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Excitotoxic hippocampal neuron loss following sustained electrical stimulation of the perforant pathway in the mouse.

Prolonged electrical stimulation of the perforant pathway in the rat evokes epileptiform discharges in dentate granule cells and irreversibly damages hilar neurons. In this in vivo study, we demonstrate that similar perforant path stimulation in C57Bl/6 mice causes the same pattern of hippocampal neuron loss. Therefore, this mouse model of seizure-induced hippocampal injury can be used for a wide variety of studies in genetically altered conditions not available in rats.

Animals↗

Alteration in the pattern of nerve terminal protein immunoreactivity in the perforant pathway in Alzheimer's disease and in rats after entorhinal lesions.

Neurons in layer II of the entorhinal cortex consistently develop neurofibrillary tangles in Alzheimer's disease (AD). Experimental neuroanatomical studies have shown that these neurons give rise to the perforant pathway, a major excitatory projection to the hippocampal formation, which terminates in a discrete pattern in the outer portion of the molecular layer of the dentate gyrus. The distribution of two nerve terminal associated proteins, synaptophysin and NT75, was studied in the molecular layer of the dentate gyrus in AD and control cases to determine whether Alzheimer neuronal pathology is associated with loss of synaptic markers. In parallel studies, the effect of ablation of the entorhinal cortex in rats was evaluated. In AD as compared to controls, a decrease in synaptophysin immunostaining was evident in the terminal zone of the perforant pathway. NT75 nerve terminal immunostaining was too weak to interpret in the human hippocampal formation. Both synaptophysin and NT75 immunoreactivity were found in association with some neuritic plaques. In rats, entorhinal lesions resulted in diminished immunoreactivity for both synaptophysin and NT75 in the perforant pathway terminal zone. These results suggest that nerve terminal protein loss is a concomitant feature of neuronal pathology in AD.

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Influence of raphe nuclei on neuronal transmission from perforant pathway through dentate gyrus.

1. In chronically prepared, freely moving rats, electrical stimulation was applied to the perforant pathway and monosynaptic responses were recorded extracellularly in the ipsilateral dentate gyrus. In some tests a stimulus was also applied to the median raphe nucleus (mr) prior to activating the perforant pathway. Experiments were performed during two behavioral conditions: slow-wave sleep (SWS) and the still, alert state (SAL). Two varieties of evoked responses were recorded: those due to synchronous firing of neuronal action potentials (evoked action potentials or EAPs) and those produced by excitatory synaptic activity (evoked synaptic potentials or ESPs). 2. As reported previously (38), perforant path stimulation elicited EAPs of greater magnitude during SWS than during SAL. The application of a prior stimulus to mr (prestimulation) markedly increased the already elevated EAPs observed during SWS. The EAPs during SAL were unaffected by prestimulation. 3. The minimum delay time (time between mr and perforant path stimuli) at which the augmentation of the EAPs appeared during SWS was approximately 5 ms. The augmentation reached a maximum at delay times of 25-40 ms and was present up to a delay time of 150 ms. 4. As in former experiments (38), ESPs recorded in the molecular layer of the dentate gyrus after perforant path stimulation were found to be greater during SAL than during SWS. Prestimulation of mr had no significant effect on the ESPs at any level of the molecular layer during either SWS or SAL. 5. The perforant path afferent volley was recorded at high gain in the dentate gyrus. Its amplitude was found to be solely dependent on perforant path stimulus intensity and not on behavioral state or the prestimulation of mr. 6. In preparations anesthetized with Chloropent (82% chloral hydrate, 18% pentobarbital; Fort Dodge Laboratories, Fort Dodge, IA), prestimulation was applied at each of a number of loci within the pons and medulla, including mr, As in SWS, prestimulating mr resulted in augmented EAPs with a minimum delay time of 5 ms. Similar augmented responses were observed when stimulation was applied at other raphe nuclei (dorsal raphe, pontis, magnus, and pallidus), but there was no augmentation when stimulation was applied at other brain stem sites. Threshold stimulus intensities for producing augmented EAPs in the raphe nuclei were less than 30 microA. 7. In freely moving animals it was first established that the EAP responses during SWS were markedly greater than during SAL. Midline lesions were then made at the rostrocaudal level of mr. Following the lesions, there was no longer any significant difference in the magnitude of the EAPs recorded during the two behaviors. 8. These findings suggest that tonic influences arising from raphe nuclei during SWS may be involved in the facilitation of neuronal transmission through the dentate gyrus observed during this behavioral state.

Animals↗

Is aspartic acid the neurotransmitter of the perforant pathway?

In order to determine whether an amino acid may act as a neurotransmitter in the perforant pathway we examined the effect of lesion of rat entorhinal cortex on the concentrations of various amino acids in the hippocampus proper and fascia dentata. Only the aspartic acid content was found significantly decreased after the lesion. This decreases is not due to a loss from target cells of the perforant pathway, but rather to a loss from its degenerating terminals.

Amino Acids↗

Alterations of ubiquitin immunoreactivity in the hippocampal formation after perforant pathway lesion.

Immunohistochemical techniques were employed to examine the changes in free ubiquitin within the hippocampus 1, 3, 7, 14, and 30 days after a unilateral perforant pathway lesion occurred in the rat brain. Immunoreactivity for ubiquitin was remarkably decreased in the cell body and proximal dendrites of neurons throughout the hippocampus ipsilateral to the lesion at 1 day post-lesion. At 3 days post-lesion, ubiquitin immunoreactivity was recovered in interneurons in the whole hippocampus as well as in mossy cells in the hilar region, although granule cells in the dentate gyrus and pyramidal cells in the CA1 subfield remained unlabeled, and pyramidal cells in the CA3 subfield demonstrated only weak immunoreactivity. In addition, we observed an increase in ubiquitin immunolabeling of the hilar neuropil ipsilateral to the lesion at 1 and 3 days post-lesion, and a decrease in immunolabeling in the inner portion of the molecular layer at 3 days post-lesion. All these alterations were transient, and by 7 days post-lesion, ubiquitin immunoreactivity was indistinguishable in the hippocampus ipsilateral to the lesion, compared to the controls. Immunoblot analysis also revealed a decrease in the amount of ubiquitin in the hippocampus ipsilateral to the lesion 1 and 3 days post-lesion. These data suggest that deafferentation of the perforant pathway results in transient reduction in free ubiquitin of the hippocampus, and that the ubiquitin system is involved in hippocampal plasticity following perforant lesions.

Animals↗

Alterations of GABA(A)beta2/3 immunoreactivity in the dentate gyrus after perforant pathway lesion.

Immunocytochemical techniques were employed to examine the changes in the GABA receptor subunits beta2/3 within the dentate gyrus of the rat brain 1, 3, 7, 14, 30 and 90 days after a unilateral perforant pathway lesion. Three days post-lesion we observed a decrease in beta2/3 immunolabeling in the inner molecular layer of the dentate gyrus followed by a comparable decrease in the outer molecular layer 7 days post-lesion. These decreases were transient; 30 and 90 days post-lesion, beta2/3 immunolabeling appeared similar to controls in the inner portion of the molecular layer, while in the outer region the labeling was increased. In this latter region we also observed a dense band of AChE fibers. Following survival times of 3 days we observed a diffuse staining of the neuropil in the hilar region, and a dense amorphous accumulation of peroxidase reaction product in the polymorphic region. These responses were transient and by 14 days the hilar/polymorphic region appeared indistinguishable from controls. These data suggest a unique pattern of immunoabeling in the molecular and polymorphic region in response to perforant pathway lesion. A putative explanation for this response is discussed.

Acetylcholinesterase↗

Nitric oxide synthase immunoreactivity in the rat hippocampus after status epilepticus induced by perforant pathway stimulation.

Nitric oxide has recently been implicated in mediation of neuronal excitotoxicity and damage. This study aimed at elucidating the changes in the expression of neuronal isoform of nitric oxide synthase (nNOS) in the hippocampus after status epilepticus induced by perforant pathway stimulation. nNOS-immunoreactivity (nNOS-ir) and neuronal damage, assessed by silver staining, were evaluated separately in different hippocampal subfields 2 weeks after induction of status epilepticus. Perforant pathway stimulation resulted in an increase in the number of nNOS-immunoreactive neurons in the stratum radiatum of the CA1 and CA3 subfields of the hippocampus proper, and the hilus of the dentate gyrus. The morphology and distribution of the nNOS-ir neurons resembled that of interneurons. No correlation of the number of nNOS-ir neurons to the neuronal damage score was observed. Our results suggest that status epilepticus provokes a de novo expression of nNOS protein, and the nNOS expressing neurons may be selectively resistant to epileptic brain injury.

Animals↗

Influence of neurons of the parafascicular region on neuronal transmission from perforant pathway through dentate gyrus.

We have previously reported that activation of an ascending brainstem pathway by stimulation of the median raphe nucleus (MR) influences neuronal transmission from the perforant pathway through the dentate gyrus in a behaviorally dependent manner. In particular, stimulation of the MR markedly facilitated such transmission when applied during slow-wave sleep (SWS), but was ineffective when applied during the still-alert state (SAL). We present here evidence for a relay in this circuit located rostral to the MR in cells proximal to the fasciculus retroflexus (PF, parafascicular region). In contrast to stimulation of the MR, stimulation of the PF facilitates neuronal transmission from the perforant pathway through the dentate gyrus during both SWS and SAL indicating the presence of a gate at or proximal to the PF that is preferentially closed during SAL.

Animals↗

Reticular formation influence on neuronal transmission from perforant pathway through dentate gyrus.

Electrical stimulation of the perforant pathway discharges granule cell synchronously, giving rise to a characteristic evoked potential in the granule cell layer termed here the evoked action potential or EAP. In freely moving rats, we applied 3 pulses of low intensity electrical stimulation to the medullary reticular formation prior to the application of the perforant path pulse. The effect of prior reticular formation stimulation was a marked augmentation of the normal EAP response to the perforant path stimulus. The augmentation was dependent on the behavioral state of the experimental animal (it occurred during slow-wave sleep but not during still, alert behavior) and was eliminated by anesthetic agents. The latency of EAP augmentation effect (minimum effective time interval between application of the reticular formation stimulus and the perforant path pulse) was 13--18 msec. In order to localize the sites in the medullary reticular formation from which EAP augmentation could be elicited, threshold currents for producing the effect were determined during dorso-ventral penetrations of a reticular formation stimulating electrode. EAP augmentation was elicited at low stimulus currents from a relatively broad region of the reticular formation. It was also noted that reticular formation stimulation which produced EAP augmentation always elicited one or more motor responses of the neck, back, face or vibrissae. Subsequent investigation of the pathways underlying these motor responses suggested that the effect of reticular formation stimulation on granule cell excitability was mediated by a polysynaptic pathway, the first segment of which was a projection to cells of nucleus gigantocellularis of the caudal medulla.

Afferent Pathways↗

GABA(A) receptor gamma subunits in the hippocampus of the rat after perforant pathway lesion.

Immunohistochemical and Western blotting techniques were employed to examine the alterations in immunostaining of the gamma-amino butyric acid (GABA) receptor subunits gamma 1/3 and 2 within the hippocampus of the rat brain at 1, 3, 7, 14, and 30 days after a unilateral perforant pathway lesion. At 1, 3, and 7 days post-lesion, we observed a remarkable decrease in gamma 1/3 neuropil staining in the deafferented zone (i.e., the outer molecular layer of the dentate gyrus ipsilateral to the lesion), although at 3 and 7 days post-lesion, staining intensity was considerably recovered. At 14 days post-lesion, the gamma 1/3 immunostaining was indistinguishable from that of controls and it appeared yet more robust at 30 days post-lesion. We also observed a slight decrease in gamma 2 neuropil staining until 7 days post-lesion, and an increase in gamma 2 staining at 30 days post-lesion. Western blot analysis demonstrated data that was relatively consistent with our immunohistochemical observations, although gamma 3 was hardly detectable. Our study suggests that gamma subunits of the GABA(A) receptor in the dentate gyrus display a plastic response to the deafferentation of the perforant pathway.

Animals↗

Degenerative terminals of the perforant pathway are human alpha-synuclein-immunoreactive in the hippocampus of patients with diffuse Lewy body disease.

We investigated the hippocampal pathology in diffuse Lewy body disease (DLBD) using alpha-synuclein immunohistochemistry. Ubiquitin-positive intrahippocampal structures caused by the degeneration of terminal axons of the perforant pathway were observed to be alpha-synuclein immunoreactive. These alpha-synuclein-positive degenerative terminals contained granulo-filamentous or vesiculo-tubular components similar to those of Lewy bodies (LB) immunoelectron microscopically, suggesting that alpha-synuclein may abnormally aggregate into filamentous or membranous cytoskeletal components including neurofilaments and synaptic vesicles in DLBD. A 'dying back' degenerating process due to a blockage of axonal transport may explain why the degenerative terminals and LB share similar alpha-synuclein-positive components, but the origin cells of the perforant pathway contain only a few LB.

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Functional re-establishment of the perforant pathway in organotypic co-cultures on microelectrode arrays.

Co-cultures of entorhinal cortex (EC) and dentate gyrus (DG) explants are a useful model system to study the formation and stabilization of axonal projections. We adapted this model system to EC-DG co-cultures on microelectrode arrays (MEA) for the characterization of axonal projections on a functional level for days and weeks. EC and DG explants were placed on MEA to allow for the reconstitution of perforant pathway projections. Connections formed were characterized by morphological and electrophysiological analyses to verify characteristic features of perforant pathway signal transmission. Morphological analysis reveals proper projection of EC neurons into the molecular layer of the DG. Examination of synaptic transmission after high frequency stimulation imply unidirectional connections that used glutamate receptors of the AMPA/kainate type as main mediators of excitatory signal transmission. The system was evaluated by the introduction of the NCAM binding peptide C3d. In accordance with in vivo and in vitro experiments C3d modulated signal transmission by NCAM-related mechanisms resulting in morphological re-arrangements.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Activity of the conformationally rigid 2-amino-4-phosphonobutanoic acid (AP4) analogue (RS)-1-amino-3-(phosphonomethylene)cyclobutane-1-carboxylic acid (cyclobutylene AP5) on evoked responses in the perforant pathway of rat hippocampus.

The highly rigid and conformationally extended 2-amino-4-phosphonobutanoic acid (AP4) analogue (RS)-1-amino-3-(phosphonomethylene)-cyclobutane-1-carboxylic acid (cyclobutylene AP5) was synthesized and found to inhibit evoked responses in the rat lateral perforant path (LPP) with an IC50 of 41 (+/- 1.5 S.E.M.) microM and the medial perforant pathway with an IC50 of 218 (+/- 3.7 S.E.M.) microM. Furthermore, paired pulse potentiation experiments suggest that cyclobutylene AP5 acts, in part, at a presynaptic site in the LPP. Thus, cyclobutylene AP5 appears to act in a similar manner to L-AP4 in the perforant pathway. These data support the hypothesis that L-AP4 assumes an extended conformation at the L-AP4 receptor of the LPP.

Animals↗

Physiological identification and analysis of dentate granule cell responses to stimulation of the medial and lateral perforant pathways in the rat.

Stimulation of the dorsomedial or ventrolateral perforant pathways resulted in quantitatively different extracellularly recorded EPSPs in the fascia dentata of the rat. The two potentials differed in latency to peak and in width at half amplitude in a manner consistent with the different locus of termination of the two pathways on the granule cell dendrites. Both potentials were able to follow brief stimulus trains of 100 Hz, which suggests that they are monosynaptic. Medially elicited responses had their peak negativity approximately 100 to 180 micrometer deeper in the molecular layer than laterally elicited responses. Stimulation at short intervals along a dorsomedial to ventrolateral track in the angular bundle yielded a step function rather than a continuum of EPSP peak latency and half-width, in agreement with Hjorth-Simonsen's ('72) evidence for the separateness of the two pathways. Both pathways were able to induce granule cell discharge. Laterally elicited spikes, however, were delayed. Stimulation at intermediate locations frequently elicited double spikes from the granule cell population. Population spikes elicited by either pathway were inhibited for as long as 100 msec after a single discharge. Both pathways showed facilitation with double stimuli at short intervals, and both showed post-tetanic potentiation lasting at least 30 minutes. Under conditions where it could be shown that the two pathways at least partially converged onto the same granule cells, the response of one pathway did not increase when long lasting potentiation was induced on the other.

Animals↗

Reduction of adenosine A1-receptors in the perforant pathway terminal zone in Alzheimer hippocampus.

The cells of origin of the perforant pathway are destroyed in Alzheimer's disease (AD). In rat the adenosine A1-receptors are specifically localized on the perforant path terminals in the molecular layer of the dentate gyrus. In the present study the density of A1-receptors in the hippocampus of Alzheimer's disease (AD) patients (n = 9) and non-dement controls (n = 3) has been investigated autoradiographically with [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPDPX) as the ligand probe. In AD hippocampi binding of [3H]CPDPX was greatly reduced in the outer two thirds of the dentate gyrus molecular layer, likely due to the degeneration of the perforant path. Binding of [3H]CPDPX was not significantly altered in other parts of the AD hippocampus, e.g. the CA1 and the CA3, in spite of a pronounced cellular pathology and reduced N-methyl-D-aspartate (NMDA) receptor densities, assessed as strychnine insensitive [3H]glycine autoradiography. This contrasts with the presumed localization on dendrites of pyramidal neurons of A1 receptors within the CA1 and the CA3.

Adenosine↗

Activation of the dentate gyrus by stimulation of the contralateral perforant pathway: evoked potentials and long-term potentiation after ipsi- and contralateral induction.

Rats were chronically implanted with stimulation electrodes in the perforant pathway (pp) bilaterally and a recording electrode in the dentate gyrus (DG) unilaterally. Evoked field potentials (EPs) were recorded upon alternating stimulation of the pp on both sides, and long-term potentiation (LTP) was induced. Besides the EP after ipsilateral stimulation, an EP with a latency of approximately 5.5-6.5 ms was also seen upon stimulation of the contralateral pp. This potential was reversibly abolished during pentobarbital anesthesia and irreversibly after lesioning of the ipsilateral angular bundle. Paired-pulse facilitation and paired-pulse depression, depending on interstimulus interval and intensity, were also observed. Therefore, this long-latency potential could be characterized as polysynaptic and induced perhaps by transsynaptic activation via the ipsilateral entorhinal cortex. Ipsilateral tetanization induced strong E/S potentiation of both the ipsilaterally and contralaterally evoked EP, but with different time courses. Tetanization of the contralateral pp did not induce LTP of the ipsilaterally induced EP in the first 4 h. But afterwards a late and slowly developing potentiation occurred. The contralaterally induced EP also showed potentiation of the population spike, which was not immediately detectable but developed slowly over time. The results can be interpreted such that, after stimulation of the pp, the DG on the opposite side cannot only be activated via the weak crossed entorhinal projection but also transsynaptically via an entorhino/entorhinal connection.

Animals↗

Perforant pathway activation of hippocampal CA1 stratum pyramidale neurons: electrophysiological evidence for a direct pathway.

Electrophysiological techniques were used to investigate the effect of stimulating the perforant pathway (PP) on pyramidal neurons in the CA1 region of the hippocampal slice. Stimulation of the PP evoked both field potentials and single unit discharges in the pyramidal cell layer of the CA1 region. Several lines of experimental evidence suggest that the CA1 response does not involve granule or CA3 neurons: (i) movement of the recording electrode in the CA1 region away from the site of stimulation in the PP and closer to the CA3 region increased the latency of the evoked potential; (ii) the sum of latencies of the individual pathways in a trisynaptic circuit - from the PP to granule cells to CA3 neurons to CA1 neurons - was 2-3 times longer than the latency of the PP evoked response recorded in the CA1 region: (iii) lesioning the mossy fiber pathway or excising the CA3 region did not inhibit the CA1 response to PP stimulation. Other experimental results suggest that the PP activation of CA1 pyramidal neurons involves a direct synaptic pathway: (i) PP stimulation evoked potentials with similar latencies in the dentate gyrus and the CA1 region; (ii) the CA1 response was abolished in a Krebs' solution containing low calcium/high magnesium; (iii) excising a portion of the CA1 region between the stimulating electrode in the PP and the CA1 recording electrode, but sparing the PP, did not abolish the CA1 response; (iv) electrolytic lesions of the PP abolished the CA1 response to PP stimulation, but did not affect the CA1 response to stimulation of the CA3 region. The data suggest that fibers in the PP make direct synaptic connection with pyramidal neurons in the CA1 region of the hippocampus.

Animals↗

Examining the gateway to the limbic system with diffusion tensor imaging: the perforant pathway in dementia.

Current treatments for Alzheimer's disease (AD) are only able to slow the progression of mental deterioration, making early and reliable diagnosis an essential part of any promising therapeutic strategy. In the initial stages of AD, the first neuropathological alterations occur in the perforant pathway (PP), a large neuronal fiber tract located at the entrance to the limbic system. However, to date, there is no sensitive diagnostic tool for performing in vivo assessments of this structure. In the present bimodal magnetic resonance imaging (MRI) study, we examined 10 elderly controls, 10 subjects suffering from mild cognitive impairment (MCI), and 10 AD patients in order to evaluate the sensitivity of diffusion tensor imaging (DTI), a new MRI technique, for detecting changes in the PP. Furthermore, the diagnostic explanatory power of DTI data of the PP should be compared to high-resolution MRI volumetry and intervoxel coherences (COH) of the hippocampus and the entorhinal cortex, two limbic regions also involved in the pathophysiology of early AD. DTI revealed a marked decrease in COH values in the PP region of MCI (right side: 26%, left side: 29%, as compared to controls) and AD patients (right side: 37%, left side: 43%, as compared to controls). Reductions in COH values of the PP region were significantly correlated with cognitive impairment. DTI data of the PP zone were the only parameter differing significantly between control subjects and MCI patients, while the volumetric measures and the COH values of the hippocampus and the entorhinal cortex did not. DTI of medial temporal brain regions is a promising non-invasive tool for the in vivo diagnosis of the early/preclinical stages of AD.

Aged↗