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Summary: Purpose: The functional significance of gap-junction (GJ) channels in seizure susceptibility and induction and maintenance of seizures in the developing rat brain was investigated on the 4-aminopyridine (4-AP) in vivo epilepsy model.
Methods: In electrophysiological experiments, GJs were manipulated with a blocker or opener before induction or at the active epileptic foci between postnatal days 9 and 28 (P9–28). Semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) amplification was used to measure the levels of connexin (Cx) 26, 32, 36, and 43 mRNAs at the untreated cortex or epileptic foci.
Results: The basic electrocorticogram (ECoG) and Cx messenger RNA (mRNA) expression patterns exhibited characteristic maturation; the 4-AP–induced epileptiform activity correlated well with these changes. Cx mRNA expressions were significantly upregulated around P16 (except for Cx26). The Cx26, 36, and 43 gene inducibility was highest around P16 and then declined significantly. In the youngest animals, the GJ opener induced rhythmic synchronous cortical activity. On maturation, the seizures became focalized and periodic; the discharges accelerated their amplitude and frequency increase. A transient decrease (P13–14) and then increase (P15–16) in seizure susceptibility were followed by a tendency to periodicity and focalization.
Conclusions: The study suggests that GJ communication is involved in rhythm genesis and synchronization of cortical activity and may enhance the epileptogenicity of the developing brain.
Clinical experience and various experimental data indicate that the developing nervous system is more sensitive than the mature one to different convulsive effects (1–5). Although the physiological factors underlying this differential epileptogenicity have not been fully clarified, the higher susceptibility of the immature brain can be explained by certain characteristic neurobiologic features. The developing brain exhibits a high metabolic rate, abundant neuronal and synaptic networks, the overexpression of receptors and enzymes, the depolarizing effect of γ-amino-acid, the hypersynchrony of neuronal circuits, and enhanced synaptic plasticity (6,7). In addition, the immature cerebral cortex and hippocampus have higher densities of excitatory amino acid receptors and gap junction (GJ) channels as compared with the adult organs (4,8).
Intercellular communication via GJ channels is an important form of cell-to-cell communication in early brain development (8–14). Electrical coupling via GJ channels has been reported both between pairs of inhibitory neurons and among inhibitory and excitatory neurons during the early postnatal days in the rat cortex (10). Moreover, the incidence of coupling between neurons and glia has been observed at this early age in rats (8,12,15,16). It is believed that a possible correlation exists between the high seizure susceptibility of the immature brain and the elevated communication through the GJ channels (4). However, the role of GJ coupling in epilepsy in the adult and the developing nervous system is still not fully understood.
Accordingly, the purpose of the present study was to investigate the functional significance of GJ channels in the epileptogenicity and seizure susceptibility of the immature mammalian brain. With this aim, we made use of the K+ channel blocker 4-aminopyridine (4-AP)-induced epileptiform activity in rats between postnatal days 9 and 28 (P9–28). This neocortical seizure model is appropriate for studying epileptiform activity in the developing nervous system, because infants and children often have seizures of neocortical origin (17).
We investigated whether the intensity of GJ communication influences the process of development of epilepsy in normal brain tissue (which is called epileptogenesis) and the possible contribution of GJ communication in the induction, manifestation, and propagation of seizures at already established epileptic foci (ictogenesis) at different developmental points. By combining electrophysiology and pharmacology, we examined the effects of the functional state of the GJ channels modified by the local application of either a blocker or opener on the basic electrocorticogram (ECoG) and on the seizure activity of the neocortex of the developing brain. Further, the developmental expression levels and the plastic changes induced in the connexin (Cx) 26, 32, 36, and 43 messenger RNA (mRNA) levels by epileptiform activity were examined in the rat neocortex by me means of semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis. This work is an extension of our earlier studies carried out on adult animals (18–20).
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With regard to recent findings that imply a role for GJ channels in the synchronization of neuronal activity, in the present study, we focused on possible roles of GJ channels in the epileptogenicity and seizure susceptibility of the immature mammalian brain. We carried out electrophysiologic experiments, combined with pharmacologic manipulations of the GJ channels, and measured developmental and 4-AP–induced synchronous activity-dependent plastic changes in the Cx26, 32, 36, and C43 mRNA levels in the rat neocortex in different postnatal developmental stages. The epileptic cortical activity induced by 4-AP is accompanied by specific quantitative alterations in the different Cx mRNA levels and suggests a cause–effect relation between the electrical communication pathway and epileptiform synchronization. Our RT-PCR results revealed a highly diverse basic expression pattern for the Cx26, 32, 36, and 43 genes and showed quantitative alterations in their expression during the epileptogenic process in an age-related manner. Each of the Cxs examined displayed a unique basic pattern of postnatal development with a common feature: a marked alteration in their expression levels was found at around P16. Expressions were significantly upregulated at around this age for all the followed genes except Cx26. The Cx26 mRNA level was characterized by a marked decrease after the first 2 weeks.
During the postnatal development, the epileptiform activity induced strong, subtype-specific changes in the levels of expression of all the examined Cx genes: the transcript level of Cx43 was gradually elevated up to P23, the mRNA level of Cx36 showed a transient increase with a marked peak expression around P16, whereas the mRNA levels of Cx32 and 26 demonstrated only very modest age-dependent changes after repeated seizures. The inducibility of the Cx26, 36, and 43 genes was highest at around P16 and had significantly declined by P23. Upregulation of Cx26 and Cx45 mRNA was detected in neuronal cells undergoing apoptotic cell death in vulnerable regions such as hippocampus, amygdala, and some thalamic nuclei, whereas Cx36 was downregulated after the kainate-induced in vivo model of status epilepticus (13). In our 4-AP–induced seizure model, characterized by isolated, repeating seizures, although some kind of neuronal and astroglial damage was detected (23) approximately at the time point of the sample collection for mRNA analysis, apoptotic cell death is not characteristic in the neocortex.
Pharmacologic manipulation of the GJ channels differentially affects the basic ECoG and the induction and maintenance of seizures in different postnatal developmental stages. In the basic ECoG, we found a characteristic inherent sinusoid-like, generalized pattern, restricted until P12. The ability of the cortex to express sustained wave activity around these postnatal days has already been reported (11). The widespread, synchronous rhythmic activity in the basic ECoG was markedly enhanced by 4-AP (Fig. 4) and strongly abolished by carbenoxolone (Figs. 2–4). GJ blockers were shown earlier to abolish horizontal wave propagation and to eliminate epileptiform waves completely in young animals (11). In contrast, the GJ opener TMA alone induced considerable rhythmic, highly synchronous seizure-like activity in the P9–13 animals, which was apparently not modified by 4-AP (Figs. 3 and 4). The failure of 4-AP to modify the TMA-induced cortical activity suggests that the targets of the two drugs may overlap each other and supports GJ involvement in the widespread, synchronous rhythmic activity at this age. 4-AP in P9–12 animals induced a rhythmic activity, characterized by the generalized occurrence of sustained seizure-like, relatively slow potentials, with variable amplitudes and low frequencies. On maturation, the seizure discharges became faster, with increasing amplitude and frequency.
The onset of spontaneous and the 4-AP– and/or TMA-induced generalized synchronous activity could be correlated in part with the extensive cell coupling by the GJ channels (12,24), overwhelming the poorly developed chemical synaptic systems (5) in the first 2 weeks in the rat neocortex. Under physiologic conditions, we measured relatively high levels of Cx26 and 43 mRNA at P10–14, and the presence of Cx 36 mRNA was also detected, although at a lower level (Fig. 7). In time, the level of Cx26 mRNA somewhat declined, whereas the Cx43 expression progressively increased up to P23, and the Cx36 transcript level increased transiently, with peak expression at around P16 (Fig. 7).
The coupling of combinations of pyramidal and nonpyramidal cells and between neurons and glia, mediated by Cx26, 36, and 43, has been reported at this early age in rats (8,10,12). Besides the dominating homotypic coupling, a heterotypic form of coupling is also involved in connecting pyramidal and nonpyramidal neurons, or neurons and astrocytes at P7 and P14 (12). In networks where large numbers of neurons transmit electrical signals directly through GJ channels, the temporal heterogeneity of the discharges decreases, the synchrony thereby being enhanced (25).
The generalized synchronized, rhythmic pattern of both the spontaneous and the epileptiform activity, and their sensitivity to pharmacologic manipulation of the GJ channels on P9–12, may indicate that GJ channels in the immature cortex link neurons and glia cells into extensive networks that may allow electrical activity to spread over long distances. The horizontal propagation of waves has been correlated with the presence of dendrodendritic GJ channels during the first 10–12 postnatal days in rats (11,24,26). Furthermore, GJ channels are also reported to be capable of producing large functional clusters of coupled neurons in vertical columns, serving to synchronize the activity of several cortical layers (24,27).
The complex role of GJ channels at this early age could additionally be related to the relatively low number of chemical synapses (5). Neurons with intrinsic bursting properties that are frequently presumed to be involved in synchronous, rhythmic activity are not present in the sensorimotor cortex of rats in the first 2 weeks (28).
In the first 2 weeks after birth, pyramidal cells have been shown to use predominantly Cx26, whereas the nonpyramidal cells may equally use both Cx26 and 43 for the formation of GJ channels (12). Cx26 has been reported both in the astrocytes and in the neurons of the developing brain and spinal cord (29,30), with the highest expression prenatally and during the first 3 weeks of postnatal life (8,12,24). In addition, more recently, Cx45 gene expression has also been reported in neurons with genetic approach during embryogenesis, with a peak of expression at P1, and declined subsequently during brain development (13,31). However, Cx45 protein is widely expressed in many developing and mature nonneural cell types beside selected subsets of neurons. Conversely, Cx36 may be of particular interest, as is expressed exclusively in neurons and is present both in the young neurons and also in adult cortex (31). Nevertheless, it is possible, that Cx45 might play certain role in some of the epileptiform events of the young animals described in this article, either participating in neuronal–neuronal or in neuronal–glia communication mediated by GJs, which should be the subject of further analysis.
The mRNA level of Cx36 underwent a transient increase, with a marked peak expression at ∼P16 in our study. The level of developmental change and its time course for Cx36 exhibits region specificity (32). In the cerebral cortex, a progressive increase was observed, with peaks between 7 and 16 days (9,32–36). In the early stages of postnatal development, Cx36 is detectable even in neuronal populations that are devoid of Cx36 mRNA in the adult stage (32). Upregulation of Cx36 has been linked to juvenile myoclonic epilepsy (37).
The astrocytic Cx43 is present in the cortex throughout the period of development. (16,29,32,38,39). Astrocytes in adults compose an astrocytic syncytium which gives physical and metabolic support to the neurons (40,41). In the first 2 postnatal weeks, astrocytes are more likely to couple to neurons than to other astrocytes (12) through GJ channels, and they may promote the synchrony of spontaneously active neural networks (30).
Recent findings have lent support to the concept that the Cxs, although redundant, may play a significant role in unstable, transient cell–cell contacts (42). With regard to the relatively high level and unspecific occurrence of the Cxs, it seems justified to presume that the extensive communication through the GJ channels in the first 2 weeks may play a key role in the enhanced seizure susceptibility and mediate not only the induction, but also the propagation of rhythmic synchronous activity. Consequently, the decline of this feature later could be explained by the decreased frequency of GJ coupling between the neurons as the cells mature and in parallel by the formation of more specific neuronal networks involving excitatory and inhibitory chemical synapses (5,12,24).
The spontaneous rhythmic pattern that was described at P9–12 was not manifested in the basic ECoG (Figs. 1 and 2), and it was not induced by TMA pretreatment in the animals after (Fig. 1). In addition, we observed a transient reduction in the epileptogenicity of the animals during a brief developmental window at around P13–14 (Fig. 4). The sudden disappearance of the rhythmic features of the basic electrocortical activity and the inability of carbenoxolone and TMA to influence the basic cortical activity (Figs. 3–5) suggest a weaker GJ involvement in this developmental stage. We detected a decreased expression of Cx26 by P15–23 (Fig. 7), whereas the level of expression of Cx43 gradually increased. The expressions of these Cxs probably became restricted to specific cell types after P16, contributing to more-specific neuronal networks. These observations confirm the findings of earlier experiments, in which the glutamate-independent coordinated activity decreased and a neurobiotin tracer indicated a low incidence of neuronal coupling at around P15 (11,12). Although the basal level of Cx43 mRNA was increased in the P17 and P23 animals, it was markedly elevated by the epileptiform activity (Fig. 7). This increment of Cx43 mRNA could be an indication of a more-specific astrocytic activity and adjustability to the elevated neuronal firing activity. Although the oligodendrocyte-specific Cx32 mRNA (43) was not detected at P10, it was dramatically induced by the convulsant 4-AP at this age (Fig. 7, P10). At later ages (P14–23), the basic level became detectable and gradually increased with age. As a consequence, the relative inducibility of this gene progressively decreased (Fig. 7).
The lack of manifestation of the fastest seizure discharges in animals before P13–14 could be related to the relative scarcity of chemical synaptic connections and the slow nature of synaptic potentials at this age (5). Accordingly, the increase in the frequency and the appearance of a faster discharge in older animals could be explained in terms of the observation that the number of excitatory synapses significantly increased during the second postnatal week, and these synapses are able to follow activation faithfully at high frequencies (5). In addition, the actual number and ratio of the open versus closed GJ channels can modulate the frequency of the discharges (20). The application of the GJ opener TMA at already active epileptic focus at most developmental points enhanced generalized synchronization and increased the duration of ictal events, resulting in the cortical activity pattern that is characteristic of young animals with abundant GJ communication (Figs. 3–5). These observations confirm our earlier findings in adult animals showing that epileptiform activity can upregulate the expression of Cxs 26, 32, 36, and 43 mRNAs, indicated also by the strong efficacy of pharmacologic manipulation of GJ communication by carbenoxolone and TMA (18–20). However, the inducibility of the Cx mRNAs examined here revealed some subtype specificity at the different developmental time points.
One possible explanation of the transiently elevated epileptogenicity of young animals at around P16–17 (Fig. 4) could be the appearance of the first intrinsic bursting neurons during the third postnatal week (5,44) and/or the transient hyperactivity of the N-methyl-d-aspartate–mediated neurotransmission and/or the immature stage of γ-aminobutyric acid (GABA)ergic inhibition (7). Because the percentage of neurons with intrinsic bursting capacity is high in early postnatal life (45,46), these cells can act as cellular pacemakers coupled by GJ channels (7).
In addition, although the basic level of neuron-specific Cx26 mRNA gradually decreased with age, the other neuron-specific Cx36 expression was significantly increased and approached the highest level at around P16 (Fig. 7). The elevated level of GJ communication in scattered subpopulations of cells express Cx36 mRNA can substantially contribute to the elevated seizure activity observed at this age. A recent in vitro study based on abrupt maturation at the end of the second postnatal week of synchronous activity among electrically coupled, low-threshold spiking inhibitory interneurons revealed that such activity was absent on earlier postnatal days (14). The strong synchronizing ability of this inhibitory cell network may contribute to the increased seizure susceptibility of rats after the second postnatal week.
All measured Cx mRNA levels exhibited an obvious increase after 60-min periods of seizure activity, indicating that, besides the increased efficacy of excitatory chemical neurotransmission, electrical coupling through the GJ channels may also contribute to the transiently elevated level of epileptogenicity of P15–16 animals. After this age, the progressive decline in seizure susceptibility could be an indication of the fine-tuning of local synaptic connectivity and the pruning back of some excess of the chemical and electrical synapses, in parallel with the development of the fully active inhibitory GABAA prune-back receptor system (5,7). The incidence of coupling between excitatory and inhibitory cells declines with age (10,47,48), and in the adult, most electrical coupling exists between homogeneous cell types, a condition that may diminish the susceptibility of the neural networks to synchronization (27).