the reactor-detector system in biomedical applications is illustrated by the measurement of increases of rat striatal extracellular Ch following cardiac arrest.

Interruptions of early cortical development affect limbic association areas and social behaviour in rats; possible relevance for neurodevelopmental disorders.

Deficits in social behaviour are found in several neuropsychiatric disorders with a presumed developmental origin. Adequate social behaviour may rely importantly on the associative integration of new stimuli with previously stored, related information. The limbic allocortex, in particular the entorhinal region, is thought to support this kind of processing. Therefore, in the present study, gestating dams were treated with methylazoxymethanol acetate (MAM) on one of gestational days nine to twelve, to interrupt neuronal proliferation in the entorhinal region of the developing foetuses. Effects of prenatal MAM administration on social behaviour were evaluated in adult animals. As the entorhinal cortex has been implicated by some studies in spatial memory, effects on this function were also investigated. Following the behavioural studies, brain morphology was screened for effects of MAM. Our results show moderate to severe social impairment in MAM-treated animals, depending on the exact timing of prenatal exposure. By contrast, spatial reference and working memory were not importantly affected in any group. Analysis of brain morphology in the MAM-treated offspring supported maldevelopment of the entorhinal cortex and revealed mild abnormalities also in some connected limbic and limbic affiliated structures, such as the perirhinal and ectorhinal cortex, the anterior cingulate cortex and the medial septum-diagonal band region. Findings are discussed with respect to entorhinal cortex function, and with regard to their relevance for psychiatric disorders with a putatively neurodevelopmental pathogenesis, such as schizophrenia.

Prolonged c-Jun expression in the basolateral amygdala following bulbectomy: possible implications for antidepressant activity and time of onset.

Olfactory bulbectomy is a well established animal model of depression. Neurochemical and behavioral alterations observed following olfactory bulbectomy, are due, in part, to the neurodegeneration of specific brain structures. Amygdaloid dysfunction in particular, is known to play a substantial role in the syndrome of the olfactory bulbectomized rat. The present study examined both short- and long-term alterations in immediate early gene expression, tyrosine hydroxylase and serotonin immunoreactivity, and classical silver staining, following olfactory bulbectomy in the basolateral amygdala. The results indicated no consistent change in Fos expression observed over the experimental period. Following bulbectomy, long term (up to 64 days post-lesion) Jun expression, not coincident with silver staining, was observed in the basolateral nucleus. The basolateral nucleus was also intensely immunoreactive for serotonin at this timepoint post-bulbectomy. Thus, following bulbectomy long term alterations in Jun expression occurs in the serotonin rich basolateral amygdala. As a site of action for antidepressant compounds, alterations at the immediate early gene level in this region may have implications both for the model, and antidepressant drug action therein.

Olanzapine-induced Fos expression in the rat forebrain; cross-tolerance with haloperidol and clozapine

Acute administration of the atypical antipsychotic drug olanzapine (5 mg kg(-1 i.p.) increased the number of Fos-positive cells moderately in the prefrontal cortex and the striatum; more pronounced were the effects in the nucleus accumbens, the lateral septum, the hypothalamic paraventricular nucleus and the amygdala. The acutely-induced Fos responses of olanzapine were significantly reduced in all brain areas investigated after a 3-week treatment period, indicating the development of tolerance. Through evaluation of cross-tolerance we investigated whether the effects of olanzapine, haloperidol and clozapine on Fos expression and on plasma corticosterone are mediated by the same or by different mechanisms. Cross-tolerance between olanzapine and either haloperidol or clozapine was assessed by the administration of a challenge dose of olanzapine to rats, that were pretreated for 3 weeks with either the same drug, with saline (1 ml kg(-1) day(-1), haloperidol (1 mg kg(-1) day(-1) or clozapine (20 mg kg(-1) day(-1). A competitive dose of olanzapine in long-term haloperidol-treated rats showed cross-tolerance in the rostral part of the cingulate cortex, the dorsomedial and the dorsolateral striatum, the nucleus accumbens and the lateral septum. Cross-tolerance between olanzapine and clozapine, however, was limited to limbic nuclei, including the prefrontal cortex, the lateral septum, the hypothalamic paraventricular nucleus and the amygdala, with minor effects in the mid- and caudal parts of the cingulate cortex. Interesting are the common effects in the lateral septum, possibly an important target for antipsychotic efficacy. Olanzapine administration induced elevated levels of plasma corticosterone and cross-tolerance was seen in haloperidol- and clozapine-pretreated rats.

Modulation of rotational behavior in healthy volunteers by cortisol administration

Asymmetrical turning behavior is an established indicator of asymmetrical dopaminergic activity and thought to be a manifestation of hemispatial neglect. We set out to find converging support for the hypothesis that cortisol modulates frontal dopaminergic asymmetrical activity, and hence dopaminergically mediated approach behaviors, by studying the effect of cortisol administration on turning behavior in healthy subjects. Both when our subjects attempted to rotate through a target angle (twice 360 degrees) in a clockwise or anticlockwise direction with reduced sensory input, cortisol induced a relative clockwise turning bias. Furthermore, this effect interacted with scores on novelty seeking, a putative indicator of individual differences in dopaminergic function: subjects scoring higher on novelty seeking demonstrated a smaller or no increase after cortisol administration in their clockwise turning bias. The results provide converging support for the hypothesis that cortisol modulates frontal dopaminergic asymmetrical activity. As we discuss, they further point to the possible involvement of the insula in cortisol effects.

Lack of cross-tolerance between haloperidol and clozapine towards Fos-protein induction in rat forebrain regions

We investigated whether the acute effects of haloperidol and clozapine on Fos expression in the rat forebrain are mediated by the same receptors through evaluation of cross-tolerance, particularly in the commonly affected areas. Acutely administered haloperidol (1 mg/kg. i.p.) and clozapine (20 mg/kg, i.p.) induce regionally different (e.g., the striatum, the hypothalamic paraventricular and supraoptic nuclei, and the central amygdala) and overlapping (e.g., the nucleus accumbens and the lateral septum) patterns of Fos-protein distribution in the rat forebrain. After long-term treatment, part of the acute effects of these drugs disappears in most brain areas, except in the lateral septum, the hypothalamic paraventricular and supraoptic nuclei and the amygdala following haloperidol administration. Cross-tolerance between haloperidol and clozapine was determined by administering a challenge dose of the one antipsychotic, following a 21-day pretreatment with the same or the other drug or saline. In none of the investigated brain regions was cross-tolerance towards Fos-protein induction found after haloperidol challenge in the clozapine-treated rats. Conversely, a competitive dose of clozapine in long-term haloperidol-treated rats showed cross-tolerance in the lateral septum, while the common effect of the drugs in both the dorsomedial and the dorsolateral parts of the striatum was very small. These findings indicate that, for the major part, the responses to haloperidol and clozapine are mediated by different receptors, even in brain areas that are affected by both drugs.

Limited participation of 5-HT1A and 5-HT2A/2C receptors in the clozapine-induced Fos-protein expression in rat forebrain regions

Through the development of tolerance following long-term clozapine treatment, we investigated whether 5-HT(1A) and 5-HT(2A/2C) receptors participate in the clozapine-induced Fos-protein expression in the rat forebrain. Tolerance exists when the acutely increased Fos responses to a challenge dose of the 5-HT(1A) and 5-HT(2A/2C) agonists 1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane-hydrochloride (DOI) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), respectively, given simultaneously to rats, are attenuated after 3-week clozapine (20 mg kg(-1) day(-1) i.p.) pretreatment. As compared to the acute effects of clozapine, the Fos responses to concomitant administration of the 5-HT receptor agonists DOI (2.5 mg kg(-1) i.p. ) and 8-OH-DPAT (2.5 mg kg(-1) i.p.) were more pronounced in the prefrontal cortex, the nucleus accumbens core and the dorsomedial and ventromedial striatum, areas in which clozapine (20 mg kg(-1) i. p.) exhibited marginal effects. In the hypothalamic paraventricular nucleus, both clozapine and DOI/8-OH-DPAT induced a remarkably high number of Fos-positive nuclei. Long-term clozapine pretreatment attenuated the acutely induced Fos expression of the 5-HT receptor agonists in the nucleus accumbens core, the dorsomedial and ventromedial parts of the striatum and the lateral septum, indicating (partial) common sites of action of the agents in these brain regions. No tolerance was found in the nucleus accumbens shell and the hypothalamic paraventricular nucleus and the central amygdala, suggesting that the clozapine-induced Fos responses, though distinct in these regions, are independent of 5-HT receptors. The prefrontal cortex and the dorsolateral striatum indicated only a tendency towards tolerance. In addition, the involvement of the tested 5-HT receptor agonists in the clozapine-enhanced release of plasma corticosterone became apparent. The present results indicate that the clozapine-induced patterns of Fos expression in the rat forebrain can only be in part attributed to an interaction with 5-HT(1A/2A/2C) receptors.

Clozapine-induced Fos-protein expression in rat forebrain regions: differential effects of adrenalectomy and corticosterone supplement

Unlike classical antipsychotic drugs, clozapine activates the hypothalamo-pituitary-adrenal axis and induces a specific regional pattern of Fos-protein expression in the rat forebrain. Whether corticosterone plays a role in the clozapine-induced Fos response is the subject of this study. Some rats were adrenalectomized and in a number, including intact animals, a corticosterone pellet (100 mg s.c.) was implanted; after 1 week, a single dose of clozapine (20 mg kg(-1) i.p.) was administered. The clozapine-induced Fos response was not affected by adrenalectomy, apart from the nucleus accumbens shell, the subfornical organ and the supraoptic nucleus; there was an increased response in the nucleus accumbens shell, while other regions showed less Fos immunoreactivity. Implantation of the corticosterone pellet in both sham-operated and adrenalectomized animals, reduced the clozapine-induced Fos responses strongly in the hypothalamic paraventricular nucleus, the subfornical organ and possibly in the prefrontal cortex; in the supraoptic nucleus, this effect was seen only in intact animals. The effect of clozapine on plasma corticosterone levels was also diminished by supplemental corticosterone treatment. These results imply that the effects of clozapine are partially dependent upon hypothalamo-pituitary-adrenal axis integrity and activation. The efficacy of clozapine in the treatment of polydipsia and hyponatremia in chronic psychiatric patients may involve clozapine-mediated activation of the cellular activity in the subfornical organ.