The induction of c-fos mRNA was assessed using Northern blots and in situ hybridization in adult rats administered hypertonic saline (HS) and isotonic saline (IS). HS induced c-fos mRNA in magnocellular paraventricular nucleus (PVNm), parvocellular paraventricular nucleus (PVNp), supraoptic nucleus (SON), and lamina terminalis (LMT). This occurred within 5 min, peaked at 30-60 min, and disappeared by 180 min. Fos protein, detected using a specific monoclonal antibody, was maximal at 1-2 hr and disappeared 4-8 hr after HS administration. This confirms observations showing that the c-fos gene response is transient even in the presence of a continuing stimulus. In contrast, Fos-like immunoreactivity (FLI), detected using two polyclonal antisera, was observed in PVNm, PVNp, SON, and LMT for 1-24 hr during continuous osmotic stimulation. Moreover, FLI was observable in these structures for 7 d in rats administered HS and allowed to drink water ad libitum beginning 24 hr later. At times greater than 8 hr, FLI presumably represents Fos-related antigens (FRA), proteins immunologically and functionally related to Fos, whose expression is much more prolonged than authentic Fos following the osmotic stimulus. In addition to induction of c-fos expression in regions specifically involved in osmotic regulation, HS injections also induced c-fos in many other forebrain regions. In order to assess the induction of c-fos mRNA due to the "stress" of the injections, rats injected with isotonic saline were compared to uninjected controls. Isotonic saline injections induced c-fos mRNA in the PVNp, anterior hypothalamus, suprachiasmatic nucleus, cingulate gyrus, neocortex, ventral lateral septal nucleus, piriform cortex, hippocampal pyramidal and dentate granule neurons, paraventricular and intralaminar thalamic nuclei, bed nuclei of stria terminalis, cortical and medial amygdaloid nuclei, and other structures. In accord with other work, we interpret this pattern of c-fos expression to result from the stress of handling and injections. Since Fos and FRA probably bind to the promoters of target genes and regulate their expression, they likely mediate biochemical changes in the cells activated by the osmotic and stressful stimuli. Whereas the Fos signal is transient, FRA may act on target genes for the duration of the stimulus or longer.

During the past decade, substantial progress has been made in delineating clinical features of the epilepsies and the basic mechanisms responsible for these disorders. Eleven human epilepsy genes have been identified and many more are now known from animal models. Candidate targets for cures are now based upon newly identified cellular and molecular mechanisms that underlie epileptogenesis. However, epilepsy is increasingly recognized as a group of heterogeneous syndromes characterized by other conditions that co-exist with seizures. Cognitive, emotional and behavioral co-morbidities are common and offer fruitful areas for study. These advances in understanding mechanisms are being matched by the rapid development of new diagnostic methods and therapeutic approaches. This article reviews these areas of progress and suggests specific goals that once accomplished promise to lead to cures for epilepsy.

Dentate granule cells (DGCs) are the principal cell population of the hippocampal dentate gyrus, and granule cells provide the main excitation to the hippocampus proper via their mossy fibers axons. Although it is well established that granule cells express various growth factors and growth factor receptors, the functional effects of growth factors on the normal development and response to injury of granule cells are relatively unknown. To address this question, primary cultures enriched in DGCs were prepared by microdissecting hippocampal slices from neonatal rats and growing dissociated cells in defined media with added nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT4/5), ciliary neurotrophic factor, basic fibroblast growth factor (bFGF), or vehicle. The effects on cell survival and morphology were quantified by studying neuron-specific enolase-immunostained cells at various time points, plating densities, host ages, and growth factor concentrations. BDNF or bFGF significantly increased both neuronal survival and differentiation by 30-80% compared with control cultures. Maximal effects were observed at relatively longer time points (5-12 d), with younger cells (postnatal day 3-5) and at lowest plating densities. Addition of a trkB-IgG fusion protein that blocks the activity of BDNF or NT4/5 inhibited the effects of BDNF and attenuated the differentiation of cells cultured at high plating densities. Furthermore, treatment of cultures with the kinase inhibitor K252b specifically blocked the effects of BDNF, suggesting involvement of trkB (the high-affinity BDNF receptor) in BDNF-induced differentiation. These results show that growth properties of cultured neonatal DGCs are influenced by exogenously applied BDNF or bFGF in a time-, age-, and density-dependent manner. The effect of plating density suggests an endogenous expression of growth factors in these culture conditions, and this is mediated in part by endogenous BDNF acting via a tyrosine kinase receptor. Combined with previous work showing that various growth factors and their receptors are expressed by DGCs, these findings provide strong support for the hypothesis that BDNF and bFGF influence both the growth and development of DGCs in vivo.

The potential role of the nonconstitutive 72-kDa heat-shock protein (HSP72) in selective neuronal vulnerability to ischemia was studied in rats subjected to graded global ischemia. Immunocytochemistry using a monoclonal antibody against HSP72 was performed on tissue collected after 24 hr of reperfusion. The appearance of HSP72 immunoreactivity correlated in a graded fashion with those regions known to be selectively vulnerable in ischemia. That is, HSP72 was induced in only hilar interneurons and CA1 pyramidal cells following brief ischemia. After intermediate durations of ischemia, HSP72 was expressed in the CA3 neurons and cortical layers 3 and 5, and after the longest intervals, HSP72 appeared in dentate granule cells. Heat-shock protein expression preceded cell death (assessed with acid fuchsin staining) in all regions. This temporal profile suggests that the capability of neurons to express HSP72 is unlikely to account for selective vulnerability of different brain regions following ischemia; its role in neuroprotection during ischemic injury in vivo remains unknown.

The inducible 72 kDa heat shock protein (HSP72) has been shown to be protective in non-neuronal cells and neurons in culture, but its function and the control of its expression in the CNS are poorly understood. Although HSP72 is induced in neurons in vivo by neurotoxic compounds that produce seizures and neuronal damage, it is unknown if its expression is a specific response to excitation per se or to "stressful" or potentially injurious excitation, or if it is a marker or mediator of irreversible injury. We have attempted to identify the nature of the stimulus for HSP72 expression by utilizing focal electrical stimulation that can either excite or destroy postsynaptic cells, depending on the duration of afferent stimulation. Previous studies have demonstrated that intermittent stimulation of the main hippocampal afferent pathway for 24 hr evokes synchronous discharges in dentate granule cells but does not injure them. However, the same stimulation irreversibly destroys three of the four cell populations innervated by the granule cells. The three vulnerable populations are the dentate hilar mossy cells, the somatostatin/neuropeptide Y (NPY)-immunoreactive hilar neurons, and the CA3c pyramidal cells. The fourth and relatively resistant population is the GABA-immunoreactive dentate basket cells. In this study, we have localized HSP72 expression immunocytochemically in the hippocampal dentate gyrus in response to nontoxic durations of potentially neurotoxic afferent stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)