Single unit activity in lateral reticular nucleus during cortically evoked masticatory movements in rabbits

Brain Research, 337 (1985) 287-292

287

Elsevier BRE 10819

Single Unit Activity in Lateral Reticular Nucleus During Cortically Evoked Masticatory Movements in Rabbits G. MARINI and M. L. SOTGIU Istituto di Fisiologia dei Centri Nervosi, CNR, 20131 Milano (Italy)

(Accepted September 17th, 1984) Key words: lateral reticular nucleus - - mastication - - modification of neuronal activity

The activity pattern of the lateral reticular nucleus (LRN) neurones was analyzed during cortically evoked masticatory movements in anesthetized rabbits. Antidromic activation from the cerebellum and histological reconstruction of recorded neurones location from electrode tracks and microdrive readings were the criteria for neuronal identification. Tonic changes as well as rhythmic modulation of neuronal activity were found in a subpopulation of LRN cells during mastication. The same results were observed also in curarized rabbits during fictive mastication. These data support the view that LRN neurones are involved in a central mechanism controlling the masticatory movements.

INTRODUCTION The L R N is a precerebellar relay which is present in all mammals. It receives descending signals from motor centers, ascending signals from the spinal cord and cerebeUar return signals (for review see ref. 17). It appears to be heavily implicated in vestibular activity6,7. Its functional role has been seen in motor automatisms such as locomotion and scratching 2. The aim of this investigation was to examine whether the L R N is involved also in mastication which, according to current views, is a rhythmic m o v e m e n t generated by central mechanisms4,9,14,16. To this purpose the firing pattern of L R N neurones was analyzed during cortically induced mastication in rabbits. Part of these results has been published in abstract form lo. MATERIALS AND METHODS Rabbits were used since their frontolateral cortical masticatory area is particularly developed 3. Successful experiments were performed on 15 adult pigmented rabbits ( 2 - 3 kg) anesthetized with urethane

(1.4-1.6 g/kg) administered via the marginal ear vein, tracheotomized and fixed in a stereotaxic frame. The femoral artery and vein were cannulated. The temperature was kept constant at 3 7 - 3 8 °C with a heating pad. Blood pressure was monitored and prevented from falling below 80 m m Hg by Macrodex infusion. A single dose of atropine (0.05 mg/kg) was given initially. A craniotomy allowed transdural implantation of an assembly of two stainless-steel electrodes insulated except at the tip for intracortical stimulation. Placement was made in anterolateral cortex, L5. Rhythmical masticat0ry movements were evoked while using repetitive (15 Hz) stimuli at intensities < 1.5 m A , 0.5 ms duration. Rhythmic jaw movements were produced in different animals at 2.5-3.5 Hz. The L R N , ipsilateral to the cortical stimulation site, was approached from dorsally. A laminectomy at the upper cervical level and a removal of a large portion of the occipital bone was performed. Tungsten microelectrodes ( 5 - 1 0 M£2) were inserted dorsally at an angle of about 30 ° from the vertical, pointing rostrally. Extracellular recording was made in a grid of coordinates related to the midline and to the obex (L 2 - 3 , H 3.5-4.5), according to the atlas of Meessen and Olszewski 12. Responses originating

Correspondence: G. Marini, Istituto di Fisiologia dei Centri Nervosi, CNR, Via Mario Bianco 9, 20131 Milano, Italy.

0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

288 from cells were distinguished from those arising from fibers according to the well known criteria 5. For all cells poststimulus time histograms were constructed and the units which were driven by the electrical cortical stimulation were discarded. In most experiments a marking lesion was placed (30/~A cathodal for 20 s) for later histological reconstruction. Two pairs of stimulating electrodes were placed in the white matter of ipsilateral pars intermedia of the anterior lobe of the cerebellum near the entry of the ipsilateral restiform body in order to identify antidromically the L R N neurones. The criteria for antidromic activation were: (a) short and fixed latencies of response at all stimulus intensities above threshold; and (b) ability to follow frequencies higher than 200/s as exemplified in Figs. 3 and 4. Furthermore, in some experiments the m e t h o d of colliding the antidromic response with a preceding orthodromic response was used as an additional test. During re-

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cording the medulla and cerebellum were covered with paraffin oil. Electromyographic ( E M G ) activity was recorded with needle electrodes from the masseter muscle and the electroneurographic ( E N G ) activity with cuff electrodes from the hypoglossal nerve trunk, prepared in the neck and cut distally. Gallamine triethiodide (Flaxedil, 5 mg/kg/h, i.v.) was administered after data had been gathered in the unparalyzed state and the animals were artificially ventilated. Single unit activity from L R N , the E M G and E N G were stored on analog tape for further analysis. After completion of the experiment, the portion of the medulla containing the recording tracks was sectioned after freezing and serial sections (50/~m) were stained with cresyl violet for reconstructing the recording sites. By means of the marking lesions and electrode tracks each penetration was identified under the microscope and then plotted on camera lucida drawings of the brainstem sections. The depth 1o-

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Fig. 1. A: phasic LRN neurones. Phase distribution of 20 cells. The position of the burst of each neurone, plotted in relation to the normalized cycle, is shown by a horizontal line. All the neurones are put in order according to the onset of the bursts. B: tonic LRN neutones. Distribution of the time of change of discharge frequency in relation to the onset of masticatory movements (left) and of the time of recovery in relation to the end of movements (right). Height of each column in the histogram indicates the percentage of neurones that increase (open columns), decrease (dotted columns) and recover their activity in the time given on the abscissa.

289 cation of cell recordings was found from a correlation with the in vivo micrometer readings, taking into account the degree of shrinkage (about 10% as assessed by means of the marking lesions). RESULTS

A large number of spontaneously active cells were found which were not clearly activated during sequences of cortically evoked masticatory movements and were not considered in this report. One-hundred and fourteen units were unambiguously related to cortically induced mastication. Fifty percent of them being antidromically activated by ipsilateral cerebellar stimulation. In the majority of cells the spontaneous activity ranged from 7 to 40/s. In 9 cases the cells showed no spontaneous activity. Thus far, from the population of responsive units, two types of cells have been identified. Type 1 units (20 neurones)

showed a phasic modulation with masticatory rhythms. This modulation could either occur in phase with the masseter EMG, or out of phase with this activity. For each neurone, however, the position of the burst discharge was stable in successive masticatory cycles. For all these neurones the burst position in the masticatory cycle, measured between two masseter EMG and normalized to the unit, is shown in Fig. 1A. An example of type 1 neurone activity phasically modulated during masticatory movements is illustrated in Fig. 2. Note the increase in firing rate as well as the rhythmic suppression of cellular activity in the records B and C. A quantitative measure of the extent to which impulse activity was phase-locked to the masticatory movements (jaw opening and closing) was obtained from cycle histograms. In these, analysis time corresponds to the cycle duration and the probability of impulse occurrences throughout the period of the masticatory cycle is displayed

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Fig. 3. Example of tonic change of a LRN neurone activity during mastication before and after paralysis. Traces from top to bottom represent spontaneous activity of the neurone and masseter EMG (A), tonic increase during mastication monitored by masseter EMG (B), tonic increase during fictive mastication monitored by hypoglossal ENG (C), and spontaneous activity after mastication (D). In E frequency histograms in % of the control value, a: resting discharge rate (control) = 100%; b: during mastication; c: during fictive mastication; d: after the end of mastication. The vertical bars represent the S.D. of the mean. In the inset the antidromic response to cerebellar stimulation (4 pulses at 200/s). (Fig. 2D and E). Type 1 n e u r o n e s rarely (2 out of 20) were identified by their response to antidromic stimulation from the ipsilateral cerebellum. Type 2 units (94 neurones) were characterized by tonic changes of their activity during induced masticatory rhythms. In particular it was seen that 74 neurones showed an increase in discharge rate a m o u n t -

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291 of mastication or could follow it, but never outlasted it. This behavior is quantified in the histograms of Fig. lB. An example of tonic increase in activity of a type 2 cell during cortically evoked masticatory movements is shown in Fig. 3 and an example of inhibition is shown in Fig. 4F. In the sample examined, cortical stimulation below mastication threshold either had no effect on the neuronal activity or (5 units) induced opposite effects to those induced by cortically evoked masticatory movements. In Fig. 4 two of these cases are illustrated. In addition the activity of these neurones was not changed if stimuli of the same intensity used to evoke masticatory movements were applied to neighboring cortical areas. For many cells we tested LRN neurones for somatosensory input by non-painful skin stimuli applied by a probe to the face, the back, and the limbs and by applying discrete passive movements of limb joints. Furthermore, stimulating needle electrodes insulated except at the tip were placed subcutaneously in different parts of the body (forelimbs, hindlimbs, face). The results showed that neurones related to evoked masticatory movements were not responsive to these tested stimuli except for movements of the lip in a few cases. On the other hand, we observed that cells in which the activity was

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not modified by induced mastication were sometimes clearly activated by some of these stimuli. In order to rule out the possibility that the observed responses were due to proprioceptive signals from masticatory muscles, experiments were repeated in curarized rabbits. The two types of units described were observed also after paralysis. From a population of 42 responsive units, 5 units showed a phasic modulation, 37 units showed a tonic change during cortically evoked 'fictive mastication'. Out of these 37 neurones, 32 increased and 5 decreased their activity. Furthermore, in 3 cases the activity of the same neurones was recorded before and after paralysis. Fifty percent of type 2 units were identified by their response to antidromic stimulation from the cerebellum. The two types of masticatory movement-related neurones had a different distribution within the LRN. The large majority of the type 1 cells were clustered together and were encountered along the same recording tracks, at the obex level in the most dorsal portion of the nucleus, 20% of them being allocated in the boundary between L R N and the reticular formation. No preferential distribution was detected for type 2 neurones which were scattered throughout the nucleus (Fig. 5).

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292 DISCUSSION In this study a s u b p o p u l a t i o n of L R N cells has b e e n observed to fire in relation to cortically e v o k e d masticatory m o v e m e n t s . T h e s a m e results were o b s e r v e d in curarized rabbits during 'fictive mastication' confirming the view that mastication is a rhythmical m o v e m e n t g e n e r a t e d within the CNS 4,9,14,16. A s described in o t h e r studies during rhythmic movements18,19 and during ocular movements8,15, we found tonic changes as well as rhythmic m o d u l a t i o n of neuronal activity during induced mastication. T h e two types of m o d u l a t i o n s are p r o b a b l y of different functional significance, but i n t e r p r e t a t i o n remains speculative. N e u r o n e s showing a phasic m o d u l a t i o n might participate in the g e n e r a t i o n of the rhythmic activity 13 or might m e d i a t e internal f e e d b a c k signals 11 (cf. also ref. 1). N e u r o n e s showing tonic changes could express a general 'setting device' of excitability of cerebellar neurones during execution of a m o v e m e n t sequence, since they m o n i t o r the entire masticatory sequence and not the individual muscular act. REFERENCES 1 Alstermak, B., LindstrOm, S., Lundberg, A. and Sybirska, E., Integration in descending motor pathways controlling the forelimb in the cat. 8. Ascending projection to the lateral reticular nucleus from C3-C4 propriospinal neurones also projecting to forelimb motoneurones, Exp. Brain Res., 42 (1981) 282-298. 2 Arshavsky, Y. I., Gelfand, I. M., Orlovsky, G. N. and Pavlova, G. A., Messages conveyed by spinocerebellar pathways during scratching in the cat. I. Activity of neurons of the lateral reticular nucleus, Brain Research, 151 (1977) 479-491. 3 Bremer, F., Physiologie nerveuse de la mastication chez le chat et le lapin, Arch. int. Physiol., 21 (1923) 308-352. 4 Dellow, P. G. and Lund, J. P., Evidence for central timing of rhythmical mastication, J. Physiol. (Lond.), 215 (1971) 1-13. 5 Hamilton, T. C. and Johnson, J. I., Somatotopic organization related to nuclear morphology in the cuneate-gracile complex of opossum, Brain Research, 51 (1973) 125-140. 6 Kubin, L., Manzoni, D. and Pompeiano, O., Responses of lateral reticular neurons to convergent neck and macular vestibular inputs, J. Neurophysiol., 46 (1981) 48-64. 7 Kubin, L., Magherini, P. C., Manzoni, D. and Pompeiano, O., Responses of lateral reticular neurons to sinusoidal rotation of neck in the decerebrate cat, Neuroscience, 6 (1981) 1277-1290. 8 Luschei, E. S. and Fuchs, A. F., Activity of brain stem neurons during eye movements of alert monkeys, J. Neurophysiol., 35 (1972) 445-461. 9 Luschei, E. S. and Goldberg, L. J., Neuronal mechanisms of mandibular control: mastication and voluntary biting. In V. B. Brooks (Ed.), The Nervous System. IL Motor Con-

The r e p o r t e d findings suggest that L R N n e u r o n e s are involved in a m e c h a n i s m controlling the masticatory m o v e m e n t s . H o w e v e r , we cannot exclude entirely the possibility that only a p r o p o r t i o n of the phasic n e u r o n e s b e l o n g e d to the L R N ; s o m e m a y have been situated within the reticular f o r m a t i o n just dorsal from the L R N p r o p e r . F u r t h e r m o r e , the observation that cells activated by induced masticatory m o v e m e n t s are not activated by passive limb m o v e m e n t s could p r o v i d e s o m e electrophysiological evidence that a given cell p o p u l a t i o n in the L R N is specialized for a given m o t o r a u t o m a tism supporting the view that L R N e n c o d e s different aspects of m o t o r b e h a v i o r in distinct subset of neurones. ACKNOWLEDGEMENTS The authors would like to t h a n k Prof. M. W i e s e n danger for generous help and criticism during the course of this study. W e are i n d e b t e d to M. G. Orlando and U. D e G i o v a n n i for secretarial and p h o t o graphic assistance. trol, Physiological Society, Washington, 1981, pp. 1237-1274. 10 Marini, G., Sotgiu, M. L. and Wiesendanger, M., Single units activity in lateral reticular nucleus during cortically evoked mastication in rabbits, Neurosci. Lett., $7 (1983) $233. 11 McCloskey, D. I., Corollary discharges: motor commands and perception. In V. B. Brooks (Ed.), The Nervous System. H. Motor Control, Physiological Society Washington, 1983, pp. 1415-1447. 12 Meessen, H. and Olszewski, J. In S. Karger (Ed.), A Cytoarchitectonic Atlas of the Rhomboencephalon of the Rabbit, A. G. Verlag, Basel, 1949. 13 Nakamura, Y., Enomoto, S. and Kato, M., The role of the medial bulbar reticular neurones in the orbital cortically induced masticatory rhythm in cats, Brain Research, 202 (1980) 207-212. 14 Nakamura, Y., Hiraba, K., Enomoto, S. and Sahara, Y., Bulbar reticular unit activity during food ingestion in the cat, Brain Research, 253 (1982) 312-316. 15 Sparks, D. L. and Travis, R. P. Jr., Firing patterns of reticular neurons during horizontal eye movements, Brain Research, 33 (1971) 477-481. 16 Sumi, T., Modification of cortically evoked rhythmic chewing and swallowing from midbrain and pons, Jap. J. Physiol., 21 (1971) 489-506. 17 Wiesendanger, M., Cortico-cerebellar loops. In J. Massion, J. Paillard, M. Wiesendanger and W. Schultz (Eds.), Neural Coding of Motor Performance, Exp. Brain Res. (Suppl.), Springer-Verlag, Berlin, 1983, pp. 41-53. 18 Wyman, R. J., Neural generation of the breathing rhythm, Ann. Rev. Physiol., 39 (1977) 417-448. 19 Zangger, P. and Schultz, W., The activity of cells of nucleus reticularis tegmenti pontis during spontaneous locomotion in the decorticate cat, Neurosci. Lett., 7 (1978) 95-99.