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.     

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.     

Lesion-induced plasticity of central neurons: sprouting of single fibres in the rat hippocampus after unilateral entorhinal cortex lesion

In response to a central nervous system trauma surviving neurons reorganize their connections and form new synapses that replace those lost by the lesion. A well established in vivo system for the analysis of this lesion-induced plasticity is the reorganization of the fascia dentata following unilateral entorhinal cortex lesions in rats. After general considerations of neuronal reorganization following a central nervous system trauma, this review focuses on the sprouting of single fibres in the rat hippocampus after entorhinal lesion and the molecular factors which may regulate this process. First, the connectivity of the fascia dentata in control animals is reviewed and previously unknown commissural fibers to the outer molecular layer and entorhinal fibres to the inner molecular layer are characterized. Second, sprouting of commissural and crossed entorhinal fibres after entorhinal cortex lesion is described. Single fibres sprout by forming additional collaterals, axonal extensions, boutons, and tangle-like axon formations. It is pointed out that the sprouting after entorhinal lesion mainly involves unlesioned fibre systems terminating within the layer of fibre degeneration and is therefore layer-specific. Third, molecular changes associated with axonal growth and synapse formation are considered. In this context, the role of adhesion molecules, glial cells, and neurotrophic factors for the sprouting process are discussed. Finally, an involvement of sprouting processes in the formation of neuritic plaques in Alzheimer's disease is reviewed and discussed with regard to the axonal tangle-like formations observed after entorhinal cortex lesion.     

A quantitative electron microscopic study of synaptic reorganization in the rat medial habenular nucleus after transection of the stria medullaris

Synaptic plasticity has been studied electron-microscopically in the rat medial habenular nucleus by counting the numbers of different types of synapses per unit area at various times after transection of the stria medullaris on one or both sides.Over 80% of synapses in the normal medial habenula had asymmetrical thickenings, and of these most were in contact with dendritic spines, the rest were in contact with dendritic shafts. The presynaptic terminals were complex structures frequently invaginated by slender spinules from the postsynaptic element. Each presynaptic terminal made contact with a large number of dendritic spines. The remainder of the synapses involved symmetrical contacts on dendritic shafts or neuronal somata.After transection of the ipsilateral stria medullaris the presynaptic elements of both symmetrical and asymmetrical synapses showed electron-dense degeneration. Degeneration was first seen at 24 hours after operation, reached a maximum (of 19%) at two days, and disappeared by 12 days.Non-degenerating synapses fell to around 30%, of their normal level at four days and finally recovered to around 80%, of normal by more than 100 days. Differential counts for synapses on dendritic spines, shafts and cell bodies revealed different time courses for reinnervation. Thus, synapse numbers on spines began to recover sooner than those on shafts and somata. but only the number of synapses on somata returned to almost normal (98%), whilst those on spines and shafts reached only about 70%, of normal.Membrane thickenings resembling vacant postsynaptic sites were rarely (0.3%,) found in unoperated animals. They appeared after transection of the ipsilateral stria, reached a peak of around 12%, at 4 6 days, (slightly later than the degeneration), and then fell, although not quite reaching their normal low levels even at more than 100 days.The nucleus as a whole shrank by about 17% of its normal volume. This would cause the synaptic density to increase by about 12% (calculated as³ √17²%), an effect which is not of sufficient magnitude to account for the observed recovery of synapse numbers, which involves a nearly three-fold increase —i.e. from 30% to 80% normal. The temporal coincidence between the recovery of non-degenerating synapses, and the disappearance of degenerating synapses and vacant sites favours the view that the denervated sites are reinnervated by the formation of new presynaptic terminals.Under normal circumstances the contralateral stria medullaris forms few synapses in the medial habenular nucleus (0.07%, degeneration at four days after transection). However, at long-term survivals after an ipsilateral striai lesion (when the degeneration from that lesion has been completely removed), a lesion of the contralateral stria causes appreciably more degeneration (around 2%), and a considerable fall (more than 30%,) in non-degenerating synapses. Despite the (unexplained) discrepancy between these figures, the findings still suggest that at least some of the new synapses induced after denervation by an ipsilateral strial lesion are formed by axons belonging to the contralateral stria.     

Morphological changes in the rat neostriatum after unilateral 6-hydroxydopamine injections into the nigrostriatal pathway

Destruction of the dopamine-containing neurons in the rat substantia nigra results in morphological changes in the striatum which have been characterized at both the light and electron microscopic levels. After a unilateral 6-hydroxydopamine injection into the medial forebrain bundle, Golgi-impregnated medium-sized spiny neurons in the neostriatum ipsilateral to the injection had a lower density of spines on their dendrites than those on the contralateral side. A similar decrease in spine density was apparent from 12 days until at least 13.5 months after the lesion. A bilateral loss of spines occurred with increasing age regardless of the presence or absence of the nigrostriatal dopaminergic pathway. At the ultrastructural level, the general pattern of synaptic input to the Golgi-impregnated medium-sized spiny neurons was similar on both sides of the brain. The most obvious class of afferent boutons contacting these spiny neurons formed prominent asymmetrical synaptic specializations with the heads of the spines. The numbers of asymmetric synaptic profiles counted in random electron micrographs from the striata ipsilateral and contralateral to the lesion were not significantly different from each other. A small but significant increase in the length of asymmetric synaptic specialization profiles was, however, detected in the striata lacking a dopamine input.     

Synaptic reorganization in the rabbit hippocampus after lesion of commissural afferents

Summary The degeneration of commissural afferents to the hippocampus in the rabbit was studied by using the Fink-Heimer degeneration method, electron microscopy, and the combined Golgi/EM technique. The stratum oriens (CA3) was selected for quantitative electron microscopic evaluation of postlesional changes since the degeneration of commissural fibers as seen in Fink-Heimer preparations was dense throughout the width of that layer. Accordingly, in electron micrographs of stratum oriens many electron-dense degenerating boutons were found after short survival times (3 and 6 days, respectively), most of them (96%) in synaptic contact with dendritic spines. In the fine structural analysis of Golgi-impregnated CA3 pyramidal cells, spines of basal dendrites were identified as postsynaptic elements of degenerating commissural afferents in stratum oriens.Three days after the lesion, the number of intact synapses/unit area was reduced in stratum oriens of CA3 to 64% of the control; 20% of the synapses were degenerating. Thus, part of the degenerated synapses had disappeared. Evidence is provided that phagocytosis of degenerated boutons still attached to fragments of dendritic spines played a role in this process.Seven weeks after the lesion, the number of intact synapses had returned to control level, suggesting reactive growth of synaptic structures. When the ratio of spine synapses versus shaft synapses was compared with controls, no change had occurred. Thus, after an initial loss of spine synapses after short survival times, new spines have been formed in parallel with ingrowth (sprouting) of neighbouring nonlesioned 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.     

Sequential events of degeneration and synaptic remodelling in the viper optic tectum following retinal ablation. A degeneration, radioautographic and immunocytochemical study

The ultrastructural changes taking place in the retino-recipient layers of the viper optic tectum were examined between 5 and 122 days after retinal ablation. The initial degeneration of retinotectal terminals proceeds at widely different rates and is characterized by a marked degree of polymorphism in which a number of different patterns can be discerned. In the final stages of degeneration, either both the degenerating bouton and the distal portion of the postsynaptic element are engulfed by reactive glia, or, more frequently, only the degenerating terminal is eliminated and the postsynaptic differentiation remains. The free postsynaptic differentiations are reoccupied predominantly by boutons containing pleiomorphic vesicles and which are for the most part gamma-aminobutyric acid (GABA)ergic, thus forming heterologous synapses; less frequently these sites are occupied by boutons of the ipsilateral visual contingent to form homologous synapses. These two processes, both of which depend on terminal axonal sprouting, take place within the first 3 postoperative months. They are followed by a decrease in the number of heterologous synapses and a concurrent increase in the number of homologous synapses newly formed by optic boutons generated by collateral preterminal sprouting of ipsilateral retinotectal fibres. The data suggest that partial deafferentation of the optic tectum induces a transitory GABAergic innervation of free postsynaptic sites prior to the restoration of new retinal synaptic contacts.     

Differential induction of c-Fos, c-Jun and Jun B in the rat central nervous system following unilateral entorhinal cortex lesion

In order to identify some of the molecular mechanisms that occur after a central nervous system trauma, the immediate early gene encoded proteins c-Fos, c-Jun and Jun B were analysed by immunocytochemistry following unilateral entorhinal cortex lesion (controls, 30 min, 2, 5, 12 and 24 h, two, six, 10 and 14 days, four weeks and six months postlesion). In the dentate gyrus, c-Fos was induced in some supragranular neurons (30 min), massively expressed in granule cells ipsilaterally to the lesion (2 h), expressed in hilar neurons (5 h and two days) and was absent at all later stages. A basal expression of c-Jun was found in dentate granule cells of controls, which was strongly increased on the lesion side (2 h) and on the side contralateral to the lesion (12 h). c-Jun expression returned to control levels by 24 h. Jun B was induced in granule cells ipsilateral to the lesion within 2 h and was back to control levels by 5 h. In the lateral septal area, c-Fos and c-Jun were induced 30 min postlesion and decreased rapidly thereafter. In the cerebral cortex, a widespread induction of c-Fos and c-Jun occurred within 30 min after entorhinal cortex lesion and this up-regulation lasted until two days postlesion. These data indicate that electrolytic lesion of the entorhinal cortex leads to a rapid and widespread induction of c-Fos, c-Jun and Jun B. Within the denervated fascia dentata, some of these changes may be linked to the reorganization processes following the lesion. Alternatively, the alterations in immediate early gene expression reported here may be due to changes in synaptic activity or postlesional seizures which occur in this lesioning paradigm.     

Potentiation of excitatory synaptic transmission in the normal and in the reinnervated dentate gyrus of the rat

Summary Following destruction of the ipsilateral temporo-ammonic tract, which originates in the entorhinal cortex, and terminates on the granule cells of the dentate gyrus, fibers from the surviving contralateral entorhinal area proliferate forming extensive new connections with the denervated dentate granule cells. Utilizing extracellular recording techniques, we have compared the characteristics of synaptic transmission in the lesion induced afferents with the characteristics of the normal ipsilateral afferents by analyzing the responses of dentate granule cells to paired pulse activation of temporo-dentate circuitry.In the dentate gyrus of the normal rat, an extracellularly recorded EPSP evoked by stimulation of the ipsilateral entorhinal cortex is enhanced by as much as 100% by a conditioning pulse to the same afferent system. This is called paired pulse potentiation. In the reinnervated dentate gyrus, the extracellular EPSP evoked by a test stimulus delivered to the contralateral entorhinal cortex is also potentiated by a conditioning pulse. The paired pulse potentiation in the reinnervated dentate gyrus has a time course which is comparable to that of the normal ipsilateral afferent system, but the magnitude of the potentiation is somewhat less, averaging approximately 140% of control.A second manifestation of paired pulse potentiation in the normal ipsilateral temporo-dentate circuit is that more granule cells discharge in response to the second of a pair of stimuli. Potentiation of granule cell discharge, as measured by the increase in the size of the population spike in the test response, may be as much as 500% of control at the optimal interstimulus interval. In the operated animals, however, paired pulse stimulation of the lesion induced crossed temporo-dentate circuit results in little, if any, enhancement of granule cell discharge in response to the second stimulus, despite the fact that theextracellulrly recorded EPSP is potentiated.These results are discussed in relation to the similarity between the normal and the lesion induced afferents to the dentate cells, with consideration for the normal functioning of the circuit from the entorhinal cortex to the dentate gyrus.Some of 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 92717 (1974).     

The striatonigral projection and nigrotectal neurons in the rat. A correlated light and electron microscopic study demonstrating a monosynaptic striatal input to identified nigrotectal neurons using a combined degeneration and horseradish peroxidase procedure

In a light and electron microscopic study of the substantia nigra of the rat, the distribution and morphology of nigrotectal neurons and the pattern of termination of striatonigral fibres have been examined following the placement of horseradish peroxidase injections in the superior colliculus and kainic acid lesions in the dorsal striatum. In confirmation of previous findings, nigrotectal neurons which had been identified by the retrograde transport of horseradish peroxidase from the superior colliculus had mainly medium sized somata, varied from fusiform to stellate in shape and were found in mainly ventral regions of the rostral two-thirds of the substantia nigra pars reticulata. On electron microscopic examination, single and multiple (from two to six) degenerating striatonigral boutons were found in synaptic contact with the soma, proximal mainstem dendrites and small dendrites (but mainly on small dendrites) of labelled nigrotectal and unlabelled nigral neurons in the ventral region of the pars reticulata. In addition, a small number of degenerating striatonigral boutons formed axoaxonic synapses with degenerating or normal boutons which were presynaptic to nigral dendrites. Almost all of the identified striatonigral synapses were of the symmetrical type, although a few degenerating boutons established asymmetrical synaptic contacts on unlabelled dendrites. These findings provide evidence of a monosynaptic input from the dorsal striatum to nigrotectal projection neurons in the substantia nigra and thus demonstrate the existence of a bineuronal pathway from the striatum through the substantia nigra to the superior colliculus. The possible significance of the pattern of termination of striatonigral fibres in the substantia nigra is discussed with reference to the known dendritic arborization of nigral neurons.     

Electrophysiological analysis of the hippocampal projections to the entorhinal area

Responses evoked in the entorhinal area by impulse volleys originating in the ipsilateral hippocampus were analysed in the guinea-pig by means of field potential analysis. Perforant path volleys, synaptically elicited by stimulation of the dorsal psalterium of one side, were used to activate the hippocampal lamellar circuit of the same side and, through interhippocampal impulses, the hippocampal pyramidal neurons of the contralateral side. Discharge of the hippocampal pyramidal neurons was followed by a response, a fast negative deflection preceded and followed by slow waves, in the dorsal third of the ipsilateral entorhinal area. Laminar distribution of the fast negative deflection and of the time-locked unit activity suggested that excitatory synaptic effects followed by neuron discharge were generated in neurons of layers VI-II of the entorhinal area. The increasing latency of the fast negative deflection and of unit firing over the cortical depth suggested that these synaptic effects were generated in temporal sequence, going from layer VI to layer II. The entorhinal response disappeared after a lesion at the caudal border of the hippocampus interrupting the caudally-directed hippocampal efferents. The anatomy of the hippocampal and subicular projections to the entorhinal area in the guinea-pig, together with electrophysiological data obtained in recordings from the ipsilateral subiculum, suggested that the hippocampal impulses were relayed to layers VI-V of the entorhinal area by the subiculum. The delayed activation of layers IV-II was possibly mediated by intracortical connections. Double-shock experiments showed that impulses of hippocampal origin inhibited the response to dorsal psalterium volleys of entorhinal neurons giving origin to perforant path fibers. The data show that the hippocampal output activates the deep layers of the entorhinal area from which it is possibly relayed to numerous cortical and subcortical regions. Moreover, the inhibitory effects exerted on neurons originating perforant path fibers give evidence of a negative feedback control system operating in the hippocampal region.     

The projection of the visual cortex on the Clare-Bishop area in the cat

Summary Following large lesions of the cat visual cortex, the distribution of degenerating terminal boutons in the Clare-Bishop area was studied electron microscopically. Degenerating boutons were found throughout the cortical layers but mostly in layer III (51% of the total number of degenerating boutons) and layer V (24%). A smaller number of boutons were found in layers II (12%) and IV (9%), and very few in layers VI (3%) and I (1%). No degenerating terminals were observed in the upper two-thirds of layer I. Seventy-six per cent of the total degenerating boutons terminated on dendritic spines, 22% on dendritic shafts, and 2% on somata. Some degenerating boutons made synaptic contacts with somata and dendrites of nonpyramidal neurons. For example, one degenerating bouton was observed in contact with an apical dendrite of a fusiform cell. Three examples of dendritic spines, with which degenerating boutons made synaptic contacts, were found to belong to spinous stellate cells. No degenerating boutons were observed making synaptic contacts with profiles that could conclusively be traced to pyramidal cell somata.     

Synaptic input and local output of dopaminergic neurons in grafts that functionally reinnervate the host neostriatum

Summary In adult rats with a unilateral 6-hydroxydopamine-induced lesion of the nigrostriatal dopamine pathway, grafts of embryonic ventral mesencephalon can establish extensive efferent connections with the previously denervated host neostriatum and can compensate for motor and sensorimotor asymmetries induced by the lesion. The object of this study was to examine the afferent synaptic inputs to grafted dopaminergic neurons, implanted into a cortical cavity overlying the previously denervated caudate-putamen, using electron microscopic immunocytochemistry. The dopaminergic neurons of the grafts in the same animals had previously been shown to re-innervate the host neostriatum, to form synaptic connections therein and to attenuate the lesion-induced motor asymmetry that occured in response to amphetamine (Freund et al. 1985). In the light microscope, the grafts were found to contain numerous tyrosine hydroxylase-immunoreactive perikarya, dendrites, axons and axonal swellings which had distinct distributions. In addition axons and axonal swellings that were immunoreactive for either substance P or glutamate decarboxylase were present. Electron microscopic analysis of the boutons contacting tyrosine hydroxylase-immunoreactive neurons in the grafts revealed the presence of at least five distinct types of afferent synaptic boutons based on their immunochemistry, morphology, or types of membrane specialization. One type was itself immunoreactive for tyrosine hydroxylase; such synapses are extremely rare in the intact substantia nigra, none were found in the contralateral substantia nigrae or the substantia nigra of a control rat. Three of the remaining types had ultrastructural features that were similar to synaptic terminals that were immunoreactive for substance P or glutamate decarboxylase. These synapses were similar to the types of synapses found contacting dopaminergic neurons in the substantia nigra contralateral to the graft or the substantia nigra of a control rat. The results demonstrate that, in the absence of the normal extrinsic afferent inputs, the intracortical mesencephalic grafts have a well-developed local synaptic circuitry. It is suggested that local circuit regulation of dopaminergic neurons within the graft may, at least in part, be responsible for the maintenance of a normal or close to normal functional activity.     

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.     

Carbachol induces fast oscillations in the medial but not in the lateral entorhinal cortex of the isolated guinea pig brain

Fast oscillations at 25-80 Hz (gamma activity) have been proposed to play a role in attention-related mechanisms and synaptic plasticity in cortical structures. Recently, it has been demonstrated that the preservation of the entorhinal cortex is necessary to maintain gamma oscillations in the hippocampus. Because gamma activity can be reproduced in vitro by cholinergic activation, this study examined the characteristics of gamma oscillations induced by arterial perfusion or local intracortical injections of carbachol in the entorhinal cortex of the in vitro isolated guinea pig brain preparation. Shortly after carbachol administration, fast oscillatory activity at 25.2-28.2 Hz was observed in the medial but not in the lateral entorhinal cortex. Such activity was transiently associated with oscillations in the theta range that showed a variable pattern of distribution in the entorhinal cortex. No oscillatory activity was observed when carbachol was injected in the lateral entorhinal cortex. Gamma activity in the medial entorhinal cortex showed a phase reversal at 200-400 microm, had maximal amplitude at 400-500 microm depth, and was abolished by arterial perfusion of atropine (5 microM). Local carbachol application in the medial entorhinal cortex induced gamma oscillations in the hippocampus, whereas no oscillations were observed in the amygdala and in the piriform, periamygdaloid, and perirhinal cortices ipsilateral and contralateral to the carbachol injection. Hippocampal oscillations had higher frequency than the gamma activity recorded in the entorhinal cortex, suggesting the presence of independent generators in the two structures. The selective ability of the medial but not the lateral entorhinal cortex to generate gamma activity in response to cholinergic activation suggests a differential mode of signal processing in entorhinal cortex subregions.     

Characterization of input synapses on intracellularly stained neurons in hippocampal slices: an HRP/EM study

Summary This study describes the fine structure of input synapses on identified neurons in slices of the guinea pig hippocampus. For morphological identification, granule cells of the fascia dentata and pyramidal neurons of regio inferior of the hippocampus were impaled and intracellularly stained with horse-radish peroxidase (HRP). Input synapses on the HRP-stained neurons were identified in the electron microscope by the location of the synapses in inner or outer zones of the dentate molecular layer, as in the case of the synaptic contacts on injected granule cells, or by unique fine structural characteristics, as in the case of the giant mossy fiber boutons on CA3 pyramidal cells. As in tissue fixed in situ by transcardial perfusion, a large number of terminals arising from the different afferents in inner and outer zones of the dentate molecular layer were well preserved and formed synaptic contacts with small spines, large complex spines, and dendritic shafts of the HRP-filled granule cells. Mossy fiber synapses on the stained CA3 neurons were densely filled with clear vesicles, contained a few dense-core vesicles, and formed synaptic contacts with large spines or excrescences. Occasionally electrondense degenerating boutons were also found impinging on the stained dendrites and spines. The significance of the present findings for electrophysiological and pharmacological studies on brain slices is discussed.     

Lesion-induced transneuronal plasticity in the adult rat hippocampus

The process of reactive synaptogenesis has been demonstrated in several areas of the central nervous system, including the hippocampal dentate gyrus. After a complete unilateral entorhinal lesion, approximately 85% of the input to the outer two-thirds of the ipsilateral dentate molecular layer is lost. Bilateral fluctuations in synaptic density within non-denervated zones of the dentate molecular layer predict further alterations in neural circuitry at sites located transneuronally to the denervated dentate granule cells. Using quantitative electron microscopy, our study demonstrates a complete cycle of synapse loss and reacquisition within the ipsilateral but not contralateral CA4/hilus region of the hippocampal formation. This area is one of the terminal fields for the dentate granule cell mossy fiber axons. In addition the granule cell mossy fiber axons sprout during the postlesion time course and form a significantly increased number of new mossy fiber terminals within the ipsilateral and contralateral CA4/hilus area. Our results indicate that responses to brain injury may no longer be confined to a local denervated site, but probably include polyneuronal circuitry loops, which may encompass one or more areas of the central nervous system. Previous difficulties in providing a close behavioral or functional correlation to localized structural events may be explained by a more global brain response to an injury.     

Effects of neonatal intraocular colchicine on synaptogenesis and on the retention of the ipsilateral retinofugal projection within the superior colliculus

Summary A normally transient ipsilateral retinofugal projection exists in the rat but is retained following eye removal because of the loss of competitive interaction between crossed and uncrossed fibers. To further explore this phenomenon, colchicine (10^–3M) was injected into the right eye of newborn albino rats to partially suppress axonal transport in optic fibers, alter the developmental time course of retinofugal synaptic terminals and determine if this would in turn extend the period of survival of the ipsilateral projection. Measurements of the number of fibers in the nerve were also made to insure that colchicine was not lethal to the retinofugal projection. Projections into the superior colliculus were demonstrated by anterograde movement of HRP from the left eye. TMB histochemistry revealed dense labeling of the contralateral retino-recipient layers at 5 dpn in untreated or saline-injected controls. The ipsilateral projection was seen as a lighter band of activity across the colliculus which was most concentrated in the antero-medial quadrant. This pathway was transient and degenerated by 10 dpn, except for a few antero-medial fibers. Animals treated with colchicine demonstrated a retention of this pathway through 20 dpn. A concomitant quantitative analysis of synaptic development within the superior colliculus revealed populations of boutons with round (R) and flattened (F) vesicles, as well as multiple junctional (MJ) and serial (S) complexes, most of which were specialized R boutons. The various synaptic categories displayed specific ratios unique to the different stages of maturation. Intraocular colchicine reduced the ratio of R, MJ and S boutons to F terminals between 5–15 dpn (P < 0.01). By 20 dpn, the proportions of MJ and S boutons remained depressed but the normal ratio of R to F boutons was restored. Areal determinations of each synaptic profile included in the counts revealed a significant reduction in the size of MJ synaptic profiles examined in colchicine-treated animals and this may have been reflected in the slight loss of tectal volume (6–9%). Removal of the left eye and assessment of degenerating boutons showed that the expanded ipsilateral projection was not sufficiently dense to produce such a restoration. It thus appears that colchicine delayed the growth of the R population, but the effect was reversible and this category of boutons continued to develop, albeit on a later time course. Continued depression of MJ and S boutons suggests that suppression of the rate and quantity of axonally-transported substances retards the final stages of tectal synaptic differentiation, reduces their competitive advantage and allows the retention of the ipsilateral optic projection.     

The section-Golgi impregnation procedure. 2. Immunocytochemical demonstration of glutamate decarboxylase in Golgi-impregnated neurons and in their afferent synaptic boutons in the visual cortex of the cat

Sections of the cat's visual cortex were stained by an antiserum to glutamate decarboxylase using the peroxidase-antiperoxidase method; they were then impregnated by the section Golgi procedure and finally the Golgi deposit was replaced by gold. Neurons containing glutamate decarboxylase immunoreactivity were found in all layers of the visual cortex, without any obvious pattern of distribution. Fifteen immunoreactive neurons were also Golgi-impregnated and gold-toned, which enabled us to study the morphology and synaptic input of identified GABAergic neurons. These neurons were found to be heterogeneous both with respect to the sizes and shapes of their perikarya and the branching patterns of their dendrites. All the immunoreactive, Golgi-impregnated neurons had smooth dendrites, with only occasional protrusions. The synaptic input of glutamate decarboxylase-immunoreactive neurons was studied in the electron microscope. Immunoreactive neurons received immunoreactive boutons forming symmetrical synapses on their cell bodies. The Golgi-impregnation made it possible to study the input along the dendrites of immunoreactive neurons. One of the large neurons in layer III whose soma was immunoreactive was also Golgi-impregnated: it received numerous non-immunoreactive asymmetrical synaptic contacts along its dendrites and occasional ones on its soma. The same neuron also received a few boutons forming symmetrical synaptic contacts along its Golgi-impregnated dendrites; most of these boutons were immunoreactive for glutamate decarboxylase. Glutamate decarboxylase-immunoreactive boutons were also found in symmetrical synaptic contact with non-immunoreactive neurons that were Golgi-impregnated. A small pyramidal cell in layer III was shown to receive several such boutons along its somatic membrane. It is concluded that the combination of immunoperoxidase staining and Golgi impregnation is technically feasible and that it can provide new information. The present study has shown that there are many morphologically distinct kinds of aspiny GABAergic neurons in the visual cortex; that the predominant type of synaptic input to the dendrites of such neurons is from boutons forming asymmetrical synapses, but that some of the GABAergic neurons also receive a dense symmetrical synaptic input on their cell bodies, and occasional synapses along their dendrites, from the boutons of other GABAergic neurons. These findings provide a morphological basis, firstly, for a presumed powerful excitatory input to GABAergic interneurons and, secondly, for the disinhibition which has been postulated from electrophysiological studies to occur in the cat's visual cortex.