Network properties of the dentate gyrus in epileptic rats with hilar neuron loss and granule cell axon reorganization

Neuron loss in the hilus of the dentate gyrus and granule cell axon reorganization have been proposed as etiologic factors in human temporal lobe epilepsy. To explore these possible epileptogenic mechanisms, electrophysiological and anatomic methods were used to examine the dentate gyrus network in adult rats that had been treated systemically with kainic acid. All kainate-treated rats, but no age-matched vehicle-treated controls, were observed to have spontaneous recurrent motor seizures beginning weeks to months after exposure to kainate. Epileptic kainate-treated rats and control animals were anesthetized for field potential recording from the dentate gyrus in vivo. Epileptic kainate-treated rats displayed spontaneous positivities ("dentate electroencephalographic spikes") with larger amplitude and higher frequency than those in control animals. After electrophysiological recording, rats were perfused and their hippocampi were processed for Nissl and Timm staining. Epileptic kainate-treated rats displayed significant hilar neuron loss and granule cell axon reorganization. It has been hypothesized that hilar neuron loss reduces lateral inhibition in the dentate gyrus, thereby decreasing seizure threshold. To assess lateral inhibition, simultaneous recordings were obtained from the dentate gyrus in different hippocampal lamellae, separated by 1 mm. The perforant path was stimulated with paired-pulse paradigms, and population spike amplitudes were measured. Responses were obtained from one lamella while a recording electrode in a distant lamella leaked saline or the gamma-aminobutyric acid-A receptor antagonist bicuculline. Epileptic kainate-treated and control rats both showed significantly more paired-pulse inhibition when a lateral lamella was hyperexcitable. To assess seizure threshold in the dentate gyrus, two techniques were used. Measurement of stimulus threshold for evoking maximal dentate activation revealed significantly higher thresholds in epileptic kainate-treated rats compared with controls. In contrast, epileptic kainate-treated rats were more likely than controls to discharge spontaneous bursts of population spikes and to display stimulus-triggered afterdischarges when a focal region of the dentate gyrus was disinhibited with bicuculline. These spontaneous bursts and afterdischarges were confined to the disinhibited region and did not spread to other septotemporal levels of the dentate gyrus. Epileptic kainate-treated rats that displayed spontaneous bursts and/or afterdischarges had significantly larger percentages of Timm staining in the granule cell and molecular layers than epileptic kainate-treated rats that failed to show spontaneous bursts or afterdischarges. In summary, this study reveals functional abnormalities in the dentate gyri of epileptic kainate-treated rats; however, lateral inhibition persists, suggesting that vulnerable hilar neurons are not necessary for generating lateral inhibition in the dentate gyrus.     

Synaptic and axonal remodeling of mossy fibers in the hilus and supragranular region of the dentate gyrus in kainate-treated rats

Seizures evoked by kainic acid and a variety of experimental methods induce sprouting of the mossy fiber pathway in the dentate gyrus. In this study, the morphological features and spatial distribution of sprouted mossy fiber axons in the dorsal dentate gyrus of kainate-treated rats were directly shown in granule cells filled in vitro with biocytin and in vivo with the anterograde lectin tracer Phaseolus vulgaris leucoagglutinin (PHAL). Sprouted axon collaterals of biocytin-filled granule cells projected from the hilus of the dentate gyrus into the supragranular layer in both transverse and longitudinal directions in kainate-treated rats but were not observed in normal rats. The sprouted axon collaterals projected into the supragranular region for 600-700 microm along the septotemporal axis. Collaterals from granule cells in the infrapyramidal blade crossed the hilus and sprouted into the supragranular layer of the suprapyramidal blade. Sprouted axon segments in the supragranular layer had more terminal boutons per unit length than the axon segments in the hilus of both normal and kainate-treated rats but did not form giant boutons, which are characteristic of mossy fiber axons in the hilus and CA3. Mossy fiber axons in the hilus of kainate-treated rats had more small terminal boutons, fewer giant boutons, and there was a trend toward greater axon length compared with mossy fibers in the hilus of normal rats. With the additional length of supragranular sprouted collaterals, there was an overall increase in the length of mossy fiber axons in kainate-treated rats. The synaptic and axonal remodeling of the mossy fiber pathway could alter the functional properties of hippocampal circuitry by altering synaptic connectivity in local circuits within the hilus of the dentate gyrus and by increasing the divergence of the mossy fiber terminal field along the septotemporal axis.     

Nitric oxide scavenging by hemoglobin or nitric oxide synthase inhibition by N-nitro-L-arginine induces cortical spreading ischemia when K+ is increased in the subarachnoid space

We investigated the combined effect of increased brain topical K+ concentration and reduction of the nitric oxide (NO.) level caused by nitric oxide scavenging or nitric oxide synthase (NOS) inhibition on regional cerebral blood flow and subarachnoid direct current (DC) potential. Using thiopental-anesthetized male Wistar rats with a closed cranial window preparation, brain topical superfusion of a combination of the NO. scavenger hemoglobin (Hb; 2 mmol/L) and increased K+ concentration in the artificial cerebrospinal fluid ([K+]ACSF) at 35 mmol/L led to sudden spontaneous transient ischemic events with a decrease of CBF to 14+/-7% (n=4) compared with the baseline (100%). The ischemic events lasted for 53+/-17 minutes and were associated with a negative subarachnoid DC shift of -7.3+/-0.6 mV of 49+/-12 minutes' duration. The combination of the NOS inhibitor N-nitro-L-arginine (L-NA, 1 mmol/L) with [K+]ACSF at 35 mmol/L caused similar spontaneous transient ischemic events in 13 rats. When cortical spreading depression was induced by KCl at a 5-mm distance, a typical cortical spreading hyperemia (CSH) and negative DC shift were measured at the closed cranial window during brain topical superfusion with either physiologic artificial CSF (n=5), or artificial CSF containing increased [K+]ACSF at 20 mmol/L (n=4), [K+]ACSF at 3 mmol/L combined with L-NA (n=10), [K+]ACSF at 10 mmol/L combined with L-NA (five of six animals) or [K+]ACSF at 3 mmol/L combined with Hb (three of four animals). Cortical spreading depression induced longlasting transient ischemia instead of CSH, when brain was superfused with either [K+]ACSF at 20 mmol/L combined with Hb (CBF decrease to 20+/-20% duration 25+/-21 minutes, n=4), or [K+]ACSF at 20 mmol/L combined with L-NA (n=19). Transient ischemia induced by NOS inhibition and [K],ACSF at 20 mmol/L propagated at a speed of 3.4+/-0.6 mm/min, indicating cortical spreading ischemia (CSI). Although CSH did not change oxygen free radical production, as measured on-line by in vivo lucigenin-enhanced chemiluminescence, CSI resulted in the typical radical production pattern of ischemia and reperfusion suggestive of brain damage (n=4). Nimodipine (2 microg/kg body weight/min intravenously) transformed CSI back to CSH (n=4). Vehicle had no effect on CSI (n=4). Our data suggest that the combination of decreased NO. levels and increased subarachnoid K+ levels induces spreading depression with acute ischemic CBF response. Thus, a disturbed coupling of metabolism and CBF can cause ischemia. We speculate that CSI may be related to delayed ischemic deficits after subarachnoid hemorrhage, a clinical condition in which the release of Hb and K+ from erythrocytes creates a microenvironment similar to the one investigated here.     

Differential regulation by MK801 of immediate-early genes, brain-derived neurotrophic factor and trk receptor mRNA induced by a kindling after-discharge

Transient changes in immediate-early genes and neurotrophin expression produced by kindling stimulation may mediate secondary downstream events involved in kindling development. Recent experiments have demonstrated conclusively that both kindling progression and mossy fibre sprouting are significantly impaired by administration of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801. To further examine the link between kindling, changes in gene expression and the NMDA receptor, we examined the effects of MK801 on neuronal induction of immediate-early genes, brain-derived neurotrophic factor (BDNF) and trk receptor mRNA expression produced by a single electrically induced hippocampal after-discharge in rats. The after-discharge produced a rapid (after 1 h) increase in Fos, Jun-B, c-Jun, Krox-24 mRNA and protein and Krox-20 protein in dentate granule neurons and a delayed, selective expression of Fos, Jun-D and Krox-24 in hilar interneurons. MK801 pretreatment produced a very strong inhibition of Fos, Jun-D and Krox-20 increases in dentate neurons but had a much smaller effect on Jun-B and c-Jun expression. MK801 did not inhibit Krox-24 expression in granule neurons or the delayed expression of Fos, Jun-D and Krox-24 in hilar interneurons. BDNF protein and trk B and trk C mRNA expression were also strongly induced in dentate granule cells 4 h following an after-discharge. MK801 abolished the increase in BDNF protein and trk B, but not trk C mRNA in granule cells at 4 h. These results demonstrate that MK801 differentially regulates the AD-increased expression of a group of genes previously identified as being likely candidates for an involvement in kindling. Because MK801 significantly retards the development of kindling and mossy fibre sprouting, it can be argued that those genes whose induction is not significantly attenuated by MK801 are unlikely to play an important role in the MK801-sensitive component of kindling and the changes in neural connectivity (mossy fibre sprouting) associated with kindling. Conversely, the role in kindling of those genes whose expression was significantly attenuated by MK801 (Fos, Jun-D, Krox-20, trkB and BDNF) requires further examination.     

Synaptic reorganization in explanted cultures of rat hippocampus

Due to loss of afferent innervation, synaptic reorganization occurs in organotypic hippocampal slice cultures. With extra- and intracellular recordings, we confirm that the excitatory loop from the dentate gyrus (DG) to CA3 and further to CA1 is preserved. However, hilar stimulation evoked antidromic population spikes in the DG which were followed by a population postsynaptic potential (PPSP); intracellularly, an antidromic spike with a broad shoulder or EPSP/IPSP sequences were induced. Synaptic responses were blocked by glutamate receptor antagonists. Stimulation of CA1 induced a PPSP in DG. Dextranamine stained pyramidal cells of CA1 were shown to project to DG. After removal of area CA3, DG's and mossy fibers' (MF) stimulation still elicited PPSPs and EPSP/IPSP sequences in area CA1 which disappeared when a cut was made through the hippocampal fissure. During bicuculline perfusion, hilar stimulation caused EPSPs in granule cells and spontaneous and evoked repetitive firing appeared even after its isolation from areas CA3 and CA1. Collateral excitatory synaptic coupling between granule cells was confirmed by paired recordings. Besides the preservation of the trisynaptic pathway in this preparation, new functional synaptic contacts appear, presumably due to MF collateral sprouting and formation of pathways between areas CA1 and DG.     

GABA-receptor-independent dorsal root afferents depolarization in the neonatal rat spinal cord

Dorsal root afferent depolarization and antidromic firing were studied in isolated spinal cords of neonatal rats. Spontaneous firing accompanied by occasional bursts could be recorded from most dorsal roots in the majority of the cords. The afferent bursts were enhanced after elevation of the extracellular potassium concentration ([K+]e) by 1-2 mM. More substantial afferent bursts were produced when the cords were isolated with intact brain stems. Rhythmic afferent bursts could be recorded from dorsal roots in some of the cords during motor rhythm induced by bath-applied serotonin and N-methyl--aspartate (NMDA). Bilaterally synchronous afferent bursts were produced in pairs of dorsal roots after replacing the NaCl in the perfusate with sodium-2-hydroxyethansulfonate or after application of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline with or without serotonin (5-HT) and NMDA. Antidromic afferent bursts also could be elicited under these conditions by stimulation of adjacent dorsal roots, ventrolateral funiculus axons, or ventral white commissural (VWC) fibers. The antidromic bursts were superimposed on prolonged dorsal root potentials (DRPs) and accompanied by a prolonged increase in intraspinal afferent excitability. Surgical manipulations of the cord revealed that afferent firing in the presence of bicuculline persisted in the hemicords after hemisection and still was observed after removal of their ventral horns. Cutting the VWC throughout its length did not perturb the bilateral synchronicity of the discharge. These findings suggest that the activity of dorsal horn neurons is sufficient to produce the discharge and that the bilateral synchronicity can be maintained by cross connectivity that is relayed from side to side dorsal to the VWC. Antagonists of GABAB, 5-HT2/5-HT1C, or glutamate metabotropic group II and III receptors could not abolish afferent depolarization in the presence of bicuculline. Depolarization comparable in amplitude to DRPs, could be produced in tetrodotoxin-treated cords by elevation of [K+]e to the levels reported to develop in the neonatal rat spinal cord in response to dorsal root stimulation. A mechanism involving potassium transients produced by neuronal activity therefore is suggested to be the major cause of the GABA-independent afferent depolarization reported in our study. Possible implications of potassium transients in the developing and the adult mammalian spinal cord are discussed.     

Evidence from simultaneous intracellular recordings in rat hippocampal slices that area CA3 pyramidal cells innervate dentate hilar mossy cells

1. Simultaneous intracellular recordings of area CA3 pyramidal cells and dentate hilar "mossy" cells were made in rat hippocampal slices to test the hypothesis that area CA3 pyramidal cells excite mossy cells monosynaptically. Mossy cells and pyramidal cells were differentiated by location and electrophysiological characteristics. When cells were impaled near the border of area CA3 and the hilus, their identity was confirmed morphologically after injection of the marker Neurobiotin. 2. Evidence for monosynaptic excitation of a mossy cell by a pyramidal cell was obtained in 7 of 481 (1.4%) paired recordings. In these cases, a pyramidal cell action potential was followed immediately by a 0.40 to 6.75 (mean, 2.26) mV depolarization in the simultaneously recorded mossy cell (mossy cell membrane potentials, -60 to -70 mV). Given that pyramidal cells used an excitatory amino acid as a neurotransmitter (Cotman and Nadler 1987; Ottersen and Storm-Mathisen 1987) and recordings were made in the presence of the GABAA receptor antagonist bicuculline (25 microM), it is likely that the depolarizations were unitary excitatory postsynaptic potentials (EPSPs). 3. Unitary EPSPs of mossy cells were prone to apparent "failure." The probability of failure was extremely high (up to 0.72; mean = 0.48) if the effects of all presynaptic action potentials were examined, including action potentials triggered inadvertently during other spontaneous EPSPs of the mossy cell. Probability of failure was relatively low (as low as 0; mean = 0.24) if action potentials that occurred during spontaneous activity of the mossy cell were excluded. These data suggest that unitary EPSPs produced by pyramidal cells are strongly affected by concurrent synaptic inputs to the mossy cell. 4. Unitary EPSPs were not clearly affected by manipulation of the mossy cell's membrane potential. This is consistent with the recent report that area CA3 pyramidal cells innervate distal dendrites of mossy cells (Kunkel et al. 1993). Such a distal location also may contribute to the high incidence of apparent failures. 5. Characteristics of unitary EPSPs generated by pyramidal cells were compared with the properties of the unitary EPSPs produced by granule cells. In two slices, pyramidal cell and granule cell inputs to the same mossy cell were compared. In other slices, inputs to different mossy cells were compared. In all experiments, unitary EPSPs produced by granule cells were larger in amplitude but similar in time course to unitary EPSPs produced by pyramidal cells. Probability of failure was lower and paired-pulse facilitation more common among EPSPs triggered by granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Low toxicity and high antitumour activity of daunomycin by conjugation to an immunopotential amphoteric branched polypeptide

Gamma oscillations (approximately 40 Hz) were induced in transverse hippocampal slices by tetanic stimulation of CA1 and/or subiculum. Tetanic stimulation of each site elicited population gamma oscillations in the surrounding tissue <400 micro(m) away. Stimulation of CA1 alone could evoke activity at both CA1 and subiculum. Subicular stimulation, however, did not transmit to CA1. When the rostral end of CA1 was stimulated, gamma oscillations transmitted across <1.5 mm of silent CA1 before reappearing in the subiculum. Tetanic stimulation of CA1 increased [K+]o to 8.2 +/- 1.5 mM (mean +/- SE). The location of the peak increase corresponded to the site of local gamma generation. Silent areas of CA1 experienced smaller [K+]o increases, to 4.9 +/- 0.7 mM. The subiculum, which generated gamma, remained at the baseline 3.0 mM. Although fluctuations in [K+]o may have an impact on the generation of gamma rhythms, they are not necessary for them. Gamma oscillations had similar frequencies in CA1 and subiculum (40.4 +/- 2.9 and 43.9 +/- 3.1 Hz, respectively). When present in both, the oscillations typically were phase locked with the subiculum lagging by 5.4 +/- 1.8 ms. When both CA1 and subiculum were stimulated the lag decreased by 28%. These delays approximate those expected for the conduction velocity of axons between the two regions, here estimated at 0.52 +/- 0.07 m/s. Transmission of gamma oscillations from CA1 to subiculum was blocked by the focal addition of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor antagonist, 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione, to the subiculum. Oscillations induced in CA1 by local tetanic stimulation were blocked by focal application of the gamma-aminobutyric acid-A (GABAA) receptor antagonist, bicuculline, to CA1. Focal application of bicuculline to the subiculum blocked gamma due to subicular stimulation but not that due to CA1 stimulation. Bath-applied bicuculline disrupted subicular gamma evoked by subicular stimulation and led to a transient period of epileptiform responses before completely blocking responses. The further addition of the GABAB receptor antagonist, CGP 55845A, reversed this block, restoring the epileptic discharges evoked by tetanic stimulation. This suggests that the subiculum differs from hippocampal CA3 and neocortex, in having a powerful GABAB receptor-dependent mechanism to prevent epileptic discharges. The subiculum generates gamma rhythms both in response to local stimulation and to gamma rhythms evoked in CA1. Subicular gamma differs from that in CA1 in the presence of population spike doublets rather than singlets on many cycles. In both areas, generation of gamma by local stimulation depends on GABAA receptors, suggesting that the subiculum shares the interneuronal network mechanism we proposed for CA1.     

Abnormal targeting of developing hippocampal mossy fibers after epileptiform activities via L-type Ca2+ channel activation in vitro

The hippocampal mossy fibers, which originate from the dentate granule cells, develop mainly in the early postnatal period and are involved in numerous pathological processes. In this study, hippocampal slices prepared from premature rats were cultivated in the presence of convulsants to evaluate the influences of epileptiform activities on mossy fiber ontogeny. Electrophysiological and histochemical analyses revealed that prolonged hyperexcitability inhibited proper growth of the mossy fibers and caused ectopic innervation to the stratum oriens and the dentate molecular layer. These phenomena were prevented by pharmacological blockade of L-type Ca2+ channels, which did not affect convulsant-evoked ictal bursts. After single-pulse stimulation of the stratum granulosum in the slices cultured under paroxysmal conditions, the dentate gyrus displayed excessive excitation, but synaptic transmission to the CA3 region was hypoactive. However, brief repetitive stimulation elicited delayed epileptiform discharges in the CA3 region that were inhibited by an NMDA receptor antagonist. Chronic treatment with an L-type Ca2+ channel blocker ameliorated such aberrant neurotransmissions. These results suggest that ictal neuron activities at the developmental stage of the mossy fibers bring about the errant maturation associated with hippocampal dysfunction, which may form a cellular basis for the sequelae of childhood epilepsy, including chronic epilepsy or cognitive deficits. Thus I propose that L-type Ca2+ channel blockers can ameliorate the aversive prognosis of childhood epilepsy.     

Effects of barium on stimulus-induced rises in [K+]o in juvenile rat hippocampal area CA1

Immature glia may not be able to buffer K+ ions released during neuronal activity. Therefore, we investigated entorhinal-hippocampal slices of juvenile rats (ages P15-18 and P22-26) using a perfusion medium containing 2 mM BaCl2 in order to block glial inward rectifying and leak potassium channels. In contrast to adult animals, rises in [K+]o in slices from juvenile animals elicited by repetitive alvear stimulation were not augmented by Ba2+. Ba2+ effects on fast field potentials, slow field potentials and the applied current sink source distribution were roughly similar as in adult rats. We conclude that the capacity to buffer large quantities of K+ ions by mechanisms involving Ba2+-sensitive K+ channels has not yet developed in juveniles.     

Mouse cerebellar granule cell differentiation: electrical activity regulates the GABAA receptor alpha 6 subunit gene

GABAA receptor alpha6 subunit gene expression marks cerebellar granule cell maturation. To study this process, we used the Deltaalpha6lacZ mouse line, which has a lacZ reporter inserted into the alpha6 gene. At early stages of postnatal cerebellar development, alpha6-lacZ expression is mosaic; expression starts at postnatal day 5 in lobules 9 and 10, and alpha6-lacZ is switched on inside-out, appearing first in the deepest postmigratory granule cells. We looked for factors regulating this expression in cell culture. Membrane depolarization correlates inversely with alpha6-lacZ expression: granule cells grown in 25 mM [K+]o for 11-15 d do not express the alpha6 gene, whereas cultures grown for the same period in 5 mM [K+]o do. This is influenced by a critical early period: culturing for >/=3 d in 25 mM [K+]o curtails the ability to induce the alpha6 gene on transfer to 5 mM [K+]o. If the cells start in 5 mM [K+]o, however, they still express the alpha6-lacZ gene in 25 mM [K+]o. In contrast to granule cells grown in 5 mM [K+]o, cells cultured in 25 mM [K+]o exhibit no action potentials, mEPSCs, or mIPSCs. In chronic 5 mM [K+]o, factors may therefore be released that induce alpha6. Blockade of ionotropic and metabotropic GABA and glutamate receptors or L-, N-, and P/Q-type Ca2+ channels did not prevent alpha6-lacZ expression, but inhibition of action potentials with tetrodotoxin blocked expression in a subpopulation of cells.     

Postnatal development of low [Mg2+] oscillations in neocortex

One form of rhythmic activity intrinsic to neocortex can be induced in slices of adult somatosensory cortex by lowering [Mg2+]o to unblock N-methyl--aspartate (NMDA) receptors. It has been suggested that a population of intrinsically burst-firing (IB) neurons that are unique to cortical layer 5 may play a role in the rhythmic activity seen under these conditions. Whole cell patch-clamp and field-potential recordings in slices of somatosensory cortex from neonatal rats were used to study the development of IB cells and the development of 0 [Mg2+] oscillations. IB cells were not encountered before postnatal day 12 (P12) in layer 5, but from P13 to P19 an increasing proportion of cells had IB properties. Recordings from cells at P7, P17, and P19 in 0 [Mg2+] indicate that dramatic changes occur postnatally in 0 [Mg2+]-induced activity. At P7, cells largely showed trains of single action potentials. In contrast, at P19, cells showed organized bursts of rhythmic activity lasting 0.5-5 s separated by periods of relative quiescence. Cells recorded at P17 were found to have less organized rhythmic activity than cells from P19 cortex. Field-potential recordings in 0 [Mg2+] made at P7 showed infrequent and slowly occurring field depolarizations, whereas field-potential recordings at P19 consisted of spontaneous bursts of 4-12 Hz oscillations identical to those observed in the adult. Application of NE, which inhibits burst-firing of layer 5 IB cells, significantly altered the discharge pattern of 0 [Mg2+] oscillations at P19. These data suggest that the maturation of one type of rhythmic network activity intrinsic to neocortex is influenced by the development of the membrane properties of a single cell type.     

Effects of hyposmolar solutions on membrane currents of hippocampal interneurons and mossy cells in vitro

Whole cell voltage-clamp recordings in rat hippocampal slices were used to investigate the effect of changes in extracellular osmolarity on voltage-activated potassium currents. Currents were evoked from oriens/alveus (O/A) interneurons, hilar interneurons, and mossy cells. Hyposmolar external solutions produced a significant potentiation of K+ current recorded from O/A and hilar interneurons, but not from mossy cells. Hyposmolar solutions also dramatically potentiated the spontaneous excitatory postsynaptic currents recorded from mossy cells. These results suggest that hippocampal excitability can be modulated by the complex actions exerted by changes in extracellular osmolarity.     

Granule cell mRNA levels for BDNF, NGF, and NT-3 correlate with neuron losses or supragranular mossy fiber sprouting in the chronically damaged and epileptic human hippocampus

This study determined in temporal lobe epilepsy patients if there were correlations among hippocampal granule cell expression of neurotrophin mRNAs, aberrant supragranular mossy fiber sprouting, and neuron losses. Consecutive surgically resected hippocampi (n = 9) and comparison tissue from autopsies (n = 3) were studied for: 1. Granule cell mRNA levels using in situ hybridization for brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3); 2. neo-Timm supragranular mossy fiber sprouting; and 3. Ammon's horn neuron densities. Clinically, patients were classified into those with hippocampal sclerosis (HS; n = 7) and non-HS cases (i.e., mass lesions and autopsies; n = 5). Results showed that compared to non-HS cases, HS patients showed increased granule cell mRNA levels for BDNF, NGF, and NT-3 (p = 0.035, p = 0.04, p = 0.045 respectively; one-tail directional test). Moreover, granule cell BDNF mRNA levels correlated inversely with Ammon's horn neuron densities (p = 0.02) and correlated positively with greater supragranular mossy fiber sprouting (p = 0.02). NGF mRNA levels correlated inversely with Ammon's horn neuron densities (p = 0.02), and NT-3 mRNA levels correlated inversely with age at surgery (p = 0.04) and correlated positively with greater mossy fiber sprouting (p = 0.026). These results indicate in the chronically damaged human hippocampus that granule cells express neurotrophin mRNAs, and mRNA levels correlate with either hippocampal neuron losses or aberrant supragranular mossy fiber sprouting. These data support the hypothesis that in the epileptic human hippocampus, there may be pathophysiologic associations among mossy fiber synaptic plasticity, hippocampal neuron damage, and granule cell mRNA neurotrophin levels.     

Spatial stability of extracellular potassium ion and blood flow distribution in rat cerebral cortex after permanent middle cerebral artery occlusion

Extracellular potassium ion activity ([K+]o) increases precipitously during brain ischemia when blood flow falls below threshold values less than approximately 15 mL/100 g/min. This flow threshold for increase of [K+]o occurs also in focal ischemia producing gradient from ischemic core to adjacent normally perfused brain. In this study we investigated the spatial and temporal stability of extracellular potassium ion and blood flow gradients after permanent middle cerebral artery occlusion (MCAO) in rats. [K+]o and regional CBF were measured, respectively, with K+-sensitive and polarographic hydrogen-sensitive microelectrodes at different cortical locations in the middle cerebral artery distribution region. Spatial assessment of [K+]o and regional CBF was conducted at 30, 90, and 180 minutes after MCAO. [K+]o in the more lateral cortex (core) increased from near 3 mmol/L before MCAO to greater than 50 mmol/L and was associated with flow values less than 25% of pre-ischemic levels. Measurements medial to the core (penumbra) indicated progressively decreasing levels of [K+]o and improvement of CBF. There was a tendency for [K+]o in penumbral zones to decrease toward normal levels with time, but there was little dissipation of [K+]o in core regions. In contrast, the spatial CBF profile remained remarkably constant for the entire recording period. Thus, unlike infarction which has been reported to expand with time after focal ischemia, the spatial [K+]o disturbance tends to contract primarily due to decreasing [K+]o with time in the penumbra. Thus, steady state levels of [K+]o after focal ischemia may not be a valuable predictor of cell viability.     

Dynorphin and the kappa 1 ligand [3H]U69,593 binding in the human epileptogenic hippocampus

The distribution of dynorphin (DYN), one of its binding sites (kappa 1 receptor) and their relationship to neuronal loss and granule cell hyperexcitability was examined in hippocampi from patients with temporal lobe epilepsy (TLE). In hippocampi that were not the seizure focus (mass associated temporal lobe epilepsy, MaTLE; and paradoxical temporal lobe epilepsy, PTLE) DYN-like immunoreactivity was localized in the dentate granule cells and their mossy fiber terminals within the hilus and area CA3. In hippocampi that were the seizure focus (MTLE), 89% showed an additional band of immunoreactivity confined to the inner molecular layer (IML) of the dentate gyrus, representing recurrent mossy fiber collaterals. In 11% of MTLE patients no staining was found in the IML (MTLE/DYN-). The MTLE/DYN- hippocampi were also characterized by a significantly lower degree of cell loss than in MTLE hippocampi in the dentate granule cell layer, the hilus and CA3. Both MTLE and MTLE/DYN- hippocampi showed evoked epileptiform bursting in granule cells while MTLE showed greater polysynaptic EPSPs and spontaneous excitatory activity. Thus granule cell recurrent collateral sprouting may account for only some aspects of hyperexcitability. In 30% of the MTLE group, hilar neurons of a variety of morphological types expressed DYN immunoreactivity in their somata and dendrites. The density of [3H]U69,593 binding sites in MaTLE and PTLE patients was highest in areas CA1 and the subiculum-regions having little or no DYN-staining. In the dentate molecular layer, hilus and CA3--regions with the most DYN immunoreactivity--there was a low density of ligand binding. The significance of this transmitter/receptor mismatch is yet unknown.     

Epilepsy induced collateral sprouting of hippocampal mossy fibers: does it induce the development of ectopic synapses with granule cell dendrites?

In the present study, using Golgi and electron microscopy techniques, experimentally induced epilepsy (kindling and kainate treatment) elicited collateral sprouting of mossy fibers in rat hippocampus. Collateral branches invade the hilus, cross the granule cell layer, and distribute throughout the inner third of the molecular layer. These newly developed collaterals may acquire the typical features of mossy fibers including giant fiber varicosities (mousses), although the mean surface of these mousses was thinner in these collaterals than in terminal branches. Granule cell dendrites may develop giant thorny excrescences, suggesting that the targets of these collaterals are granule cells. Giant synaptic boutons appear in the inner third of molecular layer of epileptic rats. These boutons acquire the morphological features of mossy fiber boutons and made multiple synaptic contacts with dendritic spines. The analysis of the profile types suggests that some of the newly developed collateral mossy fibers made hypotrophic synaptic contacts.     

A comparison of impulsive and instrumental subgroups of batterers

The physiological interactions between the dentate gyrus (DG) and CA3 were studied in urethane-anesthetized rats by using field potential recording and current source density (CSD) analysis. Stimulation of CA3b resulted in a short-latency (<2.5-ms onset latency) antidromic population spike in both the DG and CA3c. An excitation (current sink) at the middle molecular layer (MML) was observed at 3-ms latency, possibly mediated by the backfiring of perforant path fibers that projected to both DG and CA3. CA3 stimulation also resulted in a sink at the dendritic layers of CA3c, which was likely mediated by excitatory CA3 recurrent collaterals. It was inferred that the DG was excited at the inner molecular layer (IML) after stimulation near the CA3b/CA3c border. This IML excitation (sink) probably resulted from orthodromic CA3 or hilar projections to the IML and not from mossy fiber backfiring. The IML and the CA3c dendritic sinks were blocked by an intracerebroventricular injection of a non-N-methyl-D-aspartate receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione, but not by a gamma-aminobutyric acid type A (GABA(A)) receptor antagonist, bicuculline. CA3b stimulation evoked population spike bursts (3-7-ms latency) in both DG and CA3c when GABA(A) inhibition was suppressed by bicuculline, thus confirming that the excitatory afferents project from CA3b to DG and CA3c. A CA3 conditioning stimulus pulse given 30-200 ms before a perforant-path test pulse increased the amplitude of the perforant-path-evoked DG population spike (as compared with the test response without conditioning). After a moderate-intensity stimulation of CA3, a late (<20-ms latency) excitation of the MML of the DG was found. The late DG excitation was blocked by procaine injection at the medial perforant path, suggesting its origin from the medial entorhinal cortex. In conclusion, rich interactions between CA3 and other hippocampal structures were studied quantitatively by CSD analysis in vivo. We infer that CA3 provides an early excitatory feedback path to DG through recurrent collaterals or hilar interneurons and a late feedback through the medial entorhinal cortex.     

Consequences of neonatal seizures in the rat: morphological and behavioral effects

Whereas neonatal seizures are a predictor of adverse neurological outcome, there is controversy regarding whether seizures simply reflect an underlying brain injury or can cause damage. We subjected neonatal rats to a series of 25 brief flurothyl-induced seizures. Once mature the rats were compared with control littermates for spatial learning and activity level. Short-term effects of recurrent seizures on hippocampal excitation were assessed by using the intact hippocampus formation preparation and long-term effects by assessing seizure threshold. Brains were analyzed for neuronal loss, sprouting of granule cell axons (mossy fibers), and neurogenesis. Compared with controls, rats subjected to neonatal seizures had impaired learning and decreased activity levels. There were no differences in paired-pulse excitation or inhibition or duration of afterdischarges in the intact hippocampal preparation. However, when studied as adults, rats with recurrent flurothyl seizures had a significantly lower seizure threshold to pentylenetetrazol than controls. Rats with recurrent seizures had greater numbers of dentate granule cells and more newly formed granule cells than the controls. Rats with recurrent seizures also had sprouting of mossy fibers in CA3 and the supragranular region. Recurrent brief seizures during the neonatal period have long-term detrimental effects on behavior, seizure susceptibility, and brain development.