Search

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., (Copyright 1999 Academic Press.)

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.

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.

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.

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)

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.

Previous [3H]thymidine studies in Nissl-stained sections in rats established that the substantia nigra pars compacta and the ventral tegmental area originate sequentially according to an anterolateral to posteromedial neurogenetic gradient. We investigated whether that same pattern is found in mice in the dopaminergic neurons in each of these structures. Using tyrosine hydroxylase immunostaining combined with [3H]thymidine autoradiography, the time of origin of dopaminergic midbrain neurons in the retrorubral field, the substantia nigra pars compacta, the ventral tegmental area, and the interfascicular nucleus was determined in postnatal day 20 mice. The dams of the experimental animals were injected with [3H]thymidine on embryonic days (E) 11-E12, E12-E13, E13-E14, and E14-E15. The time of origin profiles for each group indicated significant differences between populations. The retrorubral field and the substantia nigra pars compacta arose nearly simultaneously and contained the highest proportion of neurons, 49 to 37%, generated on or before E11. Progressively fewer early-generated neurons were found in the ventral tegmental area (20%), and the interfascicular nucleus (8.5%). In addition, anterior dorsolateral neurons in the substantia nigra and ventral tegmental area were more likely to be generated early than the posterior ventromedial neurons. These findings indicate that mouse and rat brains have nearly identical developmental patterns in the midbrain, and neurogenetic gradients in dopaminergic neurons are similar to those found in Nissl studies in rats.

The dopamine (DA) uptake system was investigated in the mesostriatal system of normal and weaver mutant mice, which lose mesencephalic DA neurons, as well as in weaver mutants with ventral mesencephalic grafts to the striatum. Assays of [3H]DA uptake in striatal synaptosomal fractions in vitro and autoradiography of [3H]mazindol binding in brain sections were carried out in wild-type mice (+/+) and in the two hemispheres of homozygous weaver mutants (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. Net [3H]DA uptake, expressed as pmol/mg-protein/2-min, was on the average 50.6 in the striatum of wild-type mice, 7.9 in the non-grafted, and 10.1 in the transplanted striatum of weaver mutants. [3H]DA uptake in wild-type mice differed significantly from both the grafted and non-grafted weaver striata (P < 0.001). Paired comparisons for [3H]DA uptake between right and left sides of recipient weaver mice showed a significant side effect (P < 0.02), the right side being 28-38% higher than the left side [mean of all individual (R-L)/L values]. The results of amphetamine-induced turning behavior tests were compared with the biochemical findings. Mice with grafts to the right side rotated an average of 22 turns to the left and 7 turns to the right during the five one-minute sessions; the mean value L/(L + R) was 64%. A plot of (L-R) rotations against (R-L) [3H]DA uptake gave a correlation coefficient of 0.552 (P < 0.05), indicating that animals with a strong rotational bias to the left tended to have higher [3H]DA on the right. Similarly, the animals that were used for [3H]mazindol binding autoradiographic studies displayed on the average 72% rotations to the left side. In the [3H]mazindol binding data, non-grafted weaver mutants showed the severest depletion relative to wild-type in the dorsomedial and dorsolateral caudate-putamen (86% and 87%, respectively). Mice with unilateral grafts to the right side showed an increase in [3H]mazindol binding signal in the transplanted side of 40-64% (depending on dorsoventral topography) over the contralateral, non-grafted side. These findings attest to the functional effects of the grafts at the anatomical, biochemical, and behavioral levels. The parallel measurements of motor performance and DA uptake in the same animals offers an index of behavioral recovery as a function of transmitter-related activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Using in situ hybridization histochemistry with [32P]oligonucleotide probes, we studied the cellular localization of RNA transcripts for amyloid beta-protein precursor (beta APP), growth-associated phosphoprotein-43 (GAP-43) and microtubule-associated protein 2 (MAP2) in the mesostriatal system of normal (+/+) and weaver (wv/wv) mutant mice, which lose mesencephalic dopamine neurons. In addition, expression of the same messages was studied in ventral mesencephalic cell suspensions transplanted to the weaver striatum. Transcripts encoding GAP-43, MAP2 and isoforms beta APP695, beta APP714 and beta APP751 were present in normal substantia nigra and progressively reduced in weaver substantia nigra; such a reduction was correlated with dopamine neuron loss. The survival of dopamine neurons in unilateral intrastriatal grafts was documented by methamphetamine-induced rotational asymmetry tests and by tyrosine hydroxylase immunocytochemistry. High hybridization signals were obtained for GAP-43, MAP2, beta APP695, beta APP714 and beta APP751 RNA transcripts in the grafted tissue; the beta APP770 species--normally seen in striatum and not substantia nigra--was not expressed in the grafts, but it was present in the recipient striatum. Following immunocytochemical labelling with antibodies, GAP-43 and MAP2 immunoreactivities were seen in cell processes in the grafts and surrounding tissue, whereas beta APP immunoreactivity was mainly found in grafted cell bodies. These results suggest that the transplanted mesencephalic cells mature very similarly to those in the normal substantia nigra, expressing different mRNAs that are normally present in the ventral midbrain and which are reduced in the weaver mutant as a consequence of dopamine neuron loss.

Dissociated cell suspensions were prepared from the ventral midbrain of normal mouse foetuses and stereotaxically implanted into the neostriatum of 2-3 months old homozygous weaver mutant mice, which are severely deficient in dopamine. In tests of amphetamine-induced turning behaviour 60 days after grafting, recipient animals displayed a rotational bias opposite to the grafted side. Prior to perfusion, which was carried out at 80 days after transplantation surgery, the grafted striata of the weaver recipients were deprived of their intrinsic mesostriatal dopamine input by local injections of 6-hydroxydopamine into the ipsilateral substantia nigra in order to selectively study the innervation derived from the graft. Grafts were found to contain an estimated 100-700 tyrosine hydroxylase immunoreactive neurones. An ultrastructural analysis demonstrated that both axons and dendrites immunoreactive for tyrosine hydroxylase extended from the graft into the recipient striatum. In the host striatum proximal to the graft (i.e. at a distance of 0.0-0.5 mm from the graft) the proportion of dendrites to axons was about 1:2, whereas distal to the graft (i.e. at a distance of 0.5-1.0 mm) it was 1:20. Graft-derived tyrosine hydroxylase immunoreactive axons were primarily found in apposition with unlabelled dendrites or spines of the recipient striatum (greater than 90%). Graft-derived dopaminergic dendrites received synaptic input from unlabelled axon terminals and were opposed to the unlabelled somata of striatal neurones in a few instances. In conclusion, this study shows that mesencephalic cell suspensions survive in the weaver striatum and provide a functional dopamine innervation which comprises both axonal and dendritic processes.

Dopamine (DA) D2 receptor binding is increased in the striatum of 5-6 months old weaver mutant mice (Kaseda et al. 1987). This may occur in response to the loss of DA neurons in the midbrain and the decrease in DA content in the striatum of homozygous mutants. One purpose of the present study was to determine if the diminished DA innervation is associated with changes in D2 receptors at earlier ages and if the increase in DA D2 receptor binding seen at 5-6 months is a lasting phenomenon. Specific [3H]spiperone binding was measured in the dorsolateral (DL), dorsomedial (DM) and ventrolateral (VL) striatum and in the nucleus accumbens (AC) of homozygous weaver mutant mice (wv/wv), heterozygous littermates (wv/+) and wild-type controls (+/+). Mice were studied at 20 days and 1, 3, 6, 9 and 12 months of age. The difference in specific [3H]spiperone binding in DL striatum between wv/wv and +/+ mice was significantly greater at 6 months than the difference at 1 month and at 12 months of age. Foetal ventral mesencephalic grafts survive and establish functional innervation in the striatum of weaver mice as shown by the induction of a contralateral turning bias (Low et al. 1987). The second aim of the present studies was to determine if such grafts would also reverse the increase in DA D2 receptor binding in the striatum. Aspiration cavities were prepared in the cortex of weaver mice, and ventral mesencephalic tissue from E14-E15 +/+ foetuses was subsequently placed on the surface of the right dorsal striatum when the recipients were 3 months old.(ABSTRACT TRUNCATED AT 250 WORDS)

A recently introduced quantitative radioautographic technique was used to characterize the striatal dopaminergic deficit in weaver mutant mice and to evaluate the extent of DA reinnervation resulting from cell suspension grafts of fetal ventral mesencephalic tissue. Brain slices from normal mice and unilaterally grafted weaver mice were incubated in [3H]DA, in the presence of desipramine and pargyline 3-5 months after graft surgery. Semi-thin sections from the fixed and resinembedded slices were subsequently exposed on tritium sensitive film and afterwards dipped in nuclear emulsion for light microscope radioautography. Alternative slices were embedded in Epon for post-embedding tyrosine hydroxylase (TH) immunocytochemistry. The grain density of the film radioautographs matched well the distribution of TH positive fibers. Both methods revealed an almost complete absence of DA axons in the dorsomedial quadrant of the weaver neostriatum and an increasing density of DA innervation towards the ventrolateral areas. In the light microscope radioautographs, only the ventral striatum (i.e. nucleus accumbens and olfactory tubercle) and a narrow ventral and periventricular zone of the caudate-putamen were covered by silver grain clusters typical of DA varicosity labeling. Such labeled varicosities were nevertheless found in reduced numbers the lateral portion of both nucleus accumbens and the olfactory tubercle. The remaining neostriatum was overlaid by diffuse silver grains. suggesting a deficient DA uptake and storage mechanism in the residual DA fibers in this region. Immunocytochemistry using antibodies specific for DA or TH provided further evidence that the residual DA innervation in the weaver neostriatum was biochemically defective. Weaver mice with grafts of ventral mesencephalic tissue in the right neostriatum showed an amphetamine-induced rotational bias to the contralateral side, which was not seen in the sham-operated animals. In contrast to the intrinsic weaver neostriatal DA innervation, DA fibers of graft origin exhibited the normal, clustered type of varicosity labeling. The computerized image analysis of silver grain density in film radioautographs was calibrated by counting these labeled varicosities in selected areas of light microscope radioautographs from the same sections. Results showed a mean DA reinnervation of neostriatal tissue surrounding the graft of about 20%, in some cases up to 80%, of the density seen in wild type mice, with a gradual decrease with distance up to 1-1.4 mm from the graft. The ventral parts of the neostriatum, which contained higher numbers of residual intrinsic DA fibers, were much more sparsely reinnervated than the dorsal and dorsomedial areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Weaver mutant mice are characterized by a decrease in striatal dopamine (DA), which is associated with a progressive loss of DA neurones in the substantia nigra. This mutant thus provides the opportunity to examine the functional effects of DA neurones grafted to the striatum in a genetic model of parkinsonism. Ventral mesencephalic tissue from normal foetuses was placed on the surface of the right dorsal striatum of adult weaver mutants. After grafting, animals were tested for methamphetamine-induced circling behaviour. Mutants with DA containing grafts displayed a significant circling bias toward the left, non-grafted side. Mutants without grafts did not display any rotational bias to either side. These results demonstrate that grafted DA containing neurones establish a functional innervation of the weaver striatum and suggest that grafting of neural tissue is a viable approach in restoring function in genetic degenerative disorders of the nigrostriatal system.

Ventral mesencephalic anlagen survive following grafting to the striatum of weaver mutant mice and reinnervate the dopamine-depleted basal ganglia of the recipients. The aim of the present study was to examine the pattern of connectivity established by graft-deriving dopamine afferents in the host striatum. Grafts were obtained from normal embryos at a gestational age of 14-15 days and implanted into a surgical cavity overlying the dorsal striatum of adult weaver recipients. Tissue was processed for electron microscopic immunocytochemistry using a primary antiserum against tyrosine hydroxylase. At the time of examination, recipient weaver mutants were 8.5 months old and the grafts had survived for 4.5 months. Grafts were found to contain an estimated 100-1000 tyrosine hydroxylase immunoreactive neurons. Tyrosine hydroxylase immunoreactive fibres, displaying characteristic varicosities, innervated the dorsal striatum to a depth of 1000 micron. In the non-grafted striatum, 8% of the contacts of tyrosine hydroxylase immunoreactive nerve terminals were junctional. That proportion contrasted with the corresponding value of normal animals, which is 27%. In the grafted striatum, 29% of the contacts were junctional. That percentage approximated the value found in normal animals. By applying a stereological correction, it can be estimated from those numbers that the true proportion of junctional contacts in the non-grafted striatum of 8.5-month-old mutants may be 26%, whereas that in the grafted side may be 91%, which is close to the normal situation. The majority of contacts in the reinnervated striatum (84%) were made with dendrites and spines. However, the proportion of total axosomatic contacts in the reinnervated striatum was twice as high as that found in the striatum of normal animals, and the proportion of junctional synapses was three times higher than that found normally. We conclude that: (1) in spite of a genetically determined degenerative process, the dorsal neostriatum of weaver mutant mice is receptive to synaptic investment by dopamine afferents originating in normal donor tissue. (2) In repopulating the denervated weaver striatum, graft-deriving dopamine afferents display a connectional selectivity, i.e. they establish synaptic relations preferentially with those cellular domains that are normally innervated by dopamine nerve terminals. In this context, it is possible that dopamine fibres originating in the grafts invest postsynaptic sites that had either been vacated from the intrinsic dopamine input or had never received such an input. (3) The striatal connectivity following transplantation may retain features of immaturity as suggested by t

Attempts to reconstruct the damaged nigrostriatal pathway in experimental models of Parkinson disease have thus far been carried out in animals with neurotoxically induced dopamine deficiency. The present study establishes the weaver (wv/wv) mutant mouse as a genetic model of chronic striatal dopamine denervation by demonstrating a marked decrease of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta. Moreover, grafts of embryonic ventral mesencephalon taken from genetically normal mice and transplanted into the lateral ventricle of adult weaver mutants can survive and grow in the mutant host environment, express tyrosine hydroxylase immunoreactivity, and reinnervate the target regions of the recipient. These results provide evidence of integration of graft and host tissue and suggest that transplantation of dopamine neurons may be effectively applied to overcome nigrostriatal degeneration of genetic etiology.