MPTP treatment of common marmosets impairs proteasomal enzyme activity and decreases expression of structural and regulatory elements of the 26S proteasome.

Dysfunction of the ubiquitin-proteasome system occurs in the substantia nigra (SN) in Parkinson's disease (PD). However, it is unknown whether this is a primary cause or a secondary consequence of other components of the pathogenic process. We have investigated in nonhuman primates whether initiating cell death through mitochondrial complex I inhibition using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) altered proteasomal activity or the proteasomal components in the SN. Chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolase (PGPH) activating of 20S proteasome were decreased in SN homogenates of MPTP-treated marmosets compared to naïve animals. Western blotting revealed a marked decrease in the expression of 20S-alpha subunits, but no change in 20S-beta subunits in the SN of MPTP-treated marmoset compared to naïve animals. There was a marked decrease in the expression of the proteasome activator 700 (PA700) and proteasome activator 28 (PA28) regulatory complexes. The 20S-alpha4 subunit immunoreactivity was decreased in the nucleus of colocalized tyrosine hydroxylase (TH)-positive cells of MPTP-treated animals compared to naïve animals but no difference in the intensity of 20S-beta1i subunit staining. Immunoreactivity for PA700-Rpt5 and PA28-alpha subunits within surviving TH-positive cells of MPTP-treated marmoset was reduced compared to naïve controls. Overall, the changes in proteasomal function and structure occurring follow MPTP-induced destruction of the SN in common marmosets were very similar to those found in PD. This suggests that altered proteasomal function in PD could be a consequence of other pathogenic processes occurring in SN as opposed to initiating cell death as previously suggested.

Proteasomal activity in brain differs between species and brain regions and changes with age.

Age-related increase in protein oxidation in brain coupled to an impairment of proteasomal activity may underline neuronal loss but differences in susceptibility between species and brain regions remain unexplained. We now investigate differences in proteasomal activity, measured as chymotrypsin-, trypsin- and peptidylglutamyl-like hydrolysing activities between brain regions in rats, mice and common marmosets. In aged rats and mice, proteasomal activity was decreased in the cortex, striatum, cerebellum, globus pallidus and substantia nigra overall when compared to young animals. However, in the aged brain only chymotrypsin-like activity was decreased in the cortex and the globus pallidus while only trypsin-like activity was reduced in the cerebellum. In contrast, in the striatum, both chymotrypsin-like and trypsin-like activities were reduced and in the substantia nigra, all the three catalytic activities of proteasome were significantly impaired. Chymotrypsin-like and trypsin-like activities were significantly higher in all the brain regions of marmosets compared to those of mice and rats. Peptidylglutamyl-like activity was only significantly higher in the cerebellum and striatum of marmoset compared to rodents. The data suggest that there is higher proteasome activity in common marmoset brain compared to rat and mouse and that the basal ganglia are more prone to an age-related decrease in proteasomal activity. This may explain the involvement of altered ubiquitin-proteasome system activity in Parkinson's disease and the relationship to ageing.

Alterations in expression of dopamine receptors and neuropeptides in the striatum of GTP cyclohydrolase-deficient mice.

The hph-1 mice have defective tetrahydrobiopterin biosynthesis and share many neurochemical similarities with l-dopa-responsive dystonia (DRD) in humans. In both, there are deficiencies in GTP cyclohydrolase I and low brain levels of dopamine (DA). Striatal tyrosine hydroxylase (TH) levels are decreased while the number of DA neurones in substantia nigra (SN) appears normal. The hph-1 mouse is therefore a useful model in which to investigate the biochemical mechanisms underlying dystonia in DRD. In the present study, the density of striatal DA terminals and DA receptors and the expression of D-1, D-2, and D-3 receptors, preproenkephalin (PPE-A), preprotachykinin (PPT), and nitric oxide synthase (NOS) mRNAs in the striatum and nucleus accumbens and nigral TH mRNA expression were examined. Striatal DA terminal density as judged by specific [3H]mazindol binding was not altered while the levels of TH mRNA were elevated in the SN of hph-1 mice compared to control (C57BL) mice. Total and subregional analysis of the striatum and nucleus accumbens showed that D-2 receptor ([3H]spiperone) binding density was increased while D-1 receptor ([3H]SCH 23390) and D-3 receptor ([3H]7-OH-DPAT) binding density was not altered. In the striatum and nucleus accumbens, expression of PPT mRNA was elevated but PPE-A mRNA, D-1, D-2 receptor, and nNOS mRNA were not changed in hph-1 mice compared to controls. These findings suggest that an imbalance between the direct strionigral and indirect striopallidal output pathways may be relevant to the genesis of DRD. However, the pattern of changes observed is not that expected as a result of striatal dopamine deficiency and suggests that other effects of GTP cyclohydrolase I deficiency may be involved.

Modafinil prevents the MPTP-induced increase in GABAA receptor binding in the internal globus pallidus of MPTP-treated common marmosets.

The psychostimulant drug modafinil induces a reversal of motor deficits in MPTP treated primates and prevents MPTP toxicity to substantia nigra but its mechanism of action is not clear. In common marmosets acutely treated with MPTP in the presence or absence of modafinil, we have studied changes in GABA(A) receptor binding in the basal ganglia. MPTP treatment had no effect on [(3)H]-flunitrazepam (FNZ) binding density in the striatum or external globus pallidus (GPe) but increased [(3)H]-FNZ binding density in the internal globus pallidus (GPi). Administration of modafinil (10-100 mg/kg) with MPTP did not alter [(3)H]-FNZ binding density in the striatum or GPe. Low doses of modafinil (10 and 30 mg/kg) had no effect on the increased [(3)H]-FNZ binding density in the GPi but high dose modafinil (100 mg/kg) significantly decreased [(3)H]-FNZ binding density in GPi. These findings suggest that modafinil can selectively alter GABA binding density in the GPi either by preventing MPTP-induced toxicity or through an action on striatal output pathway related to its antiparkinsonian activity and its ability to inhibit MPTP toxicity.

Alterations in striatal neuropeptide mRNA produced by repeated administration of L-DOPA, ropinirole or bromocriptine correlate with dyskinesia induction in MPTP-treated common marmosets.

Chronic administration of L-DOPA to MPTP-treated common marmosets induces marked dyskinesia while repeated administration of equivalent antiparkisonian doses of ropinirole and bromocriptine produces only mild involuntary movements. The occurrence of dyskinesia has been associated with an altered balance between the direct and indirect striatal output pathways. Using in situ hybridisation histochemistry, we now compare the effects of these drug treatments on striatal preproenkephalin-A (PPE-A) and adenosine A(2a) receptor mRNA expression as markers of the indirect pathway and striatal preprotachykinin (PPT) mRNA and preproenkephalin-B (PPE-B, prodynorphin) mRNA expression as markers of the direct pathway.The equivalent marked losses of specific [3H]mazindol binding in the striatum of all drug treatment groups confirmed the identical nature of the nigral cell loss produced by MPTP treatment. MPTP-induced destruction of the nigro-striatal pathway markedly increased the level of PPE-A mRNA in the caudate nucleus and putamen and decreased the levels of PPT and PPE-B mRNA relative to normal animals. Repeated treatment with L-DOPA for 30 days produced marked dyskinesia but had no effect on the MPTP-induced increase in PPE-A mRNA in the caudate nucleus and putamen. In contrast, L-DOPA treatment normalised the MPTP-induced decrease in the level of PPT and PPE-B mRNA. Repeated treatment with ropinirole produced little or no dyskinesia but markedly reversed the MPTP-induced elevation in PPE-A mRNA in the caudate nucleus and putamen. However, it had no effect on the decrease in PPT or PPE-B mRNA. Similarly, bromocriptine treatment which induced only mild dyskinesia attenuated the MPTP-induced elevation in PPE-A mRNA in the caudate nucleus and putamen with no effect on reduced striatal PPT or PPE-B mRNA. Neither MPTP treatment nor treatment with L-DOPA, bromocriptine or ropinirole had any effect on adenosine A(2a) receptor mRNA in the striatum. These patterns of alteration in striatal PPE-A and PPT and PPE-B mRNA produced by L-DOPA, bromocriptine and ropinirole show differential involvement of markers of the direct and indirect striatal output pathways related to improvement of locomotor activity and mirror the relative abilities of the drugs to induce dyskinesia.

6-Hydroxydopamine lesioning differentially affects alpha-synuclein mRNA expression in the nucleus accumbens, striatum and substantia nigra of adult rats.

The effect of a unilateral 6-hydroxydopamine (6-OHDA) lesion and/or repeated administration of levodopa (L-DOPA) to normal and 6-OHDA-lesioned rats on alpha-synuclein mRNA expression was investigated by in situ hybridization histochemistry. A 6-OHDA lesion of the nigro-striatal pathway alone, confirmed by the loss of nigral tyrosine hydroxylase mRNA expression, markedly decreased alpha-synuclein mRNA in the lesioned substantia nigra (SN). In contrast, the levels of alpha-synuclein mRNA in the denervated striatum and nucleus accumbens were not altered. Chronic administration of L-DOPA to normal or 6-OHDA-lesioned rats had no effect on alpha-synuclein mRNA expression in the SN, striatum or nucleus accumbens. These data confirm that alpha-synuclein is localized in the nigro-striatal tract but that its gene expression is not regulated by dopamine.

Chronic high dose L-DOPA alone or in combination with the COMT inhibitor entacapone does not increase oxidative damage or impair the function of the nigro-striatal pathway in normal cynomologus monkeys.

Parkinson's disease (PD) is characterised by a loss of pigmented dopaminergic neurones in the zona compacta of substantia nigra. The mechanisms underlying nigral cell death remain unknown but may involve oxidative damage. There has been concern that L-DOPA treatment may accelerate nigral pathology in PD through chemical and enzymatic oxidation to reactive oxygen species. In the present study, we examined tissues from normal macaque monkeys treated for 13 weeks with high doses of L-DOPA (in combination with the peripheral decarboxylase inhibitor, carbidopa) and/or the COMT inhibitor, entacapone. Plasma was analysed for changes in protein carbonyls as a marker of oxidative damage to protein. Cortical tissue was examined for changes in levels of protein carbonyls, lipid peroxidation and oxidative damage to DNA. The integrity of the nigro-striatal pathway was assessed by nigral tyrosine hydroxylase mRNA levels and specific [(3)H]mazindol binding to dopaminergic terminals in caudate-putamen. No alterations in plasma protein carbonyls were observed in any treatment group. An increase was found in the levels of protein carbonyls, lipid peroxidation and 5-OH uracil, but not other products of oxidative DNA damage, in cerebral cortex of monkeys treated with L-DOPA plus carbidopa or with L-DOPA plus carbidopa and entacapone but this was only statistically significant in the latter group. There was no change in nigral tyrosine hydroxylase mRNA levels or specific striatal [(3)H]mazindol binding in brain tissue from monkeys treated with either L-DOPA plus carbidopa or L-DOPA plus carbidopa and entacapone. The results show that in the normal monkeys L-DOPA does not provoke marked oxidative damage even at high doses, and that there is little or no potentiation of its effects by entacapone. Neither L-DOPA plus carbidopa nor L-DOPA plus carbidopa and entacapone led to obvious damage to the nigro-striatal pathway.

Chronic high dose L-dopa treatment does not alter the levels of dopamine D-1, D-2 or D-3 receptor in the striatum of normal monkeys: an autoradiographic study.

To assess the role of dopamine receptors in the genesis of dyskinesia, we have used quantitative autoradiography to determine the effect of chronic L-dopa administration on dopamine D-1 (using [3H]SCH 23390), D-2 (using [3H]spiperone) and D-3 (using [3H]7-OH-DPAT) receptor binding levels in the striatum of dyskinetic or non-dyskinetic monkeys. Total and subregional striatal analysis showed no difference in D-1, D-2 or D-3 receptor binding in the caudate and putamen between monkeys receiving high dose L-dopa treatment with marked dyskinesia and those without dyskinesia compared to untreated animals. It thus appears unlikely that changes in dopamine receptor expression are a primary cause of L-dopa induced dyskinesia. Rather, a functional dissociation of D-2 receptor coupling to co-expressed enkephalin/adenosine-2a receptor activity in the striato-GPe indirect pathway may be more important in the development or expression of L-dopa-induced involuntary movements.

Up-regulation of secretoneurin immunoreactivity and secretogranin II mRNA in rat striatum following 6-hydroxydopamine lesioning and chronic L-DOPA treatment.

Destruction of the nigro-striatal pathway in Parkinson's disease and treatment with L-DOPA lead to persistent alterations in basal ganglia output pathways that are poorly characterised. Differential display mRNA analysis was used to study the effects of 6-hydroxydopamine-induced lesions of the medial forebrain bundle on gene expression in the rat striatum. One up-regulated cDNA identified in two independent groups of 6-hydroxydopamine-lesioned animals was cloned and sequence analysis showed 97% homology to secretogranin II. Differential up-regulation of secretogranin II following 6-hydroxydopamine lesioning was confirmed in a further group of 6-hydroxydopamine-lesioned rats using TaqMan real time quantitative reverse transcription-polymerase chain reaction. Following chronic L-DOPA treatment of 6-hydroxydopamine-lesioned rats, secretogranin II mRNA was further up-regulated to a similar degree to that observed for preproenkephalin A mRNA expression. Immunohistochemical analysis confirmed the increase in secretogranin II peptide levels in striatal neurones in 6-hydroxydopamine-lesioned rats following chronic L-DOPA treatment. The increase in secretogranin II mRNA occurring following destruction of the nigro-striatal pathway and chronic L-DOPA treatment may result in an increase in secretoneurin levels, which could be important for the regulation of striatal output pathways.

Increased cannabinoid CB1 receptor binding and activation of GTP-binding proteins in the basal ganglia of patients with Parkinson's syndrome and of MPTP-treated marmosets.

Recent evidence obtained in rat models of Parkinson's disease showed that the density of cannabinoid CB1 receptors and their endogenous ligands increase in basal ganglia. However, no data exists from post-mortem brain of humans affected by Parkinson's disease or from primate models of the disorder. In the present study, we examined CB1 receptor binding and the magnitude of the stimulation by WIN55,212-2, a specific CB1 receptor agonist, of [35S]GTPgammaS binding to membrane fractions from the basal ganglia of patients affected by Parkinson's disease. In Parkinson's disease, WIN55,212-2-stimulated [35S]GTPgammaS binding in the caudate nucleus, putamen, lateral globus pallidus and substantia nigra was increased, thus indicating a more effective activation of GTP-binding protein-coupled signalling mechanisms via CB1 receptors. This was accompanied by an increase in CB1 receptor binding in the caudate nucleus and the putamen, although no changes were observed in the lateral globus pallidus and the substantia nigra. Because Parkinson's disease patients had been chronically treated with l-DOPA, brains were studied from normal common marmosets and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated animals with and without chronic L-DOPA treatment. MPTP-lesioned marmosets had increased CB1 receptor binding in the caudate nucleus and the putamen compared to control marmosets, as well as increased stimulation of [35S]GTPgammaS binding by WIN55,212-2. However, following l-DOPA treatment these parameters returned towards control values. The results indicate that a nigro-striatal lesion is associated with an increase in CB1 receptors in the basal ganglia in humans and nonhuman primates and that this increase could be reversed by chronic l-DOPA therapy. The data suggest that CB1 receptor blockade might be useful as an adjuvant for the treatment of parkinsonian motor symptoms.

Antiparkinsonian and neuroprotective effects of modafinil in the mptp-treated common marmoset.

The psychostimulant drug, modafinil, protects rodents against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity, striatal ischemia and partial transection of the nigro-striatal pathway. We now report on the ability of modafinil to reverse motor disability in MPTP-treated common marmosets and to prevent MPTP-induced nigral cell death in this species. In the initial experiments, adult common marmosets were treated with MPTP to produce stable motor deficits. The subsequent administration of modafinil (10, 30 or 100 mg/kg/day, p.o.) produced a dose-dependent reversal of motor disability. In a subsequent experiment, normal common marmosets were concurrently treated with 10, 30 or 100 mg/kg of modafinil once daily by gavage during acute MPTP administration (daily for 5 days), continuing for 2 weeks after the last dose of MPTP. Modafinil dose-dependently prevented the decline in motor activity normally produced by MPTP treatment. MPTP treatment caused a 76% loss of nigral tyrosine-hydroxylase-immunoreactive cells in placebo-treated animals, and this was dose-dependently prevented by modafinil. At the highest dose (100 mg/kg/day) of modafinil, there was no significant loss of tyrosine-hydroxylase-immunoreactive cells in the substantia nigra compared with normal animals. MPTP treatment also reduced striatal dopamine uptake sites by 95%, as measured by specific [3H]-mazindol binding, compared with normal controls. Modafinil treatment dose-dependently reduced the loss of specific [3H]-mazindol binding. Behavioural and morphological evidence in the present study indicate a potential antiparkinsonian and neuroprotective role for modafinil, which may form a new pharmacological approach to the treatment of Parkinson's disease.

Alterations in preproenkephalin and adenosine-2a receptor mRNA, but not preprotachykinin mRNA correlate with occurrence of dyskinesia in normal monkeys chronically treated with L-DOPA.

Chronic treatment with L-DOPA induces dyskinesia in patients with Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-treated monkeys, but is not thought to do so in normal humans or primates. However, we have shown that chronic oral high dose L-DOPA administration, with the peripheral decarboxylase inhibitor, carbidopa and with or without the peripherally acting catechol-O-methyl transferase (COMT) inhibitor, entacapone, to normal macaque monkeys for 13 weeks induced dyskinesia in a proportion of animals. In the present study, in situ hybridization histochemistry was used to investigate the effect of chronic L-DOPA administration on the activity of the direct and indirect striatal output pathways by measuring striatal preprotachykinin (PPT), preproenkephalin-A (PPE-A) and adenosine-2a (A2a) receptor gene expression in these monkeys. Overall there was no significant difference in striatal PPT, PPE-A and A2a receptor mRNA levels between normal animals and all L-DOPA (plus carbidopa and/or entacapone)-treated animals irrespective of whether or not dyskinesia occurred. However, when the level of PPE-A and A2a receptor mRNA was analysed in eight monkeys displaying marked dyskinesias as a result of L-DOPA (plus carbidopa with or without entacapone) treatment, there was a significant increase in PPE-A and A2a receptor mRNA message levels in the striatum compared with animals receiving identical treatment, but displaying few or no involuntary movements, and compared with normal controls. There was no difference in striatal PPT mRNA levels in monkeys exhibiting severe dyskinesia compared with those showing little or no dyskinesia after L-DOPA treatment or to normal controls. These results suggest that prolonged L-DOPA treatment alone has no consistent effect on either the direct or indirect pathways, as judged by striatal PPT, PPE-A or A2a receptor mRNA levels in normal monkeys. However, in monkeys exhibiting marked dyskinesia resulting from chronic L-DOPA treatment, abnormal activity is detected in the indirect striato-pallidal output pathway, as judged by striatal PPE-A and A2a receptor mRNA levels, indicating an imbalance between the direct and indirect striatal pathway which may explain the emergence of dyskinesia in these animals.

Chronic L-DOPA treatment increases striatal cannabinoid CB1 receptor mRNA expression in 6-hydroxydopamine-lesioned rats.

The effect of a unilateral 6-hydroxydopamine (6-OHDA) lesion of the left medial forebrain bundle and 3 weeks treatment with L-DOPA of normal and 6-OHDA lesioned rats on CB1r mRNA expression was investigated by in situ hybridization. A 6-OHDA lesion of nigrostriatal pathway alone, confirmed by the loss of nigral tyrosine hydroxylase mRNA, did not alter CB1r mRNA levels in the dopamine depleted striatum. Similarly, chronic L-DOPA treatment of normal rats had no effect on striatal CB1r mRNA expression. In contrast, chronic L-DOPA treatment of 6-OHDA-lesioned rats significantly increased CB1r mRNA expression in the denervated striatum. These results suggest that the CB1r activity may be altered by L-DOPA's action and this may be related to the treatment of Parkinson's disease.

High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.

The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy.

Glutathione depletion in rat brain does not cause nigrostriatal pathway degeneration.

Nigral cell death in Parkinson's disease (PD) may involve oxidative stress and mitochondrial dysfunction initiated by a decrease in reduced glutathione (GSH) levels in substantia nigra. L-buthionine-(S,R)-sulphoximine (BSO; 4.8 and 9.6 mg/kg/day), an irreversible inhibitor of gamma-glutamyl cysteine synthetase, was chronically infused into the left lateral ventricle of rats over a period of 28 days and markedly reduced GSH concentrations in substantia nigra (approx. 59% and 65% in 4.8 and 9.6 mg/kg/d BSO respectively) and the striatum (approx. 63% and 80% in 4.8 and 9.6 mg/kg/d BSO respectively). However, the number of tyrosine hydroxylase (TH)-positive cells in substantia nigra was not altered by BSO-treatment compared to control animals. Similarly, there was no difference in specific [3H]-mazindol binding in the striatum and nucleus accumbens of BSO-treated rats compared to control rats. In conclusion, depletion of GSH following chronic administration of BSO in the rat brain does not cause damage to the nigrostriatal pathway and suggests that loss of GSH alone is not responsible for nigrostriatal damage in PD. Rather, GSH depletion may enhance the susceptibility of substantia nigra to destruction by endogenous or exogenous toxins.

Altered motor function and graft survival produced by basic fibroblast growth factor in rats with 6-OHDA lesions and fetal ventral mesencephalic grafts are associated with glial proliferation.

Basic fibroblast growth factor (bFGF) promotes the survival and growth of dopaminergic neurons. We have investigated the effect of treating fetal ventral mesencephalon with bFGF or nerve growth factor (NGF) on the subsequent survival and function of grafted dopaminergic neurons implanted into the striatum of rats with a unilateral 6-hydroxydopamine lesion. Animals implanted with fetal ventral mesencephalon mixed with bFGF, but not with NGF, displayed more rapid compensation in motor behavior up to 9 weeks after graft implantation. bFGF, but not NGF, produced an increase in the number of tyrosine hydroxylase (TH)-positive neurons, a larger graft volume, and longer neurite outgrowth in comparison to control grafts. bFGF also increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes within the grafts. The increased number of GFAP-positive astrocytes induced by bFGF correlated with the increase in the number of TR-positive neurons, with graft volume and with neurite outgrowth. These findings support the use of bFGF to promote the functional integration of fetal ventral mesencephalon grafts into the denervated host brain and and suggest that its action may be indirectly mediated through glial cells.

Partial reversal of increased preproenkephalin messenger ribonucleic acid (mRNA) and decreased preprotachykinin mRNA by foetal dopamine cells in unilateral 6-hydroxydopamine-lesioned rat striatum parallels functional recovery.

In situ hybridization histochemistry was used to investigate the expression of striatal preproenkephalin and preprotachykinin messenger ribonucleic acid (mRNA) in rats with 6-hydroxydopamine lesions of the nigrostriatal pathway followed 4 weeks later by implantation of foetal dopamine cells into the denervated striatum. Striatal dopamine deafferentation caused an (+)-amphetamine-induced rotational asymmetry, an increase in striatal preproenkephalin mRNA message, and a decrease in striatal preprotachykinin mRNA message relative to control animals. Two months after grafting a foetal ventral mesencephalon suspension, there was reversal of the rotational asymmetry to (+)-amphetamine. At this time the increase in striatal preproenkephalin mRNA was significantly attenuated and the decrease in preprotachykinin mRNA was partially reversed compared to animals with a 6-hydroxydopamine lesion alone. Subregional analysis showed the attenuation of the increase in preproenkephalin mRNA to occur in dorsolateral, dorsomedial and ventromedial, but not ventrolateral, striatal subdivisions. The partial reversal of the decreased preprotachykinin mRNA density after grafting was only statistically significant in the DM and VM subdivisions. These results demonstrate graft-induced partial recovery of striatal function, as judged by preproenkephalin and preprotachykinin mRNA levels, within 2 months of transplantation.

Regenerative and other responses to injury in the retinal stump of the optic nerve in adult albino rats: transection of the intracranial optic nerve.

The response to injury of the proximal (retinal) stump of the intracranially transected optic nerve in adult rats has been studied by electron microscopy. The central part of the retinal stump of the optic nerve underwent severe ischaemic damage resulting in the formation by 3 days postoperation (dpo) of a cone-shaped region of necrotic tissue which extended from a base occupying most of the cross-sectional area of the nerve at the cut end to an apex within the intraorbital part of the nerve and only 2-3 mm from the eyeball. A mixture of apparently viable and dead or dying cells and axons was present in an intermediate zone surrounding the ischaemic core. Apparently intact nerve fibres occupied most of the periphery of the optic nerve. Small bundles of sprout-like axons were seen in the intermediate zone at 3 dpo, and by 5 dpo such sprouts were present at the periphery of the degenerative core. By 7 dpo, the sprouts were also found in the centre of the degenerative core, accompanied by astrocyte processes. The number of axonal sprouts present in the degenerative core and intermediate zone was much higher at 2 and 4 wk postoperation (wpo) than at 7 dpo, then declined gradually by 6 and 8 wpo. These results show that intracranial transection of the rat optic nerve produces extensive degeneration in the proximal stump and effectively produces an intraorbital axotomy of many retinal ganglion cells. Nevertheless, surviving axons display the ability to produce regenerative sprouts which persist for considerably longer than those produced after intraorbital injury.

Chronic L-DOPA treatment differentially regulates gene expression of glutamate decarboxylase, preproenkephalin and preprotachykinin in the striatum of 6-hydroxydopamine-lesioned rat.

The effect of a unilateral 6-hydroxydopamine lesion of the medial forebrain bundle in rats and subsequent L-DOPA treatment for eight weeks on preproenkephalin, preprotachykinin and glutamate decarboxylase (M(r) 67,000) gene expression in the striatum was investigated by in situ hybridization. A 6-hydroxydopamine lesion of the medial forebrain bundle markedly increased the level of preproenkephalin messenger RNA (+66%) and modestly elevated the level of glutamate decarboxylase (M(r) 67,000) messenger RNA (+36%) in the denervated striatum, but caused a decrease in the level of preprotachykinin messenger RNA (-54%) relative to the intact striatum and to sham-lesioned control animals. Treatment with L-DOPA (200 mg/kg/24 h) for eight weeks reduced but did not abolish the 6-hydroxydopamine lesion-induced elevation of preproenkephalin messenger RNA and slightly reduced the elevation of glutamate decarboxylase (M(r) 67,000) messenger RNA in denervated striatum relative to intact side and control groups. However, L-DOPA treatment almost completely reversed the decrease in preprotachykinin messenger RNA caused by 6-hydroxydopamine lesioning when compared to intact side and control groups. The effect of L-DOPA on the gene expression of preproenkephalin and glutamate decarboxylase (M(r) 67,000) differs from the increase in striatal enkephalin content and glutamate decarboxylase activity previously found following L-DOPA treatment. In contrast, L-DOPA reversed the changes in preprotachykinin messenger RNA, reflecting previously reported increases in substance P content. The findings provide new evidence that chronic L-DOPA treatment differentially affects direct striatonigral and indirect striatopallidal pathways at the molecular level.

Regenerative and other responses to injury in the retinal stump of the optic nerve in adult albino rats: transection of the intraorbital optic nerve.

The proximal stump of the optic nerve was examined by electron microscopy from 1 d to 8 wk (dpo/wpo) after intraorbital transection. At 1 dpo a layer of axonal, cytoplasmic and myelin debris approximately 15 microns thick was present at the cut end. A zone approximately 25 microns thick of abnormal and partly degenerate tissue composed of many swollen axons filled with organelles of predominantly abnormal appearance lay between the zone of debris and more proximal levels of the optic nerve, which retained a normal appearance. The earliest putative axonal sprouts were seen at 1 dpo in this zone. By 2 dpo, bundles of small nonmyelinated axons containing microtubules, almost certainly axonal sprouts, had grown out from more proximal regions of the proximal stump and extended as far as its cut end. By 3 dpo, large numbers of axonal sprouts, as well as large numbers of macrophages and newly formed blood vessels, were seen close to the cut end of the proximal stump. Glial cells were not seen to accompany these early outgrowing bundles of axonal sprouts. By 5 dpo, the number of sprouts and macrophages had increased; many bundles of sprouts were now in contact with the surface of astrocytes, which were partly covered by basal lamina. At 7 dpo most of the macrophages had disappeared from the most distal part of proximal stump and bundles of axonal sprouts, associated with astrocytes, which in some cases had penetrated and were fasciculating such bundles, were present at the cut end. The regenerating axonal sprouts in the scar-like tissue at the distal end of the proximal stump of the optic nerve declined in numbers sharply at 2 wpo and only a few sprout-like axonal profiles were present by 8 wpo. Thus while ultimately abortive the early regenerative response is vigorous and involves the outgrowth of a large number of axonal sprouts in the first week after injury.