Key words : cerebellar afferents, retrograde transport, hypoglossal motoneurons

                    The cerebellum of the brain is well known to play an important role in the control and coordination of movements, however, the precise neuronal mechanisms which underlie the function continue to be subject of active research investigations. In 1977, with the method of the retrograde axonal transport of Horseradish Peroxidase (HRP), Kotchabhakdi and Walberg was probably the first to discover that neurons in several cranial nerve motor nuclei, including the hypoglossal nucleus, which controls the movements of the tongue, project their axons as afferents to specific regions of the cerebellum. The objective of the present study is to investigate the cell of origin of cerebellar afferents from neurons in the hypoglossal nucleus in the rats. The method of double retrograde axonal transport of florescence tracers was applied to find out whether these afferents originate from large alpha (a) motoneurons innervating the tongue muscles or from other interneurons in the motor nucleus.

                    Experimental Procedure : : Florescence tracers were stereotactically injected in 20 Wistar rats anesthetized with pentobarbital(50 mg/kg). 0.3 Microliter of 10% Solution of Dextran Tetramethy Rhodamine Biotin (Micro-Ruby or 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. 25 microliters of 3% solution of Fluoro-Gold (FG) in PBS was injected into the ventral part of the body of the tongue. 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, mounted on glass slides, and examined under the epifluorescence or MRC600 Confocal microscope. The presence of both single or double retrograde labeled neurons in the hypoglossal nucleus was photographed, stored and printed out as computer image files. The distribution of single and double-labeled neurons was mapped on standard diagrams of the rat brainstem for further analysis.

                    Results, Discussion and Conclusion: Neurons labeled only with MR retrogradely transported from the injection sites in the cerebellum were found bilaterally in the middle and caudal regions of the hypoglossal nucleus as well as other previously known brainstem nuclei. These MR labeled neurons appear to represent only a small population of the entire hypoglossal neurons. Neurons labeled only with FG retrogradely transported from the injection in the tongue muscles were numerous, distributed only ipsilaterally among almost the entire population of corresponding motoneurons which innervate the tongue muscles. A smaller proportion of FG labeled neurons within the hypoglossal nucleus were also double labeled with MR, indicating that they project their axon collaterals to both the tongue muscles and the cerebellum. The double-labeled neurons in the rat were small to medium in size and scattered among the population of predominantly large alpha (a) motoneurons labeled with FG. The findings provide clear evidence that a small population of motoneurons in the hypoglossal nucleus of the rat project their axon collaterals to the cerebellum. and the tongue muscles. However, these cerebellar afferent neurons in the rat seem unlikely to originate from large a- motoneurons as previously reported in the cat and monkeys. These neurons may play a role in monitoring or controlling tongue movements during chewing, licking, buccal cleaning, sucking, swallowing, respiration, and motor speech.

Acknowledgement: Supported by Collaboration between Mahidol University and 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 318.)

  HYPOTHALAMIC NEURONS, INITIALLY RES-PONDING ONLY TO THE SIGHT
  OF FOOD, RESPOND TO THE SIGHT OF WATER AFTER
  INTRACEREBROVENTRICULAR ADMINISTRA-TION OF DIPSOGENIC
  AGENTS : A CLINICAL TEST IN BEHAVING CONSCIOUS SHEEP (NO. 921)

Suriyaphun Mungarndee, Basil Arthur Baldwin, Naiphinich Kotchabhakdi

Key words : hypothalamus, zona incerta, extracellular recording, angiotensin- II, sheep

                    Extracellular single-unit recordings were made in the lateral hypothalamus (LH) and zona incerta (ZI) of conscious sheep (n=6) during the visual presentation (V_P) of food or water. Initially, the sheep were hungry but not thirsty. The effects of intracerebroventricular (icv) administration of 0.85M NaCl plus 200ng Angiotensin-II (ANG-II), which induces intense thirst, revealed that they became strongly responsive to the V_P of water under the influences of the dipsogenic agents (P<0.001). Thus, the neuronal response (n=12/236) could be altered by changing the animal’s dominant motivational state from hunger to thirst. It was suggested that they might be neurons responsive to the Vp of whatever the animal currently needs. The icv administration, during recording from neurons in conscious animals, of neuropeptides capable of inducing food or water intake, will be of value in the study of the neurophysiology of ingestive behavior.

                    Experimental Procedure: Intact adult ewes (Ovis aries) were prepared for microelectrode (mE) recording. Hypertonic solution of 0.85M NaCl and 200ng ANG-II were administrated icv. Units found responding with a significant change in firing rate to the V_P of food or water compared to its spontaneous baseline (S_b) were selected to record. The S_b-firing rate of the targeted neurons was recorded for 35s before the fixed 5s period was acquired. Neuronal signals from the mE were amplified up to 20K by an ac pre-amplifier and were filtered with analog electronic filtering, with a band-pass cut-off between 500-5000 Hz. The signals were sent to an A/D interface for on-line analysis. The units were identified and isolated by a graphical clustering method. The animals were presented with each of the clinical stimuli in a randomized order. Statistical comparisons were made between the neuronal firing rate (Hz) during the various components of the clinical test during the S_b 5s-period. For a single neuron responding to the V_P of substances, it was required to display > 50% change in firing rate compared with S_b and a statistically significant was determined by ANOVA and Wilcoxon test post hoc. At the end of each experiment, small lesions were made at the