Somatostatin, cholecystokinin and neuropeptide Y mRNAs in normal and weaver mouse brain.

The distribution of mRNAs encoding for somatostatin, cholecystokinin and neuropeptide Y was determined by in situ hybridization histochemistry in the weaver (wv/wv) mouse, a model of dopamine deficiency as well as in normal (+/+) controls. Weaver mutants did not show any appreciable departure from the normal pattern of expression for mRNA encoding for neuropeptide Y. In contrast, an 82% increase in mRNA encoding for somatostatin was observed in the reticular thalamic nucleus, whereas increases in the order of 20-87% were observed in different hypothalamic nuclei of the weaver brain. In addition, a 47-103% increase of the hybridization signal encoding for cholecystokinin was observed in the cerebral cortex, hippocampus and thalamus of the weaver brain. It can be assumed that the elevated and region-specific somatostatin and cholecystokinin levels observed in the weaver brain may be due to a secondary or compensatory response under conditions of altered neurotransmitter levels.

AMPA receptor subunit RNA transcripts and [(3)H]AMPA binding in the cerebellum of normal and pcd mutant mice: an in situ hybridization study combined with receptor autoradiography.

The expression of AMPA receptor subunit mRNAs and the binding of [(3)H]AMPA were studied in the cerebellum of normal and "Purkinje cell degeneration" ( pcd) mutant mouse. In the pcd cerebellum, [(3)H]AMPA binding was decreased significantly in both the molecular and granule cell layers by 63% and 36%, respectively. In those mutants, GluRA, GluRB and GluRC mRNAs were not detected in the Purkinje cell layer, and the levels of GluRB and GluRD mRNAs were significantly decreased in the granule cell layer by 16% and 57%, respectively. Cerebellar grafts transplanted into the pcd cerebellum expressed only GluRB and GluRC mRNAs, suggesting that donor cells express the appropriate subunits normally expressed by Purkinje neurons. Our results, firstly, support the idea that the expression of the GluRA subunit in Golgi epithelial cells may depend upon the sustained interaction with adjacent Purkinje cells, and secondly, suggest that granule cells which are more resistant to transsynaptic death may express higher levels of GluRB mRNA.

Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease.

Parkinson's disease is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. We now report that minocycline, a semisynthetic tetracycline, recently shown to have neuroprotective effects in animal models of stroke/ischemic injury and Huntington's disease, prevents nigrostriatal dopaminergic neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Minocycline treatment also blocked dopamine depletion in the striatum as well as in the nucleus accumbens after MPTP administration. The neuroprotective effect of minocycline is associated with marked reductions in inducible NO synthase (iNOS) and caspase 1 expression. In vitro studies using primary cultures of mesencephalic and cerebellar granule neurons (CGN) and/or glia demonstrate that minocycline inhibits both 1-methyl-4-phenylpyridinium (MPP(+))-mediated iNOS expression and NO-induced neurotoxicity, but MPP(+)-induced neurotoxicity is inhibited only in the presence of glia. Further, minocycline also inhibits NO-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) in CGN and the p38 MAPK inhibitor, SB203580, blocks NO toxicity of CGN. Our results suggest that minocycline blocks MPTP neurotoxicity in vivo by indirectly inhibiting MPTP/MPP(+)-induced glial iNOS expression and/or directly inhibiting NO-induced neurotoxicity, most likely by inhibiting the phosphorylation of p38 MAPK. Thus, NO appears to play an important role in MPTP neurotoxicity. Neuroprotective tetracyclines may be effective in preventing or slowing the progression of Parkinson's and other neurodegenerative diseases.

Partial restoration of striatal GABAA receptor balance by functional mesencephalic dopaminergic grafts in mice with hereditary parkinsonism.

Levels of inhibitory amino acid receptors were studied in the weaver (wv/wv) mouse model of dopamine (DA) deficiency after unilateral intrastriatal transplantation of fetal mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935. The average increase in [3H]GBR 12935 binding in grafted dorsal striatum compared to nongrafted wv/wv striatum was 60% 3 months after grafting. Autoradiography of 8 nM [3H]flunitrazepam and 12 nM [3H]muscimol binding was carried out to visualize the distribution of GABAA receptors in +/+ mice and in recipient weaver mutants. A 17% increase in [3H]flunitrazepam binding and a 20% increase in [3H]muscimol binding was found in the nongrafted dorsal striatum of weaver mutants compared to +/+. The functional mesencephalic grafts had a partial normalizing effect on both [3H]flunitrazepam and [3H]muscimol binding in the dorsal striatum of the weaver recipients. The normalization brought about by the grafts was around 20% for [3H]flunitrazepam binding and more than 40% for [3H]muscimol binding. The results are discussed in the context of the important interaction between the converging glutamatergic corticostriatal and DAergic nigrostriatal pathways in controlling the functional GABAergic output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.

[3H]CNQX and NMDA-sensitive [3H]glutamate binding sites and AMPA receptor subunit RNA transcripts in the striatum of normal and weaver mutant mice and effects of ventral mesencephalic grafts.

Levels of excitatory amino acid receptors were studied in the weaver mouse model of DA deficiency after unilateral intrastriatal transplantation of E12+/+ mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935 (average increase in grafted dorsal striatum compared to nongrafted side, 60%). Autoradiography of 80 nM [3H]CNQX and 100 nM NMDA-sensitive [3H]glutamate binding was carried out to visualize the topography of non-NMDA and NMDA receptors, respectively, in +/+ mice and in recipient weaver mutants 3 months after grafting. Increases of 30% or more were found for [3H]CNQX binding in the dorsal nongrafted weaver striatum compared to +/+, and a further 6-9% increase in grafted weaver compared to nongrafted side. The added increase of non-NMDA receptors in the transplanted striatum might be explained by a presence of such receptors on DA presynaptic endings of graft origin. A 20% increase in NMDA-sensitive [3H]glutamate binding was measured in the dorsal nongrafted weaver striatum compared to +/+. NMDA-sensitive [3H]glutamate binding in the transplanted side of weaver mutants tended to be slightly higher in all areas of the striatal complex compared to the nongrafted side, without reaching conventional levels of statistical significance. Using in situ hybridization histochemistry with synthetic 32p labeled oligonucleotide probes, we investigated RNA transcripts encoding the four AMPA receptor subunits. RNA transcripts in the striatum are seen with a decreasing signal intensity in the following order: GluRB > GluRA > GluRC > GluRD. The weaver caudate-putamen shows a 12% increase in GluRA subunit mRNA compared to +/+, whereas mesencephalic neuron transplantation leads to slight increases (3%) in the levels of GluRB mRNA in the nucleus accumbens. The results are placed in the context of the important interaction between the converging glutamatergic corticostriatal and the DAergic nigrostriatal pathways in controlling the functional output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.

Rate of neuronal fallout in a transsynaptic cerebellar model.

Quantitative analyses of transsynaptic granule cell death subsequent to the genetically determined degeneration of Purkinje cells in the cerebellum of pcd/pcd mutant mice show that granule neuron fallout follows a typical mathematical pattern of elemental decay. Biological and theoretical connotations are discussed in light of the empirical observations and a simulation model.

MAP2 and GAP-43 expression in normal and weaver mouse cerebellum: correlative immunohistochemical and in situ hybridization studies.

MAP2 is a major microtubule-associated brain protein, selectively localized in dendrites; growth-associated phosphoprotein GAP-43 is a neuron-specific protein associated with axonal outgrowth. In adult cerebellum, both of these proteins and their corresponding RNA transcripts are most strongly expressed by granule cells. Using immunocytochemistry with antibodies and in situ hybridization histochemistry with [32P] labeled oligonucleotide probes, we examined the cellular localization of MAP2, GAP-43 and their mRNAs in the cerebellum of control and weaver (wv/wv) mutant mice, which exhibit massive granule cell death. In wild-type (+/+) mice, MAP2 immunoreactivity was seen in neuronal somata and dendrites of the granule cell layer; GAP-43 immunoreactivity was present in molecular layer, corresponding to the distribution of parallel fibres. Transcripts encoding MAP2 and GAP-43 were localized in the layer of the granule cell somata. In heterozygous weaver mice (wv/+), which feature an intermediate degree of granule cell loss, MAP2 immunoreactivity was localized in the granular layer, and the pattern of GAP-43 immunostaining was also similar to +/+, the only difference being a thinner molecular layer. Heterozygotes had an anatomical pattern of MAP2 and GAP-43 mRNA hybridization qualitatively similar to that of the wild-type with some deviations in signal intensity. In homozygous weaver mutants (wv/wv), MAP2 immunoreactivity was extremely weak in the area beneath Purkinje cells and a certain GAP-43 immunoreactivity was seen in the upper part of cerebellar cortex. Hybridization signals for MAP2 and GAP-43 mRNAs were minimal. The reported alterations in regional pattern of MAP2 and GAP-43 expression in mutant mice offer a molecular correlate of dendritic and axonal protein gene transcription pertinent to the neuropathological manifestations of certain forms of heredodegenerative ataxia.

Cerebellar grafts partially reverse amino acid receptor changes observed in the cerebellum of mice with hereditary ataxia: quantitative autoradiographic studies.

We used quantitative autoradiography of [3H]CNQX (200 nM), [3H]muscimol (13 nM), and [3H]flunitrazepam (10 nM) binding to study the distribution of non-NMDA and GABA(A) receptors in the cerebellum of pcd mutant mice with unilateral cerebellar grafts. Nonspecific binding was determined by incubation with 1 mM Glu, 200 microM GABA, or 1 microM clonazepam, respectively. Saturation parameters were defined in wild-type and mutant cerebella. In mutants, non-NMDA receptors were reduced by 38% in the molecular layer and by 47% in the granule cell layer. The reduction of non-NMDA receptors in the pcd cerebellar cortex supports their localization on Purkinje cells. [3H]CNQX binding sites were visualized at higher density in grafts that had migrated to the cerebellar cortex of the hosts (4.1 and 11.0 pmol/mg protein, respectively, at 23 and 37 days after grafting) than in grafts arrested intraparenchymally (2.6 and 6.2 pmol/mg protein, respectively, at 23 and 37 days after grafting). The pattern of expression of non-NMDA receptors in cortical vs. parenchymal grafts suggests a possible regulation of their levels by transacting elements from host parallel fibers. GABA(A) binding levels in the grafts for both ligands used were similar to normal molecular layer. Binding was increased in the deep cerebellar nuclei of pcd mutants: the increase in [3H]muscimol binding over normal was 215% and the increase in [3H]flunitrazepam binding was 89%. Such increases in the pcd deep cerebellar nuclei may reflect a denervation-induced supersensitivity subsequent to the loss of Purkinje axon terminal innervation. In the deep nuclei of pcd mutants with unilateral cerebellar grafts, [3H]muscimol binding was 31% lower in the grafted side than in the contralateral nongrafted side at 37 days after transplantation; [3H]flunitrazepam binding was also lower in the grafted side by 15% compared to the nongrafted side. Such changes in GABA(A) receptors suggest a significant, albeit partial, normalizing trend of cerebellar grafts on the state of postsynaptic supersensitive receptors in the host cerebellar nuclei.

Atrophy and loss of dopaminergic mesencephalic neurons in heterozygous weaver mice.

The phenotypic effect of the weaver mutation in the ventral midbrain of homozygous mutants is associated with the progressive loss of dopaminergic neurons. To discover whether the number of mesencephalic dopaminergic cells is altered in weaver heterozygotes (wv/+), we studied mice between 20 and 365 days of age. We counted tyrosine hydroxylase (TH)-immunopositive cells in the substantia nigra (SN), retrorubral nucleus (RRN), and ventral tegmental area (VTA), and measured cross-sectional areas of neuronal somata in the SN of wv/+ and age-matched wild-type controls (+/+). The number of TH-positive cells in the wv/+ ventral midbrain was on average 13% lower than normal. Cell loss was detected selectively in the SN (12%) and VTA (23%). The areas of somatic profiles in the wv/+ nigral neurons were on average reduced by 9.8%. The neuronal losses in the SN and VTA correlated with a 13.8% reduction in dopamine level in the ventral striatum in wv/+ mice at 14-16 months of age. Our findings imply that a single dose of the weaver gene in the mouse is associated with cellular damage leading to a chronic deficiency in the mesostriatal dopaminergic system.

Amelioration of the behavioral phenotype in genetically ataxic mice through bilateral intracerebellar grafting of fetal Purkinje cells.

We have previously applied neural grafting to "Purkinje cell degeneration" mutant mice (gene symbol pcd, mouse chromosome 13), a model of recessively inherited cerebello-olivary atrophy, to create appropriate interactions between wild-type and mutant cells in elucidating gene effects on the involved neuron populations and to address issues of the structural integration of donor Purkinje cells into the disrupted cerebellar loop. Behaviorally, pcd homozygotes manifest ataxic signs beginning at 3-4 wk of age. The functional effects of cerebellar transplants on motor performance have long remained an open question. The aim of the present study was to determine the recovery of motor responses in pcd mutants in a battery of behavioral tasks after bilateral transplantation of cerebellar cell suspensions (prepared from wild-type mice) into the parenchyma of the deep cerebellar nuclei of the hosts, according to a protocol that emphasizes the reconstruction of the missing inhibitory cortico-nuclear projection. With this approach, the denervated deep nuclei of the host receive a new Purkinje axonal innervation; further, most transplanted Purkinje cells end up occupying cortical localities anyway and display a correct dendritic tree orientation toward the pia. Motor coordination and fatigue resistance were assessed in a rotarod treadmill apparatus, a behavioral paradigm useful in studying various brain abiotrophies and treatments, including developmental perturbations of the cerebellar cytoarchitecture. Locomotor activity was quantified by the number of squares mice crossed as they moved about in an open-field matrix. Grafted pcd mice performed significantly better than sham-operated mutants in both of these tasks. Moreover, graft-recipient mice were able to sustain their abdomen above the floor on their limbs during movement, contrasting to the typical lowered, widened stance of sham-operated pcd mutants. These findings clearly demonstrate that bilateral transplants of fetal Purkinje cells have functional effects on motor performance in the pcd model of hereditary cerebellar ataxia.

The cerebellar model of neural grafting: structural integration and functional recovery.

A synopsis is presented of the recent history of cerebellar tissue transplantation over the past 25 years. The properties of growth and differentiation of cerebellar grafts placed intraocularly or intracranially are reviewed, as well as the interaction of heterotopic and orthotopic grafts with the host brain. Particular emphasis is placed on the use of ataxic mouse mutants as recipients of donor cerebellar tissue for the correction of their structural deficits and the functional recovery of behavioural responses.

Grafted cerebellar cells in a mouse model of hereditary ataxia express IGF-I system genes and partially restore behavioral function.

Fetal grafts of normal cerebellar tissue were implanted into the cerebellum of Purkinje cell degeneration mutant mice (pcd/pcd), a model of adult-onset recessively inherited cerebello-olivary atrophy, in an attempt at correcting their cellular and motor impairment. Donor cerebellar cells engrafted in the appropriate sites, as evidenced by the pattern of expression of insulin-like growth factor-I (IGF-I) system genes. Bilateral cerebellar grafts led to an improvement of motor behaviors in balance rod tests and in the open field, providing evidence for functional integration into the atrophic mouse cerebellum and underscoring the potential of neural transplantation for counteracting the human cerebellar ataxias.

Graft-induced restoration of function in hereditary cerebellar ataxia.

Fetal cerebellar cell suspensions, prepared from wild-type (+/+) mice, were implanted bilaterally into the cerebellum of Purkinje cell degeneration' (pcd) mutant mice, a model of adult-onset recessively inherited cerebellar ataxia, to study the functional effects of the grafts on motor coordination and fatigue resistance in a rotating rod treadmill paradigm. The viability of transplanted Purkinje cells was verified with immunocytochemistry for calbindin-D28k and for glutamate receptor 2/3 subunits and with in situ hybridisation histochemistry for insulin-like growth factor I mRNA, biochemical markers normally expressed by Purkinje cells in the cerebellum. Sham injections of vehicle did not appreciably modify the performance of pcd mutants in the rota-rod tests. On the other hand, bilateral cerebellar grafts led to a 3.5-fold increase in the time period that recipient pcd mice were able to stay on the rotating drum based on the comparison of mean scores (of three trials) or a 5.5-fold increase based on the comparison of maximum scores (of the three trials). These findings provide evidence for a motor enhancement in the pcd mouse model of hereditary cerebellar ataxia following intracerebellar transplantation of primordial Purkinje cells.

Selective vulnerability of late-generated dopaminergic neurons of the substantia nigra in weaver mutant mice.

In homozygous weaver (wv/wv) mutant mice, nearly 50% of the dopaminergic substantia nigra neurons degenerate by postnatal day 20. We have now determined that the total number of dopaminergic neurons in the ventral midbrains of a litter of obligatory homozygous weaver pups and a litter of normal wild-type control pups indicates that no significant differences are present between groups at birth. To test the hypothesis that the subsequent degeneration of these neurons is linked to their time of origin, [3H]thymidine autoradiography was combined with tyrosine hydroxylase immunocytochemistry to construct neurogenetic timetables on postnatal day 20 in wild-type mice and weaver homozygotes. Both groups have the same span of neurogenesis but have statistically different proportions of neurons generated on specific days. In wild-type mice, more than half of the dopaminergic neurons originate on or after embryonic day 12. In contrast, over two-thirds of the surviving dopaminergic neurons in homozygous weaver mice originate on or before embryonic day 11. Our data suggest that the weaver gene does not interfere with the generation of dopaminergic neurons, but it preferentially kills late-generated dopaminergic neurons between birth and postnatal day 20.

Altered IGFBP5 gene expression in the cerebellar external germinal layer of weaver mutant mice.

The IGF system components play important roles in cerebellar development as demonstrated by their specific spatial-temporal expression. IGF-I, type I IGF receptor (IGFR-I), IGFBP2 and IGFBP5 mRNA are localized in distinct cell populations, and all are expressed at the highest levels at the peak of Purkinje cell growth, active synaptogenesis and dendritic formation. To understand IGF-I's action at the cellular level, in situ hybridization was employed to investigate the distribution of IGF system gene transcripts in the cerebellum of weaver mutant mice (wv/wv). Although located ectopically, the surviving Purkinje cells express IGF-I mRNA at the same level in wv/wv as in +/+. No alteration in the cellular distribution or mRNA levels was observed with IGFBP2, or IGFR-I mRNAs. However, the pattern of IGFBP5 expression is altered in the external germinal layer of wv/wv mice. Not only is IGFBP5 expressed by more granule cell precursors of wv/wv cerebellum, but its mRNA level is 2.3 fold that of +/+. The altered IGFBP5 gene expression in granule cell precursors may modulate the interaction of IGF-I with IGFR-I in ways that contribute to their massive death occurring in the development of wv/wv cerebellum.

Transplantation of mesencephalic cell suspensions from wild-type and heterozygous Weaver mice into the denervated striatum: assessing the role of graft-derived dopaminergic dendrites in the recovery of function.

The Weaver (wv) mutation leads to a loss of mesencephalic dopamine cells and nigrostriatal dopamine axons in homozygosity (wv/wv) and to a deficiency of nigral dopaminergic dendrites without a concomitant loss of dopamine cell somata or axons in heterozygosity (wv/+). Previous studies have shown that grafts of foetal dopamine cells from wild-type (+/+) donors can survive when implanted into the wv/wv striatum, supply both an axonal and a dendritic innervation to the host, establish synaptic connections with host striatal neurons, and bring about a functional recovery evidenced by rotational asymmetry tests. The aims of the present study were to examine whether wv/+ dopamine cells maintain a "dendrite-poor" phenotype after transplantation to the denervated striatum, and to compare their functional effects with those of wild-type (+/+) grafts in reversing amphetamine-induced turning behaviour. To that end, +/+ and wv/+ ventral mesencephalic tissue (dissected out from E10-E12 foetal mice and made into a cell suspension by enzymatic and mechanical dissociation) was stereotactically grafted into the right striatum of either wv/wv hosts or +/+ hosts subjected in advance to 6-OHDA lesions of the right substantia nigra. Viability and morphology of grafted neurons were assessed by tyrosine hydroxylase immunocytochemistry on serial sections of the host forebrains. Dopamine cell bodies survived in comparable numbers in the grafts regardless of donor genotype; however, grafts of either genotype contained fewer dopaminergic cells when they were hosted in the wv/wv striatum as compared to the striatum of +/+ mice with 6-OHDA lesions. Despite the survival of cell somata, the dendritic arborisation of wv/+ cells was strikingly poorer than that of +/+ cells in grafts placed into both host types, most likely reflecting their in situ phenotypic abnormality. Recipient wv/wv mice with +/+ and wv/+ grafts exhibited 88% and 83% left rotations, respectively; 6-OHDA hosts with +/+ and wv/+ grafts showed 178% and 165% reversals of asymmetry, respectively. The differences between the effects of +/+ and wv/+ grafts were not statistically significant.(ABSTRACT TRUNCATED AT 400 WORDS)

Ventral mesencephalic grafts in the neostriatum of the weaver mutant mouse: structural molecule and receptor studies.

Mesencephalic cell suspensions were prepared from E12 wild-type (+/+) mouse embryos and stereotaxically implanted into the dorsal neostriatum of weaver mutant mice (wv/wv), which have a genetic mesostriatal dopamine (DA) deficiency. Survival of DA neurons in the grafts was documented by tyrosine hydroxylase (TH) immunocytochemistry. Axon growth was monitored by immunocytochemistry using a battery of antibody markers, and the cellular localization of structural protein and receptor RNA transcripts was studied by in situ hybridization histochemistry using [32P]oligonucleotide probes. The cell suspension grafts exhibited strong immunoreactivity for neural cell adhesion molecule (N-CAM), growth-associated phosphoprotein GAP-43, microtubule-associated protein 2 (MAP2), beta-amyloid protein precursor (beta APP), and phosphorylated neurofilament epitopes (clone SMI-31); intermediate-to-high levels of immunoreactivity were seen for synaptophysin. High levels of hybridization were found in the grafts for the RNA transcripts of GAP-43, MAP2, and isoforms beta APP695, beta APP714 and beta APP751 of the beta APP. No hybridization signal was detected in the grafts for DA D2 or neurotensin receptor mRNAs, both of which are normally expressed by nigral DA neurons. DA receptor autoradiography using the D2/D3 agonist [3H]CV 205-502 as a ligand showed no binding in the transplants, indicating an apparent abnormality of grafted cells; neurotensin binding sites, labeled with [125I]neurotensin, were visualized in the suspensions, indicating the possibility that receptors could be present but that RNA message levels might be too low to allow detection. These findings offer a molecular correlate of axonal, dendritic and structural protein expression by transplanted mesencephalic neurons; further, they suggest that specific functional properties of grafted nigral cells are maintained after transplantation, while other aspects of their cellular biology may be compromised.

Amelioration of the behavioral phenotype in weaver mutant mice through bilateral intrastriatal grafting of fetal dopamine cells.

Weaver mutant mice lose more than two-thirds of their nigral dopamine neurons. Behaviorally, weaver homozygotes display tremor, gait instability, and toppling over to the sides after a few steps. The recovery of functional responses was determined in a battery of behavioral tests in weaver mutants after bilateral transplantation of mesencephalic cell suspensions (prepared from wild-type mice) to the striatum. Equilibrium was tested by the time mice were able to stay on a suspended balance rod before falling off. Locomotor coordination was measured by the number of times mice toppled over to the sides as they moved about in an open-field matrix. Locomotor activity was quantified by the number of square crossings in an open-field matrix. Grafted weaver mutants performed significantly better than non-grafted mutant mice in all of these three tasks. The findings clearly demonstrate that bilateral transplants of foetal DA cells enhance motor performance in the weaver model of chronic nigrostriatal DA deficiency.