Time-dependent changes in commissural field potentials in the dentate gyrus following lesions of the entorhinal cortex in adult rats.

Previous neuroanatomical work has shown that lesions of the entorhinal cortex in adult rats cause the commissural projections to spread from their normally restricted locus in the inner molecular layer approximately 40-50 mum into the outer molecular layer (that is, into the zone deafferented by the lesion). In the present study we measured the effects of the entorhinal lesion on the distribution of short-latency potentials elicited by commissural stimulation in the molecular layer. Studies with animals tested at various times after the lesion and with a preparation that permitted recording from the same rat at several post-lesion intervals both indicated that the commissural response spread 100-150 mum towards the deafferented outer molecular layer, while the maximum response spread 50-100 mum. These effects were first detectable by 9 days after the lesion and were fully developed by 15 days post-lesion. These findings suggest that the growth of the commissural system seen after entorhinal lesions results in the rapid formation of functional terminals and are discussed in relationship to the behavioral consequences of brain lesions.     

Enhanced but delayed axonal sprouting of the commissural/associational pathway following a combined entorhinal cortex/fimbria fornix lesion

From previous lesion studies of the hippocampus it has been reported that axons of the commissural/associational pathway expand their termination zone in the molecular layer of the dentate gyrus by 20–25% in response to loss of input from the entorhinal cortex. However, although much is known about the response of the commissural/associational pathway with regard to extent, latency, and speed of the reinnervation response following an entorhinal cortex lesion, little is known about how the loss of additional afferent systems might modulate this response. To address this issue, we examined at 14, 30, and 45 days postlesion, the sprouting of commissural/associational afferents following either a unilateral fimbria fornix transection, a unilateral entorhinal cortex lesion, or combined lesions of both the entorhinal cortex and the fimbria fornix. Loss of septal innervation to the hippocampus was assessed using the cholinesterase stain, whereas sprouting from the commissural/associational pathway was determined from Holmes fiber-stained sections. In addition, the Timms stain was used to examine the time course of the loss of terminal fields of the various zinc-containing afferent systems within the hippocampus. Following the removal of input to the hippocampus via the fimbria fornix transection, there was no evidence of sprouting of the commissural/associational fibers into the deafferented portion of the dentate gyrus. In contrast, rats receiving an entorhinal cortex lesion showed a significant increase (28%) in the width of the commissural/associational fiber plexus that was present by 14 days postlesion. By comparison, the magnitude of the expansion of the commissural/associational fiber plexus was significantly larger after lesioning both the entorhinal cortex and the fimbria than after the entorhinal cortex lesion alone (45% vs. 28%). In addition, the expansion of the commissural/associational fiber plexus that was not increased at 14 days postlesion but was significantly increased at 30 days postlesion. The delay in the sprouting of the commissural/associational pathway coincided with the time course of loss of zinccontaining fibers in the outer molecular layer of the dentate gyrus as assessed with the Timms stain. These results suggest that the magnitude and time course for the sprouting of axons from the commissural/associational pathway into the partially deafferented hippocampus of the adult rat is lesion dependent and that the effect of the loss of input from the entorhinal cortex can be modulated and enhanced by the concomitant depletion of input from the fimbria fornix. © 1995 Willy-Liss, Inc.     

Histochemical evidence of altered development of cholinergic fibers in the rat dentate gyrus following lesions: II. Effects of partial entorhinal and simultaneous multiple lesions

It has been concluded previously that the septohippocampal fibers which project to the rat dentate gyrus extend or branch in the denervated area of the molecular layer following a complete ipsilateral entorhinal lesion. The septohippocampal fibers thus appear to replace some of the perforant fibers which degenerate as a result of the lesion. The reactive fibers eventually become localized to a much smaller and more superficial area after lesions of immature rats than after lesions made in adulthood. To determine whether this difference in the response results from a selective reaction to loss of the lateral perforant path in the immature rat, various portions of the entorhinal cortex were removed at the age of 11 days, and the cholinergic septohippocampal fibers were visualized by acetylcholinesterase histochemistry. An alternative possibility, that the difference between immature and adult rats is attributable to an interaction with other reactive afferents, was tested by removing other sources of input (the contralateral entorhinal cortex, contralateral hippocampal formation or both) along with the ipsilateral entorhinal cortex at the age of 11 days and then demonstrating the septohippocampal fibers histochemically. Lesions of the lateral part of the ipsilateral entorhinal cortex (source of the lateral perforant path) at 11 days of age evoked a septohippocampal reaction along the outer edge of the molecular layer, where the lateral perforant path fibers normally terminate. This result matched that produced by a complete entorhinal lesion. Lesions of the medial entorhinal cortex evoked no obvious reaction. In contrast, the septohippocampal fibers in adult rats proliferated in the denervated area of the molecular layer after lesions of either part of the entorhinal cortex. Combining lesions of other sources of innervation to the dentate gyrus with an ipsilateral entorhinal lesion at 11 days of age did not alter the response of septohippocampal fibers, as determined histochemically. Neither did the septohippocampal fibers react to removal of commissural afferents alone. The response at any age was unaffected by prior or subsequent removal of the contralateral entorhinal cortex. These results indicate that in immature rats the septohippocampal fibers respond only to loss of the lateral perforant path, but these same fibers can later react to loss of any part of the perforant path. They are regarded as support for the hypothesis that the reactive septohippocampal fibers preferentially interact with dendritic growth cones. Our results do not support explanations based on a hypothetical attraction between septohippocampal and crossed perforant path fibers (which react in the same area) or on competition with commissural fibers (which reinnervate an adjacent area). We suggest further that proximity to the degenerating elements does not in itself determine the pattern of reinnervation after lesions of the central nervous system.     

Comparison of commissural sprouting in the mouse and rat fascia dentata after entorhinal cortex lesion

Reactive axonal sprouting occurs in the fascia dentata after entorhinal cortex lesion. This sprouting process has been described extensively in the rat, and plasticity-associated molecules have been identified that might be involved in its regulation. To demonstrate causal relationships between these candidate molecules and the axonal reorganization process, it is reasonable to analyze knockout and transgenic animals after entorhinal cortex lesion, and because gene knockouts are primarily generated in mice, it is necessary to characterize the sprouting response after entorhinal cortex lesion in this species. In the present study, Phaseolus vulgaris-leucoagglutinin (PHAL) tracing was used to analyze the commissural projection to the inner molecular layer in mice with longstanding entorhinal lesions. Because the commissural projection to the fascia dentata is neurochemically heterogeneous, PHAL tracing was combined with immunocytochemistry for calretinin, a marker for commissural/associational mossy cell axons. Using both techniques singly as well as in combination (double-immunofluorescence) at the light or electron microscopic level, it could be shown that in response to entorhinal lesion mossy cell axons leave the main commissural fiber plexus, invade the denervated middle molecular layer, and form asymmetric synapses within the denervated zone. Thus, the commissural sprouting response in mice has a considerable translaminar component. This is in contrast to the layer-specific commissural sprouting observed in rats, in which the overwhelming majority of mossy cell axons remain within their home territory. These data demonstrate an important species difference in the commissural/associational sprouting response between rats and mice that needs to be taken into account in future studies.     

Accelerated rates of synaptogenesis by “sprouting” afferents in the immature hippocampal formation

Light and electron microscopic methods were used to study the rate with which undamaged afferents sprout into and form synapses within denervated dendritic zones in the immature rat brain. The middle and outer molecular layers of the dentate gyrus were deafferented by ablation of the ipsilateral entorhinal cortex in 14-day-old rats, and the extension of the commissural projections, which are normally restricted to the inner molecular layer, into the denervated territory was studied by light microscopic autoradiographic tracing methods. Collateral growth was noted as early as 13 hours after the lesion and was found to reach throughout the middle and outer molecular layers by 48 hours postlesion. Quantitative analyses (grain counts) revealed that the addition of commissural fibers and terminals to the denervated zones proceeded extremely rapidly up to 72 hours after the removal of the entorhinal cortex, but slowed markedly thereafter. Electron microscopic procedures were used to assess the rate at which synapses formed during this period and yielded the following information: 1) The density of intact synapses fell to below 20% of normal values within 20 hours of the lesion, 2) reinnervation began before 30 hours postlesion, and 3) the rate at which synapses were added to deafferented middle molecular layer was much more rapid from 20–96 hours postlesion than is observed in this zone during normal development. Furthermore, the sprouting of the inner molecular layer afferents into the middle molecular layer did not retard the pace of synaptogenesis in their normal target region. These results suggest that intrinsic limitations in the capacity of axons and dendrites are not responsible for determining the rate of synaptogenesis in the developing hippocampus. It is proposed instead that existing synapses (or terminals and spines) tend to suppress the formation of new contacts such that the local density of connections regulates the speed with which further innervation occurs.     

The effects of neonatal 6-hydroxydopamine treatment on morphological plasticity in the dentate gyrus of the rat following entorhinal lesions

Lesions of the entorhinal cortex, which is the major source of afferents to the outer two thirds of the molecular layer of the dentate gyrus, induce an expansion of the commissural projection, which is normally restricted to the inner third, and an intensification of acetylcholinesterase staining in the outer portion of the layer; these changes are thought to be due to the sprouting of the commissural fibers and certain cholinergic afferents to the dentate gyrus, respectively. We have studied these sequelae of entorhinal lesions in young adult rats which had been treated with 6-hydroxydopamine (6-OHDA) as neonates, in order to determine whether in the absence of its normal noradrenergic input, morphological plasticity in the dentate gyrus would be altered either in magnitude or extent. In animals treated with 6-OHDA, the levels of noradrenaline in the hippocampal formation were reduced by 93%. Despite this, there was clear evidence for an expansion of the commissural projection following entorhinal lesions, as judged both autoradioraphically and in Timm-stained material. Similarly, the intensification of acteylcholinesterase staining in the outer part of the molecular layer appeared as marked as after comparable lesions in untreated animals. From these observations it would appear that in the dentate gyrus, at least, morphological plasticity does not require the presence of an intact noradrenergic innervation.     

Stereological analysis of the reorganization of the dentate gyrus following entorhinal cortex lesion in mice

Denervation of the dentate gyrus by entorhinal cortex lesion has been widely used to study the reorganization of neuronal circuits following central nervous system lesion. Expansion of the non-denervated inner molecular layer (commissural/associational zone) of the dentate gyrus and increased acetylcholinesterase-positive fibre density in the denervated outer molecular layer have commonly been regarded as markers for sprouting following entorhinal cortex lesion. However, because this lesion extensively denervates the outer molecular layer and causes tissue shrinkage, stereological analysis is required for an accurate evaluation of sprouting. To this end we have performed unilateral entorhinal cortex lesions in adult C57BL/6J mice and have assessed atrophy and sprouting in the dentate gyrus using modern unbiased stereological techniques. Results revealed the expected increases in commissural/associational zone width and density of acetylcholinesterase-positive fibres on single brain sections. Yet, stereological analysis failed to demonstrate concomitant increases in layer volume or total acetylcholinesterase-positive fibre length. Interestingly, calretinin-positive fibres did grow beyond the border of the commissural/associational zone into the denervated layer and were regarded as sprouting axons. Thus, our data suggest that in C57BL/6J mice shrinkage of the hippocampus rather than growth of fibres underlies the two morphological phenomena most often cited as evidence of regenerative sprouting following entorhinal cortex lesion. Moreover, our data suggest that regenerative axonal sprouting in the mouse dentate gyrus following entorhinal cortex lesion may be best assessed at the single-fibre level.     

The distriution of the commissural-associational afferents of the dentate gyrus after perforant path lesions in one-day-old rats

Lesions were made in the entorhinal cortex of one-day-old rats and the distribution of the axons in the dentate gyrus molecular layer studied with the Holmes silver stain when the animals reached adulthood. The commissural-associational projections, normally restricted to the inner dendritic zones, spread evenly throughout the molecular layer ipsilateral to the lesion. This pattern of aberrant growth is markedly different from that which occurs after entorhinal lesions placed in 7-day-old rats. The results are discussed in terms of the factors that dictate the topography of developing afferents.     

Reactive plasticity in the dentate gyrus following bilateral entorhinal cortex lesions in cynomolgus monkeys

Hippocampal structural plasticity induced by entorhinal cortex (EC) lesions has been studied extensively in the rat, but little comparable research has been conducted in primates. In the current study we assessed the long-term effects of bilateral aspiration lesions of the EC on multiple markers of circuit organization in the hippocampal dentate gyrus of young adult monkeys (Macaca fascicularis). Alternate histological sections were processed for the visualization of somatostatin and vesicular acetylcholine transporter (VAChT) immunoreactivity and acetylcholinesterase histochemistry (AChE). The markers revealed the distinct laminar organization of dentate gyrus circuitry for stereology-based morphometric quantification. Consistent with findings in rats, the volume of the somatostatin-immunopositive outer molecular layer (OML), innervated by projections from the EC, was decreased by 42% relative to control values. The inner molecular layer (IML) displayed a corresponding volumetric expansion in response to denervation of the OML as measured by AChE staining, but not when visualized for quantification by VAChT immunoreactivity. Nonetheless, stereological estimation revealed a 36% increase in the total length of VAChT-positive cholinergic fibers in the IML after EC damage, along with no change in the OML. Together, these findings suggest that despite substantial species differences in the organization of hippocampal circuitry, the capacity for reactive plasticity following EC damage, previously documented in rats, is at least partly conserved in the primate dentate gyrus.     

Rapid expression and transport of embryonic N-CAM in dentate gyrus following entorhinal cortex lesion: Ultrastructural analysis

Neural cell adhesion molecules are known to be important in axon guidance and synapse formation in the developing brain. The embryonic form of neural cell adhesion molecule (eN-CAM) is reexpressed in the outer molecular layer (OML) of the dentate gyrus following entorhinal cortex (ERC) lesion. Ultrastructural analysis revealed localization of eN-CAM to the membrane of granule-cell dendritic membranes and occasionally axons within the denervated zone. Because eN-CAM is expressed rapidly (within 2 days) after ERC lesion, we were interested in the temporal sequence of expression. Denervated hippocampi (12, 15, 24, and 48 hours post-ERC lesion) were stained with anti-eN-CAM and processed for immunoelectron microscopy. At 12 hours, there was no evidence of staining for eN-CAM. By 15 hours after lesion, membranes of both dendrites and axons throughout the molecular layer exhibited moderate eN-CAM staining, and dendritic cytoplasm was heavily labeled. Twenty-four hours following lesion, plasma membrane staining of eN-CAM on both axons and dendrites had increased in intensity within the OML, whereas membrane eN-CAM staining was diminished in the inner molecular layer (IML), and the intradendritic cytoplasmic staining disappeared. By 48 hours after lesion, eN-CAM staining had disappeared from the IML but remained intense and widely distributed in the OML. These findings suggest a rapid transport of de novo synthesized protein. A generalized reaction appears to occur immediately following denervation, and eN-CAM is up-regulated in the complete expanse of the dendritic membrane, despite the fact that only the OML is denervated. The newly up-regulated eN-CAM is rapidly withdrawn or disappears from the membrane in the (nondenervated) IML over the 24–48 hours postlesion. The brain rapidly responds to injury at the cellular level in the denervated zone in preparation for renervation by axon sprouting. © 1994 Wiley-Liss, Inc.     

Muscarinic acetylcholine receptor immunoreactivity after hippocampal commissural/associational pathway lesions: Evidence for multiple presynaptic receptor subtypes

The present study addressed the hypothesis that differential localization of m1–m4 subtypes in the inner one-third of the dentate molecular layer is due to selective presynaptic expression of the receptor proteins on the hippocampal commissural/associational pathways. Physical and chemical lesions of the commissural and associational pathways were used to denervate afferent terminals in the inner one-third of the molecular layer, and fluid injections were used to lesion granule cells, their postsynaptic target. Immunocytochemistry utilizing muscarine acetylcholine receptor (mAChR) subtype-specific antibodies was used to identify changes in expression patterns in the molecular layer postlesion. m1 immunoreactivity in the molecular layer did not change after commissural/associational pathway lesions. m2 immuno-reactivity in the inner one-third of the molecular layer was attenuated only after lesions involving the associational pathway. In contrast, m3 and m4 immunoreactivity in the inner one-third of the molecular layer was almost completely abolished by lesions of both pathways simultaneously. Granule cell lesions greatly attenuated m1 and m3 immunoreactivity in the molecular layer, with little to no diminution of m2 and m4 immunoreactivity. The results indicate that, in the inner one-third of the molecular layer, m1 and m3 are mainly postsynaptic on granule cells, whereas m2 and m4 are presynaptic on the commissural/associational pathways. This study provides direct anatomical evidence for the diversity of molecular subtypes presynaptically on the commissural/associational pathways. J. Comp. Neurol. 380:382–394, 1997. © 1997 Wiley-Liss, Inc.     

Sprouting of central noradrenergic fibers in the dentate gyrus following combined lesions of its entorhinal and septal afferents

Virtually all of the afferents to the hippocampal formation undergo collateral sprouting after removal of adjacent afferent systems. However, the central noradrenergic (NA) afferents, which demonstrate a remarkable propensity for regeneration and sprouting in other regions of the brain, have not been found to sprout in the denervated hippocampal formation. The present study was designed to determine if the pattern of innervation by NA fibers in the dentate gyrus of adult rats can be altered by interruption of the other major afferents. The innervation pattern of NA fibers was examined in the dentate gyrus 4 weeks after removal of the ipsilateral and/or contralateral entorhinal afferents and/or transection of the fimbria-fornix and supracallosal stria. The noradrenergic identity of the fibers was indicated by immunoreactivity for dopamine beta hydroxylase (DBH) and peripheral sympathetic fibers were demonstrated by immunoreactivity for nerve growth factor receptor (NGFr), which did not stain cholinergic fibers in this application. In control brains, the noradrenergic innervation of the dentate molecular layer was light and uniform across the width of the layer. Transection of the perforant path (ipsilateral entorhinal afferents) or ventral hippocampal commissure (contralateral entorhinal afferents) resultd in a significant increase in innervation density in the outer half of the molecular layer, and the combination of these two lesions produced the greatest increase. In those brains with transection of the ipsilateral and contralateral entorhinal afferents, the denervated dentate gyrus had a nearly twofold increase in density of DBH-immunoreactive fibers within the outer half of the molecular layer. These fibers tended to course parallel to the pial surface rather that perpendicular as in control sections. Transection of the fimbria-fornix alone had no affect on the innervation pattern of DBH-ir fibers in the molecular layer. When the fimbria-fornix was transected in combination with both of the other lesions, an overall increase in innervation density occurred, but there was no further increase in the difference between the inner and outer halves of the molecular layer. No NGFr-immunoreactive fibers were observed in the molecular layer in any of the brains, indicating that the DBH-immunoreactive fibers in this region were not of peripheral origin. It is concluded that removal of the ipsi- and contralateral entorhinal afferents to the dentate gyrus results in the sprouting of central NA fibers in the outer half of the molecular layer. © 1994 Wiley-Liss, Inc.     

Transneuronal changes in dendrites of GABAergic parvalbumin-containing neurons of the rat fascia dentata following entorhinal lesion

The perforant path fibers from the entorhinal cortex form synapses with both granule cells and GABAergic, parvalbumin-containing (PARV) nongranule cells. The authors recently reported a persistent reduction of PARV-positive dendrites in the termination zones of entorhinal fibers in the hippocampus proper and fascia dentata after lesion of the entorhinal cortex. In the present study the authors analyzed the effects of de-entorhination on the ultrastructure of postsynaptic PARV-positive dendrites in the molecular layer of the fascia dentata. PARV immunocytochemistry was performed 2, 8, 55, and 360 days after an ipsilateral entorhinal lesion and, for comparison, 10 days after an ipsilateral fimbria-fornix transection that disconnects the hippocampus from its septal and commissural afferents. Two days after entorhinal lesion, the authors observed swelling of the tissue close to the hippocampal fissure. Adjacent distal dendritic tips of PARV-positive dendrites in the former perforant path termination zone persisted 55 days after entorhinal lesion and could still be observed after postlesional survival times for 1 year. Degenerating axon terminals were still present 55 days following lesion and PARV-positive dendrites exhibited abnormal invaginations. Fimbria transection did not result in similar dendritic changes in PARV-positive neurons. The results indicate a long-lasting process of reorganization in the molecular layer of the fascia dentata followin entorhinal lesion and persisting changes in the morphology of PARV-immunoreactive dendrites. Entorhinal fibers seem to play a specific role for the maintenance of these dendrites, since similar changes did not occur following removal of septal and commissural fibers.     

The organization of the embronic and early postnatal murine hippocampus. II. Development of entorhinal,commissural, and septal connections studied with the lipophilic tracer DiI

We have analyzed the early development of the main hippocampal afferents in the mouse. Following injections of the lipophilic tracer 1–1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchorate (DiI) in the entorhinal cortex, entorhinal axons were observed for the first time inthe hippocampus at E15, in the white matter, At E17, entorhinal fibers arborized within the stratum lacunosum-moleculare. At subsequent stages entorhinal axons formed dense networks that were restricted to their appropriate termination zone in the lacunosum-moleculare. The first axons invading the fascia dentata were noticed at E19, their density increasing at later stages. These axons were mainly present in the outer molecular layer. This onset of entorhinohippocampal projections was corroborated by retrograde labeling data after injections in the hippocampus. Commissural fibers first entered the contralateral hippocampus at E18, their number increasing at the following stages. Commissural axons arborized within the stratum oriens and radiatum in the hippocampus proper. In the fascia dentata, the earliest commissural fibers were seen at P2, terminating in the inner zone of the molecular layer and in the hilus. We conclude that developing entorhinal and commissural axons show a high degree of laminar specificity from the earliest stages of formation, which is compatible with the notion that distinct subsets of early maturing neurons populating the hippocampal plexiform layers may attract particular fiber systems. Hippocamposeptal fibers develop at E15, before the first septal fibers can be detected in the hippocampus. These early hippocamposeptal fibers originated from nonpyramidal neurons and terminated in the medial septal area, which is the main source of septal afferents to the hippocampus. In contrast, septohippocampal fibers were not seen in the hippocampus until E17. At perinatal stages, the hippocamposeptal connection reshapes, sending axons to the dorsolateral septal area as the innervation of the medial septum becomes less conspicuous. This sequence suggests that hippocampal neurons pioneer the formation of septohippocampal connections. © 1994 Wiley-Liss, Inc.     

The effect of collateral sprouting on the density of innervation of normal target sites: implications for theories on the regulation of the size of developing synaptic domains

The ‘commissural’ innervation of the denate gyrus molecular layer has been analyzed in normal adult rats and in those in which the ipsilateral entorhinal cortex had been removed by aspiration at 14 days post-natal. This ablation severely deafferents the distal two-thirds of the molecular layer and induces ‘sprouting’ by the commissural afferents which are normally restricted to the more proximal dendritic zone. It was the objective of the present study to employ quantitative electron microscopy to determine (1) the extent of synaptic recovery in the deafferented field; (2) the magnitude of the contribution by the commissural fibers to the reinnervation of the deafferented field; and (3) if sprouting by the commissural projections causes a reduction in the density of the terminal field they generate in their normal target region.The synaptic density of the neonatally deafferented middle molecular layer was found to have returned to near control levels by adulthood. Degeneration studies performed in the adult revealed that commissural endings were located in equivalent numbers in the inner and middle molecular layers of rats in which the entorhinal cortex had been removed at 14 days post-natal; in normal rats (i.e. no neonatal surgery) the commissural terminals were found only in the inner molecular layer. Furthermore, and most importantly, the density of commissural terminals in the inner molecular layer was virtually identical in the ‘sprouted’ and control rats. Thus the tremendous areal expansion of the commissural terminal field which occurs after early deafferentation of the distal parts of the granule cell dendrites was not accompanied by any loss of input to the normal target of this afferent. Therefore, sprouting in this system represents an exaggeration of normal growth rather than a redistribution of a fixed population of endings. The relevance of these findings to theories concerned with the regulation of axonal growth and terminal proliferation during development is discussed.     

The response of the associational afferents to the dentate gyrus to simultaneous or sequential elimination of the commissural and entorhinal afferents

Previous studies have shown that following the removal of the commissural afferents to the dentate gyrus, the ipsilateral association afferents, which are normally distributed within the same region of the molecular layer, sprout new collateral branches and in time occupy essentially all the vacated synaptic sites. It is also known that when the entorhinal afferents to the dentate gyrus are interrupted the associational and commissural fibers can both undergo a similar phase of reactive synaptogenesis and give rise to new collaterals which extend for some distance into the denervated zone. Since the associational fibers can sprout after the removal of either the commissural or entorhinal afferents experiments were designed to determine their capacity for sprouting in newborn and young adult rats when both groups of afferents were eliminated either simultaneously or sequentially (i.e., after an interval of 8 weeks). The resulting changes in the terminal field of the associational afferents were assessed, two months after the last deafferentation, by measuring in autoradiographs the width of the zone occupied by the associational afferents labeled with [3H]proline, and by estimating the volume of this region in Timm-stained sections. The results indicate that under these conditions the associational afferents are capable of expanding their terminal field not only to occupy essentially all of the synaptic sites made available by the elimination of commissural fibers, but also to occupy a significant proportion of the space vacated by the removal of the entorhinal afferents. This suggests that the capacity of the associational afferents for reactive synaptogenesis is greater than that expressed after either commissural of entorhinal lesions alone.     

Lesion-induced synapse reorganization in the hippocampus of cats: sprouting of entorhinal, commissural/associational, and mossy fiber projections after unilateral entorhinal cortex lesions, with comments on the normal organization of these pathways.

This study evaluates whether three forms of sprouting occur in the hippocampus of the cat following unilateral entorhinal cortex (EC) lesions: (1) sprouting of projections from the EC contralateral to the lesion; (2) sprouting of the commissural/associational system; and (3) sprouting of mossy fibers. Tract tracing techniques were used to define the normal organization of the entorhinal cortical projection system, the commissural/associational (C/A) systems, and the mossy fiber projections in normal cats. The same techniques were then used to evaluate whether there were changes in these projections in animals with long-standing unilateral EC lesions. The projections from the entorhinal cortex were evaluated autoradiographically following injections of 3H proline into the entorhinal area. The projections of the C/A system were traced using the Fink-Heimer technique after lesions of the hippocampal commissures, and by using autoradiographic techniques after injections of 3H proline into the hippocampus. The distribution of mossy fibers was evaluated using the Timm's stain. The results reveal that unilateral lesions of the EC in cats lead to the same sorts of sprouting that have been described in rats. There is: (1) an increase in the density of the crossed projection from the surviving EC to the contralateral dentate gyrus that had been deprived of its normal EC inputs; (2) an expansion of the terminal field of the C/A projection system into portions of the molecular layer of the dentate gyrus normally occupied by EC projections; and (3) an increase in supragranular mossy fibers in some animals. The mossy fiber sprouting was especially prominent when the lesions encroached upon the hippocampus. The studies also reveal additional details about the normal organization of hippocampal pathways in cats. The most important points are: (1) there is a crossed projection from the entorhinal cortex to the contralateral dentate gyrus; and (2) there is a complex laminar organization of the commissural and associational terminal fields in the molecular layer of the dentate gyrus that appears to be related to the point of origin of the projections along the septotemporal axis of the hippocampus. This heretofore unrecognized aspect of the laminar organization of C/A terminations has important implications for the temporal competition hypothesis, which has been advanced to account for the development of these afferent systems.     

Antibody to NGF inhibits collateral sprouting of septohippocampal fibers following entorhinal cortex lesion in adult rats.

We have used an antiserum raised against mouse 2.5S NGF to examine the involvement of endogenous neurotrophins in the collateral sprouting of septohippocampal fibers in the adult rat brain. The antiserum was administered intraventricularly. Immunocytochemical techniques indicated that the injected antibodies penetrated into brain tissue that included the basal forebrain, cortex, striatum, corpus callosum, and hippocampus. Unilateral lesioning of the entorhinal cortex was done to evoke the sprouting of the cholinergic septohippocampal fibers. At 8 days postlesion, the sprouting was much advanced, as evidenced by an increase in density of the acetylcholinesterase (AChE) staining in the outer molecular layer (OML) of the dentate gyrus and by the associated increase in the absolute number of AChE-positive fibers in the OML. As well, there was a widening of the inner molecular layer (IML), interpreted as being due to sprouting of noncholinergic axons in that region. In rats injected daily with anti-NGF or anti-NGF Fab fragments, no increase in AChE density, or in the population of AChE-positive fibers, was observed in the OML. In contrast, the widening of the IML seemed to be unaffected by the anti-NGF treatment. No changes were observed in the AChE related parameters in the dentate gyrus of nonlesioned animals treated similarly for 8 days with anti-NGF; there was, however, a decrease of choline acetyltransferase (ChAT) immunostaining in the ChAT-positive cells of the basal forebrain. Our findings and the confirmation that our polyclonal anti-NGF also recognizes other members of the NGF neurotrophin family, specifically brain-derived neurotrophic factor and neurotrophin-3, indicate that at least one of these neurotrophins plays a key role in the collateral sprouting of the cholinergic septohippocampal fibers (but not that presumed to occur within the IML) following an entorhinal cortex lesion.     

The tracing study of developing entorhino-hippocampal pathway

The entorhino-hippocampal pathway is the major excitatory input from neurons of the entorhinal cortex on both ipsilateral and contralateral hippocampus/dentate gyrus. This fiber tract consists of the alvear path, the perforant path and a crossed commissural projection. In this study, the histogenesis and development of the various subsets of the entorhino-hippocampal projection have been investigated. DiI, DiO, Fast Blue tracing and calretinin immunocytochemistry as well as were carried out with pre and postnatal rats at different developmental stages. The alvear path and the commissural pathway start to develop as early as embryonic day E16, while the first perforant afferents reach the stratum lacunosum-moleculare of the hippocampus at E17 and at outer molecular layer of the denate gyrus at postnatal day 2. Retrograde tracing with DiI identifies entorhinal neurons in layer II-IV as the developmental origin of the entorhino-hippocampal pathway. Furthermore, calretinin immunocytochemistry revealed transitory Cajal-Retzius cells in the stratum lacunosum-moleculare of the hippocampus from E16. DiI labeling of entorhinal cortex fibers and combined calretinin-immunocytochemistry reveal a close relationship between Cajal-Retzius cells and entorhinal afferents. This temporal and spatial relationship suggests that Cajal-Retzius cell serves as a guiding cue for entorhinal afferents at early cortical development.     

Dissociation of the immunoreactivity of synaptophysin and GAP-43 during the acute and latent phases of the lithium-pilocarpine model in the immature and adult rat

RATIONALE: Lithium-pilocarpine-induced status epilepticus (SE) generates neuronal lesions in the limbic forebrain, cerebral cortex and thalamus that lead to circuit reorganization and spontaneous recurrent seizures. The process of reorganization in regions with neuronal damage is not fully clarified. METHODS: In the present study, we evaluated by immunohistochemistry the early reorganization during the latent period with two neuronal markers, synaptophysin and growth-associated protein 43 (GAP-43) in rats subjected to SE at PN21 and as adults. RESULTS: Synaptophysin immunoreactivity increased between 24 h and 3 weeks post-SE in regions with severe and rapidly occurring neuronal loss, namely thalamus, amygdala, piriform and entorhinal cortices. GAP-43 expression decreased at 1 and 3 weeks in the same regions. The immunoreactivity of synaptophysin and GAP-43 increased in the inner molecular layer of dentate gyrus from 24 h after SE, and decreased in the outer molecular layer from 72 h after SE. These changes likely result from the death of hilar neurons and the reduction of the input from the entorhinal cortex. In 21-day-old rats that experience less SE-induced neuronal loss, increased immunoreactivity of synaptophysin was only found in piriform and entorhinal cortex while no changes occurred in GAP-43 expression. CONCLUSION: These findings suggest that there is an age-related relation between the extent and rapidity of the process of neuronal death and the expression of these markers. Synaptophysin appears to be a more sensitive marker of plasticity induced by SE than GAP-43.