Reduced locomotor responses to cocaine in ghrelin-deficient mice.

Ghrelin, an orexigenic hormone produced by the stomach, increases food intake and enhances the locomotor and rewarding effects of cocaine. Consistent with these behavioral effects, ghrelin increases dopamine cell activity in the mesolimbic system resulting in elevated levels of dopamine release and turnover in target regions such as the ventral striatum. In the current study, we examined the psychostimulant effects of acute and daily cocaine in mice with targeted deletion of the ghrelin gene (ghrelin knockout; KO) and that of their wild-type (WT) littermates. We hypothesized that ghrelin-KO mice would be hyporesponsive to the effects of cocaine as reflected in attenuated locomotor activity following both acute and chronic injections, and that this would be correlated with striatal dopamine and dopamine metabolite concentrations. Results show that the locomotor stimulating effect of cocaine (10 mg/kg) was decreased in ghrelin-KO mice as compared with their WT littermates. In addition, repeated daily injection of cocaine resulted in gradual increases in locomotor activity in WT mice, an effect that was attenuated in ghrelin-KO mice. These behavioral effects were correlated with changes in dopamine utilization in the striatum of WT mice that were not seen in ghrelin-KO mice unless these were pretreated with ghrelin. These data suggest that ghrelin is important for normal function of the mesolimbic dopaminergic system, potentially modulating both dopamine release and reuptake.

Embryonic substantia nigra grafts in the mesencephalon send neurites to the host striatum in non-human primate after overexpression of GDNF.

In spite of partial success in treating Parkinson's disease by using ectopically placed grafts of dopamine-producing cells, restoration of the original neuroanatomical circuits, if possible, might work better. Previous evidence of normal anatomic projections from ventral mesencephalic (VM) grafts placed in the substantia nigra (SN) has been limited to neonatal rodents and double grafting or bridging procedures. This study attempted to determine whether injection of a potent growth-promoting factor, glial cell line-derived neurotrophic factor (GDNF), into the target regions or placement of fetal striatal co-grafts in the nigrostriatal pathway might elicit neuritic outgrowth to the caudate nucleus. Four adult St. Kitts green monkeys received embryonic VM grafts into the rostral mesencephalon near the host SN, and injections of adeno-associated virus 2 (AAV2)/GDNF or equine infectious anemia virus (EIAV)/GDNF into the caudate. Three adult monkeys were co-grafted with fetal VM tissue near the SN and fetal striatal grafts (STR) 2.5 mm rostral in the nigrostriatal pathway. Before sacrifice, the striatal target regions were injected with the retrograde tracer Fluoro-Gold (FG). FG label was found in tyrosine hydroxylase-labeled neurons in VM grafts in the SN of only those monkeys that received AAV2/GDNF vector injections into the ipsilateral striatum. All monkeys showed FG labeling in the host SN when FG labeling was injected on the same side. These data show that grafted dopaminergic neurons can extend neurites to a distant target releasing an elevated concentration of GDNF, and suggest that grafted neurons can be placed into appropriate loci for potential tract reconstruction.

Embryonic substantia nigra grafts show directional outgrowth to cografted striatal grafts and potential for pathway reconstruction in nonhuman primate.

Transplantation of embryonic dopamine (DA) neurons has been tested as a therapy for Parkinson's disease. Most studies placed DA neurons into the striatum instead of the substantia nigra (SN). Reconstruction of this DA pathway could serve to establish a more favorable environment for control of DA release by grafted neurons. To test this we used cografts of striatum to stimulate growth of DA axons from embryonic SN that was implanted adjacent to the host SN in African green monkeys. Embryonic striatum was implanted at one of three progressive distances rostral to the SN. Immunohistochemical analysis revealed DA neuron survival and neuritic outgrowth from the SN grafts at 12-36 weeks after grafting. Each animal showed survival of substantial numbers of DA neurons. Most fibers that exited SN grafts coursed rostrally. Striatal grafts showed evidence of target-directed outgrowth and contained dense patterns of DA axons that could be traced from their origin in the SN grafts. A polarity existed for DA neurites that exited the grafts; that is, those seen caudal to the grafts did not appear to be organized into a directional outflow while those on the rostral side were arranged in linear profiles coursing toward the striatal grafts. Some TH fibers that reached the striatal grafts appeared to arise from the residual DA neurons of the SN. These findings suggest that grafted DA neurons can extend neurites toward a desired target over several millimeters through the brain stem and caudal diencephalon of the monkey brain, which favors the prospect of circuit reconstruction from grafted neurons placed into appropriate locations in their neural circuitry. Further study will assess the degree to which this approach can be used to restore motor balance in the nonhuman primate following neural transplantation.

AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons.

Neural transplantation offers the potential of treating Parkinson's disease by grafting fetal dopamine neurons to depleted regions of the brain. However, clinical studies of neural grafting in Parkinson's disease have produced only modest improvements. One of the main reasons for this is the low survival rate of transplanted neurons. The inadequate supply of critical neurotrophic factors in the adult brain is likely to be a major cause of early cell death and restricted outgrowth of fetal grafts placed into the mature striatum. Glial derived neurotrophic factor (GDNF) is a potent neurotrophic factor that is crucial to the survival, outgrowth and maintenance of dopamine neurons, and so is a candidate for protecting grafted fetal dopamine neurons in the adult brain. We found that implantation of adeno-associated virus type 2 encoding GDNF (AAV2-GDNF) in the normal monkey caudate nucleus induced overexpression of GDNF that persisted for at least 6 months after injection. In a 6-month within-animal controlled study, AAV2-GDNF enhanced the survival of fetal dopamine neurons by 4-fold, and increased the outgrowth of grafted fetal dopamine neurons by almost 3-fold in the caudate nucleus of MPTP-treated monkeys, compared with control grafts in the other caudate nucleus. Thus, the addition of GDNF gene therapy to neural transplantation may be a useful strategy to improve treatment for Parkinson's disease.

Prenatal cocaine exposure enhances responsivity of locus coeruleus norepinephrine neurons: role of autoreceptors.

Children exposed to cocaine during gestation have a higher incidence of neurobehavioral deficits. The neurochemical bases of these deficits have not been determined, but the pharmacology of cocaine and the nature of the abnormalities suggest that disruptions in catecholaminergic systems may be involved. In the current study, we used a rat model of prenatal cocaine exposure to examine the impact that this exposure has on the locus coeruleus (LC) noradrenergic system in offspring. Pregnant rats received twice-daily i.v. injections of cocaine (3 mg/kg) or saline between gestational days 10 and 20, and progeny were tested as juveniles. Exposure to a mild stressor elevated an index of norepinephrine turnover in the prefrontal cortex and also increased Fos expression in tyrosine hydroxylase-positive LC neurons in rats exposed to prenatal cocaine but not in rats exposed to prenatal saline. No change in the number of tyrosine hydroxylase-positive neurons in the LC was observed between the two prenatal treatment groups. Specific binding of [125I]-para-iodoclonidine, a radioligand with specificity for high affinity alpha2A-adrenergic receptors, was decreased in the LC of rats exposed to prenatal cocaine compared with prenatal saline controls. As alpha2-adrenergic receptors on LC norepinephrine neurons function as autoreceptors, their down-regulation by prenatal cocaine exposure provides a plausible mechanism for the observed heightened reactivity of norepinephrine neurons in these animals. These data indicate that prenatal cocaine exposure results in lasting changes to the regulation and responsivity of rat LC norepinephrine neurons. A similar dysregulation of LC norepinephrine neurons may occur in children exposed to cocaine during gestation, and this may explain, at least partly, the increased incidence of cognitive deficits that have been observed in these subjects.

Neural stem cells implanted into MPTP-treated monkeys increase the size of endogenous tyrosine hydroxylase-positive cells found in the striatum: a return to control measures.

Neural stem cells (NSC) have been shown to migrate towards damaged areas, produce trophic factors, and replace lost cells in ways that might be therapeutic for Parkinson's disease (PD). However, there is very little information on the effects of NSC on endogenous cell populations. In the current study, effects of implanted human NSC (hNSC) on endogenous tyrosine hydroxylase-positive cells (TH+ cells) after treatment with 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) were explored in nonhuman primates. After MPTP damage and in PD, the primate brain is characterized by decreased numbers of dopamine neurons in the substantia nigra (SN) and an increase in neurons expressing TH in the caudate nucleus. To determine how implanted NSC might affect these cell populations, 11 St. Kitts African green monkeys were treated with the selective dopaminergic neurotoxin, MPTP. Human NSC were implanted into the left and right caudate nucleus and the right SN of eight of the MPTP-treated monkeys. At either 4 or 7 months after NSC implants, the brains were removed and the size and number of TH+ cells in the target areas were assessed. The results were compared to data obtained from normal untreated control monkeys and to the three unimplanted MPTP-treated monkeys. The majority of hNSC were found bilaterally along the nigrostriatal pathway and in the substantia nigra, while relatively few were found in the caudate. In the presence of NSC, the number and size of caudate TH+ cells returned to non-MPTP-treated control levels. MPTP-induced and hNSC-induced changes in the putamen were less apparent. We conclude that after MPTP treatment in the primate, hNSC prevent the MPTP-induced upregulation of TH+ cells in the caudate and putamen, indicating that hNSC may be beneficial to maintaining a normal striatal environment.

Prenatal exposure to cocaine reduces the number and enhances reactivity of A10 dopaminergic neurons to environmental stress.

Prenatal exposure to cocaine has been shown to result in poor cognitive performance in the resulting offspring in humans and laboratory animals. The underlying biochemical changes that contribute to these behavioral effects are not known but have been proposed to involve changes in dopaminergic function. In these studies, we exposed rats to cocaine in utero using the clinically relevant intravenous model and report a mean loss of 24.8% of the tyrosine hydroxylase immunoreactive, presumed dopaminergic, neurons in the A10, but not A9 and A8, cell groups of the young adult offspring. Additionally, in prenatal cocaine-exposed rats dopaminergic neurons in the ventral, midline A10, and lateral A9 regions demonstrated a hyperreactivity to environmental stress, as measured by activation of the immediate-early gene, Fos. Mild, intermittent footshock did not further increase the number of dopamine neurons expressing Fos in prenatal cocaine-exposed rats, as it did in the prenatal saline controls. Because the exposure to cocaine took place during development, other potential changes in dopaminergic and nondopaminergic neuronal systems could result from the cocaine-induced reduction in numbers of A10 dopamine neurons. We hypothesize that a perinatal loss of A10 dopamine neurons, and subsequent developmental changes, contributes to a dysregulation of the adult mesoprefrontal system, resulting in the reported cognitive deficits.

Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration.

Previously, we observed that injection of an adenoviral (Ad) vector expressing glial cell line-derived neurotrophic factor (GDNF) into the striatum, but not the substantia nigra (SN), prior to a partial 6-OHDA lesion protects dopaminergic (DA) neuronal function and prevents the development of behavioral impairment in the aged rat. This suggests that striatal injection of AdGDNF maintains nigrostriatal function either by protecting DA terminals or by stimulating axonal sprouting to the denervated striatum. To distinguish between these possible mechanisms, the present study examines the effect of GDNF gene delivery on molecular markers of DA terminals and neuronal sprouting in the aged (20 month) rat brain. AdGDNF or a control vector coding for beta-galactosidase (AdLacZ) was injected unilaterally into either the striatum or the SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the side of vector injection. Two weeks postlesion, rats injected with AdGDNF into either the striatum or the SN exhibited a reduction in the area of striatal denervation and increased binding of the DA transporter ligand [(125)I]IPCIT in the lesioned striatum compared to control animals. Furthermore, injections of AdGDNF into the striatum, but not the SN, increased levels of tyrosine hydroxylase mRNA in lesioned DA neurons in the SN and prevented the development of amphetamine-induced rotational asymmetry. In contrast, the level of T1 alpha-tubulin mRNA, a marker of neuronal sprouting, was not increased in lesioned DA neurons in the SN following injection of AdGDNF either into the striatum or into the SN. These results suggest that GDNF gene delivery prior to a partial lesion ameliorates damage caused by 6-OHDA in aged rats by inhibiting the degeneration of DA terminals rather than by inducing sprouting of nigrostriatal axons.

Nicotine receptor inactivation decreases sensitivity to cocaine.

The reinforcing properties of nicotine and psychomotor stimulants are thought to be mediated through the mesolimbic dopamine (DA) system. This study investigates the role of high affinity nicotinic acetylcholine receptors (nAChRs) in cocaine place preference and examines some neurochemical changes in the mesolimbic DA system that might account for the interaction between nicotine and cocaine. 5 mg/kg is the lowest dose of cocaine able to condition a place preference in C57Bl/6 mice. Co-treatment with the nicotinic antagonist mecamylamine (1.0 mg/kg) disrupted place preference to 5 mg/kg cocaine. In addition, mice lacking the high affinity nAChR containing the beta2 subunit showed decreased place preference to 5 mg/kg cocaine, although higher doses of cocaine could condition a place preference in these knock out animals. In contrast, co-administration of a low dose of nicotine (0.2 mg/kg) potentiated place preference to a subthreshold dose of cocaine (3 mg/kg). DA turnover was monitored in several brain regions using tissue levels of DA and its primary metabolite DOPAC as an indication of DA release. Wild type mice showed decreased DA turnover following treatment with 5 mg/kg cocaine; whereas, this response was not seen in mice lacking the beta2 subunit of the nAChR. Induction of chronic fos-related antigens by cocaine was also reduced in mutant mice as compared to their wild type siblings, implying that downstream actions of cocaine were also affected by inactivation of the high affinity nAChR. These data indicate that activation of the high affinity nAChR may contribute to cocaine reinforcement.

Estrogen is essential for maintaining nigrostriatal dopamine neurons in primates: implications for Parkinson's disease and memory.

There are sexual differences in several parameters of the nigrostriatal dopamine neurons, as well as in the progression of diseases associated with this system, e.g., Parkinson's disease and dementia. These differences, as well as direct experimental data in rodents, suggest that gonadal hormones play a role in modulating this system. To determine whether circulating estrogen might have long-term effects by altering the number of dopamine neurons, the density of dopamine neurons was calculated in the compact zone of the substantia nigra of male and intact female short- (10 d) and longer-term (30 d) ovariectomized and short- and longer-term ovariectomized but estrogen-replaced nonhuman primates (African green monkeys). Furthermore, the number of tyrosine hydroxylase-expressing neurons, the total number of all types of neurons, and the volume of the compact zone of the substantia nigra were calculated in 30 d ovariectomized and in 30 d ovariectomized and estrogen-replaced monkeys. Unbiased stereological analyses demonstrated that a 30 d estrogen deprivation results in an apparently permanent loss of >30% of the total number of substantia nigra dopamine cells. Furthermore, the density calculations showed that brief estrogen replacement restores the density of tyrosine hydroxylase-immunoreactive cells after a 10 d, but not after a 30 d, ovariectomy. Moreover, the density of dopamine cells is higher in females than in males. These observations show the essential role of estrogen in maintaining the integrity of the nigral dopamine system, suggest a new treatment strategy for patients with Parkinson's disease and with certain forms of memory-impairing disorders, and provide another rationale for estrogen replacement therapy for postmenopausal women.

Divergent effects of putative anxiolytics on stress-induced fos expression in the mesoprefrontal system of the rat.

Previously, we reported that R(+)HA-966, a weak partial agonist for the glycine/NMDA receptor, and guanfacine, a noradrenergic alpha2 agonist, have anxiolytic-like actions on the biochemical activation of the mesoprefrontal dopamine neurons and fear-induced behaviors. Here, we examined these two putative anxiolytic agents, both with primary actions independent of GABAergic systems, for their ability to alter stress-induced Fos-like immunoreactivity in the mesoprefrontal cortex and in tyrosine hydroxylase-stained, presumed dopaminergic, neurons in the ventral tegmental area. The benzodiazepine agonist, lorazepam, and partial agonist, bretazenil, were also tested in this footshock paradigm [10 x 0.5 sec, 0.8 mA paired with a 5-sec tone]. In saline-treated rats, footshock resulted in an increase in Fos-li in the prelimbic and infralimbic cortices and tyrosine hydroxylase-labeled cells in the ventral tegmental area. Treatment with lorazepam or bretazenil prevented the stress-induced activation in Fos-li nuclei in all regions of the medial prefrontal cortex and in dopaminergic neurons in the ventral tegmental area. In contrast, the actions of the novel anxiolytic-like agents on stress-induced Fos-li were different than those observed with benzodiazepine agonists. Both putative anxiolytics, R(+)HA-966 and guanfacine, did not reduce, but significantly enhanced the stress-induced Fos-li in the prelimbic region of the medial prefrontal cortex. Additionally, treatment with R(+)HA-966 completely blocked, while guanfacine attenuated, the stress-induced increase in the number of Fos-li, TH-li cells in the ventral tegmental area. These results indicate that the putative anxiolytics, R(+)HA-966 and guanfacine, have actions on the stress-sensitive mesoprefrontal system which appear distinct from those of traditional anxiolytics.

Inflammatory responses and their impact on beta-galactosidase transgene expression following adenovirus vector delivery to the primate caudate nucleus.

An E1, E3 deleted adenovirus vector, serotype 5, carrying the marker gene LacZ was bilaterally microinfused into the caudate nuclei of 10 St Kitts green monkeys. The location and number of cells expressing transgene and host immunologic response were evaluated at 1 week (n = 2) and 1 month (n = 8) following vector infusion. A large number of cells expressed beta-galactosidase in some monkeys, exceeding 600000 in one monkey, but no expression was seen in three of 10. All monkeys had positive adenoviral antibody titers before vector infusion, indicating the possibility of previous exposure to some adenovirus, but only one showed a significant increase in titer afterwards. Inflammatory cell markers revealed an inverse correlation between transgene expression and the extent of inflammatory response. Dexamethasone administered immediately before and for 8 days following vector delivery, however, had no effect on transgene expression. The demonstration of significant inflammatory responses in the brain of some individual primates, including demyelination, indicates the need for new generations of adenovirus vectors, or the successful suppression of inflammatory responses, before this vector is suitable for non-cytotoxic clinical applications in the CNS.

An antisense oligonucleotide reverses the footshock-induced expression of fos in the rat medial prefrontal cortex and the subsequent expression of conditioned fear-induced immobility.

The immediate-early genes, including c-fos, have been proposed to be involved in learning and memory. In this report, we examine stress-induced Fos-like immunoreactivity (Fos-li) in subregions of the prefrontal cortex during a conditioned fear paradigm. During the acquisition phase, the rats were conditioned to fear a formerly neutral tone by pairing the tone with a mild footshock. The rats were then tested for fearful behavior by reexposure to the tone without additional footshock. During acquisition, Fos-li was increased in the medial prefrontal cortex (infralimbic and prelimbic) but not the anterior cingulate and M1 motor cortex. However, during the extinction phase, no significant increase in Fos-li was observed in any region. These findings indicate that acquisition, but not extinction, of conditioned fear is associated with an increase in Fos-li in subregions of the medial prefrontal cortex. In other animals, an antisense oligonucleotide directed against the c-fos mRNA was injected into the infralimbic/prelimbic cortex 12 or 72 hr before the acquisition session. Antisense treatment given 12, but not 72, hr earlier suppressed Fos production without altering behavior during the acquisition session. Three days after the acquisition session, rats were tested for fearful behavior as before. The antisense oligonucleotide blockade of Fos production during acquisition was associated with a significantly less fearful response during the extinction session. These results support a role for Fos in the medial prefrontal cortex during the acquisition of aversive learning.

In vivo expression of therapeutic human genes for dopamine production in the caudates of MPTP-treated monkeys using an AAV vector.

An adeno-associated virus (AAV) vector, expressing genes for human tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC), demonstrated significantly increased production of dopamine in 293 (human embryonic kidney) cells. This bicistronic vector was used to transduce striatal cells of six asymptomatic but dopamine-depleted monkeys which had been treated with the neurotoxin MPTP. Striatal cells were immunoreactive for the vector-encoded TH after stereotactic injection for periods up to 134 days, with biochemical effects consistent with dopamine biosynthetic enzyme expression. A subsequent experiment was carried out in six more severely depleted and parkinsonian monkeys. Several TH/aadc-treated monkeys showed elevated levels of dopamine near injection tracts after 2.5 months. Two monkeys that received a beta-galactosidase expressing vector showed no change in striatal dopamine. Behavioral changes could not be statistically related to the vector treatment groups. Toxicity was limited to transient fever in several animals and severe hyperactivity in one animal in the first days after injection with no associated histological evidence of inflammation. This study shows the successful transfection of primate neurons over a period up to 2.5 months with suggestive evidence of biochemical phenotypic effects and without significant toxicity. While supporting the idea of an in vivo gene therapy for Parkinson's disease, more consistent and longer lasting biochemical and behavioral effects will be necessary to establish the feasibility of this appraoch in a primate model of parkinsonism.

Effects of D2 dopamine receptor antagonists on Fos protein expression in the striatal complex and entorhinal cortex of the nonhuman primate.

Recent studies have reported that acute administration of dopamine D2 receptor antagonists increases expression of the immediate early gene c-fos in the rat striatal complex. There have been no corresponding studies of the effects of D2 antagonists in primate species. Since all clinically effective antipsychotic drugs share D2 receptor antagonism, it is important to define the extent to which these drugs may alter expression of c-fos or its protein product, Fos, in primates. We therefore examined the effects of administration of two D2 receptor antagonists, haloperidol and metoclopramide, on Fos expression in the striatum and temporal cortices of the vervet monkey. Metoclopramide does not appear to possess significant antipsychotic efficacy but potently produces extra-pyramidal side effects, while haloperidol is an effective antipsychotic drug that produces extrapyramidal side effects. Both drugs increased the number of Fos-like immunoreactive (Fos-li) neurons in the caudate nucleus and putamen; the numbers of Fos-li neurons in these regions were increased in both the patch and matrix compartments. Haloperidol but not metoclopramide increased the number of Fos-li neurons in the nucleus accumbens shell. Similarly, haloperidol but not metoclopramide increased the number of Fos-li neurons in the entorhinal cortex. Neither drug altered Fos expression in the inferior temporal cortex. These data suggest that the dorsolateral caudate nucleus and putamen may be sites at which D2 receptor antagonists elicit extrapyramidal side effects, and the nucleus accumbens shell and entorhinal cortex may be loci at which the therapeutic actions of antipsychotic drugs are manifested.

Restoration of dopamine transporter density in the striatum of fetal ventral mesencephalon-grafted, but not sham-grafted, MPTP-treated parkinsonian monkeys.

Transplantation of fetal dopamine neurons to the adult striatum potentially offers a means to reverse the striatal dopamine deficiency that characterizes Parkinson's disease. Many investigations in rodents have supported the hope that neural grafting may be a useful treatment for parkinsonism. However, clinical studies have generally produced more modest improvements in motor abnormalities than observed in lower species. It is possible that the number of fetal dopamine neurons that survive transplantation is insufficient to restore dopaminergic innervation of the large human striatum to a level where striking recovery is obtained. In fact, there has been no quantitative study of graft outgrowth to indicate what portion of the dopamine-depleted striatum might be reinnervated with present techniques. Furthermore, it has been speculated that regeneration of the host dopamine system in response to the implantation surgery may play an important role in the beneficial effects of neural grafting in primates. The present study used nine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian monkeys to investigate these issues. Sham implantation procedures produced no increase in either dopamine transporter density (measured by quantitative autoradiography) or tissue dopamine concentration (measured by HPLC) in the striatum of MPTP-treated monkeys. In sham-grafted and nonimplanted MPTP-treated monkeys, the striatal dopamine concentration was reduced by 99%, based on analysis of 16 sampled sites in the caudate nucleus and putamen of each monkey. No behavioral recovery was seen in the sham-grafted and nonimplanted MPTP-treated groups. In contrast, transplantation of fetal dopamine neurons to the caudate nucleus or putamen of MPTP-treated monkeys resulted in a significant elevation of dopamine transporter density and dopamine levels in the grafted striatal nucleus. Each grafted MPTP-treated monkey received ventral mesencephalon dopamine neurons from one donor harvested during putative neurogenesis. Donor ventral mesencephalon was divided equally and implanted into six sites either in the caudate nucleus or putamen. One graft site in each monkey was examined by dopamine transporter autoradiography. In sections in which graft fibers were present, a mean of one-third of the volume of the grafted nucleus was occupied by an elevated density of dopamine transporters. This increase in dopamine transporter density was defined to be at least 5-10% of the control density. However, full behavioral recovery was not observed in the grafted MPTP-treated group. These data provide no support for the hypothesis that regeneration of the host dopamine system occurs in response to a sham implantation procedure in severely parkinsonian monkeys. The current study illustrates the power of the applied techniques for delineating the relationship between the level of host dopamine depletion, the extent of graft-induced dopaminergic restoration, and behavioral recovery.

Sham surgery does not ameliorate MPTP-induced behavioral deficits in monkeys.

Parkinsonism has been reported to improve following transplantation of fetal mesencephalic tissue into the striatum of MPTP-exposed monkeys and humans and in patients with idiopathic Parkinson's disease. While there is good evidence for the survival of grafted tyrosine hydroxylase (TH)-positive cells in animal studies, it is not known whether they produce neuronal effects that account for behavioral improvement after transplantation or whether spontaneous or graft-induced changes in the host striatum are at least partly responsible. Are neuronal synaptic connections and dopamine release necessary, or would "toenails and talcum powder" do the job equally well? We have addressed these questions by studying several types of implantation surgeries, including sham surgery, the implantation of cerebellar tissue, and the implantation of mesencephalic TH-positive fetal tissue of various gestational ages into the striatum. Adult male African green monkeys received systemic MPTP administration (cumulative doses of 2.0-2.5 mg/kg) prior to these stereotaxic surgical manipulations. Subjects were matched for quantitative behavioral deficits prior to surgery. Subjects were examined and assessments made by "blinded" observers who scored individual spontaneous and elicited behaviors. Observers were trained and tested repeatedly for inter-rater reliability. A "parkinsonian summary score" derived and determined using a principal component factor analysis of a large sample of data from MPTP-treated and normal monkeys of the same species was used to assess behavior. Postmortem brain tissue was prepared for biochemical analysis of dopamine concentrations and TH immunohistochemical studies. The most dramatic improvement was seen in monkeys with "early" (< 4 cm fetal crown rump length) surviving substantia nigra grafts in the caudate nucleus. Some behavioral improvements were seen in MPTP-treated sham-operated monkeys, cerebellar-grafted monkeys, and "later" (> 14 cm fetal crown rump length) substantia nigra-grafted monkeys. These changes in monkeys which did not have surviving dopamine-producing grafts probably represent the recovery capacity of MPTP-treated host brain during this time interval since un-operated subjects showed similar changes. More variable effects were seen with substantia nigra grafts in the putamen. The most consistent correlate of behavioral improvement in all experimental groups was elevation in dopamine concentrations near the grafts compared with a distant striatal location which is believed to represent the depletion without the effects of the grafts. While these data do not establish the precise mechanism of action, they point to a hierarchy of factors which provide increasingly larger restorative effects, including sprouting of host neurons and increased dopamine production by grafted fetal dopamine neurons. Sham surgery appears to be significantly less effective than early fetal mesencephalic tissue which survives and releases dopamine.

Noradrenergic activity in anticipatory nausea.

Two studies were conducted to examine the hypothesis that noradrenergic activity is a cause of the anticipatory nausea associated with cancer chemotherapy. In the first study concentrations of plasma 3-methoxy-4-hydroxyphenyl-glycol (MHPG) on day 1 of cycle 5 of initial chemotherapy were significantly higher in patients with than without anticipatory nausea. To determine whether elevated MHPG reflected a clinically significant causative role for noradrenergic activity in anticipatory nausea, we conducted a randomized, double-blind, placebo-controlled, crossover trial of clonidine for anticipatory nausea. At a dose of clonidine that produced significant side effects and reductions of plasma MHPG, anticipatory nausea was improved only marginally. These studies do not support a causative role for noradrenergic activity in anticipatory nausea that can be reduced by clonidine with an acceptable therapeutic index.

Grafting of fetal substantia nigra to striatum reverses behavioral deficits induced by MPTP in primates: a comparison with other types of grafts as controls.

Fetal substantia nigra (SN) cells were transplanted into the caudate nucleus (CN) of four vervet monkeys (Cercopithecus aethiops sabaeus) that had been treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP treatment appears to produce a syndrome similar to that observed in patients with idiopathic Parkinson's disease. Normal and parkinsonian behaviors were quantitated by trained observers 5 days/week. Twenty-eight behaviors based on previous factor analyses were individually scored and rated. Parkinsonian signs included freezing, head and limb tremor, difficulty in eating, delayed initiation of movement, poverty of movement, tremor that stopped with intention, decreased response to threats, and lying immobile in the cage. These signs were combined to give an overall rating of parkinsonism. A summary measure of 'normal' healthy behavior was also examined, including such behaviors as yawning, scratching, self-grooming, shifting, and eating. Overall ratings of parkinsonism increased and those of healthy behavior decreased after MPTP. In the 4 monkeys grafted with fetal SN cells into the CN, behavior returned to pre-treatment levels by the time of sacrifice (2, 5, or 7.5 months after grafting). Three control subjects were transplanted with either SN cells into an inappropriate brain site (cortex) or inappropriate, non-dopaminergic, cells (cerebellar) into the CN. Subjects were also compared with three control animals that did not receive MPTP but received cryopreserved or fresh SN and other cells into the CN. Only MPTP-treated subjects that received SN cells into the CN showed evidence of a reversal of the MPTP syndrome after transplantation. In addition, grafting in animals that were not MPTP-treated did not appear to affect behavior. This paper reports the specific behavioral effects of severe MPTP toxicity that were or were not reversed after transplantation and suggests that only fetal SN cells grafted into the CN may be able to reverse behavioral deficits in MPTP-treated monkeys.

Improvements in MPTP-induced object retrieval deficits and behavioral deficits after fetal nigral grafting in monkeys.

Improvements in MPTP-induced deficits were only found in subjects that received fetal substantia nigra transplants into the caudate nucleus. The MPTP-induced deficits were assessed using an object retrieval task that examined cognitive and subtle motor performance and by behavioral observation to determine the overall status of the subjects. Subjects that were also moderately or severely impaired by MPTP administration but that received inappropriate donor cells or implant sites (cerebellum to CN or SN to cortex) did not show any evidence of behavioral recovery. These subjects could not respond on the task in the months after grafting and were sacrificed, showing no improvements in parkinsonian signs or healthy behavior signs, up to 5-6 months after surgery. Grafting of SN cells into the striatum of non-MPTP lesioned subjects failed to modify normal behavior or induce abnormal behavior determined by our 2 behavioral assessment methods. In those monkeys that received the appropriate transplants, TH immunohistochemistry revealed that cells of the fetal substantia nigra grafted into the caudate nucleus survived and extended neurites into the host striatum. Indeed, grafted dopamine neurons were often associated with appreciable innervation of the caudate nucleus and appeared to be well incorporated into the host brain. In contrast, examination of the striatum of subjects in the inappropriate-graft group (e.g., cerebellar cells grafted into the caudate) showed no evidence of TH staining within the graft or host caudate nucleus. This indicated that there was no evidence of dopamine neurons present in the grafted tissue and that the mere presence of a fetal graft did not appear to induce sprouting in these MPTP-treated subjects. Although behavioral recovery occurred in only those monkeys that received appropriate transplants (fetal SN to host CN) and not in those that received inappropriate grafts (fetal cerebellum to CN or fetal SN to cortex), the CSF HVA levels did not distinguish those monkeys with improved parkinsonism from those that remained severely parkinsonian. The finding that in some SN-CN grafted subjects reported here, there was evidence of increased dopamine and lowered HVA/dopamine ratio in the vicinity of the SN grafts (cf. Elsworth et al., 1990b) is consistent with the hypothesis that graft-derived or graft-induced dopamine production is responsible for behavioral recovery. In addition, the finding that CSF HVA levels in non-MPTP lesioned subjects were unchanged by fetal SN grafts further indicates that CSF HVA levels may not be sufficiently sensitive to changes in central dopamine production to reflect release of dopamine from relatively small grafts that may, in lesioned subjects, modify behavior.(ABSTRACT TRUNCATED AT 400 WORDS)