Inhibitory postsynaptic potentials evoked in hypoglossal motoneurons by lingual nerve stimulation

EXPERIMENTAL

NEUROLOGY

75, 103-l 11 (1982)

Inhibitory Postsynaptic Potentials Evoked in Hypoglossal Motoneurons by Lingual Nerve Stimulation MITSURU

TAKATA’

Department of Physiology. School of Dentistry, Tokushinta University, Kuranwto-do, Tokushima, Japan Received May 7. 1981: revision received August 19, 1981 The percent magnitude of a short- and a long-lasting inhibitory postsynaptic potential (IPSP) produced in tongue retractor and protruder motoneurons by lingual nerve stimulation was studied in cats. In the retractor motoneurons, stimulation of the &&lateral lingual nerve produced primarily the short-lasting IPSP, and the neurons had received synaptic input primarily from the afferent fibers in the contralateral lingual nerve generating the long-lasting IPSP. In the protruder motoneurons, in contrast, there was no pronounced difference. in the IPSP patterns evoked by stimulation of the ipsilateral and contralateral lingual nerve.

INTRODUCTION In reports of intracellular recordings from hypoglossal motoneurons innervating either retractor or protruder muscles of the tongue (R- and PMns), it was suggested that inhibitory postsynaptic potentials (IPSPs) evoked by lingual nerve stimulation were formed by a combination of a short- and a long-lasting IPSP ( 11, 15). When double shocks separated by a lOO-ms interval were applied to the lingual nerve, the second shock produced only a short-lasting IPSP, suggesting that the first shock depressed the ability of the neuron to produce the long-lasting IPSP in reAbbreviations: IPSP-inhibitory postsynaptic potential; R-Mn, P-Mn-retractor, protruder motoneuron; ipsi-L, contra-L-ipsilateral, contralateral lingual nerve; S-IPSP, LIPSPshort-, long-lasting inhibitory postsynaptic potential; SI, L%-percentage magnitude of short-, long-lasting IPSP. ’ The author wishes to thank Dr. R. W. McCarley, Laboratory of Neurophysiology, Harvard Medical School, for his helpful criticism in preparing this manuscript. This study was supported in part by grant-in-aid for scientific research 56370029 from the Japan Ministry of Education, Science and Culture. 103 00144886/82/010103-09$02.00/0 copyrillht 0 1982 by Academii AlllightSdrrpmduaimiU~yfOrmrc&md.

Prm, IDC.

104

MITSURU

TAKATA

sponse to the second shock (15). This finding enabled us to separate the short-lasting component of lingually-induced IPSPs in hypoglossal motoneurons. In this study, we measured the percentage magnitude of the short- and long-lasting components of the IPSPs induced in R- and P-Mns by lingual nerve stimulation. The synaptic linkage of ipsilateral and contralateral lingual nerve afferent fibers on R- and P-Mns are discussed. MATERIALS

AND

METHODS

Fifteen adult cats weighing 2.5 to 3.5 kg were used. They were anesthetized with sodium pentobarbital (Nembutal, 30 mg/kg, i.p.) and immobilized by i.v. injection of gallamine triethiodide. In some cases the cats were decerebrated and decerebellated, but in most cases only a small caudal part of the cerebellum was sucked out to enable micropipet penetrations into the hypoglossal nucleus. A pneumothorax was made and respiration was maintained artificially. Fibers of the medial branch of the cat hypoglossal nerve innervate the protruder muscles and those of the lateral branch, the retractor muscles. A sleeve electrode was used to stimulate the cut central ends of the medial and the lateral branches of the hypoglossal nerves (protruder and retractor fibers), and the lingual nerves of either side. The threshold-stimulus (XT) for the lingual ner.ti was determined by recording the incoming nerve volley at the semilunar ganglion (14). In the present experiments the lingual nerve was stimulated at an intensity five times the nerve threshold (5 XT). Glass micropipets filled with 2 M potassium citrate were used for intracellular recordings. Electrode resistance was between 15 and 20 ma. Potentials were recorded with a direct-coupled amplifier. RESULTS Inhibitory Postsynaptic Potentials in Hypoglossal Motoneurons from Lingual Nerve Stimulation. It was reported that IPSPs produced in many P-Mns by stimulation of either the ipsilateral or the contralateral lingual nerve (ipsi-L or contra-L) were formed by a combination of short- and of long-lasting IPSPs (S-IPSPs and L-IPSPs) (11, 15). As in the case of the P-Mns, two IPSP components were produced also in the R-Mns by stimulation of the lingual nerve (Fig. 1A). Figure 1A, shows an antidromic spike of an R-Mn evoked by stimulation of the retractor fibers. The IPSPs induced by ipsi-L stimulation are shown in b,. After i.v. administration of strychnine (0.1 mg/kg), stimulation of the ipsi-L (b2) produced only a long-lasting (strychnine-insensitive) IPSP. By subtracting the long-lasting IPSP (b2) from the control IPSP (b,), the time course of the short-lasting

LINGUALLY

INDUCED

IPSPs

105

(strychnine-sensitive) IPSP was obtained (&, open circles). The duration of the S-IPSP was about 80 ms, and the solid circles in A, show the time course of the L-IPSP. As illustrated in &, the amplitude of the S-IPSP was about two times larger than that of the L-IPSP. When double shocks separated by a 90-ms interval were applied to the ipsi-L after blockage of the S-IPSP by strychnine, no IPSPs were produced by the second shock (Fig. 1B, ). In Bz and B3 are shown responses of this R-Mn to double shocks of the ipsi-L, when double shocks separated by a 140-ms and a 240-ms interval were applied to the ipsi-L after the administration of strychnine. Short- and Long-Lasting Inhibitory Postsynaptic Potentials in Retractor Motoneurons. To measure the percentage magnitude of the S- and the L-IPSP components of the lingually induced IPSP, we conducted the following experiment (see results in Fig. 1C): Double shocks separated by a IOO-ms interval at 5.0 XT were applied to the ipsi-L to produce the IPSPs shown in Cl. The percentage magnitude of the S- and the L-IPSP components of lingually induced IPSPs evoked in this R-Mn by ipsi-L stimulation was measured from the tracings illustrated in Cz. Tracings 1 and 2 indicate an IPSP produced by the first and second shock applied to the ipsi-L. By subtracting tracing 2 from tracing 1, we obtained tracing 3 (dashed line), which indicates the time course and magnitude of the LIPSP. The marks S and L denote the maximum amplitude of the S-IPSP (tracing 2) and the L-IPSP (tracing 3). We then calculated the percentage magnitude of the S-IPSP (S%) and the L-IPSP (L’S) components of the lingually induced IPSP by the formula: S/(&s + L) x 100 = %

and

100 - % = L%,

where S = the maximum amplitude of the short-lasting IPSP (mV) and L = the maximum amplitude of the long-lasting IPSP (mV). By this calculation, the percentage magnitude of the S- and the L-IPSP in this RMn was determined to be 61 and 3996, respectively. In the present study 200 R-Mns were examined. The % and the L% in 43 R-Mns obtained from a single experiment are illustrated in Fig. 2. In these experiments, the maximum amplitude of the S- and the L-IPSP components was obtained by application of double shocks separated by a IOO-ms interval to the lingual nerve. The histograms on the left side labeled Ipsi Ling.N. indicate the % (open column with abscissa S) and the L% (solid column with abscissa L) of the IPSPs evoked by ipsilateral lingual nerve stimulation, and those on the right side labeled Contra Ling.N. are the % and the LI of the IPSPs evoked by contralateral lingual nerve stimulation. The ordinate N indicates the number of cells. From the histograms it was found that, in the IPSPs evoked by ipsi-L stimulation, the average of the % and the L% was 70.18% (N = 42) and 29.82% (N = 42),

MITSURU

106

TAKATA

> E a-i

\ \ \

I 2

\ \ \

\ ;

LINGUALLY

INDUCED

IPSPs

107

respectively (arrow). With respect to the IPSPs elicited by contralateral lingual nerve stimulation, it was found that the average of the S% and L% was 20.57% (N = 36) and 79.43% (N = 36), respectively (arrow). It was also found that in 7 of 43 explored R-Mns, the contra-L stimulus produced only an L-IPSP. These results indicate that the R-Mns responding with two components of IPSPs to lingual nerve stimulation are principally innervated by afferent fibers in the ipsi-L, which generate the S-IPSP, and by fibers in the contraL, which generate the L-IPSP. Short- and Long-Lasting Inhibitory Postsynaptic Potentials in Protruder Motoneurons. In the present study 250 P-Mns were examined. Figure 3 illustrates the S% and the L% calculated for the 32 P-Mns obtained from a single experiment by the same method as for R-Mns. From the histograms it was found that, in the IPSPs set up by ipsi-L stimulation, the average of the S% and the L% was 52.83% (N = 31) and 47.17% (N = 31), respectively (arrow). In the IPSPs set up by contra-L stimulation, the average of the S% and the L% was 51.33% (N = 31) and 48.67% (N = 31), respectively (arrow). These results indicate that P-Mns responding with two components of IPSPs to lingual nerve stimulation receive afferent fibers from the bilateral lingual nerves to generate the S- and the L-IPSP. Synaptic Linkage of Ipsilateral and Contralateral Lingual Nerve Afferent Fibers to Retractor Motoneurons. As reported in the P-Mns (1 l), the relative magnitude of IPSPs evoked in the R-Mns by stimulation of the ipsi-L and the contra-L was measured. The relative magnitude of the S- and the L-IPSP indicates the relationship between IPSPs produced by ipsi-L stimulation and those by contra-L stimulation. The relative magnitude of the S-IPSP (so/o) and the L-IPSP (1%) was calculated by Contralateral

S/ipsilateral

S X 100 = s% contralateral

and Llipsilateral

L X 100 = I%,

where S = the maximum amplitude of the short-lasting IPSP (mV) and L = the maximum amplitude of the long-lasting IPSP (mV). FIG. 1. Two components of IPSPs in retractor motoneurons. A.--antidromic spike, AsIPSPs elicited by ipsilateral lingual nerve stimulation, b,-control IPSPs, br-3 min after injection of strychnine (0.1 mg/kg, i.v.), &-tracings of the short-lasting (open circles) and the long-lasting IPSP (solid circles). B-IPSPs set up by double shocks after the administration of strychnine: BL,2, and a-application of double shocks separated by 90-, 140-, and 240-ms intervals to the ipsilateral lingual nerve. C-IPSPs set up by double shocks: &--stimuli of the @lateral lingual nerve, C2-tracings of the short-lasting (tracing 2) and the long-lasting IPSP (tracing 3). Tracing 1 denotes IPSPs elicited by ipsilateral lingual nerve stimulation. Marks S and L indicate the maximum amplitude of the short- and the long-laating IPSP.

108

MITSURU

FIG. 2. Percentage magnitudes of motoneurons. The abscissas S and L open and solid columns, respectively. indicates the average of the .!i’% and

TAKATA

short (5’%)- and long (LW)-lasting IPSPs in retractor indicate the SPJOand the LI, which are graphed by the The ordinate N indicates the number of cells. An arrow L%. For further details, see text.

In Fig. 4A and B are shown the relative magnitudes of the short- and long-lasting IPSPs for each member of the R-Mn pool investigated. In these experiments, the maximum amplitude of the S- and the L-IPSP was obtained by the application of double shocks separated by a lOO-ms interval to the lingual nerve. Figure 4A indicates that in 9/25 R-Mns, contra-L stimulation did not produce an S-IPSP, and that the S-IPSP produced by ipsi-L stimulation was always larger in amplitude than that from contraL stimulation. With respect to the L-IPSP, in 20/25 R-Mns, the L-IPSP produced by contra-L stimulation was larger in amplitude than that produced by ipsi-L stimulation (Fig. 4B). DISCUSSION It is known that the medial branch of the hypoglossal nerve supplies the tongue protruder muscles (genioglossus, geniohyoid, transverse, and vertical intrinsic muscles) and the lateral branch supplies the tongue retractor

LINGUALLY

INDUCED

109

IPSPs

FIG. 3. sR6,and L% in protruder motoneurons. The abscissas S and L indicate the S% and the L%, which are graphed by the open and solid columns, respectively. The ordinate N indicates the number of cells. An arrow indicates the average of the 9% and LW.

muscles (hypoglossus, styloglossus, infrahyoid, and longitudinal intrinsic muscles) (7, 8). In two components of IPSPs (the S- and the L-IPSP) produced in hypoglossal motoneurons by stimulation of the lingual nerve, it was already

w75

00

0

l

0 300M-

OOOO O

00

25.

o- , 1

0

zw-

00 O"O

00

10

l5-

l -... -em**

lOO-

0 Od,

5

l *ee

20

25 CELL No.

1

, 5

10

15

20

t 25 CELLNo.

FIG. 4. In retractor motoneurons, the relative magnitude of the short- and the long-lasting IPSPs plotted against the cell number is shown in A and B, respectively. For further details, see text.

110

MITSURU

TAKATA

shown that the S-IPSP was reversed to a depolarizing potential by membrane hyperpolarization and that strychnine administration blocked only the S-IPSP. In contrast, the L-IPSP was selectively blocked by picrotoxin and no reversal point of the L-IPSP was obtained upon hyperpolarization of the membrane ( 15). By displacing the membrane potential toward large hyperpolarization, no notable change was seen in the amplitude of the LIPSP (15), indicating that this long-lasting hyperpolarization is not a manifestation of disfacilitation. The finding that spikes induced in hypoglossal motoneurons by imposed currents were depressed during the L-IPSP ( 15) suggests that this hyperpolarization is also postsynaptic in nature. With respect to the ionic mechanism for the L-IPSP, it was found before that no measurable conductance increase could be detected during the L-IPSP ( 12), as reported in trigeminal motoneurons (13). From those findings it was suggested that the synapses for the L-IPSP are preferentially situated in the dendritic region of the cell (15), and that inhibitory synapses for the S-IPSP are probably located predominantly on the somatic region of the cell (12, 15). In the present study it was found that, when double shocks separated by about lOO-ms intervals were applied to the lingual nerve after blockage of the S-IPSP by strychnine, no IPSPs were produced by the second shock, indicating that no L-IPSP is generated by the second shock when double shocks separated by about lOO-ms intervals were applied to the lingual nerve. It was elucidated before that, when double shocks separated by a IOO-ms interval were applied to the lingual nerve after blockage of the LIPSP by picrotoxin, the first and second shocks produced the S-IPSP of identical size and duration (15). The results enabled us to measure the maximum amplitude of the S- and the L-IPSP by application of double shocks separated by a lOO-ms interval to the lingual nerve. In the present experiments, it was revealed that no pronounced differences were found between the patterns of IPSPs elicited in the P-Mns by stimulation of either the ipsi-L or the contra-L. The situation was quite different in the R-Mns, where ipsilateral lingual nerve stimulation elicited primarily a SIPSP and contralateral lingual nerve stimulation elicited primarily a LIPSP. This suggests that the R-Mns may utilize these different inputs in the production of complex tongue movements. With respect to localization of inhibitory synapses, several studies reported that the picrotoxin-sensitive synapses occur preferentially on dendrites, and the strychnine-sensitive synapses are primarily on soma (1, 4, 6, 15). Functionally, this suggests the possibility that dendritic inhibitory synapses might reduce the effectiveness of dendritic excitatory synapses, but that somatic inhibitory synapses would reduce the effectiveness of all synapses on the neuron (2, 3, 5, 9, 15). In some R-Mns, stimulation of the

LINGUALLY

INDUCED

IPSPs

111

contra-L produced only an L-IPSP. The muscles which are innervated by these R-Mns are unknown, but it may be that the reduction of the effectiveness of excitatory synapses by inhibitory synapses located on the dendritic region of these R-Mns may be involved in producing complex tongue movements, as was suggested for spinal motoneurons (10). REFERENCES 1. ALTMANN, H., G. TEN. BRUGGENCATE, AND U. SONNHOF. 1972. Differential strength of action of glycine and GABA in hypoglossus nucleus. PflUgers Arch. 331: 90-94. 2. BARRETT, J. N., AND W. E. GRILL. 1974. Influence of dendritic location and membrane properties on the effectiveness of synapses on cat motoneurones. J. Physiol. (London) 239: 325-345. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

BARRETT, J. N. 1976. Motoneuron dendrites: role in synaptic integration. Pages 17 1- 189 in J. W. MOORE, Ed.. Membranes, Ions and Impulses. FASEB and Plenum, New York/London. BRUGGENCATE, G. TEN., AND U. SONNHOF. 1971. Glycine and GABA actions in hypoglossus nucleus and blocking effects of strychnine and picrotoxin. Experientia 27: 1109. GELFAN, S.. G. KAO, AND D. S. RUCHKIN. 1970. The dendritic tree on spinal neurons. J. Comp. Neural. 139: 385-412. KELLERTH. J. O., AND A. J. SZUMSKI. 1966. Effects of picrotoxin on stretch-activated postsynaptic inhibitions in spinal motoneurones. Acta Physiol. Scand 66: 146-156. KRAMMER, E. B., T. RATH, AND M. F. LISCHKA. 1979. Somatotopic organization of the hypoglossal nucleus: a HRP study in the rat. Brain Res. 170: 533-537. LEWIS, P. R., B. A. FLUMERFELT, AND C. C. D. SCHUTE. 1971. The use of choline&erase techniques to study topographical localisation in the hypoglossal nucleus of the rat. J. Anaf. 110: 203-213. ODUTOLA, A. G. 1976. Cell grouping and Golgi architecture of the hypoglossal nucleus of the rat. Exp. Neural. 52: 356-371. SCHEIBEL, M. E., AND A. B. SCHEIBEL. 1969. Terminal patterns in cat spinal cord. III. Primary afferent collaterals. Brain Res. 13: 413-443. TAKATA, M. 1979. Lingually induced postsynaptic potentials of the hypoglossal motoneurons. J. Dent. Res. 58: 2279. TAKATA, M. In press. Synaptic linkage of lingual nerve afferents to hypoglossal motoneurons. In Y. KAWAMURA AND R. DUBNER, Eds., Oral-Facial Sensory and Motofunctions. Quintessence Pub. Co., Tokyo. TAKATA, M., AND S. FUJITA, 1979. The properties of lingually induced IPSPs in the masseteric motoneurons. Brain Rex 168: 648-65 1. TAKATA, M., K. ITO, AND Y. KAWAMURA. 1975. Inhibition of hypoglossal motoneurons by stimulation of the jaw-opening muscle afferents. Jap. J. Physiol. 25: 453-465. TAKATA, M., AND K. OGATA. 1980. Two components of inhibitory postsynaptic potentials evoked in hypoglossal motoneurons by lingual nerve stimulation. Exp. Neurol. 69: 299310.