Dynamic Regulation of the Activated, Autophosphorylated State of Ca2+/Calmodulin-Dependent Protein Kinase II by Acute Neuronal Excitation In Vivo

Abstract: Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) has been implicated in various neuronal functions, including synaptic plasticity. To examine the physiological regulation of its activated, autophosphorylated state in relation to acute neuronal excitation in vivo, we studied the effect of electroconvulsive treatment in rats on CaMKII activity and in situ autophosphorylation levels. As early as 30 s after the electrical stimulation, a profound but transient decrease in its Ca²⁺/calmodulin-independent activity, as well as in the level of its autophosphorylation at Thr²⁸⁶ (α)/Thr²⁸⁷ (β) measured by using phosphorylation state-specific antibodies, was observed in homogenate from hippocampus and parietal cortex, which was reversible in 5 min. In the later time course, a moderate, reversible increase, which peaked at around 60 min after the electrical stimulation, was observed in parietal cortex but not in hippocampus. The early-phase decrease was found to occur exclusively in the soluble fraction. In addition, partial translocation of CaMKII from the soluble to the particulate fraction seems to have occurred in this early phase. Thus, the activated, autophosphorylated state of CaMKII is under dynamic and precise regulation in vivo, and its regulatory mechanisms seem to have regional specificity.     

Dynamics of Ca(2+)/calmodulin-dependent protein kinase II following acute myocardial ischemia-translocation and autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase (CaMK) family is responsive to changes in the intracellular Ca(2+) concentration. However, their functions have not been well established in the ischemia/reperfusion heart. The effects of myocardial ischemia on CaMKII, the most strongly expressed form, were investigated using isolated rat hearts. Rat hearts were rendered globally ischemic by stopping perfusion for 15 min, and then reperfused, heart ventricles being analyzed in each phase. Western blotting detected a decrease in the cytosolic and concomitant increase in the particulate fraction of CaMKII following transient ischemia. Redistribution to the cytosol was revealed on reperfusion. Northern blot showed CaMKII gene expression decreased by ischemia. Furthermore, autoradiography and confocal immunohistochemical findings provided autophosphorylation of CaMKII in the cytosol, ischemia causing decrease, with gradual recovery on reperfusion. These results indicate a transient partial translocation of CaMKII accompanied by kinase activity, with residual myocardial CaMKII undergoing autophosphorylation during ischemia and reperfusion, demonstrating two different characteristic dynamics of CaMKII.     

Increased levels of messenger RNAs for neurotrophic factors in the brain during kindling epileptogenesis.

Kindling, induced by repeated subconvulsive electrical or chemical stimulations leads to progressive and permanent amplification of seizure activity, culminating in generalized seizures. We report that kindling induced by electrical stimulation in the ventral hippocampus leads to a marked and transient increase in mRNA for NGF and BDNF in the dentate gyrus, the parietal cortex, and the piriform cortex. BDNF mRNA increased also in the pyramidal layer of hippocampus and in the amygdaloid complex. No change was seen in the level of HDNF/NT-3 mRNA. The increased expression of NGF and BDNF mRNAs was not influenced by pretreatment with the NMDA receptor antagonist MK801, but was partially blocked by the quisqualate, AMPA receptor antagonist NBQX. The presumed subsequent increase of the trophic factors themselves may be important for kindling-associated plasticity in specific neuronal systems in the hippocampus, which could promote hyperexcitability and contribute to the development of epileptic syndromes.     

Electrical activity of the rat brain following antenatal hypoxia

EEG and EPs in the visual and parietal neocortical areas and the hippocampus were studied in freely behaving rats at the age of two-three months after antenatal hypoxia. Increase of spectral power in delta and theta bands and its decrease in alpha and beta bands in the background EEG and in responses to a protracted light stimulation were observed in experimental animals in comparison to control ones. The most pronounced changes were observed in the parietal cortex and hippocampus. The character of changes in latencies and the share of individual EP components recorded point to an accelerated excitation reverberation in neuronal networks in response to afferent stimuli and to a prolongation of after-discharges in the parietal cortex and hippocampus testifying to peculiarity of information processing in these brain structures. On the basis of other authors' data, certain parallelism is supposed to exist between the electrophysiological parameters in experimental animals and some groups of children with mental retardation.     

Metabolic Changes in Cerebral Cortex, Hippocampus, and Cerebellum During Sustained Bicuculline-Induced Seizures

Abstract— The objective of the present experiments was to study metabolic correlates to the localization of neuronal lesions during sustained seizures. To that end, status epilepticus was induced by i.v. administration of bicuculline in immobilized and artificially ventilated rats, since this model is known to cause neuronal cell damage in cerebral cortex and hippocampus but not in the cerebellum. After 20 or 120 min of continuous seizure activity, brain tissue was frozen in situ through the skull bone, and samples of cerebral cortex, hippocampus, and cerebellum were collected for analysis of glycolytic metabolites, phosphocreatine (PCr), ATP, ADP, AMP, and cyclic nucleotides. After 20 min of seizure activity, the two “vulnerable” structures (cerebral cortex and hippocampus) and the “resistant” one (cerebellum) showed similar changes in cerebral metabolic state, characterized by decreased tissue concentrations of PCr, ATP, and glycogen, and increased lactate concentrations and lactate/ pyruvate ratios. In all structures, though, the adenylate energy charge remained close to control. At the end of a 2-h period of status epilepticus, a clear deterioration of the energy state was observed in the cerebral cortex and the hippocampus, but not in the cerebellum. The reduction in adenylate energy charge in the cortex and hippocampus was associated with a seemingly paradoxical decrease in tissue lactate levels and with failure of glycogen resynthesis (cerebral cortex). Experiments with infusion of glucose during the second hour of a 2-h period of status epilepticus verified that the deterioration of tissue energy state was partly due to reduced substrate supply; however, even in animals with adequate tissue glucose concentrations, the energy charge of the two structures was significantly lowered. The cyclic nucleotides (cAMP and cGMP) behaved differently. Thus, whereas cAMP concentrations were either close to control (hippocampus and cerebellum) or moderately increased (cerebral cortex), the cGMP concentrations remained markedly elevated throughout the seizure period, the largest change being observed in the cerebellum. It is concluded that although the localization of neuronal damage and perturbation of cerebral energy state seem to correlate, the results cannot be taken as. evidence that cellular energy failure is the cause of the damage. Thus, it appears equally probable that the pathologically enhanced neuronal activity (and metabolic rate) underlies both the cell damage and the perturbed metabolic state. The observed changes in cyclic nucleotides do not appear to bear a causal relationship to the mechanisms of damage.     

Age-dependent targeting of protein phosphatase 1 to Ca2+/calmodulin-dependent protein kinase II by spinophilin in mouse striatum

Mechanisms underlying age-dependent changes of dendritic spines on striatal medium spiny neurons are poorly understood. Spinophilin is an F-actin- and protein phosphatase 1 (PP1)-binding protein that targets PP1 to multiple downstream effectors to modulate dendritic spine morphology and function. We found that calcium/calmodulin-dependent protein kinase II (CaMKII) directly and indirectly associates with N- and C-terminal domains of spinophilin, but F-actin can displace CaMKII from the N-terminal domain. Spinophilin co-localizes PP1 with CaMKII on the F-actin cytoskeleton in heterologous cells, and spinophilin co-localizes with synaptic CaMKII in neuronal cultures. Thr286 autophosphorylation enhances the binding of CaMKII to spinophilin in vitro and in vivo. Although there is no change in total levels of Thr286 autophosphorylation, maturation from postnatal day 21 into adulthood robustly enhances the levels of CaMKII that co-immunoprecipitate with spinophilin from mouse striatal extracts. Moreover, N- and C-terminal domain fragments of spinophilin bind more CaMKII from adult vs. postnatal day 21 striatal lysates. Total levels of other proteins that interact with C-terminal domains of spinophilin decrease during maturation, perhaps reducing competition for CaMKII binding to the C-terminal domain. In contrast, total levels of α-internexin and binding of α-internexin to the spinophilin N-terminal domain increases with maturation, perhaps bridging an indirect interaction with CaMKII. Moreover, there is an increase in the levels of myosin Va, α-internexin, spinophilin, and PP1 in striatal CaMKII immune complexes isolated from adult and aged mice compared to those from postnatal day 21. These changes in spinophilin/CaMKII interactomes may contribute to changes in striatal dendritic spine density, morphology, and function during normal postnatal maturation and aging.     

Cholinergic Deficit Induced by Ethylcholine Aziridinium (AF64A) Transiently Affects Somatostatin and Neuropeptide Y Levels in Rat Brain

The question whether during the process of cholinergic degeneration somatostatin- and/or neuropeptide Y-containing neurons in rat hippocampus and cortex react to the withdrawal of cholinergic function was addressed. After bilateral intracerebroventricular injection of the cholinotoxin ethylcholine aziridinium (AF64A; 1 or 2 nmol/ventricle) in rats, the activity of choline acetyltransferase (ChAT) started to decline in the hippocampus within 24 h. The reduction of ChAT activity reached its maximum within 4 days (34 and 55% after 1 and 2 nmol of AF64A/ventricle, respectively) and persisted during the observation period of 14 days. In the parietal cortex, ChAT activity decreased by 23% 4 days after 2 nmol of AF64A/ventricle. The loss in ChAT activity was accompanied by a transient decline in the levels of somatostatin and a transient increase in the levels of neuropeptide Y in both brain areas. In the hippocampus, the reduction in somatostatin content was most pronounced after 2 days (by 22 and 33% after 1 and 2 nmol of AF64A/ventricle, respectively). Within 14 days, somatostatin levels returned to control values. Neuropeptide Y levels increased slightly by approximately 25% of control values in the hippocampus. The changes described were present in both the dorsal and ventral subfields of the hippocampus. Similar but less pronounced changes in levels of both neuropeptides were observed in the parietal cortex. The present data provide further evidence for a close neuronal interrelationship between cholinergic and somatostatin- and/or neuropeptide Y-containing neurons in rat hippocampus and parietal cortex.     

CaMKII inactivation by extracellular Ca(2+) depletion in dorsal root ganglion neurons

A mechanism by which Ca(2+)/CaM-dependent protein kinase (CaMKII) is autophosphorylated by changes in extracellular calcium in the absence of detectable changes in cytoplasmic [Ca(2+)] has been identified. We find that when the external Ca(2+) concentration ([Ca(2+)](O)) is lowered, Ca(2+) is released from intracellular stores to maintain a constant cytoplasmic Ca(2+) level, gradually depleting the endoplasmic Ca(2+) stores. Accompanying the store-depletion is a rapid decrease in CaMKII activity. Approximately 25% of the measured CaMKII autophosphorylation in DRG neurons in culture can be regulated by Ca(2+) flux from intracellular stores caused by manipulating [Ca(2+)](O), as shown by blocking refilling of store-operated Ca(2+)-channels with SK&F 96365, Ruthenium Red, and a partial block with Ni(2+). Blocking voltage-gated Ca(2+)-channels with either isradipine or SR 33805, had no effect on CaMKII autophosphorylation induced by restoring Ca(2+)(O) to normal after depleting the intracellular Ca(2+) stores. These results show that removal of Ca(2+)(O) has profound effects on intracellular Ca(2+) signaling and CaMKII autophosphorylation, in the absence of measurable changes in intracellular Ca(2+). These findings have wide-ranging significance, because [Ca(2+)](O) is manipulated in many experimental studies. Moreover, this explanation for the paradoxical changes in CaMKII phosphorylation in response to manipulating [Ca(2+)](O) provides a possible mechanism linking activity-dependent depletion of Ca(2+) from the synaptic cleft to a protein kinase regulating many neuronal properties.     

Decreased Resting Functional Connectivity after Traumatic Brain Injury in the Rat

Traumatic brain injury (TBI) contributes to about 10% of acquired epilepsy. Even though the mechanisms of post-traumatic epileptogenesis are poorly known, a disruption of neuronal networks predisposing to altered neuronal synchrony remains a viable candidate mechanism. We tested a hypothesis that resting state BOLD-fMRI functional connectivity can reveal network abnormalities in brain regions that are connected to the lesioned cortex, and that these changes associate with functional impairment, particularly epileptogenesis. TBI was induced using lateral fluid-percussion injury in seven adult male Sprague-Dawley rats followed by functional imaging at 9.4T 4 months later. As controls we used six sham-operated animals that underwent all surgical operations but were not injured. Electroencephalogram (EEG)-functional magnetic resonance imaging (fMRI) was performed to measure resting functional connectivity. A week after functional imaging, rats were implanted with bipolar skull electrodes. After recovery, rats underwent pentyleneterazol (PTZ) seizure-susceptibility test under EEG. For image analysis, four pairs of regions of interests were analyzed in each hemisphere: ipsilateral and contralateral frontal and parietal cortex, hippocampus, and thalamus. High-pass and low-pass filters were applied to functional imaging data. Group statistics comparing injured and sham-operated rats and correlations over time between each region were calculated. In the end, rats were perfused for histology. None of the rats had epileptiform discharges during functional imaging. PTZ-test, however revealed increased seizure susceptibility in injured rats as compared to controls. Group statistics revealed decreased connectivity between the ipsilateral and contralateral parietal cortex and between the parietal cortex and hippocampus on the side of injury as compared to sham-operated animals. Injured animals also had abnormal negative connectivity between the ipsilateral and contralateral parietal cortex and other regions. Our data provide the first evidence on abnormal functional connectivity after experimental TBI assessed with resting state BOLD-fMRI.     

Hippocampal neurons and glia in epileptic EL mice

Reactive changes in hippocampal astrocytes are frequently encountered in association with temporal lobe epilepsy in humans and with drug or kindling-induced seizures in animal models. These reactive changes generally involve increases in astrocyte size and number and often occur together with neuronal loss and synaptic rearrangements. In addition to producing astrocytic changes, seizure activity can also produce reactive changes in microglia, the resident macrophages of brain. In this study, we examined the effects of recurrent seizure activity on hippocampal neurons and glia in the epileptic EL mouse, a natural model of human multifactorial idiopathic epilepsy and complex partial seizures. Timm staining was used to evaluate infrapyramidal mossy fiber organization and the optical dissector method was used to count Nissl-stained neurons in hippocampus of adult (about one year of age) EL mice and nonepileptic C57BL/6J (B6) and DDY mice. Immunostaining forglial fibrillary acidic protein (GFAP) and Iba1, an actin cross-linking molecule restricted to macrophages and microglia, was used to evaluate astrocytes and microglia, respectively. The EL mice experienced about 25–30 complex partial seizures with secondary generalization during routine weekly cage changing. No significant differences were found among the mouse strains for Timm staining scores or for neuronal counts in the CA1 and CA3 pyramidal fields or in the hilus. However, the number of GFAP-positive astrocytes was significantly elevated in the stratum radiatum and hilus of EL mice, while microglia appeared hyper-ramified and were more intensely stained in EL mice than in the B6 or DDY mice in the hilus, parietal cortex, and pyriform cortex. The results indicate that recurrent seizure activity in EL mice is associated with abnormalities in hippocampal astrocytes and brain microglia, but is not associated with obvious neuronal loss or mossy fiber synaptic rearrangements. The EL mouse can be a useful model for evaluating neuron-glia interactions related to idiopathic epilepsy.     

Modification of presynaptic CaM kinase II affinity for ATP in hippocampus after long term blockade of serotonin reuptake

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is markedly enriched at synapses, where it is involved in the control of synaptic transmission, transmitter release and synaptic plasticity. CaMKII has also been found to be involved in the long-term action of antidepressants on post-receptor signaling mechanisms, because monoamine reuptake inhibitors induced an increase in autophosphorylation and activity of the kinase in nerve terminals of hippocampus. To study whether changes in the amount of enzyme or kinetic changes, due to posttranslational modifications, are responsible for kinase activation in nerve terminals, alpha-CaMKII level and kinetic constants of the autophosphorylation reaction as a function of ATP concentration were measured in presynaptic cytosol from hippocampus. Treatment with two serotonin reuptake inhibitors did not change the level of presynaptic kinase or the Vmax of autophosphorylation reaction. Instead the Km of the kinase for ATP was decreased 2.8-fold with fluvoxamine and 3.5-fold with paroxetine, implying an increase in the affinity for ATP. This result represents the first finding of changes in kinetic constants of a major brain enzyme after treatment with antidepressant drugs.     

Regulation of neuronal excitability in Drosophila by constitutively active CaMKII

The ability of calcium/calmodulin-dependent protein kinase II (CaMKII) to become calcium independent after autophosphorylation makes this enzyme a temporal marker of neuronal activity. Here we show that the calcium-independent form of CaMKII has unique effects on larval viability, locomotion, and neuronal excitability in Drosophila. Expression of constitutively active T287D, but not calcium-dependent T287A, mutant CaMKII in Drosophila neurons resulted in decreased viability, behavioral defects, and failure of action potential propagation. The actions of T287D may be mediated, at least in part, by increased potassium conductances. Expression of T287D CaMKII also stimulated an increase in the number of boutons at the larval neuromuscular junction, but did not affect the mechanics of release. This study defines a role for autophosphorylation of CaMKII in the regulation of multiple neuronal functions including the intrinsic properties of neurons.     

CaMKII, an enzyme on the move: regulation of temporospatial localization

Calcium-calmodulin-dependent protein kinase II (CaMKII) is an important regulator of neuronal and behavioral plasticity. Studies in which the subcellular distribution of CaMKII has been altered argue that targeting of this enzyme to specific subcellular compartments is crucial to many of its roles. Understanding how a very abundant enzyme can achieve specificity of action over time and space requires an understanding of the functional diversity of the enzyme and its distribution. In this review we will discuss how structurally distinct isozymes, splice isoforms, and autophosphorylation states of CaMKII can affect kinase activity and localization. We will focus on the fast activity-dependent synaptic localization of the kinase and its association with postsynaptic proteins. The ability of enzyme activation to regulate protein-protein interactions with these binding partners and the potential for such binding interactions to regulate CaMKII activity in novel ways may represent new paradigm for CaMKII regulation.     

Ketamine Self-Administration Elevates αCaMKII Autophosphorylation in Mood and Reward-Related Brain Regions in Rats

Modulation of αCaMKII expression and phosphorylation is a feature shared by drugs of abuse with different mechanisms of action. Accordingly, we investigated whether αCaMKII expression and activation could be altered by self-administration of ketamine, a non-competitive antagonist of the NMDA glutamate receptor, with antidepressant and psychotomimetic as well as reinforcing properties. Rats self-administered ketamine at a sub-anesthetic dose for 43 days and were sacrificed 24 h after the last drug exposure; reward-related brain regions, such as medial prefrontal cortex (PFC), ventral striatum (vS), and hippocampus (Hip), were used for the measurement of αCaMKII-mediated signaling. αCaMKII phosphorylation was increased in these brain regions suggesting that ketamine, similarly to other reinforcers, activates this kinase. We next measured the two main targets of αCaMKII, i.e., GluN2B (S1303) and GluA1 (S831), and found increased activation of GluN2B (S1303) together with reduced phosphorylation of GluA1 (S831). Since GluN2B, via inhibition of ERK, regulates the membrane expression of GluA1, we measured ERK2 phosphorylation in the crude synaptosomal fraction of these brain regions, which was significantly reduced suggesting that ketamine-induced phosphorylation of αCaMKII promotes GluN2B (S1303) phosphorylation that, in turn, inhibits ERK 2 signaling, an effect that results in reduced membrane expression and phosphorylation of GluA1. Taken together, our findings point to αCaMKII autophosphorylation as a critical signature of ketamine self-administration providing an intracellular mechanism to explain the different effects caused by αCaMKII autophosphorylation on the post-synaptic GluN2B- and GluA1-mediated functions. These data add ketamine to the list of drugs of abuse converging on αCaMKII to sustain their addictive properties.     

Decreased Protein Kinase C Activity During Cerebral Ischemia and After Reperfusion in the Adult Rat

The possible activation of protein kinase C (PKC) during total cerebral ischemia was investigated in the rat. Translocation of PKC activity from the soluble to the particulate fraction was used as an index of PKC activation. There was a drop in the proportion of particulate PKC activity from 30% for controls to 20% by 30 min of ischemia (p less than 0.01). By 20 min of cardiac arrest, there was a 40% decline of the total cellular PKC activity (p less than 0.01). This was not accompanied by an increase in activator-independent activity, a finding indicating PKC was not being converted to protein kinase M. These data suggest that PKC was not activated during ischemia, but rather that ischemia causes a reduction in cellular PKC activity. Translocation of PKC activity to the particulate fraction was not observed in the cerebral cortex or hippocampus of reperfused brain for up to 6 h of recovery following 11-13 min of total cerebral ischemia. The level of total, soluble, and particulate PKC activity in the cerebral cortex was reduced (p less than 0.05), corresponding to the decrease observed by 15 min of ischemia without reflow. A similar decline in activity was also observed in the hippocampus. No increase in activator-independent activity was observed. These data suggest that PKC was inhibited during cerebral ischemia and that this reduced level of PKC activity was maintained throughout 6 h of recovery. We conclude that pathological activation of PKC was not responsible for the evolution of ischemic brain damage.     

Reduced calcium/calmodulin-dependent protein kinase II activity in the hippocampus is associated with impaired cognitive function in MPTP-treated mice

Parkinson's disease (PD) patients frequently reveal deficit in cognitive functions during the early stage in PD. The dopaminergic neurotoxin, MPTP-induced neurodegeneration causes an injury of the basal ganglia and is associated with PD-like behaviors. In this study, we demonstrated that deficits in cognitive functions in MPTP-treated mice were associated with reduced calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and impaired long-term potentiation (LTP) induction in the hippocampal CA1 region. Mice were injected once a day for 5days with MPTP (25mg/kg i.p.). The impaired motor coordination was observed 1 or 2week after MPTP treatment as assessed by rota-rod and beam-walking tasks. In immunoblotting analyses, the levels of tyrosine hydroxylase protein and CaMKII autophosphorylation in the striatum were significantly decreased 1week after MPTP treatment. By contrast, deficits of cognitive functions were observed 3-4weeks after MPTP treatment as assessed by novel object recognition and passive avoidance tasks but not Y-maze task. Impaired LTP in the hippocampal CA1 region was also observed in MPTP-treated mice. Concomitant with impaired LTP induction, CaMKII autophosphorylation was significantly decreased 3weeks after MPTP treatment in the hippocampal CA1 region. Finally, the reduced CaMKII autophosphorylation was closely associated with reduced AMPA-type glutamate receptor subunit 1 (GluR1; Ser-831) phosphorylation in the hippocampal CA1 region of MPTP-treated mice. Taken together, decreased CaMKII activity with concomitant impaired LTP induction in the hippocampus likely account for the learning disability observed in MPTP-treated mice.     

Developmental Iodine Deficiency and Hypothyroidism Reduce Phosphorylation of Calcium/Calmodulin-Dependent Kinase II in the Rat Entorhinal Cortex

Iodine is essential for the synthesis of triiodothyronine (T₃) and thyroxine (T₄). Iodine deficiency leads to inadequate thyroid hormone. Hypothyroidism induced by iodine deficiency during gestation and postnatal period leads to cognitive deficits in learning and memory. However, the mechanism underlying these deficits is unclear. Calcium-dependent calmodulin kinase II (CaMKII) known as a potential memory molecule regulates important neuronal functions including learning and memory. Recent studies have shown that hypothyroidism alters phosphorylation of CaMKII in hippocampus or even in sympathetic ganglia of rats. Though the entorhinal cortex (EC) is an important functional structure within the neuronal network responsible for learning and memory, little is known about the effect of hypothyroidism on phosphorylation of CaMKII in the EC. Here, we report that iodine deficiency and propylthiouracil treatment through gestation and lactation reduce phosphorylation of CaMKII in the EC of pups. The increase of calcineurin, as well as reduction of neurogranin and calmodulin, may account for the reduced phosphorylation of CaMKII induced by developmental iodine deficiency and hypothyroidism. These findings in the EC may contribute to understanding the mechanisms that underlie impairment of learning and memory induced by developmental iodine deficiency and hypothyroidism.     

Down-Regulation of AMPA Receptor Subunit GluR2 in Amygdaloid Kindling

Abstract: Alterations in glutamatergic transmission are postulated to be important in kindling and epilepsy. The levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits (GluR1, 2, and 4) were compared in amygdalakindled and sham-operated animals using subunit-specific antibodies and quantitative western blotting. Four limbic regions were examined: limbic forebrain, piriform cortex/amygdala, hippocampus, and entorhinal cortex. When subunit levels were examined 24 h after the last stage 5 seizure, levels of GluR2 were found to be selectively reduced in limbic forebrain (30%) and piriform cortex/amygdala (25%), with no changes in other regions examined. In addition, no changes in the other subunits were observed in any region. The decrease in GluR2 that was observed in kindled animals at 24 h was no longer present at 1 week and 1 month after the last stage 5 seizure. Because the GluR2 subunit uniquely determines the calcium permeability of these receptors and because the piriform cortex has been implicated as a source of excitatory drive for limbic seizures, reduced GluR2 expression may be important in increasing neuronal excitability in kindling-induced epilepsy, or may reflect a compensatory mechanism resulting from kindling.     

Decreased Muscarinic Acetylcholine Receptor Number in the Central Nervous System of the Tottering (tg/tg) Mouse

The tottering mouse (tg/tg) is a single-locus mutant, phenotypically characterized by the development of epilepsy associated with distinct electroencephalographic abnormalities. Because of reported alterations in muscarinic receptor (mAChR) number in various seizure states, mAChR density was examined in discrete brain regions of tottering (tg/tg) and coisogenic wild-type (+/+) mice. Saturation binding experiments revealed a widespread decrease in membrane mAChR density in the CNS of adult tottering (tg/tg) mice as compared with age-matched control wild-type (+/+) mice. The decrease was most pronounced in the hippocampus, where tg/tg mice exhibited a 40-60% reduction in mAChR density with no change in the affinity of the receptor for antagonists or agonists. At postnatal day 10, before the reported onset of electroencephalographic abnormalities, 114 and 65% increases in mAChR density were observed in the tg/tg hippocampus and cortex, respectively. Following the development of seizure activity at postnatal day 22, mAChR density in the tg/tg hippocampus was reduced by 29%. No change in brain mAChR density was seen in adult heterozygotes (+/tg), which do not develop electroencephalographic or seizure abnormalities. These results indicate that the development of reduced mAChR number in the CNS of the tg/tg mouse is secondary to abnormal neuronal activity, providing further support for the hypothesis that membrane depolarization can cause a decrease in neuronal mAChR density.