Epileptic afterdischarge in the hippocampal-entorhinal system: current source density and unit studies

The contribution of the various hippocampal regions to the maintenance of epileptic activity, induced by stimulation of the perforant path or commissural system, was examined in the awake rat. Combination of multiple-site recordings with silicon probes, current source density analysis and unit recordings allowed for a high spatial resolution of the field events. Following perforant path stimulation, seizures began in the dentate gyrus, followed by events in the CA3-CA1 regions. After commissural stimulation, rhythmic bursts in the CA3-CA1 circuitry preceded the activation of the dentate gyrus. Correlation of events in the different subregions indicated that the sustained rhythmic afterdischarge (2-6 Hz) could not be explained by a cycle-by-cycle excitation of principal cell populations in the hippocampal-entorhinal loop. The primary afterdischarge always terminated in the CA1 region, followed by the dentate gyrus, CA3 region and the entorhinal cortex. The duration and pattern of the hippocampal afterdischarge was essentially unaffected by removal of the entorhinal cortex. The emergence of large population spike bursts coincided with a decreased discharge of interneurons in both CA1 and hilar regions. The majority of hilar interneurons displayed a strong amplitude decrement prior to the onset of population spike phase of the afterdischarge. These findings suggest that (i) afterdischarges can independently arise in the CA3-CA1 and entorhinal dentate gyrus circuitries, (ii) reverberation of excitation in the hippocampal-entorhinal loop is not critical for the maintenance of afterdischarges and (iii) decreased activity of the interneuronal network may release population bursting of principal cells.     

Interneurons in the hippocampal dentate gyrus: an in vivo intracellular study

Interneurons in the dentate area were characterized physiologically and filled with biocytin in urethane-anaesthetized rats. On the basis of axonal targets the following groups could be distinguished. (i) Large multipolar interneurons with spiny dendrites in the deep hilar region densely innervated the outer molecular layer and contacted both granule cells and parvalbumin-positive neurons (hilar interneuron with perforant pathway-associated axon terminals; HIPP cells). (ii) A pyramidal-shaped neuron with a cell body located in the subgranular layer innervated mostly the inner molecular layer and the granule cell layer (hilar interneuron with commissural-associational pathway-associated axon terminals; HICAP cell). It contacted both granule cells and interneurons. Axon collaterals of HIPP and HICAP neurons covered virtually the entire septo-temporal extent of the dorsal dentate gyrus. (iii) Calbindin-immunoreactive neurons with horizontal dendrites in stratum oriens of the CA3c region gave rise to a rich axon arbor in strata oriens, pyramidale and radiatum and innervated almost the entire extent of the dorsal hippocampus, with some collaterals entering the subicular area (putative trilaminar cell). (iv) Hilar basket cells innervated mostly the granule cell layer and to some extent the inner molecular layer and the CA3c pyramidal layer. HIPP and trilaminar interneurons could be antidromically activated by stimulation of the fimbria. Only the HICAP cells could be monosynaptically discharged by the perforant path input. All interneurons examined showed phase-locked activity to the extracellularly recorded theta/gamma oscillations or to irregular dentate electroencephalogram spikes. These observations indicate that the interconnected interneuronal system plays a critical role in coordinating population of the dentate gyrus and Ammon's hom.     

Electrophysiological and pharmacological characterization of the direct perforant path input to hippocampal area CA3

Monosynaptic perforant path responses evoked by subicular stimulation were recorded from CA3 pyramidal cells of rat hippocampal slices. These monosynaptic responses were isolated by using low intensities of stimulation and by placing a cut through the mossy fibers. Perforant path-evoked responses consisted of both excitatory and inhibitory components. Excitatory postsynaptic currents (EPSCs) were mediated by both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidreceptors (AMPAR) and N-methyl--aspartate receptors (NMDAR). Inhibitory postsynaptic currents consisted of gamma-aminobutyric acid-A (GABAA-) and -B (GABAB)-receptor-mediated components. At membrane potentials more positive than -60 mV and at physiological [Ca2+]/[Mg2+] ratios, >30% of perforant path evoked EPSC was mediated by NMDARs. This value varied as a function of the membrane voltage and external [Mg2+]. Two types of responses were observed after low-intensity stimulation of the perforant path. The first type of response showed paired-pulse facilitation and was reduced by 2-amino-4-phosphonobutyric acid (AP4). The second type of response showed paired-pulse depression and was reduced by baclofen. Electrophysiological and pharmacological characteristics of these two types of responses are similar to the properties of lateral and medial perforant path-evoked EPSPs in the dentate gyrus.     

Endogenous dynorphins inhibit excitatory neurotransmission and block LTP induction in the hippocampus

Although anatomical and neurochemical studies suggest that endogenous opioids act as neurotransmitters, their roles in normal and pathophysiological regulation of synaptic transmission are not defined. Here we examine the actions of prodynorphin-derived opioid peptides in the guinea-pig hippocampus and show that physiological stimulation of the dynorphin-containing dentate granule cells can release endogenous dynorphins, which then activate kappa 1 opioid receptors present in the molecular layer of the dentate gyrus. Activation of kappa 1 receptors by either pharmacologically applied agonist or endogenously released peptide reduces excitatory transmission in the dentate gyrus, as shown by a reduction in the excitatory postsynaptic currents evoked by stimulation of the perforant path, a principal excitatory afferent. In addition, released dynorphin peptides were found to block the induction of long-term potentiation (LTP) at the granule cell-perforant path synapse. The results indicate that endogenous dynorphins function in this hippocampal circuit as retrograde, inhibitory neurotransmitters.     

Construction and overexpression in Escherichia coli of genetically engineered derivatives of penicillin-binding protein 2'' of Staphylococcus epidermidis

This paper reports a study of long-term potentiation (LTP) of perforant path synapses in CA1. Using rat hippocampal slices with CA3 and the dentate gyrus removed, stimulation of the perforant path evoked a population excitatory postsynaptic potential (pEPSP) that was negative-going in s. lacunosum-moleculare of CA1. High-frequency conditioning stimulation of the perforant pathway induced LTP of the perforant path pEPSP in slices disinhibited by the GABAA receptor antagonist bicuculline methiodide (20 microM). Conditioning of the perforant pathway in normal medium, however, failed to induce LTP. Potentiation of the perforant path pEPSP in the presence of bicuculline lasted at least 1 h, was specific to the tetanized pathway, and based on a threshold property, appeared associative in nature.     

Hippocampal interneurons expressing glutamic acid decarboxylase and calcium-binding proteins decrease with aging in Fischer 344 rats

Aging leads to alterations in the function and plasticity of hippocampal circuitry in addition to behavioral changes. To identify critical alterations in the substrate for inhibitory circuitry as a function of aging, we evaluated the numbers of hippocampal interneurons that were positive for glutamic acid decarboxylase and those that expressed calcium-binding proteins (parvalbumin, calbindin, and calretinin) in young adult (4-5 months old) and aged (23-25 months old) male Fischer 344 rats. Both the overall interneuron population and specific subpopulations of interneurons demonstrated a commensurate decline in numbers throughout the hippocampus with aging. Interneurons positive for glutamic acid decarboxylase were significantly depleted in the stratum radiatum of CA1, the strata oriens, radiatum and pyramidale of CA3, the dentate molecular layer, and the dentate hilus. Parvalbumin interneurons showed significant reductions in the strata oriens and pyramidale of CA1, the stratum pyramidale of CA3, and the dentate hilus. The reductions in calbindin interneurons were more pronounced than other calcium-binding protein-positive interneurons and were highly significant in the strata oriens and radiatum of both CA1 and CA3 subfields and in the dentate hilus. Calretinin interneurons were decreased significantly in the strata oriens and radiatum of CA3, in the dentate granule cell and molecular layers, and in the dentate hilus. However, the relative ratio of parvalbumin-, calbindin-, and calretinin-positive interneurons compared with glutamic acid decarboxylase-positive interneurons remained constant with aging, suggesting actual loss of interneurons expressing calcium-binding proteins with age. This loss contrasts with the reported preservation of pyramidal neurons with aging in the hippocampus. Functional decreases in inhibitory drive throughout the hippocampus may occur due to this loss, particularly alterations in the processing of feed-forward information through the hippocampus. In addition, such a profound alteration in interneuron number will likely alter inhibitory control of excitability and neuronal synchrony with behavioral states.     

LTP in the lateral perforant path is beta-adrenergic receptor-dependent

Norepinephrine induces an activity-independent long-lasting depression of synaptic transmission in the lateral perforant path input to dentate granule cells, whereas high frequency stimulation induces activity-dependent long-term potentiation (LTP). We investigated the role of endogenous activation of beta-adrenergic receptors in LTP of the lateral and medial perforant paths under conditions affording selective stimulation of these pathways in the rat hippo-campal slice. Propranolol (1 microM), a beta-receptor antagonist, blocked LTP induction of both lateral and medial perforant path-evoked field excitatory postsynaptic potentials. The results indicate a broad requirement for norepinephrine in different types of synaptic plasticity, including activity-independent depression and activity-dependent LTP in the lateral perforant path.     

Development of microglia in the postnatal rat hippocampus

During the prenatal development of the hippocampus, microglial cell precursors progressively occur in all subfields in accordance with known ontogenetic gradients of the region (Dalmau et al., J. Comp. Neurol. 1997a;377:70-84). The present study follows the regional distribution of these microglial cell precursors and their morphological differentiation in the rat hippocampus from birth to postnatal (P) day 18. The results demonstrate that the cellular differentiation and the subregional distribution of microglia follow the specific developmental gradients of the different parts of Ammon's horn and the dentate gyrus. Microglial cell distribution in the dentate gyrus is thus delayed compared with that in Ammon's horn. The appearance of microglia in the hippocampal subregions and differentiation of cell precursors into adult microglia occur earlier at temporal levels than at septal levels. Distribution of microglial cells follows an outside-to-inside pattern from the hippocampal fissure to the main cell layers in either Ammon's horn or the dentate gyrus. Meanwhile, the resident microglial cells located in the stratum oriens and dentate hilus at birth also increase in number and gradually disperse throughout the whole tissue of the two layers with age. In Ammon's horn, microglial differentiation occurs earlier in CA3 than in CA1. In the dentate gyrus, microglia appear earlier in relation to the external limb than to the internal limb, largely following a lateral-to-medial gradient. The differentiation and appearance of microglia in the various hippocampal and dentate subregions often correspond to the developmental stage of intrinsic and extrinsic afferent nerve fiber projections. Finally, in both Ammon's horn and the dentate gyrus, cells resembling reactive microglia are also observed and, in particular, in the perforant path projections from P9 to P18, suggesting their participation not only in phagocytosis of dead cells but also in axonal elimination and/or fiber reorganization.     

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.     

Circuit-specific alterations of N-methyl-D-aspartate receptor subunit 1 in the dentate gyrus of aged monkeys

Age-associated memory impairment occurs frequently in primates. Based on the established importance of both the perforant path and N-methyl-D-aspartate (NMDA) receptors in memory formation, we investigated the glutamate receptor distribution and immunofluorescence intensity within the dentate gyrus of juvenile, adult, and aged macaque monkeys with the combined use of subunit-specific antibodies and quantitative confocal laser scanning microscopy. Here we demonstrate that aged monkeys, compared to adult monkeys, exhibit a 30.6% decrease in the ratio of NMDA receptor subunit 1 (NMDAR1) immunofluorescence intensity within the distal dendrites of the dentate gyrus granule cells, which receive the perforant path input from the entorhinal cortex, relative to the proximal dendrites, which receive an intrinsic excitatory input from the dentate hilus. The intradendritic alteration in NMDAR1 immunofluorescence occurs without a similar alteration of non-NMDA receptor subunits. Further analyses using synaptophysin as a reflection of total synaptic density and microtubule-associated protein 2 as a dendritic structural marker demonstrated no significant difference in staining intensity or area across the molecular layer in aged animals compared to the younger animals. These findings suggest that, in aged monkeys, a circuit-specific alteration in the intradendritic concentration of NMDAR1 occurs without concomitant gross structural changes in dendritic morphology or a significant change in the total synaptic density across the molecular layer. This alteration in the NMDA receptor-mediated input to the hippocampus from the entorhinal cortex may represent a molecular/cellular substrate for age-associated memory impairments.     

Galanin modulation of seizures and seizure modulation of hippocampal galanin in animal models of status epilepticus

We examined the role of hippocampal galanin in an animal model of status epilepticus (SE). Control rats showed abundant galanin-immunoreactive (Gal-IR) fibers in the dentate hilus, whereas no Gal-IR neurons were observed. Three hours after the onset of self-sustaining SE (SSSE), induced either by intermittent stimulation of the perforant path for 30 min (PPS) or by injection of lithium and pilocarpine, Gal-IR fibers disappeared in the hilus and remained absent for up to 1 week afterward. Twelve hours after the induction of SE by PPS or 3 hr after pilocarpine administration, Gal-IR neurons appeared in the hilus; these neurons increased in number after 1 d and gradually declined 3 and 7 d later. Galanin concentration in the hippocampus, measured by ELISA, significantly decreased on the plateau of SSSE and increased 24 hr after PPS. Galanin (0.05 nmol) injected into the hilus prevented the induction of SSSE, and 0.5 nmol of galanin stopped established SSSE. These effects were attenuated by galanin receptor antagonists (M35 > M40 >/= M15). 2-Ala-galanin (5 nmol), a putative agonist of galanin type 2 receptors, prevented but was unable to stop SSSE. M35 facilitated the development of SSSE when given before PPS. We suggest that hippocampal galanin acts as an endogenous anticonvulsant via galanin receptors. SE-induced galanin depletion in the hippocampus may contribute to the maintenance of seizure activity, whereas the increase of galanin concentration and the appearance of galanin-immunoreactive neurons may favor the cessation of SSSE. The seizure-protecting action of galanin SSSE opens new perspectives in the treatment of SE.     

Altered inhibition of dentate granule cells during spatial learning in an exploration task

To investigate the extent to which inhibitory interneurons control impulse flow through the dentate gyrus during spatial learning in an exploration task, dentate field potentials were recorded in response to paired stimulation of the perforant path while rats rested or explored. Recurrent inhibition of the granule cells was measured as the reduction of the second waveform when a population spike was present in the first. Both the population spike and the field EPSP (fEPSP) were suppressed at interstimulus intervals shorter than approximately 40 msec. Consistent differences were observed between potentials recorded at equivalent brain temperature in the exploration and resting (reference) conditions. During exploration, the fEPSP of the second (test) waveform was reduced further compared with reference potentials with a similar response to the first (conditioning) stimulus. This reduction was observed only when the first pulse elicited a population spike. The population spike of the second waveform was facilitated compared with reference potentials with similar fEPSP slopes. These observations suggest that exploration is coupled to increased inhibition on the perforant-path terminals or the dendrites of the granule cells, whereas the inhibition on the somata is decreased. The two phenomena were not correlated and followed different time courses. The suppression of the fEPSP decayed gradually, although it was still present at 15 min, whereas the facilitation of the population spike was stable. Together, these changes, which likely involve different populations of interneurons, may focus and amplify incoming signals from the entorhinal cortex.     

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.     

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.     

Distribution of parvalbumin-containing interneurons in the hippocampus of the gerbil--a qualitative and quantitative statistical analysis

In the gerbil (Meriones unguiculatus) hippocampal formation, the calcium-binding protein parvalbumin (PV) shows a unique species-specific distribution: it is present in the perforant path from the entorhinal cortex to the stratum molecular of the dentate are and cornu ammonis. A possible relation of this to the seizure-sensitivity of gerbils has been suggested. In addition, as in other species, PV is contained in a subpopulation of GABAergic nerve cells of the gerbil hippocampus. The characteristics of these PV-containing neurons are here described. Distribution and shape of the PV-positive neurons in general agreed with the features described for rat hippocampus with two notable exceptions: in CA2 PV-containing perikarya were densely crowded and gave rise to an intense immunoreactive plexus around the pyramidal cells and, in CA1, the number of stained neurons was variable, often much lower than in rats and occasionally not a single PV-positive neuron was present. In parasagittal brain sections of the lateralities 1.0, 1.6 and 2.2 mm from the midline, obtained from 27 male gerbils, the number of PV-containing neurons was determined. The data set obtained in CA3 and dentate area resembled unimodal distributions, while in CA1 a bimodal frequency distribution was present. Since parametric and non-parametric correlation tests rely on a unimodal distribution of the data set, they gave falsely significant values in CA1. The bimodal distribution suggests that, with respect to the PV-containing interneurons in CA1, two different populations of gerbils were included in our sample, those with many positive neurons and those with only a few. Since the nerve terminal staining is preserved also in those gerbils with only a few positive perikarya in CA1, it seems possible that an unknown factor influenced PV expression and storage in the soma. Sex, age, seasonal or circadian rhythm or quality of immunocytochemical staining did not influence the outcome of the quantitative analysis. However, a relation of the expression of the high affinity calcium buffering PV in interneurons and the individual seizure sensitivity of the gerbil is considered.     

Time-dependent changes in neurotrophic factor mRNA expression after kindling and long-term potentiation in rats

We compared the time-dependent changes in messenger ribonucleic acid (mRNA) levels for two neurotrophic factors after amygdala-kindled seizures and hippocampal long-term potentiation (LTP) in rats in vivo. The brain-derived neurotrophic factor (BDNF) mRNA levels in the bilateral granule cell layer of the dentate gyrus, increased significantly 1-4 h after stage 5 kindled seizures. Nerve growth factor (NGF) mRNA levels increased throughout the bilateral limbic regions more gradually than those of BDNF mRNA. The maximum levels in the dentate gyrus ipsilateral to stimulation (BDNF mRNA: 493%, NGF mRNA: 199% of control levels) occurred 2 h after seizures. As observed with kindling, BDNF and NGF mRNA expression increased in the dentate gyrus ipsilateral to stimulation also increased following LTP induced by the perforant path stimulation, with maximum levels occurring 2 h and 4 h, respectively, after stimulation, when they reached 284% and 189% of the control levels, respectively. These results suggest that BDNF and NGF are involved in enhancement of synaptic efficacy in the granule cells of the dentate gyrus in the hippocampus in kindling, not related to the neuronal excitability associated with seizure activity.     

Biomechanics of human common carotid artery and design of novel hybrid textile compliant vascular grafts

Entorhinal cortex lesion (ECL) leads to anterograde degeneration of perforant path axons and is known to induce a rapid and intense reaction of astrocytes and microglial cells in the deafferented dentate gyrus. Phagocytosis of degenerating axons involves the establishment and maintenance of cell-matrix and cell-cell interactions by activated glial cells. It was thus our aim to investigate whether the process of axon phagocytosis is accompanied by the expression of adhesion molecules on activated microglial cells or reactive astrocytes, as such molecules mediate bot cell-matrix and cell-cell interactions. We found that the integrin adhesion molecules leukocyte function antigen-1 (LFA-1), very late antigen-4 (VLA-4), and the ligand for LFA-1, intercellular adhesion molecule-1 (ICAM-1), were expressed on microglial cells accumulating in the outer molecular layer of the deafferented dentate gyrus. This upregulation of adhesion molecule expression on microglial cells showing morphological criteria of activation occurred rapidly following ECL, reached its peak at 3 days post lesion (dpl), and gradually returned to control levels after 9 dpl. Astrocytes were never labeled by antibodies directed against these adhesion molecules. Prelabeling of the perforant path with a fluorescent tracer and subsequent ECL led to phagocytosis of fluorescent-labeled axonal debris by cells that were located in the outer molecular layer and showed typical microglial morphology. Double-fluorescence labeling demonstrated that microglial cells engaged in the phagocytosis of axonal debris expressed LFA-1, VLA-4, and the LFA-1-ligand ICAM-1. In conclusion, our results demonstrate that anterograde degeneration of perforant path axons results in adhesion molecule expression on activated microglial cells engaged in axon phagocytosis. The expression of such molecules could represent a mechanism that retains activated microglia in areas of axonal degeneration and perhaps enables the interaction of microglial cells with each other or with other immunocompetent cells.     

Altered hemostasis in male rats following administration of the ACAT inhibitor SKF-99085

We have previously found that high-frequency stimulation of the medial amygdala (MeA) induces long-term potentiation (LTP) of the population spike in the perforant path-dentate granule cell synapses of anesthetized rats. In the present study, we investigated the influence of MeA stimulation on the relationship between the population excitatory postsynaptic potential (pEPSP) and population spike in the dentate gyrus. High-frequency stimulation of the MeA produced a leftward shift of the E-S curve, in which population spike amplitude was plotted against pEPSP slope at various stimulus intensities. MeA-induced population spike LTP was also observed under blockade of GABAergic inhibition with picrotoxin. These results suggests that MeA stimulation leads to a long-lasting change in the internal firing characteristics of the dentate granule cells.     

The regulation of hippocampal dynorphin by neural/neuroendocrine pathways: models for effects of aging on an opioid peptide system

Previous research has demonstrated increased messenger RNA expression and peptide content in an opioid system localized to hippocampal dentate granule cells in aged rats. This altered regulation of dynorphin was correlated with the emergence of an age-related impairment in spatial learning. Considerable evidence exists for additional effects of aging on systems that provide input to the dynorphin-containing dentate granule cells. Such changes have been well documented for loss of perforant path innervation from entorhinal cortex, deterioration in septohippocampal cholinergic neurons, and high amounts of glucocorticoids that have, among their targets, receptors located in the dentate gyrus. Similar to the effects of aging on hippocampal dynorphin, age-related changes in each of these systems correlate with the severity of spatial learning impairment in aged rats. This raises the possibility that dysregulation of dynorphin in the aged brain is a reactive response to antecedant change(s) in this circuitry, a hypothesis that was examined by separately manipulating in young rats the three neural/neuroendocrine systems identified above. Of the three models examined only removal of the perforant path reproduced the effect of aging on dynorphin in the hippocampal formation. An immunotoxin was used in Experiment 1 to selectively remove septo-hippocampal cholinergic neurons in young rats. No alteration in hippocampal opioid peptides was produced by this treatment. Experiment 2 examined effects of exposure to excess corticosterone. Adrenalectomized rats exhibited a significant decrease in hippocampal dynorphin-A (1-8) content, which was reversed by corticosterone replacement at a concentration approximating normal basal levels. Dynorphin-A (1-8) content, however, was not reliably increased by exposure to excess corticosterone. In contrast, perforant path removal was found to reproduce the effect of aging on dynorphin content; either aspiration of the entorhinal cortex or knife-cut transections of the perforant path reliably increased hippocampal dynorphin content. These results support the conclusion that age-related deterioration in the septohippocampal cholinergic system and evaluated exposure to corticosterone are not sufficient to induce an elevation in hippocampal dynorphin content. Only removal of the perforant path innervation was found to reproduce the elevation in hippocampal dynorphin content observed in aged rats with hippocampal-dependent learning impairment.     

Ultrastructural evidence for bouton proliferation in the partially deafferented dentate gyrus of the adult rat

A quantitative morphological study of the changes in the dentate gyrus molecular layer in response to the removal of perforant path afferents was made utilizing electron microscopic techniques. Alterations in 1. the population of remaining afferents, 2. glial cells, and 3. granule cell dendrites are reported. The major observation was an increase in intact bouton density in the region of denervation which began at 5 days post-lesion and continued through 11 days post-lesion, the longest post-lesion survival time studied.