The lateral neostriatum is necessary for compensatory ingestive behaviour after intravascular dehydration in female rats.

Aberrant striatal function results in an array of physiological symptoms, including impaired consummatory and regulatory behaviours, which can lead to weight loss and dehydration. It was hypothesised, therefore, that cell loss in the neostriatum may contribute to altered fluid intake by regulating physiological signals related to dehydration status. To test this theory, rats with lesions of the lateral neostriatum and sham controls underwent a series of physiological challenges, including the experimental induction of intracellular and intravascular dehydration. No baseline differences in prandial or non-prandial drinking were observed, nor were differences in locomotor activity evident between groups. Furthermore, intracellular dehydration increased water intake in lesion rats in a manner comparable to sham rats. Interestingly, a specific impairment was evident in lesion rats after subcutaneous injection of poly-ethylene glycol was used to induce intravascular dehydration, such that lesion rats failed to adapt their water intake to this physiological change. The results suggest that the striatal lesions resulted in regulatory dysfunction by impairing motivational control over compensatory ingestive behaviour after intravascular hydration, while the physiological signals related to dehydration remain intact. Loss of these cells in neurodegenerative disorders, such Huntington's disease, may contribute to regulatory changes evident in the course of the disease.

Intrastriatal excitotoxic lesion or dopamine depletion of the neostriatum differentially impairs response execution in extrapersonal space.

Dysfunction of the neostriatum, a primary feature of several neurodegenerative disorders, including Parkinson's disease and Huntington's disease, has been found to result in impaired localisation of, and reaction to, contralateral stimuli. On the basis of previous findings, it is hypothesised that, with increasing eccentricity of the response option, striatal cell loss may impair response localisation at the furthest levels of eccentricity, whereas dopamine (DA) depletion may not impact adversely upon responses executed in this extrapersonal space. In order to elucidate more fully the function of the striatum, the present study examined the differential impact of unilateral DA depletion or excitoxic lesion on response execution in ipsilateral and contralateral space at up to four levels of eccentricity. The results confirmed that, after both types of striatal dysfunction, the sensory ability to detect stimuli remains intact, whereas the ability to direct responses in absolute contralateral space is impaired. Distinct differences in the profiles of impairment were, however, evident, with a marked increase in response omissions observed after DA depletion, which may reflect decreased motivational processing, and recovery of function observed in rats with excitotoxic lesions, which suggests the ability to re-learn. Furthermore, the data demonstrate that, after cell loss, responding in near contralateral space is controlled by competing striata, whereas responding in extrapersonal space relies on the contralateral hemisphere. These results have implications for understanding the role of the striatum in egocentrically defined response localisation, as well as for unravelling the behavioural impact of striatal cell loss or aberrant DA transmission observed in neurodegenerative diseases.

Context-driven changes in L-DOPA-induced behaviours in the 6-OHDA lesioned rat.

Both contralateral rotational behaviour and dyskinetic abnormal involuntary movements (AIMs) are induced by the administration of l-DOPA in the unilateral 6-OHDA lesioned rat model of Parkinson's disease. Since rotational responses can be conditioned to environmental cues we have investigated the extent to which drug-induced AIMS may also be conditioned by exteroceptive cues and experience. In Experiment I, 6-OHDA lesioned rats received repeated daily injections of l-DOPA either in their home cage (control) or in association with a brief (20 mins) exposure to the rotometers (paired). To assess conditioning, all animals then received two tests in the rotometer bowls. Following injection of saline the paired group both rotated more contralaterally and displayed manifest AIMs, neither of which were exhibited by the control rats. Moreover, following injection of l-DOPA, the paired group showed a trend for increased AIMs compared to controls. Two further studies provided longer exposure to the conditioning environments in counterbalanced designs. Although, using these parameters, re-exposure in the presence of saline did not induce context-dependent AIMs, a strong context-specific component of the sensitised response to l-DOPA was seen; chronic administration of drug produced a significantly stronger behavioural response in animals paired with a particular environment for drug administration than controls. This data suggests that part of the sensitisation of behavioural responding to l-DOPA administration is not solely a pharmacological phenomenon, but is also conditioned to the environmental context in which the drug is administered. This has clear implications for the clinical observation and experimental measurement of drug-induced dyskinesia in Parkinson's disease patients and animal models.

Functional integration of striatal allografts in a primate model of Huntington's disease.

Huntington's disease is an autosomal dominant, inherited disorder that results in progressive degeneration of the basal ganglia (especially the neostriatal caudate nucleus and putamen) and other forebrain structures and is associated with a clinical profile of movement, cognitive and psychiatric impairments for which there is at present no effective therapy. Neuropathological, neurochemical and behavioral features of the disease can all be reproduced in experimental animals by local injection of excitotoxic or metabolic toxins into the neostriatum. All these features of the disease can be alleviated, at least in rats, by transplantation of embryonic striatal tissue into the degenerated striatum, which was the basis for commencing the first clinical trials of striatal transplantation in Huntington's patients. However, although rat striatal xenografts may temporarily reduce apomorphine-induced dyskinesias in monkeys, there has been no demonstration that allograft techniques that work well in rats translate effectively to the much larger differentiated striatum of primates. Here we demonstrate good survival, differentiation and integration of striatal allografts in the primate neostriatum, and recovery in a test of skilled motor performance. Long-term graft survival in primates indicates probable success for clinical transplants in Huntington's disease; in addition, our data suggest that graft placement has a direct influence on the pattern and extent of functional recovery.

Challenges facing quantification of rat locomotion along beams of varying widths.

Optoelectronic motion capture systems have been widely used to investigate temporal gait parameters in humans and animals in order to understand function and behavioural attributes of different pathologies, e.g. Parkinson's disease (PD). The aim of the present paper was to investigate the practicality of utilising this system to investigate the effects of a unilateral 6-hydroxydopamine (6-OHDA) lesion on rat locomotion while walking on beams of varying widths (graduated, narrow, and wide). Temporal gait parameters of ten male Lister Hooded rats (five controls and five hemiparkinsonian) were observed using passive markers placed in locations that were representative of their four limbs and their body axis. The results demonstrate that marker-based motion capture can provide an effective and simple approach to quantifying temporal gait parameters for rat models of PD. They also reveal how the width of the path affects the locomotion in both experimental cohorts. Such measurements can be compared with human motion analysis to explore correlations between the animal model and human behaviour, which is an important step for translational medicine.

A simple breeding protocol for the procurement of accurately staged rat donor embryos for neural transplantation.

Obtaining accurately staged rat embryos can be difficult because of the variety of breeding protocols employed and because precise staging cannot be confirmed until excision of the embryos from the dam. The detection of estrus, pairing of animals, and confirmation of pregnancies is generally left to commercial suppliers, as in-house breeding can be laborious and unpredictable. Here we describe a simple, reliable in-house breeding protocol for the generation of accurately staged embryos as assessed by measurements of average crown to rump length (CRL).

Aged rats: recovery of motor impairments by intrastriatal nigral grafts.

Dissociated cell suspensions, prepared from the substantia nigra and septal regions of rat embryos, can be grafted to the depths of the caudate-putamen and hippocampus of aged rats. The grafts were rich in dopamine-containing and acetylcholinesterase-positive neurons and had produced extensive new dopaminergic and cholinergic terminal networks in the host neostriatum and hippocampus, respectively. The intrastriatal dopaminergic grafts were associated with a significant improvement in motor coordination in the aged rats. This result suggests that the intracerebral grafting technique may provide a new tool for exploring the role of dopaminergic and cholinergic deficits in the neurological and behavioral impairments associated with aging.

Nigral transplants reinnervating the dopamine-depleted neostriatum can sustain intracranial self-stimulation.

Transplants of embryonic substantia nigra reinnervated the striatum and were able to sustain intracranial self-stimulation in rats with brain lesions induced by 6-hydroxydopamine. Dopaminergic drugs and alterations in current intensity produced typical changes in response rates. Animals with electrodes implanted into cortical grafts or into the denervated striatum failed to exhibit self-stimulation. These findings suggest that transplanted dopamine neurons convey specific, temporally organized information axonally to the striatum.

Intrahippocampal septal grafts ameliorate learning impairments in aged rats.

Grafts of fetal septal tissue rich in cholinergic neurons were implanted as a dissociated cell suspension into the depth of the hippocampal formation in aged rats with severe impairments in spatial learning abilities. After 2 1/2 to 3 months, the rats with grafts, but not the controls, had improved their performance in a spatial learning test. Their improvement was due, at least in part, to an increased ability to use spatial cues in the task. In all animals the grafts had produced an extensive acetylcholinesterase-positive terminal network in the surrounding host hippocampal formation. Thus, the action of cholinergic neurons in the graft onto elements in the host hippocampal circuitry may be a necessary, but perhaps not sufficient, prerequisite for the observed functional recovery.

Membrane permeability coefficients of murine primary neural brain cells in the presence of cryoprotectant.

Neural cells isolated from the brain have a number of research and clinical applications, including transplantation to patients with neurodegenerative conditions. Tissue supply is one of the major limiting factors to clinical transplantation. Cryopreservation of primary neural cells would improve supply, aid in organisation of transplantation surgery and facilitate research. To date, cryopreservation using standard methods has resulted in reduced yield and/or viability of primary neural tissue. In order to optimise freezing protocols specifically for such cells, the non-osmotic volume (V(b)), water permeability (L(p)) and permeability to cryoprotectant (P(cpa)) were determined. Murine foetal brain tissue from the ganglionic eminence (GE), ventral mesencephalon (VM), or neocortical mantle (Ctx) was trypsinised to a single cell suspension. To determine V(b,) cell volume was measured after exposure to anisotonic solutions of sucrose (150-1500 mOsmol/kg). L(p) (mum/min.atm) and P(cpa) (mum/s) were determined for GE cells by measuring cell volume during exposure to 1.5 mol/l cryoprotectant. Cell volume was determined using an electronic particle counting method. V(b) was 27% for Ctx and GE, and 30% for VM. The osmotic response of GE cells was similar in the presence of propane-1,2-diol and dimethyl sulphoxide. In the presence of ethylene glycol, cell volume decrease was greater on initial exposure to cryoprotectant and recovery slower. Differences in L(p,) but not P(cpa), were found between cryoprotectants. The present results provide key parameters for optimisation of freezing protocols for cryopreservation of primary foetal brain tissues for application in neural cell transplantation.

Differential expression of immediate early genes in the hippocampus and spinal cord.

We have demonstrated that immediate early genes can be differentially activated within the central nervous system. We examined the effects of tetanic stimulation in the hippocampus and of noxious sensory stimulation of the spinal cord on the expression of eight immediate early genes. Induction of long-term potentiation (LTP) in the dentate gyrus resulted in an increase in mRNA and protein for NGFI-A (also termed Zif/268, Egr-1, or Krox 24), and less consistently for jun-B mRNA. No increase was seen for c-fos, NGFI-B, c-jun, jun-D, SRF, or PC4 mRNAs. Blockade of the NMDA receptor prevented the induction of both LTP and NGFI-A mRNA in the dentate gyrus. However, commissural stimulation, which prevented the induction of LTP, resulted in bilateral activation of all the genes examined, including NGFI-A. No change was seen in animals trained in a water maze. These results suggest that no simple relationship exists between LTP, spatial learning, and immediate early gene induction. Stimulation of sensory fibers resulted in an increase in mRNA for NGFI-A, c-fos, SRF, NGFI-B, and c-jun in spinal cord neurons. Blockade of the NMDA receptor had no effect on immediate early gene induction in the spinal cord.

Recovery of functional deficits following early donor age ventral mesencephalic grafts in a rat model of Parkinson's disease.

It has previously been reported that dopaminergic grafts derived from early donor age, embryonic age 12-day-old (E12) rat embryos produced a fivefold greater yield of dopamine neurons than those derived from conventional E14 donors. The present study addresses whether E12 grafts are able to ameliorate lesion-induced behavioral deficits to the same extent as E14 grafts. In a unilateral rat model of Parkinson's disease, animals received grafts derived from either E12 or E14 donor embryos, dispersed at four sites in the lesioned striatum. Both E12 and E14 grafts were able to induce recovery on both amphetamine and apomorphine rotation tests, and to ameliorate deficits in the cylinder, stepping test, and corridor tests, but were unable to restore function in the paw reaching task. E12 grafts were equivalent to E14 grafts in their effects on lesion-induced deficits. However, E12 grafts resulted in cell yields greater than previously reported for untreated primary tissue, with mean TH-positive cell counts in excess of 25,000 neurons, compared with E14 TH cell counts of 4000-5000 cells, representing survival rates of 75% and 12.5%, respectively, based on the expected adult complement. The equivalence of graft induced behavioral recovery between the two graft groups is attributed to a threshold number of cells, above which no further improvement is seen. Such high dopamine cell survival rates should mean that multiple, functioning grafts can be derived from a single embryonic donor, and if similar yields could be obtained from human tissues then the goal of one embryo per patient would be achieved.

Ascorbic acid increases the number of dopamine neurons in vitro and in transplants to the 6-OHDA-lesioned rat brain.

The inadequate survival of dopamine neurons following intracerebral transplantation is in part attributed to the generation of reactive oxygen species and subsequent oxidative stress. To address this, we investigated whether the antioxidant ascorbic acid (vitamin C) had any effect on the yields of dopamine neurons derived from E14 rat ventral mesencephalic cells in vitro and in grafts. Following in vitro differentiation in medium containing ascorbic acid at concentrations ranging from 20 to 100 microM, significantly more neurons were immunopositive for the marker of mesencephalic dopamine neurons, tyrosine hydroxylase (TH), when compared to standard differentiation conditions containing no ascorbic acid. Mesencephalic cell suspensions supplemented with 100 microM ascorbic acid were also transplanted into unilateral 6-OHDA-lesioned rats and behavioral rotation was assessed at 2, 4, and 6 weeks posttransplantation. Grafts pretreated with ascorbic acid contained significantly more surviving dopamine neurons compared to nontreated grafts. However, no significant difference in rotation score was observed, with both groups showing a reversal and overcompensation of rotational bias. In addition, no evidence of neurogenesis of nigral dopamine neurons was observed in transplant groups. While the increased number of dopamine neurons observed in our study following ascorbic acid treatment may reflect a selective survival effect, our in vitro results suggest that ascorbic acid may act to increase the number dopamine neurons, both in culture and following transplantation, by stimulating dopaminergic differentiation of neural precursors from the fetal ventral mesencephalon.

A rat embryo staging scale for the generation of donor tissue for neural transplantation.

In rat models of Parkinson's and Huntington's diseases, embryonic neural cells obtained from embryos of specified ages can be implanted into the brain to partially restore both physiology and function. However, in litters produced using overnight mating protocols (often from commercial suppliers), the embryonic age can be difficult to determine precisely. As a result, embryonic size based on crown to rump length (CRL) is usually a more reliable method of embryo staging than the day of mating. This approach is not without difficulty. There are a number of rat staging scales in the literature, none of which deal with donor ages younger than E13, and there are discrepancies between scales at some donor ages. In the present article, we have devised a short mating-period protocol to produce precisely aged embryos. We show that CRL is a highly accurate, reproducible index of donor age and we present an updated embryonic staging scale for Sprague-Dawley (CD) rats that includes donor ages younger than those previously reported.

Functional integration of neural grafts in Parkinson's disease.

Functional imaging in a Parkinson's patient with a neural transplant shows that the graft is still functional after ten years, and that dopamine from the graft can bind to postsynaptic sites.

Improved survival of young donor age dopamine grafts in a rat model of Parkinson's disease.

In an attempt to improve the survival of implanted dopamine cells, we have readdressed the optimal embryonic donor age for dopamine grafts. In a rat model of Parkinson's disease, animals with unilateral 6-hydroxydopamine lesions of the median forebrain bundle received dopamine-rich ventral mesencephalic grafts derived from embryos of crown to rump length 4, 6, 9, or 10.5 mm (estimated embryonic age (E) 11, E12, E13 and E14 days post-coitus, respectively). Grafts derived from 4 mm embryos survived poorly, with less than 1% of the implanted dopamine cells surviving. Grafts derived from 9 mm and 10.5 mm embryos were similar to those seen in previous experiments with survival rates of 8% and 7% respectively. The best survival was seen in the group that received 6 mm grafts, which were significantly larger than all other graft groups. Mean dopamine cell survival in the 6 mm group (E12) was 36%, an extremely high survival rate for primary, untreated ventral mesencephalic grafts applied as a single placement, and more than fivefold larger than the survival rate observed in the 10.5 mm (E14) group. As E12 ventral mesencephalic tissues contain few, if any, differentiated dopamine cells we conclude that the large numbers of dopamine cells seen in the 6 mm grafts must have differentiated post-implantation. We consider the in vivo conditions which allow this differentiation to occur, and the implications for the future of clinical trials based on dopamine cell replacement therapy.

SMAD transcription factors are altered in cell models of HD and regulate HTT expression.

Transcriptional dysregulation is observable in multiple animal and cell models of Huntington's disease, as well as in human blood and post-mortem caudate. This contributes to HD pathogenesis, although the exact mechanism by which this occurs is unknown. We therefore utilised a dynamic model in order to determine the differential effect of growth factor stimulation on gene expression, to highlight potential alterations in kinase signalling pathways that may be in part responsible for the transcriptional dysregulation observed in HD, and which may reveal new therapeutic targets. We demonstrate that cells expressing mutant huntingtin have a dysregulated transcriptional response to epidermal growth factor stimulation, and identify the transforming growth factor-beta pathway as a novel signalling pathway of interest that may regulate the expression of the Huntingtin (HTT) gene itself. The dysregulation of HTT expression may contribute to the altered transcriptional phenotype observed in HD.

Cholinergic grafts, memory and ageing.

Neural grafts rich in cholinergic neurones can survive transplantation to the neocortex or hippocampus in rats. Such grafts have the capacity to ameliorate a variety of functional deficits associated both with explicit lesions that deafferent the neocortex or hippocampus and with natural ageing. The transplantation technique enhances our understanding of the involvement of forebrain cholinergic systems in normal cognitive functions (including memory) and of the role of cholinergic degeneration in the dysfunctions associated with ageing. It is unlikely, however, that these observations will extend to a therapeutic strategy for dementia using neural transplantation, because the human diseases (at least in the case of Alzheimer's disease and multi-infarct dementia) involve widespread degeneration of other populations of cortical neurones that are not so amenable to functional transplantation as the diffuse forebrain cholinergic systems.

The basal forebrain-cortical cholinergic system: interpreting the functional consequences of excitotoxic lesions.

Ibotenic acid and kainic acid lesions of the basal forebrain induce profound deficits in performance on a wide variety of tasks involving discrimination learning and memory. These observations have been widely taken to reflect damage of cholinergic projections from the nucleus basalis magnocellularis (NBM) to the neocortex, and to provide an animal model of dementia. However, injections of the toxins quisqualic acid and, more recently, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) into the same site, which produce at least as extensive cholinergic cell loss, induce only marginal impairments on the same range of cognitive tasks. Further analysis suggests that the cholinergic regulation of the neocortex may influence specific aspects of discrimination learning and visual attention. Conversely, we propose that many of the functional consequences of ibotenic acid lesions on mnemonic tasks cannot be attributed to disruption of basal forebrain cholinergic systems, but may instead result from damage in the globus pallidus to corticostriatal output pathways.

Huntington's disease: animal models and transplantation repair.

Recent identification of the gene for Huntington's disease is currently attracting widespread attention. While having importance for predictive testing and the potential of elucidating the underlying disease process, this discovery does not yet provide any advances for therapeutic intervention. Here we review recent advances in the development of improved animal models of Huntington's disease and strategies for its repair. Novel toxins may better mimic the neuropathology, and provide important clues about the underlying metabolic disorder, of the human disease. In addition, recent experiments into the cellular morphology, development and function of striatal cell transplants in both rats and monkeys are now indicating the prospect of viable strategies for structural repair in this disorder.