Perforant Path Projection Research Articles

Effects of Prenatal Ethanol Exposure on Phospholipase C‐β1 and Phospholipase A2 in Hippocampus and Medial Frontal Cortex of Adult Rat Offspring

Previous studies in our laboratory using a rat model of fetal alcohol exposure (FAE) suggest that FAE-induced behavioral deficits are, in part, linked to neurochemical and electrophysiological deficits in long-term potentiation (LTP) in the entorhinal cortical perforant path projection to the hippocampal formation. Several findings suggest that signal-activated phospholipase C (PLC) and phospholipase A2 (PLA2) are critical to the induction and maintenance of LTP. Thus, alterations in phospholipid metabolism may play a significant role in the LTP deficits observed in FAE offspring. To test this hypothesis, we measured PLC-beta 1 and PLA2 activities in the hippocampus and medial frontal cortex of adult rats prenatally exposed to ethanol. PLC-beta 1 activities were significantly decreased by 20 to 30% in both the hippocampus and medial frontal cortex of FAE rats, compared with ad libitum and pair-fed controls. Total Ca(2+)-dependent PLA2 activity was 25% lower in the medial frontal cortex of FAE rats, but did not significantly differ from controls in the hippocampal formation. Approximately 30% of the measured activity in both the medial frontal cortex and hippocampal formation of ad libitum and pair-fed animals was associated with an 85 kDa cytosolic PLA2 form. Cytosolic PLA2 activities were significantly reduced in both the medial frontal cortex and hippocampal formation of FAE rats, compared with controls. These changes in Ca(2+)-dependent PLA 2 and PLC-beta 1 activities, coupled with reports of FAE-induced deficits in protein kinase C activity, indicate that prenatal exposure to moderate quantities of ethanol causes profound and long-lasting deficits in the cellular signaling mechanisms associated with activity-dependent synaptic plasticity and memory formation.

Morphological and Immunophenotypic Microglial Changes in the Denervated Fascia Dentata of Adult Rats: Correlation with Blood–Brain Barrier Damage and Astroglial Reactions

The reactions of microglial and astroglial cells to anterograde axonal degeneration were studied in the fascia dentata of adult rats from 1 to 42 days after removal of the entorhinal perforant path projection. The observations focused on the kinetics of glial activation in terms of induction of immunomolecules on the glial cells and the possible correlation between these changes and lesion-induced extravasation of plasma constituents. Normal and activated microglial cells were identified by immunohistochemical visualization of the constitutively expressed complement type 3 receptor (CR3/CD11b). Activated microglial cells were stained immunohistochemically for the inducible major histocompatibility complex (MHC) antigen class I and class II and the leukocyte common antigen (LCA/CD45). Astroglial cells were identified by immunohistochemical staining for glial fibrillary acidic protein (GFAP). Blood–brain barrier (BBB) conditions were primarily evaluated by immunohistochemical staining for extravasated Immunoglobulin G (IgG), but also by intravenously injected horseradish peroxidase (HRP) and Evans Blue. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path terminal zones displayed an increase in CR3 immunoreactivity, changes of morphology and an induced expression of MHC antigen class I. At the same time there was a hitherto undescribed leakage of IgG through the BBB (albeit without detectable extravasation of HRP and Evans Blue). One day later microglial cells also expressed LCA, but MHC antigen class II was not induced under these degenerative conditions. The activation of microglial cells occurred prior to a noticeable hypertrophy of astroglial cells and increase in GFAP immunoreactivity, as this first became evident on Postlesional Day 2. From the results we conclude (1) that perforant path axonal degeneration induces an endothelial transcytosis of blood-borne IgG by mechanisms which cannot be envisioned by conventional HRP tracer methods and (2) that the early activation of both microglial cells and astroglial cells is likely to be initiated and later influenced by both axonal degeneration and extravasated plasma constituents. The demonstration of an early induction of immunomolecules on activated microglial cells and extravasation of blood borne molecules might moreover form the basis for a correspondingly early intervention aiming to regulate microglial immunomolecule, cytokine, and growth factor gene expression in the affected areas.

The perforant path projection to hippocampal area CA1 in the rat hippocampal-entorhinal cortex combined slice.

1. The perforant path projection from layer III of the entorhinal cortex to CA1 of the hippocampus was studied within a hippocampal-entorhinal combined slice preparation. We prevented contamination from the other main hippocampal pathways by removal of CA3 and the dentate gyrus. 2. Initially the projection was mapped using field potential recordings that suggested an excitatory sink in stratum lacunosum moleculare with an associated source in stratum pyramidale. 3. However, recording intracellularly from CA1 cells, stimulation of the perforant path produced prominent fast GABAA and slow GABAB IPSPs often preceded by small EPSPs. In a small number of cells we observed EPSPs only. 4. CNQX blocked excitatory and inhibitory responses. This indicated the presence of an intervening excitatory synapse between the inhibitory interneurone and the pyramidal cell. 5. Focal bicuculline applications revealed that the major site of GABAA inhibitory input was to stratum radiatum of CA1. 6. The inhibition activated by the perforant path was very effective at reducing simultaneously activated Schaffer collateral mediated EPSPs and suprathreshold-stimulated action potentials. 7. Blockade of fast inhibition increased excitability and enhanced slow inhibition. Both increases relied upon the activation of NMDA receptors. 8. Perforant path inputs activated prominent and effective disynaptic inhibition of CA1 cells. This has significance for the output of hippocampal processing during normal behaviour and also under pathological conditions.

Synaptic and intrinsic properties of neurons of origin of the perforant path in layer II of the rat entorhinal cortex in vitro.

Layer II of the entorhinal cortex (EC) provides the first step in the hippocampal trisynaptic loop via the perforant path projection to the dentate gyrus. While a great deal is known about this projection and the properties of the dentate granule cells, much less information is available concerning the properties of and synaptic inputs to the cells of origin of the pathway in layer II. The present experiments have employed a slice preparation of the rat EC to study the intrinsic membrane properties and synaptic organization of layer II neurons. Two types of neurons could be identified electrophysiologically. The majority were designated type I and displayed a pronounced time-dependent inward rectification in the hyperpolarizing direction. Type II displayed little evidence of this characteristic. However, morphological examination suggested that both types were spiny stellate neurons projecting via the perforant path. Synaptic responses of both types displayed evidence of excitatory inputs mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. In general, however, at low frequencies the responses were dominated by inhibitory inputs mediated by both GABAA and GABAB receptors. At higher frequencies the bias was shifted much more toward excitation. The contribution of synaptic and intrinsic properties of layer II neurons to the processing capabilities of the EC is discussed.

Microglial and astroglial reactions to anterograde axonal degeneration: a histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions.

The reaction of microglial and a stroglial cells to anterograde axonal degeneration was studied in the fascia dentata of adult rats at various timepoints after removal of the entorhinal perforant path projection. Microglial cells were identified by histochemical staining for nucleoside diphosphatase (NDPase) at light and electron microscopical levels. Astroglial cells were stained immunocytochemically for glial fibrillary acidic protein (GFAP). Activated astroglial cells and some microglial cells also stained immunocytochemically for the intermediate filament protein vimentin. Phagocytotic activity was detected by histochemical staining for acid phosphatase. The postlesional connective reorganization of the cholinergic septohippocampal projection was monitored by histochemical staining for acetylcholinesterase. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path zones of the fascia dentata and the adjacent neuropil reacted by shortening and coarsening of processes and an increase in NDPase reactivity. These changes occurred prior to a noticeable increase in GFAP immunoreactivity and hypertrophy of astroglial cells (first evident on postlesional day 2) or sprouting of cholinergic septohippocampal fibres (first evident on day 3). There was evidence of an early, local proliferation of microglial cells in the denervated perforant path zones and migration into these zones of microglial cells from adjacent intact areas. The specific accumulation of strongly stained microglial cells within the denervated parts of the dentate molecular layer persisted for at least 4 weeks, while the astroglial reaction subsided at 3 weeks. The results demonstrate an early activation of microglial cells by axonal degeneration, and indicate that these cells may play a pivotal, inductive role in the subsequent glial and neural events.

Opioid receptor-dependent long-term potentiation at the lateral perforant path-CA3 synapse in rat hippocampus

The involvement of opioid receptors in the induction of long-term potentiation (LTP) was investigated in the lateral and medial perforant path projections to area CA3 of the hippocampus in anesthetized rats. The opioid receptor antagonist naloxone (10 nmol), applied to the hippocampal CA3 region 10 min prior to tetanization, blocked the induction lateral perforant path-CA3 LTP induced by high-frequency stimulation. By contrast, LTP induction in medial perforant path-CA3 was not attenuated by a 10 nmol quantity of naloxone. (+)-Naloxone (10 nmol), the inactive stereoisomer of naloxone, was without effect on the induction of lateral perforant path-CA3 LTP. Naloxone applied l h following LTP induction did not reverse established lateral perforant path-CA3 LTP, indicating that opioid receptors are involved in the induction but not the maintenance of LTP in this pathway. LTP of medial perforant path responses developed immediately, while LTP of lateral perforant path responses was slow to develop. The latter pattern is similar to the time course of the development of LTP observed at the mossy fiber-CA3 synapse and suggests that lateral and medial perforant path synapses may use distinct mechanisms of both induction and expression of LTP. These data extend previous findings demonstrating opioid receptor-dependent mechanisms of LTP induction at both the mossy fiber-CA3 synapse and the lateral perforant path-dentate gyms synapse. We suggest that lateral perforant path and mossy fiber synapses may utilize similar, opioid receptor-dependent, mechanisms of LTP induction and expression.

Xenografts of mouse hippocampal tissue, exchange of laminar and neuropeptide specific nerve connections with the host rat brain

Immature hippocampal and fascia dentata tissue from embryonic and newborn C57 mice was grafted to the hippocampal region of newborn Kyoto rats. The age of the donor mice varied from embryonic day 13 to the day of birth, and the recipient rats from the day of birth up to 2 days. After survival times of from 5 weeks to 1 year the recipient brains were histologically processed for the tracing of host-xenograft connections by silver staining and electron microscopy of anterograde degeneration, AChE histochemistry, immunohistochemical demonstration of the neuropeptides CCK add enkephalin, and the histochemical Timm sulphide silver method, as well as stained by ordinary cell and fiber stains. The survival of the xenografts depended on the donor age, with less than 10% survival for newborn donors and 60–69% for E13–16 donors. The surviving xenografts developed an organotypic organization and retained a mouse-specific CCKreactivity in the associational hilodentate system and the dentate mossy fibers. Judged by their positive AChE histochemistry most xenografts received a host rat cholinergic projection when placed in normal cholinoreceptive areas, including areas outside the normal reach of the septo-hippocampal system like the neocortex. Xenografts encroaching on the trajectory of the host rat commissural and perforant path projections or their terminal fields in fascia dentata received laminar and neuropeptide specific host projections. Electron microscopy of host rat perforant path fibers traced to the xenograft dentate molecular layer confirmed the laminar distribution and revealed numerous asymmetric synaptic contacts with spines. An efferent xenograft projection of CCK-reactive mouse mossy fibers into the host CA3 mossy fiber layer demonstrated that this cross-species, mouse to rat innervation also applied to the normal developmental rules, despite the, for the rat abnormal, CCK-content. The formation of laminar and neuropeptide specific mouse-rat nerve connections demonstrates the potentials of intracerebral neuronal grafting in basic and applied neurobiological research by providing new experimental models for the analysis of developmental and functional interactions between nerve cells.

Exploratory behavior and reaction to novelty in rats with hippocampal perforant path systems disrupted.

In this article, exploratory behavior and reaction to novelty were investigated in rats with either disruption of the total hippocampal perforant path projection (TPP), the medial perforant path (MPP), or lateral perforant path (LPP). All three experimental groups displayed increased locomotor activity, and the LPP group was even more active than the TPP and MPP groups. The TPP and LPP animals made more rearings than the control rats. The MPP and LPP groups exhibited less exploration of familiar objects than the TPP and control groups. TPP animals appeared unable to recognize novelty, LPP rats showed decreased preference for novelty, and MPP rats had only a slight deficit in recognition of novelty. It is suggested that perforant path disruptions reduce the hippocampal formation's access to sensory information of neocortical origin.

Intracephalic transplants of freeze-stored rat hippocampal tissue.

The survival and cellular and connective organization of intracephalic transplants of developing, freeze-stored rat hippocampal tissue were examined. Blocks of tissue containing the hippocampus and fascia dentata were obtained from late embryonic (E16-E22) and early postnatal rats (P0-P4) and immersed in a tissue culture medium with 10% of the cryoprotective agent DMSO, frozen at a cooling rate of approximately 1 degree C/minute, and stored for 1-226 days in liquid nitrogen. After quick thawing and washing out of the DMSO the tissue blocks were transplanted to the brain of adult rats. From 2 weeks to 3 months later the recipient brains were processed histologically. The cellular and connective organization of the transplants and their interaction with the host brains were analyzed after thionin cell staining, Timm's staining for hippocampal and dentate afferents, immunohistochemical staining for enkephalin-, CCK-, and somatostatin-reactive neurons and afferents, AChE staining for cholinergic afferents, and silver stains for fiber architectonics and tracing of connections by anterograde axonal degeneration. Freeze-storage narrowed the range of donor ages with good transplant survival. The best surviving hippocampal and dentate transplants thus came from 17-21-day-old embryos. There was no correlation between the length of storage and survival. Structurally the transplants of stored tissue were more frequently fragmented than the transplants of fresh tissue when located outside the brain parenchyma in the brain ventricles. This was in accordance with the results of a previous study of grafts of freeze-stored and fresh hippocampal tissue placed in the anterior eye chamber. Despite the decrease in survival and the tendency for fragmentation many well-structured and organotypically organized hippocampal and dentate transplants were recovered corresponding to the donor ages E19-E21. In addition to the main cell types (granule cells and pyramidal cells) the freeze-stored transplants also contained peptidergic nerve cells reacting for CCK, somatostatin, and enkephalin. The organization of the intrinsic nerve connections and the exchange of connections with the host brain were similar for transplants of stored and fresh tissue. Besides the consistent innervation of the hippocampal and dentate transplants by host cholinergic afferents monitored by AChE staining, several appropriately located dentate transplants thus sent mossy fibers to the host CA3. Others received host perforant path projections. A CA3-associated transplant projection to the denervated perforant path zones in the host fascia dentata was also observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Complementary effects of unilateral striatal dopamine depletion and amphetamine infusions on a lateralized visual reaction time task in rats

When rats trained to forage in one environment are exposed to a second, highly similar environment, their hippocampal place code exhibits a partial remapping in the new environment that becomes more complete with repeated exposures (Shapiro, Tanila, Eichenbaum, Hippocampus 7 (6) (1997) 624–642, Bostock, Muller, Kubie, Hippocampus 1 (2) (1991) 193–206). If the perforant path projection to CA3 functions as a pattern completion mechanism, and the DG projection via the mossy fibers performs pattern separation (O'Reilly, McClelland, Hippocampal conjunctive encoding, storage, and recall: avoiding a trade-off, Hippocampus 4 (6) (1994) 661–682), then partial remapping can be understood as the combined effect of these two projections. We investigated learning rules that could be responsible for the gradual separation of two maps, and found that, while simple Hebbian learning and Hebbian covariance learning would not produce the separation effect, BCM learning was one rule that would.

Perforant pathway lamination in the dentate gyrus is unaffected by prenatal ethanol exposure

The entorhinal (perforant path) projection to the dentate gyrus was labeled with an anterograde horseradish peroxidase method to test whether prenatal exposure to ethanol affected the normal development of afferent lamination. Mean ethanol consumption of the ethanol-consuming dams was 12.7 g/kg±0.3 g per day during days 1–21 of gestation. Adult offspring of normal and pair-fed controls as well as ethanol-exposed rats were analyzed. Computer-assisted image analysis of the entorhinal terminal field organization revealed no permanent changes in the development of the afferent lamination pattern in the dentate gyrus molecular layer in spite of the heavy in utero ethanol exposure.

Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat

The immunohistochemical localization of calcium-binding protein (CaBP) in the cerebellum, hippocampal formation and olfactory bulb of the rat was examined using rabbit anti-human or sheep anti-chick antisera purified by affinity chromatography. CaBP-like immunoreactivity was observed within the somata and dendrites of: (1) cerebellar Purkinje cells; (2) dentate granule cells, CA1 pyramidal cells and scattered interneurons in the stratum radiatum of the hippocampus; (3) periglomerular cells in the olfactory bulb. Staining was conspicuously absent in certain major cell types in each of these structures including cerebellar granule cells, hippocampal pyramidal cells in the CA3 region and both mitral and granule cells in the olfactory bulb. Immunoreactive fibers in the cerebellum presumably corresponding to climbing fiber inputs from the inferior olive and efferent projections to the deep cerebellar nuclei, were also observed. In the hippocampus intense staining was present in the mossy fiber projection to the CA3 region and in the terminal regions of the perforant path projection from entorhinal cortex.

Physiological compensation for loss of afferent synapses in rat hippocampal granule cells during senescence.

1. The effects of senescence on the input-output characteristics of the perforant path projection to granule cells of the fascia dentata were studied in rats using extracellular techniques in vivo, and both extra- and intracellular recording in vitro. 2. Senescent animals exhibited a significant reduction in the perforant path excitatory synaptic field potential at all stimulus intensities tested. This was associated with a reduction in the size of the afferent fibre response, although there was no apparent change in the threshold for fibre activation. These data support the anatomical literature which indicates a loss of afferent synapses with advanced age. 3. For a given magnitude of afferent fibre response, however, the old animals exhibited a larger synaptic field potential, suggesting that the remaining synapses were in fact more powerful. Furthermore, the magnitude of the extracellular population spike, an index of the number of discharging granule cells, was greater in the old animals when plotted as a function of extracellular e.p.s.p. amplitude. 4. Intracellular recording from a total of 190 granule cells in the transverse hippocampal slice preparation revealed a 17% reduction in the voltage threshold for synaptically elicited granule cell discharge, and a 13% reduction in the latency of the action potential in old compared to young rats. Resting potentials, action potential amplitudes, whole neurone time constants, the relations between applied current and input resistance, and the discharge threshold following depolarizing current pulses, were not different between age groups. 5. These data indicate that granule cells could partly compensate for a loss of synapses during senescence by an increase in their electrical responsiveness to synaptic activation and possibly by an increase in synaptic efficacy.

Factors specifying the development of synapse number in the rat dentate gyrus: effects of partial target loss

The development of the dentate gyrus has been studied under conditions of partial reduction of granule cell number. Neonatal rats were subjected to X-irradiation, a procedure which reduces the number of granule cells to 20% of control values. In X-irradiated rats, quantitative analyses were performed on cells in the entorhinal cortex which give rise to the perforant path projection to the dentate granule cells, and on the remaining, undamaged dentate granule cells. These residual cells were examined morphologically for possible hyperdevelopment in comparison to granule cells from control animals. Granule cells in X-irradiated animals were similar to granule cells in control animals with respect to dendritic structure and synaptic density. The number of neurons in both the medial and lateral entorhinal cortices in X-irradiated animals appeared normal until day 12, at which time a selective reduction in cell numbers became apparent. By day 30, 25–55% of the cells of origin of the perforant path were absent in X-irradiated animals. It is hypothesized that these cells are subject to retrograde transynaptic degeneration as a result of target removal. Further, it appears that granule cells play an important role in determining the density of their innervation.

Locomotor and avoidance behavior in rats with partial or total hippocampal perforant paths sections

Bilateral partial damage to the hippocampal perforant paths in rats produced impaired two-way active avoidance performance, while the behavior in open field, passive and one-way active avoidance remained unchanged. Total disruption of the perforant path projection resulted in increased open field activity and impaired one-way active avoidance, but normal passive avoidance behavior. The results are discussed in terms of reduced sensory information to the hippocampal formation.