Apoptosis in cultured hNT neurons.

Programmed cell death (apoptosis) is an important mechanism shaping the size of different cell populations within the developing nervous system. In our study we used the NT2/D1 clone originally established from the Ntera 2 cell line to investigate the baseline levels of apoptosis in cultured postmitotic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retinoic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might affect baseline apoptotic rate in differentiating hNT neurons. Second, we investigated whether cultured hNT neurons, previously shown to possess dopaminergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we found that the postmitotic hNT neuronal cells exposed to RA demonstrated significantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rapidly dividing NT2 precursor cell line (3.6-4.4%) at both studied (1 and 5 days in vitro, DIV) time points. Similar apoptotic nuclear morphology, including a variable extent of nuclear fragmentation was observed in all examined hNT cultures. On the other hand, the incidence of apoptotic nuclei was rare in cultures of NT2 precursors not subjected to RA treatment. Combined immunocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining revealed dopaminergic hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors were not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of postmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors implicated in the etiology of Parkinson's disease (PD), as well as to screen numerous pharmacological treatments that may slow or prevent dopaminergic deterioration.

Microcarrier enhanced survival of human and rat fetal ventral mesencephalon cells implanted in the rat striatum.

The transplantation of tissue containing dopamine-producing cells into the mammalian central nervous system is an emerging treatment for Parkinson's disease, despite relatively poor survival of implanted tissue. Recent evidence has suggested that Cytodex microcarriers enhance the survival of dopaminergic rat chromaffin cells transplanted into the rat striatum in the absence of immunosuppression. The current study was undertaken to evaluate the survival of rat and human fetal ventral mesencephalic neurons (VM) implanted alone or after attachment to microcarriers in the striatum of rats without immunosuppression. Rat fetal VM neurons demonstrated enhanced survival in the rat striatum when transplanted on microcarriers, compared to their transplantation alone during the 3-mo period examined in the present study. Transplants of human fetal VM neurons on microcarriers also survived remarkably well in the rat striatum without systemic immunosuppression. In contrast, human fetal VM cells transplanted alone into the rat striatum did not survive without systemic immunosuppression. There was no evidence of TH fiber sprouting in the vicinity of any transplant site. These data indicated that Cytodex microcarriers provide enhanced survival of both rat allograft and human xenograft fetal mesencephalic cells in the rat striatum without the necessity of systemic immunosuppression, perhaps by inducing a unique neuron-glia environment.

Asymmetrical motor behavior in rats with unilateral striatal excitotoxic lesions as revealed by the elevated body swing test.

Severe degeneration of basal ganglia neurons, particularly the intrinsic neurons of the striatum, is the major underlying neuropathology implicated in clinical attributes of Huntington's disease (HD). The excitotoxin-lesioned striatum provides a useful model for evaluating behavioral parameters of HD. Animals with unilateral excitotoxic lesions exhibit asymmetrical rotational behavior in response to dopamine agonists, such as apomorphine. However, the observed behavior is a pharmacological reaction, and subject to sensitization effects. A behavioral test using undrugged animals may demonstrate a more natural response of the animals to the lesion effects. Recently, we have developed the 'drug-free' elevated body swing test (EBST), and demonstrated that hemiparkinsonian rats exhibited significant biased swing activity. In the present study, we observed that animals with unilateral intrastriatal 3-nitropropionic acid or quinolinic acid lesions displayed a significant biased swing activity with the direction ipsilateral to the lesioned side of the brain. This ipsilateral swing corresponded to the ipsilateral rotational behavior exhibited by the lesioned animals when challenged with apomorphine. The present results demonstrated that the EBST is a sensitive measure for characterizing asymmetrical behavior in animals with striatal lesions.

Systemic 3-nitropropionic acid: behavioral deficits and striatal damage in adult rats.

Previous animal studies have demonstrated that systemic administration of 3-nitropropionic acid (3-NP) leads to neuropathological changes similar to those seen in Huntington's disease (HD). Recently, we reported hypoactivity in 6- and 10-week old rats treated with systemic 3-NP (IP, 10 mg/kg/day) once every 4 days for 28 days. Although these behavioral results seem to differ from the observed hyperactivity in most excitotoxic models of HD, 3-NP may provide a better model of juvenile onset and advanced HD. In the present study, older rats were similarly treated with 3-NP to further characterize the reported age dependency of striatal neuronal death caused by 3-NP. Hypoactivity was observed in 14- and 28-week old rats with the latter demonstrating more profound features. The present study also provided the first direct evidence of a 3-NP effect on passive avoidance behavior. Experimental and control animals showed no significant difference in daytime acquisition and retention of a passive avoidance task. However, when the retention tests were conducted during the night time (in contrast to previous daytime tests), 3-NP-treated animals exhibited significant retention deficits. In addition, the neuropathological effects of 3-NP were determined by Nissl, AChE and NADPH-diaphorase histochemistry. Metabolic activity was studied using cytochrome oxidase activity as an index. Results revealed striatal glial infiltration, loss of intrinsic striatal cholinergic neurons, but some sparing of large AChE positive neurons, minimal damage of NADPH-diaphorase-containing neurons, and very slight, if any, alterations in cytochrome oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)