Kiyoshi Ando

Transplantation of human neural stem cells for spinal cord injury in primates

Recent studies have shown that delayed transplantation of neural stem/progenitor cells (NSPCs) into the injured spinal cord can promote functional recovery in adult rats. Preclinical studies using nonhuman primates, however, are necessary before NSPCs can be used in clinical trials to treat human patients with spinal cord injury (SCI). Cervical contusion SCIs were induced in 10 adult common marmosets using a stereotaxic device. Nine days after injury, in vitro‐expanded human NSPCs were transplanted into the spinal cord of five randomly selected animals, and the other sham‐operated control animals received culture medium alone. Motor functions were evaluated through measurements of bar grip power and spontaneous motor activity, and temporal changes in the intramedullary signals were monitored by magnetic resonance imaging. Eight weeks after transplantation, all animals were sacrificed. Histologic analysis revealed that the grafted human NSPCs survived and differentiated into neurons, astrocytes, and oligodendrocytes, and that the cavities were smaller than those in sham‐operated control animals. The bar grip power and the spontaneous motor activity of the transplanted animals were significantly higher than those of sham‐operated control animals. These findings show that NSPC transplantation was effective for SCI in primates and suggest that human NSPC transplantation could be a feasible treatment for human SCI.

Isolation and transplantation of dopaminergic neurons generated from mouse embryonic stem cells

Embryonic stem (ES) cells differentiate into dopamine (DA)-producing neurons when co-cultured with PA6 stromal cells, but the resulting cultures contain a variety of unidentified cells. In order to label live DA neurons in mixed populations, we introduced a GFP reporter under the control of the tyrosine hydroxylase (TH) gene promoter into ES cells. GFP expression was observed in TH-immunoreactive cells that differentiated from the ES cells that carried the TH-GFP reporter gene. DA neurons expressing GFP were sorted from the mixed cell population by fluorescence-activated cell sorting of cells exhibiting GFP fluorescence, and the sorted GFP(+) cells obtained were transplanted into a rat model of Parkinsons disease. Some of these cells survived and innervated the host striatum, resulting in a partial recovery from parkinsonian behavioral defects. This strategy of isolation and transplantation of ES-cell-derived DA neurons should be useful for cellular and molecular studies of DA neurons and for clinical application in the treatment of Parkinsons disease.

Correlation between quantitative imaging and behavior in unilaterally 6-OHDA-lesioned rats

We evaluated correlation between neurochemical and functional alterations of the nigrostriatal dopaminergic system in rat brains lesioned with 6-hydroxydopamine (6-OHDA), that model hemi-Parkinsons disease (PD), by using three different quantitative in vivo and in vitro methods. Rats unilaterally lesioned with different doses of 6-OHDA underwent two types of in vivo experiments: (1) a rotational behavioral study with methamphetamine (MAP) or apomorphine (APO); and (2) a positron emission tomography (PET) study with [11C]PE2I (radioligand for dopamine transporters) or [11C]raclopride (radioligand for dopamine D2 receptors). An in vitro autoradiographic study with the same radioligands was also conducted. The number of rotations after the MAP or APO injection increased with increased doses of 6-OHDA. The in vitro and in vivo binding of [11C]PE2I dose-dependently decreased in response to the 6-OHDA injections, while that of [11C]raclopride dose-dependently increased. There was a significant negative hyperbolic correlation between the number of rotations after MAP injection and the binding of [11C]PE2I. In contrast, there was a significant positive linear correlation between the number of rotations after APO injections and the binding of [11C]raclopride. These results robustly reveal a molecular pharmacological basis of parkinsonian symptoms in animal models of PD, and indicate the utility and validity of in vivo PET measurements in assessing pre- and post-synaptic dopaminergic functions.

Profile of drug effects on temporally spaced responding in rats.

A differential reinforcement of low rate schedule was used with rats to test 15 psychotropic drugs. The computer analysis was based on interresponse time (IRT). Mean IRT, IRT standard deviation, median IRT, IRT midrange, modal IRT, frequency of modal IRT, and an efficiency index, in addition to numbers of responses and reinforcements and the IRT histogram were obtained for each rat in each drug test. An increase in number of responses and a peak shift to shorter IRTs in the histograms were observed with amphetamine, methamphetamine, priradrol and nicotine, as reported by many other investigators. Decrease in IRT midrange and less change in number of responses were observed with diazepam and chlordiazepoxide. Long pauses were found with LSD-25, 2,5-dimethoxy-4-methylamphetamine (DOM) and mescaline. In a factor analysis, the following main factors were obtained. High values in factor loading a1 were observed with chlorpromazine, chlordiazepoxide, pentobarbital, imipramine, nialamide, LSD-25, DOM and mescaline. With these drugs, mean IRT and IRT standard deviation were also high. Values for a2, were high with amphetamine, methamphetamine, pipradrol and nicotine. High a3 values were observed in some rats with chlorpromazine, diazepam, chlordiazepoxide, pentobarbital, pipradrol and caffeine. The changes in a3 values were correlated with changes in the IRT midrange. These results may be valuable in classifying new compounds in drug screening programs as being of the amphetamine type, nicotine type, diazepam type of LSD-25 type.

Cigarette smoking in rhesus monkeys

In the present pilot study, an attempt was made to shape and maintain cigarette smoking behavior in rhesus monkeys both with and without the simultaneous use of other reinforcers. Initially, 14 monkeys were trained to suck air and puff on cigarettes using sweetened liquid reinforcer. After smoking had been established, the sweetened liquid reinforcement was removed. Smoking without this reinforcement, referred to as ‘voluntary smoking,’ was then observed during 20-h daily sessions. Of 14 monkeys studied, 2 have engaged in voluntary smoking for 2 years or longer. The maximum figures recorded for any single 20-h session were 3,271 puffs (20 cigarettes) in one monkey and 16,384 puffs (47 cigarettes) in the other. Although the baseline variability of smoking by these monkeys was quite high, low-nicotine and nicotine-free cigarettes seemed to lead to clear decreases in smoking. In 2 other monkeys that did not perform voluntary smoking, smoking was reestablished under a randomtime or a tandem schedule for sweetened liquid reinforcement. Within this situation (‘schedule-controlled smoking’) schedule manipulations also led to changes in intake of cigarrette smoke. The voluntary smoking model described in the present paper should be useful for studying the factors involved in initiating and maintaining smoking behavior and for studying the psychopharmacological effects of smoking, while the schedule-controlled smoking model should be useful for studying the physiological effects of smoking and for studying the relationship of smoking with various disease entities.

Parkinson Disease: Diffusion MR Imaging to Detect Nigrostriatal Pathway Loss in a Marmoset Model Treated with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

PURPOSE To investigate the use of diffusion-tensor imaging (DTI) to detect denervation of the nigrostriatal pathway in a nonhuman primate model of Parkinson disease (PD) after treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MATERIALS AND METHODS This study was approved by the institutional committee for animal experiments. DTI was performed in marmosets (n = 6) by using a 7-T magnetic resonance (MR) imager before and 10 weeks after administration of MPTP. Fixed brains of a normal marmoset and a marmoset model of PD (n = 1) were analyzed by using microscopic tractography. Tyrosine-hydroxylase staining of dopaminergic neurons and three-dimensional histologic analysis also were performed in normal marmosets (n = 2) and a PD marmoset model (n = 2) to validate the course of the nigrostriatal pathway revealed at tractography. Statistical analysis of voxel-based and post hoc region-of-interest analyses of DTI maps was performed by using a paired t test. RESULTS At voxel-based analysis of DTI before and after treatment, MPTP-treated marmoset brains showed significantly increased axial and radial diffusivity in the bilateral nigrostriatal pathway (P < .05, false discovery rate corrected). The largest area of significantly increased diffusivity was an area of axial diffusivity in the right hemispere (177 mm(3)) that corresponded to the location of dopaminergic neurodegeneration at histologic evaluation. Region-of-interest analysis revealed a 27% increase in axial diffusivity in the right hemisphere (1.198 mm(2)/sec ± 0.111 to 1.522 mm(2)/sec ± 0.118; P = .002). Three-dimensional histologic analysis with tyrosine-hydroxylase staining showed the course of the nigrostriatal pathway and degeneration in the PD marmoset model as the absence of a tyrosine-hydroxylase stained region. Microscopic tractography showed that the connection of the substantia nigra to the striatum followed the same course as the nigrostriatal pathway and fewer fiber tracts in the PD marmoset model. CONCLUSION DTI and microscopic tractography showed the loss of fiber structures of the nigrostriatal pathway in the marmoset model of PD. The results of this study provide a potential basis for the use of DTI in the clinical diagnosis of PD.

Role of Ventral Striatal Dopamine D1 Receptor in Cigarette Craving

BACKGROUND Several theories of cigarette craving suggest that dopaminergic function in the ventral striatum plays an important role. The objective of this study was to determine correlations between craving-related brain activation and dopamine D1 receptor (D1R) binding in smokers. METHODS Twelve smokers and 12 nonsmoking controls underwent [(15)O]H(2)O-positron emission tomography activation study and D1R-binding study using [(11)C]SCH 23390, and the correlations between receptor binding and cue-induced regional cerebral blood flow (rCBF) changes were assessed. Consecutive D1R-binding changes were examined during a period of 6 months of postsmoking abstinence in five smokers. RESULTS Cue-induced activation was observed in the left ventral striatum including the nucleus accumbens in smokers. D1R binding in the ventral striatum showed a negative relationship with cue-induced craving and rCBF changes. D1R binding was significantly low in smokers, and there was a trend of increase after smoking abstinence. CONCLUSIONS D1R binding and cue-induced rCBF changes in the ventral striatum suggest the important role of D1R in this region in cigarette craving.

PET Analysis of Dopaminergic Neurodegeneration in Relation to Immobility in the MPTP-Treated Common Marmoset, a Model for Parkinson's Disease

Background Positron Emission Tomography (PET) measurement was applied to the brain of the common marmoset, a small primate species, treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The marmoset shows prominent Parkinson’s disease (PD) signs due to dopaminergic neural degeneration. Recently, the transgenic marmoset (TG) carrying human PD genes is developing. For phenotypic evaluations of TG, non-invasive PET measurement is considered to be substantially significant. As a reference control for TG, the brain of the MPTP-marmoset as an established and valid model was scanned by PET. Behavioral analysis was also performed by recording locomotion of the MPTP-marmoset, as an objective measure of PD signs. Methodology/Principal Findings Marmosets received several MPTP regimens (single MPTP regimen: 2 mg/kg, s.c., per day for 3 consecutive days) were used for PET measurement and behavioral observation. To measure immobility as a central PD sign, locomotion of marmosets in their individual living cages were recorded daily by infrared sensors. Daily locomotion counts decreased drastically after MPTP regimens and remained diminished for several months or more. PET scan of the brain, using [11C]PE2I as a ligand of the dopamine (DA) transporter, was performed once several months after the last MPTP regimen. The mean binding potential (BPND) in the striatum (putamen and caudate) of the MPTP-marmoset group was significantly lower than that of the MPTP-free control group (n = 5 for each group). In the MPTP-marmosets, the decrease of BPND in the striatum closely correlated with the decrease in locomotion counts (r = 0.98 in putamen and 0.91 in caudate). Conclusion/Significance The present characterization of neural degeneration using non-invasive PET imaging and of behavioral manifestation in the MPTP marmoset mimics typical PD characteristics and can be useful in evaluating the phenotype of TG marmosets being developed.

Brain regions mediating the discriminative stimulus effects of nicotine in rats.

Abstract: The involvement of cerebral regions in the discriminative stimulus (DS) effects of nicotine was studied using rats. Substitution tests with nicotine administered into the medial prefrontal cortex, nucleus accumbens, and ventral tegmental area, all of which are located on the mesolimbocortical dopaminergic neurons, and into the dorsal hippocampus and medial habenular nucleus, which possess high densities of nicotinic cholinergic receptors, were conducted in rats trained to discriminate nicotine (0.5 mg/kg sc) from saline solution in a two‐lever, food‐reinforced, operant task. Nicotine administered into the medial prefrontal cortex substituted for nicotine (0.5 mg/kg sc), whereas nicotine administered into the nucleus accumbens and ventral tegmental area partially substituted for sc injected nicotine. However, nicotine administered into the dorsal hippocampus and medial habenular nucleus did not substitute for sc injected nicotine. These results suggest that the medial prefrontal cortex is primarily involved in the DS effects of nicotine, whereas the nucleus accumbens and ventral tegmental area are partially involved.

Medial prefrontal cortex is involved in the discriminative stimulus effects of nicotine in rats.

Abstract  Rationale: Central nicotinic receptors have been reported to be involved in the discriminative stimulus (DS) effects of nicotine. Objectives: The purpose of the present study was to investigate the role of the medial prefrontal cortex (mPFC) and the medial habenular nucleus (mHb) in the DS effects of nicotine. Methods: Substitution tests with nicotine administered into mPFC and mHb were conducted in rats trained to discriminate nicotine (0.5 mg/kg, SC) from saline in a two-lever, food reinforced, operant task. Results: Nicotine (40 µg) administered into mPFC substituted for nicotine (0.5 mg/kg, SC), whereas nicotine administered into mHb did not. Conclusions: Together with our previous study indicating that the nucleus accumbens and the ventral tegmental area are partially involved in the DS effects of nicotine, the present study suggests that mPFC is primarily involved in the DS effects of nicotine.