Olivopontocerebellar atrophy

To study the pathophyhsiology of olivopontocerebellar atrophy (OPCA), we destroyed inferior olive nuclei of male Wistar rats using 3-acetyl pyridine (3-AP)+ harmaline+ niacinamide. These rats showed a sluggish and ataxic gait. To elucidate the relationship between thyrotropin releasing hormone (TRH) in the Purkinje cell of cerebellum and the inferior olive nucleus, we investigated the concentrations of TRH in the cerebellar cortex, nuclei, and medulla oblongata including the inferior olive nuclei using radioimmunoassay method as well as TRH receptor in the Purkinje cells of cerebellum using immunohistochemical method. All statistical comparisons were done using non-parametric tests (Mann-Whitney U-test). We found that two weeks after the treatment, TRH concentrations in the cerebellar cortex as well as nuclei were significantly lower than in the controls but no significant difference in the medulla oblongata was observed between 3-AP treated rats and controls. Moreover, four weeks after the treatment, TRH-receptor positive Purkinje cell counts were significantly fewer than in the controls. These results suggest that TRH in the Purkinje cell of cerebellum may play a role in the ataxic gait observed in the rats whose inferior olive were destroyed. [Neurol Res 2000; 22: 401-403]

Keywords: TRH; TRH receptor; Purkinje cell; inferior olive; 3-AP; OPCA

INTRODUCTION

The climbing fiber afferents are a structure unique to the cerebellar cortex. They originate, presumably solely, from the inferior olive and make an extensive, excitatory synaptic contact with dendrites of cerebellar Purkinje cells. The importance of the climbing fiber afferents in cerebellar functions has been emphasized in connection with the learning process which may occur in the cerebellar cortex. Destruction of inferior olive induces rapid depression in synaptic action of cerebellar Purkinje cells1-6.

The pathophysiology of the rat whose inferior olive are destroyed is supposed to be a model for understanding patients with olivopontocerebellar atrophy (OPCA)7,8. It is reported that thyrotropin-releasing hormone (TRH) might produce inhibitory effects on the Purkinje cells which, in turn, might have excitatory or disinhibitory effects9. We have previously reported that in patients with OPCA, TRH concentrations in the cerebellar cortex and olivary nucleus were significantly lower than in the control cases10. We also reported the presence of TRH receptor in the Purkinje cells of rat cerebellum using the immunohistochemical method11.

Llinas et al.12 have reported that 3-acetyl pyridine (3AP) plus harmaline plus niacinamide produced a more selective destruction of inferior olive in rat. Thus, we produced inferior olive destroyed rats by the method of Llinas et al.12 and studied TRH concentrations in the cerebellar cortex, nuclei and medulla oblongata including the inferior olive using radioimmunoassay13. In addition, TRH receptor in the Purkinje cells of the cerebellum was investigated using immunohistochemical method11.

MATERIALS AND METHODS

Inferior olive destroyed rats

Male Wistar rats of age 6 weeks weighing about 170180 g were used. According to the method of Llinas et al.12. we-gave an intraperitoneal injection of 3-AP of 75 mg kg^sup -1^ body weight, and a subsequent injection of harmaline (15 mg kg^sup -1^) 3 h later. An injection of niacinamide (300 mg kg^sup -1^ ) was done 4.5 h after the 3-AP injection.

TRH concentration

Rats anesthetized with chloroform were decapitated and brains were removed using information from the atlas of Paxinos and Watson14, and divided into the following respective regions: cerebral cortex, striatum, thalamus, brain stem and cerebellum. The cerebellum was also divided into the cerebellar cortex and nuclei. The brain stem was divided into the pons and the medulla oblongata. Brain tissues were homogenized in 0.1 N HCI by sonication. After centrifugation at 3000 rpm for 15 min at 4degC, supernatants were dried under a stream of air in a hot water bath (60degC). AntiTRH antibody was obtained by the method previously reported15.

Assay procedure

Standard solution or extracted samples (100 (mu)I), antibody (100 (mu)l, diluted 1 :5000), TRH-^sup 125^l (100 (mu)l), and diluent (400 (mu)I) were added to 10x75 mm plastic tubes at 4degC. After reaction at 4degC for 24 h, 100 (mu)l) of diluted anti-rabbit-gamma-globulin sheep serum was added to all tubes. All tubes were incubated for an additional 24 h at 4degC, and centrifuged at 3000 rpm for 30 min. The supernatants were removed by aspiration, and the radioimmunoactivity of precipitates was counted with an automatic gamma counter.

TRH receptor staining and immunohistochemical method

According to the method of Mitsuma et al.11, we investigated TRH receptor in rats using an antibody against TRH-receptor which was raised in rabbits immunized with a conjugate of synthetic TRH-receptor protein fragment (TRH-receptor 15-28) to bovine albumin.

The rats were anesthetized with sodium pentobarbital and transcardially perfused with 0.01 % glutaraldehyde and 4% paraformaldehyde in Bouin's solution (pH 7.2). The brain was removed and post-fixed for an additional hour at 4degC, then cut at 4 um thickness using a vibratome. Immunohistochemical treatment was performed by the avidin-biotin complex (ABC) method using Vecstatin kits (Vector Laboratories Inc., Burlingam, CA, USA). The primary antibody was used after dilution (1 : 50).

TRH positive cell count

After staining, TRH positive Purkinje cell counts were done under light-microscopy at a magnification of 100x. Cell numbers were calculated for each group. Each sample was surveyed in five different sections and counted respectively. Means and SEM were calculated for each sample.

To determine TRH concentrations and TRH positive Purkinje cell counts, all statistical comparisons were done using non-parametric tests (Mann-Whitney U-test). p

RESULTS

Clinical features of 3-AP treated rats

After the inferior olive destruction, the rats became grossly ataxic and showed tremor-like involuntary movements, but after several hours, they recovered some of their motor performance after this acute phase of general motor disturbance. However, their movements remained sluggish and a distinctive gait disorder was noted. This gait was characterized by an exaggerated flexion of the limbs and an abnormal shift of body weight from one side to the other side. The rats showed no obvious non-cerebellar abnormalities.

TRH concentrations

TRH concentrations in the cerebellar cortex as well as in the nuclei of 3-AP treated rats were significantly lower than in the control rats (Tables 1 and 2). No significant difference in TRH concentrations in the medulla oblongata was observed between 3-AP treated rats and control rats (Table 3).

TRH positive Purkinje cell counts

As we have previously reported", the specific TRH receptor-positive Purkinje cells were visualized as shown in Figure 1. Significant staining was detected in the neural perikarya of Purkinje cells (Figures 1 and 2). TRH positive Purkinje cells were counted 2 weeks, 4 weeks, 5 weeks, and 6 weeks after 3-AP treatment. At two weeks later, TRH positive Purkinje cell counts did not show a significant difference between 3-AP treated rats and control rats (Table 4). Thereafter, TRH positive Purkinje cells in the 3-AP treated rats were significantly fewer than in the control rats. Moreover, morphological changes in the TRH positive Purkinje cells were also observed (Table 4, Figure 2).

DISCUSSION

We produced destruction of inferior olive nuclei in rats using 3-AP according to the method of Llinas et aL 2. 3-AP treated rats showed a sluggish and ataxic gait. In this study, we found that 2 weeks after treatment, TRH concentrations in the cerebellar cortex as well as nuclei of these rats were significantly lower than in the control rats. However, TRH concentration in the medulla oblongata showed no significant difference between 3AP treated rats and control rats. Moreover, we found that TRH positive Purkinje cell counts had significantly decreased 4 weeks after 3-AP treatment.

We have previously investigated the concentrations of TRH in three patients with OPCA'. In this study, we found that in two patients with OPCA, TRH concentrations in the cerebellar cortex and olivary nucleus were lower than in the control cases. In contrast, in one patient with OPCA, TRH concentration in the dentate nucleus was higher than in the controls. In addition, it is reported that TRH might electrophysiologically produce inhibitory effects on Purkinje cells which, in turn, might have excitatory or disinhibitory effects9. These results support the contention that TRH in the cerebellum of patients with OPCA play a pathophysiological role.

Nadi et al.16 reported that 14 days after injection of 3AP, the level of aspartate was lower than the control rats. Climbing afferents fibers have been reported to synapse with Purkinje cell. Hence, if the decrease in aspartate is related to a specific pool in the climbing fibers, these data suggest a selective decrease of aspartate in the cerebellar cortex of rats injected with 3-AP. Taken together with Nadi's report, our results suggest that TRH as well as aspartate in the cerebellum play a role in the pathophysiology of neurological deficits resulting from the destruction of the rat inferior olive nuclei.

ACKNOWLEDGEMENTS

We are very grateful to Mrs Makoto Naruse and Minoru Fukayama for their technical assistance and encouragement.

REFERENCES

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Koshin Adachi, Bo N. Rhue, Mei Li, Masayuki Izumi, Hiroshi Osano and Terunori Mitsuma

Fourth Department of Internal Medicine, Aichi Medical University, Yazako, Japan

Correspondence and reprint requests to: Koshin Adachi, Fourth Department of Internal Medicine, Aichi Medical University, 21 Karimata, Yazako, Nagakute-cho, Aichi-gun, Aichi-ken 480-1103, Japan. Accepted for publication September 1999.

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