Re-establishment of electrophysiologically functional entorhinal cortical input to the dentate gyrus deafferented by ipsilateral entorhinal lesions: Innervation by the contralaeral entorhinal cortex

Summary We have unilaterally ablated the entorhinal cortex of the developing rat, thereby removing the major synaptic input to the ipsilateral dentate gyrus. We have then examined the efferent projections of the remaining contralateral entorhinal cortex to determine if these might reoccupy the synaptic territory vacated by the ipsilateral entorhinal fibers. By placing lesions in the remaining contralateral entorhinal cortex, and tracing the resulting degeneration products, we show that the contralateral entorhinal projection is reorganized, establishing an anomalous terminal projection to the dentate gyrus deafferented by the initial lesion. The result is a contralateral entorhinal innervation of the dentate gyrus which normally receives only ipsilateral entorhinal afferents.In addition, we investigate the functional capacity of these anomalous contralateral projections. We show that stimulation of the entorhinal cortex in the normal animal never results in short latency activation of the granule cells contralateral to the stimulating electrode, whereas in the lesioned animals, the contralateral entorhinal stimulation results in a short latency, apparently monosynaptic, evoked potential in the dentate gyrus which had been deprived of its ipsilateral entorhinal innervation. Furthermore, this stimulation results in the discharge of cells in the granule cell layer.Therefore, following unilateral entorhinal lesion, the remaining contralateral entorhinal cortex extends its efferent projection to establish electrophysiologically functional synapses with the granule cells deafferented by the initial lesions.The material in this paper was included in a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of California at Irvine, Irvine Calif.     

Growth of a new fiber projection in the brain of adult rats: Re-innervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions

Summary Ablation of the entorhinal cortex of the rat removes the major synaptic input to the granule cells of the ipsilateral dentate gyrus. Following unilateral entorhinal lesions in adult rats, we have examined the efferent projections of the remaining contralateral entorhinal cortex to determine if these might sprout to re-innervate the deafferented dentate gyrus. Autoradiographical tracing of the fiber projections of the remaining contralateral entorhinal cortex 60 days following lesions indicates that new fibers sprout and grow for several hundred microns into the denervated regions, to terminate on portions of the granule cell dendrites which would normally receive ipsilateral entorhinal afferents.These re-innervating fibers form electrophysiologically functional synaptic connections with the denervated dentate granule cells. In the normal animal, unilateral stimulation of the entorhinal cortex does not result in short latency activation of the contralateral dentate gyrus whereas following ipsilateral entorhinal lesions, re-innervation by contralateral entorhinal afferents is reflected electrophysiologically by the appearance of a new short latency evoked potential to contralateral entorhinal stimulation. By field potential analysis, we demonstrate that this new short latency evoked potential is a reflection of mono-synaptic activation of the denervated dentate granule cells by the re-innervating contralateral entorhinal fibers.In addition, the time course of contralateral entorhinal re-innervation is determined electrophysiologically. The new short latency response to contralateral entorhinal stimulation appears as early as 9 days post-lesion, matures functionally between 9 and 15 days, and after 15 days, remains apparently undiminished for as long as 200 days. This implies that the new synapses formed in response to a deafferenting lesion are formed rapidly and remain permanently capable of activating the dentate granule cells which had been deprived of ipsilateral entorhinal input.The material in this paper was included in a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of California at Irvine, Irvine, California 92664.     

Mossy fiber sprouting in the fascia dentata after unilateral entorhinal lesions: quantitative analysis using computer-assisted image processing

Axon sprouting typically occurs in a brain region that has been partially denervated. The present study demonstrates, quantitatively, evidence for sprouting outside the region of deafferentation. A modification of the Timm sulfide silver histochemical method was used to monitor an increase in the mossy fiber terminal field in the fascia dentata of adult rats following severe deafferentation of the outer three-fourths of stratum moleculare by unilateral entorhinal lesions. Computer-assisted image processing techniques were used to quantify mossy fiber sprouting. In stratum granulosum, and to a lesser extent in the deep (supragranular) portion of stratum moleculare (areas separated from the zone of deafferentation), there was a three-fold increase in the area of mossy fiber staining on the side of the lesion compared to the non-operated side (and unoperated animals). Much of the increased staining was located near the tip of the infrapyramidal (ventral) blade of the fascia dentata. Since mossy fiber sprouting apparently occurs in the absence of degeneration-produced synaptic dilution in that region, it may represent an example of post-lesion growth initiated by conditions fundamentally different from those normally believed to induce sprouting.     

Specific patterns of fibre outgrowth from transplants to host mice hippocampi, shown immunohistochemically by the use of allelic forms of Thy-1

Foetal mouse hippocampal primordia from mice homozygous for the Thy-1.1 allele were transplanted into the hippocampal region of adult histocompatible mice, homozygous for the Thy-1.2 allele. After survival periods of two months to one year the transplants consisted of a discrete tissue mass, well fused with the host, and distinguished from it by an intense Thy-1.1 immunoreactivity of the neuropil. The host hippocampus was entirely negative for Thy-1.1 immunoreactivity except for well defined projections arising from the transplant. These projections had three different patterns of distribution in the host: (1) a mossy-fibre-like distribution (to the stratum lucidum of field CA3); (2) a fimbria-like distribution (to the stratum oriens and stratum radiatum of fields CA1 and CA3), and (3) a commissural/association-like distribution (to the inner stratum moleculare of the dentate gyrus). Analysis of the position of the transplants and their component cell types indicated that, for each of the three distribution patterns, three conditions had to be fulfilled: (1) the transplant had to contain the appropriate type of cell (granule cells for mossy fibre projections, and larger pyramid-like cells for the other two); (2) the transplant neurons giving rise to the projection had to lie in direct contact with the host field to be innervated, and (3) the terminal field had to be specifically denervated. Thus, for three fibre systems the patterns of transplant-to-host projections observe rules of organization comparable to those of the normal hippocampal circuitry. This implies that in the adult host brain there remain (or there can be elicited) the molecular cues capable of establishing patterns of neuronal connectivity comparable to those formed during normal development.     

Vascular endothelial growth factor and its receptor Flk-1 are expressed in the hippocampus following entorhinal deafferentation

Vascular endothelial growth factor (VEGF) has been thought of as a mitogen that promotes proliferation of endothelial cells and as a neurotrophic factor that stimulates neurogenesis and axonal growth in both peripheral and central nervous systems. To investigate the potential involvement of VEGF in the lesion-induced reorganization in the brain, the expression changes of VEGF and its receptor Flk-1 were analyzed in the mouse hippocampus after transections of the entorhinal afferents. In situ hybridization and immunohistochemistry showed the time-dependent expression upregulation of VEGF mRNA and protein in the entorhinally denervated hippocampal stratum lacunosum-moleculare and dentate outer molecular layer, which initiated by 3 days postlesion, reached its maximum at 7-15 days postlesion, still persisted by 30 days postlesion for protein, and recovered to the normal levels at 30 days postlesion for mRNA and at 60 days postlesion for protein. Double labeling of VEGF and glial fibrillary acidic protein revealed that VEGF-expressing cells in the denervated areas were reactive astrocytes. Semi-quantitative RT-PCR analysis showed that VEGF receptor Flk-1 mRNA was also time-dependently upregulated in the deafferented hippocampus with its maximal elevation at 7-15 days postlesion while the Flt-1 mRNA levels remained unchanged at any time point we examined. Immunohistochemistry analysis also displayed the upregulation of Flk-1 protein in the denervated stratum lacunosum-moleculare and outer molecular layer with a time course similar to that of VEGF mRNA upregulation. Flk-1 receptors were found to be expressed not only by reactive astrocytes but also by neurites, which most likely belong to sprouting axons by 7 days postlesion and regrowing dendrites by 15-30 days postlesion. From these data we suggest that the spatiotemporal upregulation of VEGF and Flk-1 in the hippocampus is induced by entorhinal deafferentation and that VEGF may be involved in the structural reorganization in the deafferented hippocampus via directly or indirectly promoting neurite growth.     

Polysynaptic activation of the dentate gyrus of the hippocampal formation: An olfactory input via the lateral entorhinal cortex

Summary The possibility that olfactory input is transmitted to specific subregions of the hippocampal formation via the entorhinal cortex was investigated electrophysiologically by analyzing the laminar profiles of potentials evoked in the hippocampal formation by stimulation of the lateral olfactory tract (LOT). LOT stimulation resulted in long latency (14–20 ms) evoked responses in the dentate gyrus of the hippocampal formation ipsilateral to the stimulation. The variable long latency of these responses and their inability to follow stimulus rates of 40/s suggested that these potentials reflected polysynaptic activation. Analysis of the laminar profiles of the evoked potentials indicated that the responses originated from a synaptic field localized in the outer portion of the stratum moleculare of the dentate gyrus, a terminal distribution which overlaps that of the lateral entorhinal cortical (LEC) projection to the dentate gyrus. Lesions of the LEC eliminated the long latency responses in the dentate gyrus evoked by LOT stimulation. In addition, a conditioning pulse delivered either to the LOT or to the LEC produced paired pulse potentiation of the response elicited by subsequent stimulation of the other structure. No evidence was found to indicate that responses were generated in regio superior of the hippocampus proper following LOT stimulation. Taken together, these results suggest that stimulation of the LOT activates the dentate gyrus of the hippocampal formation by multisynaptic pathways which relay through the lateral portion of the entorhinal area. This finding is discussed with regard to entorhinal cortical organization and the known olfactory projections to the LEC.Some of this material was presented in abstract form at the 7th Annual Meeting of the Society for Neuroscience, 1977     

An electrophysiological analysis of chronic ethanol neurotoxicity in the dentate gyrus: Distribution of entorhinal afferents

Summary This study investigated the persistent effects of chronic ethanol consumption on the distribution of entorhinal afferents to stratum moleculare of the dentate gyrus. Rats were maintained on ethanol, or sucrose-containing liquid diets, for a period of 20 weeks but were withdrawn from the special diet for at least 8 weeks prior to acute electrophysiologic recordings. One-dimensional laminar analyses were obtained by stepping a microelectrode in 25 m increments across both the dorsal and ventral blades of the dentate gyrus and sampling the field potentials at each point. Current-source density (CSD) was calculated from the field potential data.Electrical stimulation of the angular bundle elicited a short-latency, negative field potential covering the outer 2/3 of the molecular layer. CSD analysis revealed a major current sink in stratum moleculare bounded by a major current source, localized to the hilus, granule cell layer, and proximal stratum moleculare, and a minor current source localized to the outer molecular layer. Chronic ethanol treatment resulted in (1) a significant shrinkage of the spatial extent of the current sink in stratum moleculare, (2) a significant reduction in the distance from the peak inward synaptic current to the granule cell layer and (3) no change in the distance from the proximal inversion point to the granule cell layer. Taken together, these results indicate a loss of entorhinal afferents in the outer molecular layer. Coupled with available anatomical evidence, these results suggest that chronic ethanol treatment produces a preferential loss of lateral entorhinal afferents to the dentate gyrus.Supported by funds from the Medical Research Service of the Veterans Administration, NIAAA Grant AA-00200 to Don W. Walker, NIAAA predoctoral fellowship AA-05153 to WCA and NINCDS grant NS-10229 to William Brownell     

Synaptic proliferation in the auditory cortex of the young adult rat following callosal lesions

Summary The long-term effects of partial deafferentation in the neocortex of adult rats were studied in four-month old rats in which the corpus callosum had been completely sectioned when they were one-month old. Quantitative light microscopy was used to identify morphological changes in the auditory cortex resulting from the loss of established callosal connections. Particular attention was directed at those cortical layers known to receive the heaviest callosal projection (layers II and III) and at neurons known to be postsynaptic to callosal afferents (layer V pyramidal neurons). The comparative analysis of both semithin plastic sections and Golgi-impregnated material from long-term, callosally-lesioned rats and age-matched control animals reveals no differences in the overall cortical thickness, the thickness of cortical layers, the numbers of neurons or the density of spines along apical dendrites of layer V pyramidal neurons. However, as a result of the callosal lesion, large diameter apical dendrites are significantly thinner in the callosally deafferented cortex and there is a small increase in the number of neuroglial cells in the deeper cortical layers.To determine whether another system of afferents to the auditory cortex spreads into the deafferented callosal domain, geniculate lesions were made in long-term, callosally-lesioned animals and age-matched controls. The terminal projection patterns of thalamic afferents were compared using the Fink-Heimer technique and quantitative electron microscopy. Normally in the auditory cortex there is only a small region of overlap between the terminal projection fields of callosal afferents and thalamic afferents, the latter projecting chiefly to layer IV and low layer III. However, three months after callosal lesions, thalamic axons had proliferated superficially into part of the callosal domain. Furthermore, in the normal auditory cortex after geniculate lesions, there were three rostrocaudally oriented bands of relatively dense thalamocortical terminal degeneration separated by regions of less dense degeneration. In the doubly lesioned animals these bands of degeneration were less distinct due to a proliferation of thalamic axons into the regions characterized by sparse projections.Part of this study was done by SF in partial fulfillment of the requirements for the degree of Master of Arts, Boston University, Boston, MA, USA.     

Localization of two major GABA(A) receptor subunits in the dentate gyrus of the rat and cell type-specific up-regulation following entorhinal cortex lesion

GABA(A) receptor subunits show a specific regional distribution in the CNS during development and in the adult animal. In the hippocampal formation, individual subsets of GABAergic interneurons are highly immunoreactive for the alpha1-subunit, whereas granule and pyramidal cells show a strong expression of the alpha2-subunit. Using confocal microscopy and digital image analysis, we demonstrate that in the dentate gyrus the alpha1-subunit immunolabeling appears in differently sized clusters. The large clusters, which are confined to dendrites of interneurons, show no alpha2 labeling, whereas the smaller ones coincide with alpha2-subunit-positive clusters. In the molecular layer, the clusters of both alpha-subunits co-localize with the anchoring protein gephyrin. In the granule cell layer and hilus, we found alpha1- and alpha2-subunit-positive clusters which were devoid of gephyrin labeling. Lesions of the medial entorhinal cortex led to the deafferentation of dendrites in the middle molecular layer of the dentate gyrus. This resulted in a significantly increased concentration of alpha2-subunit-positive clusters. We also observed an increase of alpha1-subunit immunolabeling in the deafferented area. We found no change in the co-localization between alpha1 and alpha2, and no significant change in the number of large alpha1-positive clusters along individual dendritic segments of interneurons. In a previous study, we demonstrated that calbindin-immunoreactive dendrites of granule cells revealed a significant increase in gephyrin immunoreactivity following lesion, whereas parvalbumin-positive dendrites showed no such alterations. The predominant localization of small gephyrin clusters in dendrites of granule cells, which was also described in this study, leads to the conclusion that the increase of the alpha2-subunit-positive clusters, demonstrated in the present study, indicates that, following entorhinal cortex lesion, new GABAergic synapses may be formed and that they contact predominantly granule cell dendrites.     

Evidence for the sprouting of entorhinal afferents into the "hippocampal zone" of the molecular layer of the dentate gyrus.

Summary Fragments of the dentate gyrus containing portions of the stratum granulosum and the overlying stratum moleculare were isolated from their commissural, associational and hypothalamic inputs in 11 and 28-day-old rats. Six weeks later the distribution of the entorhinal afferents to the isolated portion of the stratum moleculare was determined autoradiographically by the injection of ³H-proline into the medial entorhinal area, and the appearance of the molecular layer was examined in Timm's sulfide silver preparations. It is evident from our material that under these circumstances the entorhinal afferents to the dentate gyrus can extend right up to the stratum granulosum and that the normal staining pattern that characterizes the hippocampal zone of the molecular layer in Timm's preparations may disappear and be replaced, in part, by a narrow zone of dense, mossy fiber-like staining.The simplest interpretation of these findings is that fibers from the medial entorhinal area are capable of sprouting into the deafferented hippocampal zone of the dentate gyrus. However, the observation that entorhinal afferents may occupy the entire thickness of the molecular layer does not, by itself, establish this point because in every case examined the whole molecular layer was shrunken. While this calls for some caution in the interpretation of the findings, our results suggest that there is no special barrier to the growth of entorhinal fibers and that the inner part of molecular layer of the dentate gyrus is capable of receiving afferents other than those normally present.This work was supported in part by grants NS-10943 and EY-01255 from the National Institutes of Health     

Axonal transport and axon sprouting in the adult rat dentate gyrus: An autoradiographic study

In response to an entorhinal lesion, the commissural and associational afferents to the dentate gyrus have been shown to expand beyond their normal terminal zone into the area denervated by the entorhinal lesion. The present study has investigated the axonal transport of [³H]-labeled proteins in the commissural and associational projections following an entorhinal lesion. Injections of [³H]proline, [³H]leucine or [³H]fucose were given in the vicinity of the commissural and associational cells of origin before, immediately subsequent to, or at 5 to 15 days after the entorhinal lesion. The disposition of previously- or newly-synthesized proteins was examined in the commissural and associational terminal field at different times after an entorhinal lesion by light-microscopic autoradiography. The expanded commissural and associational zone was labeled whenever injections were given on, or before, the day of the entorhinal lesion and the width of the expanded zone increased with the time after the lesion. However, in contrast to the expanded associational projection, the expanded commissural projection was usually not labeled when the injections were made near the time that afferent reactivity commences (approximately 5 days after the lesion) and the animals were killed 2–4 days later. The expanded commissural projection was labeled, however, if injections were made at a later stage of afferent reactivity and the animals were killed 1 day later.The selective labeling of the expanding associational zone, when animals receive injections near the period of afferent reactivity and are killed 2–4 days later, appears to be due to the ability of labeled, slowly transported material to mark selectively the sprouting associational axons. The failure to label the expanding commissural zone in this experimental situation, is ascribed to the failure of labeled, fast-axonally transported materials to mark the sprouting commissural axons.Pre-labeling the normal commissural/associational projections several days in advance of the entorhinal lesion did not alter the subsequent labeling pattern of the expanding commissural/associational zone. Thus, proteins present in cells at the time of the lesion participate in the formation of axonal sprouts.     

Ontogeny of the noradrenergic innervation of the rat hippocampal formation

Summary The noradrenergic (NA) innervation of the rat hippocampal formation arrives embryonically into a structure in which cytogenesis and cell migration are still active processes. At embryonic day 18 (E18) the first fluorescent axons appear in the septal end of CA3 at the boundary of the marginal zone and cortical plate, the future stratum lucidum. By birth axons invade the subiculum and also course along the septo-temporal axis in a longitudinal associational system in stratum moleculare of CA3. The innervation of the area dentata increases significantly by postnatal day four (P4). The innervation pattern throughout the dentate and Ammon's horn is fairly complete by P10. High affinity uptake of ³H-NA also matures embryonically and correlates postnatally with the extent of innervation estimated by fluorescence histochemistry. The levels of endogenous NA develop more slowly, showing only 60–80% of older adult values by P48.Compared to the maturation of other hippocampal afferents, the NA innervation is extremely precocious. It is localized in areas which could allow it to have significant trophic functions during early stages of histogenesis. In addition, its presence in the rapidly developing structure may contribute to its eventual distribution in a relatively less organized terminal pattern than that of the later-arriving entorhinal, commissural and septal afferents.     

Histochemical changes in enzymes of energy metabolism in the dentate gyrus accompany deafferentation and synaptic reorganization

The dentate gyrus of adult rats was examined histochemically for cytochrome oxidase and lactate dehydrogenase activity after unilateral lesions of the entorhinal cortex. In normal animals, synaptic terminal fields of the perforant pathway from the entorhinal cortex show high levels of cytochrome oxidase activity (the other two-thirds dentate molecular layer), whereas terminal zones of the commissural and associational fibers show high levels of lactate dehydrogenase activity (the inner one-third dentate molecular layer). Lesions of the entorhinal cortex result in a significant reduction in staining for cytochrome oxidase in the deafferented outer molecular layer of the dentate gyrus. The changes become prominent at 16-24 h after the lesion and persist until 90 days, the longest post-lesion survival time studied. In the non-deafferented inner zones ipsilateral to the lesion, there is an increase in staining for cytochrome oxidase and lactate dehydrogenase at 24 h post-lesion that disappears by days 2-4. From 8 to 90 days post-lesion, the band of high reactivity for lactate dehydrogenase in the inner molecular layer spreads approximately 40 microns into the overlying deafferented zone. This expansion parallels the expansion of the commissural and associational terminal fields into the adjacent deafferented molecular layer. Thus, lesion-induced synaptogenesis in the dentate gyrus is accompanied by a corresponding change in enzyme activity. The results indicate that the pattern of activity of enzymes involved in energy metabolism in the dentate gyrus depends on the distribution of pathway-specific synaptic input.     

The effect of unilateral and bilateral removal of the entorhinal cortex on the glucose utilization in various hippocampal regions in the rat

In the present study lesion-induced changes in function of various hippocampal regions, as reflected by the metabolic rate of glucose, were measured by means of quantitative autoradiography, 4 days after unilateral or bilateral surgical removal of the entorhinal cortex. The greatest decrease (45%) was seen in the stratum lacunosum moleculare of the CA1, whereas a lesser decline (34%) was seen in the molecular layer of the dentate gyrus, stratum lucidum of the CA3 (31%) and the stratum radiatum of the CA1 (36%). These findings support the view that in addition to the indirect trisynaptic temporo-ammonic pathway, there is a functionally active direct pathway.     

Electrophysiological analysis of the dorsal hippocampal commissure projections to the entorhinal area

Synaptic effects evoked in the entorhinal area by dorsal hippocampal commissure (dorsal psalterium) projections were analysed in anesthetized adult guinea-pigs by means of a field potential analysis. Stimuli applied to the caudal part of the dorsal psalterium evoked a complex response in the dorsal third of the entorhinal area. The early part of the entorhinal response consisted of a slow wave interrupted by a spike potential. The electrophysiological characteristics and the laminar distribution of the slow wave and of the spike potential, together with the presence of time-locked unit activity, suggested that dorsal psalterium projections evoke monosynaptic excitatory postsynaptic potentials leading to cellular discharge in radially oriented neurons of layers II and III. The commissural fibers responsible for these effects originate in the contralateral presubiculum. The early part of the entorhinal response was followed by three waves in close temporal sequence. These waves were polysynaptically generated and associated with excitatory and inhibitory synaptic effects. Inhibition was demonstrated for the monosynaptically generated spike potential. Whether these effects were mediated by intracortical circuits and/or extrinsic projections cannot be stated from the present results. Causal relations were observed between the entorhinal monosynaptic response and that evoked by dorsal psalterium stimulation in the ipsilateral dentate gyrus, previously shown to be relayed by perforant path fibers. The results indicate that presubicular commissural projections to the entorhinal area monosynaptically activate neurons of the perforant pathway, whose discharge brings about activation of the ipsilateral dentate gyrus.     

Projections from the presubiculum and the parasubiculum to morphologically characterized entorhinal-hippocampal projection neurons in the rat

The relations between the inputs from the presubiculum and the parasubiculum and the cells in the entorhinal cortex that give rise to the perforant pathway have been studied in the rat at the light microscopical level. Projections from the presubiculum and the parasubiculum were labeled anterogradely, and, in the same animal, cells in the entorhinal cortex that project to the hippocampal formation were labeled by retrograde tracing and subsequent intracellular filling with Lucifer Yellow. The distribution and the number of appositions between the afferent fibers and hippocampal projection neurons in the various layers of the entorhinal cortex were analyzed. The results show that layers I–IV of the entorhinal cortex contain neurons that give rise to projections to the hippocampal formation. The morphology of these projection neurons is highly variable and afferents from the presubiculum and the parasubiculum do not show a preference for any specific morphological cell type. Both inputs preferentially innervate the dendrites of their target cells. However, presubicular and parasubicular projections differ with respect to the layer of entorhinal cortex they project to. The number of appositions of presubicular afferents with cells that have their cell bodies in layer III of the entorhinal cortex is 2–3 times higher than with cells in layer II. In contrast, afferents from the parasubiculum form at least 2–3 times as many synapses on the dendrites of cells located in layer II than on neurons that have their cell bodies in layer III. Cells in layers I and IV of the entorhinal cortex receive weak inputs from the presubiculum and parasubiculum. Not only is the presubiculum different from the parasubiculum with respect to the distribution of projections to the entorhinal cortex, they also differ in their afferent and efferent connections. In turn, cells in layer II of the entorhinal cortex differ in their electrophysiological characteristics from those in layer III. Moreover, layer II neurons give rise to the projections to the dentate gyrus and field CA3/CA2 of the hippocampus proper, and cells in layer III project to field CA1 and the subiculum. Therefore, we propose that the interactions of the entorhinal-hippocampal network with the presubiculum are different from those with the parasubiculum.     

Factors affecting survival and outgrowth from transplants of entorhinal cortex

The purpose of this study was to examine injury-related effects on the survival and growth of entorhinal tissues transplanted to the adult rat entorhinal area. Embryonic entorhinal cortex was transplanted to the angular bundle region of adults either immediately, or 8-10 days, after severing specific host projections. Graft survival (Nissl stain) and connectivity (acetylcholesterase stain and retrograde labeling with wheat germ agglutinin-horseradish peroxidase) were examined two months post-transplantation. Grafts transplanted 8-10 days after severing the angular bundle were large and contained many cells which innervated the hippocampal formation. Grafts transplanted immediately after severing the angular bundle were small, did not integrate well with the host tissues, and failed to innervate the hippocampal formation. Grafts transplanted without producing any prior lesion, or following lesions which did not damage host entorhinal projections, were intermediate in size, but failed to innervate the hippocampal formation. The data demonstrate that: (1) introducing a delay between the lesion and implant surgeries can significantly enhance graft survival and the establishment of transplant-to-host projections, and (2) transplant-derived innervation of the host is significantly impaired when host homologous fibers are intact. These findings suggest that environmental factors, induced specifically by the destruction of host homologous fibers, are responsible for the differences in transplant survival and connectivity observed.     

Autoradiographic evidence that septohippocampal fibers reinnervate fascia dentata denervated by entorhinal lesion during development

Summary the projection from the ventromedial septum to the fascia dentata was investigated autoradiographically in normal adult rats and in adult rats whose entorhinal cortex had been removed unilaterally at the age of 11 days. In the fascia dentata of normal rats and in the fascia dentata contralateral to the entorhinal lesion septohippocampal fibers and terminals were distributed just below and, to a lesser extent, just above the granular layer. The molecular layer above the supragranular zone was lightly and more or less uniformly innervated. Ipsilateral to the entorhinal lesion, however, the outer part of the dentate molecular layer received an anomalously dense septal projection (average of 3–4 times the contralateral projection). The entorhinal lesion did not consistently affect the density of this projection in any other lamina. These results confirm that septohippocampal fibers increase their density of innervation when synaptic sites are made available by degeneration of lateral perforant path fibers during development. This represents a net increase in total septal innervation of the fascia dentata, not merely a change in the distribution of the projection among its target zones.     

Differential regulation of Ca(2+)-calmodulin stimulated and Ca(2+)-insensitive adenylyl cyclase messenger RNA in intact and denervated mouse hippocampus

The Ca(2+)-calmodulin stimulated AC1 and Ca(2+)-insensitive AC2 are major isoforms of adenylyl cyclase, playing an important role in synaptic plasticity in the mammalian brain. We studied the pattern of expression of AC1 and AC2 genes in the hippocampus of C57BL/6 mice. We found that there were differences in their patterns of distribution in the dentate gyrus. AC1 messenger RNA was detected both in the dentate granule cell bodies and the corresponding molecular field whereas AC2 messenger RNA was preferentially distributed in the dentate granule cell layer, suggesting that AC1 and AC2 messenger RNA are differentially regulated in the dentate gyrus. In order to examine the regulation of AC1 and AC2 expression in response to synaptic deafferentation and reinnervation, the distribution patterns of the two AC messenger RNA in the hippocampal fields and the parietal cortex were analysed 2, 5, 9 and 30 days following an unilateral entorhinal cortex lesion. Interestingly, we found significantly reduced levels of AC1 hybridization signal following the lesion whereas the level of AC2 messenger RNA remained unaffected in all lesioned groups. The changes in AC1 messenger RNA were transient, with a maximal reduction at five days postlesion, and were restricted to the granule cell bodies and stratum moleculare of the deafferented dentate gyrus. No significant change in AC1 messenger RNA levels was detected in other hippocampal fields nor for any other postlesion times studied.These findings suggest that, at least in the dentate gyrus, messenger RNA for AC1 and AC2 might be differentially compartmentalized in cell bodies and dendritic fields. The activity-dependent regulation of AC1 messenger RNA levels by afferent synapses may provide an elegant mechanism for achieving a selective local regulation of AC1 protein, close to its site of action.