Apoptotic and necrotic cell death following kindling induced seizures.

The study was designed to determine which type of cell death occurs following kindling induced seizures, and to determine which neurons die. For this purpose seizures were kindled from the entorhinal cortex. Following a range of 5-85 stage 5 seizures, rats were sacrificed, and the tissue was prepared for analysis. The TUNEL and silver impregnation methods were used to identify apoptotic or necrotic cell death, respectively. These methods were subsequently combined with immunocytochemistry, to determine if diseased neurons expressed somatostatin or the NMDA receptor (NMDAR1). The tissue analysis demonstrated that following kindling induced seizures, 1) hippocampal and extrahippocampal neurons die, 2) some neurons die through apoptosis, others through necrosis, and 3) some of the diseased neurons express somatostatin, others the NMDAR1 and that both subpopulations of neurons are present at hippocampal and extrahippocampal sites.

Effects of generalized convulsive seizures on corticotropin-releasing factor neuronal systems.

Most stressors generate a set of endocrine and neural adaptations that form a stress response. The corticotropin-releasing factor neurons of the paraventricular nucleus of hypothalamus integrate endocrine and neural inputs, and cause a cascade of events with resultant increased levels of pituitary adrenocorticotropic hormone and adrenal hormones. Although activation of the hypothalamic-pituitary-adrenal axis is associated with a large variety of stressors, the effects of seizures on hypothalamic corticotropin-releasing factor neurons are essentially unknown. The goal of the present study was to elucidate the effects of generalized convulsive seizures on distinct and separate corticotropin-releasing factor cell populations in brain. Seizure-activated neurons were identified immunocytochemically through their expression of the Fos protein. Seizures were induced by intraperitoneal injection of kainic acid. In the paraventricular nucleus, the vast majority of corticotropin-releasing factor-like parvocellular neurons also expressed Fos-like protein following seizure elicitation. This response was specific to corticotropin-releasing factor neurons of the paraventricular nucleus, as corticotropin-releasing factor neurons in central nucleus of the amygdala or bed nucleus of the stria terminalis did not simultaneously localize Fos following seizures.

Activation of oxytocin-containing neurons of the paraventricular nucleus (PVN) following generalized seizures.

Due to the complex nature of generalized limbic seizures, marked disturbances in physiological homeostasis occur. Accompanying the motor manifestations which characteristically are associated with generalized limbic seizures, alterations in neuroendocrine, behavioral, and autonomic functions may be observed. The paraventricular nucleus (PVN) of the hypothalamus is known to play a significant role in such neuronal responses to stressful stimuli; however, the effect of seizures on hypothalamic neurons is unknown. We have used the immunocytochemical detection of the Fos protein to anatomically identify neurons in the PVN which are activated following generalized limbic seizures. To induce seizures, rats received intraperitoneal injections of kainic acid or were kindled from the entorhinal cortex. We have demonstrated that elicitation of generalized limbic seizures induces a dramatic number of neurons in the PVN to express the Fos protein. Numerous Fos-immunolabeled neurons were identified in both the parvicellular and magnocellular component of the PVN. In the latter, this study clearly reveals a preferential and selective activation of oxytocin-containing neurons, and it extends and supports the hypothesis that oxytocin plays a role in the body's response to specific stress paradigms. Data suggest that an activation of the oxytocin neuronal system may be part of the adaptive mechanism that enables the hypothalamus to modulate and maintain an adequate response to stressors (e.g., generalized seizures) to regain homeostasis.

Oxytocin and vasopressin mRNA expression in rat hypothalamus following kainic acid-induced seizures.

In this study, the regulation of hypothalamic oxytocin and vasopressin messenger RNA expression following the induction of seizures was investigated by in situ hybridization. Following kainic acid-induced seizures, a significant increase in oxytocin messenger RNA in the paraventricular nucleus was demonstrated at 1.5 h, one and two weeks; its level decreased at three weeks and was significantly increased again at four weeks; at eight weeks the messenger RNA level still remained higher than that of controls. Vasopressin messenger RNA in the paraventricular nucleus was increased significantly only at 1.5 h following induction of seizures. The oxytocin messenger RNA level in the supraoptic nucleus was also increased early at 1.5 h and later at four weeks following seizures; however, these increases did not last as long as those in the paraventricular nucleus. Vasopressin messenger RNA in the supraoptic nucleus was also increased after the initial seizures; however, its messenger RNA level vacillated up and down throughout the post-seizure times studied. The earliest significant increase of vasopressin messenger RNA was at one week after seizures, and there was a late significant increase of vasopressin messenger RNA at three weeks after seizures. The present study demonstrates that following kainic acid-induced seizures both, the oxytocin and vasopressin messenger RNA expressions, were up-regulated and these up-regulations were long-term events. The increase of oxytocin messenger RNA in the paraventricular nucleus was more persistent than the others. The pattern of messenger RNA up-regulation was different for oxytocin and vasopressin, and different in the paraventricular nucleus and supraoptic nucleus. These different patterns of messenger RNA elevations suggest that the different components of the rat hypothalamus were regulated differentially by kainic acid-induced seizures.

The kindling-activated neuronal network: recruitment of somatostatin-synthesizing neurons.

The present study demonstrates the anatomical extent of the kindling-activated neuronal network in general, and specifically the recruitment of extrahippocampal somatostatin (SST)-synthesizing neurons into this network. It has been known that SST neurons of the hippocampal formation are activated during episodes of seizure, however, it was not known if this activation was a local event or extended to other areas in the brain. We were therefore interested in determining if and which SST neurons outside the hippocampal formation might be recruited into this seizure-activated neuronal network. Using the kindling model of seizure elicitation, expression of the Fos protein in activated, depolarized neurons was utilized to identify seizure-activated neurons. Subsequently, the mRNA for SST was identified through in situ hybridization in the same tissue section, allowing the identification of seizure-activated, SST-synthesizing neurons. The results show that: (a) the majority of SST-synthesizing neurons in the forebrain and diencephalon became activated during the kindling development; (b) their recruitment into the kindling-activated neuronal network occurred progressively; and, (c) these SST-synthesizing neurons represented a component of the kindling-activated neuronal network throughout the development of kindling-induced seizures.

The substantia nigra pars reticulata, seizures and Fos expression.

The induction of the proto-oncogene c-fos has been used extensively to identify spatially distributed neural systems activated by seizures. The substantia nigra pars reticulata (SNpr) has been implicated as a critical structure in neural networks involved in the modulation of seizure expression, yet the SNpr has not been reported to express Fos following seizures induced in a variety of seizure paradigms. In this study we determined whether (1) the temporal characteristics of Fos induction in the SNpr were different than those of other brain areas following kindled seizures, (2) neurons in the SNpr possess the cellular machinery to express Fos, (3) Fos can be induced in SNpr by direct electrical stimulation, and (4) Fos expression is induced in the SNpr following kainate or pilocarpine-induced status epilepticus. Results indicate that Fos is not induced in SNpr at any time point (1-12 h) after kindled seizures, and that serum response factor, a constitutively expressed nuclear protein necessary for Fos expression, is present in SNpr neurons. Results further indicate that Fos expression in the SNpr is induced following either direct electrical stimulation or pilocarpine status, but not status elicited by kainate. We conclude that, in so far as the SNpr represents a critical structure for modulating seizure expression, seizure activity does not represent a sufficient stimulus to induce Fos in SNpr neurons. Further, the neural networks defined by Fos expression following seizure may be incomplete, and should be interpreted conservatively.

Activation of somatostatin-synthesizing neurons in the hippocampal formation through kindling-induced seizures.

The present study was designed to determine if and to what extent somatostatin (SST) synthesizing neurons of the hippocampal formation are activated during seizures, elicited through kindling of the perforant pathway. Tissue was used and analyzed from animals which had experienced a single after discharge, or a stage 3 or stage 5 seizure. The protein expression of the oncogene c-fos in activated, depolarizing neurons was utilized to identify seizure-activated SST-synthesizing neurons. Combined immunocytochemical and in situ hybridization methods were used to identify these double-labeled, Fos protein, and SST mRNA-containing neurons. The results were quantified and compared across seizure stages. The resulting data demonstrate that at every stage of seizure development, a majority of SST-synthesizing neurons is activated, but that these activated SST mRNA-containing neurons represent only a minority of all seizure-activated, Fos-expressing neurons in the hippocampal formation. The data further reveal a numerical hierarchy in which the majority of double-labeled neurons is present in the hilus of the dentate, followed by the stratum oriens of CA1. It is concluded that SST-synthesizing neurons represent an integral component of the kindling activated neuronal network and, since the SST synthesizing neurons represent the minority of all seizure-activated neurons in the hippocampal formation, that this neuronal network is likely to be of considerable neurochemical complexity.

Seizure-induced activation of enkephalin- and somatostatin-synthesizing neurons.

The extent of the neuronal network that is activated by kainic acid-induced seizures was anatomically identified and neurochemically characterized. Seizure-activated neurons were identified through the immunocytochemical demonstration of Fos protein in neuronal nuclei. These seizure-activated neurons were characterized by determining if they contained the mRNA for somatostatin or enkephalin, using in situ hybridization procedures. The results demonstrate that a majority of enkephalin- and somatostatin-synthesizing neurons expressed the Fos protein following seizures and that they represent a major component of the kainic acid-induced, seizure-activated neuronal network.

Enkephalin, substance P, and serotonin axonal input to c-fos-like immunoreactive neurons of the rat spinal cord.

Mustard oil, which stimulates small diameter afferents, was used to evoke the expression of the oncogene c-fos in the lumbar spinal cord. C-fos-like immunoreactivity was concentrated in, but not limited to, neuronal nuclei of laminae I and II of the lumbar dorsal horn. Double-label immunocytochemistry was used to determine if neurons which expressed c-fos-like immunoreactivity received axonal input from enkephalin-, substance P- or serotonin-immunoreactive neurons. The analysis of vibratome and semithin plastic-embedded tissue sections demonstrated that the majority of c-fos-like immunoreactive neurons received input from enkephalin-, substance P- or serotonin-immunoreactive axonal varicosities.

ACTH and enkephalin axonal input to paraventricular neurons containing c-fos-like immunoreactivity.

The presence of c-fos like protein in neuronal nuclei has been observed in several areas of the central nervous system. It is associated with activation of these neurons by specific stimuli, in particular stressful stimuli. The present study investigated the expression of c-fos-like immunoreactivity in the paraventricular nucleus (PVN) of the hypothalamus following stimulation of small diameter, nociceptive afferents in the hindfoot of the rat. The afferent innervation to these c-fos containing PVN neurons was examined in order to identify putative neurotransmitters which might modulate the activity of stress responsive, i.e. c-fos containing neurons in the PVN. Adrenocorticotropic releasing hormone (ACTH), enkephalin (ENK), and corticotropin releasing factor (CRF), peptides whose functions have been related to the regulation of stress behavior, were selected to investigate their potential innervation of c-fos immunolabeled neurons. Analysis of immunocytochemically double-labeled vibratome and semi-thin plastic-embedded sections revealed that ACTH and ENK immunoreactive axonal varicosities were present in close anatomical proximity to a substantial number of parvocellular PVN neurons that contained c-fos in response to noxious stimulation. Few c-fos containing PVN neurons were apposed by CRF axonal varicosities. The resulting data show that a large number of c-fos immunoreactive PVN neurons, though not the majority, is innervated by ACTH and ENK. This suggests that the activity of stress responsive, c-fos expressing neurons can substantially be modulated and regulated by ACTH and ENK and to a lesser degree by CRF.

Coexistence of CRF peptide and oxytocin mRNA in the paraventricular nucleus.

Several studies have reported coexistences of peptides in parvocellular neurons of the paraventricular nucleus (PVN). However, the coexistence of peptides in the magnocellular PVN is less clear. Controversy exists in particular about the coexistence of corticotropin-releasing factor (CRF) and oxytocin (OX). Although these peptides are present in distinct areas of the PVN, some overlap may exist. This study investigated a potential coexistence of OX and CRF in magno- and parvocellular PVN. The data demonstrate with clarity that neurons containing both the mRNA for OX and the peptide CRF are present in subpopulations of magnocellular and parvocellular neurons of the PVN.

Coexistence of corticotropin-releasing factor and enkephalin in the paraventricular nucleus of the rat.

Corticotropin-releasing factor and enkephalin-containing neurons are found in the paraventricular nucleus of the rat hypothalamus. Their immunocytochemical distribution suggests that a subpopulation of neurons of the paraventricular nucleus might contain both peptides. The present study describes the coexistence of corticotropin-releasing factor and enkephalin in parvicellular neurons of the paraventricular nucleus. Immunocytochemical labeling of peptides was combined with in situ hybridization of mRNA to visualize peptide and mRNA labeling in the same tissue section. This resulted in several observations: (1) neurons labeling for the peptide corticotropin-releasing factor also contain the mRNA to synthesize corticotropin-releasing factor, (2) neurons labeling for the peptide enkephalin also contain the mRNA to synthesize the peptide enkephalin, (3) a subpopulation of corticotropin-releasing factor neurons contains the mRNA to synthesize enkephalin, and (4) a subpopulation of enkephalin neurons contains the mRNA to synthesize corticotropin-releasing factor. A large percentage of enkephalin immunoreactive neurons contains corticotropin-releasing factor mRNA, whereas a smaller percentage of corticotropin-releasing factor immunoreactive neurons contains enkephalin mRNA. These double-labeled neurons are present throughout the rostral-caudal extent of the paraventricular nucleus; the majority of these neurons is present in the medial parvicellular paraventricular nucleus.

Mediation of changes in paraventricular vasopressin and oxytocin mRNA content to the medullary vagal complex and spinal cord of the rat.

Retrograde tracing was combined with in situ hybridization to demonstrate that a small percentage of neurons of the paraventricular hypothalamic nuclei (PVN) which project to the spinal cord or the medullary vagal complex contain the mRNA to produce the peptides vasopressin or oxytocin. These projection neurons respond to salt loading with an upregulation of mRNA for these peptides. The present study provides an anatomical basis for a direct regulatory influence of PVN neurons on preganglionic neurons in the spinal cord and medulla.

Electrical stimulation of the rat ventral midbrain elicits antinociception via the dorsolateral funiculus.

The pain-suppressive effects of focal electrical stimulation of sites throughout the ventral midbrain were examined in awake rats. Chronic bipolar electrodes were implanted in medial and lateral regions of the midbrain. Current thresholds for suppression of the tail-flick reflex in response to noxious heat were determined for both a biphasic and a monophasic stimulation parameter at each site. Stimulation of areas throughout the ventral midbrain produced tail-flick suppression (TFS), but no one area was consistently effective in all animals. Monophasic and biphasic stimulation were qualitatively equal in the duration of TFS and the distribution of effective sites. The production of TFS was not correlated with other behavioral reactions to brain stimulation. TFS appeared to be mediated by non-opiate pathways since naloxone administration (10 mg/kg) had no discernible effect on the production of TFS. The current threshold for producing TFS was extremely variable over both short (one half hour) and long (one week) intervals. The incidence of TFS from previously effective sites was significantly less following bilateral dorsolateral funiculus (DLF) lesions, indicating that the antinociceptive effects of ventral midbrain stimulation are mediated by this spinal pathway.

Distribution of GAD-immunoreactive neurons in the first (SI) and second (SII) somatosensory cortex of the monkey.

The distribution of neurons immunoreactive for glutamic acid decarboxylase (GAD), the synthesizing enzyme of gamma-aminobutyric acid (GABA), was examined in the first (SI) and second (SII) somatosensory cortex of monkeys. GAD-like immunoreactive (GAD-LI) somata and puncta were present in all layers of SI and SII. All GAD-LI somata were identified as non-pyramidal neurons and were most numerous in layer IV of SI and in layer III of SII. Layer IV of SI also contained the highest density of GAD-LI puncta. In SII, GAD-LI puncta were distributed more homogeneously and did not show a dense band of labelled puncta in layer IV. The major and minor diameters of GAD-LI somata in SII ranged from 6.9 to 26.2 micron and from 6.2 to 19.0 micron, respectively. The major diameters of GAD-LI somata in SII were significantly smaller than those in SI in layers I, III and IV. Differences between the distributions of GAD-LI puncta and somata in SI and SII may be accounted for by differences in the number and/or distribution of different types of GABAergic neurons. Functional differences of neurons in SI and SII may be related to the differences in GABAergic inhibitory mechanisms and reflected in the distribution of GABAergic neurons.

Immunocytochemical analysis of noradrenaline, substance P and enkephalin axonal contacts on serotonin neurons in the caudal raphe nuclei of the cat.

The present study investigates the anatomical basis for interactions between serotonin immunoreactive neurons in nuclei raphe magnus and pallidus, and either noradrenaline, substance P (SP) or enkephalin immunoreactive axonal varicosities. Using a double-label immunocytochemical method, we found that each of these neurochemicals could be localized to axons which contacted serotonin immunoreactive neurons. The frequency and location of these inputs differed in nuclei raphe magnus and pallidus. SP immunoreactive varicosities formed the greatest number of contacts. These findings suggest that serotonin-containing neurons in the caudal raphe nuclei receive input from multiple putative neurotransmitters.

Inhibition of the responses of cat dorsal horn neurons to noxious skin heating by stimulation in medial or lateral medullary reticular formation.

Responses of single lumbar dorsal horn units to noxious radiant heating (50 degrees C, 10 s) of glabrous footpad skin were recorded in cats anesthetized with sodium pentobarbital and 70% nitrous oxide. The heat-evoked responses of 37/40 units were reduced during electrical stimulation (100 ms trains, 100 Hz, 3/s, 25-600 microA) in the medullary nucleus raphe magnus (NRM) and/or in laterally adjacent regions of the medullary reticular formation (MRF). Inhibition was elicited by stimulation in widespread areas of the medulla, but with greatest efficacy at ventrolateral sites. The magnitude of inhibition increased with graded increases in medullary stimulation intensity. Mean current intensities at threshold for inhibition or to produce 50% inhibition were higher for NRM than for MRF sites. Units' responses to graded noxious heat stimuli increased linearly from threshold (42-43 degrees C) to 52 degrees C. During NRM (5 units) or ipsilateral MRF stimulation (7 units), responses were inhibited such that the mean temperature-response functions were shifted toward higher temperatures with increased thresholds (1.5 degrees and 1 degree C, respectively) and reduced slopes (to 60% of control). Contralateral MRF stimulation had a similar effect in 4 units. Inhibitory effects of NRM and MRF stimulation were reduced (by greater than 25%) or abolished in 4/6 and 5/12 units, respectively, following systemic administration of the serotonin antagonist methysergide. Inhibitory effects from NRM, ipsi- and contralateral MRF were reduced or abolished in 2/9, 4/8 and 6/9 cases, respectively, following systemic administration of the noradrenergic antagonist phentolamine. These results confirm and extend previous studies of medullospinal inhibition and the role of monoamines, and are discussed in terms of analgesic mechanisms.