Two types of inhibitory postsynaptic potentials in the hypoglossal motoneurons

Progress in NeurobiologyVol. 40, pp. 385 to 41I, 1993 Printed in Great Britain.All rights reserved

0301-0082/93/$15.00 © 1992PergamonPress Ltd

TWO TYPES OF I N H I B I T O R Y POSTSYNAPTIC POTENTIALS IN THE H Y P O G L O S S A L M O T O N E U R O N S MITSURU TAKATA Department of Physiology, School of Dentistry, Tok~hima University, Tokushima 770, Japan (Received 3 March 1992)

CONTENTS !. Introduction 2. Stereotyped series of postsynaptic potentials in hypoglossal motoneurons 2.1. Short-lasting IPSP 2.2. Effect of strychnine on the short-lasting IPSP 2.3. Long-lasting IPSP 2.4. Effect of picrotoxin on the long-lasting IPSP 2.5. Membrane potential dependence of the long-duration hyperpolarizing potential 2.6. Membrane potential dependence of the EPSP 2.7. Localization of excitatory and inhibitory synapses 3. Two components of IPSPs in hypoglossal motoneurons 3.1. Lingually induced PSPs in tongue protruder and retractor motoneurons 3.2. Separation of two components of IPSPs by strychnine administration 3.3. Separation of two components of IPSPs by double shocks 4. Percentage magnitude of the S- and the L-IPSP in hypoglossal motoneurons 4.1. S% and L% in lingually induced IPSPs 4.2. S% and L% in inferior alveolar-induced IPSPs 5. Axotomized hypoglossal motoneurons 5.1. Firing of axotomized motoneurons 5.2. Lingually induced PSPs in axotomized motoneurons 5.3. Cortically induced PSPs in axotomized motoneurons 5.4. Disjunction of presynaptic contacts on axotomized motoneurons 6. Physiological characteristics on reinnervating and nonreinnervating hypoglossal motoneurons 6.1. Synaptic potentials in reinnervating and nonreinnervating motoneurons 6.2. Repetitive firing on reinnervating and nonreinnervating motoneurons 6.3. Recovery of the firing behaviors and synaptic efficacy in motoneurons 7. Percentage magnitude of the S- and the L-IPSP in reinnervating and nonreinnervating hypoglossal motoneurons 7.1. S% and L% in reinnervating motoneurons 7.2. S% and L% in nonreinnervating motoneurons 7.3. S% and L% in motoneurons after the self-union operation 7.4. Maintenance and rearrangements of synaptic contacts Acknowledgements References

1. INTRODUCTION The hypoglossal nucleus consists of large multipolar motoneurons which give rise to fibers innervating the intrinsic and extrinsic muscles of the tongue and control tongue movements associated with orofacial functions such as mastication (Dubner et al., 1978), deglutition (Tomomune and Takata, 1988; see review by Miller, 1982) and phonation (Gracco and Abbs, 1988; Barlow and Farley, 1989). The hypoglossal nerve of the cat divides into the medial and the lateral branches. The fibers of the medial branch supply the tongue protruder muscles (genioglossus, geniohyoid, transverse and vertical intrinsic muscles) and the lateral branch supply the tongue retractor muscles (hyoglossus, styloglossus, infrahyoid and longitudinal intrinsic muscles) (Lewis et al., 1971).

385 386 386 387 388 388 389 389 39O 391 391 392 392 394 394 394 396 396 397 397 399 399 399 400 401 402 402 404 405 405 407 407

Therefore, hypoglossai motoneurons can be divided into two groups: protruder motoneurons (P-Mns) innervating protruder muscles and retractor motoneurons (R-Mns) innervating retractor muscles of the tongue (Morimoto et al., 1968). The morphology of hypoglossal motoneurons has been investigated at the light and electron microscopic level (Odutola, 1976; Boone and Aldes, 1984a; Aides and Boone, 1985), and the use of horseradish peroxidase as a sensitive neuronal labeling method demonstrated the somatotopic organization of the hypoglossai nucleus (Krammer et al., 1979; Uemura et al., 1979; Chibuzo and Cummings, 1982). Boone and Aides (1984b) demonstrated five types of synaptic terminals in the hypoglossal nucleus of the rat and classified them as follows: (I) S-boutons (spherical vesicles with an asymmetrical synapses); (2) F-boutons (flattened

385

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M. TAKATA

vesicles with a symmetrical synapse); (3) P-boutons (pleomorphic admixture of flattened and spherical vesicles with a symmetrical synapse); (4) C-boutons (pleomorphic vesicles with a subsynaptic cistern); and (5) T-boutons (spherical vesicles with an asymmetrical synapse and subsynaptic dense bodies). In order to understand complex and coordinated movements of the tongue the integration of synaptic inputs on hypogiossal motoneurons must be examined.

2. STEREOTYPED SERIES O F POSTSYNAPTIC POTENTIALS IN HYPOGLOSSAL MOTONEURONS

Intracellular recordings from cat hypoglossal motoneurons innervating either protruder or retractor muscles of the tongue (P- and R-Mns) revealed that stimulation of the lingual nerve or the inferior alveolar nerve elicits a stereotyped series of postsynaptic potentials. These include: (1) an excitatory postsynaptic potential (EPSP), (2) a subsequent short-lasting inhibitory postsynaptic potential (IPSP) which is blocked by the administration of strychnine, and (3) a long-lasting IPSP which is relatively insensitive to the injected current (Takata and Tomomune, 1987). Recently, in hippocampal pyramidal ceils it was shown that stimulation of afferents in the stratum radiatum evokes a late hyperpolarizing potential which was not reduced by 7-aminobutyric acid (GABA) antagonists, in addition to a GABA-IPSP (Dunwiddie et al., 1980; Nicoll et al., 1980; Thalmann and Ayala, 1982; Newberry and Nicoll, 1984).

The following studies were performed to present the properties of postsynaptic potentials evoked in cat hypogiossal motoneurons by stimulation of the cerebral cortex and the peripheral nerves. 2.1. SHORT-LASTINGIPSP The postsynaptic potentials (PSPs) evoked in 182 cat hypoglossal motoneurons by stimulation of the cerebral cortex (Cx), the inferior alveolar nerve (inf. air. n.) or the lingual nerve were explored. In a motoneuron innervating the protruder muscles of the tongue (P-Mn) illustrated in Fig. 1A, stimulation of the Cx (Fig. lAb) the inf. air. n. (Fig. IA~) or the lingual nerve (Fig. lAd) produced an IPSP with a latency of 2.5 msec, 4.0 msec or 4.0 msec from stimulus artifact. The threshold-stimulus for the lingual nerve and the inf. alv. n. was determined by recording the incoming nerve volley at the semilunar ganglion (Takata, 1979), and both nerves were stimulated with an intensity of 5 times the nerve threshold. A stimulation site to the cortical surface examined was in the "face area" of Woolsey's Ms I region (Woolsey, 1958) as already pointed out by Porter (1967). Figure IAa shows an antidromic spike of a P-Mn on stimulation of the medial branch of the hypoglossal nerves. The relation between the amplitude of cortically or peripherally induced hyperpolarizing synaptic potentials and of the membrane potential displacements was examined with a recording micropipette filled with 2 Mpotassium citrate. The responses to 5 trials were averaged and the results are shown in the averaged records. Figures lA~d denote

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FIG. 1. An S-IPSP in a P-Mn. A: Effect of membrane polarization, a: Antidromic spike, b, c and d: Reversal of a cortically, an inferior alveolar- and a lingually induced hyperpolarizing synaptic potential. B: Effect of chloride ions. a: Antidromic spike. The records b~, c~, d~ and b2, c,, d2 show a cortically, an inferior alveolar- and a lingually-induced IPSP before and after an increase in the intracellular concentration of chloride ions. b~, c3and d3: Extracellular records corresponding to the records b_,,c2and d2. (From Takata and Tomomune, 1987.)

387

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the membrane potential dependence of cortically, inferior alveolar and lingually induced hyperpolarizing synaptic potentials. Numerals denote the amounts of injected current in nanoamperes (nA). At the resting membrane level (O nA) stimulation of the Cx, the inf. alv. n. or the lingual nerve evoked a hyperpolarizing synaptic potential. By displacing the membrane potential toward hyperpolarization, these hyperpolarizing synaptic potentials were decreased in amplitude ( - 1 and - 2 n A ) and reversed to a depolarizing potential ( - 3 nA). By displacement of the membrane potential to depolarization, these hyperpolarizing synaptic potentials were increased in amplitude (2 and 3 nA). The reversal point of these IPSPs was obtained at the same hyperpolarized membrane potential. These IPSPs were relatively sensitive to the injected current and reversed to a depolarizing potential by membrane hyperpolarization by the injection of - 2 nA into a ceil, suggesting that the short-lasting IPSP (S-IPSP) is generated at the soma (Takata and Ogata, 1980; Takata and Tomomune, 1986). In the next studies, the effect of an increase of the intracellular concentration of chloride ions on the cortically, the inferior alveolar and the lingually induced IPSP in the same cell was examined (Fig. 1B). When chloride ions were injected from a recording micropipette filled with 3 M potassium chloride into a P-Mn, an S-IPSP set up by stimulation of the Cx, the

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inf. alv. n. and the lingual nerve was reversed to a depolarizing potential (Fig. 1B (b:, c2 and d 2 )), just like the IPSP in other motoneurons (Coombs et aL, 1955; Llin/ts and Baker, 1972). The records b3, c3 and d 3 show extracellular records corresponding to the records b2, c2 and d2, respectively. After the reversed postsynaptic potentials, chloride-insensitive hyperpolarizing postsynaptic potentials were still detected. 2.2.

EFFECT OF STRYCHNINE ON THE

SHORT-LASTINGIPSP The effect of strychnine on the IPSP was examined and the results are shown in Fig. 2A. As illustrated in a sample record, IPSPs produced in a P-Mn by stimulation of the Cx (b0, the inf. alv. n. (b2) or the lingual nerve (b3) were composed of two components. After the administration of strychnine (0.1 mg/kg), stimulation of the Cx, the inf, alv. n. or the lingual nerve produced EPSPs and spikes followed by an enhanced hyperpolarizing potential, illustrated in c~, c2 and c3, respectively. The amplitude of this hyperpolarizing potential was larger than that of the summated afterhyperpolarization (AHP). As already reported (Takata et aL, 1979), the A H P amplitude increases linearly until the number of antidromic spikes increases up to 4 and remains at 5 mV in size despite a further increase in the spike number. The results demonstrated that two components of IPSPs

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FIG. 2. Inhibition of an S-IPSP in P-Mns by the injection of strychnine. A: Effect of strychnine, a: Antidromic spike. The PSPs produced by stimulation of the Cx, the inf, alv. n. and the lingual nerve before (b I, b2and b3) and after (cj, c, and c3) the injection of strychnine are shown, d I, d2 and d3: Field potentials. B: Membrane conductance changes, a, and b2: Conductance increase during a cortically and lingually induced strychnin-insensitive hyperpolarizing potential. The resting membrane resistance is shown in a t and b a. C: Effect of membrane polarization on a cortically induced strychnine-insensitive hyperpolarizing potential. (From Takata and Tomomune, 1987.) JPN 40,3--F

388

M. TAKATA

(strychnine-sensitive and -insensitive IPSP) were produced in hypoglossal motoneurons by stimulation of the Cx, the inf. alv. n. or the lingual nerve and strychnine administration blocked only the S-IPSP (glycine-IPSP). -

2.3. LONG-LASTINGIPSP To investigate the property of a strychnine-insensitive IPSP, we examined the effects of membrane polarization on the IPSP in the cell responding with a small EPSP, followed by predominant IPSPs to a single shock delivered to the lingual nerve or the Cx after the injection of strychnine. In such a cell the properties of a strychnine-insensitive hyperpolarizing potential were explored and the results are shown in Fig. 2B and C. The membrane conductance changes during a strychnine-insensitive hyperpolarizing potential were measured by conventional pulse techniques. In neurons it is possible to demonstrate an increase in conductance during a strychnine-insensitive IPSP when tested by measuring the voltage drop produced by small constant current pulses injected into the cell (Gustafsson et al., 1978). By injecting a brief hyperpolarizing pulse (3 nA, 7 msec) into a P-Mn, the membrane resistance was measured, illustrated in Fig. 2B. As seen, a conductance increase could be detected during a cortically (a2) and lingually induced strychnine-insensitive hyperpolarizing potential (b2). A resting membrane resistance is shown in the records a~ and b~, respectively. In a P-Mn illustrated in Fig. 2C, the membrane potential dependence of a strychnine-insensitive hyperpolarizing potential produced by cortical stimulation was examined. After the injection of strychnine, an EPSP followed by IPSPs was evoked, illustrated at the resting membrane level (0 nA). By displacing the membrane potential toward depolarization (4, 6 and 8 nA), a strychnine-insensitive hyperpolarizing potential was increased in amplitude, and the amplitude of a depolarizing synaptic potential was decreased. By displacement of the membrane potential to hyperpolarization, the amplitude of a depolarizing component was increased, and a hyperpolarizing component was decreased in amplitude ( - 2 , - 4 , - 6 , - 8 and - 1 0 n A ) . The reversal point of a strychnine-insensitive IPSP could not be measured by the injection of - 1 0 n A into a cell, suggesting that inhibitory synapses producing a strychnine-insensitive IPSP (long-lasting IPSP, L-IPSP) are located on dendritic branches. 2.4. EFFECTOF PICROTOXINON THE LONG-LASTINGIPSP The effect of i. v. administered picrotoxin on a strychnine-insensitive IPSP was explored in P-Mns and the results are demonstrated in Fig. 3. After the injection of strychnine, stimulation of either the Cx or the lingual nerve produced an EPSP, which generated spikes, followed by a strychnine-insensitive IPSP (B~ and Ca), because the excitatory influence of cortical or lingual nerve stimulation was masked by a strychnine-sensitive IPSP. The records Bb~ and Cb show the responses of this cell to a single shock delivered to the Cx and the lingual nerve 50 min after the injection of

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FIG. 3. Effect of picrotoxin on a strychnine-insensitive hyperpolarizing potential in a P-Mn. A: Antidromic spike. Ba and C~: Cortically and lingually induced PSPs after the injection of strychnine. Bbm and Cb: Responses to a single shock delivered to the Cx and the lingual nerve 50 min after the injection of picrotoxin. Responses of the cell to a single shock delivered to the Cx 60 min after the injection of picrotoxin are shown in Bb2, D~ and D2: AHP produced by a single and a brief train (5 msec interval) shock. E: Duration of cortically induced EPSPs (ordinate) as a function of the time after the injection of picrotoxin (abscissa). The results in A-E are from the same cell. (From Takata and Tomomune, 1987.)

picrotoxin (3 mg/kg, i. v.). In both cases the injection of picrotoxin elongated the duration of depolarizing synaptic potentials, and stimulation of either the Cx or the lingual nerve evoked 4 spikes or 2 spikes in this cell, suggesting that a strychnine-insensitive IPSP evoked in P-Mns by cortical or lingual nerve stimulation is assumed to be, in part the GABA-IPSP. The resting membrane potential of a P-Mn demonstrated in Fig. 3 remained stable for 62 min at 58 mV after the injection of picrotoxin, The results obtained from responses of a P-Mn to cortical stimulation after the injection of picrotoxin are summarized in Fig. 3E, in which the duration of depolarizing synaptic potentials (ordinate) was plotted against the time after the injection of picrotoxin (abscissa). Responses to 3-7 trials were recorded and are demonstrated by open circles, An apparent increase in the duration of cortically induced depolarizing synaptic potentials occurred after the injection of picrotoxin, reaching a plateau (about 37 msec) at 45 min after the injection and stayed nearly constant. Responses of the cell to a single shock delivered to the Cx 60 min after the injection of picrotoxin are shown at slower sweep speeds in the record Bb2. In the presence of a GABA antagonist, a single shock delivered to the Cx elicited a burst of spikes (4 spikes) followed by an enhanced hyperpolarizing potential. The peak amplitude of this enhanced long-duration hyperpolarizing potential (L-HP) after the administration of picrotoxin was about two times greater than that of a hyperpolarizing potential before the

389

I N H I B I T O R Y POSTSYNAPT1C P O T E N T I A L S

administration of picrotoxin. In addition, the amplitude of the L-HP was larger than that of the summated AHP elicited by a brief train of action potentials, illustrated in D2. The record D, shows the AHP elicited by a single shock of the medial branch of the hypoglossal nerves.

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2.5. MEMBRANEPOTENTIALDEPENDENCEOF THE LONG-DURATIONHYPERPOLARIZINGPOTENTIAL We have demonstrated that strychnine-insensitive hyperpolarizing potentials were formed by a combination of the GABA-IPSP and the L-HP. In the following study, the membrane potential dependence of an L-HP was examined in a P-Mn and the results are shown in Fig. 4. The records b l , ct and d, show EPSPs followed by an L-HP 120min after the injection of picrotoxin in cats treated with strychnine by stimulation of the Cx, the inf. alv. n. or the lingual nerve. In the experiment both the lingual and the inf. alv. n. were stimulated with an intensity of 20 times the nerve threshold. Figure 4B shows the amplitude change of the L-HP set up by cortical (open circles) or lingual nerve (filled circles) stimulation as a function of the amount of injected hyperpolarizing current. As illustrated, an L-HP produced by cortical or lingual nerve stimulation remained relatively insensitive to hyperpolarization, suggesting that inhibitory synapses for the L-HP are preferentially situated in the dendritic region of the hypoglossal motoneurons. 2.6. MEMBRANEPOTENTIALDEPENDENCEOF TIIEEPSP In order to examine the membrane potential dependence of cortically induced EPSPs, it is necessary to prevent contamination by hyperpolarizing synaptic potentials following EPSPs. As illustrated in Fig. 5, after blockage of an S-IPSP by the administration of strychnine i. v., stimulation of either the Cx or the inf. alv. n. produced EPSPs, which generates spikes, followed by an L-IPSP (A2 and A3). When a

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mv FIG. 4. A long-duration hyperpolarizing potential in a P-Mn. A: Long-duration hyperpolarizing potential, a: Antidromic spike, b~, c t and d~: Long-duration hyperpolarizing potential (L-HP) produced by stimulation of the Cx, the inf. alv. n. and the lingual nerve 120 min alter the injection of picrotoxin in cats treated with strychnine, b2,C 2 and d2: Field potentials. B: Amplitude change of the L-HP set up by cortical (open circles)or lingual nerve (filledcircles)stimulation as a function of the amount of injected hyperpolarizing current. The results in A and B are Prom differentcells. (From Takata and Tomomune, 1987.)

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FiG. 5. Membrane potential dependence of an EPSP in a P-Mn. A: Suppression of the IPSP. At: Antidromic spike. Cortically induced and inferior alveolar-induced PSPs after the administration of strychnine are shown in A2and A3. A4: Suppression of cortically induced IPSPs when a single shock was delivered to the inf. alv. n. preceding cortical stimulation by 100 msec. B: Effect of membrane polarization on an EPSP. Cortically induced IPSPs were suppressed by a shock delivered to the inf. alv. n. preceding cortical stimulation by 100 msec. C: Change in amplitude of a cortically induced (open circles) and an inferior alveolar-induced EPSP (open triangles) as a function of the amount of injecting depolarizing current. The results in A-C are from the same cell. (From Takata and Tomomune, 1987.) single shock was delivered to the inf. alv. n. preceding a test Cx volley by 100msec, cortically induced L-IPSPs were effectively suppressed, while excitation was unaffected (A,). As a result, the duration of a cortically induced EPSP was elongated from 20 to about 37 msec, suggesting that the inf. alv. n. shock suppressed the ability of the neuron to produce L-IPSPs in response to the Cx shock. In the next study, the relation between the amplitude of a cortically induced EPSP and the membrane potential displacements was examined and the results are shown in Fig. 5B and C. As illustrated in Fig. 5B, the responses to 5 trials were averaged. In these averaged records a cortically induced GABA-IPSP was suppressed by a single shock delivered to the inf. alv. n. preceding a test Cx volley by 100msec. Numerals denote the amounts of injected current in nanoamperes. By displacing the membrane potential toward depolarization, both cortically and inferior alveolar-induced depolarizing synaptic potentials were decreased in amplitude. The results are represented graphically in Fig. 5C, in which the amplitude change of a cortically induced (open circles) and an inferior alveolar-induced EPSP (open triangles) was plotted against the amount of injected current. Since an abortive spike was superimposed on an EPSP in the record at a 2 and 4 nA depolarizing current, the amplitude of an EPSP was measured from the record at a 6 nA depolarizing current. As a spike was seen when the membrane was hyperpolarized by injecting inward direct current ( - 8 nA), the change in amplitude of an EPSP under hyperpolarization could not be examined. Finally, we have demonstrated that the amplitude of both a cortically induced EPSP and an inferior alveolar-induced

390

M. TAKATA

EPSP was decreased greatly with depolarization and reached nearly zero by injecting outward direct current with 26 nA into a cell. 2.7. LOCALIZATION OF EXCITATORY AND INHIBITORY SYNAPSES

In the major fraction of P-Mns it has been reported that lingual nerve stimulation produced the IPSP or the complex postsynaptic potentials that appeared to consist of both EPSPs and IPSPs at differing latencies (Takata, 1981). In addition, we have demonstrated that IPSPs consisting of a short- and long-lasting component (the S- and L-IPSP) were produced in some P-Mns by stimulation of either the lingual nerve or the inf. alv. n. (Takata and Ogata, 1980; Takata and Tomomune, 1986). In two components of IPSPs it was shown that the S-IPSP was reversed to a depolarizing potential by membrane hyperpolarization and the L-IPSP was relatively insensitive to the injected current. From those findings it was suggested that the L-IPSP is generated in dendrites, while the S-IPSP is generated at the soma (Fig. 6.). In addition, the long-duration hyperpolarizations have also been observed in neurons of the olfactory cortex, and Satou et al. (1982) presented evidence that this hyperpolarization may be due to an increase in potassium conductance. Recently, the role of the S-IPSP on P-Mns was studied and we found that the IPSP elicited in a genioglossus motoneuron during swallowing was sensitive to chloride ions and to membrane polarization, suggesting that inhibitory synapses situated on the somatic region on the genioglossus motoneurons play an important role in swallowing (Tomomune and Takata, 1988). In the spinal cord the location of excitatory synapses formed by a single group la axon with a motoneuron has been investigated by horseradish peroxidase injection into the Ia axon (Burke et al., Cx, P

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FIG. 6. Synaptic contacts on hypoglossal motoneurons. A, B and C: Inhibitory interneurons. D: Excitatory interneurons. Mn: Hypoglossal motoneuron. Cx and P: Cortex and peripheral nerves. Filled and open triangle: Inhibitory and excitatory synapses. 1 and 2: Strychnine-sensitive synapses. 3 and 4: Picroto×in-sensitive synapses. 5: Strychnine and picrotoxin-insensitive synapses. 1, 3 and 5: Inhibitory synapses generating the S-IPSP, the L-IPSP and the L-HP. 6: Excitatory synapses generating the EPSP.

1979; Brown and Fyffe, 1981). It was demonstrated that group Ia synapses are located on both the proximal dendrite and distal dendritic branches, and the number of synaptic contacts per motoneuron range from 2-5 with a mean of 3.8 (Brown and Fyffe, 1981) or from 3-18 with an average of 7 (Burke et al., 1979; see review by Redman, 1990). With respect to the membrane potential dependence of polysynaptic EPSPs, Klee (1975) demonstrated that polysynaptic EPSPs increased greatly with hyperpolarization and showed some decrease with depolarization. Llin~s and Sugimori (1980) found that a climbing fiberevoked EPSP in a Purkinje cell was reversed to a hyperpolarizing potential by outward direct current injection (22.1 nA), while recording intracellularly from a cerebellar slice. Recently, Hestrin et al. (1990) have studied the properties of the excitatory postsynaptic current (EPSC) in the CA 1 region of rat hippocampus in vitro and demonstrated voltage-dependent properties of the hippocampal EPSC. They also demonstrated that hippocampal EPSCs are consistent with relative contribution of N M D A (the Nmethyl-D-aspartate) and non-NMDA components, whereas the EPSCs onto Purkinje cells are mediated by a non-NMDA type (Perkel et al., 1990). In experiments we have demonstrated that when a single shock was delivered to the inf. alv. n. preceding a test Cx volley by 100 msec, the duration of a cortically induced EPSP was elongated from 20 to about 37 msec, suggesting that inf. alv. n. stimulation suppressed the cortically induced IPSP, while the EPSP was unaffected. Therefore, we could test the membrane potential dependence of the cortically induced EPSP and found that the cortically induced EPSP was relatively sensitive to the membrane polarization, suggesting that excitatory synapses for the cortically induced EPSP are situated more proximally on the soma-dendritic tree in the hypoglossal motoneurons. From the basis of bouton and vesicle size, shape and morphological features, five types of synaptic boutons were identified in hypoglossai motoneurons on the rat (Boone and Aides, 1984b). They were S-boutons (spherical vesicles), F-boutons (flattened vesicles), P-boutons, C-boutons and T-boutons. Boone and Aides (1984b) demonstrated that Sboutons were the predominant type found on dendrites, and F-boutons were more prevalent on motoneuron somata. From the sensitivity of PSPs to the injected current, it is suggested that inhibitory synapses generating the S-IPSP (synapse marked 1 in Fig. 6) are directly chiefly at motoneuron somata and excitatory inputs (synapse marked 6 in Fig. 6) largely onto the dendritic tree. If one accepts the argument on the relation between the shape of synaptic vesicles and the functional activity of the terminal, F- and S-boutons are related to inhibitory and excitatory neurotransmission (Uchizono, 1965; Gottlieb and Cowan, 1972; Nakajima and Reese, 1983). After blockage of the S-IPSP (glycine-IPSP) by strychnine administration, stimulation of the Cx, the inf. alv. n. or the lingual nerve produced an EPSP, which generated spikes, followed by an enhanced hyperpolarizing potential, indicating that strychnine probably disinhibits the inhibitory interneurons responsible for generating the GABA-IPSP (synapse marked 2 in Fig. 6), as reported in the trigeminal

INHIBITORY POSTSYNAPTICPOTENTIALS

motoneurons (Takata and Fujita, 1979). We have demonstrated that strychnine-insensitive hyperpolarizing potentials were formed by a combination of the GABA-IPSP (L-IPSP) and the L-HP. From the sensitivity of the GABA-IPSP to the injected current, it is suggested that inhibitory synapses generating the L-IPSP (synapse marked 3 in Fig. 6) are situated proximally on the dendritic tree. Inhibitory neurotransmitter, glycine and GABA, associated with F-boutons (Matus and Dennison, 1971) and pleomorphic vesicles (McLaughlin et aL, 1974) have also been demonstrated in the hypoglossal nucleus (Okada et al., 1971; Zarbin et al., 1981). By light and electron microscopic immunochemistry with an antibody directed against GABA, Takasu el al. (1987) investigated the location of inhibitory synapses on the hypoglossal nucleus of the monkey, and suggested that GABAergic inhibition in the monkey hypoglossal nucleus occurs mainly on the dendrites of the motoneurons. In addition, in the hypoglossal nucleus small neurons have been described in the monkey (Cooper, 1981) and rat (Boone and Aides, 1984a). With respect to the properties of these small neurons, Takasu and Hashimoto (1988) demonstrated by a combined Golgi-electron microscopic technique that small neurons in the hypoglossal nucleus in the rat are a short-axoned Golgi type II interneuron and suggested that these neurons are GABAergic inhibitory interneurons. In addition, we have demonstrated that a single shock delivered to the Cx, the inf. alv. n. or the lingual nerve elicited a burst of spikes followed by an enhanced L-HP in the presence of GABA antagonists (Curtis et al., 1971a, b), suggesting that picrotoxin administration probably disinhibits the inhibitory interneurons responsible for generating the L-HP (synapse marked 4 in Fig. 6). In the hippocampal pyramidal cells it was also demonstrated that bicuculline administration increases the size of the late hyperpolarizing potential, and suggested that bicuculline blocks GABA-mediated inhibition on interneurons, and thereby increases the excitation of interneurons generating the late hyperpolarizing potential (Newberry and Nicoll, 1984). It is known that the effect of GABA is mediated by at least two classes of receptors, GABAA and GABAB (Hill and Bowery, 1981). Bowery et al. (1984) have proposed that GABAA receptors correspond to the site that is antagonized by bicucuiline, whereas GABAB receptors are bicuculline-insensitive but specifically activated by baclofen. Yoon and Rothman (1991) examined the effects of GABA and baclofen on preand postsynaptic membrane conductances in dissociated rat hippocampal cells and demonstrated that the presynaptic GABA B effect responsible for synaptic modulation has a pharmacological profile similar to the postsynaptic GABA B effect. In neocortical pyramidal cells, it was demonstrated that a short-latency, fast inhibition (the f-IPSP), antagonized by bicuculline and picrotoxin, is identical to the inhibition which is thought to be mediated by GABA (Krnjevic, 1984) and is mediated by an increase in chloride conductance. In contrast, it was demonstrated that a long-lasting, more prolonged inhibition (the I-IPSP) is mediated by a relatively small increase in conductance which is probably

39 !

potassium-specific (Avoli, 1986; Howe et ai., 1987), and it is not reduced by bicuculline and picrotoxin. Also, Connors et al. (1988) concluded that in pyramidal cells in in vitro neocortical slice, the f-IPSP provides robust suppression of activity with fine temporal control, and the I-IPSP increases the threshold for spiking activity and decreases adapted firing rates. The fact that the time course and amplitude of the L-HP were slower and larger than that of the AHP and picrotoxin administration increased the size of the L-HP suggest that the L-HP is probably a hyperpolarizing potential produced by interneurons. In the hippocampal pyramidal cells Newberry and Nicoll (1984) proposed that a large portion of the synapses responsible for generating the late hyperpolarizing potential terminate on the dendrites of pyramidal cells. In the present study we have demonstrated that the L-HP is relatively insensitive to the injected current, suggesting that the synapses for the L-HP are preferentially situated in the dendritic region of the hypoglossal motoneurons (synapse marked 5 in Fig. 6). If synaptic contacts producing the EPSP, the LIPSP and L-HP are located on dendritic branches at different electronic distances from the soma, then the propagation of the synaptic potential generated at these contacts to the soma would undergo different amounts of electronic attenuation. Functionally, it is suggested 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. The reduction of the effectiveness of excitatory synapses by inhibitory synapses situated on the dendritic region of the cell may be involved in producing complex tongue movements.

3. TWO COMPONENTS OF IPSPs IN HYPOGLOSSAL MOTONEURONS 3.1. LINGUALLYINDUCEDPSPs IN TONGUEPROTRUDER AND RETRACTORMOTONEURONS The patterns of postsynaptic potentials (PSPs) produced in the P- and the R-Mns by lingual nerve stimulation were explored. In some cases, depolarization of the membrane potential by penetration of a micropipette into a cell masked EPSPs. In such a case, by injecting a constant hyperpolarizing current into a cell, the membrane potential was kept at the resting membrane potential. From the patterns of PSPs produced in the P-Mns by lingual nerve stimulation, the P-Mns were divided into three types. In the first and the second type of P-Mns, lingual nerve stimulation produced an IPSP and an IPSP-IPSP sequence. An EPSP-IPSP sequence was produced in the third type of P-Mns. In 250 explored P-Mns, the percentage of neurons responding with the IPSP, the IPSP-IPSP sequence and the EPSP-IPSP sequence to lingual nerve stimulation was 40%, 30% and 30%, respectively (Takata, 1981b). As for the case of P-Mns, the R-Mns were also divided into three types from the PSPs set up by

392

M. TAKATA

lingual nerve stimulation. There were an EPSP, an E P S P - I P S P sequence and an I P S P - I P S P sequence. In 200 explored R-Mns, the percentage of neurons responding with the EPSP, the E P S P - I P S P sequence and the I P S P - I P S P sequence was 7%, 68% and 25%, respectively (Takata, 1981b). 3.2. SEPARATIONOF TWO COMPONENTS OF IPSPs BY STRYCHNINE ADMINISTRATION In the P-Mns responding with the I P S P - I P S P sequence to lingual nerve stimulation, separation of the S- and the L-IPSP by strychnine administration was carried out. The IPSPs produced in a P-Mn by stimulation of the ipsilateral lingual nerve (ipsi-L) are demonstrated in Fig. 7A. When the ipsi-L (aj) was stimulated with stimulus intensities of four times the nerve threshold (4T), which is supramaximal for A# fibers, double IPSPs were produced in this P-Mn. After the injection of strychnine intravenously (0.08 mg/kg), an S-IPSP set up by ipsi-L stimulation was depressed. Records a 2 and a3 show the postsynaptic potentials immediately and 3 min after the injection of strychnine. The record of a3 is shown at slower sweep speeds in a4. After the administration of strychnine the rising phase of IPSPs became slower, and 3 min after the injection an S-IPSP was completely blocked, and only an L-IPSP remained, as illustrated in a3. In Ab is shown the IPSPs evoked in a P-Mn by ipsi-L stimulation before (tracing A) and after (tracing B) the

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It was reported that IPSPs produced in many P-Mns and R-Mns by stimulation of either the ipsilateral or the contralateral lingual nerve (ipsi-L or contra-L) were formed by a combination of the S- and the L-IPSP. To investigate the properties of each of the two components of the IPSPs, responses of a R - M n to double shock stimulation of the ipsi-L were examined (Fig. 8A). Record A~ shows an antidromic spike of a A

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injection of strychnine. The time course of the S-IPSP can be reconstructed by subtracting the amplitude of tracing B from that of tracing A, as illustrated in Ac by open circles. Here are shown the two components of IPSPs set up by ipsi-L stimulation. The postsynaptic potential demonstrated by solid circles denotes the time course of an L-IPSP. F r o m these tracings it was found that the S-IPSP with a time-to-peak of about 8 msec was about 70 msec in duration and that the time-to-peak of the L-IPSP was about 15 msec. 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. 7B). Record Ba shows an antidromic spike of an R - M n 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 produced only an L-IPSP (b2). By subtracting the L-IPSP (b2) from the control IPSP (b 0, the time course of the S-IPSP was obtained (B c, open circles). The duration of the S-IPSP was about 80 msec, and the solid circles in Be show the time course of the L-IPSP.

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FIG. 7. Two components of IPSPs in protruder and retractor motoneurons. A: IPSPs in a P-Mn. Series a shows the PSPs after ipsi-L stimulation. 1: Control IPSPs. 2 and 3: Immediately and 3 min after injection of strychnine. 4: Records of a 3 at slower sweep speeds, b: Tracings of IPSPs before (A) and after (B) injection of strychnine, c: Time course of the short-lasting (open circles) and the long-lasting IPSP (solid circles). B: 1PSPs in a R-Mn. a: Antidromic spike, b: IPSPs elicited by ipsi-L stimulation, h Control lPSPs. 2:3 min after injection of strychnine, c: Tracings of the short-lasting (open circles) and the long-lasting IPSP (solid circles). (From Takata and Ogata, 1980; Takata, 1982.)

FIG. 8. Separation of two components of 1PSPs in the R-Mns. A: Responses to double shocks of the ipsi-L, a: Antidromic spike, b: Responses to double shocks before (b 0 and after (b2) injection of strychnine, c~: Tracings of 1PSPs before (A) and after (B) injection of strychnine, c2: Time course of the IPSPs. Tracing A shows the IPSP evoked by the second shock before injection of strychnine, and tracing B shows the responses to double shocks after the injection of strychnine. By subtracting the tracing B from tracing A in c~, the time course of the S-IPSP (open circles) was obtained. B: Effect of picrotoxin, a~: Responses of a R-Mn to double shocks of the ipsi-L, a 2 and a3: PSPs 5 and 8 min after injection of picrotoxin, b: 15min after injection, demonstrated with high amplification. (From Takata and Ogata, 1980.)

INHIBITORY POSTSYNAPTICPOTENTIALS

R-Mn. When two shocks separated by a 50-msec interval were applied to the ipsi-L, the first shock produced a large IPSP with a latency of about 3.5 msec, but only a small IPSP was caused by the second shock (b~). After the administration of strychnine (0.08 mg/kg), the first shock produced a longlasting, strychnine-insensitive IPSP with a latency of about 5 msec, but the second shock did not produce any postsynaptic potential (b2). In the tracings of ct are shown the postsynaptic potentials before (A) and after (B) the injection of strychnine. By subtracting the amplitude of (B) from that of (A), the potential illustrated by open circles in c2 was obtained. As already noted, this potential was an S-IPSP. It was noted that the time course of an S-IPSP (open circles in cz) was similar to that of the postsynaptic potential (tracing A in c2) set up by the second shock before the administration of strychnine. Tracing B in c2 shows responses of an L-IPSP to double shock stimulation. These results suggested that, when two shocks were applied to the lingual nerve, the second shock produced only an S-IPSP. In the following study the effect of picrotoxin on the IPSPs produced in a R-Mn by double shocks was examined (Fig. 8B). When double shocks with an 80-msec interval were applied to the ipsi°L, the second shock produced only an S-IPSP (a~). Records a: and a 3 show responses 5 and 8min after the

a-~

, Jlomv ~omsec

393

administration of picrotoxin (7 mg/kg, i. v.). It was found that the postsynaptic potentials set up by the first shock gradually decreased in size, with little change in the second IPSP (az and a3). Responses recorded with high amplification 15 min after the injection of picrotoxin are shown in Bb. The first and second shock produced IPSPs of identical size and duration. In the next study, the IPSPs evoked in a P-Mn by double shocks of the inf. alv. n. were studied and results are demonstrated in Fig. 9. Record A a shows an antidromic spike of a P-Mn. The relation between the amplitude of inferior alveolar-induced hyperpolarizing potentials and of the membrane potential displacements was examined and results are shown in A b. Numerals denote the amounts of injected current in nanoamperes (nA). At the resting membrane level (0 hA), inf. aiv. n. stimulation evoked a hyperpolarizing potential. By displacing the membrane potential toward hyperpolarization, an inferior alveolarinduced hyperpolarizing potential was decreased in amplitude ( - l and - 2 nA) and reversed to a depolarizing potential ( - 3 nA). By displacement of the membrane potential to depolarization, this hyperpolarizing potential was increased in amplitude (1, 2, and 3 nA). Previous studies reported that when double shocks separated by 70-100msec intervals were applied to the lingual nerve, the second shock

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FIG. 9. Inferior alveolar-induced IPSPs. A: Effect of membrane polarization, a: Antidromic spike of a P-Mn. b: Dependence of inferior alveolar-induced PSPs on the membrane potential. Numerals denote the amount of injected current in hA. B: Two components of IPSPs in a P-Mn. a: Antidromic spike, b~ and bz: Responses to double shocks of the ipsilateral and contralateral inferior alveolar nerve, b3and b4: Field potentials corresponding to the record b~ and b2. C: IPSPs in a P-Mn set up by application of double shocks to the ipsilateral inferior alveolar nerve, a I and a2. Before and after injection of strychnine, a3: Field potentials corresponding to the record az. b: Tracings of the PSPs before (A) and after (B) injection of strychnine, in which the PSP illustrated by the open circles was obtained by subtracting tracing B from A. D: PSPs in a R-Mn. al: Responses to double shocks of the ipsilateral inferior alveolar nerve, a2 Field potentials, b~: Responses of a R-Mn to double shocks after injection of strychnine, bz: Field potentials. (From Takata and Tomomune, 1986.)

394

M. TAKATA

produced only an S-IPSP because the first shock depressed the ability of the neuron to produce an L-IPSP in response to the second shock (Takata, 1982; Takata and Ogata, 1980). In the present study, separation of the two components of IPSPs produced in a P-Mn was performed by application of double shocks to either the ipsilateral or the contralateral inf. alv. n. (ipsi-inf. alv. n. or contra-inf, alv. n.). Results are shown in Fig. 9B. An antidromic spike is shown in B~. When two shocks separated by an 80-msec interval at 5.0 x T were applied to the ipsi- or the contra-inf, alv. n. (bj and b2), the first shock produced a large IPSP with a latency of about 4.0 msec from stimulus artifact, but only a small IPSP was evoked by the second shock in this cell. In the records illustrated in Bb, responses to five trials were averaged. The records b 3 and b 4 show extracellular records corresponding to b~ and b2, respectively. To test the blockage of an S-IPSP by strychnine, the effect of strychnine on the two components of IPSPs was examined and results are shown in Fig. 9C. After the administration of strychnine (0.1 mg/kg i. v.), the first shock produced only an L-IPSP and the second shock did not produce any postsynaptic potential (a2). The record a~ shows the responses of the same P-Mn to double shocks before the administration of strychnine. The results illustrated in a~ and a 2 are demonstrated at high magnification in Cb, in which the tracings A and B correspond to the record a~ (before the injection of strychnine) and a2 (after the injection of strychnine). In Ch, the time course of an IPSP, illustrated by open circles, was obtained by subtracting tracing B from tracing A, Note that the time course of an IPSP was similar to that of an IPSP produced by the second shock before the injection of strychnine. A sample record of a R-Mn responding with mainly an S-IPSP to inf. alv. n. stimulation is shown in a~ of Fig. 9D. When double shocks separated by a 90-msec interval at 5.0 x T were applied to the ipsi-inf, alv. n., both the first and second shocks produced a large IPSP in this cell. In this record, responses to five trials were averaged. In a R-Mn illustrated in b~ of Fig. 9D, after the administration of strychnine, the first shock produced an L-IPSP, but the second shock did not produce any postsynaptic potential when double shocks were applied to the ipsi-inf, alv. n. The results suggested that, when two shocks separated by 80-90 msec intervals were applied to the inf. alv. n. the second shock produced only an S-IPSP. Therefore, it was possible to obtain the time course of an L-IPSP by subtracting the IPSP produced by the second shock from the IPSP produced by the first shock.

4. PERCENTAGE MAGNITUDE OF THE S- AND THE L-IPSP IN HYPOGLOSSAL MOTONEURONS 4.1. S% AND L% IN LINGUALLYINDUCEDIPSPs 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. 10): double shocks separated by a

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FIG. 10. Two components of IPSPs in retractor motoneurons. A: IPSPs set up by double shocks of the ipsilateral lingual nerve. B: Tracings of the short-lasting (tracing 2) and the long-lasting IPSP (tracing 3). Tracing 1 denotes IPSPs elicited by the first shock of the ipsi-L Marks S and L indicate the maximum amplitude of the S- and the L-IPSP. (From Takata, 1982.) 100-msec interval at 5.0 × T were applied to the ipsi-L to produce the IPSPs in a R-Mn shown in A. 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 B. 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 I, we obtained tracing 3 (dashed line), which indicates the time course and magnitude of the L-IPSP. 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%) components of the lingually induced IPSP by the formula:

S/(S + L) x 100 = S% and 1 0 0 - S% = L%, where S = the maximum amplitude of the S-IPSP (mV) and L = the maximum amplitude of the L-IPSP (mV). By this calculation, the percentage magnitude of the S- and the L-IPSP in this R-Mn was determined to be 61 and 39%, respectively. The S% and the L% in 42 R-Mns obtained from a single experiment are illustrated in Fig. I1A. In these experiments, the maximum amplitude of the Sand the L-IPSP components was obtained by application of double shocks separated by a 100-msec interval to the lingual nerve. The histograms on the left side labeled Ipsi Ling. N. indicate the S% (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 S% and the L% 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 S% and the L% was 70% (N = 42) and 30%

INHIBITORY

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4.2. S % AND L % IN INFERIOR ALVEOLAR-INDUCEDIPSPs

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395

POTENTIALS

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%s %S L Fro. I I. Percentage magnitude of lingually induced short (S %)- and long (L %)-lasting IPSPs. The abscissae S and L indicate the S% and the L%, which are graphed by the open and solid columns. The ordinate N indicates the number of cells. An arrow indicates the average of the S% and L%. A: Retractor motoneurons. B: Protruder motoneurons. (From Takata, 1982.)

(N =42), respectively (arrow). With respect to the IPSPs elicited by contra-L stimulation, it was found that the average of the S % and L % was 21% ( N = 3 6 ) and 79% ( N = 3 6 ) , respectively (arrow). It was also found that in 7 of 42 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 contra-L, which generate the L-IPSP. In the following study P-Mns were examined. Figure 11B illustrates the S % and the L % calculated for the 31 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 53% ( N = 3 1 ) and 47% ( N = 3 1 ) , respectively (arrow). In the IPSPs set up by contra-L stimulation, the average of the S % and the L % was 51% (N = 31) and 49% (N = 31), respectively (arrow). These results indicate that the 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.

As reported on the lingually induced IPSP, the percentage magnitude of the S-IPSP (S%) and the L-IPSP (L%) components of the inferior alveolarinduced IPSP was calculated. Figures 12A and 12B illustrated the S % and the L % calculated for the R-Mns and P-Mns. The maximum amplitude of the S- and the L-IPSP in inferior alveolar-induced IPSPs was obtained by application of double shocks separated by 80-90 msec intervals to the inf. alv. n. The histograms on the left labeled IPSlINFALV N. indicate the S % (open column with abscissa S) and the L % (solid column with abscissa L) of the IPSPs evoked by ipsi-inf, alv. n. stimulation, and those on the right labeled CONTRA INF ALV N are the S% and the L% of the IPSPs evoked by contra-inf, alv. n. stimulation. The ordinate N indicates the number of cells. From the histograms obtained from R-Mns (Fig. 12A), we found that (in the IPSPs set up by ipsi-inf, alv. n. stimulation) the average of the S % and the L % was 59% (N = 22) and 41% (N = 22), respectively (arrow). With respect to the IPSPs evoked by contrainf. alv. n. stimulation, the average of the S % and the L % was 64% (N = 18) and 36% (N = 18), respectively (arrow). The results indicate that the R-Mns responding with two components of IPSPs to inf. alv.

A

N

N

N

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FIG. 12. Percentage magnitude of inferior alveolar-induced S- and L-IPSPs. The abscissae S and L indicate the S% and the L %, which are graphed by the open and solid columns. The ordinate N indicates the number of cells. An arrow indicates the average of the S% and L%. A: Retractor motoneurons. B: Protruder motoneurons. (From Takata and Tomomune, 1986.)

396

M.

TAKATA

n. stimulation received afferent fibers from the bilateral inferior alveolar nerves to generate the S- and the L-IPSP. From the histograms obtained from P-Mns (Fig. 12B), it was found that, in the IPSPs set up by ipsi-inf, alv. n. stimulation, the average of the S % and the L% was 23% (N =42) and 77% (N =42), respectively (arrow). With respect to the IPSPs evoked by contra-inf, alv. n. stimulation, we found that the average of the S % and the L % was 30% (N = 20) and 70% (N = 20), respectively (arrow). The results indicate that the P-Mns responding with two components of IPSPs to inf. alv. n. stimulation were principally innervated by afferent fibers in both the ipsi- and contra-inf, alv. n. which generate the L-IPSP.

changes of the cell body (Brattg~rd et al., 1957; Watson, 1965; Cull, 1974; Wooten et al., 1978; Rotter et al., 1979; Aldskogius et al., 1980, 1984; Arvidsson and Aldskogius, 1982; Aldskogius and Sevensson, 1988; Snider and Thanedar, 1989). From morphological studies on axotomized hypogiossal motoneurons, it is expected that recording of PSPs at selected postoperative times, when reduced incidence of synapses would be restricted to somatic regions in axotomized hypoglossal motoneurons, enables us to separate inhibitory synapses located on cell somata and dendrites. However, the precise mechanism underlying the loss of efficacy at chemical synapses in axotomized hypoglossal motoneurons is unclear, and it is not known whether muscle reinnervation itself is necessary for synapse recovery. 5.1. FIRINGOF AXOTOMIZEDMOTONEURONS

5. AXOTOMIZED HYPOGLOSSAL MOTONEURONS Morphological studies have revealed that the majority of hypoglossal motoneurons exhibited chromatolysis at the end of the first week following axotomy (Flumerfelt and Lewis, 1975) and several quantitative studies have shown that by 1 week after axotomy there is a loss of about 50% of the synapses on the hypoglossal motoneurons (Watson, 1965; Sumner and Watson, 1971; Sumner and Sutherland, 1973; Cull, 1975; Sumner, 1975a, b, c; Rotter et al., 1979). Moreover, in the hypoglossal motoneurons axotomy is followed by chemical and morphological

2 "= 2'

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At the first step the firing of protruder motoneurons innervating the protruder tongue muscles was investigated. The axotomized P-Mns were identified from the antidromic spike evoked by stimulation of the previously severed protruder fibers. Responses of an axotomized P-Mn 7th postoperative day to double shock stimulation of the protruder fibers are shown in Fig. 13A. When two shocks separated by 5 msec (A4) and 4 msec intervals (A3) were applied to the protruder fibers, the second antidromic volley produced a spike. When the interval of double shocks was shortened to 3 msec (A2), the second shock still produced an antidromic spike,

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FIG. 13. Axotomized protruder motoneurons. A: Failure of separation of the initial segment from the soma-dendritic component. B: Lingually induced PSPs in a control P-Mn in unoperated side. h Antidromic spike. 2 and 3: Responses to double shock stimulation of the ipsi-L and the contra-L. C: Lingually induced PSPs in two different P-Mns 24 days after axotomy (C~ and C0. 1: Antidromic responses. 2 and 3: Responses to double shock stimulation of the ipsi-L and the contra-L. D: Lingually induced PSPs in two different P-Mns 40 days after axotomy (D~ and Db). 1: Antidromic responses. 2 and 3: Responses to a stimulus of the ipsi-L and the contra-L. (From Takata et al., 1980; Takata and Nagahama, 1983.)

INHIBITORY POSTSYNAPTICPOTENTIALS

and the inflexion was less prominent than in normal motoneurons. Even when the interval of double shocks was shortened to 2.5 msec (At), no separation of the initial segment (IS) from the soma-dendritic (SD) component was obtained, indicating that axotomized P-Mns have a high safety factor for impulse transmission from the initial segment to the somadendritic membrane, as already reported in axotomized spinal motoneurons (Eccles et al., 1958; Kuno and Llinfis, 1970b). In a majority of the P-Mns in normal cats there is no spontaneous activity, however, 20 out of 49 P-Mns examined on the 12th to 21st postoperative day fired spontaneously, and some neurons fired with grouped discharges (Takata et al., 1980). In chromatolyzed spinal motoneurons it has been reported that spikelike potential responses were often seen riding on monosynaptic EPSPs evoked by muscle afferent volleys and such partial responses seemed to originate from dendrites at some distance from the cell body (Kuno and Liinfis, 1970a). The finding that many P-Mns fired with double or burst discharges spontaneously after section of the hypoglossal nerves indicates that the axotomized motoneurons may also have multiple sites for the initiation of spikes as noted in chromatolyzed spinal motoneurons (Kuno and Llinfis, 1970a). 5.2. LINGUALLY INDUCED PSPs IN AXOTOMIZED MOTONEURONS

With respect to a disjunction of presynaptic contacts on axotomized rat hypoglossal motoneurons, Sumner (1975a) observed shrinkage of dendrites, and a reduced incidence of dendritic synapses. Sumner and Sutherland 0973) have also found that boutons with symmetrical synapses decreased in numbers from 7-35 days on cell somata, and from 14-49 days in the neuropil. Morphological work has also revealed that after transection of the lingual nerve there are no changes on bouton numbers as compared with numbers of synaptic boutons in normal hypoglossal motoneurons, but the restoration of bouton numbers from detachment of synapses after axotomy was inhibited significantly by lingual nerve division (Cull, 1975). Responses of P-Mns 24 days after axotomy to double shock stimulation of the ipsi-L and the contra-L were explored (Fig. 13C). Figure 13B shows responses of a P-Mn in the unoperated side (control P-Mn) to double shock stimulation of the ipsi-L (B2) and the contra-L (B3). Record B~ shows an antidromic spike of a control P-Mn. Results obtained from two different P-Mns in the operated side (axotomized P-Mn) in the same animal are shown in Fig. 13Ca and Cb. In P-Mns 24 days after axotomy, stimulation of the previously severed fibers produced a large late depolarization following an antidromic spike (a~ and bt). Responses of these two axotomized P-Mns to double shock stimulation of the ipsi-L (a2 and b2) and the contra-L (a3 and b3) at 70msec intervals are illustrated. In many axotomized P-Mns stimulation of the lingual nerve produced an EPSP-IPSP sequence. In a P-Mn illustrated in Cb, an EPSP-IPSP sequence was produced by stimulation of either the ipsi-L (b2) or the contra-L (b3). Results

397

indicate that the amplitude of an S-IPSP produced in control P-Mns was about 2.0 times greater than that in axotomized P-Mns. By subtracting the amplitude of an IPSP set up by the second shock from that of an IPSP set up by the first shock, the maximum amplitude of an L-IPSP was measured (Takata, 1982). From this measurement it was found that synaptic efficacy of inhibitory synapses for the LIPSP was also declined in the P-Mns 24 days after axotomy. In the present experiments, satisfactory recordings were obtained from 20 P-Mns having an antidromic spike of 60-75 mV in both the unoperated and the operated side, and the maximum amplitude of lingually induced IPSPs in control P-Mns (n = 20) was about 1.7 times (average) greater than that in axotomized P-Mns (n = 20). In P-Mns 40 days after axotomy, an EPSP-IPSP sequence was produced in 16 out of 17 explored P-Mns by stimulation of the lingual nerve, and records obtained from two different P-Mns are shown in Fig 13D~ and Db. In P-Mns 40 days after axotomy, stimulation of the previously severed protruder muscle fibers produced a large late depolarization following an antidromic spike (at and b~). Responses of these two axotomized P-Mns to stimulation of the ipsi-L (a2 and b2) and the contra-L (a3 and b3) are illustrated. We have demonstrated that the maximum amplitude of lingually-induced IPSPs in control PMns is always larger than that of axotomized P-Mns. In the present study, it was found that the amplitude of the S- and the L-IPSP produced in the P-Mns 24 days after axotomy by lingual nerve stimulation was significantly reduced in size as compared with control P-Mns. With respect to the S-IPSP, the amplitude of the S-IPSP produced in control P-Mns was about 1.7 times greater than that of axotomized P-Mns. This finding suggests that synaptic efficacy of inhibitory synapses for the S-IPSP in the ipsilateral lingual nerve pathway may be declined in the P-Mns 24 days after axotomy. In the P-Mns 24 days after axotomy, it was also demonstrated that the amplitude of the L-IPSP produced in control P-Mns was greater than that of axotomized P-Mns, suggesting that synaptic efficacy of inhibitory synapses for the LIPSP may be declined in the P-Mns 24 days after axotomy. In most P-Mns 40 days after axotomy a large EPSP and a spike was produced by stimulation of either the ipsi- or the contra-L, indicating that synaptic efficacy of inhibitory synapses was declined. The results suggest that in hypoglossal motoneurons, axotomy is followed by the decline of synaptic efficacy of inhibitory rather than of excitatory synapses as found in axotomized trigeminal motoneurons (Takata and Nagahama, 1986). 5.3. CORTICALLY INDUCED PSPs IN AXOTOMIZED MOTONEURONS

Anatomical investigations have settled the presence of axonal projections from the cerebral cortex to the trigeminal sensory nucleus (Walberg, 1957; Kuypers, 1958, 1960; Wold and Brodal, 1973, 1974). Following lesions of the orbital and procreate gyri, Wold and Brodai (1973) found that the cortical fibers from both gyri terminate mainly in the ventral most part of the nucleus interpolaris. In addition, they have also

398

M. TAKATA

demonstrated that the degenerating fibers from the primary sensorimotor and the primary motor cortical areas were mainly found contralaterally and appear to end in the rostral part of the trigeminal complex, in the nucleus principalis and the rostral part of the nucleus oralis (Wold and Brodal, 1974). In addition, there is no anatomical and physiological evidence on monosynaptic projections from the cerebral cortex or the periphery to the hypoglossal motoneurons (Szentfigothai, 1948; Walberg, 1957; Kuypers, 1958; Porter, 1967). In the present experiments, we have demonstrated that the latency of cortically induced EPSP and IPSP was about 2.0. msec (_+0.2msec) and 2.5msec (_+0.2msec), suggesting that cortically induced EPSP and IPSP is probably disynaptic and trisynaptic as the shortest pathway. The PSPs produced in axotomized P-Mns on the fight side by stimulation of the orbital gyrus on the left side were explored (Fig. 14). A stimulating electrode to the cortical surface in operated cats was fixed at the orbital gyrus as demonstrated in A. Even when dealing with polysynaptic PSPs, it is possible to make qualitative estimates of synaptic potentials in normal and axotomized P-Mns. However, such a comparison of PSP pattern demands that the membrane potential of the cells being compared be within the same range. Therefore, the results are based on data obtained from normal and axotomized P-Mns having an antidromic spike of 60-70 inV.

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FIG. 14. Cortically induced postsynaptic potentials in protruder motoneurons. A: Cortically induced PSPs in normal P-Mns. Stimulating site of the orbital gyrus is shown by a dot indicated by an arrow. B, C and D: Cortically induced PSPs in P-Mns 30 (B), 40 (C) and 80 days (D) after axotomy. Histogram showing the number of P-Mns responded with an EPSP (Type A), EPSPs with a short- and a long-latency component followed by IPSPs (Type B), a short-lasting EPSP followed by predominent IPSPs (Type C) and an IPSP (Type D). The ordinate N indicates the number of ceils. (From Takata and Nagahama, 1984.)

The PSPs produced in normal P-Mns on the right side by stimulation of the orbital gyrus on the left side are summarized in A. The relative frequency of the three types of cortically induced PSPs recorded in a sample of 46 normal P-Mns are illustrated in histogram form. The three types of cortically induced PSPs are an EPSP (open, Type A), a short-latency EPSP followed by predominant IPSPs (hatched, Type C) and an IPSP (dotted, Type D), respectively. It is evident that the major fraction of the total sample of normal P-Mns studied showed the IPSPs and a short-latency EPSP followed by predominant IPSPs. Cortically induced PSPs in P-Mns 30 days after axotomy are explored. In 4 out of 14 explored P-Mns, stimulation of the orbital gyrus produced two components of EPSPs (a short- and long-latency component) followed by an IPSP. In normal P-Mns, there were no P-Mns responding with two components of EPSPs composed of a short- and a long-latency component to cortical stimulation. Therefore, the P-Mns responding with two components of EPSPs to cortical stimulation were grouped as the Type B. The relative frequencies of the four types of cortically induced PSPs recorded in 14 P-Mns 30 days after axotomy are shown in the histogram in B. It is evident that the number of P-Mns responded with two components of EPSPs (filled, Type B) to stimulation of the cortical gyrus increased. On the 40th postoperative day, in 27 out of 68 explored P-Mns two components of EPSPs followed by IPSPs were produced by stimulation of the orbital gyrus. As shown in the histogram in C, it is evident that cortical stimulation produced two components of EPSPs followed by IPSPs (filled, Type B) in about 40% of explored P-Mns 40 days after axotomy. In the next studies, the PSPs produced in P-Mns 60 and 80 days after axotomy by cortical stimulation were examined. On the 60th postoperative day, in 11 out of 21 explored P-Mns cortical stimulation produced only EPSPs and the relative frequencies of P-Mns responding with a short-latency EPSP followed by predominant IPSPs was decreased. The results illustrated in D were obtained from 43 P-Mns 80 days after axotomy by stimulation of the orbital gyrus. In the histogram of D it is evident that the major fraction (20 of 43 P-Mns, or about 46%) of the total sample of explored P-Mns showed two components of EPSPs followed by IPSPs (filled, Type B). By comparing the peak amplitude of cortically induced IPSPs in normal P-Mns with that in P-Mns 80 days after axotomy, it was found that the average of the peak amplitude of the IPSP in normal P-Mns (n = 31) was about 1.5 times greater than that of P-Mns 80 days after axotomy (n = 7). In axotomized hypoglossal motoneurons, Sumner (1975b) showed that boutons with spherical vesicles decreased in number after axotomy, whereas those with flat vesicles did not, and suggested that affinity is lost between excitatory boutons and their postsynaptic sites after axotomy, while inhibitions are unaffected. In the present experiments, however, we have demonstrated that cortical stimulation produced two components of EPSPs (a short- and a long-latency component) followed by IPSPs in axotomized P-Mns (30, 40 and 46% of explored P-Mns 30, 40 and 80 days after axotomy, respectively). In

INHIBITORY POSTSYNAPTICPOTENTIALS

normal P-Mns it is evident that the major fraction of P-Mns studied showed the IPSPs (about 67% of the total sample) and a short-latency EPSP followed by predominant IPSPs (about 30% of the total sample). In normal P-Mns the long-latency EPSP is probably depressed by the generation of a predominant IPSP. From the present findings, it is probably safe to assume that in cortically induced PSPs of axotomized hypoglossal motoneurons the efficacy of inhibitory synapses is diminished, while excitation is unaffected. The fact that two components of EPSPs were produced in the major fraction of axotomized P-Mns is probably due to the reduction of numbers of inhibitory synapses from axotomized P-Mns. However, the changes of synaptic contacts made by the corticobular fibers in the interneurons impinging on hypoglossal motoneurons are still unknown. 5.4. DISJUNCTION OF PRESYNAPTIC CONTACTS ON AXOTOMIZEDMOTONEURONS In cats, sectioning the axon causes a disjunction of presynaptic contacts on axotomized motoneurons. This reactive deafferentation is one of the responses of nerve cells to axotomy including spinal motoneurons (see review by Mendell, 1984), cranial motoneurons (Sumner and Sutherland, 1973; Watson, 1974; Sumner, 1975a; Baker et aL, 1981; Takata and Nagahama, 1983). In axotomized spinal motoneurons it was elucidated that a monosynaptic EPSP is significantly reduced in size, and inhibitory synapses located on the cell body are also blocked, as are the somatic excitatory synapses (Eccles et al., 1958; Kuno and Llin~s, 1970a; Kuno et al., 1974a; see review by Mendell, 1984). In the spinal motoneurons Kuno and Llin~is (1970b) also demonstrated that both Ia EPSP and proximally located IPSPs by la inhibitory interneurons were affected by axotomy. Moreover, it is generally agreed that after axotomy the motoneuronal input resistance tends to increase (Gustafsson, 1979; Gustafsson and Pinter, 1984b; Pinter and Vanden-Noven, 1989) and that dendritic geometry is altered. Therefore, alterations in postsynaptic membrane properties of axotomized motoneurons could contribute to synaptic responses. Furthermore, Jacob and Berg (1987, 1988) suggested that in axotomized neurons reduced neurotransmitter sensitivity of the postsynaptic membrane could contribute to decreased EPSP. In addition, in many neurons studied, axotomy produces a preferential depression of excitatory potentials and a selective stripping of proximally located boutons containing spherical vesicles (Blinzinger and Kreutzberg, 1968; Hamberger et al., 1970; Matthews and Nelson, 1975; Sumner, 1975b). However, in axotomized hypoglossal motoneurons, we have demonstrated that in lingually and cortically induced PSPs the efficacy of inhibitory synapses is diminished, while excitation is unaffected (Takata and Nagahama, 1983, 1984). In axotomized masseteric motoneurons (Mass. Mns) Takata and Nagahama (1986) also demonstrated that long-lasting EPSPs followed by IPSPs were produced when the cerebral cortex or the lingual nerve was stimulated, suggesting that the efficacy of inhibitory synapses is

399

greatly reduced while that of excitatory synapses is unaffected. As already reported in normal Mass. Mns it has been shown that a single shock delivered to either the cerebral cortex or the lingual nerve induced IPSPs (Nakamura et aL, 1967; Takata and Fujita, 1979; Kubo et al., 1981) and that excitation of the inferior alveolar nerve evoked multiphasic responses, the IPSP-EPSP-IPSP sequence (Baranyi and Chase, 1984). Moreover, Delgado-Garcia et al. (1988) demonstrated that following axotomy the disynaptic inhibition of cat abducens motoneurons induced by ipsilateral vestibular nerve stimulation either disappeared or was reduced for 5-30 days. But, disynaptic activation produced by contralateral VIIIth nerve stimulation was apparently not affected. These changes were accompanied by a decrease of axosomatic pleomorphic synaptic endings and no changes were observed in either the number or distribution of synaptic endings on proximal and distal dendrites. With respect to a disjunction of presynaptic contacts in axotomized motoneurons, it was finally elucidated that a monosynaptic EPSP was significantly reduced in size, but disynaptic and polysynaptic activation was not affected. However, a disynaptic and polysynaptic IPSP was significantly reduced (Kuno and Llimis, 1970a; Kuno et al., 1974a, b; Takata and Nagahama, 1983, 1984, 1986; DelgadoGarcia et al., 1988). While there is a lot of information about the physiological and morphological aspects of axotomy-induced deafferentation (see review by Titmus and Faber, 1990), the underlying mechanisms for the decline of synaptic efficacy of chemical synapses are still unknown.

6. P H Y S I O L O G I C A L CHARACTERISTICS ON REINNERVATING AND NONREINNERVATING HYPOGLOSSAL MOTONEURONS In adult cats, sectioning the hypoglossal nerves causes a disjunction of presynaptic contacts on axotomized hypoglossal motoneurons (Takata, 1981a; Takata and Nagahama, 1983). In the following study, we have attempted to demonstrate whether or not in the tongue protruder motoneurons whose axons had been cut but allowed to regenerate to make functional contact with the tongue retractor muscles (foreign muscles), the percentage magnitude of the S- and the L-IPSP components in the IPSPs set up by stimulation of either the lingual nerve or the inferior alveolar nerve is changed. However, it is not known whether muscle reinnervation itself is necessary for synapse recovery. 6.1. SYNAPTIC POTENTIALS IN REINNERVATING AND NONREINNERVATINGMOTONEURONS At first, in order to present the synaptic efficacy on reinnervating and nonreinnervating hypoglossal motoneurons, synaptic potentials in the P-Mns 126 days after their cut axons were reunited to the tongue retractor muscle (foreign muscles), the styloglossus muscle, were explored and results are shown in Fig. 15. As illustrated in Aa and Ba, a motoneuron was identified from the antidromic spike evoked by stimulation of the previously sutured axons in the

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FIG. 15. PSPs in P-Mns after cut axons were reunited to the foreign muscle. A: Reinnervating P-Mn. a: Antidromic spike. Unitary muscle activity in the styloglossus muscle (b0 followed an induced spike in a P-Mn (b2). An inferior alveolar-induced IPSP is shown in % c2: Field potentials, d: Effect of membrane polarization on an inferior alveolar-induced IPSP. B: Nonreinnervating P-Mn. a: Antidromic spike. No muscle responses were produced in the styloglossus muscle (b 0 by an induced spike in a P-Mn (b2). An inferior alveolar-induced IPSP is shown in c~. c2: Field potentials. (From Takata et al., 1990.) styloglossus muscle. To ensure the correct identification of reinnervating P-Mns, muscle responses in the styloglossus muscle produced by an induced spike of the cell by application of a constant depolarizing current across the membrane were recorded. When the cell illustrated in Fig. 15A was stimulated with depolarizing current (b2) unitary muscle activity in the styloglossus muscle followed the cell spike onefor-one with constant latency (b~), indicating that this P-Mn reinnervated the tongue retractor muscles (foreign muscles), the styloglossus muscle. In this reinnervating P-Mn, stimulation of the inf. alv. n. produced an IPSP, illustrated in Ac. The relation between the amplitude of inferior alveolar-induced hyperpolarizing synaptic potentials and the membrane potential displacements was examined and results are shown in series d. By displacing the membrane potential towards hyperpolarization, a hyperpolarizing synaptic potential was decreased in amplitude ( - 4 nA) and reversed to a depolarizing potential ( - 8 nA). By displacement of the membrane potential to depolarization, a hyperpolarizing synaptic potential was increased in amplitude (6 nA). In 23 out of 54 explored cells, unitary muscle activity was produced by an induced spike of the cell. The latency of unitary muscle activity was measured from a peak of the neuron spike and the values ranged from 2.8-4.5msec (n =23). In nine reinnervating cells, synaptic potentials produced by inf. alv. n. stimulation were examined. As a result, an IPSP was produced in seven cells and small EPSPs followed by large IPSPs were produced in the remaining two cells. In the cell illustrated in Fig. 15B, no muscle responses were produced (b0 by an induced spike of the cell (b2), indicating that this cell failed to reinnervate the styloglossus muscle. In this cell stimulation of the inf. alv. n. produced and IPSP (Be). In 31 out of 54 explored cells no muscle responses were produced by an induced spike of the cell. In 18 nonreinnervating cells synaptic potentials produced by stimulation

of the inf. alv. n. were examined. As a result, an IPSP was produced in 13 cells and an EPSP followed by IPSPs was produced in five cells. Finally, we found that there were no differences on the patterns of PSPs and the amplitude of IPSPs produced in reinnervating and nonreinnervating P-Mns by inferior alveolar nerve stimulation, suggesting that the recovery of the synaptic efficacy of inhibitory synapses is timedependent rather than muscle reinnervation and that the synapse recovery is not dependent on retrograde trophic messages from muscle. 6.2. REPETITIVEFIRING ON REINNERVATINGAND NONREINNERVATINGMOTONEURONS The effect of axotomy that occurs in motoneurons is a reduction in the amplitude and duration of the AHP which follows an action potential (Kuno and Llin/ts, 1970a; Gustafsson, 1979). These changes may be indicative of a transient response to axotomy. The application of a constant depolarizing current across the membrane gives rise to repetitive discharges, whose frequency is dependent upon the current strength. In order to investigate the firing properties of hypoglossal motoneurons, the relationship between the firing frequency and the magnitude of the injected depolarizing current was tested. In Fig. 16 are shown the typical results obtained from a P-Mn of a normal cat. Repetitive firing of the P-Mn responding to increasing amounts of injected depolarizing currents is illustrated (Ab). In B is graphically represented a relationship between the firing frequency (impulses/see) and the amount of injected depolarizing current (nA). This frequencycurrent ( f - l ) relationship plotted for the first (open circles) and the second interspike intervals (filled circles) revealed two distinct slopes (the primary and the secondary range) as is already known for cat spinal and trigeminal motoneurons (Kernell, 1965; Baldissera and Gustafsson, 1974; Calvin, 1974; Takata et al,, 1982). In this normal P-Mn it was found that the slope of the primary and the secondary range was 5 imp/see per nA and 12 imp/see per nA, respectively.

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FIG. 16. The relationship between firing frequency and injected depolarizing current in a normal protruder motoneuron. A~: Antidromic spike. Ab: Repetitive firing of this P-Mn evoked by an injecting current. The numerals denote the amount of injected current in nanoamperes (nA). B: Frequency-current relationship for the first (open circles) and second interspike intervals (filled circles). (From Takata et al., 1980.)

INHIBITORY POSTSYNAPTICPOTENTIALS

In order to investigate the firing properties of axotomized motoneurons, t h e f - I relation in reinnervating and nonreinnervating P-Mns was tested. Repetitive firing in P-Mns 126 days after their cut axons were reunited to the styloglossus muscle was examined and results are shown in Fig. 17. In a P-Mn illustrated in A, unitary muscle activity in the styloglossus muscle (aj) followed the cell spike (a2) one-for-one, indicating that this P-Mn reinnervated the styloglossus muscle. In this reinnervating P-Mn, the relationship between the firing frequency and the amount of injected depolarizing current was examined (Ab). Repetitive firing of this cell responding to increasing amounts of injected depolarizing current is illustrated in C. The minimum intensity of depolarizing current required to elicit a single action potential (rheobase) was determined and the rheobase of this cell was 2 nA (2-5 nA, n = 15). In this P-Mn, the f - I relation plotted from the first interspike intervals revealed two distinct slopes. The slope of the primary and the secondary range was 12 (+2.5, n = 10) and 45 ( + 5.5, n = 10) imp/sec per nA, illustrated by open circles in C. In a P-Mn illustrated in B, no muscle responses were produced (a0 by an induced spike of the cell (a2). As illustrated in Bb, this nonreinnervating P-Mn shows sustained firing to maintain pulses of intraceilularly injected depolarizing currents. T h e J - I relation plotted from the first interspike intervals of a nonreinnervating P-Mn is represented by filled circles in C. 6.3. RECOVERYOF THE FIRING BEHAVIORSAND SYNAPTIC EFFICACY IN MOTONEURONS

It is accepted that the mean input resistances of cat spinal motoneurons increase after axotomy (Kuno et al., 1974a, b; Gustafsson and Pinter, 1984b;

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401

Foehring et al., 1986b; Sernagor et al., 1986), especially in the F-type motoneurons where there is a two-fold or more increase in input resistance after axotomy (Gustafsson, 1979; Gustafsson and Pinter, 1984a, b; Pinter and Vanden-Noven, 1989). In addition, Gustafsson and Pinter (1984b) suggested that axotomy-induced transformations occurred in F-type motoneurons, while the S-type motoneurons remained relatively unchanged. Moreover, the high input conductance of the F-type motoneurons has been associated with the relatively expansive dendritic trees. It was reported that in hypoglossal motoneurons dendritic retraction occurred after nerve section (Sumner and Watson, 1971; Sumner 1975a, b, c) and Laiwand et aL (1988) observed the increased input resistance has been attributed to a reduction in cell body diameter in axotomized vagal motoneurons. Moreover, it was reported that the enhanced soma-dendritic excitability after axotomy appeared to result from increased concentrations of functional voltage-sensitive Na ÷ channels in the soma-dendritic membranes (Seragor et al., 1986; Titmus and Faber, 1986). However, in cat abducens motoneurons (Delgado-Garcia et al., 1988), mammalian spinal (Czeh et al., 1977) or cranial (Gallego et al., 1987) sensory neurons, and sympathetic ganglion cells (Purves, 1975; Kelly et al., 1986; Gordon et al., 1987) axotomy tends to have no effect on the input resistance. Especially in cat abducens motoneurons Baker et al. (1981) and Delgado-Garcia et al. (1988) reported that abducens motoneurons appeared to become less excitable after axotomy and this characteristic reduced IS excitability was not accompanied by changes in input resistance. In hypoglossal motoneurons the physiological characteristics of the response to axotomy are similar to those for spinal motoneurons, in that P-Mns become more excitable following axotomy. Axotomized P-Mns have a higher sensitivity to injected current and increased spontaneous activity, indicating that these characteristics are dependent on lowered current and voltage thresholds for spike initiation (Takata et al., 1980). Furthermore, the temporal separation of the IS and SD spike components was also decreased following axotomy. As one of characteristic signs of axotomized P-Mns, the large delayed depolarization following an antidromic spike was seen, suggesting that axotomyinduced excitability changes result from lowered soma-dendritic threshold for excitation, leading to re-excitation of the soma by dendritic spikes (Heyer and Llinfis, 1977; Takata et al., 1980). Changes in the firing behaviors of axotomized motoneurons have been assessed by injecting intracellular current pulses. It is generally agreed that the currents underlying the duration of the AHP are important for neural discharge frequency, and the effect of axotomy that occurs in motoneurons is a reduction in the amplitude and duration of the AHP (Kuno and Llinfis 1970a; Heyer and Llinfis, 1977; Gustafsson, 1979; Gustafsson and Pinter, 1984b; Foehring et al., 1986b). Recently, it was demonstrated that axotomy induces a reduction in two types of K+-current, the Ca2+-dependent K-current and the A-current governing the firing behavior of the motoneurons (Yarom et al., 1985b; Laiwand et al., 1988).

402

M.

TAKATA

With respect to the firing behaviors, it was demonstrated that in reinnervating P-Mns fired in a sustained manner by injecting depolarizing currents into the cell, the f - I relation revealed two distinct slopes (the primary and the secondary range). In contrast, a distinct breakpoint in the f - I relation was missing in nonreinnervating motoneurons as demonstrated in axotomized motoneurons (Heyer and Llinfis, 1977; Takata and Nagahama, 1986; Takata et al., 1990). Therefore, the recovery of processes that control rhythmical firing of motoneurons is probably dependent on muscle reinnervation, as it is suggested that AHP duration is regulated by retrograde trophic messages from muscles (Kuno and Llinfis 1970b; Czeh et al., 1978). It is generally agreed that all of membrane characteristics (axon conduction velocity, rheobase, sag conduction, input resistance, membrane time constant and AHP duration) are modified by axotomy and are not restored if motoneurons do not reinnervate muscle fibers (Kuno et al., 1974b; Foehring et al., 1986a, b; Pinter and Vanden-Noven, 1989; see review by Titmus and Faber, 1990). It was also reported that synaptic contacts onto axotomized motoneurons occurs after muscle reinnervation (Sumner and Sutherland, 1973) and Gordon (1983) has proposed that reinnervation is important for functional recovery. However, Takata et al. (1990) demonstrated that in hypoglossal motoneurons 126 days after their cut axons were reunited to the tongue muscle there were no differences on the patterns of postsynaptic potentials produced in reinnervating and nonreinnervating motoneurons by peripheral nerve stimulation, suggesting that the recovery of the synaptic efficacy of inhibitory synapses is timedependent rather than muscle reinnervation, however, the recovery of processes that control rhythmical firing of motoneurons is probably dependent on muscle reinnervation, as reported in trigeminal motoneurons (Takata et al., 1990).

motoneurons show little or no preference for their former fibers. In the following experiments, we have attempted to demonstrate whether or not in the P-Mns whose axons had been cut but allowed to regenerate to make functional contact with the tongue retractor muscles (foreign muscles), the percentage magnitude of the Sand the L-IPSP components in the IPSPs set up by stimulation of either the lingual nerve or the inf. alv. n. is changed. 7.1. S % AND L % iN REINNERVATINGMOTONEURONS Recently, it was demonstrated that following nine months self-reinnervation, 89% of regenerated spinal motoneurons were in the normal range with respect to the AHP half-decay time (Foehring et al., 1986a, 1987). Therefore, experiments were performed on a cat at the 262nd postoperative day after the medial branch innervating protruder muscles were sutured to the lateral branch innervating retractor muscles (axon-union). By stimulation of the previously sutured axons, retraction of the tongue was observed, indicating that sutured axons made nerve-muscle contacts. The degree of reinnervation was estimated by the ratio of the maximum tongue retraction evoked by stimulation of the lateral branch in intact side (open circles) to that evoked by stimulation of sutured axons in operated side (closed circles) (Fig. 18A). With increasing stimulus strength, the magnitude of tongue retraction gradually increased, reaching a maximum at 9 V and remained constant in spite of further increase of stimulus strength. The inset record shows a maximal retraction of the tongue A 1=~

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7. PERCENTAGE MAGNITUDE OF THE SAND THE L-IPSP IN REINNERVATING AND NONREINNERVATING HYPOGLOSSAL MOTONEURONS In half of the explored P-Mns 126 days after their cut axons were reunited to the styloglossus muscle (foreign muscle), an induced spike of the cell produced unitary muscle activity in the foreign muscle, indicating that an explored cell made nerve-muscle contacts. However, it is evident that in the remaining cells no unitary muscle activity was evoked by an induced spike of the cell, indicating that these motoneurons failed to regenerate to the foreign muscle to which they were directed by suturing. It was also demonstrated that in spinal motoneurons the innervation ratio reached the maximum value within 50-60 days after the self-union and never reached 100% following 150 days after the self-union operation (Kuno and Llinfis, 1970b). Furthermore, lp and Vrbora (1983) showed that in leg muscles either soleus' own nerve or a foreign nerve was eventually able to innervate soleus muscle, indicating that

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FIG. 18. Degree of reinnervation and synaptic potentials in protruder motoneurons 262 days after axon-union. A: Tongue retraction produced by stimulation of the lateral branch in intact side (open circles) and the sutured axons in operated side (closed circles). Inset record shows a maximal retraction of the tongue. B: IPSPs. Record a shows an initial segment component and a soma-dendritic component. Responses to double shocks of the ipsilateral lingual nerve or inferior alveolar nerve are shown in b~ and c a. Records b~ and c, show field potentials. C: Membrane potential dependence of lingually induced PSPs. Numerals denote the amount of injected current in nanoamperes (nA). (From Takata et al., 1991.)

403

INHIBITORY POSTSYNAPTIC POTENTIALS

produced by stimulation of either the lateral branch in intact side or the previously sutured axons. From this test, the percentage magnitude of the retraction of the tongue by stimulation of sutured axons was determined as 56% (the degree of reinnervation). The following results were obtained from the PMns reinnervating the tongue retractor muscle with 56% reinnervation. A P-Mn was identified from the antidromic spike evoked by stimulation of the previously sutured axons, illustrated in a of Fig. 19A. A summation of the A H P was explored by using a short train of antidromic stimuli at 4 msec intervals and results are shown in b of Fig. 19A. As illustrated in b]-bs, the A H P amplitude increased linearly until the number of spikes increased up to four and thereafter it ceased to increase despite a further increase of the spike number. The peak amplitude of the summated A H P in a cell was about 5 mV and this value was within the range obtained from normal cells (Takata et al., 1979). In the following study, the relationship between the firing frequency and the amount of injected depolarizing current was examined and results are shown in c of Fig. 19A. Repetitive firing of a cell responding to increasing amounts of injected depolarizing current is illustrated, and the f - I relation is graphically represented in Fig. 19B by open circles. The f - I relation plotted from the first interspike intervals revealed two distinct slopes. In this P-Mn it was demonstrated that the slope of the primary and the secondary range was 11 and 29imp/sec per nA, respectively, indicating that this cell made nervemuscle contacts. The following results were obtained in a cat 262 days after axon-union with 56% reinnervation. When two shocks separated by 90msec intervals were applied to the lingual nerve (Fig. 18Bb) or the inf. alv. n. (Fig. 18Be), the first shock produced a large IPSP

A

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with a latency of 4msec from stimulus artifact, but only a small IPSP was evoked by the second shock. The antidromic spike is shown in Ba. When double shocks of 4 msec intervals were applied to the sutured axons, the second shock separated an initial segment component from a soma-dendritic component, suggesting that this cell made nerve-muscle contacts. In the next step, the relation between the amplitude of lingually induced hyperpolarizing potentials and of the membrane potential displacement was examined and results are shown in Fig. 18C. In the records, responses to five trials were averaged and numerals denote the amounts of injected currents in nanoamperes (nA). At the resting membrane level (0 nA), lingual nerve stimulation evoked a hyperpolarizing potential. By displacing the membrane potential toward hyperpolarization, a lingually induced hyperpolarizing potential was reversed to a depolarizing potential ( - 6 and - 1 0 n A ) , indicating that this hyperpolarizing potential is an IPSP. As already reported (Takata and Ogata, 1980; Takata, 1982; Takata and Tomomune, 1986), the percentage magnitude of the S- and the L-IPSP set up by lingual nerve and inf. alv. n. stimulation in a P-Mn, illustrated in Fig. 18Bb and Be, was determined to be 70 and 30%, and 60 and 40%, respectively. The percentage magnitude of the S-IPSP (S %) and the L-IPSP (L %) components of lingually induced or inferior alveolar-induced IPSPs was calculated for 55 P-Mns in a cat 262 days after axon-union with 56% reinnervation and results are illustrated in Fig. 20. Figure 20Aa and Ab shows the average of S% in the IPSPs produced in normal P-Mns and R-Mns by ipsilateral lingual nerve (closed arrow) and inf. alv. n. stimulation (open arrow). In normal P-Mns (Fig. 20Aa) the average of S % in lingually induced and inferior alveolar-induced IPSPs was 53 and 23%

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404

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FIG. 20. Percentage magnitude of S- and L-IPSPs in protruder motoneurons 262 days after axon-union with 56% reinnervation. The abscissae S and L indicate S% and L%, which are graphed by the open and closed columns. The ordinate N indicates the number of cells. A: S% in normal protruder (a) and retractor motoneurons (b). Open and closed arrow indicates the average of S% in inferior alveolarinduced and lingually induced IPSPs. B: IPSPs produced by ipsilateral lingual nerve (Ling. N.) stimulation. C: IPSPs produced by ipsilateral inferior alveolar nerve (Inf. alv. N.) stimulation. The closed arrow in B and the open arrow in C denote the average of S% and L%. (From Takata et al., 1991.)

(closed and open arrows), and in normal R-Mns (Fig. 20Ab) the average was 70 and 59% (closed and open arrows), respectively. In Fig. 20B the histograms labeled lingual nerve indicate S % (open column with abscissa S) and L % (closed column with abscissa L) of the IPSPs evoked by ipsilateral lingual nerve stimulation in operated animals. The ordinate N indicates the number of cells. The histograms illustrated in Fig. 20C show S % and L % of the IPSPs evoked by ipsilateral inf. alv. n. stimulation in operated animals. F r o m the histograms it was determined that in the IPSPs set up by lingual nerve stimulation, the average of S % and L % (closed arrow) was 68% (N = 55) and 32% (N = 55), respectively. With respect to the IPSPs evoked by inf. alv. n. stimulation, we found that the average of S % and L % (open arrow) was 57% (N = 55) and 43% (N = 55), respectively. The results suggest that the percentage magnitude of the S- and the L-IPSP components in the IPSPs produced in the tongue protruder motoneurons reinnervating the tongue retractor muscle was rearranged to appear like that exhibited by the tongue retractor motoneurons that normally supply that muscle.

7.2. S % AND L % IN NONREINNERVATING MOTONEURONS

some P-Mns, when double shocks with 3-msec intervals were applied to the sutured axons, a failure of separation of the initial segment component from the soma-dendritic component was seen (Fig. 2lB,) and the f - I relation in this P-Mn is represented in Fig. 19B by closed circles. In addition, in some P-Mns in this animal, the large delayed depolarization following an antidromic spike was evoked by the application of a threshold-stimulus to the sutured axons (Fig. 21Ca). The results suggest that these P-Mns

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In the following studies, S % and L % of the IPSPs evoked on P-Mns 197 days after axon-union by ipsilateral lingual nerve and inf. alv. n. stimulation were measured. The degree of reinnervation in the sample was 6% (Fig. 21A), indicating that many P-Mns failed to regenerate to make functional contact with the foreign muscle. A maximal retraction of the tongue produced by stimulation of either the lateral branch in the intact side or the previously sutured axons is illustrated in Fig. 21A, and A h. In

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FIG. 21. Degreeof reinnervation and synaptic potentials in protruder motoneurons 197 days after axon-union with 6% reinnervation. A: Maxima[ retraction of the tongue produced by stimulation of the lateral branch in intact side (a) and the sutured axons (b). B and C: IPSPs. Record a in B and C shows a response to doable shocks and a single shock of the sutured axons. Responses to double shocks of the ipsilateral lingual nerve or inferior alveolar nerve are shown in b~ and c r Records b 2 and c 2 show field potentials. (From Takata et al., 1991.)

INHIBITORY POSTSYNAPTIC POTENTIALS

failed to regenerate to make functional contact with muscles. As already demonstrated, axotomized motoneurons have a high safety factor for impulse transmission from the initial segment to the somadendritic membrane, and produce the large delayed depolarization following an antidromic spike (Takata et al., 1980). The IPSPs evoked in P-Mns with characteristic signs of axotomized motoneurons by stimulation of the ipsilateral lingual nerve and inf. air. n. are shown in b and c of Fig. 21B and C. S % set up in a P-Mn, illustrated in B, by lingual nerve and inf. alv. n. stimulation was 54% and 33%, respectively. In addition, S % set up in a P-Mn, illustrated in C, by lingual nerve and inf. alv. n. stimulation was determined to be 65% and 26%, respectively. S % and L % were measured in a cat 197 days after axon-union with 6% reinnervation, in which 51 P-Mns were recorded, and results are illustrated in Fig. 22. The histograms in B and C indicate S % and L % of the IPSPs evoked by ipsilateral lingual nerve and inf. alv. n. stimulation. From the histograms it was determined that in the IPSPs set up by lingual nerve stimulation, the average of S % and L % (closed arrow in B) was 57% (N =51) and 43% (N =51), respectively. With respect to the IPSPs evoked by inf. alv. n. stimulation, we found that the average of S % and L % (open arrow in C) was 29% (N = 51) and 71% (N = 51), respectively. In A, and Ab is shown the average of S % in the IPSPs produced in normal P-Mns and R-Mns by ipsilateral lingual nerve and inf. alv. n. stimulation. The results indicate that S % and L % in the IPSPs set up in P-Mns of an operated animal with 6% reinnervation by ipsilateral lingual nerve and inf. alv. n. stimulation was in the range of that exhibited by the P-Mns that normally supply that muscle.

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7.3. S % AND L % IN MOTONEURONSAFTER THE SELF-UNION OPERATION The following experiments were done on P-Mns at the 24th and the 40th postoperative day after their cut axons were reunited to their own muscles. The responses in 32 P-Mns at the 24th postoperative day obtained from a single experiment are examined. In the IPSPs set up by ipsi-L stimulation, the averages of S % and L % were 42% (n = 32) and 58% (n --32), respectively, indicating that S % and L % in these samples was in the range of that exhibited by the P-Mns that normally supply that muscle. In comparison with the decrease of the S-IPSP in the P-Mns 24 days after axotomy, in the P-Mns after their cut axons were reunited to muscles, the decrease was less prominent. In order to further study the synaptic efficacy, IPSPs on the P-Mns at the 40th postoperative day after their cut axons were reunited to their own muscles were studied and results obtained from a single experiment are examined. In the IPSPs set up by ipsi-L stimulation, the average of S % and L % were 40% (n = 4 6 ) a n d 60% (n =46), respectively, indicating that S % and L % in these samples was in the range of that exhibited by the P-Mns that normally supply that muscle. 7.4. MAINTENANCEAND REARRANGEMENTSOF SYNAPTIC CONTACTS With evidence that axotomy triggers in RNA and protein synthesis (BrattgArd et al., 1958; Grafstein and McQuarrie, 1978; Aldskogius et al., 1980; Austin, 1985), it is suggested that axotomy-induced modifications in membrane excitability (newly synthesized Na + channel) may reflect alterations in metabolic activity of the neuron which result in the reorganization of the neural membrane (Baker et al., 1981).

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S0 %% 10C L $ L S S S FIG. 22. Percentage magnitude of S- and L-IPSPs in protruder motoneurons 197 days after axon-union with 6% reinnervation. The abscissae S and L indicate S% and L%, which are graphed by the open and closed columns. The ordinate N indicates the number of cells. A: S% in normal protruder (a) and retractor motoneurons (b). Open and closed arrows indicate the average of S% in inferior alveolar-induced and lingually induced IPSPs. B: IPSPs produced by ipsilateral lingual nerve (Ling. N.) stimulation. C: IPSPs produced by ipsilateral inferior alveolar nerve (Inf. alv. N.) stimulation. The closed arrow in B and the open arrow in C denote the average of S% and L%. (From Takata et al., 1991.)

406

M. TAKATA

The light microscopic observation of choline acetyltransferase (CHAT) immunoactivity demonstrated that hypoglossal motoneurons are innervated by ChAT immunoreactive terminals (Connaughton et al., 1986) in the same way as are spinal cord motoneurons (Armstrong et al., 1983; Houser et al., 1983; Satoh et al., 1983). Connaughton et al. (1986) also demonstrated location of CHAT, substance P and enkephalin in the hypoglossal nucleus of the rat and suggested that possible candidates include the raphe nuclei for substance P and propriobullar interneurones for ChAT (Saper and Loewy, 1980; Travers and Norgren, 1983). Following transection of the hypoglossal nerve a reduction of ChAT and acetylcholine esterase (ACHE) is demonstrated in the hypoglossal nucleus (Watson, 1971; Fonnum et al., 1973; Gottesfeld and Fonnum, 1977; Wooten et al., 1978; Davidoff and Schulze, 1988). There is evidence that acetylcholine (ACh) accumulates inside the neuron after axotomy (Evans and Saunders, 1967; Fonnum et al., 1973). Yarom et aL (1985a) demonstrated in slices of vagal motoneurons that intracellular injection of ACh blocks both the voltage-dependent and Ca2+-dependent K conductances, thereby increasing Ca 2÷ currents and prolonging the action potential, and the effects of ACh are reversible. However, externally applied ACh was not effective. In axotomized P-Mns an increased Ca 2+ influx by accumulation of ACh inside the cell may relate as a function of a quaternary ammonium compound to produce a large delayed depolarization following an antidromic spike. In addition, it was also demonstrated that muscarinic receptors are found on hypoglossal motoneurons (Wamsley et al., 1981), and these receptors disappear after axotomy (Rotter et al., 1977; Rotter et aL, 1979). Moreover, it has recently been again demonstrated that the intensity and the number of ChAT-labeled cells were reduced on axotomized hypoglossal motoneurons. This decrease was maximal two weeks postoperative and by 30 days many of the motoneurons had begun to re-express the enzyme (Armstrong et al., 1991). Armstrong et al. (1991) also demonstrated that by 90 days post-transection ChAT immunoreactivity in the vast majority of motoneurons completely recovered to the normal level. Snider and Thanedar (1989) suggest that this reversibility presumably reflects the reinnervation of the nerve with its target. However, there were no differences on the patterns of inferior alveolar-induced postsynaptic potentials produced in reinnervating and nonreinnervating motoneurons 126 days after their cut axons were reunited to the tongue muscle, suggesting that the recovery of the synaptic efficacy of inhibitory synapses is time-dependent rather than muscle reinnervation (Takata et al., 1990). With respect to the maintenance of synapses, it has been postulated that retrograde transport of some material to the neuron from its periphery is essential for the maintenance of the contacts that it receives. As a t r o p h i c factor N G F (nerve growth factor) has been shown to be important for the regulation of preganglionic synaptic inputs to sympathetic postganglionic neurons (Purves and Njh, 1976, 1978; Purves and Lichtman, 1978). In superior cervical ganglion cells, a disjunction of presynaptic contacts

on axotomized neurons was diminished if exogenous N G F was supplied to the ganglion cells, suggesting that one of the roles of the target tissue is to supply a tropic factor (Nj~ and Purves, 1978). Moreover, in sympathetic ganglion cells, it was suggested that a tropic substance which is released by ganglion cells may be an important factor which controls the synaptic rearrangements occurring after partial denervation of the superior cervical ganglion (Mahler and Nj~, 1984). In addition, axotomy-like reactive deafferentation could be provoked by injection of ganglion cells with antiserum to N G F (Nj~ and Purves, 1978; Purves and Nj~, 1978; Purves and Lichtman, 1978). In axotomized hypoglossal motoneurons it was also demonstrated that in contrast to the decrease in ChAT immunoreactivity, transection of the hypoglossal nerves resulted in the expression of N G F receptor immunoreactivity within the lesioned motoneurons (Armstrong et aL, 1991). Armstrong et al. (1991) also demonstrated that the number of N G F receptor-labeled neurons decreases sharply by 15 days, and no N G F receptor immunoreactivity is detected in the hypoglossal nucleus following 30 or 90 day survival times. Recently, Moix et al. (1991) demonstrated that topical application of vincristine to the hypoglossal nerve blocks axonal transport of W G A (wheat germ agglutinin) and causes loss of ChAT but no appearance of N G F receptor. Vincristine is known as a microtubule inhibitor that blocks fast axonal transport (Grafstein and Forman, 1980). Furthermore, Wood et al. (1990) demonstrated in motoneurons that crushing the hypoglossal nerve was a strong stimulus to N G F receptor expression but transection was not. They suggested that N G F receptor induction resulted from local damage to the nerve, In axotomized hypoglossal motoneurons the function of N G F receptor is not known, but Armstrong et al. (1991) suggest that the regeneration and/or survival of these motoneurons might be influenced by NGF. In addition, we have no experimental evidence on the relation between the recovery of synaptic contacts and the N G F receptor induction in axotomized hypoglossal motoneurons. In sympathetic ganglion cells and spinal motoneurons, it was revealed that axotomy causes loss of functional inputs (Matthews and Nelson, 1975; Mendell et al., 1976; Purves, 1975) and the death of many injured neurons (Purves, 1975; Smolen, 1983) and that there is an increase in the number of synaptic inputs to the surviving neurons. However, Aldskogius and Thomander (1986) studied the organization of the facial motor nucleus and reported that after trarisection of the facial nerve at the adult stage, no significant loss of motoneurons occurred. Armstrong et al. (1991) also demonstrated no loss of hypoglossal motoneurons after transection of the hypoglossal nerves in rats. Recently, Kashihara et al. (1988) reported that in rats four days after birth the majority of axotomized motoneurons die if target contact is prevented, whereas, in rats two to four weeks after birth, section of the sciatic nerve is known to cause no loss of the motoneurons (Schmalbruch, 1984; Sheard et al., 1984). Moreover, ZiskindConhaim and Presley (1991) demonstrated in rats that intercostal nerve transection in neonates results

INHIBITORYPOSTSYNAPTICPOTENTIALS in a significant loss of motoneurons, and most muscle reinnervation is by intact motoneurons that originally did not innervate the denervated muscle. The experiments on the synaptic replacement in adult cats have been performed in the red nucleus by Tsukahara (1978). They have shown that synapses formed by corticoruber fibers rearrange after elimination of the input from cerebellum or peripheral manipulations, suggesting that the new corticorubral synapses are formed in the soma membrane in the neuron. In spinal motoneurons it is demonstrated that the return of motoneuron properties following reinnervation is not target specific (Kuno et aL, 1974b), but Foehring et al. 0987, 1988) found that M G motoneurons innervating soleus muscle had properties different from those of M G motoneurons reinnervating M G or L G muscle, therefore, functional connection with a similar muscle is sufficient for restoration of normal motoneuronal properties. In the cat forelimb, Tsukahara and Fujito (1976) have demonstrated that antagonist cross-reinnervation has been shown to cause synaptic reorganization in the red nucleus. In the hypoglossai motoneurons we have demonstrated that the percentage magnitude of a short- and a long-lasting inhibitory postsynaptic potential in the inhibitory postsynaptic potentials produced in the tongue protruder motoneurons, whose axons had been cut but allowed to regenerate to make functional contact with the tongue retractor muscles, by lingual nerve or inferior alveolar nerve stimulation, was rearranged to appear like that exhibited by the tongue retractor motoneurons that normally supply that muscle (Takata et aL, 1991). Acknowledgements--This study was supported by grant-inaids for scientific research from the Japan Ministry of Education, Science and Culture.

REFERENCES ALDES, L. D. and BOONE, T. B. (1985) Organization of projections from the principal sensory trigeminal nucleus to the hypoglossal nucleus in the rat: an experimental light and electron microscopic study with axonal tracer techniques. Expl Brain Res. 59, 16-29. ALDSKOGIUS,H. and SVENSSON,M. (1988) Effect on the rat hypoglossal nucleus of vinblastine and colchicine applied to the intact or transected hypoglossal nerve. Expl NeuroL 99, 461-473. ALDSKOGIUS,H. and THOMANDER,L. (1986) Selective reinnervation of somatotopically appropriate muscles after facial nerve transection and regeneration in the neonatal rat. Brain Res. 375, 126-134. ALDSKOGIUS,H., BARRON,K. D. and REGAL,R. (1980) Axon reaction in dorsal motor vagal and hypoglossal neurons of the adult rat. Light microscopy and RNA-cytochemistry. J. comp. Neurol. 193, 165-177. ALDSKOGIUS,H., BARRON,K. D. and REGAL,R. (1984) Axon reaction in hypoglossal and dorsal motor vagal neurons of adult rat: Incorporation of (3H) leucine. Expl NeuroL 85, 139-151. ARMSTRONG, D. M., SAPER, C. B., LEVEY, A. I., WAINER, B. H. and TEARY,R. D. (1983) Distribution of cholinergic neurons in rat brain: Demonstrated by the immunocytochcmical localization of choline acctyltransfcrase. J. comp. NeuroL 216, 53~8.

407

ARMSTRONG. D. M., BRADY, R., HERSH, L. B., HAYES, R. C. and WILEY, R. G. (1991) Expression of choline acctyltransferase and nerve growth factor receptor within hypoglossal motoneurons following nerve injury.Expl Brain Res. 304, 596--607. ARVlDSSON,J. and ALDSKOGIUS,H. (1982) Effect of repeated hypoglossal nerve lesions on the number of neurons in the hypoglossal nucleus of adult rats. Expl Neurol. 75, 520-524. AUSTIN,L. (1985) Molecular aspects of nerve regeneration. In: Alterations of Metabolites in the Nervous System, Handbook of Neurochemistry, Vol. 9, pp. 1-45. Ed. A. LAJTnA. Plenum Press: New York. AVOLI, M. (1986) Inhibitory potentials in neurones of the deep layers of the/n vitro neocortical slice. Brain Res. 370, 165-170. BAKER, R., DELGADO-GARCIA,J. and MCCREA, R. (1981) Morphological and physiological effects of axotomy on cat abducens motoneurons. In: Lesion-Induced Neuronal Plasticity in Sensorimotor Systems, pp. 51-63. Eds. W. PRECHTand H. FOLHR. Springer: Berlin. BALDISSERA,F. and GUSTAFSSON,B. (1974) Firing behavior of a neurone model based on the afterhyperpolarization conductance time course and algebraical summation. Adaptation and steady state firing. Acta physiol, scand. 92, 27-47. BARANYI, A. and CHASE, M. H. (1984) Ethanol-induced modulation of the membrane potential and synaptic activity of trigeminal motoneurons during sleep and wakefulness. Brain Res. 307, 223-245. BARLOW, S. and FARLEY, G. (1989) Neurophysiology of speech. In: Neurobiology of Speech, Language and Hearing, pp. 146-200. Eds. D. KUCHN, M. LEMMEand J. BAUMGARTNER.Little Brown: Boston. BLINZINGER,K and KREUTZBERG,K. (1968) Displacement of synaptic terminals from regenerating motoneurons by microglial cells. Z. Zellforsch. 85, 145-157. BOONE, T. B. and ALDES,L. D. (1984a) The ultrastructure of two distinct neuron populations in the hypoglossal nucleus of the rat. Expl Brain Res. 54, 321-326. BOONE,T. B, and ALI)ES, L. D. (1984b) Synaptology of the hypoglossal nucleus in the rat. Expl Brain Res. 57, 22-32. BOWERY,N. G., PRICE, G. W., HUDSON,A. L., HILL, D. R., WALKIN,G. P. and TURNBULL,M. J. (1984) GABA receptor multiplicity: Visualization of different receptor types in the mammalian CNS. Neuropharmacology 23, 219-231. BRATTGARD, S.-O., EDSTROM, J.-E. and HYDEN, H. (1957) The chemical changes in regenerating neurons. J. Neurochem. 1, 316-325. BRATTG.~RD, S.-O., HYDEN, H. and SJOSTRANO,J. (1958) Incorporation of orotic acid-~4C in regenerating single nerve cells. Nature 182, 801-802. BROWN, A. G. and FYFFE, R. E. W. (1981) Direct observations on the contacts made between Ia afferent fibers and ~t-motoneurons in the cat's lumbosacral spinal cord. J. Physiol., Lond. 313, 121-140. BURKE, R. E., WALMSLEY,B. and HODGSON,J. A. (1979) H a P anatomy of group la afferent contacts on alpha motoneurons. Brain Res. 160, 347-352. CALVIN,W. H. (1974) Threes modes of repetitive firing and the role of threshold time course between spikes. Brain Res. 69, 341-346. CHIBUZO, G. A. and CUMMINGS,J. F. (1982) An enzyme tracer study of the organization of the somatic motor center for the innervation of different muscles of the tongue: Evidence for two sources. J. comp. Neurol. 205, 273-281. CONNAUGHTON, M., PRIESTLEY,J. V., SOFRONIEW.M. V., ECKENSTEIN, F. and CUELLO, A. C. (1986) Inputs to motoneurons in the hypoglossal nucleus of the rat: Light and electron microscopic immunocytocbemistry for choline acetyltransfcrase, substance P and cnkephalins using monoclonal antibodies. Neuroscience 17, 205-224.

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CONNORS,B. W., MALENKA,R. C. and SILVA,L. R. (1988)

FONNUM,F., FRIZELL,M and SJOSTRAND,J. (1973) Trans-

Two inhibitory postsynaptic potentials, and GABA a and GABAb receptor-mediated responses in neocortex of rat and cat. J. Physiol., Lond. 406, 443-468. COOMBS,J. S., ECCLES,J. C. and FATT, P. (1955) The specific ionic conductances and the ionic movements across the motoneuronal membrane that produce the inhibitory post-synaptic potential. J. Physiol., Lond. 130, 326-373. COOPER, M. H. (1981) The hypoglossal nucleus of the primate: A Golgi study. Neurosci. Lett. 21, 249-254. CULL, R. E. (1974) Role of nerve-muscle contact in maintaining synaptic connections. Expl Brain Res. 20, 307-310. CULL, R. E. (1975) Effect of sensory nerve division of the afferent synapses of axotomized motor neurones. Expl Brain Res. 22, 421-425. CURTIS, D. R., DUGGAN, A. W., FELIX, D. and JOHNSTON, A. R. (1971a) Bicuculline an antagonist of GABA and synaptic inhibition in the spinal cord of the cat. Brain Res. 32, 69-96. CURTIS, D. R., DUGGAN,A. W., FELIX, D., JOHNSTON,A. R. and MCLENNAN, H. (1971b) Antagonism between bicuculline and GABA in the cat brain. Brain Res. 33, 57-73. CZEH, G., KUDO,N. and KUNO,M. (1977) Membrane properties and conduction velocity in sensory neurones following central or peripheral axotomy. J. PhysioL, Lond. 270, 165 180. CZEH, G., GALLEGO, R., KUDO, N. and KUNO, M. (1978) Evidence for the maintenance of motoneuron properties by muscle activity. J. Physiol., Lond. 281, 239-252. DAVIDOFF, M. S. and SCHULZE, W. (1988) Coexistence of GABA- and choline acetyltransferase (ChAT)-like immunoreactivity in the hypoglossal nucleus of the rat. Histochemistry 89, 25-33. DELGADO-GARCIA,J. M., DEE POZO, F., SPENCER,R. F. and BAKER, R. (1988) Behavior of neurons in the abducens nucleus of the alert cat-III. Axotomized motoneurons. Neuroscience 24, 143 160. DUBNER, R., SESSLE,B. J. and STOREY,A. T. (1978) The Neuronal Basis of Oral and Facial Function. Plenum Press: New York. DUNWIDDIE,T., MUELLER,A., PALMER,i . , STEWART,J. and HOTTER, B. (1980)Electrophysiological interactions of enkephalins with neuronal circuitry in the rat hippocampus. i. Effects on pyramidal cell activity. Brain Res. 184, 311 330. ECCLES, J. C., LIBET, B. and YOUNG, R. R. (1958) The behaviour of chromatolysed motoneurons studied by intracellular recording. J. Physiol., Lond. 143, 11-40. EVANS, C. A. N. and SAUNDERS,N. R. (1967) The distribution of acetylcholine in normal and regenerating nerves. J. Physiol., Lond. 192, 79-92. FLUMERFELT, B. A. and LEWIS, P. P. (1975) Cholinesterase activity in the hypoglossal nucleus of the rat and the changes produced by axotomy: a light and electron microscopic study. J. Anat. 119, 309-331. FOEHRING, R. C., SYPERT, G. W. and MUNSON, J. B. (1986a) Properties of self-reinnervated motor units of medial gastrocnemius of cat. I. Long-term reinnervation. J. Neurophysiol. 55, 931-946. FOEHRING, R. C., SYPERT,G. W. and MUNSON,J. B. (1986b) Properties of self-innervated motor units in medial gastrocnemius of cat: If. Axotomized motoneurons and the time course of recovery. J. Neurophysiol. 55, 947-965. FOEHRING, R. C., SYPERT,G. W. and MUNSON,J. B. (1987) Motor-unit properties following cross-reinnervation of cat lateral gastrocnemius and soleus muscles with medial gastrocnemius nerve. If. Influence of muscle on motoneurons. J. Neurophysiol. 57, 1227-1245. FOEHRING, R. C., SYPERT,G. W. and MUNSON,J. B. (1988) Interactions between motoneurons and the muscle fibers they innervate. In: The Current Status of Peripheral Nerve Regeneration, pp. 287-296. Eds. T. GORDON,R. STEINand P. A. SMITH. Alan R. Liss: New York.

port, turnover and distribution of choline acetyltransferase and acetylcholinesterase in the vagus and hypoglossal nerves of the rabbit. J. Neurochem. 21,

1109-1120. GALLEGO, R., IVORRA,I. and MORALES,A. (1987) Effects of central or peripheral axotomy on membrane properties of sensory neurones in the petrosal ganglion of the cat. J. Physiol., Lond. 391, 39-56. GORDON, T. (1983) Dependence of peripheral nerves on their target organs. In: Somatic and Autonomic Nerve Muscle Interactions, pp. 289-325. Eds. Y. VRBOVA,G. BURNSTOCK, R. CAMPBELL and R. GORDON. Elsevier: Amsterdam. GORDON, T., KELLY, M. E. M., SANDERS,E. J., SHAPIRO,J. and SMITHP. A. (1987) The effects of axotomy on bullfrog sympathetic neurones. J. Physiol., Lond. 392, 213-229. GOTTESFELD,Z. and FONNUM,F. (1977) Transmitter synthesizing enzymes in the hypoglossal nucleus and cerebellumeffect of actylpyridine and surgical lesions. J. Neurochem. 28, 237-239. GOTTLIEB, D. I. and COWAN, W. M. (1972) On the distribution of axonal terminals containing spheroidal and flattened synaptic vesicles in the bippocamus and dentate gyrus of the rat and cat. Z. Zellforsch. 129, 413-429. GRAFSTEIN, B. and FORMAN,D. S. (1980) lntracellular transport of neurons. Physiol. Rev. 60, 167-283. GRAFSTEIN, B. and MCQUARRIE, E. I. (1978) Role of the nerve cell body in axonal regeneration. In: Neuronal Plasticity, pp. 155-195. Ed. C. W. COTMAN. Raven Press: New York. GRACCO,V. and ABBS,J. (1988) Central patterning of speech movements. Expl Brain Res. 71, 515-526. GUSTAFFSON, B. (1979) Changes in motonuerone electrical properties following axotomy. J. Physiol., Lond. 293, 197-215. GUSTAFSSON,B. and PINTER, M. J. (1984a) Relations among passive electrical properties of lumbar ~t-motoneurones of the cat. J. Physiol., Lond. 356, 401-431. GUSTAFSSON, B. and P1NTER, M. J. (1984b) Effects of axotomy on the distribution of passive electrical properties of cat motoneurones. J. Physiol., Lond. 356, 433-442. GUSTAFSSON, B., LINDSTROM, S. and TAKATA, M. (1978) Afterhyperpolarization mechanism in the dorsal spinocerebellar tract cells of the cat. J. Physiol., Lond. 275, 283-301. HAMBERGER,A., HANSSON,H. A. and SJOSTRAND,J. (1970) Surface structure of isolated neurons: Detachment of nerve terminals during axon regeneration. J. Cell Biol. 47, 319-331. HERSTRIN, S., NICOLL, R. A., PERKEL, n. J. and SAH, P. (1990) Analysis of excitatory synaptic action in the rat hippocampus using whole-cell recording from thin slices. J. Physiol., Lond. 442, 203 225. HEYER, C. B. and LLINAS, R. (1977) Control of rhythmic firing in normal and axotomized cat spinal motoneurons. J. Neurophysiol. 40, 480-488. HILL, D. R. and BOWERY, N. G. (1981) 3H-baclofen and 3H-GABA bind to bicuculline-insensitive GABA Bsites in rat brain. Nature 290, 149-152. HOUSER, C. R., CRAWFORD, G. D., BARBER, R. P., SALVATERRA,P. M. and VAUGHN,J. E. (1983) Organization and morphological characteristics of cholingeric neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase. Brain Res. 266, 9%119. HOWE, J. R., SUTOR, B. and ZIEGLGANSBERGER,W. (1987) Characteristics of long-duration inhibitory postsynaptic potentials in rat neocortical neurons in vitro. Cell. taBlet. Neurobiol. 7, 1-18. IP, M. C. and VRBORA,G. (1983) Reinnervation of the soleus muscle by its own or an alien nerve. Neuroscience 10, 1463-1469.

INHIBITORY POSTSYNAPTICPOTENTIALS JACOB, M. H. and BERG, D. K. (1987) Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion. J. Cell Biol. 105, 1847-1854. JACOB, M. H. and BERG, D. K. (1988) The distribution of acetylcholine receptors in chick ciliary ganglion neurons following disruption of ganglionic connections. J. Neurosci. 8, 3838-3849. KASHIHARA,Y., KUNO, M. and MIYATA,Y. (1988) Cell death of axotomized motoneurones in neonatal rats, and its prevention by peripheral reinnervation. J. Physiol., Lond. 386, 135-148. KELLY, M. E. M., GORDON, T., SHAPIRO,J. and SMITH,P. A. (1986) Axotomy affects calcium sensitive potassium conductance in sympathetic neurones. Neurosci. Lett. 67, 163-168, KERNELL, D. (1965) The adaptation and the relation between discharge frequency and current strength of cat lumbosacral motoneurones stimulated by lasting injected currents. Acta physiol, scand. 65, 65-73. KLEE, M. E. (1975) Differences between monosynaptic and polysynaptic excitatory post-synaptic potentials in cat motoneurons. In: Perspectives in Neurobiology, pp. 261-271. Ed. M. SANTINI. Raven Press: New York. KRAMMER, E. B., RATH, T. and LISCHKA, M. F. (1979) Somatotopic organization of the hypoglossal nucleus: a HRP study in the rat. Brain Res. 170, 533-537. KRNJEVlC, K. (1984) Neurotransmitters in cerebral cortex: A general account. In: Functional Properties of Cortical Cells, pp. 39-62. Eds. E. G. JOHN and A. PETERS. Plenum Press: New York. KUBO, Y., ENOMOTO, S. and NAKAMURA,Y. (1981) Synaptic basis of orbital cortically induced rhythmical masticatory activity of trigeminal motoneurons in immobilized cats. Brain Res. 230, 97-110. KUNO, M. and LLINAS, R. (1970a) Enhancement of synaptic transmission by dendritic potentials in chromatolysed motoneurons of the cat. J. Physiol., Lond. 210, 807-821. KUNO, M. and LLINAS, R. (1970b) Alterations of synaptic action in chromatolysed motoneurones of the cat. J. Physiol., Lond. 210, 823-838. KUNO, M., MIYATA,Y. and MUNoZ-MARTINEZ,E. J. (1974a) Differential reaction of fast and slow ct-motoneurones to axotomy. J. Physiol., Lond. 240, 725-739. KUNO, M,, MIYATA, Y. and MUNOZ-MARTINEZ, E. J. (1974b) Properties of fast and slow ct-motoneurons following motor reinnervation. J. Physiol., LoAd. 242, 273-288. KUYPERS, H. G. J. M. (1958) An anatomical analysis of corticobulbar connexions to the pons and lower brain stem in the cat. J. Anat. 92, 198-218. KUYFERS, H. G. J. M. (1960) Central cortical projections to motor and somatosensory cell groups. An experimental study in the rhesus monkey. Brain 83, 161-184. LAIWAND, R., WERMAN, R. and YAROM, Y. (1988) Electrophysiology of degenerating neurones in the vagal motor nucleus of the guinea-pig following axotomy. J. Physiol., Lond. 404, 749-766. LEWIS, P. R., FLUMERFELT,B. A. and SCHUTE,C. C. D. (1971) The use of cholinesterase techniques to study topographical localization in the hypoglossal nucleus of the rat. J. Anat. 110, 203-213. LLINAS, R. and BAgER, K. (1972) A chloride-dependent inhibitory postsynaptic potential in cat trochlear motoneurons. J. Neurophysiol. 35, 484-492. LLINAS, R. and SUGIMORI,M. (1980) Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices. J. Physiol., Lond. 305, 171-195. MAHLER, J. and N)/~, A. (1984) Rearrangement of synapses on guinea-pig sympathetic ganglion cells after partial interruption of the preganglionic nerve. J. Physiol., Lond. 348, 43-56.

409

MATTHEWS, M. R. and NELSON, V. H. (1975) Detachment of structurally intact nerve endings from chromatolytic neurons of rat superior cervical ganglion during the depression of synaptic transmission induced by postganglionic axotomy. J. Physiol., LoAd. 245, 91-135. MATUS, A. I. and DENNISON, M. E. (1971) Autoradiographic localization of tritiated glycine at 'flat-vesicle' synapses in the spinal cord. Brain Res. 32, 195-197. MCLAUGHLIN, B. J., WOOD, J. G., SAITO, K., BARBER, R., VAUGHN,J, E,, ROBERTS,E. and Wu, J. Y. (1974) The fine structural localization of glutamic decarboxylase in synaptic terminals of rodent cerebellum. Brain Res. 76, 377-39 I. MENDELL, L. M. (1974) Modifiability of spinal synapses. Physiol. Rev. 64) 260-324. MENDELL, L. M., MUNSON, J. B. and SCOTT, J. G. (1976) Alterations of synapses on axotomized motoneurons. J. Physiol., Lond. 256, 67-79. MILLER, A. J. (1982) Deglutition. Physiol. Rev. 62, 129-184. Molx, L. J., GI~mSON,D. M., ARMSTRONG,D. M. and WILEY, R. G. (1991) Separate signals mediate hypoglossal motor neuron response to axonal injury. Brain Res. 564, 176-180. MORIMOTO, T., TAKATA, M. and KAWAMURA, Y. (1968) Effect of lingual nerve stimulation of hypoglossal motoneurons. Expl Neurol. 22, 174-190. NAKAJIMA, Y. and R~.SE, T. S. (1983) Inhibitory and excitatory synapses in crayfish stretch receptor organs studied with direct rapid freezing and freeze-substitution. J. comp. Neurol. 213, 66-73. NAKA~tJRA,Y., GOLDBERG,L. J. and CLEMENTE,C. D. (1967) Nature of suppression of the masseteric monosynaptic reflex induced by stimulation of the orbital gyrus of the cat. Brain Res. 6, 184-198. NEWBERRY, N. R. and NICOLL, R. A. (1984) A bicucullineresistant inhibitory post-synaptic potential in rat hippocampal pyramidal cells in vitro. J. Physiol., Lond. 348, 239-259. NICOLL, R. A., ALGER, B. E. and JArlR, C. E. (1980) Enkephalin blocks inhibitory pathways in the vertebrate CNS. Nature 287, 22-25. NJ.~, A. and PURVES,P. (1978) The effects of nerve growth factor and its antiserum on synapses in the superior cervical ganglion of the guinea-pig. J. Physiol., Lond. 277, 53-75. ODUTOLA,A. B. (1976) Cell grouping and Golgi architecture of the hypoglossal nucleus of the rat. Expl Neurol. 52, 356-371. OgADA, Y., NITSCH-HASSLER, C., KIM, J. S., BAg, I. J. and HASSLER, R. (1971) Role of gamma-aminobutyric acid (GABA) in the extrapyramidal motor system. I. Regional distribution of GABA in rabbit, rat, guinea pig and baboon CNS. Expl Brain Res. 13, 514-518. PERKEL, D. J., HESTRIN, S., SAH, P. and NICOLL, R. A. (1990) Excitatory synaptic currents in cerebellar Purkinje cells. Proc. R. Soc. Lond. B Biol. Sci. 241, 116-121. PINTER, M. J. and VANOEN-NOWN, S. (1989) Effects of preventing reinnervation on axotomized spinal motoneurons in the cat. I. Motoneuron electrical properties. J. Neurophysiol. 62, 311-324. PORTER, R. (1967) Cortical actions on hypoglossal motoneurones in cats: a proposed role for a common internuncial cell. J. PhysioL, Lond. 193, 295-308. PURVES, D. (1975) Functional and structural changes in mammalian sympathetic neurones following interruption of their axons. J. Physiol., Lond. 252, 429--463. PURVES, D. and LICHTMAN, J. W. (1978) Formation and maintenance of synaptic connections in autonomic ganglia. Physiol. Rev. 58, 821-862. PURVES, D. and NJ.~, A. (1976) Effect of nerve growth factor on synaptic depression following axotomy. Nature 260, 535-536.

410

M. TAKATA

PURVES, D. and NJ£, A. (1978) Trophic maintenance of synaptic connections in autonomic ganglia. In: Neuronal Plasticity, pp. 27-47. Ed. C. W. COTMAN. Raven Press: New York. REDMAN,S. (1990) Quantal analysis of synaptic potentials in neurons of the central nervous system. Physiol. Rev. 70, 165-198. ROTTER, A., BIRDSALL,N. J. M., BURGEN, A. S. V., FIELD, P. M. and RAISMAN, G. (1977) Axotomy causes loss of muscarinic receptors and loss of synaptic contacts in the hypoglossal nucleus. Nature 256, 734-735. ROTTER, A., BIRDSALL, N. J. M., BURGEN, A. S. V., FIELD, P. M., SMOLEN, A. and RAISMAN,G. (1979) Muscarinic receptors in the central nervous system on the rat--IV. A comparison of the effects of axotomy and deafferentation on the binding of [3HI propylbenzilycholine mustard and associated synaptic changes in the hypoglossal and pontine nuclei. Brain Res. Rev. 1, 207-224. SAPER, C. B. and LOEWY, A. D. (1980) Efferent connections of the parabrachial nucleus in the rat. Brain Res. 197, 291 317. SATOH, K., ARMSTRONG,D. M. and FIBIGER,H. C. (1983) A comparison of the distribution of central cholinergic neurons as demonstrated by acetylcholinesterase pharmacohistochemistry and choline acetyltransferase immunohistochemistry. Brain Res. Bull. II, 693-720. SATOU, M., MORI, K., TAZAWA,Y. and TAKAGI,S. F. (1982) Two types of postsynaptic inhibition in pyriform cortex of the rabbit: fast and slow inhibitory postsynaptic potentials. J. Neurophysiol. 48, 1142-1156. SCHMALBRUCH, H. (1984) Motoneuron death after sciatic nerve section in newborn rats. J. comp. Neurol. 22,4, 252 258. SERNAGOR, E., YAROM,Y. and WERMAN, R. (1986) Sodiumdependent regenerative responses in dendrites of axotomized motoneurons in the cat. Proc. natn. Acad. Sci. U.S.A. 83, 7966-7970. SHEARD, P., MCCAIG, C. D. and HARRIS,A. J. (1984) Critical periods in rat motoneuron development. Derl Biol. 102, 21-31. SMOLEN,A. J. ( 19833 Retrograde transneuronal regulation of the afferent innervation to the rat superior cervical sympathetic ganglion. J. Neurocytol. 12, 27-45. SNIDER, W. D. and THANEDAR,S. (1989) Target dependence of hypoglossal motor neurons during development and in maturity. J. comp. Neurol. 279, 489-498. SUMNER, B. E. H. (1975a) A quantitive analysis of the responses of presynaptic boutons to postsynaptic motor neuron axotomy. Expl Neurol. 46, 605~15. SUMNER, B. E. H. (1975b) A quantitative analysis of boutons with different types of synapses in normal and injured hypoglossal nuclei. Expl Neurol. 49, 406-418. SUMNER, B. E. H. (1975c) A quantitative study of subsurface cisterns and their relationships in normal and axotomized hypoglossal neurones. Expl Brain Res. 22, 175 183. SUMNER, I. E. H. and SUTHERLAND,F. 1. (1973) Quantitative electron microscopy on the injured hypoglossal nucleus in the rat. J. Neurocytol. 2, 315-328. SUMNER, B. E. H. and WATSON,W. E. (19713 Retraction and expansion of the dendritic tree of motor neurones of adult rats induced in t:itro. Nature 233, 273 275. SZENTAGOTHAI, J. (1948) Anatomical considerations of monosynaptic reflex arcs. J. Neurophysiol. II, 445-453. TAKASU, N. and HASHIMOTO, P. H. (1988) Morphological identification of an interneuron in the hypoglossal nucleus of the rat: A combined Golgi-electron microscopic study. J. comp. Neurol. 271,461-471. TAKASU, N., NAKATANI, T., ARIKUNI, T. and KIMURA, H. (19873 Immunocytochemical localization of 7-aminobutyric acid in the hypoglossal nucleus of the macaque monkey, Macaca fuscata: A light and electron microscopic study. J. comp. Neurol. 263, 42 53.

TAKATA,M. (1979) Postsynaptic potentials in the jawopening motoneurons by stimulation of the trigeminal nerves. Brain Res. 163, 161-164. TAKATA, M. (1981a) Lingually induced inhibitory postsynaptic potentials in hypoglossal motoneurons after axotomy. Brain Res. 22,4, 165-169. TAKATA, M. (1981b) Synaptic linkage of lingual nerve afferents to hypoglossal motoneurons, In: Oral-Facial Sensory and Motor Functions, pp. 25-36. Eds. Y. KAWAMURAand R. DUaN~. Quintessence: Tokyo. TAKATA, M. (1982) Inhibitory postsynaptic potentials evoked in hypoglossal motoneurons by lingual nerve stimulation. Expl Neurol. 75, 103-11 I. TAKATA, M. and FUJITA, S. (1979) The properties of lingually induced IPSPs in the masseteric motoneurons. Brain Res. 168, 648-651. TAKATA, M. and NAGAHAMA,T. (1983) Synaptic efficacy of inhibitory synapses in hypoglossal motoneurons after transection of the hypoglossal nerves. Neuroscience 10, 23--29. TAKATA, M. and NAGAHAMA,T. (1984) Cortically induced postsynaptic potentials in hypoglossal motoneurons after axotomy. Neuroscience 13, 855-862. TAKATA, M. and NAGAHAMA, T. (1986) Cortically and lingually induced postsynaptic potentials in trigeminal motoneurons after axotomy. Expl Brain Res. 61, 272-279. TAKATA,M. and OGATA,K. (1980) Two components of inhibitory postsynaptic potentials evoked in hypoglossal motoneurons by lingual nerve stimulation. Expl Neurol. 69, 299-310. TAKATA, M. and TOMOMUNE,N. (19863 Properties of postsynaptic potentials evoked in hypoglossal motoneurons by inferior alveolar nerve stimulation. Expl Neurol. 93, 117 127. TAKATA, M. and TOMOMUNE, N. (19873 Properties of sterotyped series of postsynaptic potentials in hypoglossal motoneurons. Brain Res. 426, 358-366. TAKATA, M., FUJITA, S. and SHOHARA, E. (1979) Postsynaptic potentials in the hypoglossal motoneurons set up by hypoglossal nerve stimulation. Jap. J. Physiol. 29, 49-60. TAKATA, M., SHOHARA,E. and FUJITA, S. (19803 The excitability of hypoglossal motoneurons undergoing chromatolysis. Neuroscience 5, 413-419. TAKATA, M., FUJITA,S. and KANAMORI,N. (1982) Repetitive firing in trigeminal mesencephalic tract neurons and trigeminal motoneurons. J. Neurophysiol. 47, 23-30. TAKATA, M., TOMOMUNE, N., NAGAHAMA,T., TOMIOKA, S. and NAKAJO, N. (1990) Synaptic efficacy of inhibitory synapses and repetitive firing in the reinnervating trigeminal and hypoglossal motoneurons. Neuroscience 36, 785 792. TAKATA, M., TOMOMUNE,N. and TOMIOKA,S. (1991) Synaptic efficacy of inhibitory synapses in the reinnervating hypoglossal motoneurons. Neuroscience 44, 757-763. THALMANN, R. n . and AYALA, F. F. (1982) A late increase in potassium conductance follows synaptic stimulation of granule neurons of the dentate gyrus. Neurosci. Lett. 29, 243-248. TITMUS, M. J. and FABER,D. S. (1986) Altered excitability of goldfish Mauthner cell following axotomy. II. Localization and ionic basis. J. Neurophysiol. 55, 1440-1454. TITMUS, M. J. and FAaER, D. S. (19903 Axotomy-induced alterations in the electrophysiological characteristics of neurons. Prog. Neurobiol. 35, 1-51. TOMOMUNE, N. and TAKATA, M. (19883 Excitatory and inhibitory postsynaptic potentials in cat hypoglossal motoneurons during swallowing. Expl Brain Res. 71, 262 272. TRAVERS,J. B. and NORGREN, R. (19833 Afferent projections to the oral motor nuclei in the rat. J. comp. Neurol. 220, 280-298.

INHIBITORY POSTSYNAPTICPOTENTIALS TSUKAHARA,N. (1978) Synaptic plasticity in the red nucleus. In: Neuronal Plasticity, pp. 13-130. Ed. C. W. COTMAN. Raven Press: New York. TSUKAH^RA, N. and FtJm'O, Y. (1976) Physiological evidence of new synapses from cerebrum in the red nucleus neurons following cross-union of forelimb nerves. Brain Res. 106, 184-188. UCHIZONO, K. (1965) Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat. Nature 207, 642-643. UEMURA, M., MATSUDA, K., Kosm, M., TAKEUCHI, Y., M^TSUSmMA, R. and MmUNO, N. (1979) Topographical arrangement of hypoglossal motoneurons: An HRP study in the cat. Neurosci. Left. 13, 99-104. WALBERO, F. (1957) Do the motor nuclei of the cranial nerves receive corticofugal fibers? An experimental study in the cat. Brain 80, 597~i05. WAMSLEY, J. K., LEWIS, M. S. and KUHAR, M. J. (1981) Autoradiographic localization of muscarinic cholinergic receptors in rat brain stem. J. Neurosci. 1, 176-191. WATSON, W. E. (1965) An autoradiographic study of the incorporation of nucleic acid precursors by neurons and glia nerve regeneration. J. Physiol., Lond. 180, 740-753. WATSON, W. E. (1971) Quantitative observations upon acetylcholine hydrolase activity of nerve cells after axotomy. J. Neurochem. 13, 1549-1550. WATSON, W. E. (1974) Cellular responses to axotomy and to related procedures. Br. reed. Bull. 30, 112-115. WOLD, J. E. and BRODAL, A. (1973) The projection of cortical sensorimotor regions onto the trigeminal nucleus in the cat. An experimental anatomical study. Neurobiology 3, 353--375. WOLD, J. E. and BROOAL,A. (1974) The cortical projection of the orbital and procreate gyri to the sensory trigeminal

411

nuclei in the cat. An experimental anatomical study. Brain Res. 65, 381-395. WOOD, S. J., PmTCHARD, J. and SOFRONmW, M. V. (1990) Re-expression of nerve growth factor receptor after axonal injury recapitulates a developmental event in motor neurons: differential regulation when regeneration is allowed or prevented. Eur. J. Neurosci. 2, 650-657. WOOLSEY, C. N. (1958) Patterns of sensory representation in the cerebral cortex. In: Biological and Biochemical Bases of Behavior, pp. 63-81. Eds. H. F. HARLOW and C. N. WOOLSEY. The University of Wisconsin Press: Madison. WOOTEN, G. F., PARK, D. H., JOH, T. H. and REIS, D. J. (1978) Immunochemical demonstration of reversible reduction in choline acetyltransferase concentration in rat hypoglossal nucleus after hypoglossal nerve transection. Nature 275, 324-325. YAROM, Y., BRACHA, O. and WERMAN, R. (1985a) lntracellular injection of acetylcholine blocks various potassium conductances in vagal motoneurons. Neuroscience 16, 739-752. YAROM, Y., SUGIMORI, M. and LLINAS, R. (1985b) Ionic currents and firing patterns of mammalian vagal motoneurons in vitro. Neuroscience 16, 719-737. YOOM, K.-W. and ROTHMAN,S. i . (1991) The modulation of rat hippocampal synaptic conductances by baclofen and ),-aminobutyric acid. J. Physiol., Lond. 442, 377-390. ZARBIN, M. A., WAMSLEY, J. K. and KUHAR, M. J. (1981) Glycine receptor: light microscopic autoradiographic localization with [~H]-strychnine. J. Neurochem. 1, 532-547. ZISKIND-CONHAIM, L. and PRESLEY,S. (1991) Reinnervation of developing rat muscle by non-axotomized motoneurons. J. comp. Neurol. 313, 725-734.