Glial and neuronal expression of the Inward Rectifying Potassium Channel Kir7.1 in the adult mouse brain

Inward Rectifying Potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis and neuronal excitability. The most recently described Kir subtype is Kir7.1, which is known as a K⁺ transporting subtype. Earlier studies localised Kir7.1 to subpopulations of neurones in the brain. However, the pattern of Kir7.1 expression across the brain has not previously been examined. Here, we have determined neuronal and glial expression of Kir7.1 in the adult mouse brain, using immunohistochemistry and transgenic mouse lines expressing reporters specific for astrocytes [glial fibrillary acidic protein‐enhanced green fluorescent protein (GFAP‐EGFP], myelinating oligodendrocytes (PLP‐DsRed), oligodendrocyte progenitor cells (OPC, Pdgfra‐creER^T2/Rosa26‐YFP double‐transgenic mice) and all oligodendrocyte lineage cells (SOX10‐EGFP). The results demonstrate significant neuronal Kir7.1 immunostaining in the cortex, hippocampus, cerebellum and pons, as well as the striatum and hypothalamus. In addition, astrocytes are shown to be immunopositive for Kir7.1 throughout grey and white matter, with dense immunostaining on cell somata, primary processes and perivascular end‐feet. Immunostaining for Kir7.1 was observed in oligodendrocytes, myelin and OPCs throughout the brain, although immunostaining was heterogeneous. Neuronal and glial expression of Kir7.1 is confirmed using neurone‐glial cortical cultures and optic nerve glial cultures. Notably, Kir7.1 have been shown to regulate the excitability of thalamic neurones and our results indicate this may be a widespread function of Kir7.1 in neurones throughout the brain. Moreover, based on the function of Kir7.1 in multiple transporting epithelia, Kir7.1 are likely to play an equivalent role in the primary glial function of K⁺ homeostasis. Our results indicate Kir7.1 are far more pervasive in the brain than previously recognised and have potential importance in regulating neuronal and glial function.     

Neuronismo y reticulismo: neuronal-glial circuits unify the reticular and neuronal theories of brain organization

The neuronal doctrine, which shaped the development of neuroscience, was born from a long-lasting struggle between reticularists, who assumed internal continuity of neural networks and neuronists, who defined the brain as a network of physically separated cellular entities, defined as neurones. Modern views regard the brain as a complex of constantly interacting cellular circuits, represented by neuronal networks embedded into internally connected astroglial syncytium. The neuronal-glial circuits endowed with distinct signalling cascades form a 'diffuse nervous net' suggested by Golgi, where millions of synapses belonging to very different neurones are integrated first into neuronal-glial-vascular units and then into more complex structures connected through glial syncytium. These many levels of integration, both morphological and functional, presented by neuronal-glial circuitry ensure the spatial and temporal multiplication of brain cognitive power.     

Cellular localization of the uptake of [3H]taurine and [3H]β-alanine in cultures of the rat central nervous system

The cellular localization of the uptake of [³H]taurine and [³H]β-alanine was studied in cultures of rat central nervous system using autoradiography. In brain stem and spinal cord cultures, both amino acids were taken up by neurones and glial cells. In cerebellar cultures, [³H]taurine was accumulated by all glial cells and by a small number of neurones, whereas [³H]β-alanine was only taken up by glial elements. The uptake of both amino acids was sodium and temperature dependent, indicating an active transport mechanism.The results provide further support for the hypothesis that taurine and β-alanine are neurotransmitters in the mammalian central nervous system.     

Mechanisms of glial retraction in the hypothalamo-neurohypophysial system of the rat

It has been known for more than twenty years that changes in glial coverage of magnocellular neurones in the hypothalamo-neurohypophysial system accompany activation of those neurones. This led to the so-called 'glial retraction hypothesis.'However, until recently, little has been established as to how this structural plasticity of astrocytes develops. This paper will explore a number of hypotheses and supporting data concerning these changes.     

Differences in transductional tropism of adenoviral and lentiviral vectors in the rat brainstem

Adenoviral vectors (AVVs) and lentiviral vectors (LVVs) are highly useful research tools which can be used to investigate the function of specific cell phenotypes in the brain. The transductional tropism of viral vectors has a critical impact upon the transgene expression in different brain areas. This largely depends on the properties of the viral particles, which for AVVs are most commonly analogous to the serotype 5 adenovirus and, in the case of LVVs, are determined by the envelope used for pseudotyping, for example the vesicular stomatitis virus coat (VSVG). We have created a matching set of shuttle plasmids that allow a one-step transfer of an entire expression cassette between the backbones of AVVs and LVVs. This has permitted a fair assessment of the impact of the vector type on tropism for both AVVs and LVVs. Thus, the aims of this study were twofold: (i) to develop and demonstrate the validity of a transgene 'swap' system between AVVs and LVVs; and (ii) using this system, to assess the tropism of AVVs and LVVs for neuronal versus glial cell types. We have constructed AVVs and VSVG-coated LVVs to express monomeric red fluorescent protein (mRFP) driven by the human cytomegalovirus promoter (hCMV). Transgene expression in neurones and glia in the hypoglossal and dorsal vagal motor nuclei of the rat brainstem was compared by determining the colocalization with immunostaining for the neuronal marker NeuN (neuronal nuclear antigen) and the glial marker GFAP (glial fibrillatory acidic protein). We found that 55% of mRFP-expressing cells transduced with AVVs were immunopositive for GFAP, while only 38% were NeuN-immunoreactive. In contrast, when the same expression cassette was delivered by VSVG-coated LVVs, the neurone/glia ratio of mRFP expression was reversed with 56% of mRFP-positive cells identified as neurones and 26% as glia. Thus, the present study provides compelling evidence that VSVG-coated LVVs significantly shift transgene expression towards neurones while transduction with AVVs favours glia.     

Growth hormone as a neuronal rescue factor during recovery from CNS injury.

There is growing evidence to suggest that growth hormone plays a role in the growth and development of the CNS. Specifically, growth hormone has been implicated in promoting brain growth, myelination, neuronal arborisation, glial differentiation and cognitive function. Here we investigate if growth hormone has a role in the recovery from an unilateral hypoxic-ischaemic brain injury. Using moderate (15 min hypoxia) and severe (60 min hypoxia) models of hypoxic-ischaemia in juvenile rats and standard immunohistochemical techniques, we found intense growth hormone-like immunoreactivity present within regions of cell loss by 3 days (P<0.05). Growth hormone-like immunoreactivity was observed on injured neurones, myelinated axons, glial cells within and surrounding infarcted tissue and on the choroid plexus plus ependymal cells within the injured hemisphere. The pattern of immunoreactivity suggests that (a) growth hormone (or a growth hormone-like substance) is transported via the cerebrospinal fluid and (b) that growth hormone (or a growth hormone-like substance) is acting in a neurotrophic manner specifically targeted to injured neurones and glia.To test this hypothesis we treated a moderate hypoxic-ischaemic brain injury with 20 microg of rat growth hormone by intracerebroventricular infusion starting 2 h after injury (n=12/group). After 3 days the animals were killed and the extent of neuronal loss quantified. Growth hormone treatment reduced neuronal loss in the frontoparietal cortex (P<0.001), hippocampus (P<0.01) and dorsolateral thalamus (P<0.01) but not in the striatum. This spatial distribution of the neuroprotection conveyed by growth hormone correlates with the spatial distribution of the constitutive neural growth hormone receptor, but not with the neuroprotection offered by insulin-like growth factor-I treatment in this model. These results suggest that some of the neuroprotective effects of growth hormone are mediated directly through the growth hormone receptor and do not involve insulin-like growth factor-I induction.In summary, we have found that a growth hormone-like factor increased in the brain in the days after injury. In addition, treatment with growth hormone soon after an hypoxic-ischaemic injury reduced the extent of neuronal loss. These results further suggest that a neural growth hormone axis is activated during recovery from injury and that this may act to restrict the extent of neuronal death.     

Structural plasticity in the hypothalamic supraoptic nucleus at lactation affects oxytocin-, but not vasopressin-secreting neurones

Magnocellular neurones in the supraoptic nucleus of the hypothalamus are usually separated by neuropil and glial elements. In lactating animals, however, the surface membranes of many neurosecretory somata and dendrites are frequently in direct apposition, without any glial interposition. A significant number of such neurones are also bridged by the same presynaptic terminal ("double synapses"). As the supraoptic nucleus is composed of two types of neurosecretory cell, secreting either oxytocin or vasopressin, we carried out comparative quantitative analyses on identified supraoptic neurones of virgin and lactating rats to determine which neurones were affected by the structural changes and to what extent. The neurones were identified in: (i) normal and Brattleboro homozygote rats by electron microscopic immunocytochemistry (pre- and post-embedding procedures) using antisera raised against oxytocin, vasopressin and oxytocin-related neurophysin I, and (ii) in homozygous Brattleboro rats by their neuronal content of approximately 170 nm neurosecretory granules. We report here that, in virgin animals under normal conditions, a small proportion of both types of neurone show neuronal appositions. At lactation, neuronal appositions are far more numerous and extensive, as are "double synapses". These changes affect exclusively the oxytocinergic neurones. The increased appositions cannot result solely from glial retraction because the hypertrophied oxytocin cells have a greater absolute, though smaller proportional, coverage by glial processes than cells in the control animals. From the present observations, and those obtained in chronically dehydrated animals (see accompanying article), it is clear that the plastic changes in the supraoptic nucleus are closely related to the activity of its oxytocinergic neurones. During lactation, these structural modifications may serve to facilitate and maintain the characteristic synchronized electrical activity of these neurones at milk ejection. On a few occasions, we also found appositions between one oxytocinergic and one vasopressinergic neurone, which may account for the rare cases of electrophysiological interactions between the two types of cell.     

Osmotic stimulation causes structural plasticity of neurone-glia relationships of the oxytocin but not vasopressin secreting neurones in the hypothalamic supraoptic nucleus

A comparative quantitative analysis was carried out on identified supraoptic neurones of male and female Wistar and Long Evans rats under normal conditions and after chronic osmotic stimulation, and in homozygous Brattleboro rats suffering from diabetes insipidus. The neurones were identified by immunocytochemical or morphological means. Osmotic stimulation resulted in significant increases in the number and extent of direct neuronal appositions and in the number of presynaptic terminals contacting two neurosecretory cells simultaneously ("double" synapses). In the supraoptic nuclei of both sexes these increases were restricted to the oxytocin secreting neurones. In Brattleboro homozygous rats treated with vasopressin, the proportion of oxytocinergic neurones in apposition was not modified, but the number of appositions per soma profile decreased as did the incidence of "double" synapses. In nuclei of osmotically stimulated rats, increase in cell volume affected both types of neurosecretory cell and was accompanied by an increase of the absolute extent of glial coverage. However, the extent of glial coverage of the oxytocinergic neurones did not match the hypertrophy of the cells, resulting in a decrease in their relative glial coverage, compared to normal hydrated animals. The increased neuronal appositions, therefore, cannot result simply from a retraction of glial processes. The structural reorganization of the oxytocinergic system observed during chronic osmotic stimulation was as extensive as that observed at lactation. Moreover, the changes were as extensive in Wistar as in Brattleboro lactating rats, although the latter have an added osmotic stimulus. This implies that lactation and osmotic stimulation do not produce additive effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in extracellular potassium concentration produced by neuronal activity in the central nervous system of the leech

1. Experiments were made on single neurones and glial cells in the central nervous system of the leech to study the accumulation of K that occurs in the extracellular spaces around neurones as a result of impulse activity.2. The resting potential of a neurone is too insensitive to be used for the estimation of small changes in K concentration. The undershoot of the action potential, however, provided a reliable indicator of the K accumulation that occurs around a neurone during activity.3. After a single impluse the amplitude of the undershoot of a second action potential was decreased; the effect corresponded to a peak increase in K concentration of about 0.8 mM/l. immediately after the spike and declined exponentially with a time constant of about 100 msec. With trains of impulses the K concentration increased exponentially, again with a time constant of about 100 msec. The final value of K depended on the frequency and could build up to about double the normal concentration of 4 mM/l.4. The build-up of K was markedly reduced when the extracellular space surrounding a neurone was enlarged by removing its glial investment.5. Synchronous, repetitive activation of groups of neurones caused a slow depolarization of neighbouring glial cells in the C.N.S. of the leech, similar to that observed in amphibia and mammals. The change in glial membrane potential was also used to estimate the changes in K concentration and these values agreed with measurements derived from the undershoot.6. Increases of K concentration in the bathing fluid of the same order as those caused by neural firing markedly affected the frequency of ;spontaneous' neuronal discharges and synaptic potentials occurring within certain neurones in the C.N.S.7. The possible effects of physiologically occurring increases of K concentration on integration are discussed.     

Loss of function of glial gap junctions may cause severe cognitive impairments in schizophrenia

A generalized cognitive deficit is at the core of schizophrenia. It is hypothesized that a loss of function of glial gap junctions may cause severe cognitive impairment in schizophrenia. Glial gap junctions are electrical channels that may register the neuronal activation frequencies of glial–neuronal compartments by generating gap junction plaques. The various proteins (connexins) of gap junctions may be capable to differentiate between the operation qualities of the cognate synapses defined by the neurotransmitter types. Thus, the brain is capable of distinguishing between different cognitive qualities (domains or categories). If the function of glial gap junction proteins is lost, the brain is incapable to distinguish between same and different qualities of information processing. Dependent on the brain regions affected, this disorder may be responsible for severe cognitive impairment in schizophrenia. Finally, general approaches for testing the hypothesis are outlined.     

Erythropoietin protects against apoptosis and increases expression of non-neuronal cell markers in the hypoxia-injured developing brain

Erythropoietin (EPO) is a cytokine hormone with cytoprotective effects in many tissues including the brain. Although the benefits of administration of recombinant human EPO (rhEPO) for neonatal hypoxic brain injury have been demonstrated in neuronal tissue, the effect on non-neuronal cell populations is unclear. We tested the hypothesis that rhEPO would not only protect neuronal cells but also glial cells at a stage of brain development where their maturation was particularly sensitive, and also protect the vasculature. This was evaluated in a rat model of hypoxic injury. 1000 IU/kg rhEPO was delivered intraperitoneally at the start of 4 h hypoxia or normoxia. Treatment groups of neonatal rats (day of birth, at least N = 10 per group) were as follows: normoxia; normoxia plus rhEPO; hypoxia (8% FiO(2) delivered in temperature-controlled chambers); and hypoxia plus rhEPO. Day of birth in rats is equivalent to human gestation of 28-32 weeks. The effects of rhEPO administration, especially to non-neuronal cell populations, and the associated molecular pathways, were investigated. Apoptosis was increased with hypoxia and this was significantly reduced with rhEPO (p < 0.05). The neuronal marker, microtubule-associated protein-2, increased in expression (p < 0.05) when apoptosis was significantly reduced by rhEPO. In addition, compared with hypoxia alone, rhEPO-treated hypoxia had the following significant protein expression increases (p < 0.05): the intermediate filament structural protein nestin; myelin basic protein (oligodendrocytes); and glial fibrillary acidic protein (astrocytes). In conclusion, rhEPO protects the developing brain via anti-apoptotic mechanisms and promotes the health of non-neuronal as well as neuronal cell populations at a time when loss of these cells would have long-lasting effects on brain function.     

Autoradiographic evidence for endothelin receptors on astrocytes in cultures of rat cerebellum, brainstem and spinal cord.

The cellular localization of binding sites for 125I-endothelin-1 and -3 (ET-1, ET-3) was studied in explant cultures of rat cerebellum, brain stem and spinal cord by means of autoradiography. The majority of astrocytes in these cultures expressed binding sites for both ET-1 and ET-3. There was a difference in the intensity of labelling between glial cells in the same culture. Some cells revealed intense radioactivity whereas neighbouring astrocytes were only slightly labelled. Besides glial cells, cerebellar neurones presumably Purkinje cells and granule cells as well as medium-sized and large neurones in brain stem and spinal cord cultures showed binding sites for both peptides. Our results provide strong evidence for the existence of ET receptors on astrocytes.     

Postnatal development of the dorsal lateral geniculate nucleus in the normal and enucleated albino mouse

Summary Quantitative data (numbers of neurones and glial cells, total volumes, internuclear volumes) were obtained during normal development and after bilateral and unilateral enucleation at birth in the dorsal lateral geniculate nucleus (LGN_d) of the mouse, at 5, 10, 30, 60, and 180 days postnatal.During normal development there is a neuronal loss of about 30% up to 30 days, at which age the total number of neurones stabilises at around 17,000. Glial proliferation and an increase in the volume of LGN_d continues at least to 180 days.More severe degenerative effects were found after bilateral than after unilateral enucleation. At 180 days, bilateral enucleation leads to a neuronal loss of 27% compared to the controls, with a glial deficit of 53% and a decrease in the volume of LGN_d of 57%.Degenerative effects were very different in LGN_d contralateral or ipsilateral to enucleation in monocularly enucleated mice, due to the extensive crossing of the retinal fibres. At 180 days, we found a deficit of 10% in the numbers of neurones and glial cells, in the ipsilateral LGN_d compared to normal: the volume of LGN_d was slightly less (3%) than in controls. The contralateral LGN_d after unilateral enucleation behaved like LGN_d after bilateral enucleation until 60 days. At 180 days, some minor modifications were found, showing an additional neuropil decrease of 13% and an additional neuronal loss of 6% in the bilaterally enucleated LGN_d compared to the unilaterally enucleated contralateral LGN_d.The time-course of degeneration both after bilateral and unilateral enucleation was discussed.     

Cellular orientation of atrial natriuretic peptide in the human brain

Many peptide hormones and neurotransmitters have been detected in human neuronal tissue. The localisation of atrial natriuretic peptide (ANP) in the human brain was considered to be both interesting and relevant to the understanding of neurochemistry and brain water–electrolyte homeostasis. This vasoactive peptide hormone has been localised in rat and frog neuronal tissue. In the present study, we report the immunohistochemical localisation of ANP in autopsy samples of human brain tissue employing the avidin–biotin–peroxidase complex technique, using an antibody against a 28 amino acid fragment of human ANP. The most intense staining of immunoreactive ANP was detected in the neurones of preoptic, supraoptic and paraventricular nuclei of the hypothalamus, epithelial cells of the choroid plexus and ventricular ependymal lining cells. Immunoreactive neurones were also observed in the median eminence, lamina terminalis, infundibular and ventromedial nuclei of the hypothalamus, and in neurones of the brain stem, thalamic neurones and some neurones of the caudate nucleus. The network of ANP cells in numerous hypothalamic centres may regulate the salt and water balance in the body through a hypothalamic neuro-endocrine control system. ANP in the brain may also modulate cerebral fluid homeostasis by autocrine and paracrine mechanisms.     

Is neuronal communication with NG2 cells synaptic or extrasynaptic?

NG2-expressing glial cells (NG2 cells) represent a major pool of progenitors able to generate myelinating oligodendrocytes, and perhaps astrocytes and neurones, in the postnatal brain. In the last decade, it has been demonstrated that NG2 cells receive functional glutamatergic and GABAergic synapses mediating fast synaptic transmission in different brain regions. However, several controversies exist in this field. While two classes of NG2 cells have been defined by the presence or absence of Na(+) channels, action potential firing and neuronal input, other studies suggest that all NG2 cells possess Na(+) conductances and are the target of quantal neuronal release, but are unable to trigger action potential firing. Here we bring new evidence supporting the idea that the level of expression of Na(+) conductances is not a criterion to discriminate NG2 cell subpopulations in the somatosensory cortex. Surprisingly, recent reports demonstrated that NG2 cells detect quantal glutamate release from unmyelinated axons in white matter regions. Yet, it is difficult from these studies to establish whether axonal vesicular release in white matter occurs at genuine synaptic junctions or at ectopic release sites. In addition, we recently reported a new mode of extrasynaptic communication between neurones and NG2 cells that relies on pure GABA spillover and does not require GABAergic synaptic input. This review discusses the properties of quantal neuronal release onto NG2 cells and gives an extended overview of potential extrasynaptic modes of transmission, from ectopic to diffuse volume transmission, between neurones and NG2 cells in the brain.     

Postnatal development of the visual cortex of the mouse after enucleation at birth

Summary Quantitative data in the neocortex up to the age of 180 days (neuronal densities, number of neurones, glial cells, dendritic intersections and spines) were compared in normal mice and mice enucleated at birth.Bilateral enucleation induced an increase of neuronal density in all cortical layers of areas 17, 18a, and 41, the supragranular layers II–III being more affected than layers IV–VI. This was noticed in layer II 10 days after the operation and was maximal in all layers between 30 and 60 days; at 180 days there was some return to normal of the neuronal density in all layers. The total number of neurones and glial cells were the same in the bilaterally enucleated mice as in the controls. No reaction in dendritic branching was evident for pyramids of layers III and V in areas 17 and 41 after bilateral enucleation. In contrast the number of spines was reduced on the apical dendrites of pyramids from layers III and V in area 17, but not in area 41.After unilateral enucleation the reaction was less severe and delayed compared with bilateral enucleation, the first signs of increase of neuronal density appearing 30 days after the lesion in the contralateral hemisphere. The contralateral areas 17 and 18a were more affected than the ipsilateral ones and area 41 showed no change compared to the control. As after bilateral enucleation, layers IV and V were least affected by unilateral enucleation in both ipsi- and contralateral cortices.These results suggest that deafferentation in an immature system affects the development of all cortical layers but with a greatest intensity in supragranular layers, which are not the main direct targets of thalamo-cortical input.Supported by the Swiss National Research Foundation, Grants no. 3-641-71 and 3-434-74     

Autoradiographic localization of 3H-gamma-aminobutyric acid in the medial hypothalamus

Summary Tritium labelled gamma-aminobutyric acid (³H-GABA) was infused into the third ventricle of rats with normal or deafferented hypothalamus and the distribution of the label was studied by light and electron microscopic autoradiography.In control as well as deafferented hypothalamus a few neurones accumulated radioactivity, while the majority was unlabelled. Characteristic clusters and rows of silver grains were observed in the neuropil of several regions probably indicating labelled cell processes and terminal axons. Electron microscopy showed that at least some of the clusters were over axon terminals with synaptic vesicles. ³H-GABA accumulated also in the ependyma and glial elements.The results suggest that in the medial hypothalamus there is a preferential uptake of GABA in some neurones and nerve fibers; at least some of these are hypothalamic interneurones. This supports the hypothesis that some hypothalamic neurones and nerve endings may use GABA as a transmitter.     

Long‐term changes in the ipsilateral substantia nigra after transient focal cerebral ischaemia in rats

Transient focal cerebral ischaemia can cause neuronal damage in remote areas, including the ipsilateral thalamus and subsutantia nigra, as well as in the ischaemic core. In the present study, we investigated long‐term changes in the ipsilateral substantia nigra from 1 up to 20 weeks after 90 min of transient focal cerebral ischaemia in rats, using tyrosine hydroxylase (TH), neuronal nuclei (NeuN), Iba‐1, glial fibrillary acidic protein (GFAP) and brain‐derived neurotrophic factor (BDNF) immunostaining. These results show that transient focal cerebral ischaemia in rats can cause a severe and prolonged neuronal damage in the ipsilateral striatum. Our results with TH and NeuN immunostaining also demonstrate that the atrophy of the ipsilateral substantia nigra after transient focal cerebral ischaemia was not static but progressive. Furthermore, our double‐labelled immunohistochemical study suggests that BDNF released by GFAP‐positive astrocytes may play a key role in the survival of dopaminergic neurones in the ipsilateral substantia nigra at the chronic stage after transient focal cerebral ischaemia, although the areas of the ipsilateral substantia nigra are decreased progressively after ischaemia. Thus our study provides further valuable information for the pathogenesis of neuronal damage after transient focal cerebral ischaemia.     

Gliomas and seizures

Glial neoplasms account for nearly 50% of all adult primary brain tumors. They originate from glial cells in the brain and/or spinal cord and include low-grade diffuse astrocytomas, anaplastic-astrocytomas, and glioblastomas. Of all brain tumors, glioblastoma multiforme (GBM) is the most aggressive and is characterized by rapid glial cell growth, resistance to radio- and chemo- therapies, and relentless infiltration and spreading throughout the central nervous system (CNS). In glioblastomas, primary tumor growth and CNS invasion are associated with the activation of complex structural molecular and metabolic changes within the tumor tissue, which profoundly affect the surrounding neuronal networks and may in part explain induction of epilepsy. In fact, epileptic seizures are very common among patients with glial tumors, reaching nearly 50% in glioblastoma patients and almost 90% in low-grade astrocytomas. The overall hypothesis presented here discusses the possibility that the aberrant tumor cell metabolism may act directly on neuronal network, and this leads to seizure susceptibility. Further invasion and growth of the malignant glial cells exacerbate this initial pathologic state which promotes recurrent seizures (epileptogenesis).