Adult Rat Neostriatum Research Articles

Erratum: Sustained induction of neuronal addition to the adult rat neostriatum by AAV4-delivered noggin and BDNF

Correction to: Gene Therapy (2012) 19, 483–493; doi:10.1038/gt.2011.114; published online 15 September 2011 Since the online publication of this paper the authors have noticed that they omitted to mention support by the New York State Stem Cell Research Program (NYSTEM) in the Acknowledgements section.

Sustained induction of neuronal addition to the adult rat neostriatum by AAV4-delivered noggin and BDNF

Intraventricular ependymal infection by adenoviruses expressing brain-derived neurotrophic factor (BDNF) and noggin is sufficient to induce the heterotopic recruitment of new medium spiny neurons to the adult neostriatum, from endogenous subependymal neural progenitor cells. This approach was found to slow disease progression and extend survival in an R6/2 mouse model of Huntington's disease (HD). However, the practical therapeutic value of this strategy is limited by the transient expression and immunogenicity of adenoviral vectors. In addition, it has been unclear whether sustained overexpression of BDNF and noggin would yield similarly sustained neuronal production and striatal recruitment, or whether progenitor depletion or tachyphylaxis might supervene to limit the therapeutic potential of this approach. To address these issues, we used adeno-associated virus serotype 4 (AAV4), an ependymotrophic vector that is neither immunogenic nor neurotoxic, to achieve sustained BDNF and noggin expression. Using AAV4, we found that BDNF and noggin achieved levels sufficient to initiate and maintain, for at least 4 months, ongoing neuronal addition to the neostriatum and olfactory bulb. Over this period, we noted no diminution of treatment-associated neuronal recruitment from resident progenitors. AAV4:BDNF and noggin-induced neuronal addition may thus provide a means to provide longlasting and persistent striatal neuronal replacement in conditions of striatal neuronal loss, such as HD.

Gonadectomy and hormone replacement exert region- and enzyme isoform-specific effects on monoamine oxidase and catechol-O-methyltransferase activity in prefrontal cortex and neostriatum of adult male rats

Sex differences and gonadal hormone influences are well known for diverse aspects of forebrain amine and indolamine neurotransmitter systems, the cognitive and affective functions they govern and their malfunction in mental illness. This study explored whether hormone regulation/dysregulation of these systems could be related to gonadal steroid effects on catechol-O-methyltransferase and monoamine oxidase which are principal enzymatic controllers of forebrain dopamine, serotonin and norepinephrine levels. Driven by male over female differences in cortical enzyme activities, by male-specific associations between monoamine oxidase and catechol-O-methyltransferase gene polymorphisms and cognitive and dysfunction in disease and by male-specific consequences of gene knockouts in mice, the question of hormone sensitivity was addressed here using a male rat model where prefrontal dopamine levels and related behaviors are also known to be affected. Specifically, quantitative O-methylation and oxidative deamination assays were used to compare the activities of catechol-O-methyltransferase's soluble and membrane-bound isoforms and of monoamine oxidase's A and B isoforms in the pregenual medial prefrontal cortex and dorsal striatum of male rats that were sham operated, gonadectomized or gonadectomized and supplemented with testosterone propionate or with estradiol for 28 days. These studies revealed significant effects of hormone replacement but not gonadectomy on the soluble but not the membrane-bound isorfom of catechol-O-methyltransferase in both striatum and cortex. A significant, cortex-specific testosterone—but not estradiol—attenuated effect (increase) of gonadectomy on monoamine oxidase's A but not B isoform was also observed. Although none of these actions suggest potential roles in the reguation/dysregulation of prefrontal dopamine, the suppressive effects of testosterone on cortical monoamine oxidase-A that were observed could have bearing on the increased incidence of cognitive deficits and symptoms of depression and anxiety that are repeatedly observed in males in conditions of hypogonadalism related to aging, other biological factors or in prostate cancer where androgen deprivation is used as a neoadjuvant treatment.

Long-Term, Stable Differentiation of Human Embryonic Stem Cell-Derived Neural Precursors Grafted into the Adult Mammalian Neostriatum

Stem cell grafts have been advocated as experimental treatments for neurological diseases by virtue of their ability to offer trophic support for injured neurons and, theoretically, to replace dead neurons. Human embryonic stem cells (HESCs) are a rich source of neural precursors (NPs) for grafting, but have been questioned for their tendency to form tumors. Here we studied the ability of HESC-derived NP grafts optimized for cell number and differentiation stage prior to transplantation, to survive and stably differentiate and integrate in the basal forebrain (neostriatum) of young adult nude rats over long periods of time (6 months). NPs were derived from adherent monolayer cultures of HESCs exposed to noggin. After transplantation, NPs showed a drastic reduction in mitotic activity and an avid differentiation into neurons that projected via major white matter tracts to a variety of forebrain targets. A third of NP-derived neurons expressed the basal forebrain-neostriatal marker dopamine-regulated and cyclic AMP-regulated phosphoprotein. Graft-derived neurons formed mature synapses with host postsynaptic structures, including dendrite shafts and spines. NPs inoculated in white matter tracts showed a tendency toward glial (primarily astrocytic) differentiation, whereas NPs inoculated in the ventricular epithelium persisted as nestin(+) precursors. Our findings demonstrate the long-term ability of noggin-derived human NPs to structurally integrate tumor-free into the mature mammalian forebrain, while maintaining some cell fate plasticity that is strongly influenced by particular central nervous system (CNS) niches.

Adenovirally expressed noggin and brain-derived neurotrophic factor cooperate to induce new medium spiny neurons from resident progenitor cells in the adult striatal ventricular zone.

Neurogenesis from endogenous progenitor cells in the adult forebrain ventricular wall may be induced by the local viral overexpression of cognate neuronal differentiation agents, in particular BDNF. Here, we show that the overexpression of noggin, by acting to inhibit glial differentiation by subependymal progenitor cells, can potentiate adenoviral BDNF-mediated recruitment of new neurons to the adult rat neostriatum. The new neurons survive at least 2 months after their genesis in the subependymal zone and are recruited primarily as GABAergic DARPP-32+ medium spiny neurons in the caudate-putamen. The new medium spiny neurons successfully project to the globus pallidus, their usual developmental target, extending processes over several millimeters of the normal adult striatum. Thus, concurrent suppression of subependymal glial differentiation and promotion of neuronal differentiation can mobilize endogenous subependymal progenitor cells to achieve substantial neuronal addition to otherwise non-neurogenic regions of the adult brain.

Expression of regeneration-related molecules in injured and regenerating striatal and nigral neurons.

Peripheral nerve grafts in the neostriatum promote axonal regeneration from restricted classes of CNS neuron, principally cells in the substantia nigra pars compacta (SNpc) and striatal cholinergic interneurons. We have examined the molecular responses of CNS neurons induced to regenerate axons by tibial nerve grafting to the neostriatum of adult rats. Brain sections were probed for mRNAs for the transcription factor c-jun, and the cell recognition molecule CHL1, or immunoreacted for TrkA or p75, 1 day to 29 weeks after grafting (dpo; wpo). In unoperated rats, scattered neurons throughout the neostriatum showed weak signals for CHL1 mRNA and slightly stronger signals for c-jun mRNA. Cells of similar appearance strongly expressed TrkA but possessed little p75. By 1 dpo, many neostriatal neurons of various sizes and GFAP + glial cells near the host/graft interface had upregulated CHL1 mRNA, c-jun mRNA and p75. Most of the larger (20-25 microm diameter) CHL1 mRNA+ cells were also TrkA+, indicating that they were NGF-sensitive cholinergic interneurons. From two weeks postgrafting, high levels of CHL1 and c-jun mRNAs and p75 in the neostriatum were confined to a few presumptive cholinergic interneurons; p75+ cells were also TrkA+ and were larger than TrkA+ neurons on the contralateral side. Retrograde labelling showed that most p75+ and some TrkA+ neurons regenerated axons through the graft. Neurons in the SNpc showed a moderate to strong signal for CHL1 mRNA, weaker signal for c-jun mRNA, and no p75 or TrkA. Some SNpc cells upregulated c-jun mRNA after graft implantation, although they did not upregulate CHL1 mRNA, p75 or TrkA. Since neostriatal neurons which regenerate axons into grafts express receptors for NGF, and grafts mimic the effects of NGF treatment on these cells, sensitivity to graft-derived NGF may be a determinant of their high regenerative capacity. The finding that c-jun and CHL1 are consistently expressed by CNS neurons induced to regenerate their axons strongly supports the idea that these molecules are directly involved in axonal regeneration.

LONG TERM PRODUCTION OF REACTIVE OXYGEN SPECIES DURING PERINATAL ASPHYXIA IN THE RAT CENTRAL NERVOUS SYSTEM: EFFECTS OF HYPOTHERMIA

The formation of oxygen-derived free radicals in hypoxic and ischemic/reperfused brains has been proposed as an important step that links brain injury to neuronal death. Previously, we have demonstrated that reactive oxygen species (ROS) production was significantly increased in rat neostriatum during acute perinatal asphyxia (PA) in pups. In this article, we have studied the time course of ROS production in the neostriatum and neocortex of adult rats subjected to PA using electron spin resonance spectrometry (ESR) in order to record ROS production. Further more, we analyzed the actions of hypothermia on ROS release in pups and adult rats. We used for this study 6-month-old rats that suffered sub-severe and severe PA when they were pups. The most significant production of ROS was detected either in the neostriatum or neocortex at 19 and 20 min of PA. Hypothermia during 20 and 100 min at 15°C prevented ROS formation either in pups and adult rats. These data further support the concept that free radicals may contribute to the brain injury alterations and that hypothermia can prevent long-term sequelae induced by PA.

GABA promotes survival but not proliferation of parvalbumin-immunoreactive interneurons in rodent neostriatum: an in vivo study with stereology.

Amino-acid neurotransmitters regulate a wide variety of developmental processes in the mammalian CNS including neurogenesis, cell migration, and apoptosis. In order to investigate the role of GABA in early development of forebrain interneurons, we determined the survival of parvalbumin-immunoreactive GABAergic interneurons in the adult rat striatum following prenatal exposure to either GABA(A) receptor agonist or antagonist. Unbiased stereology was used to quantify parvalbumin-immunoreactive neuron number in the neostriatum of adult rats exposed to the drugs in utero, and the results were compared to pair-fed or vehicle controls. Embryos were exposed to the GABA(A) antagonist (bicuculline) or agonist (muscimol) during previously defined proliferative or post-proliferative periods for parvalbumin-immunoreactive interneurons. Unbiased stereology using the optical fractionator was used to estimate the total number of parvalbumin-immunoreactive neurons in neostriatum of experimental and control rats. No significant alteration in parvalbumin-immunoreactive neuron number was observed in rats treated with either bicuculline (1 or 2mg/kg/day) or muscimol (1mg/kg/day) during the proliferative phase. Administration of bicuculline during the post-proliferative phase significantly reduced parvalbumin-immunoreactive neuron number in the neostriatum. A concomitant decrease in neostriatal volume was also observed, suggesting that the effect is not restricted to parvalbumin-immunoreactive interneurons. Positional analysis revealed loss of normal regional distribution gradients for parvalbumin-immunoreactive neurons in neostriatum of rats exposed to bicuculline in the embryonic post-proliferative phase. This data collectively suggests that GABA promotes survival but not proliferation of parvalbumin-immunoreactive progenitors. GABA may also promote migration of subpopulations of interneurons that ultimately populate the ventral telencephalon.

Influence of Environment on the Efficacy of Intrastriatal Dopaminergic Grafts

Functional recovery is influenced by experience. The aim of the present work was to examine the effects of “enriched” environment (EE) versus an “impoverished” environment on the anatomical and functional integration of intrastriatal dopaminergic grafts. These influences were studied using a paradigm where grafting was performed before the dopamine-depleting lesion. Dopaminergic grafts were implanted into the left neostriatum of adult male rats. In the enriched group, grafted rats were housed collectively and were trained on different behavioral tests following grafting. In contrast, impoverished grafted rats were housed individually and not further manipulated. Ten weeks after grafting, the mesotelencephalic dopaminergic pathway was destroyed unilaterally to the grafted side and different behaviors were followed for 7 months. Grafting prior to lesioning had no prophylactic effects on the performance as the graft did not prevent the onset of the lesion-induced impairments. However, under EE conditions, a graft effect was manifested in the reduction of drug-induced rotation and on the indices of bias as tested by a spatial alternation test. No positive graft effects were observed in the skilled paw reaching test. Grafted rats raised under impoverished conditions performed in a fashion indistinguishable from the control lesioned animals on most measures of behavior. A beneficial effect of EE conditions was observed on survival of TH-positive neurons within the grafts. The results suggest that survival of grafted neurons, and the reduction of the magnitude of particular behavioral impairments, can be optimized by increasing the complexity of the subject's environment.

Alterations in the turnover rate of dopamine D1 but not D2 receptors in the adult rat neostriatum after a neonatal dopamine denervation

Adult rats that were treated with intracerebral ventricular injection of 6-hydroxydopamine (6-OHDA) as neonates exhibit a profound loss of nigrostriatal dopamine innervation in addition to a variety of other neurochemical and anatomical changes, including alterations in the number of neostriatal D1 and D2 receptor binding sites. In the present study, the turnover of neostriatal dopamine D1 and D2 receptors was measured in rats previously treated with 6-OHDA or ascorbic acid vehicle as neonates at various time intervals after peripheral N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 10 mg/kg) administration. Dopamine receptors were labelled with [ 3H]SCH23390 (D1) and [ 3H]raclopride (D2), while the degree of dopamine denervation was assessed by the measurement of neostriatal dopamine, homovanillic acid and dihydroxyphenylacetic acid content. Two days after acute EEDQ treatment, the maximal binding capacity ( B max) of [ 3H]SCH23390 and [ 3H]raclopride binding was significantly decreased to 58 and 32% of control values, respectively, without any significant alteration in their equilibrium dissociation constants ( K d). A time-dependent increase in the density of [ 3H]SCH23390 and [ 3H]raclopride binding was observed in both treatment groups following a single dose of EEDQ. The rate of recovery of D1 receptors was significantly slower in the 6-OHDA-lesioned animals as compared to controls with a half-life of 103 compared to 53 h, respectively. No differences were observed in the rate of recovery of D2 receptors in these two treatment groups. These data are consistent with the findings of decreased expression of D1 receptors in neonatal 6-OHDA-lesioned rats owing to decreased receptor synthesis, and further suggest that in this model the up-regulation of D2 receptors is a result of a post-transcriptional mechanism, such as an increased rate of post-synthetic maturation.

Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat neostriatum: A quantitative autoradiographic and immunocytochemical analysis

Dopamine (DA) axon terminals (varicosities) in the neostriatum of adult rats were examined for shape, size, content, synaptic incidence, type of junction, synaptic targets, and microenvironment after electron microscopic identification either by [3H]DA uptake autoradiography or by immunocytochemistry with monoclonal antibodies against DA-glutaraldehyde-protein conjugate. Both approaches yielded comparable results. Whether they were from the paraventricular or the mediodorsal neostriatum, respectively, the [3H]DA-labeled and DA-immunostained varicosities were generally oblong and relatively small; more than 60% contained one or more mitochondria. Sixty to seventy percent were asynaptic, and 30–40% were endowed with a synaptic membrane differentiation (junctional complex), as inferred by stereological extrapolation from single thin sections (both approaches) or observed directly in long, uninterrupted series of thin sections (immunocytochemistry). The synaptic DA varicosities always displayed symmetrical junctions: 67% with dendritic branches, 30% with dendritic spines, and 2–3% with neuronal cell bodies. DA varicosities juxtaposed to one another were frequent. Other axonal varicosities were more numerous in the immediate vicinity of DA varicosities than around randomly selected, unlabeled terminals. The respective microenvironments of DA and unlabeled varicosities also showed enrichment in the preferred synaptic targets of both groups of varicosities, with dendritic branches for DA and dendritic spines for the unlabeled ones. These data suggest a dual mode of operation that is diffuse as well as synaptic for the nigrostriatal DA system. In such a densely DA-innervated brain region, they also dead to the hypothesis that a basal level of extracellular DA might be maintained permanently around every tissue constituent and, thus, contribute to the mechanisms of action, properties, and functions (or dysfunctions) of DA within the neostriatum itself and as part of the basal ganglia circuitry. © 1996 Wiley-Liss, Inc.

Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat neostriatum: a quantitative autoradiographic and immunocytochemical analysis.

Dopamine (DA) axon terminals (varicosities) in the neostriatum of adult rats were examined for shape, size, content, synaptic incidence, type of junction, synaptic targets, and microenvironment after electron microscopic identification either by [3H]DA uptake autoradiography or by immunocytochemistry with monoclonal antibodies against DA-glutaraldehyde-protein conjugate. Both approaches yielded comparable results. Whether they were from the paraventricular or the mediodorsal neostriatum, respectively, the [3H]DA-labeled and DA-immunostained varicosities were generally oblong and relatively small; more than 60% contained one or more mitochondria. Sixty to seventy percent were asynaptic, and 30-40% were endowed with a synaptic membrane differentiation (junctional complex), as inferred by stereological extrapolation from single thin sections (both approaches) or observed directly in long, uninterrupted series of thin sections (immunocytochemistry). The synaptic DA varicosities always displayed symmetrical junctions: 67% with dendritic branches, 30% with dendritic spines, and 2-3% with neuronal cell bodies. DA varicosities juxtaposed to one another were frequent. Other axonal varicosities were more numerous in the immediate vicinity of DA varicosities than around randomly selected, unlabeled terminals. The respective microenvironments of DA and unlabeled varicosities also showed enrichment in the preferred synaptic targets of both groups of varicosities, with dendritic branches for DA and dendritic spines for the unlabeled ones. These data suggest a dual mode of operation that is diffuse as well as synaptic for the nigrostriatal DA system. In such a densely DA-innervated brain region, they also lead to the hypothesis that a basal level of extracellular DA might be maintained permanently around every tissue constituent and, thus, contribute to the mechanisms of action, properties, and functions (or dysfunctions) of DA within the neostriatum itself and as part of the basal ganglia circuitry.

Ultrastructural characterization of the acetylcholine innervation in adult rat neostriatum

The ultrastructural features of acetylcholine axon terminals (varicosities) in adult rat neostriatum were characterized by electron microscopy after immunostaining with a sensitive monoclonal antibody against rat choline acetyltransferase. Several hundred single sections from these varicosities were analysed for shape, size and content, presence of a synaptic membrane specialization, and composition of the microenvironment. An equivalent number of unlabeled varicosities selected at random from the same micrographs were similarly examined. The immunostained varicosity profiles were relatively small and seldom showed a junctional membrane specialization. Stereological extrapolation to the whole volume of these varicosities indicated that less than 10% were synaptic. Far fewer dendritic spines were juxtaposed to these predominantly asynaptic profiles than to their unlabeled counterparts. This difference seemed imputable to the low synaptic incidence of the acetylcholine varicosities and was consistent with the view that these are randomly distributed in relation to surrounding elements. The bulk of the data was suggestive of volume transmission. This raised the possibility that, in such a densely innervated area, a basal level of acetylcholine is permanently maintained around all cellular elements, contributing to the modulatory properties of this transmitter. This basal level of acetylcholine could also serve as a regulatory signal controlling the expression of different receptor subtypes in neurons, glia and blood vessels.

Dopamine receptor alterations correlate with increased GABA levels in adult rat neostriatum : effects of a neonatal 6-hydroxydopamine denervation

The effects of neonatal intracerebroventricular 6-hydroxydopamine (6-OHDA) injection on the densities of dopamine (DA) receptors and GABA levels were determined in the rostral neostriatum of adult rats. Measurement of GABA turnover indicated that increased tissue GABA in the DA-lesioned neostriatum is a consequence of higher GABA synthesis rate (205%). Binding experiments with [ 3H]SCH23390 (D 1 receptors) and [ 3H]raclopride (D 2 receptors) point to a correlation between tissue GABA content and altered DA receptors. Three months after the lesion there was a 27% decrease in D 1 receptors and a 22% increase in D 2 receptors. In control neostriatum, GABA levels were inversely related to D 2 receptors and this relationship was reversed after 6-OHDA treatment. In contrast, the positive correlation between GABA and D 1 receptors remained unchanged after the lesion. Irreversible blockade of DA receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) decreased both D 1 and D 2 sites (73–87%) in both control and lesioned neostriatum, but increased GABA levels by 25% only in animals which have received 6-OHDA just after birth. Following acute inhibition of DA synthesis or of DA catabolism, GABA levels remained unchanged. The present results indicate that DA depletion by itself is not the cause for the increase in GABA levels. The augmented GABAergic activity following neonatal 6-OHDA is seemingly influenced primarily by DA receptor status; presumably, changes in D 2 receptor properties during maturation may be a principal cause for an increase in neostriatal GABA content.

Long distance pathways of diffusion for dextran along fibre bundles in brain. Relevance for volume transmission.

Texas Red-labelled dextran with a mol. wt of 3000 g mol-1, a marker for the extracellular space, was injected unilaterally into the neostriatum of adult rats (0.3-30 micrograms microliter-1) and its distribution evaluated 1 min to 5 h later. Diffusion in the neuropil was observed with clearance starting after 30 min. After 10-15 min strong labelling along the myelinated fibre bundles was observed in the entire neostriatum. After about 20 min the labelling along the fibres reached into the corpus callosum and the overlaying deep layers of the cerebral cortex. A marked cellular uptake and accumulation of labelled dextran was found in putative perivascular pericytes. Thus, in the living brain preferential extracellular fluid pathways for diffusion exist, especially along fibre bundles, which allow the exchange of chemical signals between two distant regions. These may represent extracellular fluid pathways for volume transmission.

Synaptic reorganisation in the rat striatum after dopaminergic deafferentation: an ultrastructural study using glutamate decarboxylase immunocytochemistry.

The ultrastructure of GABAergic and non-GABAergic synapses in the adult rat neostriatum was examined 6-8 months after unilateral removal of the nigrostriatal dopaminergic pathway by 6-hydroxydopamine injection into the medial forebrain bundle. GABAergic profiles were identified by preembedding glutamate decarboxylase (GAD) immunocytochemistry performed on parasagittal vibratome sections. In three representative fields of the striatum, the nature and number of boutons and their postsynaptic partners were determined and the differences between the striata ipsi- and contralateral to the lesion analyzed. The percentage of GAD-immunoreactive boutons was increased from 23% on the intact side to 28% on the lesioned side. In addition, the GABAergic boutons underwent significantly more multiple contacts with several independent postsynaptic profiles, preferentially with dendritic spines. This could reflect a lesion-induced sprouting of local GABAergic axon terminals. On the other hand, although the vast majority of GABAergic boutons underwent synaptic contacts with dendrites (77% vs. 80%), the number of boutons per dendrite or per dendritic circumference remained unchanged. Thus, the higher frequency of GABAergic boutons may simply reflect the loss of the dopaminergic nerve endings, without a heterosynaptic replacement by GABAergic boutons. The deafferentation also induced structural changes of the postsynaptic profiles. Some dendritic spines had a shortened neck; others were completely integrated in the dendrite which now contained a spine apparatus and was contacted by boutons with the features of axospinous synapses. The spine retraction resulted in a quantitative decrease in the number of spines. Analysis of the synaptic curvature revealed that only spines with a flat contact zone were lost. Concurrently, the number of dendrites was increased, of the GAD-containing in particular, suggesting that the denrites of GABAergic interneurons tend to elongate and/or arborize. Taken together, the results of the present study show that the dopaminergic denervation caused a remodeling of the postsynaptic neurons. The relative increase of the number of GABAergic boutons and their synaptic contacts suggests that an altered wiring of the intrinsic GABAergic system contributes to the changes in the striatal output activity.

Quantification of the serotonin hyperinnervation in adult rat neostriatum after neonatal 6-hydroxydopamine lesion of nigral dopamine neurons

Light microscope autoradiography after uptake and storage of tritiated serotonin (5-HT) in brain slices was used to count 5-HT axon terminals (varicosities) in the 5-HT-hyperinnervated neostraitum of adult rats subjected to neonatal 6-hydroxydopamine treatment and age-matched, normal controls. After correction for incomplete autoradiographic exposure and for section thickness, the results were expressed in millions of varicosities per mm 3 of tissue. Control values ranged from 4.8 in the rostral to 6.3 in the caudal neostriatum (5.8 at intermediate level), for an average of 5.6.The corresponding values in 5-HT-hyperinnervated tissue ranged from 9.7 to 7.7 (8.8 at intermediate level), for an average of 8.7 and increases of 102%, 52% and 22% above control in the rostral, intermediate and caudal neostriatum, respectively (average increase of 55%). These data confirmed the predilection of the 5-HT hyperinnervation for the rostral neostriatum and demonstrated its presence in the caudal neostriatum also.

The localization of an antibody to STEP in embryonic striatal tissue grafts.

We used immunohistochemical staining with antibodies against the novel protein striatal enriched phosphatase (STEP) to investigate the internal organization of grafts of embryonic striatal tissues implanted in the ibotenic acid-lesioned neostriatum of adult rats. STEP immunoreactivity was found in discrete patches within the grafts, which colocalized with areas designated as 'patch' zones when stained for the enzyme acetylcholinesterase and with antibodies against tyrosine hydroxylase and DARPP-32. As previously hypothesized, the pattern of STEP immuno-reactivity in embryonic striatal tissue grafts provides further indication that the patch zones are indeed comprised of striatal like cell populations. The novel protein STEP provides a sensitive and precise marker for this compartment within the grafts.

Localization of the mRNAs for the short and long isoforms of the dopamine D2 receptor in embryonic striatal grafts: predominance of the short isoform.

The expression of mRNA coding for the long (D2L) and short (D2S) isoforms of the dopamine D2 receptor was measured by in situ hybridization histochemistry in embryonic striatal tissue grafts implanted into the ibotenic acid-lesioned neostriatum of adult rats. The intact, lesioned and grafted neostriata were evaluated 3 months after transplantation using 35S-labelled 'antisense' oligonucleotide probes (45-mer) specific for each mRNA isoform. In the adult neostriatum each mRNA isoform exhibited a strong lateral-to-medial gradient in expression in terms of both the number of expressing cells and the hybridization signal (grain density) per cell. Although there were no significant differences in the numbers of cells expressing the two isoforms in each striatal compartment, the hybridization signal per cell for D2S was significantly greater than that for D2L in the lateral and central striatum. The mRNA expression of each isoform was markedly reduced by the lesions. In the striatal grafts, the expression of both D2L and D2S occurred in 'patches' that corresponded to patches of intense acetylcholinesterase activity, and which are believed to involve reaggregation of striatal-like tissues within the graft. In contrast to the situation in the intact neostriatum the striatal grafts contained significantly greater numbers of cells expressing D2S mRNA; however, similar to the findings in the intact striatum the mRNA signal per cell was significantly greater than that for D2L mRNA. These results indicate that the D2 receptor isoforms are differentially expressed in mature striatal tissue grafts, and suggest that the dopamine regulation of striatal grafts via D2 signal transduction mechanisms may be similar to that observed in the normal neostriatum.

Host Serotonin Axons Innervate Intrastriatal Ventral Mesencephalic Grafts After Implantation in Newborn Rats

This study investigated the potential of immature and adult serotonin (5-HT) neurons for axonal growth into intrastriatal grafts of ventral mesencephalic tissue. Implantation of dissociated fetal (embryonic days 14-15) ventral mesencephalic tissue was carried out in immature [postnatal days (P) 5-14] and adult rat neostriatum. The brains were processed 2-6 months later for dopamine and 5-HT immunocytochemistry. A few grafts implanted into adult and P7 recipients contained small numbers of cotransplanted 5-HT cell bodies. These also displayed a rich network of 5-HT axons, even in adult rats prelesioned with 5,7-dihydroxytryptamine, indicating the graft origin of these axons. All other grafts were totally devoid of 5-HT cell bodies. After implantation in adults, such grafts contained rare 5-HT axons. In contrast, in P5-P7 recipients, they displayed many 5-HT fibres, which were uniformly distributed. Such was no longer the case after implantation in P14 recipients, which showed minimal 5-HT innervation, as in adult recipients. Processing of naïve rat brain at different ages for 5-HT immunocytochemistry showed that 5-HT axons were still clearly less numerous in the neostriatum at P21 than in adults, whereas in the substantia nigra the 5-HT innervation developed more rapidly and was comparable, at P21, to that of adults. It was concluded that 5-HT axons are able to grow into ventral mesencephalic grafts, but mainly at the fetal stage and with decreasing capacity after birth.(ABSTRACT TRUNCATED AT 250 WORDS)