Corticosterone modifies muscarinic receptor immunoreactivity in rat hippocampus.

In the present study we report the effect of corticosterone in the regulation of hippocampal muscarinic acetylcholine receptor immunoreactivity (mAChR-ir) expression in rats. Adrenalectomy (ADX) or a single injection of a mineralocorticoid antagonist RU-28318 (1.0 mg/100 g body weight (b.w.)) in adrenally intact rats 24 h prior to sacrifice revealed an increased mAChR-ir in hippocampal CA1 and CA3 areas. Corticosterone replacement (100 microg/100 g b.w.) prevented the increase in mAChR-ir of ADX animals. However, glucocorticoid receptor antagonist (RU38486) treatment in adrenally intact rats failed to affect the mAChR immunolabeling. These results point to a modulation of muscarinic receptors by corticosterone that is predominantly mediated by the mineralocorticoid receptor.

Delayed neuronal migration of protein kinase Cgamma immunoreactive cells in hippocampal CA1 area after 48 h of moderate hypoxemia in the near term ovine fetus.

The brain is uniquely sensitive to disturbances in energy and oxygen supply, particularly during the early stage of life. Since hypoxemia can indirectly activate the intracellular messenger protein kinase C (PKC), we studied the PKCgamma-immunoreaction in the fetal hippocampal CA1 region of naive (n=4), instrumented control (n=7), and instrumented hypoxemic fetuses (n=14), at a mean gestational age of 127 days. Forty-eight hours of mild to moderate hypoxemia, were followed by a 48-h recovery period. Hypoxemia resulted in an increase in carotid blood flow (137% of control), and a shift towards a higher percentage of high-voltage electrocortical activity. After recovery, the fetal brain was fixated by perfusion of both carotid arteries, sectioned and immunostained for PKCgamma. The distribution of PKCgamma-immunoreactive cells was significantly changed after 48 h of hypoxemia in that the migration of cells (from the ventricular region towards the stratum pyramidale) was delayed (p<0.01) compared to naive and instrumented control animals. In contrast to the distribution, the relative total optical density of PKCgamma-ir cells and fibres in the CA1 hippocampal area was not significant different between the animal groups. We conclude that hypoxemia delayed migration of PKCgamma-ir cells, without neuronal degeneration.

Translocation of protein kinase Cgamma occurs during the early phase of acquisition of food rewarded spatial learning.

This study describes the translocation of the brain specific protein kinase C gamma isoenzyme (PKCgamma) in the hippocampus during food rewarded spatial learning. The holeboard test was used for spatial orientation, and immunoblot analysis was used for assessment of PKCgamma in cytosolic, membrane-inserted and membrane-associated fractions. Membrane-associated PKCgamma was increased during early acquisition of spatial learning, but not in a later phase of training. This transient and apparently temporary intracellular PKCgamma translocation was only observed in the posterior but not in the anterior hippocampus, and was only detected within 10 min after termination of the learning trial. This study supports the idea that PKCgamma is significantly involved in the biochemical events underlying learning and memory, notably during the period of novel information processing. The results further promote the hypothesis that the hippocampus is specifically involved in temporal information processing, which requires the engagement of PKCgamma.

Repeated blockade of mineralocorticoid receptors, but not of glucocorticoid receptors impairs food rewarded spatial learning.

Corticosteroids from the adrenal cortex influence a variety of behaviours including cognition, learning and memory. These hormones act via two intracellular receptors, the mineralo-corticoid receptor (MR) and the glucocorticoid receptor (GR). These two receptor types display a high concentration and distinct distribution in the hippocampus, a brain region which is directly involved in the regulation of spatial orientation and learning. In this study, repeated subcutaneous administration of the mineralocorticoid receptor antagonist RU28318 (1.0 mg/100 g body weight), the glucocorticoid receptor blocker RU38486 (2.5 mg/100 g body weight), or a combination of both antagonists were investigated for their effects on working--and reference memory in morning and afternoon trials during 8 subsequent days in food rewarded spatial learning in a hole board task. Each rat received one dose of either vehicle (2% ethanol in PEG 400), RU28318, RU38486 or the combination of both antagonists directly after the first trial on training days 1, 3, 5, and 7. The experiments demonstrated that repeated blockade of mineralocorticoid receptors impairs reference memory reflected in the morning--as well as in the afternoon trial, whereas blockade of glucocorticoid receptors has little effect on this type of cognitive behaviour. Furthermore, combined blockade of MRs and GRs resulted in a decrease, in both daily trials, in reference memory as well as working memory performance. These findings suggest that in this spatial learning paradigm, the impairment of working memory required blockade of both receptor types, while reference memory performance involves predominantly the mineralocorticoid receptors.

Historical review of research on protein kinase C in learning and memory.

1. In 1977, the discovery of a new type of kinase was reported, which turned out to be a receptor for phorbol esters. Thereafter, several mechanisms regulating PKC activity and various PKC subtypes have been discovered. 2. A role for PKC in synaptic plasticity and information storage has been postulated in the mid-1980s. An important role for PKC has since been suggested in several learning and memory models, in which persistent changes in the activation of PKC outlasting the initial stimulating event are thought to be crucial. 3. A vast number of experiments have further substantiated a role of PKC in learning and memory using, molecular genetic, behavioral, pharmacological, electrophysiological or immunocytochemical approaches in the late 1980s and the 1990s. PKC research of the past decade or so of has shown some exciting aspects of the putative role of PKC in synaptic plasticity and information storage. 4. The authors have provided highlights (Table 1) on research on PKC.

Exposure to chronic psychosocial stress and corticosterone in the rat: effects on spatial discrimination learning and hippocampal protein kinase Cgamma immunoreactivity.

Previous reports have demonstrated a striking increase of the immunoreactivity of the gamma-isoform of protein kinase C (PKCgamma-ir) in Ammon's horn and dentate gyrus (DG) of rodent hippocampus after training in a spatial orientation task. In the present study, we investigated how 8 days of psychosocial stress affects spatial discrimination learning in a hole board and influences PKCgamma-ir in the hippocampal formation. The acquisition of both reference memory and working memory was significantly delayed in the stressed animals during the entire training period. With respect to cellular plasticity, the training experience in both nonstressed and stressed groups yielded enhanced PKCgamma-ir in the CA1 and CA3 regions of the posterior hippocampus but not in subfields of the anterior hippocampus. Stress enhanced PKCgamma-ir in the DG and CA3 pyramidal cells of the anterior hippocampus. In stressed animals that were subsequently trained, the PKCgamma-ir was increased in the posterior CA1 region to the same level as that found in nonstressed trained animals. Stress apparently abrogated the PKCgamma-ir training response in the CA3 region. In a second experiment, the elevation of plasma corticosterone levels to values that are found during stress did not significantly influence reference memory scores but slightly and temporarily affected working memory. The training-induced enhancement of PKCgamma-ir in the CA1 region was similar in trained and corticosterone-treated trained animals, but the learning-induced PKCgamma-ir response in the posterior CA3 area was absent after corticosterone pretreatment. These results reveal that prolonged psychosocial stress causes spatial learning deficits, whereas artificial elevation of corticosterone levels to the levels that occur during stress only mildly affects spatial memory performance. The spatial learning deficits following stress are reflected only in part in the redistribution of hippocampal PKCgamma-ir following training.

In vivo protection against NMDA-induced neurodegeneration by MK-801 and nimodipine: combined therapy and temporal course of protection.

Neuroprotection against excitotoxicity by a combined therapy with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and the L-type Ca2+ channel blocker nimodipine was examined using an in vivo rat model of NMDA-induced neurodegeneration. Attention was focused on the neuroprotective potential of this combined drug treatment before and after NMDA-exposure. NMDA was unilaterally injected in the magnocellular nucleus basalis (MBN). Neuronal damage was assessed 12 days after the NMDA-injection by measuring the reduction of cholinergic cortical fibres that originate from the MBN neurons. In controls that received no drug treatment, NMDA-exposure damaged MBN neurons such that 66% of the cholinergic terminals were lost in the ipsilateral parietal cortex. Pretreatment with a nimodipine diet (860 ppm) combined with application of MK-801 (5 mg/kg i.p.) before NMDA-exposure reduced fibre loss by 89% thereby providing a near complete neuroprotection. Combined therapy of MK-801 (5 mg/kg i.p.) and nimodipine (15 mg/kg i.p.) 8 min after NMDA-infusion reduced neuronal injury by 82%, while the same combination given 2 h after the excitotoxic treatment still yielded a 66% protection against neurotoxic damage invoked by NMDA. In conclusion, the present data show that a dual blockade of NMDA-channels and voltage-dependent calcium channels (VDCC's) up to 2 h after NMDA-exposure is able to provide a significant protection against NMDA-neurotoxicity.

Neuroprotection against NMDA induced cell death in rat nucleus basalis by Ca2+ antagonist nimodipine, influence of aging and developmental drug treatment.

In the current study the neuroprotective effect of the L-type calcium channel antagonist nimodipine in rat brain was investigated in N-methyl-D-aspartate-induced neuronal degeneration in vivo. In the present model NMDA was unilaterally injected in the magnocellular nucleus basalis and the neurotoxic impact assessed by measuring cortical cholinergic fibre loss as a percentage of fibre density of the intact control hemisphere. This procedure proved to be a reproducible model in which the degree of damage was almost linearly proportional to the NMDA dose. Neuroprotection by nimodipine was determined in a number of conditions. First, the effect of nimodipine treatment in adult animals starting two weeks prior to neurotoxic injury was compared with neuroprotection provided by perinatal treatment of the mother animals with the calcium antagonist. Surprisingly, the degree of protection was in both cases similar, yielding almost 30% reduction of fibre loss. The neuroprotective effect in adulthood of perinatal nimodipine treatment may be explained by developmentally enhanced calcium binding proteins or persistent developmental changes in calcium channel characteristics. Protection by nimodipine was also investigated in aged, 26 month old rats. Compared to young adult cases, aged animals proved to be less vulnerable to NMDA exposure, while nimodipine application was more potent, thus yielding a reduction of nearly 50% in nerve fibre damage induced by NMDA infusions. Possible mechanisms of differential calcium influx in the various experimental conditions will be discussed.

Passive avoidance training induces enhanced levels of immunoreactivity for muscarinic acetylcholine receptor and coexpressed PKC gamma and MAP-2 in rat cortical neurons.

Changes in neocortical immunoreactivity (ir) for muscarinic acetylcholine receptors (mAChRs), protein kinase C gamma (PKC gamma), microtubule-associated protein 2 (MAP-2), and the calcium-binding protein parvalbumin (PARV) induced by the performance of a one-trial passive shock avoidance (PSA) task were studied in young adult male Wistar rats. In experiment I, four groups of animals were formed: three control groups (N, naive; H, habituated but nonshocked; and S, habituated and shocked), and a fully trained group (T, habituated and shocked, followed by a retention trial 24 hr after the footshock). Compared to naive animals, the H, S, and T animals all revealed enhanced cortical ir for mAChRs, PKC gamma, and MAP-2 in discrete subsets of cortical neurons in layers 2, 3, and 5, while no changes were found for PARV. The neurons displaying enhanced levels of ir are of the pyramidal and nonpyramidal cell type and are arranged in a columnar manner. Immunofluorescent double-labeling experiments for mAChR, PKC gamma, and MAP-2 revealed that individual cortical neurons localized within the columns display enhanced ir for all three functionally related proteins. Compared to naive animals, all experimental groups revealed significant increases in the total size of cortical areas showing enhanced ir (H, S, and T over N). A further significant increase is found in animals receiving a footshock over nonshocked animals (S over H, respectively). The retention trial, however, did not induce a further increase (T over S). In some of the animals the patterns appeared to be lateralized, in either the left or right hemisphere. In order to test the role of cholinergic innervation in the induction of enhanced mAChR-ir, unilateral lesions of the nucleus basalis magnocellularis (nbm) were performed in experiment II. Apparently, an intact cholinergic innervation from the nbm is not required for the occurrence of the aforementioned columnar patterns. However, when the enhanced columnar patterns in the sensory areas of the cortex are cholinergically deprived, clear deficits in PSA performance are observed. These results indicate that although ACh is not a prerequisite for the induction of enhanced ir for mAChRs in cortical cells, such neurons demand cholinergic neurotransmission for optimal retention of the shock experience. The alterations in ir for coexpressed mAChR, PKC gamma, and MAP-2 in a discrete subset of cholinoceptive cortical neurons arranged in characteristic patterns most likely represent part of the neuronal substrate involved in functional cortical plasticity related to PSA training.