Katalin Horváth

The Influence of Postnatal Handling on Adult Neuroendocrine and Behavioural Stress Reactivity

Environmental stimuli during early stages of life can influence the development of an organism and may result in permanent changes in adult behaviour and physiology. In the present study we investigated the influence of early postnatal handling on adult neuroendocrine and behavioural stress reactivity in Wistar rats. Pups were subjected to handling from postnatal day 1–21. The young were taken from the nest every day for 15u2003min and each of the pups was handled separately. Control nests were left undisturbed. When the animals had reached an adult age of 3–4 months they were individually housed and subjected to a series of tests to measure their stress reactivity. In the first experiment we established adult behavioural coping with stressors and anxiety in the following series of tests: open field test, shock prod defensive burying test, elevated plus maze and conditioned fear test. Collectively, the data clearly indicate that handled animals are characterized by a lower stress‐induced anxiety. Yet, handled and control animals do not differ in their general way of coping with stressors. Although the lower anxiety in handled animals is often reflected in a higher activity, they are not more active per se. In a second experiment, animals were provided with a permanent jugular vein canula for repeated blood sampling to determine stress hormones: noradrenaline, adrenaline, prolactin and corticosterone. Animals were subjected to a novelty test and a conditioned fear test. The neuroendocrine response profile is consistent with the conclusion that handled animals are less anxious than controls but are not different in their general strategy of coping with stressors. The handled animals showed an attenuated adrenaline, prolactin and corticosterone response. Yet, in neither of the two tests there was a difference in noradrenaline response, a typical marker for an active coping strategy. Interestingly, the differences in neuroendocrine reactivity already appeared in response to a mild novelty challenge when there were no clear behavioural differences yet. The neuroendocrine measures are in line with the behavioural data but more sensitively reflect the differences between handled and control animals.

Action of glucocorticoids on survival of nerve cells : Promoting neurodegeneration or neuroprotection?

Extensive studies during the past decades provided compelling evidence that glucocorticoids (GCs) have the potential to affect the development, survival and death of neurones. These observations, however, reflect paradoxical features of GCs, as they may be critically involved in both neurodegenerative and neuroprotective processes. Hence, we first address different aspects of the complex role of GCs in neurodegeneration and neuroprotection, such as concentration dependent actions of GCs on neuronal viability, anatomical diversity of GC‐mediated mechanisms in the brain and species and strain differences in GC‐induced neurodegeneration. Second, the modulatory action of GCs during development and ageing of the central nervous system, as well as the contribution of altered GC balance to the pathogenesis of neurodegenerative disorders is considered. In addition, we survey recent data as to the possible mechanisms underlying the neurodegenerative and neuroprotective actions of GCs. As such, two major aspects will be discerned: (i) GC‐dependent offensive events, such as GC‐induced inhibition of glucose uptake, increased extracellular glutamate concentration and concomitant elevation of intracellular Ca2+, decrease in GABAergic signalling and regulation of local GC concentrations by 11β‐hydroxysteroid dehydrogenases; and (ii) GC‐related cellular defence mechanisms, such as decrease in after‐hyperpolarization, increased synthesis and release of neurotrophic factors and lipocortin‐1, feedback regulation of Ca2+ currents and induction of antioxidant enzymes. The particular relevance of these mechanisms to the neurodegenerative and neuroprotective effects of GCs in the brain is discussed.

Chronic Corticosterone Administration Dose-Dependently Modulates Aβ(1–42)- and NMDA-Induced Neurodegeneration in Rat Magnocellular Nucleus Basalis

The impact of glucocorticoids on β‐amyloid(1–42) (Aβ(1–42)) and NMDA‐induced neurodegeneration was investigated in vivo. Aβ(1–42) or NMDA was injected into the cholinergic magnocellular nucleus basalis in adrenalectomized (ADX) rats, ADX rats supplemented with 25%, 100%, 2×100% corticosterone pellets, or sham‐ADX controls. Aβ(1–42)‐ or NMDA‐induced damage of cholinergic nucleus basalis neurones was assessed by quantitative acetylcholinesterase histochemistry. Plasma concentrations of corticosterone and cholinergic fibre loss after Aβ(1–42) or NMDA injection showed a clear U‐shaped dose–response relationship. ADX and subsequent loss of serum corticosterone potentiated both the Aβ(1–42) and NMDA‐induced neurodegeneration. ADX+25% corticosterone resulted in a 10–90u2003nM plasma corticosterone concentration, which significantly attenuated the Aβ(1–42) and NMDA neurotoxicity. ADX+100% corticosterone (corticosterone concentrations of 110–270u2003nM) potently decreased both Aβ(1–42)‐ and NMDA‐induced neurotoxic brain damage. In contrast, high corticosterone concentrations of 310–650u2003nM potentiated Aβ(1–42)‐ and NMDA‐triggered neurodegeneration. In conclusion, chronic low or high corticosterone concentrations increase the vulnerability of cholinergic cells to neurotoxic insult, while slightly elevated corticosterone levels protect against neurotoxic injury. Enhanced neurotoxicity of NMDA in the presence of high concentrations of specific glucocorticoid receptor agonists suggests that the corticosterone effects are mediated by glucocorticoid receptors.

New Non Competitive AMPA Antagonists

New halogen atom substituted 2,3-benzodiazepine derivatives condensed with an azole ring on the seven membered part of the ring system of type 3 and 4 as well as 5 and 6 were synthesized. It was found that chloro-, dichloro- and bromo-substitutions in the benzene ring and additionally imidazole ring condensation on the diazepine ring can successfully substitute the methylenedioxy group in the well known molecules GYKI 52466 (1) and GYKI 53773 (2) and the 3-acetyl-4-methyl structural feature in 2, respectively, preserving the highly active AMPA antagonist characteristic of the original molecules. From the most active compounds (3b,i) 3b (GYKI 47261) was chosen for detailed investigations. 3b revealed an excellent, broad spectrum anticonvulsant activity against seizures evoked by electroshock and different chemoconvulsive agents indicating a possible antiepileptic efficacy. 3b was found to be highly active in a transient model of focal ischemia predictive of a therapeutic value in human stroke. 3b also reversed the dopamine depleting effect of MPTP and antagonized the oxotremorine induced tremor in mice indicating a potential antiparkinson activity.

Increased amyloid precursor protein expression and serotonergic sprouting following excitotoxic lesion of the rat magnocellular nucleus basalis: Neuroprotection by Ca2+ antagonist nimodipine

In the present study plastic neural responses to N-methyl-D-aspartate-induced excitotoxic lesions and the neuroprotective effects of the L-type voltage-dependent Ca(2+) channel antagonist nimodipine were investigated in the rat magnocellular nucleus basalis. Assessment of spontaneous behaviour in the elevated plus maze and small open-field paradigms on day 5 and day 14 post-surgery indicated anxiety and persistent hypoactivity of N-methyl-D-aspartate-lesioned rats, as compared with sham-operated controls. Nimodipine administration significantly alleviated the behavioural deficits. Quantitative histochemical analysis of acetylcholinesterase-positive fibre innervation of the somatosensory cortex and determination of the numbers of choline-acetyltransferase-positive proximal fibre branches of cholinergic projection neurons in the magnocellular nucleus basalis demonstrated a severe cholinergic deficit as a consequence of the excitotoxic lesion 14 days post-surgery. Nimodipine pre-treatment significantly attenuated the loss of cortical cholinergic innervation and preserved the functional integrity of cholinergic projection neurons in the magnocellular nucleus basalis. Double-labelling immunocytochemistry demonstrated increased amyloid precursor protein expression in shrinking and presumably apoptotic choline-acetyltransferase-positive neurons, whereas surviving cholinergic nerve cells were devoid of excessive amyloid precursor protein immunoreactivity. Moreover, as a consequence of N-methyl-D-aspartate infusion, rim-like accumulation of amyloid precursor protein-positive astrocytes was visualized in a penumbra-like zone of the excitotoxic injury. Furthermore, abundant sprouting of serotonergic projection fibres invading the damaged magnocellular nucleus basalis subdivision was demonstrated. Pharmacological blockade by the Ca(2+) antagonist nimodipine significantly attenuated both neuronal and glial amyloid precursor protein immunoreactivity and serotonergic fibre sprouting following N-methyl-D-aspartate infusion. The present data characterize plastic endogenous glial and neuronal responses in the magnocellular nucleus basalis model of acute excitotoxic brain damage. The increased amyloid precursor protein expression may indicate effective means of intrinsic neuroprotection, as secreted amyloid precursor protein isoforms are suggested to play a role in neuronal rescue following excitotoxic injury. From a pharmacological point of view, extensive sprouting of serotonergic projections in the damaged magnocellular nucleus basalis may also counteract N-methyl-D-aspartate excitotoxicity via serotonin-induced inhibition of Ca(2+) currents and membrane hyperpolarization. Hence, lesion-induced changes in spontaneous animal behaviour, such as anxiety and novelty-induced hypoactivity, may well be attributed to the considerable re-distribution of serotonergic projections in the basal forebrain. In conclusion, our present data emphasize a role of neuron-glia and neurotransmitter-system interactions in functional recovery after acute excitotoxic brain injury, and the efficacy of L-type Ca(2+) channel blockade by the selective 1,4-dihydropyridine antagonist nimodipine.

Temporal and spatial dynamics of corticosteroid receptor down-regulation in rat brain following social defeat

The experiments explored the nature and time course of changes in glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) binding in homogenates of various brain regions and pituitary of male Wistar rats following social defeat stress. One week after defeat, the binding capacity of GRs was decreased in the hippocampus and the hypothalamus while no changes were observed in the parietal cortex and the pituitary. The number of MRs remained at the same level as in undefeated rats. Three weeks postdefeat, the initially down-regulated GR returned to baseline level in the hippocampus and the hypothalamus. However, GR binding was now decreased in the parietal cortex. Severe down-regulation of MRs was detected in the hippocampal and septal tissue. The results show that brief but intense stress like social defeat induces a long-lasting down-regulation of corticosteroid receptors and that the temporal dynamics of these changes are not only differential for GRs and MRs but also for brain sites.

Oral post-lesion administration of 5-HT1A receptor agonist repinotan hydrochloride (Bay X 3702) attenuates NMDA-induced delayed neuronal death in rat magnocellular nucleus basalis

Recent evidence indicates that stimulation of postsynaptic 5-HT(1A) receptors abates excitotoxic neuronal death. Here we investigated whether oral post-lesion administration of the 5-HT(1A) receptor agonist (-)-(R)-2-[4-[[(3,4-dihydro-2H-1-benzopyran-2-yl)methyl]amino]butyl]-1,2-benzisothiazol-3(2H)-one 1,1-dioxide monohydrochloride (Repinotan HCl) attenuates N-methyl-D-aspartate (NMDA) excitotoxicity (60 nmol/microl) in the rat magnocellular nucleus basalis. Repinotan HCl (1 mg/kg) was administered from day 1, 2, 3, or 6 post-surgery twice daily for five consecutive days. This delayed drug administration protocol was employed to investigate the initiation period during which 5-HT(1A) receptor agonists may significantly influence ongoing neurodegeneration processes. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 1 mg/kg) served as reference compound. Twenty-four hours after drug delivery a small open-field test, while on day 14 post-surgery a passive avoidance test was performed. Effects of Repinotan HCl treatment on the survival of cholinergic magnocellular nucleus basalis neurons and their cortical projections were determined by quantitative acetylcholinesterase (AChE) and choline-acetyltransferase (ChAT) histochemistry. Moreover, AChE and ChAT activities were biochemically measured both in the cerebral cortex and in the magnocellular nucleus basalis. Repinotan HCl treatment markedly increased spontaneous activities in the small open-field at any time-point investigated. Improved memory performance was only demonstrated when Repinotan HCl was administered from day 1 post-lesion on wards. Repinotan HCl treatment from day 2 and 3 post-lesion on markedly attenuated both histochemical and neurochemical characteristics of NMDA excitotoxicity on cholinergic magnocellular nucleus basalis neurons and on their cortical projections. Whereas the neuroprotective profile of Repinotan HCl was superior to that of 8-OH-DPAT, oral administration of both 5-HT(1A) receptor agonists yielded largely equivalent behavioral recovery after NMDA infusion in the magnocellular nucleus basalis. In conclusion, the present data indicate the potent neuroprotective action of the 5-HT(1A) receptor agonist Repinotan HCl with a peak efficacy of delayed (2-3 day) post-lesion drug treatment in vivo. Post-lesion treatment with 5-HT(1A) receptor agonists may therefore be of significance in the intervention of neuronal damage associated with acute excitotoxic conditions.

17β-Estradiol Enhances Cortical Cholinergic Innervation and Preserves Synaptic Density Following Excitotoxic Lesions to the Rat Nucleus Basalis Magnocellularis

Estradiol exerts beneficial effects on neurodegenerative disorders associated with the decline of cognitive performance. The present study was designed to further investigate the effect of 17beta-estradiol on learning and memory, and to evaluate its neuroprotective action on cholinergic cells of the nucleus basalis magnocellularis, a neural substrate of cognitive performance. Female rats were ovariectomized at an age of 6 months. Three weeks later they received injections of either a mid-physiological dose of 17beta-estradiol or vehicle (oil), every other day for 2 weeks. The effect of estradiol on cognitive performance was tested in two associative learning paradigms. In the two-way active shock avoidance task estradiol-replaced animals learned significantly faster, while in the passive shock avoidance test no differences were observed between the experimental groups. Subsequent unilateral infusion of N-methyl-D-aspartate in the nucleus basalis magnocellularis resulted in a significant loss of cholinergic neurons concomitant with the loss of their fibers invading the somatosensory cortex. Estradiol treatment did not affect the total number of choline-acetyltransferase-immunoreactive neurons and their coexpression of the p75 low-affinity neurotrophin receptor either contralateral or ipsilateral to the lesion. In contrast, cholinergic fiber densities in estradiol-treated animals were greater both in the contralateral and ipsilateral somatosensory cortices as was detected by quantitative choline-acetyltransferase and vesicular acetylcholine transporter immunocytochemistry. However, estradiol treatment did not affect the lesion-induced relative percentage loss of cholinergic fibers. A significant decline of synaptophysin immunoreactivity paralleled the cholinergic damage in the somatosensory cortex of oil-treated animals, whereas an almost complete preservation of synaptic density was determined in estradiol-treated rats. Our results indicate that estradiol treatment enhances the cortical cholinergic innervation but has no rescuing effect on cholinergic nerve cells in the basal forebrain against excitotoxic damage. Nevertheless, estradiol may restore or maintain synaptic density in the cerebral cortex following cholinergic fiber loss. This estradiol effect may outweigh the lack of cellular protection on cholinergic cells at the functional level.

Postnatal treatment with ACTH-(4-9) analog ORG 2766 attenuates N-methyl-d-aspartate-induced excitotoxicity in rat nucleus basalis in adulthood

It has been reported that the ACTH-(4-9) analog H-Met(O(2))-Glu-His-Phe-D-Lys-Phe-OH (ORG 2766) administered in adulthood has trophic effects on neuronal tissue and when given postnatally, it can induce long-lasting changes in brain development. In the present study, we investigated whether early postnatal treatment with ORG 2766 affects adult neuronal vulnerability, i.e. the sensitivity of cholinergic neurons against excitotoxic damage. Wistar rat pups received injections of ORG 2766 or saline on postnatal days 1, 3 and 5 and were then left undisturbed until adulthood. At the age of 6 months, the animals were subjected to unilateral lesion of magnocellular basal nucleus by infusion of high dose of N-methyl-D-aspartate (NMDA). The effects of the excitotoxic insult were studied 28 hours and 12 days after the lesion by measuring both the acute cholinergic and glial responses, and the final outcome of the degeneration process. Twenty eight hours after NMDA infusion, postnatally ACTH-(4-9)-treated animals showed stronger suppression of choline-acetyltransferase immunoreactivity and increased reaction of glial fibrillary acidic protein -immunopositive astrocytes in the lesioned nucleus compared to control animals. However, 12 days post-surgery, the NMDA-induced loss of cholinergic neurons, as well as the decrease of their acetylcholinesterase -positive fibre projections in the cortex, were less in ACTH-(4-9) animals. Our data indicate that the early developmental effects of ACTH-(4-9) influence intrinsic neuroprotective mechanisms and reactivity of neuronal and glial cells, thereby resulting in a facilitated rescuing mechanism following excitotoxic injury.

Increased maternal corticosterone levels in rats: Effects on brain 5-HT1A receptors and behavioral coping with stress in adult offspring

This study examined the consequences of elevated corticosterone levels in lactating rats on their offsprings serotonergic 5-hydroxytryptamine (5-HT)1A receptor system and behavioral coping with stress. The mothers received normal drinking water or water with corticosterone, which, via the milk, enters the circulation and brains of the pups. In adulthood, the corticosterone-nursed offspring showed a consistently more passive way of coping with environmental challenges. However, they did not seem to be more anxious. Autoradiographic analysis of the 5-HT1A receptor system revealed a decrease in the adult 5-HT1A receptor binding in the hippocampal CA1 region. The results support the hypothesis that differences in behavioral coping with stress by adult rats are associated with differences in the serotonergic system. At the same time, it suggests that adult coping and its neuronal substrates are not solely determined by genes but depend on subtle developmental factors as well.