sites of affected neurons by passing dc current to deposit ferrous ions for localizing purposes. At the conclusion of all experiments, the subjects were sacrificed and the brain were perfused by 10%-formol-saline to which potassium ferrocyanide and ferriccyanide were added in order to verify the recording sites.

                    Results, Discussion and Conclusion: It was demonstrated that neurons, which initially responded only to the V_p of food in clinical tests but did not respond to water, could be made to respond to water by the icv administration of a strong dipsogenic agent. These dipsogenic stimuli, one acting by hypertonic stimuli on osmoreceptor neurons and the other on specific ANG-II receptors, were both effective in eliciting changes in the neuronal response. The results also showed that the neurons, which initially responded to the V_p of food, but not to water, returned to their previous pattern of response when the effects of the thirst-inducing stimuli had dissipated. The alternation of the neuronal response to the V_p of water by rapidly inducing thirst suggests that these neurons have their plasticity of response and that they react to the V_p of whatever the animal needs most urgently. Thus, it was concluded that the responses of these neurons are related to the motivational state of the animal.

(Presented at the 26^th Congress on Science and Technology of Thailand, 18-20 October 2000, Bangkok, Thailand and Published in the Extended Abstracts page 307.)

  DEVELOPMENT AND DISTRIBUTION OF CHO-LINERGIC AFFERENTS TO THE 
  CEREBELLUM: EXPERIMENTAL STUDY BY COMBINED DOUBLE 
  RETROGRADE AXONAL TRANSPORT WITH IMMUNO-HISTOCHEMICAL 
  STAINING OF CHOLINE ACETYL TRANSFERASE (ChAT) ENZYME (NO. 922)

Key words: cerebellar afferents, cranial nerve motor nuclei, retro grade axonal transport, immuno- histochemical Staining, choline acetly transferase enzyme, cholinergic system.

                    It is well documented that large alpha (a) motoneurons in the spinal cord and brainstem are cholinergic and release acetly choline (Ach) as neurotransmitter at all of their axon terminals. In 1977, Kotchabhakdi and Walberg were the first to report that neurons in several cranial nerve motor nuclei in the cats and monkeys project their axons as afferents directly to specific regions of the cerebellum. The findings stimulated and prompted many subsequent investigations into the neuronal origin of these cerebellar afferents, however, it was not yet known whether they originate from cholinergic alpha (a) motoneurons or from other interneurons. The objective of the present study is to investigate the development and distribution of cholinergic neurons in the motor cranial nerve nuclei which project to the cerebellum in the rats. The combined method of double retrograde axonal transport of multiple florescence tracers was applied together with immuno-histochemical staining of choline acetyl transferase (ChAT) enzyme.

                    Experimental Procedure: Florescence tracers were stereotactically injected in several groups of Wistar rats at different ages from postnatal day 20^th to adult, which were anesthetized with pentobarbital (50 mg/kg). 0.3 ml of 10% Micro-Ruby (MR) in Phosphate Buffer solution (PBS) was injected in the cerebellum, in the regions of anterior vermis(lobule I, II), posterior vermis (lobule VI, VII, IX, X), flocculus, paraflocculus, and deep cerebellar nuclei. Varying volumes between 2 to 10 ml of 3% solution of Fluoro-Gold (FG) in PBS was injected into the bellies different muscles in the head and neck. Four rats injected with only Phosphate buffer solution without florescence tracers served as the control. After the survival time of 3 days the rats were re-anesthetized then perfused with 0.9 % saline, followed consecutively by 4% paraformaldehyde, and 30 % sucrose in PBS. The brainstem and the cerebellum were removed, and sectioned transversely with a freezing microtome into 40 mmm. thick consecutive serial sections. In addition the sections were also processed with immuno-histochemical staining with monoclonal antibody against ChAT enzyme. The presence of ChAT enzyme in the neuronal cell body is demonstrated by a second antibody attached to the green fluorescence Oregon Green (OG). The sections were then mounted on glass slides, and examined under the epifluorescence or MRC600 Confocal microscope. The presence of neurons labeled with both single or double-retrograde tracers, and ChAT enzyme in the motor cranial nerve nuclei was photographed, stored and printed out as computer image files. The distributions of single and doubled-labeled neurons with or without ChAT enzyme in these nuclei were mapped onto separate standard diagrams of the rat brainstem for further analysis.

                    Results, Discussion and Conclusion: Many neurons labeled only with MR retrogradely transported from the injection sites in the cerebellum were found bilaterally and scattered sparsely in the all motor cranial nerve nuclei. Neurons labeled only with MR did not appear to have green color of OG, or they do not have ChAT enzyme. These neurons vary in size from small to medium-sized interneurons and represent only a small proportion of the entire population. Neurons labeled only with FG retrogradely transported from injection sites in the muscles were more numerous, also have green colour with the presence of ChAT enzyme, and distributed in almost the entire population of medium-sized and large motoneurons, which innervate the muscles. A smaller proportion of FG labeled neurons within these nuclei were also double-labeled with MR and have green OG color with the presence of ChAT enzyme, indicating that these cholinergic cranial motoneuons project their axon collaterals to both muscles and the cerebellum. In conclusion, the present findings provide clear evidence that a small population of cholinergic motoneurons in the cranial nerve motor nuclei of the rat project their axon collaterals directly to the cerebellum, while their main axons innervate the two muscles. In addition, cerebellar afferents also originate from other non-cholinergic interneurons within these nuclei. The findings indicate that cerebellar neuronal circuits play direct roles in monitoring and controlling lower motoneurons than previously known.

Acknowledgement: This study was supported by the research collaboration with Monash University.

(Presented at the 26^th Congress on Science and Technology of Thailand, 18-20 October 2000, Bangkok, Thailand and Published in the Extended Abstracts page 314.)