Somatic cell cloned transgenic bovine neurons for transplantation in parkinsonian rats.

Parkinson's disease symptoms can be improved by transplanting fetal dopamine cells into the putamen of parkinsonian patients. Because the supply of human donor tissue is limited and variable, an alternative and genetically modifiable non-human source of tissue would be valuable. We have generated cloned transgenic bovine embryos, 42% of which developed beyond 40 days. Dopamine cells collected from the ventral mesencephalon of the cloned fetuses 42 to 50 days post-conception survived transplantation into immunosuppressed parkinsonian rats and cells from cloned and wild-type embryos improved motor performance. Somatic cell cloning can efficiently produce transgenic animal tissue for treating parkinsonism.

Regional brain dopamine metabolism: a marker for the speed, direction, and posture of moving animals.

Brain dopamine is necessary for normal movement. To determine whether there is a precise relation between the intensity of movement and changes in brain dopamine metabolism, the investigators ran rats on straight and circular treadmills at different speeds and with different body postures. Concentrations of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid increased in the caudate and accumbens nuclei in direct relation to the speed and angular posture of the animals. Dopamine metabolism in the nucleus accumbens was more strongly linked to the speed and direction of movement, while in the caudate nucleus dopamine and 3,4-dihydroxyphenylacetic acid were affected most by posture and direction.

Segregation of human neural stem cells in the developing primate forebrain.

Many central nervous system regions at all stages of life contain neural stem cells (NSCs). We explored how these disparate NSC pools might emerge. A traceable clone of human NSCs was implanted intraventricularly to allow its integration into cerebral germinal zones of Old World monkey fetuses. The NSCs distributed into two subpopulations: One contributed to corticogenesis by migrating along radial glia to temporally appropriate layers of the cortical plate and differentiating into lamina-appropriate neurons or glia; the other remained undifferentiated and contributed to a secondary germinal zone (the subventricular zone) with occasional members interspersed throughout brain parenchyma. An early neurogenetic program allocates the progeny of NSCs either immediately for organogenesis or to undifferentiated pools for later use in the "postdevelopmental" brain.

Mice transgenic for an expanded CAG repeat in the Huntington's disease gene develop diabetes.

The autosomal dominant neurological syndrome of Huntington's disease has been modeled in transgenic mice by the expression of a portion of the human huntingtin gene together with 140 CAG repeats (the R6/2 strain). The mice develop progressive chorea with onset at approximately 9 weeks of age and with death at approximately 13 weeks. Associated symptoms include weight loss and polyuria in the absence of eating or drinking deficits. We have found that these mice have insulin-responsive diabetes. Fasting glucose was 211 + 19 mg/dl in R6/2 mice compared with 93 + 5 mg/dl in C57/B6 controls (n = 12, both groups; P < 0.01). Administration of insulin intraperitoneally led to a reduction in blood glucose. At 12.5 weeks, animals were killed and pancreas weighed and analyzed for insulin and glucagon. Pancreatic mass in R6/2 mice was the same as controls, and islets appeared normal in morphology without lymphocytic infiltration. Immunohistochemical staining showed dramatic reductions in glucagon in the alpha-cells and in insulin in the beta-cells. Direct tissue assays showed glucagon and insulin content were reduced to only 10 and 15% of controls, respectively. Diabetes has been reported as being more common in Huntington's disease and other triplet repeat disorders. The R6/2 mouse should prove useful for elucidating the mechanism of diabetes in these genetic diseases.

Insulin-like growth factor binding proteins in fetal rat mesencephalic cultures: regulation by fibroblast growth factor and insulin-like growth factor I.

In rat ventral mesencephalic cultures, IGF-I and bovine fibroblast growth factor (bFGF) act cooperatively to support the survival of dopaminergic neurons. To determine the potential role of IGFBPs in modulating the actions of IGF-I in the ventral mesencephalon, we identified the IGFBPs present in ventral mesencephalic cultures and examined their regulation by IGF-I and bFGF. In the absence of added growth factors, the major binding protein secreted from these cultures was IGFBP-2. Small amounts of IGFFBP-3 and IGFBP-4 were also detected. Addition of bFGF to the cultures increased the amounts of IGFBP-3 and IGFBP-4 released from the cells by 4.4 +/- 2.6 -fold (P < 0.1) and 11.5 +/- 3.5 -fold (P < 0.05), respectively. IGF-I, itself, had little effect on the production of IGFBPs, but when added together with bFGF increased the levels of IGFBP-3 and IGFBP-4 by 12.4 +/- 5.1 -fold (P < 0.05) and 27.4 +/- 5.3 -fold (P < 0.02), respectively. The stimulatory effect of bFGF and IGF-I on IGFBP production was apparent after a 2- to 3-day exposure of the mesencephalic cultures to the peptides. IGFBP-4, the most abundant IGFBP present in the cultures after 7 days of growth factor treatment, was immunocyto-chemically localized primarily to neurons, of which a subset were dopaminergic neurons. The addition of purified rat IGFBP-4 to the cultures in the absence of added growth factors had no effect on the survival of dopaminergic neurons, but when added with IGF-I potentiated the effect of IGF-I on neuronal survival. We propose that the up-regulation of IGFBP-4 by IGF-I and bFGF may serve to localize IGF-I to sites of action in the nervous system and thereby potentiate the neurotrophic actions of IGF-I.

Changes in brain alpha-adrenergic receptors after alpha-methyldopa administration to spontaneously hypertensive rats.

The hypotensive action of methyldopa has been linked to production of the metabolites methyldopamine and methylnorepinephrine in brain. We have studied the effect of long-term (72 hour) intravenous infusions of methyldopa to awake restrained spontaneously hypertensive rats and normotensive Wistar-Kyoto control animals to look for differences in hypotensive effect, differences in concentrations of natural and alpha-methylated catecholamines, and differences in alpha 1 and alpha 2-adrenergic receptor populations. Results described here indicate that hypertensive rats have a greater reduction in blood pressure and a larger increase in hypothalamic and brain stem methylnorepinephrine concentrations than do the normotensive animals. The methylnorepinephrine concentration reached a plateau value in hypothalamus in both strains while pons and medulla showed progressive, dose-related increases in concentration. These regional and strain differences in the metabolism of alpha-methyldopa suggest that the production of methylnorepinephrine in brain stem nuclei is most correlated with the hypotensive action of methyldopa. alpha 2 Agonist binding (p-amino-clonidine) declined in both hypothalamus and brain stem, and the fall was greater in hypertensive than in normotensive rats. alpha 1-Adrenergic receptor binding (prazosin) was increased, again more in hypertensive than in normotensive rats. The down regulation of alpha 2-adrenergic receptors and the up regulation of alpha 1-adrenergic receptors are compatible with increased alpha 2-adrenergic agonist presynaptic inhibition of catecholamine release with resultant postsynaptic alpha 1-adrenergic receptor supersensitivity. Spontaneously hypertensive rats showed greater methylnorepinephrine production, larger up regulation of alpha 1-adrenergic receptors, and greater down regulation of alpha 2-adrenergic receptors than did the normotensive animals; these changes may be physiological markers for the greater antisympathetic action of methyldopa in hypertensive animals.

Alprazolam as a neuroleptic adjunct in the emergency treatment of schizophrenia.

OBJECTIVE: While neuroleptics remain the mainstay of drug intervention in the emergency management of psychosis, a variety of agents have received study as alternatives or adjuncts to these drugs in an attempt to improve the safety and efficacy of acute treatment. The purposes of this study were to investigate the efficacy and safety of alprazolam as a neuroleptic adjunct for schizophrenic patients in psychotic relapse and to clarify the effects of combination treatment on specific aspects of the psychotic process. METHOD: Twenty-eight acutely psychotic patients with schizophrenia who were admitted to an emergency psychiatric service were randomly assigned to treatment with either haloperidol and alprazolam or haloperidol with placebo under double-blind conditions. Drug administration lasted 72 hours. RESULTS: Both groups improved significantly. The combination-treated group required significantly less medication and had 56% fewer dystonic reactions. The addition of alprazolam was most effective for symptoms of excitement and uncooperativeness, particularly in the initial hours of treatment. CONCLUSIONS: The combination of alprazolam and haloperidol seems to be the most effective for agitated patients, particularly in the first 48 hours of treatment. It may also result in fewer dystonic reactions.

Transplantation of human fetal dopamine cells for Parkinson's disease. Results at 1 year.

In an effort to improve the clinical signs of Parkinson's disease, we have implanted mesencephalic dopamine cells from a 7-week human embryo into the caudate and putamen of a 52-year-old man with Parkinson's disease. Fetal tissue was obtained from elective abortion. The woman and the patient with Parkinson's disease were unknown to each other. The woman gave specific consent and was not paid. The patient had a 20-year history of parkinsonism treated with multiple drug therapies including levodopa/carbidopa (Sinemet) every 2 1/2 hours. His symptoms were worse on the left side. For 5 months prior to transplantation, the patient underwent clinical evaluations by both a neurologist and a computer system installed in his home for daily measurement of walking and hand movements. Preoperative positron emission tomographic scanning with 6-L[18F]fluorodopa (fluorodopa) demonstrated severe dopamine depletion bilaterally. Fetal tissue was matched to the patient for ABO blood antigens, and maternal serum was screened for hepatitis B and human immunodeficiency virus type 1 prior to surgery. Fetal tissue was implanted stereotactically throughout the caudate and putamen on the right side of the brain via 10 needle tracks. The patient was not immunosuppressed. Results 12 months after surgery showed 42% improvement in left-hand speed before the first morning dose of drug and 40% greater response to drug therapy. Right-hand speed increased 15% before drug therapy and 23% after drug therapy. Reaction time was unaffected. Walking speed increased 33% after drug administration, although walking speed before the first morning dose of drugs declined 40%. Walking speed on an all-day basis improved 17%.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain contains inhibiting factors specific to the large T-antigen gene.

SV40 large T-antigen (LTa) gene-induced immortalized rat dopamine-producing nerve cells (IRB3AN27), which produce LTa protein and divide in vitro, do not divide and do not produce LTa protein when transplanted into striatum of adult rats. This suggests the presence of LTa gene-inhibiting factors in brain. Here we report that rat brain soluble fraction (SF) contains factors which specifically inhibit LTa gene activity in vitro. The brain SF inhibited LTa protein levels and the growth of IRB3AN27 cells and 2RSG cells (LTa gene-induced immortalized rat parotid acinar cells) in vitro, but it stimulated the growth of spontaneously immortalized human parotid acinar cells (2HPC8) and had no effect on the proliferation of murine neuroblastoma cells (NBP2) and rat glioma cells (C-6) in culture. In contrast, the liver SF inhibited the growth of all cell lines tested at varying degrees and thus lacked specificity with respect to LTa gene activity. The presence of specific LTa gene-inhibiting factors in the brain and general tumor growth-inhibiting factors in the liver may provide some of the mechanisms of protection against in vivo carcinogenesis.

Inhibition of proliferation and expression of T-antigen in SV40 large T-antigen gene-induced immortalized cells following transplantations.

Rat dopamine-producing nerve cells (1RB3AN27) and rat parotid acinar cells (2RSG) were immortalized by insertion of simian virus 40 (SV40) large T-antigen gene (LTa). Both of these cells divided and produced nuclear LTa in vitro. In order to assess the relationship between cell proliferation and expression of LTa in vivo, immortalized dopamine-producing nerve cells and parotid cells were grafted into the striatum and parotid gland of adult Sprague-Dawley rats, respectively. Grafted cells exhibited nuclear LTa at 1 day but not at 7 and 30 days after transplantation. At 30 days after transplantation, no tumor was found, and there was no evidence of cell division as determined by H and E staining. When the striatal areas containing the grafts were cultured, these cells did not express LTa at 4 days after plating; however, after 3 weeks, when most host cells were eliminated, the cultured grafted cells expressed LTa. After 3 months of culturing, only cells exhibiting LTa were present. These cells had the same morphology and divided with the same doubling time as 1RB3AN27 cells before grafting. Results suggest the presence of a LTa-inhibiting factor in vivo, and support the hypothesis that the expression of LTa is directly linked with proliferation of immortalized cells.

Production of terminally differentiated neuroblastoma cells in culture.

The use of fetal central nervous system (CNS) tissue in neural transplants has ethical, availability and some biological limitations. In order to overcome these problems, homogeneous populations of specific neurons in vitro should be established. Transformed nerve cells such as neuroblastoma cells (NBP2) in culture can serve as one of the sources of donor neurons in neural transplants provided they can be induced to differentiate terminally. This study showed that treatment of murine neuroblastoma (NBP2) cells with 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (R020-1724, an inhibitor of cyclic nucleotide phosphodiesterase), and ß-carotene for a period of 3 days followed by X-irradiation with 20 Gy or above produced 100% terminally differentiated cells. These differentiated NB cells had long and extensive neurites, contained elevated levels of tyrosine hydroxylase (TH) activity and very low levels of MHC class I and II antigens, and were non-tumorigenic. The viability of differentiated NB cells when determined on the criteria of attachment efficiency, re-extension of neurites and presence of TH after replating was only 9%. This was in contrast to the trypan blue exclusion technique which showed that over 90% of differentiated NB cells in culture were viable.

GDNF reduces apoptosis in dopaminergic neurons in vitro.

To explore the mechanism by which glial cell line-derived neurotrophic factor (GDNF) improves cell survival, we measured the apoptotic rate of dopamine neurons incubated with GDNF. Cultures were prepared from embryonic day 15 rat mesencephalon in medium with 5% human placental serum. GDNF reduced the rate of apoptosis in dopamine neurons from 5% to 2%. By contrast, GDNF had no effect on apoptoisis in astrocytes from embryonic mesencephalon or from neonatal cortex. Co-cultures with embryonic striatum as well as with combinations of growth factors were also tested for effects on dopamine neuron survival. Neuronal survival was maximal during co-culture with striatal cells with or without added growth factors. We conclude that GDNF inhibits apoptotic cell death in dopamine neurons.

Neural transplantation of hNT neurons for Huntington's disease.

Fetal striatal tissue transplants have been shown to restore motor deficits in rat and monkey models of Huntington's disease (HD). In the present study, using rats with unilateral striatal lesions, we compared fetal striatal tissue transplants to transplants of human NT (hNT) neurons. hNT neurons are terminally differentiated cells derived from the human NTera-2 cell line. In vitro, we have found that purified hNT neurons have a biochemical phenotype similar to that of human fetal striatal tissue. Both hNT neurons and fetal striatal tissue express mRNAs for glutamic acid decarboxylase, choline acetyltransferase, and the D1 and D2 dopamine receptors. Grafts of either hNT neurons or fetal striatal tissue into unilateral quinolinic acid-lesioned rat striatum improved methamphetamine-induced circling behavior. Sham controls showed no changes in methamphetamine-induced circling behavior. In the staircase test for skilled forelimb use, both transplant groups showed partial recovery in skilled use of the paw contralateral to the side of lesion, whereas the control animals showed continued deficits. These findings suggest that transplantation of hNT neurons may be an alternative to transplantation of fetal striatal tissue in the treatment of HD.

Immunological responses to injury and grafting in the central nervous system of nonhuman primates.

Allogeneic transplantation for the therapy of human Parkinson's disease is being considered as a viable approach at several clinical centers worldwide. As an attempt to understand the basic biology of central nervous system (CNS) transplantation, our laboratory has developed an experimental nonhuman primate model for human Parkinson's disease and carried out preliminary studies directed at evaluating the potential pathology at the graft site. In addition, studies have been conducted to examine whether such transplantation procedures lead to specific and/or nonspecific immunologic sensitization of the host or results in generalized immunosuppression. Groups of rhesus macaques (Macaca mulatta) were either controls operated (n = 6), autografted with adrenal medullary and peripheral nerve tissue (n = 3), or allografted with fetal mesencephalic tissue (n = 6). Immunohistological studies demonstrated the presence of mononuclear cell infiltrates as early as 1 wk and up to 1 yr postoperatively, although the frequency of the infiltrating cells declined with time. The infiltrates consisted of variable numbers of cells which express CD2+, CD3+, CD4+, CD8+, CD19+, CD22+, CD25+, and CD68+. There appeared to be no difference in the frequency, kinetics, or phenotype of the infiltrating cells in operative controls compared with recipients of auto- or allografts. Tissue sections obtained postoperatively showed low levels of major histocompatibility complex (MHC) Class I antigens and no detectable level of MHC-Class II antigens in neural tissue. A small aliquot of tissue from the operative site was placed in vitro with media containing interleukin-2 (IL-2), which led to the exudation and growth of mononuclear cells that were predominantly CD4+ cells. Phenotypic studies of peripheral blood mononuclear cells (PBMC) from operative controls, auto- and allograft recipient monkeys performed at varying time periods postoperatively failed to show differences in the frequencies of subsets of T-cells, B-cells, NK-cells, or monocytes. Studies on aliquots of the same PBMC failed to show major functional differences in NK-cells, LAK cells, or response to polyclonal mitogens. Finally, recipients of allogeneic mesencephalic grafts failed to show evidence of donor-specific humoral or cellular sensitization. These data indicate that transplantation of autograft adrenal or allograft fetal mesencephalic tissues in the CNS of nonhuman primate did not induce detectable donor-specific sensitization nor nonspecific immunosuppression.

Brain acetaminophen measurement by in vivo dialysis, in vivo electrochemistry and tissue assay: a study of the dialysis technique in the rat.

After peripheral injections of the electrochemically active drug acetaminophen, striatal levels of the drug were measured in 3 different ways: in vivo electrochemistry, in vivo dialysis, and tissue assay. The time course of the acetaminophen concentrations measured by in vivo dialysis paralleled the in vivo electrochemistry curve and lagged behind the peak tissue concentration. This result suggests that dialysis and electrochemistry provide equivalent measurements of extracellular fluid acetaminophen. In vitro, dialysis recovery of acetaminophen was 28% of the beaker concentration.

Wire electrodes for chronic single unit recording of dopamine cells in substantia nigra pars compacta of awake rats.

Stainless steel wire electrodes of varying sizes and coated with different insulating materials were tested in order to find a flexible fine wire which would allow the recording of small cells in deep structures of the brain in unrestrained, awake rats. Our main interest is to record from cells of substantia nigra pars compacta during locomotion. We found that stress relieved 18 micron stainless steel wire doubly insulated with lacquer and Parylene C provided the impedance, physical size, and flexibility needed to record single units during intense motor behavior.

Asymmetric dopamine and serotonin metabolism in nigrostriatal and limbic structures of the trained circling rat.

The trained circling rat model was used to investigate dopamine and serotonin metabolism in extrapyramidal and limbic structures during turning behavior. We have previously reported that dopamine turnover is increased during circling in the caudate contralateral to the circling direction in this behavioral model. We have now studied changes in dopamine and serotonin turnover in nucleus accumbens, substantia nigra and amygdala. As in the caudate, dopamine production in nucleus accumbens was selectively increased on the contralateral side after 20 min of circling. By contrast, dopamine turnover in substantia nigra exhibited a relative decline on the contralateral side. Dopamine synthesis in the amygdala was not affected by circling. Selective changes in serotonin metabolism were also seen in these brain regions. In caudate and accumbens, serotonin turnover was unaffected by circling. However, both substantia nigra and amygdala showed significant, progressive increases in serotonin metabolism in the contralateral side after 20 and 70 min of circling. These results show that extrapyramidal and limbic dopamine and serotonin metabolism are involved in turning behavior of normal animals. Multiple transmitters of the nigrostriatal pathway and the limbic system appear to interact to modulate voluntary circling behavior.

In vivo electrochemical detection of extraneuronal 5-hydroxyindole acetic acid and norepinephrine in the dorsal raphe nucleus of urethane-anesthetized rats.

In vivo electrochemical detection of endogenous neurotransmitters was done in the dorsal raphe nucleus of urethane-anesthetized male Sprague-Dawley rats. Stereotaxically implanted carbon paste electrodes were scanned using a cyclic voltammetry amplifier with semiderivative signal processing over the potential range--0.2 to +0.5 V at the rate of 10 mV/s. Two distinct peaks were observed at +0.15 V (peak 1) and +0.25 V (peak 2), respectively. Peak identification was assessed by comparing the oxidation potential observed in vivo with those observed in in vitro experiments using pure catecholamines, indoleamines and their metabolites as well as ascorbic acid. Further characterization of in vivo peaks was done by observing changes in electrochemical peaks as well as tissue neurotransmitter concentrations after pharmacological manipulations. p-Chlorophenylalanine, m-hydroxybenzylhydrazine (NSD-1015), pargyline, alpha-methyl-p-tyrosine and fusaric acid were administered in an effort to block catecholamine or serotonin synthesis or degradation. Results of these experiments revealed that peak 1 primarily represents extracellular norepinephrine, while peak 2 is primarily produced by extracellular 5-hydroxyindoleacetic acid (5-HIAA).

Overexpression of human alpha-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the appearance of intracytoplasmic inclusions called Lewy bodies (LB) in dopamine neurons in the substantia nigra and the progressive loss of these neurons. Recently, mutations in the alpha-synuclein gene have been identified in early-onset familial PD, and alpha-synuclein has been shown to be a major component of LB in all patients. Yet, the pathophysiological function of alpha-synuclein remains unknown. In this report, we have investigated the toxic effects of adenovirus-mediated alpha-synuclein overexpression on dopamine neurons in rat primary mesencephalic cultures and in a rat dopaminergic cell line - the large T-antigen immortalized, mesencephalon-derived 1RB3AN27 (N27). Adenovirus-transduced cultures showed high-level expression of alpha-synuclein within the cells. Overexpression of human mutant alpha-synuclein (Ala(53)Thr) selectively induced apoptotic programmed cell death of primary dopamine neurons as well as N27 cells. The mutant protein also potentiated the neurotoxicity of 6-hydroxydopamine (6-OHDA). By contrast, overexpression of wild-type human alpha-synuclein was not directly neurotoxic but did increase cell death after 6-OHDA. Overexpression of wild-type rat alpha-synuclein had no effect on dopamine cell survival or 6-OHDA neurotoxicity. These results indicate that overexpression of human mutant alpha-synuclein directly leads to dopamine neuron death, and overexpression of either human mutant or human wild-type alpha-synuclein renders dopamine neurons more vulnerable to neurotoxic insults.

Strands of embryonic mesencephalic tissue show greater dopamine neuron survival and better behavioral improvement than cell suspensions after transplantation in parkinsonian rats.

The success of embryonic neural transplants as a treatment for patients with Parkinson's disease has been limited by poor survival of transplanted dopamine neurons. To see if a new partially intact tissue preparation method improves survival, we have developed a technique for extruding embryonic tissue into strands. We expected this method to reduce cell damage and improve transplant survival as well as provide improved tissue delivery. We have compared transplants of tissue strands with mechanically dispersed suspensions of embryonic day 15 rat ventral mesencephalon. Tissue from ventral mesencephalon was transplanted into a single site in dopamine denervated striatum of unilateral 6-hydroxydopamine (6-OHDA) lesioned rats. To evaluate the effects of striatal cografts and growth factors on dopamine cell survival, dispersed mesencephalic cells were cotransplanted with dispersed striatal cells. Another group had dispersed mesencephalic cells cotransplanted with striatal cells incubated in the cold for 2 h with glial cell line-derived neurotrophic factor (GDNF, 100 ng/ml), insulin-like growth factor-I (IGF-I, 1500 ng/ml), and basic fibroblast growth factor (bFGF, 150 ng/ml). Behavioral improvement was assessed monthly by changes in methamphetamine-induced rotational behavior. Animals were sacrificed after 3 months, and dopamine neurons were identified by tyrosine hydroxylase (TH) immunohistochemistry. Transplants of tissue strands produced better dopamine neuron survival and led to more robust behavioral restoration than did cell suspensions even when suspensions were supported with cografts of striatal cells or pretreatment with growth factors.