Fetal homotypic transplant in the excitotoxically neuron-depleted thalamus: light microscopy

One month after an in situ injection of kainic acid into the ventrobasal thalamic complex (VB), the lesioned area is totally depleted of neurons. The present study has been undertaken to determine the cytoarchitecture and connectivity of the nucleus constructed by fetal thalamic neurons implanted into the excitotoxically lesioned area. Adult rats received an injection of kainic acid inducing a total neuronal depletion of the right lateral thalamus (including both the nucleus reticularis thalami and the lateral portion of the ventrobasal complex). One month later, homotypic neurons were taken from the dorsal thalamic primordium of rat embryos (gestational age 15-16 days), dissociated, and injected into the lesioned area as a cell suspension. After 2-4-month survival, the cytoarchitecture of the neonucleus formed by the grafted neurons within the previously neuron-depleted area was analyzed. Additionally, connectivity was analyzed in seven rats in which dorsal column nuclei and/or cortical projections to the area were labeled anterogradely with either 3H-leucine or wheat-germ agglutinin conjugated to HRP, and the animals were perfused and processed following various histological procedures (Nissl staining, autoradiographic processing, and histochemistry for visualization of peroxidase). Fetal neurons grew, differentiated, and progressively occupied the previously neuron-depleted area of the adult host CNS. They organized themselves into a neonucleus with particular cytoarchitectural features including 1) the existence of two concentric zones--a central zone containing neurons and glial cells and a marginal zone only filled with a band of glial cells, 2) an increase in cellular density compared to the intact thalamus, 3) the grouping of neurons in spherical clusters, and 4) apparent polymorphism of neuronal somata. Lemniscal and corticothalamic afferents originating from the host were observed in the neonucleus when the fetal neurons had been implanted correctly into the lesioned area but not when they had been misplaced into either normal thalamic tissue or the internal capsule. The afferents labeled from either the dorsal column nuclei or the somatosensory cortex were, however, less dense in the neonucleus than in the normal thalamus. These results are discussed with regard to the normal cytoarchitecture and connectivity of the ventrobasal complex of the rat thalamus.     

Morphological alteration of thalamic afferents in the excitotoxically lesioned striatum

Excitotoxic lesions of the neostriatum cause anatomical and biochemical changes resembling those occurring in Huntington's disease. One major characteristic of these lesions is that they acutely spare axons of passage and afferent fibers. However, evidence is accumulating that afferent axonal systems decrease their fiber density in the long-term excitotoxic lesion. Ultrastructural changes of neuron-deprived terminals may also occur. A parallel study considering changes in afferent fibers to the excitotoxically lesioned thalamus showed that, a few weeks after neuron-depletion, specific 'point-to-point' systems formed regenerating axonal growth cone-like structures. The present study used the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to determine whether specific thalamostriatal afferents form the same kind of regenerating structures following excitotoxic lesion of their target neurons. Thalamostriatal afferents decreased in density over months after lesion, but some were still labeled as long as 4 months after ibotenic acid injection. Remaining afferents formed axonal growth cone-like structures, identified at both light and electron microscopic levels, similar to those observed in the lesioned thalamus. These results demonstrate that in the striatum as in the thalamus, neuron depletion is followed by a long-term alteration of the morphology of some afferent fibers which form regenerating growth cone-like structures. These results are discussed with regard to the possible functional integration of fetal neurons transplanted into previously excitotoxically lesioned areas.     

Structural alteration and possible growth of afferents after kainate lesion in the adult rat thalamus

Afferents to the thalamic ventrobasal complex (VB) originating from the spinal cord, the dorsal column nuclei, and the somatosensory cortex were anterogradely labeled by WGA-HRP 30 days after an injection of kainic acid (KA), which produced a complete unilateral neuronal loss in the VB, the opposite side being used as a control. At the light microscopic level, there was no obvious rerouting of spinal afferents away from the lesioned areas towards unlesioned parts of VB. There was an apparent decrease in the number of lemniscal afferents to the lesioned side, which may indicate a progressive retrograde degeneration. At higher magnification, all three afferent systems studied demonstrated morphological changes, predominantly manifested by terminal swellings that reached up to 25 micron in diameter. Control experiments suggested that these morphological alterations were related neither to a direct action of the excitotoxin nor to the absence of a different afferent system but to the loss of neuronal postsynaptic targets. At the electron microscopic level, the normal ultrastructural features of VB were not observed after a KA lesion. No neuronal somata, dendrites, or normal presynaptic elements were observed. Neural elements, some of which were labeled from the somatosensory cortex or the dorsal column nuclei, were essentially of two types: varicosities and unmyelinated axonal profiles. Varicosities could be separated into two broad classes: The majority were large structures derived from large, sometimes myelinated, axons and containing a wealth of organelles. Since they were not completely surrounded by glial elements, we have denoted them unensheathed varicosities. Among the organelles, the most obvious features were vesicles and tubules of smooth endoplasmic reticulum, microtubules, mitochondria, and various lysosome-like inclusions. These unensheathed varicosities gave rise to large, mound-like protrusions containing large vacuoles and thin long protrusions either filled with neurofilaments or resembling unmyelinated axonal profiles. Others were completely surrounded by a glial sheet and were therefore called ensheathed varicosities. These ensheathed varicosities presented several characteristics typical of degenerating profiles, including neurofilamentous proliferation and morphological alterations of the mitochondria. Unmyelinated axonal profiles occupied a substantial territory in the lesioned area. They were most often grouped in bundles sometimes wrapped by glial processes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural and functional evidence for the survival of corticogeniculate neurons in kainic acid-lesioned lateral geniculate nucleus

After a kainic acid lesion in the dorsal lateral geniculate nucleus of rat, retrograde axonal transport of fluorescent dyes is blocked in corticogeniculate but not in retinogeniculate neurons. This inhibition, however, can be reversed by electrical stimulation in the subcortical white matter (Woodward and Coull, Brain Research 454 (1988) 106-115). These observations suggest that retrograde axonal transport in corticogeniculate neurons is impulse-dependent and that neuronal activity in this pathway is reduced as a consequence of the lesions. To test this we examined retrograde transport of horseradish peroxidase (HRP) and cytochrome oxidase activity in the cortex of lesioned animals. Unilateral kainic acid lesions in the geniculate inhibit the retrograde transport of HRP, but this inhibition is reversed by electrical stimulation of white matter. Moreover, histochemical staining for cytochrome oxidase activity is less intense over visual cortex on the lesioned side, implying that cortical activity in intrinsic and efferent pathways is reduced as a consequence of removal of geniculate afferents. Inasmuch as the retrograde transport of HRP is dependent upon impulse activity in neurons and is thought to be mediated by synaptic vesicle recycling, these results suggest that terminals of corticogeniculate fibers survive the kainic acid lesions in the geniculate and are capable of releasing synaptic vesicles. Ultrastructural examination of lesioned geniculates strongly supports this conclusion and reveals the presence of axon terminal profiles which are filled with small round synaptic vesicles and have membrane specializations reminiscent of synaptic contacts. These terminal profiles are presumed to be of retinal and cortical origin.     

Brain peptidases: Their possible neuronal and glial localization

Neuronal and glial localization of brain peptidases was investigated by means of the kainic acid (KA) lesion technique. Activities of 6 different peptidases were measured in the rat caudate-putamen (CP) and substantia nigra (SN) 2, 7 and 21 days after unilateral intra-CP injection with 2.5 micrograms of KA. As an indicator of KA lesion in CP, substance P content in both CP and SN was also determined. In addition, activities of the same peptidases in the primary and secondary glial cell cultures of fetal rats were measured and compared to those in CP homogenate. After the KA injection, prolyl endopeptidase (Pro-EP) activity was decreased in the lesioned CP and, to a lesser extent, in the ipsilateral SN. The activity of angiotensin-converting enzyme (ACE) in the lesioned CP was decreased with a complex time course, whereas a slow and progressive reduction was observed in the SN. Alanyl and leucyl aminopeptidase (Ala-AP and Leu-AP respectively) activities gave only small changes after the lesion; Ala-AP was decreased and Leu-AP was increased in the lesioned CP, while both were decreased in the SN. Dipeptidyl aminopeptidase (DAP) and arginyl endopeptidase (Arg-EP) activities were increased 5-fold in the CP 7 days after the KA injection. Their increases paralleled that of beta-glucuronidase, the lysosomal marker enzyme. Cultured glial cells contained only a trace amount of ACE activity. Ala-AP and Pro-EP activities were considerably lower in the glial culture cells than in the CP homogenate. In contrast, DAP and Arg-EP as well as lysosomal marker enzymes showed much higher activity in the former than in the latter. These results suggest that (1) Ala-AP and Pro-EP have large neuronal components, (2) ACE is preferencially localized in neurons and (3) DAP and Arg-EP are associated with glial lysosomal function. It is, therefore, concluded that at least a part of the brain peptidases are differentially localized in neurons and glia, and may be involved in specific neuronal or glial function.     

Decreased densities of dopamine and serotonin transporters and of vesicular monoamine transporter 2 in severely kainic acid lesioned subregions of the striatum

Summary. Fifteen days after a striatal kainic acid (KA) injection, we have examined presynaptic modifications of dopamine and serotonin terminals in the striatum through (i) autoradiographic labeling of dopamine, serotonin and vesicular monoamine transporters respectively with ³H-mazindol, ³H-citalopram and ³H-dihydrotetrabenazine, and (ii) determination of the contents in dopamine, serotonin and their metabolites. Acetylcholinesterase histochemical labeling enabled the definition of severely and moderately KA-lesioned subregions within the striatum. A significant decrease of the three transporters labeling density was observed only in the severely lesioned subregions. The strong decrease in serotonin transporter labeling revealed here has not been described until now. Besides, the striatal contents of homovanillic acid (dopamine metabolite) and 5-hydroxyindolacetic acid (serotonin metabolite) were significantly increased in the lesioned striatum. The whole data evidence an incomplete sparing of dopamine and serotonin terminals in the striatum 15 days after a KA injection, especially in the areas where the degeneration of postsynaptic neurons was the most extensive. Received May 31, 2000; accepted October 23, 2000     

The direction of apomorphine-induced rotation behavior is dependent on the location of excitotoxin in the rat basal ganglia

Adult rats received unilateral kainic acid (KA) lesions of the striatum with the anterior/posterior coordinates of the lesion at either 1.5 mm or 0.3 mm anterior to bregma. Four to six weeks after the lesion rats were placed in an open field environment and injected with apomorphine (1 mg/kg, s.c). Rats receiving the more posterior lesion (0.3 mm) rotated ipsilateral to the lesioned side of the brain. In contrast, the majority of rats receiving the more anterior (1.5 mm) placement of the lesion rotated contralateral to the lesioned side of the brain. Histological analysis of several animals receiving posterior lesions revealed damage to the hippocampus and thalamus that was not seen in the animals receiving anterior lesions. Our results are consistent with the hypothesis that the direction of apomorphine-induced rotation after excitotoxin injection into the rat basal ganglia is dependent on the location of the lesion.     

Changes in the activity of gamma-glutamyl transpeptidase induced by kainic acid and surgical lesions of the hippocampal formation in young rats

To study possible functional involvement of gamma-glutamyl transpeptidase (GGT) in glutamate transmitter metabolism we lesioned putative glutamatergic structures of the rat hippocampal formation by intracerebroventricular (i.c.v.) injection of kainic acid (KA) or by surgical CA3 axotomy. Unilateral injection of KA into the left lateral cerebral ventricle of 30-day-old rats resulted in decreased GGT activity in hippocampal areas CA3, Ca1 ipsilaterally, and in the contralateral area CA1, four hours after the induction of the chemical lesion. Four days after the injection, the enzyme activity was decreased in all hippocampal areas with the exception of the contralateral dentate gyrus. Four days after bilateral i.c.v. injection of KA, lower GGT levels were found than was seen after bilateral surgical lesion of the CA3 pyramidal cell axons (Schaffer's collaterals). The surgical lesion was followed by a decrease of GGT only in the stratum pyramidale and stratum radiatum of area CA1. In contrast to the effects in 30-day-old rats, unilateral i.c.v. injection of KA on postnatal day 12 did not alter the GGT activity in any studied hippocampal area presumably because of incomplete maturation of structures required for KA vulnerability.     

Neovascularization in kainic acid-induced lesions of rat striatum. An immunohistochemical study with laminin

Vascular changes occurring after stereotaxic injection of the excitatory neurotoxin kainic acid (KA) into the rat striatum were studied at various time intervals after the lesion using laminin immunohistochemistry. Laminin immunohistochemistry revealed marked vascular changes, including presumed neovascularization, in the lesioned striatum. Vessels with increased laminin immunoreactivity were demonstrated from 2 days up to at least 5 months following the injection. The majority was capillary vasculature, which was distributed throughout the lesioned striatum, sometimes being arranged densely along the needle tract. Fine spike-like sprouts called streamers were also loaded with laminin immunoreactivity. In contrast, laminin immunoreactivity in vessels in the control striatum was negligible. Vascular changes detected by laminin immunohistochemistry preceded but then almost paralleled gliosis demonstrated by glial fibrillary acidic protein immunohistochemistry. These results indicate that striatal injection of KA causes marked vascular changes including neovascularizations which are demonstrable by laminin immunohistochemistry.     

Host Corticostriatal Fibres Establish Synaptic Connections with Grafted Striatal Neurons in the Ibotenic Acid Lesioned Striatum

The connections between host corticostriatal afferents and neurons in intrastriatal grafts of foetal striatal tissue have been studied with electron microscopic immunocytochemistry using Phaseolus vulgaris leucoagglutinin (PHA-L) as a label of the host corticostriatal fibres. Adult rats with unilateral ibotenic acid lesions of the head of the caudate putamen received foetal cell suspension grafts from E14-15 rat embryos into the lesioned striatal area. Ten months after transplantation, multiple iontophoretic injections of PHA-L were made into the host frontal cortex. These injections labelled large numbers of corticostriatal fibres which extended across the graft - host border to form a rich axonal network mainly in the peripheral portions of the grafts. At the ultrastructural level a total of 134 PHA-L-labelled terminals were identified to form asymmetric synaptic contacts with neurons within the grafts. Of these contacts, 83% were in contact with dendritic spines, 12% with dendritic shafts, and 5% with small shafts or spines. The synaptic contacts were similar to those identified in intact regions of the host striatum that were spared by the lesion. However, the synapses in the host striatum were almost exclusively in contact with spines (98%). These results demonstrate that the corticostriatal projection, which constitutes a major source of afferent control in the normal striatum, not only extends axons into the intrastriatal striatal grafts, but also establishes synaptic connections with the implanted neuronal elements.     

Reticularis thalami afferents to the ventrobasal complex of the rat thalamus: an electron microscope study

Afferents from the nucleus reticularis thalami (RT) to the thalamic ventrobasal complex were studied in the rat by looking for degenerating terminals after selective neurotoxic lesion of RT using injections of kainic acid. Several lines of evidence are presented indicating that RT afferents terminate in the VB by F type (Gray type II) terminals and that F type terminals in the VB all depend of RT neurons.     

Maturation and fine structure of thalamic reticular neurons transplanted into the adult rat CNS.

This work was undertaken to determine whether or not cell suspension transplants provide a promising vehicle for the in vivo study of neuronal development. Cell suspensions of rat fetal tissue that contained both reticular (RT) and ventrobasal primordia were transplanted into the excitotoxically lesioned somatosensory thalamus of adult rats. The fine structure of transplanted GABAergic RT neurons that were identified by immunocytochemistry was investigated 5 days to 7 months following transplantation. The dissociation and transplantation of the tissue results in disruption of the extracellular matrix and alteration of cellular interactions which could introduce major changes in neuronal maturation. Our results show that maturation of transplanted GABAergic RT neurons was comparable to normal and that they were able to establish and receive, with one exception, usual sets of connections. In contrast, disruption of the tissue was responsible for the definitive loss of organotypic characteristics and the emergence of abnormal connections, especially synaptic connections from somatosensory afferents, the functional significance of which will be important to determine.     

Involvement of cyclin-dependent kinase-5 in the kainic acid-mediated degeneration of glutamatergic synapses in the rat hippocampus

Increased levels of glutamate causing excitotoxic damage accompany neurological disorders such as ischemia/stroke, epilepsy and some neurodegenerative diseases. Cyclin‐dependent kinase‐5 (Cdk5) is important for synaptic plasticity and is deregulated in neurodegenerative diseases. However, the mechanisms by which kainic acid (KA)‐induced excitotoxic damage involves Cdk5 in neuronal injury are not fully understood. In this work, we have thus studied involvement of Cdk5 in the KA‐mediated degeneration of glutamatergic synapses in the rat hippocampus. KA induced degeneration of mossy fiber synapses and decreased glutamate receptor (GluR)6/7 and post‐synaptic density protein 95 (PSD95) levels in rat hippocampus in vivo after intraventricular injection of KA. KA also increased the cleavage of Cdk5 regulatory protein p35, and Cdk5 phosphorylation in the hippocampus at 12 h after treatment. Studies with hippocampal neurons in vitro showed a rapid decline in GluR6/7 and PSD95 levels after KA treatment with the breakdown of p35 protein and phosphorylation of Cdk5. These changes depended on an increase in calcium as shown by the chelators 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N ′,N′‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM) and glycol‐bis (2‐aminoethylether)‐N,N,N ′,N ′‐tetra‐acetic acid. Inhibition of Cdk5 using roscovitine or employing dominant‐negative Cdk5 and Cdk5 silencing RNA constructs counteracted the decreases in GluR6/7 and PSD95 levels induced by KA in hippocampal neurons. The dominant‐negative Cdk5 was also able to decrease neuronal degeneration induced by KA in cultured neurons. The results show that Cdk5 is essentially involved in the KA‐mediated alterations in synaptic proteins and in cell degeneration in hippocampal neurons after an excitotoxic injury. Inhibition of pathways activated by Cdk5 may be beneficial for treatment of synaptic degeneration and excitotoxicity observed in various brain diseases.     

Impairment of dentate gyrus neuronal progenitor cell differentiation in a mouse model of temporal lobe epilepsy

Unilateral intrahippocampal injection of kainic acid (KA) in adult mice induces an epileptic focus replicating major histopathological features of temporal lobe epilepsy (TLE). In this model, neurogenesis is impaired in the lesioned dentate gyrus, although cell proliferation transiently is increased bilaterally in the subgranular zone (SGZ). To investigate further the relationship between epileptogenesis and neurogenesis, we compared the differentiation of cells born shortly before and after KA injection. Immunohistochemical staining for doublecortin and PSA-NCAM, two markers of young neurons, revealed a rapid downregulation of both markers ipsilaterally, whereas they were increased transiently on the contralateral side. To determine whether KA treatment directly affects neural progenitors in the SGZ, dividing cells were prelabeled with 5'-bromo-2'deoxyuridine (BrdU) treatment before unilateral injection of KA. Double staining with the proliferation marker PCNA showed that prelabeled BrdU cells survived KA exposure and proliferated bilaterally. Unexpectedly, the neuronal differentiation of these cells, as assessed after 2 weeks with doublecortin and NeuN triple-staining, occurred to the same extent as on the contralateral side. Only 5% of pre-labeled BrdU cells were GFAP-positive within the lesion. Therefore, SGZ progenitor cells committed to a neuronal phenotype before KA treatment complete their differentiation despite the rapid down-regulation of doublecortin and PSA-NCAM. These findings suggest impaired fate commitment and/or early differentiation of proliferating cells in the lesioned dentate gyrus. Loss of neurogenesis in this TLE model likely reflects an irreversible alteration of the SGZ germinal niche during development of the epileptic focus and may therefore be relevant for human TLE.     

The structural organization of the ventrobasal complex of the rat as revealed by the analysis of physiologically characterized neurons injected intracellularly with horseradish peroxidase

The ventrobasal complex (VB) of the rat thalamus contains neurons responding to non-noxious somatic stimuli as well as neurons driven exclusively by noxious stimuli. This study presents a comparison of morphological features of these two kinds of neurons. Thirteen neurons electrophysiologically characterized were impaled with the micropipette used for the recordings and intracellularly injected with horseradish peroxidase. After revealing the marker and preparation for electron microscopic procedures, 3 out of the 13 neurons were carefully studied using both the light and the electron microscope. VB neurons are stellate cells with a central rounded cell body and 6 to 10 primary dendrites which branch rapidly, giving a 'tufted' appearance. Dendrites of all orders present various types of protrusions. At the electron microscope level, 3 main kinds of synaptic profiles were observed contacting the injected neurons: small terminals with round vesicles which make asymmetrical contacts with distal dendrites; medium-sized terminals with flattened vesicles which make symmetrical contacts with dendrites of all orders and the soma; and large terminals with round vesicles which make asymmetrical contacts with primary dendrites and the soma. This study failed to reveal obvious morphological differences between functionally different VB neurons. In addition, it showed that their synaptology was apparently equivalent.     

Increased expression of glutamic acid decarboxylase mRNA in rat substantia nigra after an ibotenic acid lesion in the caudate-putamen

In situ hybridization histochemistry and RNA blots were used to study expression of glutamic acid decarboxylase (GAD) mRNA in rat caudate-nucleus and substantia nigra. In situ hybridization combined with computerized image analysis revealed that in the intact substantia nigra reticulata the cross-section area of GAD mRNA positive neurons were 25% larger in the dorsolateral part as compared with the ventromedial part. A unilateral ibotenic acid injection in caudate-putamen lesioned neurons, some of which project to the ipsilateral substantia nigra. An increased level of GAD mRNA was observed in substantia nigra ipsilateral to the lesion. Computerized image analysis of sections from in situ hybridization revealed an increase in the number of silver grains over GAD mRNA positive neurons in the dorsolateral substantia nigra reticulata ipsilateral to the lesion. However, no change was observed in the ventromedial part suggesting that GAD mRNA expression in this part of the nigra is less sensitive to inhibition by caudate-putamen afferents. In agreement with in situ experiments, RNA blots showed a 2-fold increased level of GAD mRNA in substantia nigra ipsilateral to the lesion. The increased GAD mRNA expression in the deafferented substantia nigra suggests a disinhibition of nigral GABA neurons, resulting in an increased utilization of GABA in these substantia nigra neurons.     

Synaptic organization of the rat parafascicular nucleus,with special reference to its afferents from the superior colliculus and the pedunculopontine tegmental nucleus

The synaptic organization of afferents to the parafascicular nucleus (Pf) of the thalamus was studied in rats. In the Pf, three types of axon terminals were identified: the first type was a small terminal with round synaptic vesicles forming an asymmetric synapse, the second type was a large terminal with round synaptic vesicles forming an asymmetric synapse, and the third type was a terminal with pleomorphic vesicles forming a symmetric synapse. They were named SR, LR and P boutons, respectively. In order to determine the origin of these axon terminals, biotinylated dextran amine (BDA) was injected into the main afferent sources of the Pf, the superior colliculus (SC) and the pedunculopontine tegmental nucleus (PPN). Axon terminals from the SC were both SR and LR boutons which made synaptic contacts with somata and dendrites. PPN afferents were SR boutons, which made synaptic contacts with somata and smaller dendrites. Double-labeled electron microscopic studies, in which a retrograde tracer (wheat germ agglutinin conjugated to horseradish peroxidase: WGA-HRP) was injected into the striatum and an anterograde tracer (BDA) into the SC revealed that SC afferent terminals made synapses directly with Pf neurons that projected to the striatum. Another experiment was performed to find out whether two different afferents converged onto a single Pf neuron. To address this question, two different tracers were injected into the SC and PPN in a rat. Electron microscopically, both afferent terminals from the SC and PPN made synaptic contacts with the same dendrite. Our results prove that a single neuron of the rat Pf received convergent projections from two different sources.     

Sprouting of GABAergic and mossy fiber axons in dentate gyrus following intrahippocampal kainate in the rat.

The present study examined the bilateral synaptic rearrangements of presumed gamma-aminobutyric acid (GABAergic) inhibitory axons and mossy fiber (presumed excitatory) recurrent collaterals following intrahippocampal kainic acid (KA) injection. Glutamate decarboxylase immunoreactivity (GAD-IR) was used to study inhibitory axon terminal sprouting, following 0.5 microgram KA/0.2 microliter injected unilaterally into the posterior hippocampus of rats (n = 16), with survival periods of 14, 28, and 120 days. The age-matched control animals (n = 9) received intrahippocampal 0.2 microliter saline (sham, n = 4) or no injection (normal, n = 5). To study mossy fiber synaptic rearrangements, 0.5 microgram KA/0.2 microliter volumes were injected unilaterally into the posterior hippocampus of rats (n = 10), with survival periods from 14, 28, and 120 days, and Timm sulfide-stained tissue sections were compared to age-matched sham (n = 4) or normal controls (n = 4). At 14 through 120 days after posterior KA injection, GAD-IR puncta were significantly increased in the ipsi- and contralateral inner molecular layers (IML) of the fascia dentata (FD) when compared to sham or normal controls. KA lesion also induced mossy fiber recurrent collateral sprouting into the ipsi- and contralateral FD IMLs. The loss of both the commissural and ipsilateral associational afferents to the FD apparently induced sprouting into their ipsi- and contralateral termination zones by granule cell mossy fibers and GAD-IR axons, thus establishing an abnormal circuitry near the observed pathology in the kainate model of epilepsy. Although reactive synaptogenesis of mossy fibers producing monosynaptic excitation may be one mechanism for KA epileptogenicity, the concurrent sprouting of GABAergic terminals in the same IML zone of the FD suggests that anomalous inhibitory synapses may contribute to chronic KA hippocampal hyperexcitability.     

Deafferentation-induced expansion of saphenous terminal field labelling in the adult rat dorsal horn following pronase injection of the sciatic nerve

We have examined the effect of the degeneration of sciatic nerve afferents on the distribution of saphenous terminals in the adult rat dorsal horn. Deafferentation was produced by injection into the sciatic nerve of pronase, a combination of proteolytic enzymes, which causes death of ganglion cells and degeneration of their terminal fields. The saphenous terminal fields were labelled by exposing the cut nerve to a combination of horseradish peroxidase (HRP) and wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Terminals were mainly found in the superficial dorsal horn, indicating that small-diameter afferents were heavily labelled. In one group of control animals, the normal sciatic and normal saphenous terminal fields were shown to be bilaterally symmetrical. In the experimental group, the initial injection of one sciatic nerve with pronase was followed 4 months later by bilateral HRP/WGA-HRP labelling of both saphenous nerves. In each animal, the terminal field of the saphenous nerve on the lesioned side was expanded in the medial, lateral, and caudal directions. Medially and laterally, the expanded terminal field overlapped more of the sciatic territory than normal; caudally, saphenous terminals were found in the rostral portion of the L5 segment, in an area normally filled by sciatic terminals and devoid of saphenous terminals. The expansion resulted in a total saphenous area 26% larger than the control side. Electron microscopy demonstrated that the label in both the normal and expanded territories was primarily contained in axons and terminals, with minor transneuronal labelling. Labelled terminals in the expanded areas were both simple terminals with round, clear vesicles, and glomerular terminals with multiple synaptic contacts; these terminal types resemble those previously described for primary afferents in the superficial dorsal horn. Although the preexistence of "silent" synaptic terminals in the expanded areas cannot be disproven, the data support the hypothesis that primary afferents in the adult have the potential to sprout and establish synapses when the conditions of the deafferentation are favorable.     

Intrastriatal Transplants of Embryonic Dopaminergic Neurons Counteract the Increase of Striatal Enkephalin Immunostaining but not Serotoninergic Sprouting Elicited by a Neonatal Lesion of the Nigrostriatal Dopaminergic Pathway

The aim of the our experiment was to compare the ability of intrastriatal implants of embryonic dopaminergic neurons to reverse two kinds of postlesion modification in the host brain: the change in the activity level of neurons in the denervated area and morphological modifications, e.g. collateral sprouting. The ascending dopaminergic system of 3-day-old rat pups was unilaterally lesioned by an intrahypothalamic injection of the neurotoxin 6-hydroxydopamine. This lesion has been described previously to induce an increase in the level of activity of striatal enkephalinergic neurons. The same lesion leads also to sprouting of the serotoninergic afferents in the striatum, leading to hyperinnervation of this structure. The existence of these modifications thus offers the possibility of testing the influence of grafts in one structure of the same animal on two lesion-induced reactions of different nature. A cell suspension obtained from mesencephali of embryonic day 14 rats and containing dopaminergic neurons was implanted into the denervated striatum of lesioned animals 5 days after the lesion. Nine months later the animals were killed and immunohistochemistry was performed on striatal sections using antibodies directed against tyrosine hydroxylase, methionine enkephalin and serotonin. Intensity of immunostaining (methionine enkephalin and serotonin) as well as innervation density (serotonin) was quantified through the use of a computer-assisted image analyser. The lesion led to the disappearance of striatal dopaminergic innervation. Implanted dopaminergic neurons were found scattered in the striatum and restored a dopaminergic innervation in a large portion of this structure. There was a marked increase in striatal methionine enkephalin immunostaining in lesioned animals, which was most pronounced in the dorsolateral part of the striatum (+ 150% compared to control values), while in the ventral part it was slight or non-existent. The density of striatal serotoninergic innervation was also increased by ～250% relative to control values. In grafted animals striatal enkephalin immunostaining was similar to that observed in control animals. On the other hand, the serotoninergic hyperinnervation was still present in the graft-bearing striata. These results suggest that while intrastriatal implants of embryonic dopaminergic neurons are able to counteract modifications in the functioning of local striatal neuronal systems such as the increase in enkephalinergic activity or receptor hypersensitivity occurring as a result of the lesion, they might be unable to reverse postlesion morphological modifications.