Co-grafts of muscle cells and mesencephalic tissue into hemiparkinsonian rats: behavioral and histochemical effects.

Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro. To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson's disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum. In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM). Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling. These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.

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.

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.

Intrastriatal grafting of Cos cells stably expressing human aromatic L-amino acid decarboxylase: neurochemical effects.

To study the possibility that increasing striatal activity of aromatic L-amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to L-3,4-dihydroxy-phenylalanine (L-DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos-haadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of 14CO2 from L-[14C]DOPA. The Km value for L-DOPA in intact and disrupted cells was 0.60 and 0.56 mM, respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 mM. The pH optimum for enzyme activity was 6.8. Grafting Cos-haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of L-DOPA. When measured 2 h after L-DOPA administration, the mean dopamine level in the striata of Cos-haadc-grafted animals was 2 micrograms/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 microgram/g. At 6-8 h after L-DOPA administration, striatal dopamine content in the Cos-haadc-grafted animals was 0.67 microgram/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered L-DOPA.

Conditioned medium from aged monkey fibroblasts stably expressing GDNF and BDNF improves survival of embryonic dopamine neurons in vitro.

Fibroblasts derived from the cerebral cortex of an aged Bonnet monkey (Macaca radiata) were utilized to express recombinant cDNAs encoding rat glial-cell-line-derived neurotrophic factor (GDNF) and human prepro brain-derived neurotrophic factor (BDNF) by lipofection. The cells showed stable expression and secretion of biologically active proteins. Conditioned medium from fibroblasts expressing BDNF or GDNF increased the number of surviving mesencephalic tyrosine-hydroxylase-immunoreactive neurons after 7 days in culture. The trophic effects of BDNF and GDNF were examined at two different plating densities of embryonic mesencephalic cells. At 50 000 cells/cm2 plating density, treatment of the mesencephalic cultures with BDNF-conditioned medium increased the number of tyrosine-hydroxylase-immunoreactive neurons by about 40% compared with vector-transfected control. At the same plating density, GDNF-conditioned medium increased the number of surviving tyrosine-hydroxylase-immunoreactive neurons above the vector-transfected control by 30%. When the tissue was plated at a higher density, viz., 75 000 cells/cm2, the number of tyrosine-hydroxylase-immunoreactive neurons increased by 41% with BDNF-conditioned medium, and by 56% with GDNF-conditioned medium above vector-transfected controls. Conditioned medium from cells secreting GDNF was also found to reduce the number of apoptotic tyrosine-hydroxylase-immunoreactive cells by 50%.

DNQX in the nucleus accumbens inhibits cocaine-induced conditioned place preference.

Several lines of evidence suggest that activation of both AMPA/kainate receptors and dopaminergic receptors in the nucleus accumbens may be required for psychostimulant drug induced reward. However, it has been reported that dopaminergic antagonists fail to block acquisition of conditioned place preference to cocaine. The goal of these experiments was to determine whether AMPA receptor antagonist injected into the nucleus accumbens could block conditioned place preference elicited by cocaine under conditions where dopaminergic antagonists do not inhibit acquisition of place preference. DNQX (1 microgram/0.5 microliter), injected into the nucleus accumbens just before systemic injections of cocaine (20 mg/kg i.p.) during the training sessions, attenuated the acquisition of place preference. This suggests that AMPA receptors are involved in acquisition of place preference to cocaine. By contrast, fluphenazine (2.5 micrograms/0.5 microliter), injected into the nucleus accumbens during training, did not alter cocaine-induced place preference. Analysis of locomotor activity showed that the ability of flyphenazine to inhibit cocaine-induced hyperactivity progressively decreased with each training session. These observations suggest that the failure of dopaminergic antagonists to block cocaine-induced place preference may be related to adaptations occurring following repeated exposure to these drugs. Both DNQX and fluphenazine blocked the expression of conditioned place preference in rats that had been previously trained with cocaine alone. This result suggests that both AMPA and dopaminergic receptors are involved in the expression of a conditioned place preference to cocaine.

AMPA/kainate antagonists in the nucleus accumbens inhibit locomotor stimulatory response to cocaine and dopamine agonists.

The purpose of this study was to determine whether AMPA/kainate excitatory amino acid receptors in the nucleus accumbens (NAc) play a role in the locomotor stimulation produced by cocaine and dopamine receptor agonists. The stimulation of locomotor activity produced by the systemic administration of cocaine was markedly attenuated by either the D1 receptor antagonist SCH23390 or the D2 receptor antagonist eticlopride administered directly into the NAc. This indicates that both dopaminergic receptor subtypes in the NAc are involved in the motor stimulant response to cocaine. The intra-accumbens administration of DNOX or GAMS, which have been shown to inhibit the locomotor stimulation produced by the excitatory amino acid agonist AMPA, antagonized the locomotor stimulant response to cocaine administered either systemically or directly into the NAc. DNOX and GAMS also inhibited the stimulation of locomotor activity produced by the coinjection of the D1 agonist SKF38393 and the D2 agonist quinpirole injected into the NAc of normal animals and of animals pretreated with reserpine. These results suggest that the activation of AMPA/kainate receptors in the NAc plays an important role in the locomotor stimulation produced by cocaine and directly acting dopaminergic receptor agonists. The effects produced by the activation of these receptors is independent of endogenous dopamine stores, suggesting that these receptors are located postsynaptic to the dopaminergic nerve terminals.

The NMDA receptor antagonist MK-801 elicits conditioned place preference in rats.

(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate, (MK-801) a potent noncompetitive antagonist of central NMDA receptors, has been hypothesized to have rewarding properties indicative of abuse potential. To test this hypothesis, the effects of MK-801 on the acquisition of a conditioned place preference and on locomotor activity were assessed and compared with d-amphetamine. Both MK-801 (0.03 and 0.1 mg/kg, SC) and d-amphetamine (1.0 mg/kg, SC) administration resulted in the acquisition of a conditioned place preference. However, while both amphetamine and the higher dose of MK-801 produced a behavioral activation during the training period the lower dose of MK-801 did not. These results suggest that MK-801, at doses that produce behavioral activation and below, is rewarding and therefore may have abuse potential.

Pineal physiology in microgravity: relation to rat gonadal function aboard Cosmos 1887.

For the first time pineal glands obtained from 5 male rats flown aboard an orbiting satellite (Soviet Biosatellite #1887) were analyzed for their melatonin, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), and calcium content. In addition, plasma 5-HT and 5-HIAA were measured. These parameters were compared to indicators of gonadal function: plasma testosterone concentration and spermatogonia development. Plasma melatonin was low at the time of euthanasia (lights on) and was not different among the experimental groups (flight animals, synchronous controls, and vivarium controls). Pineal calcium of flight animals was not different from ground controls. However, pineal 5-HT and 5-HIAA in the flight group were significantly higher than those in ground controls (p less than 0.05). These findings suggest a possible increase in pineal 5-HT turnover in flight animals which may result in increased melatonin secretion. Since melatonin is known to possess antigonadal properties, the alteration of pineal 5-HT turnover and its expected effects on melatonin secretion may, in part, explain the lower plasma testosterone levels (p less than 0.001) and 4-11% fewer spermatogonia cells (p less than 0.02) observed in flight animals.