Injection Of Ibotenic Acid Research Articles

Strong c-Jun/AP-1 immunoreactivity is restricted to apoptotic cells following intracerebral ibotenic acid injection in developing rats

Strong c-Jun immunoreactivity, as revealed with the antibody c-Jun/activator protein 1 (AP-1) which is raised against the amino acids 91–105 mapping with the amino terminal domain of mouse c-Jun p39, is observed in apoptotic cells, but not in necrotic cells, following intracerebral injection of ibotenic acid in the developing rat brain processed for immunohistochemistry. Immunostaining occurs in the cytoplasm and dendrites, thus suggesting impaired nuclear translocation of c-Jun in apoptotic cells. Western blotting of total brain homogenates, using the same antibody, shows a band at p39 which is more marked in treated animals than in age-matched controls. In addition, increased c-Jun N-terminal kinase 1 (JNK-1) expression, as revealed on Western blots, is found in rats treated with ibotenic acid when compared with controls. In contrast, apoptotic cells are not stained with antibodies to Jun B and Jun D. These results give further support to previous studies showing strong c-Jun expression in apoptotic cells at determinate stages of development, and emphasize that intracellular distribution of c-Jun, possible post-translational modifications of c-Jun due to phosphorylation at specific transactivation sites, and lack of associated Jun B and Jun D expression may differentiate the Jun response in apoptotic cells from other forms of cellular response involving c-Jun which are not associated with cell death.

Selective Hippocampal Lesions in Rats Disrupt Acquisition and Retention of a Positive Patterning Discrimination

Gisquet-Verrier, P. and N. El Massioui. Selective hippocampal lesions in rats disrupt acquisition and retention of a positive patterning discrimination. Physiol Behav 61(4) 577–589, 1997.—To determine the contribution of the hippocampus in the processing of a configural positive patterning discrimination (PPD) task, discrimination between reinforced presentations of a tone plus light compound stimulus and nonreinforced presentations of each of its components (TL+/T−,L−) was examined using a conditioned-suppression paradigm. In the first experiment, rats demonstrated a rapid acquisition of the PPD with an appropriate discriminative responding. Rats submitted to posttraining hippocampal lesions (using multiple injections of ibotenic acid) were no longer able to master correctly the previously solved discrimination, demonstrating significant differences in their response rates during the 2 never-reinforced elemental stimulus presentations. In Experiment II, lesioned rats were not able to correctly learn the PPD, demonstrating the same pattern of responding as in Experiment I. These rats were also severely disrupted in a radial maze elimination task. Experiment IIIa indicated that, in a simple conditioning task (T+,L+), normal rats acquired a rapid conditioned suppression for both stimuli, with the tone being slightly more susceptible to conditioning than the light stimulus. In Experiment IIIb, conditioning to the compound tone plus light stimulus led to a clear conditioning to the tone and almost no conditioning to the light, suggesting an overshadowing from the tone to the light. Similar results were obtained in rats with hippocampal lesions. These results strongly suggest that the disruption showed by rats with hippocampal lesions in the PPD task cannot be due to an alteration of the relative salience of the stimulus. The inability of rats with hippocampal lesions to solve correctly the PPD is due to difficulties in eliminating responding to some unimportant events of the situation, reflecting a deficit in selective attention processes rather than in an ability to process configural stimuli. In the discussion, the putative role of the hippocampus in selective attentional processes is more fully discussed.

Behavioral correlates of transneuronal degeneration of substantia nigra reticulata neurons are reversed by ablation of the subthalamic nucleus

In rats, acute injury of neurons in the caudate nucleus (CN) and globus pallidus (GP) by local injection of ibotenic acid (IA) or by transient forebrain ischemia has caused transneuronal cell death of neurons in the substantia nigra reticulata (SNr) weeks after the initial injury. Recently transient expression of an immediate early gene c-fos was induced specifically in neurons of the subthalamic nucleus (STN) and SNr at 36–48 h after the IA-lesions, prior to the delayed degeneration of SNr neurons. These cellular and molecular events may alter the level of inhibitory output from the basal ganglia and lead to movement disorders. To test (i) whether movement disorders occur in the early period after unilateral lesions of the CN and GP by IA-injection, and (ii) whether ablation of the STN reverses the early movement disorders, we used a modified version of Porsolt forced swim test in which the lesion-induced asymmetry of motor function becomes apparent as rotation when the animals are forced to swim. Following unilateral IA-lesions of the right CN and GP in rats, rapid contraversive rotation appeared transiently 36–48 h after the lesions, and, in turn, slow ipsiversive rotation appeared at 3–5 days postlesion. Prior ablation of the ipsilateral STN reversed these early movement disorders produced by the unilateral IA-lesions of the CN and GP and instead created persistent contraversive rotation 7–10 days after the lesions. Each phase of the dominant rotation behavior was dependent on asymmetrical limb motor activity; decreased left limb activity caused contraversive rotation, and increased left limb activity caused ipsiversive rotation. Reversal of these early movement disorders suggests that ablatin of the STN prevents the transneuronal degeneration of the SNr.

Astrocytes Grafted Into Rat Nucleus Basalis Magnocellularis Immediately After Ibotenic Acid Injection Fail to Survive and Have no Effect on Functional Recovery

In order to determine if the “trophic” properties of astrocytes makes them appropriate for use as a therapeutic agent to excitotoxic brain damage, adult male rats received grafts of cultured cerebral cortical astrocytes into the NBM immediately after infusion of ibotenic acid into the same structure. Twenty four hours after grafting and every other day for 11 days post surgery, the animals were tested for locomotor activity and habituation in an open field. Animals with NBM lesions had significantly reduced rearing activity as compared to counterparts with no lesions. Nine days after surgery, rats with NBM lesions and astrocyte grafts were as impaired in the acquisition of passive avoidance (PA) as their untreated counterparts. AH animals with ibotenic lesions were impaired on PA retention compared to rats with no lesions. There was no difference between animals that had received grafts and those that had not. Fourteen days after grafting, all brains were processed for Nissl stain, acetylcholinesterase (AChE) histochemistry, GFAP immunocytochemistry, and bisben-zamide fluorescent microscopy. Decreases in the number of neurons in the NBM as well as decreases in the density of AChE staining in the ipsilateral cortex (the area of innervation of the NBM cholinergic neurons) was evident in all animals with NBM lesions. In addition, a large number of host reactive astrocytes were seen within the NBM, its vicinity, and in the ipsilateral neocortex. Grafted astrocytes survived and integrated into the host tissue when they were grafted into the brain of intact animals but no living grafted astrocytes were found in animals injected with ibotenate. In this latter case, two weeks after grafting, instead of surviving astrocytes only fluorescent tissue ‘masses’ were seen in the NBM. surrounded by a cavity. Grafted astrocytes did not have any effect on the extension of the lesion caused by ibotenic acid infusion. These results suggest that the concentration of ibotenic acid used to injure the NBM killed not only the host cholinergic neurons but also the grafted astrocytes. The failure of astrocytes to ameliorate the behavioral deficits caused by ibotenic acid lesions of the NBM may be due to the ibotenic acid creating a lethal environment for the grafted and freshly dissociated, cultured astrocytes.

Effects of lesions in the medial prefrontal cortex on the activity of midbrain dopamine neurons

To determine whether lesions in the prefrontal cortex (PFC) alter the activity of midbrain dopamine (DA) neurons, single unit recordings were made from DA neurons in control and lesioned rats. PFC lesions, obtained by local injection of ibotenic acid into the medial PFC, had no effect on either firing rate or bursting activity of DA neurons in the ventral tegmental area (VTA). However, the number of spontaneously active DA neurons in the VTA was significantly decreased. In the substantia nigra (SN), the same lesions increased the firing rate and had no effect on either the bursting activity of the number of active DA cells. These results suggest that PFC lesions alter the activity of DA neurons. However, VTA and SN DA neurons may respond differently to PFC lesions.

OPPOSITE CHANGES IN THE EXPRESSION OF ENKEPHALIN IN THE AMYGDALA AND HYPOTHALAMUS AFTER LESIONS OF THE BED NUCLEUS OF THE STRIA TERMINALIS IN THE RAT

The expression of enkephalin in neurons of the rat forebrain was studied by in situ hybridization and immunohistochemistry after unilateral injections of ibotenic acid into the bed nucleus of the stria terminalis. Initially, we observed that the destruction of nerve cell bodies in this nucleus resulted in a prominent bilateral increase in the number of neuronal perikarya immunoreactive for [Met]enkephalin in the lateral/basolateral amygdaloid complex-especially in the anterior division of the latter nucleus-as compared with NaCl-injected rats. In a separate set of experiments, this effect was associated with a significant (two times) enhancement of the number of nerve cell bodies containing preproenkephalin A messenger RNAs in the same amygdaloid nucleus ipsilateral to the injection, as compared with controls. In the hypothalamus of both experimental and control rats, the nerve cell bodies immunoreactive for [Met]enkephalin were few since the animals were not pretreated with colchicine, and the effects of the lesion were difficult to appreciate. However, using in situ hybridization, numerous nerve cell bodies containing preproenkephalin A messenger RNAs were detected bilaterally in the perifornical area, the paraventricular (parvocellular division) and the ventromedial nuclei of the hypothalamus. In the latter nucleus, the lesion of the bed nucleus of the stria terminalis resulted in a strong decrease (about two times) in the number of labelled cell bodies as compared with the controls, whereas no significant changes were found bilaterally in the paraventricular nucleus.In agreement with some data of the literature, our results indicate that the bed nucleus of the stria terminalis plays an important role in the regulation of neuropeptide genes expression in certain regions of the limbic system. Such a role is often exerted by nerve fibres afferents to the nerve cell bodies considered. However, from numerous neuroanatomical data of the literature, it appears more probable that the induction or inhibition of the expression of enkephalin in presynaptic neurons is due to the disappearance of their postsynaptic target in the bed nucleus of the stria terminalis. Copyright © 1996 IBRO. Published by Elsevier Science Ltd.

The potential of high-resolution positron emission tomography to monitor striatal dopaminergic function in rat models of disease

The use of a recently commissioned small-diameter, high-resolution positron emission tomograph (PET) to obtain a measure of specific binding of 3 carbon-11 labelled ligands in rat striatum is described. Using cerebellum as a reference tissue, compartmental modelling was used to obtain individual estimates of striatal binding potential (defined as the ratio of rate constants to and from the specifically bound compartment) for [ 11C]raclopride (D 2 receptors), [ 11C]SCH 23390 (D 1 receptors) and [ 11C]RTI-121 (dopamine transporter). The coefficients of variation in control, anaesthetized rats were of the order of 10%. Using two models of human disease, namely striatal injection of ibotenic acid to produce postsynaptic cell loss as in Huntington's disease, and 6-hydroxydopamine injection into substantia nigra pars compacta to mimic dopaminergic terminal loss in Parkinson's disease, marked reductions in binding potential were observed for the corresponding pre- or postsynaptic markers. When the regions of interest are so small as to be of the order of the spatial resolution of the system, factors such as spill over and partial volume negate absolute quantification of tissue radioactivity. Nevertheless, the use of PET to monitor relative changes in dopaminergic integrity should be considered as a viable complement to established in vivo microdialysis and post mortem techniques.

The potential of high-resolution positron emission tomography to monitor striatal dopaminergic function in rat models of disease.

The use of a recently commissioned small-diameter, high-resolution positron emission tomography (PET) to obtain a measure of specific binding of 3 carbon-11 labelled ligands in rat striatum is described. Using cerebellum as a reference tissue, compartmental modelling was used to obtain individual estimates of striatal binding potential (defined as the ratio of rate constants to and from the specifically bound compartment) for [11C]raclopride (D2 receptors), [11C]SCH 23390 (D1 receptors) and [11C]RTI-121 (dopamine transporter). The coefficients of variation in control, anaesthetized rats were of the order of 10%. Using two models of human disease, namely striatal injection of ibotenic acid to produce postsynaptic cell loss as in Huntington's disease, and 6-hydroxydopamine injection into substantia nigra pars compacta to mimic dopaminergic terminal loss in Parkinson's disease, marked reductions in binding potential were observed for the corresponding pre- or postsynaptic markers. When the regions of interest are so small as to be of the order of the spatial resolution of the system, factor such as spill over and partial volume negate absolute quantification of tissue radioactivity. Nevertheless, the use of PET to monitor relative changes in dopaminergic integrity should be considered as a viable complement to established in vivo microdialysis and post mortem techniques.

Thalamic and basal forebrain afferents modulate the development of parvalbumin and calbindin D28k immunoreactivity in the barrel cortex of the rat.

In the adult barrel cortex of the rat the calcium-binding proteins calbindin D28k (CALB) and parvalbumin (PARV) are found in separate populations of GABAergic nonpyramidal neurons. In layers II to IV of the barrel cortex most PARV-immunoreactive neurons are likely to derive from a subpopulation of CALB-immunoreactive neurons whose CALB immunoreactivity ceases when they begin to express PARV between the second and third postnatal weeks. The aim of this study was to investigate the influence of subcortical afferents on the neurochemical differentiation of cortical PARV- and CALB-immunoreactive nonpyramidal neurons during development of the barrel cortex. We produced unilateral excitotoxic lesions with a single injection of ibotenic acid (0.5 microl, 0.05 M) in different subcortical nuclei in 7- to 8-day-old rats. Lesions involving the ventroposterior thalamic nuclei resulted in delayed development of PARV and CALB immunoreactivity in the barrel cortex. One week after ibotenic acid injections a transient decrease in the number of PARV-immunoreactive neurons in layer IV was observed, together with increased numbers of CALB-immunoreactive neurons in all cortical layers. The number of nonpyramidal neurons displaying coexistence of PARV and CALB in the lesioned hemisphere also increased compared with the numbers in the control hemisphere or control littermates. In contrast, lesions affecting the globus pallidus, zona incerta and reticular thalamic nucleus transiently increased the number of PARV-immunoreactive neurons in layers II and III, but had no effect on the number of CALB-positive cells. From 3 weeks onwards no differences were found between control and lesioned hemispheres after injections into either the ventroposterior thalamic nuclei or the magnocellular basal forebrain. These results suggest that CALB and PARV expression in nonpyramidal cortical neurons can be reversibly modulated in opposite directions by different cortical afferents during postnatal development.

Effect of ibotenic acid lesions of the omnipause neurons on saccadic eye movements in rhesus macaques.

1. Although much is known about the neurons that control saccadic eye movements, the precise manner in which they interact is still uncertain. To test the validity of competing models of the pontine saccade generator, neurotoxic lesions were made in the nucleus raphe interpositus (rip), which contains one of the principal types of saccade-related neurons, the omnipause neurons (OPNs). The correlated changes in eye movement were quantified in three juvenile rhesus macaques and compared with the results predicted by different models. 2. After the location of the OPNs was mapped, the rip was subjected to sequential, punctate pressure injections of ibotenic acid. The resulting progressive damage was correlated with changes in saccade metrics, including a decrease in peak saccadic velocity and an increase in saccade duration. 3. The damage to rip and presumably to the OPNs was not associated with a change in the animals' ability to maintain steady fixation of a stationary target. 4. The results suggest that Robinson's original local feedback model of saccade generation should be modified. Either a second integrator should be added or the concept of local feedback should be abandoned entirely. 5. The suggestion that the OPNs are primarily responsible for fixation is probably incorrect. OPNs may contribute to fixation stability along with a number of other sources.

Effects of chronic infusion of nerve growth factor (NGF) in rats with nucleus basalis magnocellularis lesion.

We attempted to evaluate the effects of bilateral injection of ibotenic acid (IA) into the nucleus basalis magnocellularis (nbm) of rats as well as the potential recovery mediated by the infusion of nerve growth factor (NGF). The lesion caused an impairment of learning and memory processes. Also, a severe depletion of choline acetyl transferase activity was detected in cortical areas. After the NGF administration, a significant reversion of these functional changes was observed. Thus, IA-lesioned rats might serve as a model for the evaluation of neurotrophic factors actions on basal forebrain damaged neurons.

Compartment analysis of cerebral glucose metabolism and in vitro glucose-metabolizing enzyme activities in the rat brain

To clarify the relationship between cerebral glucose metabolic rate constants and glucose-metabolizing enzyme activities in the cerebral cortex, we evaluated the cerebral metabolic rate of glucose (CMRGlu), metabolic rate constants of [ 18F]-2-fluoro-2-deoxy-D-glucose (FDG) and related enzyme activities in the frontal cortex under normal and glucose metabolism-suppressed conditions. Applying a three-compartment four-parameter model, metabolic rate constants were obtained by dynamic positron emission tomography with FDG, and CMRGlu was calculated based on these rate constants. The glycolytic enzyme activities were determined by in vitro biochemical assay. Three days after ibotenic acid injection into the basal forebrain, CMRGlu was decreased in the ibotenic acid-treated frontal cortex as well as k3 * (phosphorylation), while K1 * (plasma to brain) showed no remarkable change. No significant reductions of the enzyme activities except for hexokinase activity were found in the frontal cortex. Regression analysis showed a significant positive correlation between k3 * and the hexokinase activity. These results suggested that k3 * in the compartment analysis reflects hexokinase activity.

Structural basis of cortical synchronization. II. Effects of cortical lesions.

1. To understand the structural basis of the different types of interhemispheric synchronizations described in the preceding paper, we made sections of the corpus callosum and lesions of extrastriate cortex. We measured the effects of such operations on the frequency of encounter, width and strength of T, C, and H peaks in cross-correlation histograms computed from single-unit and multiunit recordings from areas 17-18 of opposite cortical hemispheres in the cat. 2. Sectioning of the corpus callosum led to a complete abolition of T and C couplings and a strong reduction of encounter rate and strength of H coupling. A section limited to the posterior half of the corpus callosum abolished T and C couplings completely. This suggests that T and C couplings are mediated by the direct reciprocal connections between visual cortical areas circulating through the posterior part of the corpus callosum. 3. The encounter rate of H peaks depended on the extent of the callosal cut. Larger lesions gave a more pronounced reduction of the number of H peaks. From this observation we conclude that H peaks are at least partially mediated by polysynaptic pathways involving widely distributed cortical regions. 4. Extensive lesions of extrastriate cortex were made by aspiration of the gray matter or injections of ibotenic acid. These lesions removed the direct inputs from cortical areas sending ipsilateral as well as contralateral inputs to the area 17-18 border region. Encounter rate and coupling strength of C and H peaks were decreased, whereas little effect was observed on T peaks. 5. These results demonstrate that all types of interhemispheric synchronization are mediated by corticocortical connections and that T and C peaks are generated by reciprocal connections between areas 17 and 18 of each hemisphere. Feedback connections play a role in mediating or facilitating the C and H types of interhemispheric synchronization.

Effects of Vamicamide on Urinary Bladder Functions in Conscious Dog and Rat Models of Urinary Frequency

Purpose To investigate the usefulness of vamicamide, (plus/minus)-(2R*, 4R*)-4-dimethylamino-2-phenyl-2-(2-pyridyl)valeramide, as a novel drug for the treatment of urinary frequency and incontinence. Materials and Methods Urinary frequency was evaluated in specially devised conscious dog and rat models by investigating the effects of the drug on urinary bladder function of these animals by cystometrography. Results In the dog model with transected hypogastric nerves, the bladder volume at micturition (bladder capacity) was less than 50 percent that of the sham-operated dog, and in the rat model with bilateral lesioning of nuclei basalis, a part of the brain, by ibotenic acid injection, bladder capacity was about 50 percent that of the sham-operated rat. Other bladder functions in both models were unchanged. In the dog model, orally administered vamicamide at 0.32 and 1.0 mg./kg. significantly increased bladder capacity and did not change residual urine volume or micturition pressure. Oxybutynin 0.10 mg./kg., one of the most popular drugs for the treatment of urinary frequency and incontinence, or atropine 0.10 mg./kg. induced significant increases in bladder capacity similarly to vamicamide at 0.32 mg./kg. In the rat model, oral vamicamide 0.32 mg./kg. also significantly increased bladder capacity and did not change micturition pressure or threshold pressure. Again, oxybutynin 0.10 mg./kg. or atropine 0.32 mg./kg. had almost the same effects as vamicamide 0.32 mg./kg. Conclusions These findings suggest that vamicamide should be useful for the treatment of urinary frequency.

Orienting-like reaction after ibotenic acid injections into the thalamic centre median nucleus in the cat.

The excitotoxin ibotenic acid was injected bilaterally into the intralaminar centre median nucleus of chronically implanted cats in order to study the effects of early excitation of centre median population on electrographic correlates of behavioral states and to compare them to those induced by injection into intralaminar centralis lateralis nucleus, previously shown. Immediately and during the first 24h after injection, highly aroused behavior with electrocortical activation, myoclonic jerks, enhancement of ocular movements and ponto-geniculo-occipital waves were observed. Surprisingly, in contrast to the increase of REM sleep episodes in centralis lateralis cases, REM sleep was obliterated. The injection sites were histologically confirmed. The different connectional properties of the two intralaminar population may explain the differential results. 5. In conclusion, the present behavioral observations and electrographic findings taken together with the known afferent and efferent projections suggest that the caudal group of intralaminar nuclei is involved in orienting-like behavior.

Deafferentation-induced c- fos gene expression in subthalamic nucleus and substantia nigra reticulata is reduced by non-NMDA receptor antagonist

Molecular events underlying the mechanism by which brain injury elicits delayed transneuronal degeneration ofneurons remote from the site of initial injury are not well understood. In rats, acute injury of the caudate nucleus (CN) and globus pallidus (GP) by local injection of excitotoxic ibotenic acid (IA) or by transient forebrain ischemia resulted in delayed cell death of neurons in the substantia nigra reticulata (SNr). To elucidate the involvement of glutamate receptor mediated hyperactivity of neurons produced by loss of inhibitory inputs in this delayed degeneration of SNr neurons, the region-specific expression of an immediate early gene, c- fos, and the effect of glutamate receptor antagonists on the c- fos expression were examined by using immunocytochemical and in situ hybridization analysis. Following unilateral IA-injection into the CN and GP, a robust expression of c- fos mRNA and Fos protein was induced specifically in neurons of both subthalamic nucleus (STN) and SNr deafferented by the IA-lesions 36 h after IA-injection. The delayed expression of Fos-protein in S,Nr neurons lasted for 48 h longer than that in STN neurons. Following unilateral IA-injection confined to the CN, an intense but short-term expression of Fos-protein was exhibited only in neurons of the deafferented SNr. c- fos mRNA and Fos protein were not expressed in neurons of the substantia nigra compacta at any time points examined. The induction of c fos mRNA and Fos protein in neurons of the: STN and SNr following IA-lesions of the CN and GP was reduced markedly by non-NMDA receptor antagonist (GYKI52466), but not by NMDA receptor antagonist (MK-801). The region-specific c fos expression implies that deprivation of inhibitory afferents (disinhibition) due to destruction of presynaptic neurons can induce increased activity of postsynaptic neurons. The effect of GYK152466 on the c- fos gene expression in neurons of the deafferented STN and SNr suggests that activation of non-NMDA receptors may be involved in a pathophysiological cascade for the transneuronal degeneration of SNr neurons.

Effects of hibernation or cryopreservation on the survival and integration of striatal grafts placed in the ibotenate-lesioned rat caudate-putamen

Tissue storage prior to intracerebral transplantation would represent a major advantage when conducting clinical transplantation trials in that the procurement of the embryonic donor tissue and the timing of neurosurgery could be planned more efficiently. In the present study, the effects of storing rat embryonic striatal tissue at either +4°C or below freezing temperature prior to grafting to the adult striatum, were assessed with regard to transplant survival, morphology and integration. Eleven days following a unilateral injection of ibotenic acid into the head of the caudate-putamen, a control group of rats received grafts of striatal primordium prepared immediately after dissection from rat embryos (embryonic day 16). A second group of rat embryonic striatal tissue was stored at 4°C (hibernation) for 5 days and then transplanted. A third group of the striatal donor tissue was cryopreserved in liquid nitrogen for 5 days before implantation surgery. Six to seven weeks following transplantation surgery, the grafts were analysed in brain sections processed for acetylcholinesterase histochemistry, DARPP-32 (dopamine and cyclic AMP regulated phosphoprotein with a molecular weight of 32 kDa) and tyrosine hydroxylase (TH) immunocytochemistry. The mean total graft volume and the relative size of the AChE-positive regions were not significantly different between the three groups. Striatal-specific graft regions, positively stained for AChE and DARPP-32, generally exhibited TH immunoreactivity, suggesting that they had received dopaminergic afferents from the host brain. We conclude that embryonic rat striatal tissue can be cryopreserved or hibernated over 5 days without significant impairment in the yield of striatal neurons following intrastriatal implantation and without markedly affecting transplant morphology.

Effects of hibernation or cryopreservation on the survival and integration of striatal grafts placed in the ibotenate-lesioned rat caudate-putamen

Tissue storage prior to intracerebral transplantation would represent a major advantage when conducting clinical transplantation trials in that the procurement of the embryonic donor tissue and the timing of neurosurgery could be planned more efficiently. In the present study, the effects of storing rat embryonic striatal tissue at either +4°C or below freezing temperature prior to grafting to the adult striatum, were assessed with regard to transplant survival, morphology and integration. Eleven days following a unilateral injection of ibotenic acid into the head of the caudate-putamen, a control group of rats received grafts of striatal primordium prepared immediately after dissection from rat embryos (embryonic day 16). A second group of rat embryonic striatal tissue was stored at 4°C (hibernation) for 5 days and then transplanted. A third group of the striatal donor tissue was cryopreserved in liquid nitrogen for 5 days before implantation surgery. Six to seven weeks following transplantation surgery, the grafts were analysed in brain sections processed for acetylcholinesterase histochemistry, DARPP-32 (dopamine and cyclic AMP regulated phosphoprotein with a molecular weight of 32 kDa) and tyrosine hydroxylase (TH) immunocytochemistry. The mean total graft volume and the relative size of the AChE-positive regions were not significantly different between the three groups. Striatal-specific graft regions, positively stained for AChE and DARPP-32, generally exhibited TH immunoreactivity, suggesting that they had received dopaminergic afferents from the host brain. We conclude that embryonic rat striatal tissue can be cryopreserved or hibernated over 5 days without significant impairment in the yield of striatal neurons following intrastriatal implantation and without markedly affecting transplant morphology.

Effects of hibernation or cryopreservation on the survival and integration of striatal grafts placed in the ibotenate-lesioned rat caudate-putamen.

Tissue storage prior to intracerebral transplantation would represent a major advantage when conducting clinical transplantation trials in that the procurement of the embryonic donor tissue and the timing of neurosurgery could be planned more efficiently. In the present study, the effects of storing rat embryonic striatal tissue at either +4 degrees C or below freezing temperature prior to grafting to the adult striatum, were assessed with regard to transplant survival, morphology and integration. Eleven days following a unilateral injection of ibotenic acid into the head of the caudate-putamen, a control group of rats received grafts of striatal primordium prepared immediately after dissection from rat embryos (embryonic day 16). A second group of rat embryonic striatal tissue was stored at 4 degrees C (hibernation) for 5 days and then transplanted. A third group of the striatal donor tissue was cryopreserved in liquid nitrogen for 5 days before implantation surgery. Six to seven weeks following transplantation surgery, the grafts were analysed in brain sections processed for acetylcholinesterase histochemistry, DARPP-32 (dopamine and cyclic AMP regulated phosphoprotein with a molecular weight of 32 kDa) and tyrosine hydroxylase (TH) immunocytochemistry. The mean total graft volume and the relative size of the AChE-positive regions were not significantly different between the three groups. Striatal-specific graft regions, positively stained for AChE and DARPP-32, generally exhibited TH immunoreactivity, suggesting that they had received dopaminergic afferents from the host brain. We conclude that embryonic rat striatal tissue can be cryopreserved or hibernated over 5 days without significant impairment in the yield of striatal neurons following intrastriatal implantation and without markedly affecting transplant morphology.

The effects of bilateral striatal lesions on the acquisition of an operant test of short term memory.

It has been previously shown that lesions of the dorsal striatum can disrupt performance on a variety of cognitive tasks related to frontal cortex function. In order to extend these studies, we have investigated the effects of bilateral striatal lesions on the acquisition of an operant test of short term memory in the delayed non-matching to position paradigm. The animals received either ibotenic acid or saline control injections into the dorsal striatum prior to training on the non-matching task. Striatal lesions retarded acquisition of the task, although with further training the lesioned rats achieved a similar level of asymptotic performance to the control animals. The lesioned rats also exhibited marked nocturnal locomotor hyperactivity when tested under conditions of food deprivation, but not when tested satiated. The results indicate that bilateral striatal lesions induce mild deficits in the acquisition of the discrimination rules involved in performance of the delayed non-matching to position task. The present study does not support a role for the neostriatum in the specific mediation of short term memory in a operant DNMTP test.