Summary:Purpose: The possible role of gap junctions in the manifestation and control of the duration of seizures was tested on the 4-aminopyridine-induced epilepsy model in rats in vivo, by using electrophysiologic, pharmacologic, and molecular biologic techniques.Methods: In electrophysiologic experiments, the functional states of the gap junctions were manipulated with a specific blocker (carbenoxolone) or opener (trimethylamine) at the already active focus of adult, anesthetized rats, 60 min after the induction of the first seizure, which was repeated spontaneously thereafter. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was used to measure the levels of connexin (Cx) 32, 43, and 36 messenger RNAs (mRNAs) prepared from the areas of the already active primary and mirror foci.Results: After repeated seizures, the expression levels of Cx32, Cx43, and Cx36 mRNAs at the epileptic foci were increased significantly. Blockade of the gap junctions with carbenoxolone shortened the duration of seizures and decreased the amplitude of the seizure discharges, whereas their opening with trimethylamine lengthened the duration and increased the amplitude. Secondary epileptogenesis was facilitated when the gap junctions were opened.Conclusions: Our findings support the idea that, in epileptic foci, the gap junctions are involved in the expression of rhythmic ictal discharges and in the control of the duration and propagation of the individual seizures in vivo. Key Words: Connexins-Gap junctions-4-AP-induced seizureCarbenoxolone-Trimethylamine.Gap junctions are dynamic structures that can be modulated by a number of intracellular and extracellular factors (1-6). The extent of coupling in the in vitro seizure models (7-10) is periodic: it is increased by alkalinization at the start of a seizure, and decreased as acidification occurs toward the end of an ictal period.Our previous work (11) revealed noteworthy upregulations of connexin (Cx)32 and Cx43 mRNAs after repeated seizures both at the primary focus (Pf) and at the mirror focus (Mf, homotopic area contralateral to the Pf). Accordingly, we examined whether manipulation of the functional state of the gap junctions with a specific blocker (carbenoxolone) or opener (trimethylamine, TMA) influences the manifestation, duration, and propagation of seizures. We also were interested in whether repeated seizures influence the expression of the Cx36 gene, coding for a gap-junction protein existing predominantly in neuronal cells of the mature brain (13).

Summary:Purpose: The selective contribution of neuronal gap junction (GJ) communication via connexin 36 (Cx36) channels to epileptogenesis and to the maintenance and propagation of seizures was investigated in both the primary focus and the mirror focus by using pharmacologic approaches with the 4-aminopyridine in vivo epilepsy model.Methods: ECoG recording was performed on anesthetized adult rats, in which either quinine, a selective blocker of Cx36, or the broad-spectrum GJ blockers carbenoxolone and octanol were applied locally, before the induction or at already active epileptic foci.Results: The blockade of Cx36 channels by quinine before the induction of epileptiform activity slightly reduced the epileptogenesis. When quinine was applied after 25-30 repetitions of seizures, a new discharge pattern appeared with frequencies >15 Hz at the initiation of seizures. In spite of the increased number of seizures, the summated ictal activity decreased, because of the significant reduction in the duration of the seizures. The amplitudes of the seizure discharges of all the patterns decreased, with the exception of those with frequencies of 11-12 Hz. The blockade of Cx36 channels and the global blockade of the GJ channels resulted in qualitatively different modifications in ictogenesis.Conclusions: The blockade of Cx36 channels at the already active epileptic focus has an anticonvulsive effect and modifies the manifestation of the 1-to 18-Hz seizure discharges. Our findings indicate that the GJ communication via Cx36 channels is differently involved in the synchronization of the activities of the networks generating seizure discharges with different frequencies. Additionally, we conclude that both neuronal and glial GJ communication contribute to the manifestation and propagation of seizures in the adult rat neocortex. Key Words: Gap junctions-Cx36-4-AP-induced seizure-EpileptogenesisIctogenesis-In vivo-Quinine-Carbenoxolone.Epilepsy is one of the most prevalent neurologic disorders worldwide, but pharmacologic therapy remains the best remedy for its treatment. One reason for the incomplete effectiveness of the currently available anticonvulsants is that they were identified by using the same classic epilepsy models, which mainly involve the same actions, without a consideration of the variations in the pathophysiologic mechanisms that result in epilepsy. Growing evidence indicates that, besides the chemical synapses, direct coupling via gap junction (GJ) channels provides a second major pathway, contributing to normal and abnormal physiologic rhythms both during development and in the adult brain (1-3).GJ channels mediating electrical signaling are involved in the physiologic synchronizing mechanism in the brain (3-5) and contribute to pathologic hypersynchrony in various in vitro (6-9) and in vivo (10-13) epilepsy models.

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1. Electrical properties of four functional classes [inactivating bursting (ib), noninactivating bursting (nib), fast spiking (fsp), and regular spiking (rsp)] of neurons in the motor cortex of conscious cats were studied with the use of intracellular voltage recording and single-electrode voltage-clamp (SEVC) techniques. Evaluations were made of action potentials and afterpotentials, current-voltage (I-V) relationships, and passive cable properties. Values of membrane potential (Vm), input resistance (RN), membrane time constant (T0), and firing threshold (T50) were also measured. The data were used to extend the electrophysiological classifications of neurons described in the companion paper. 2. Average values of Vm (from -63 to -66 mV), action-potential amplitudes (from 72 to 77 mV), and firing threshold (-54 mV) were not statistically different in different types of neurons. However, the magnitude of intracellularly injected depolarizing current required to induce spike discharge at 50% probability varied significantly (from 0.6 to 1.1 nA) among cell types. The mean RN and T0 measured at Vm varied between 8.3 and 19.8 M omega, and 7.2 and 15.1 ms, respectively, in the cell classes. 3. Action potentials were overshooting. Their mean duration at half amplitude varied from 0.25 to 0.73 ms among different cell types. Three types of action-potential configurations were distinguished. Type I action potentials found in nib and rsp neurons were relatively fast and had a depolarizing afterpotential (DAP) as well as fast and slow after hyperpolarizations (fAHPs, sAHPs). Type II action potentials found in ib and rsp cells had relatively slow rise and decay phases, DAPs, and sAHPs. Their fAHPs were small or absent. Type III action potentials were found exclusively in fsp cells, had very short durations, prominent fAHPs, but no sAHPs. 4. Steady-state I-V relationships were determined by measuring voltage responses to 0.2- to 1.0-nA hyperpolarizing, rectangular current pulses at different membrane potentials. Both RN and T0 exhibited nonlinear behavior over wide ranges of membrane potential; however, between -65 and -75 mV, the I-V relationships varied little, and they appeared constant in most cells. The steady-state values of RN increased with decreasing, and decreased with increasing the membrane potential in all but fsp cells. The I-V relationships were virtually linear in fsp neurons. 5. Transient I-V relationships were studied by measuring voltage responses to depolarizing and hyperpolarizing, rectangular current pulses of increasing amplitude from a preset membrane potential of -70 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Patterns of firing activity and characteristics of antidromic and synaptic responses to stimulation of the pyramidal tract at peduncular level [peduncular pyramidal tract (PP)] and the ventrolateral thalamic nucleus (VL) were studied in neurons of area 4 gamma of the motor cortex of awake, chronic cats using intracellular microelectrode techniques. The results offer a new functional classification of neocortical neurons based on electrophysiological properties of the 640 recorded cells. 2. Four classes of neurons were distinguished: (class i) inactivating bursting (ib) neurons (n = 60) including fast antidromic response PP (fPP) (n = 0), slow antidromic response PP (sPP) (n = 11), and no antidromic response PP cells (nPP) (n = 49); (class ii) noninactivating bursting (nib) neurons (n = 79), including fPP (n = 23), sPP (n = 0), and nPP cells (n = 56); (class iii) fast-spiking (fsp) neurons (n = 56), including fPP (n = 0), sPP (n = 0), and nPP cells (n = 56); and (class iv) regular-spiking (rsp) neurons (n = 445), including fPP (n = 96), sPP (n = 38), and nPP cells (n = 311). (Neurons in each classification were further separated by their antidromic responses to PP stimulation: fast PP (fPP) slow PP (sPP), or nPP cells, the latter not responding antidromically to electrical stimulation of the peduncle.) 3. Recurrent monosynaptic excitatory postsynaptic potentials (EPSPs) followed antidromic spikes elicited by PP stimulation in most (96%) fPP but much fewer (24%) sPP cells. In fPP cells, it was possible to separate the PP EPSPs into two monosynaptic EPSP components that were generated by other fPP and sPP cells, respectively. VL stimulation evoked monosynaptic EPSPs in 100% of fPP cells (vs. 63% of sPP cells) and antidromic action potentials in 16% of fPP cells (vs. 12% of sPP cells). 4. Firing activity consisted of single spike discharges in most PP cells; however, noninactivating bursting was observed in 19% of fPP cells, and inactivating bursting was observed in 23% of sPP cells (see below). In 18% of ib and 11% of nib/nPP neurons, VL stimulation elicited antidromic action potentials. Other bursting neurons proved to be PP cells with characteristic differences in axonal conduction velocity (see above). All PP cells among the nib cells were fPP, and all PP cells among the ib cells were sPP cells. All fsp neurons were found to be nPP cells, and none could be activated antidromically by VL stimulation. Thus the fsp pattern of discharge distinguished a unique class of nPP cells.(ABSTRACT TRUNCATED AT 400 WORDS)