Long-term increase in protein kinase C-gamma and muscarinic acetylcholine receptor expression in the cerebral cortex of amygdala-kindled rats--a quantitative immunocytochemical study.

Kindling is an animal model for epilepsy in which repeated application of an electrical stimulus to brain pathways results in an epileptic focus. The animal holds a permanent state of hyperexcitability to the stimulus for the rest of its life. Understanding the cellular and molecular processes underlying hyperexcitability could provide insight into epileptogenesis. Furthermore, it could elucidate cellular and molecular bases of synaptic plasticity in the central nervous system. In the present study the long-term effect of a kindled focus in the amygdala on the gamma-isoform of protein kinase C and the muscarinic cholinergic receptor as cellular messengers was evaluated in the cerebral cortex of rats. Following an average of 10 bilaterally generalized seizures kindling stimulation was terminated and rats were left undisturbed for approximately three months. Brains were processed by immunocytochemistry using monoclonal antibodies against protein kinase C-gamma and muscarinic cholinergic receptor protein. Digital image analysis of sections through the entire forebrain revealed an increase in optical density of both protein kinase C-gamma and the muscarinic cholinergic receptor in the piriform and entorhinal cortex of the hemisphere contralateral to the stimulation site in kindled rats. However, on the ipsilateral side no change was observed in comparison with electrode implanted nonkindled control rats. The observed increase in expression of muscarinic cholinergic receptor protein and a component of the phosphoinositide second messenger system (protein kinase C-gamma) located in specific areas of the cerebral cortex in kindled rats could serve as a basis for the permanent state of hyperexcitability in these rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel approach for studying septo-hippocampal cholinergic neurons in freely moving rats: a microdialysis study with dual-probe design.

In this study, the overflow of acetylcholine (ACh) in the septo-hippocampal system was studied using intracerebral microdialysis in freely moving rats. Dialysis probes were implanted in the ventral hippocampus and in the medial septal area (MS), including a part of the ventral limb of the diagonal band of Broca (VDB). Dialysis samples were analysed 'on-line' using HPLC with post column enzymatic conversion and electrochemical detection. Local perfusion of 1 mumol/l of the sodium-channel blocker tetrodotoxin (TTX) through the probe resulted in 94% and 92% decrease in extracellular levels of ACh in the hippocampus and the septal area, respectively. The effects of septal manipulation on the efflux of ACh in the hippocampus were studied by electrical stimulation of the septal area and by administering drugs via the septal probe. Electrical stimulation of the MS/VDB caused a 336% increase in the output of ACh in the hippocampus. Perfusion of 3 mumol/l TTX through the septal probe caused a maximal decrease of 56% in the output of ACh in the ventral hippocampus. When perfused in the MS/VDB, the excitatory amino-acid agonists N-methyl-D-aspartate (NMDA) (100 mumol/l) and kainic acid (10 mumol/l) caused an increase in the extracellular level of ACh in the hippocampus by 83% and 161%, respectively. Thus, the overflow of ACh in the hippocampus and the septal area both depend on neuronal impulse flow. The extracellular level of ACh in the hippocampus is at least partially dependent on impulse flow in septo-hippocampal fibres. Moreover, the output of ACh in the hippocampus can be manipulated by electrical and pharmacological stimulation of the MS/VDB.

Effect of different agonistic experiences on behavioural seizures in fully amygdala kindled rats.

Fully amygdala kindled rats were exposed to two different inter-male agonistic experiences in order to study the interaction between epilepsy and acute social stress. Victory experience did not influence the severity of seizure behaviour, whereas a single acute defeat modified both ictal and postictal seizure manifestations. Defeat resulted in less severe and shorter lasting motor seizures, and the accompanied postictal inhibition or behavioural depression was of shorter duration in comparison with pre-stress values. The ability of acute defeat to trigger anticonvulsant activity as implied by the weakened convulsive response is discussed.

Social conflict situations in rats differentially affect the development of amygdala kindling.

Clinical studies revealed an association between the occurrence of convulsions and stress. However, the direction of such relations and their mechanisms are not quite clear. The present study investigated the influence of stress-inducing agonistic conflict situations on the progressive process of kindling epileptogenesis induced by daily tetanic stimulation of the amygdala. Whereas repeated exposure to defeat in an agonistic setting did not affect the development of amygdala kindling, repeated victory experiences resulted in a significant retardation of the kindling process. Moreover, the kindling process was retarded to a greater extent in rats that quickly displayed offensive behavior and won the confrontation. Possible underlying mechanisms and biological substrates are discussed in terms of the ability of the individual to control or cope with the nature of the stressor.

Autosomal and Y chromosomal effects on the stereotyped response to apomorphine in wild house mice.

The behavioral response to apomorphine, a dopamine agonist, was shown to be different between a selection line characterized by Short Attack Latencies (SAL) and a selection line having Long Attack Latencies (LAL) (4). Aggressive SAL mice were more sensitive to apomorphine than nonaggressive LAL males. The aim of this research was to determine whether the stereotyped response to apomorphine is affected by the Y chromosome in the same way as it influences attack latency. For this purpose, F1 reciprocal hybrids as well as congenic lines (SAL.LY and LAL.SY) were used. The major difference between the congenic and parental lines is the nonpairing part of the Y chromosome (non-PAR). Apomorphine was injected subcutaneously at a preselected dose level of 5.0 mg/kg to induce stereotyped behavior manifested in compulsive sniffing, gnawing, and licking. Both the autosomes and the non-PAR Y chromosome affected the response to apomorphine. The effect of the autosomes was in accordance with the aggression data, whereas the effect of the non-PAR Y chromosome was different, and suggests a specific relation between dopamine systems and the non-PAR Y chromosome.

Propagation of epileptiform activity during development of amygdala kindling in rats: linear and non-linear association between ipsi- and contralateral sites.

The relationship between ipsi- and contralateral epileptiform electroencephalographic (EEG) activity was investigated in rats that were kindled daily in the amygdala. Two types of relationships--linear and non-linear associations--were studied and used to estimate time delays of EEG activity between homotopic amygdalar sites during consecutive tetanizations. The progressive development of epileptiform EEG and convulsive behaviour was accompanied by an increase in association. Maximal association values of the non-linear function were significantly higher than linear association values. The gradual development of motor seizure severity was correlated with increased non-linearity. Time delays between the two amygdalae were estimated comparably with the linear and non-linear function: 30.0 +/- 3.3 and 24.6 +/- 1.7 ms (ipsilateral leading contralateral), respectively. However, in rats displaying exclusively bilaterally generalized motor convulsions, maximal values of both functions decreased but were still significantly higher than control values of phase-randomized EEG. Corresponding positive as well as negative interhemispheric time delays were recorded during the afterdischarge. These results demonstrated a strengthened association between the ipsi- and contralateral amygdala during primary epileptogenesis induced by amygdala kindling. In contrast, development of a secondary focus in the contralateral homotopic region resulted in a weakened interhemispheric association. Secondary bilateral synchrony between the ipsi- and contralateral amygdala occurred during the evoked epileptiform EEG activity.

Amygdala kindling-induced seizures selectively impair spatial memory. 1. Behavioral characteristics and effects on hippocampal neuronal protein kinase C isoforms.

Protein kinase C (PKC) comprises a family of kinases consisting of nine subspecies that are differentially distributed in the central nervous system. This implies distinct functions. Its involvement is suggested in cellular and molecular mechanisms by which the hippocampus exerts influence on information processing. In this study, it was questioned whether abnormal activity in the neuronal substrate, particularly the hippocampal formation, induced by amygdala kindling indeed impairs spatial memory performance and correlated alpha, beta I/II, and gamma PKC subspecies expression. Rats were trained in a spatial discrimination task (SDT) and simultaneously kindled in the amygdala to induce abnormal, epileptiform activity. Control rats were only trained in the holeboard, a "free choice" maze, in which working (WM) and reference memory (RM) were simultaneously examined. Halfway through and at the end of the experiments the influence of kindling and SDT training on the immunoreactivity for PKC subspecies alpha, beta I/II, and gamma was evaluated in the hippocampal formation. Kindling resulted in a gradual increase in afterdischarge duration and motor seizure (MS) severity. Repeated SDT training ultimately resulted in an asymptotic level of WM and RM performance. As soon as generalized MSs developed, kindled rats failed to improve RM, whereas WM was not influenced. Compared to untrained rats, in trained controls PKC gamma but not PKC alpha beta I/II immunoreactivity was elevated in CA1 pyramidal and dentate gyrus granular cells. Generalized but not partial MSs abolished these alterations in PKC gamma immunoreactivity. The present data indicate that repeated training in a SDT affects the expression of PKC subspecies gamma but not of alpha or beta in the rat hippocampus. Generalized epileptiform activity impair both acquisition of new spatial RM information and PKC gamma expression. It is argued that PKC gamma plays a role in cellular mechanisms through which pathological brain activity impairs certain aspects of spatial memory.

Amygdala kindling-induced seizures selectively impair spatial memory. 2. Effects on hippocampal neuronal and glial muscarinic acetylcholine receptor.

The muscarinic acetylcholine receptor is linked via hydrolysis of phosphoinositides to the protein kinase C pathway. In a preceding paper (Beldhuis, H. J. A., H. G. J. Everts, E. A. Vander Zee, P. G. M. Luiten, and B. Bohus (1992) Amygdala kindling-induced seizures selectively impair spatial memory. 1. Behavioral characteristics and effects on hippocampal neuronal protein kinase C isoforms. Hippocampus 2:397-410), the role of different isoforms of protein kinase C in neurobiological processes associated with plasticity was studied using both a spatial learning paradigm and amygdala kindling in the rat. This study extended the findings on protein kinase C activity to the level of the muscarinic acetylcholine receptor. Rats were trained in a spatial learning paradigm and kindled simultaneously in the amygdala to develop generalized motor convulsions. Control rats were trained only in the spatial learning paradigm to acquire stable working and reference memory performance. Alteration in the expression of the muscarinic acetylcholine receptor was investigated using a monoclonal antibody to muscarinic acetylcholine receptor proteins. Trained control rats that were exposed repeatedly to the spatial learning paradigm showed an increase in immunoreactivity for the muscarinic acetylcholine receptor located in the same hippocampal regions in which the protein kinase C activity was increased. In fully kindled rats, however, this increase located in principal neurons was absent, whereas expression of muscarinic acetylcholine receptor proteins was increased in hippocampal astrocytes. Moreover, fully kindled rats showed an impairment in reference memory performance as compared to trained control rats.(ABSTRACT TRUNCATED AT 250 WORDS)