Decrease in CA3 inhibitory network activity during Theiler's virus encephalitis.

Viral infections of the central nervous system are often associated with seizures, and while patients usually recover from the infection and the seizures cease, there is an increased lifetime incidence of epilepsy. These viral infections can result in mesial temporal sclerosis, and, subsequently, a type of epilepsy that is difficult to treat. In previous work, we have shown that Theiler's murine encephalomyelitis virus (TMEV) infections in C57B/6 mice, an animal model of virus-induced epilepsy, results in changes in excitatory currents of CA3 neurons both during the acute infection and two months later, at a time when seizure thresholds are reduced and when spontaneous seizures can occur. The changes in the excitatory system differ at these two time points, suggesting different mechanisms for seizure generation. In the present paper, we examine GABAergic mediated inhibition in CA3 pyramidal cells at these two time points following TMEV infection. We found that amplitudes of sIPSCs and mIPSCs were reduced during the acute infection, but recovered at the two-month time point. These observations are consistent with previous measurements of excitatory currents suggesting different mechanisms of seizure generation during the acute infection and during chronic epilepsy.

Contributions of astrocytes to epileptogenesis following status epilepticus: opportunities for preventive therapy?

Status epilepticus (SE) is a life threatening condition that often precedes the development of epilepsy. Traditional treatments for epilepsy have been focused on targeting neuronal mechanisms contributing to hyperexcitability, however, approximately 30% of patients with epilepsy do not respond to existing neurocentric pharmacotherapies. A growing body of evidence has demonstrated that profound changes in the morphology and function of astrocytes accompany SE and persist in epilepsy. Astrocytes are increasingly recognized for their diverse roles in modulating neuronal activity, and understanding the changes in astrocytes following SE could provide important clues about the mechanisms underlying seizure generation and termination. By understanding the contributions of astrocytes to the network changes underlying epileptogenesis and the development of epilepsy, we will gain a greater appreciation of the contributions of astrocytes to dynamic circuit changes, which will enable us to develop more successful therapies to prevent and treat epilepsy. This review summarizes changes in astrocytes following SE in animal models and human temporal lobe epilepsy and addresses the functional consequences of those changes that may provide clues to the process of epileptogenesis.

Increased coupling and altered glutamate transport currents in astrocytes following kainic-acid-induced status epilepticus.

Profound astrogliosis coincident with neuronal cell loss is universally described in human and animal models of temporal lobe epilepsy (TLE). In the kainic acid-induced status epilepticus (SE) model of TLE, astrocytes in the hippocampus become reactive soon after SE and before the onset of spontaneous seizures. To determine if astrocytes in the hippocampus exhibit changes in function soon after SE, we recorded from SR101-labeled astrocytes using the whole-cell patch technique in hippocampal brain slices prepared from control and kainic-acid-treated rats. Glutamate transporter-dependent currents were found to have significantly faster decay time kinetics and in addition, dye coupling between astrocytes was substantially increased. Consistent with an increase in dye coupling in reactive astrocytes, immunoblot experiments demonstrated a significant increase in both glial fibrillary acidic protein (GFAP) and connexin 43, a major gap junction protein expressed by astrocytes. In contrast to what has been observed in resected tissue from patients with refractory epilepsy, changes in potassium currents were not observed shortly after KA-induced SE. While many changes in neuronal function have been identified during the initial period of low seizure probability in this model of TLE, the present study contributes to the growing body of literature suggesting a role for astrocytes in the process of epileptogenesis.

Differential contribution of kainate receptors to excitatory postsynaptic currents in superficial layer neurons of the rat medial entorhinal cortex.

Although in situ hybridization studies have revealed the presence of kainate receptor (KAR) mRNA in neurons of the rat medial entorhinal cortex (mEC), the functional presence and roles of these receptors are only beginning to be examined. To address this deficiency, whole cell voltage clamp recordings of locally evoked excitatory postsynaptic currents (EPSCs) were made from mEC layer II and III neurons in combined entorhinal cortex-hippocampal brain slices. Three types of neurons were identified by their electroresponsive membrane properties, locations, and morphologies: stellate-like "Sag" neurons in layer II (S), pyramidal-like "No Sag" neurons in layer III (NS), and "Intermediate Sag" neurons with varied morphologies and locations (IS). Non-NMDA EPSCs in these neurons were composed of two components, and the slow decay component in NS neurons had larger amplitudes and contributed more to the combined EPSC than did those observed in S and IS neurons. This slow component was mediated by KARs and was characterized by its resistance to either 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466, 100 microM) or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[lsqb]f[rsqb]quinoxaline-7-sulfonamide (NBQX, 1 microM), relatively slow decay kinetics, and sensitivity to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-50 microM). KAR-mediated EPSCs in pyramidal-like NS neurons contributed significantly more to the combined non-NMDA EPSC than did those from S and IS neurons. Layer III neurons of the mEC are selectively susceptible to degeneration in human temporal lobe epilepsy (TLE) and animal models of TLE such as kainate-induced status epilepticus. Characterizing differences in the complement of postsynaptic receptors expressed in injury prone versus injury resistant mEC neurons represents an important step toward understanding the vulnerability of layer III neurons seen in TLE.

Different patterns of synaptic transmission revealed between hippocampal CA3 stratum oriens and stratum lucidum interneurons and their pyramidal cell targets.

Stratum lucidum (SL) interneurons likely mediate feedforward inhibition between the dentate gyrus mossy fibers and CA3 pyramidal cells, while stratum oriens (SO) interneurons likely provide both feedforward and feedback inhibition within the CA3 commissural/associational network. Using dual whole-cell patch-clamp recordings between interneurons and CA3 pyramidal cells, we have examined SL and SO interneurons and their synapses within organotypic hippocampal slice cultures. Biocytin staining revealed different morphologies between these interneuron groups, both being very similar to those found previously in acute slices. The kinetics of IPSCs were similar between the two groups, but the reliability of synaptic transmission of SL interneuron (SL-INT) IPSCs was significantly lower than the virtually 100% reliability (non-existent failure rates) of SO-INT IPSCs. The SL-INT IPSCs also had a lower quantal content than the SO-INT IPSCs. In addition, SL-INTs were less likely than SO-INTs to innervate or to be innervated by nearby CA3 pyramidal cells. Paired-pulse stimulation at 100 ms interstimulus intervals produced similar paired-pulse depression in both interneuron synapses, despite the significantly higher failure rate of IPSCs produced by the SL-INTs compared with SO-INTs. CV analysis supported the hypothesis that paired-pulse depression was presynaptic. During repetitive, high frequency stimulation (>10 Hz for 500 ms) the two different synapses exhibited distinctly different forms of short-term plasticity: all SL interneurons displayed significant short-term facilitation (mean 113% facilitation, n=4), while, by contrast, SO interneuron synapses displayed either short-term depression (mean 42% depression, n=5 of 8) or no net facilitation or depression (n=3 of 8). These results indicate that the synaptic properties of interneurons can be quite different for interneurons in different hippocampal circuits.

Characterization of desensitization in recombinant N-methyl-D-aspartate receptors: comparison with native receptors in cultured hippocampal neurons.

In the present study we have characterized the effect of Ca2+, glycine, and agonist concentration on inactivation and desensitization in native and recombinant N-methyl-d-aspartate (NMDA) receptors. In agreement with earlier studies on neurons, we found that in the presence of saturating glycine concentrations, lowering [Ca2+]o, will decrease inactivation of NMDA receptors in cultured hippocampal neurons. However, unlike native NMDA receptors under the same recording conditions, recombinant receptors did not exhibit Ca2+-dependent inactivation. We also show that the glycine-insensitive desensitization observed in the recombinant receptors is subunit dependent, as NR1a2A and NR1a2B receptors significantly desensitized while the NR1a2C combination did not. Furthermore, we show this form of desensitization in NR1a2A receptors is due to classic agonist-induced desensitization. In addition, we demonstrate the presence of glycine-dependent desensitization in recombinant receptors. The ability of glycine to inhibit desensitization correlates to the rank order of glycine's affinity for potentiating the peak response for each subtype. Finally, using ifenprodil in the presence of high and low glycine concentrations, we present evidence that both 2A-like and 2B-like subtypes of receptors can independently coexist in single neurons.

Glycine regulation of synaptic NMDA receptors in hippocampal neurons.

1. Although glycine has been identified as a required coagonist with glutamate at N-methyl-D-aspartate (NMDA) receptors, the understanding of glycine's role in excitatory synaptic neurotransmission is quite limited. In the present study, we used the whole cell patch-clamp technique to examine the ability of glycine to regulate current flow through synaptic NMDA receptors at excitatory synapses between cultured hippocampal neurons and in acutely isolated hippocampal slices. 2. These studies demonstrate that the glycine modulatory site on the synaptic NMDA receptor is not saturated under baseline conditions and that increased glycine concentrations can markedly increased NMDA-receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal neurons in both dissociated cell culture and in slice. Saturation of the maximal effect of glycine takes place at different concentrations for different cells in culture, suggesting the presence of heterogenous NMDA receptor subunit compositions. 3. Bath-applied glycine had no effect on the time course of EPSCs in either brain slice or culture, indicating that desensitization of the NMDA receptor is not prevented by glycine over the time course of an EPSC. 4. When extracellular glycine concentration is high, all miniature EPSCs recorded in the cultured hippocampal neurons contained NMDA components, indicating that segregation of non-NMDA receptors at individual synaptic boutons does not occur.

Presence of mRNA for glutamic acid decarboxylase in both excitatory and inhibitory neurons.

Neurons in very low density hippocampal cultures that are physiologically identified as either GABAergic inhibitory or glutamatergic excitatory all contain mRNA for the gamma-aminobutyric acid (GABA) synthetic enzyme, glutamic acid decarboxylase (GAD), as detected by single cell mRNA amplification and PCR. However, consistent with the physiology, immunocytochemistry revealed that only a subset of the neurons stain for either GAD protein or GABA. A similar fraction hybridize with RNA probes for GAD65 and GAD67. Hippocampal CA1 pyramidal neurons in slice preparations, which are traditionally thought to be excitatory, also contain mRNA for GAD65 and GAD67. Hippocampal neurons in culture did not contain mRNA for two other neurotransmitter synthesizing enzymes, tyrosine hydroxylase, and choline acetyl transferase. These data suggest that in some neurons, presumably the excitatory neurons, GAD mRNA is selectively regulated at the level of translation. We propose that neurotransmitter phenotype may be posttranscriptionally regulated and neurons may exhibit transient phenotypic plasticity in response to environmental influences.

Calcium-dependent paired-pulse facilitation of miniature EPSC frequency accompanies depression of EPSCs at hippocampal synapses in culture.

Two forms of evoked neurotransmitter release at excitatory synapses between cultured hippocampal neurons have been described. After an action potential, it has been shown that transmitter initially is released synchronously, and this is followed by a period of "slow" asynchronous release. The "fast" synchronous component of release at these synapses has been found routinely to demonstrate paired-pulse and tetanic depression, whereas the short-term plasticity of asynchronous release has not been investigated. In the present experiments, we have used the whole-cell patch-clamp technique to record from pairs of neurons in a low-density hippocampal culture preparation to determine both the properties and underlying mechanisms of short-term plasticity of asynchronous release. It was found that an increase in miniature EPSC (mEPSC) frequency accompanied both single and multiple stimuli, and this mEPSC increase was facilitated during paired stimuli, even when the evoked synchronous release was depressed. In addition, both the activity-dependent depression of evoked EPSCs and facilitation of asynchronous mEPSC release were dependent on Ca accumulation in the nerve terminal. However, the Ca-dependent mechanisms underlying these two processes could be distinguished by the differential effects of two membrane-permeant calcium chelators, BAPTA-AM and EGTA-AM. Frequency-dependent depression of evoked EPSCs involves a rapid rise in intraterminal Ca, which likely triggers a process that proceeds in a Ca-independent manner, whereas the asynchronous release may be linked more directly to a sustained increase in intraterminal Ca.

Properties of inhibitory and excitatory synapses between hippocampal neurons in very low density cultures.

The whole cell patch clamp technique was used to examine the electrophysiological properties of embryonic hippocampal neurons maintained in a very low density (VLD) culture preparation. The goal of these experiments was to establish the viability of the VLD culture as a model system in which to study regulation of neurotransmission at single monosynaptic connections, in the absence of polysynaptic innervation. Depolarization of neurons in the VLD culture revealed voltage-dependent sodium, calcium, and potassium currents which were blocked with, respectively, tetrodotoxin (TTX), cobalt, and tetraethylammonium and 4-aminopyridine. When pairs of neurons were simultaneously recorded, action potentials evoked in presynaptic neurons elicited either excitatory or inhibitory postsynaptic currents (EPSCs or IPSCs, respectively). The dual component EPSCs were due to the activation of both types of postsynaptic, ionotropic glutamate receptors: N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Evoked IPSCs were due to the activation of postsynaptic gamma-aminobutyric acid (GABA) receptors. Both excitatory and inhibitory synapses exhibited short term depression in response to high frequency stimulation, although IPSCs were routinely decreased to a much greater degree than EPSCs. Spontaneous miniature EPSCs and IPSCs were found to persist in TTX, were blocked by the same pharmacological antagonists which blocked evoked responses, increased in frequency in response to hypersomotic solution, and were unaffected by changes in extracellular calcium concentration. mIPSCS were found to occur at a significantly lower frequency than mEPSCs. These experiments indicated that neurotransmission in the VLD cultures occurs in a manner consistent with the quantal hypothesis and, therefore, the VLD culture is a good model for studying excitatory and inhibitory neurotransmission between isolated pairs of neurons. In addition, these experiments, performed under comparable physiological conditions, demonstrated that there are fundamental differences underlying neurotransmitter release between excitatory and inhibitory neurons.

Paired pulse depression in cultured hippocampal neurons is due to a presynaptic mechanism independent of GABAB autoreceptor activation.

Most rapid synaptic inhibition in the vertebrate forebrain is mediated by GABA acting via GABAA and GABAB postsynaptic receptors. GABAergic neurotransmission exhibits frequency-dependent modulation; sequential inhibitory post-synaptic currents (IPSCs) evoked with interstimulus intervals between 25 msec and 4 sec routinely result in the attenuation of the amplitude of the second IPSC. This form of synaptic plasticity is known as paired pulse depression (PPD). The mechanism of PPD is presently unknown and the experiments performed in this study were designed to determine directly the location of the mechanism of PPD in hippocampal neurons maintained in low-density tissue culture. Evoked IPSCs were recorded between pairs of cultured neurons grown in relative isolation that were simultaneously being recorded with the whole-cell, patch-clamp technique. It was therefore possible to measure miniature IPSCs (mIPSCs) originating from the same synapses that were being stimulated to evoke release. PPD occurred routinely in this system, but the amplitudes of mIPSCs following IPSCs were unchanged. These results indicate that a presynaptic mechanism mediates PPD. The inability of GABAB receptor antagonists to block PPD revealed that this form of presynaptic plasticity was not due to autoinhibition of transmitter release via activation of presynaptic GABAB receptors. However, manipulations that significantly lowered the probability of release of neurotransmitter during the first action potential of a trial (e.g., lower calcium or baclofen) prevented the development of PPD. These results indicate that, under baseline conditions, the quantal content for IPSCs is relatively large for a single action potential, but the quantal content rapidly decreases, such that subsequent action potentials consistently result in much smaller IPSCs for periods as long as 4 sec.

In vitro electrophysiology of neurons in the lateral dorsal tegmental nucleus.

The lateral dorsal tegmental nucleus (LDT) provides ascending cholinergic projections to forebrain structures such as prefrontal cortex, septum, habenula, and thalamus, but relatively little is known of the physiology of LDT neurons. Intracellular recordings from LDT neurons in guinea pig brain slices found that most neurons fired action potentials either tonically or in bursts. The voltage dependent characteristics of the neurons suggest that a prolonged afterhyperpolarization due to an outward potassium current and a low-threshold calcium conductance contributed to these two modes of firing. Intracellular injections of Lucifer Yellow and subsequent staining for NADPH-diaphorase activity permitted positive identification of cholinergic neurons.

Electrophysiological properties of neurons in the lateral habenula nucleus: an in vitro study.

1. The electroresponsive characteristics of neurons in the lateral habenula were studied with intracellular recordings in a brain slice preparation of guinea pig diencephalon maintained in vitro. One hundred and two neurons met the criteria for recording stability, and of these, 18 were analyzed in detail. For these 18 neurons, the mean resting membrane potential was -61.9 mV, the mean input resistance was 124 M omega, and the mean spike amplitude of fast action potentials was 60.3 mV. 2. Lateral habenula neurons were found to have distinct patterns of activity dependent on membrane potential. At membrane potentials more positive than -65 mV, depolarization elicited trains of sodium-dependent fast action potentials. At membrane potentials more negative than -65 mV, slight depolarization elicited a tetrodotoxin-insensitive wave of depolarization, called a low-threshold spike (LTS), from which a burst of fast action potentials were triggered. The principal conductance underlying the LTS is a low-threshold calcium conductance, which is inactivated at membrane potential more positive than -65 mV and deinactivated when the membrane is hyperpolarized to potentials more negative than -65 V. 3. Upon termination of injected hyperpolarizing current, many neurons displayed oscillation in membrane potential at a frequency of 3-10 Hz, thereby generating repetitive bursts of fast spikes. 4. The pattern of neuronal activity in lateral habenula neurons was highly sensitive to slight alterations in membrane potential. The ability of these neurons to fire action potentials in two modes, tonically and in bursts, and the propensity of these neurons to dramatically alter their output in response to transient hyperpolarizing input, indicate that transmission through this relay in the dorsal diencephalic conduction system may be greatly augmented by relatively small hyperpolarizing influences on the individual neurons.

Stimulation of the lateral habenula inhibits dopamine-containing neurons in the substantia nigra and ventral tegmental area of the rat.

Neurons in the lateral habenula (LHb) of rats have efferent projections that terminate in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA), where cell bodies of dopamine-containing neurons are located. In order to study the influence of the habenula on dopaminergic activity, single-cell electrophysiological techniques were used to record unit discharge of dopamine-containing neurons in the SNC and VTA during electrical stimulation of the LHb or adjacent structures. Dopamine-containing neurons in the SNC and VTA were identified by their characteristic spike duration (greater than 2 msec), discharge rate (2-8 spikes/sec), and irregular firing pattern. Analysis of peristimulus time histograms showed that 85% of SNC cells and 91% of VTA neurons were inhibited after single pulse stimulation (0.25 mA, 0.1 msec) of the LHb. The mean time between stimulation and onset of inhibition was 11 msec (range, 2-22 msec) and mean duration of maximal suppression was 76 msec (range, 20-250 msec). Stimulation of structures adjacent to the LHb (hippocampus, lateral thalamus, medial dorsal thalamus, medial habenula) had little or no effect. Destruction of the fasciculus retroflexus, the fiber pathway that contains most habenular efferents, blocked the stimulation effects on dopamine-containing neurons. Destruction of the stria medullaris, which contains most habenular afferents, did not alter the inhibitory effect of habenular stimulation. Injection of a cytotoxin, kainic acid, in the LHb 1 week before recording sessions blocked the inhibitory consequences of habenular stimulation. These experiments show that activation of neuronal perikarya in the LHb causes orthodromic inhibition of dopamine-containing neurons in SNC and VTA via the fasciculus retroflexus.

Kainate and electrolytic lesions of the lateral habenula: effect on avoidance responses.

Male Sprague Dawley rats (n = 24), which received either bilateral electrolytic lesions, kainic acid lesions or sham treatments in the lateral habenula, were tested for acquisition of a one-way, conditioned avoidance response. Animals with electrolytic lesions failed to learn the avoidance task within 15 trials. In contrast, rats with kainic acid lesions performed as well as the control group. The results indicate that the disruption of the septal-medial habenula-interpenduncular nucleus pathway may be responsible for the observed avoidance deficit in electrolytically lesioned animals.