Neuroscience Vol. 36, No. 3, pp. 785-792,
0306-4522/90$3.00+ 0.00 Pergamon Press plc 0 1990IBRO
1990
Printed in Great Britain
SYNAPTIC EFFICACY OF INHIBITORY SYNAPSES AND REPETITIVE FIRING IN THE REINNERVATING TRIGEMINAL AND HYPOGLOSSAL MOTONEURONS M. TAKATA,* N. TOMOMUNE,T. NAGAHAMA, S. TOMIOKAand N. NAKAJO Department of Physiology, School of Dentistry, Tokushima University, Kuramoto-cho, Japan
Tokushima 770,
Abstract-The synaptic efficacy and repetitive firing in masseteric motoneurons after the self-union operation and in tongue protruder motoneurons after their cut axons were reunited to tongue retractor muscles, the styloglossus muscle, were studied in cats. To ensure the correct identification of reinnervating motoneurons, the muscle response produced by an induced spike in a motoneuron by intracellularly injected depolarizing current was recorded. In both masseteric and tongue protruder motoneurons there were no differences on the patterns of postsynaptic potentials produced in reinnervating and non-reinnervating motoneurons by peripheral nerve stimulation, suggesting that the recovery of the synaptic efficacy of inhibitory synapses is time-dependent rather than muscle reinnervation. However, the present study demonstrated that the recovery of processes that control rhythmical firing of motoneurons is probably dependent on muscle reinnervation.
reveal two distinct slopes, the primary and the secIn axotomized motoneurons it ondary range. 4,6,36*39 was found that a distinct breakpoint in they-l plots the was missing,‘3,38 and that in spinal motoneurons slopes of the f-Z curves were steeper following axotomy, and these steeper slopes were well correlated with the decreased AHP conductance.12 Thus, it is expected that axotomy will result in marked changes in the firing of motoneurons. 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.20~43 The purpose of the present paper is to present the synaptic efficacy in Mass.Mns after the self-union operation and in hypoglossal motoneurons after their cut axons were reunited to the foreign muscles.
In adult cats, sectioning the masseter and the hypoglossal nerves causes a disjunction of presynaptic contacts on axotomized masseteric and hypoglossal motoneurons.33,37,38 In hypoglossal motoneurons, morphological studies have revealed that by one week after axotomy there is a loss of about 50% of the synapses.3’q32 Sumner and Sutherland32 and Sumner” have also observed a reduced number of dendritic synapses accompanied with detachment of synapses from the soma membrane. In masseteric motoneurons (Mass.Mns), it has been demonstrated that axotomy is followed by the decline of synaptic efficacy of inhibitory rather than of excitatory underlying the synapses. 38 The precise mechanism loss of efficacy at these chemical synapses is unclear, and it is not known whether muscle reinnervation itself is necessary for synapse recovery. Another effect of axotomy that occurs in motoneurons is a reduction in the amplitude and duration of the afterhyperpolarization (AHP), which follows an action potential.i2,‘9 These changes may be indicative of a transient response to axotomy. However, the maintenance of certain intrinsic properties of motoneurons (resting potential and input resistance) is unchanged, indicating that these membrane properties are independent of target innervation.5 As is already known for cat spinal, hypoglossal and trigeminal motoneurons, the frequency-current (f-Z) functions plotted for the first interspike intervals
EXPERIMENTAL PROCEDURES All experiments were performed on 15 young, adult cats (1.75-2.1 kg). In the self-union operation, the central stump of the right masseter nerves was cut and immediately reunited with its own peripheral stump by suturing the nerve sheath with a 9/10 silk thread under sodium pentobarbital anesthesia (Nembutal, 30 mg/kg, i.p.). The left masseter nerves were left intact and animals were allowed to survive for 146 days. The hypoglossal nerve of the cat divides into the medial and the lateral branches. The fibers of the medial branch innervate the tongue protruder muscles, and those of the lateral branch the tongue retractor muscles. In experiments, the medial and the lateral branch of the right hypoglossal nerve were cut and cut medial branches were reunited with a 9/10 silk thread to the tongue retractor muscle, the styloglossus muscle, aseptically under sodium pentobarbital anesthesia (Nembutal, 30 mg/kg, i.p.). The proximal stump of the lateral branch was ligated with silk thread and the left hypoglossal nerves were left intact. In some cases regeneration was prevented by inserting the cut end of the lateral
*To whom correspondece should be addressed. AHP, afterhyperpolarization; EPSP, excitatory postsynaptic potential; f-I,frequency-current; IPSP, inhibitory postsynaptic potential; Mass.Mn, masseteric motoneuron; P-Mn, tongue protruder motoneuron; PSP, postsynaptic potential.
Abbreviations:
785
786
M. TAKATAC~
branch into polyethylene tube with a blind distal end. Animals were then allowed to survive for 126 days. In the terminal experiments, the cerebellum was sucked out under sodium pentobarbital anesthesia (Nembutal, 30mg/kg, i.p.) in order to enable penetration into the trigeminal and the hypoglossal nucleus by a micropipette. Unitary muscle activity was recorded by a wire electrode (diameter 120 pm, interelectrode distance 3 mm, insulation removed for 3 mm) implanting the masseter and the styloglossus muscle when a motoneuron was stimulated by injecting a depolarizing current. In the masseter muscle, a wire electrode was implanted into the superficial masseter muscle, the anterior and the posterior deep masseter muscle. In the animals used for the experiments on the effect of cortical stimulation on Mass.Mns a pneumothorax was made, and cats were immobilized by intravenous injection of gallamine triethiodide, and respiration was maintained artificially. The left eye was enucleated and the orbitofrontal area of the cerebral cortex was exposed. Before the administration of gallamine triethiodide, the dura was opened and a spring-mounted silver ball electrode (diameter I mm) was placed on the cortical surface and was fixed at the optimum point for inducing jaw opening-closing movements by repetitive stimuli at 50/s, the reference electrode being inserted in temporal muscles. Single shock, surface anodal stimuli (02O.S ms, below 2 mA) were applied at this site. Body temperature was maintained by means of an electric blanket thermostatically controlled from a rectal thermister. A sleeve elecrode was used to stimulate the previously sutured axons and the ipsilateral lingual nerve. Two screw electrodes implanted into the mandible were used to stimulate the ipsilateral inferior alveolar nerve. In the present experiments both the lingual nerve and the inferior alveolar nerve were stimulated at an intensity of five times the nerve threshold. In intracellular recordings. a glass micropipette
A
1
al
filled with 2 M potassium citrate ing and for current injection. between 15 and 25 MR.
was used both for recordElectrode resistance was
RESULTS
Synaptic potentials mo toneurons
in
reinnervating
masseteric
Postsynaptic potentials (PSPs) in the Mass.Mns 146 days after the central stump of the cut masseter nerve was reunited with its own peripheral stump (self-union) were explored. As illustrated in Fig. 1, the degree of reinnervation was estimated by the ratio of the maximum jaw-closure produced by stimulation of the masseter nerve in intact side (A*) to that produced by stimulation of sutured axons in operated side (A, ). From this test, the degree of reinnervation was estimated as 70% in this sample 146 days after the self-union operation. As illustrated in Fig. lB,, a Mass.Mn was identified from the antidromic spike evoked by stimulation of the previously sutured axons. To ensure the correct identification of reinnervating Mass.Mns, the following study was performed. The muscle response in the masseter muscle produced by an induced spike in a Mass.Mn by intracellularly injected depolarizing current was recorded and illustrated in Fig. lB,. When a spike was elicited in a Mass.Mn by passing brief depolarizing currents across the membrane
2 -Il-
llmm
B
2
.
w I
5mV
20mZ Fig. 1. PSPs in a reinnervating Mass.Mn. (A) Degree of reinnervation. The jaw-closure produced by stimulation of the masseter nerve in intact (2) and operated side (1) is shown. (B) Reinnervating MassMn. The record a shows an antidromic spike. Unitary muscle activity in the anterior deep masseter muscle (b,) followed an induced spike in a Mass.Mn (b,). (C) PSPs. The records a, and b, show a lingually induced and an inferior alveolar-induced PSP. The records a2 and b, show filed potentials. (D) Effect of membrane polarization. The records a and b show a lingually induced and an inferior alveolar-induced PSP.
Postsynaptic potentials in reinnervating motoneurons unitary muscle activity was evoked in the anterior deep masseter muscle (Fig. lBb,) and followed an induced spike one-for-one with a constant latency. No muscle responses were produced in the superficial masseter and the posterior deep masseter muscle by an induced spike of this MassMn, indicating that this Mass.Mn reinnervated the anterior deep masseter muscle (reinnervating motoneurons). In 18 out of 30 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 2.5 to 4.5 ms (n = 18). As illustrated in Fig. 1C in the reinnervating Mass.Mn stimulation of the lingual nerve (Fig. lC,,) or the inferior alveolar nerve (Fig. lC,,) produced large inhibitory postsynaptic potentials (IPSPs). The latency of an inferior alveolar-induced IPSP was about 3.0 ms from stimulus artifact (mean + S.D., 3.5 + 0.5 ms, n = 18). When a single shock was delivered to the lingual nerve, a small excitatory postsynaptic potential (EPSP) was seen before the generation of large IPSPs. The relation between the amplitude of lingually induced or inferior alveolarinduced hyperpolarizing potentials and the membrane potential displacements was examined and results are shown in Fig. 1D,, D, Numerals denote the amount of injected current in nanoamperes. In both cases, by displacing the membrane potential towards hyperpolarization, a hyperpolarizing synaptic potential was decreased in amplitude and reversed to a depolarizing potential. By displacement of the membrane potential to depolarization, a hyperpolarizing synaptic potential was increased in amplitude. In 12 out of 30 explored cells, no muscle responses were produced by an induced spike of the cell. A sample record is shown in Fig. 2. The antidromic spike evoked by stimulation of the previously sutured axons is shown in Fig. 2A,. Simultaneous recordings of an induced spike (Fig. 2A,,,) and muscle responses of the anterior deep masseter muscle (Fig. 2A,,) are shown in Fig. 2A,. No muscle responses were produced in the superlicial masseter, the anterior deep and the posterior deep masseter muscle by an induced spike of this Mass.Mn, indicating that this cell failed to reinnervate the masseter muscle (non-reinnervating motoneurons). In this cell stimulation of the lingual nerve or the inferior alveolar nerve produced a large IPSP (illustrated in Fig. 2B,, ,B,, ). By lingual nerve stimulation, a small EPSP followed by predominant IPSPs was produced. Finally, we found that there were no differences on the patterns of PSPs produced in reinnervating and non-reinnervating Mass.Mn by lingual or inferior alveolar nerve stimulation. In the following studies, the effect of cortical stimulation on MassMns 146 days after the selfunion operation was explored. In 36 out of 37 explored MassMns stimulation of the cerebral cortex (Fig. lB,,),
Fig. 2. PSPs in a non-reinnervating Mass.Mn. (A) Nonreinnervating Mass.Mn. The record a shows an antidromic spike. No muscle reponses were produced in the masseter muscle (b,) by an induced spike in a Mass.Mn (b,). (B) PSPs. The PSPs produced by stimulation of the lingual nerve (a,) or the inferior alveolar nerve (b,) are shown. The records a2 and b, show field potentials. (C) Effect of membrane polarization on cortically induced PSPs in a cell 146 days after the self-union operation.
produced large IPSPs. A sample record is shown in Fig. 2C. At the resting membrane level (0 nA), stimulation of the cerebral cortex produced large IPSPs. By displacing the membrane potential towards hyperpolarization, cortically induced hyperpolarizing synaptic potentials were decreased in amplitude and reversed to a depolarizing potential. By displacement of the membrane potential to depolarization, hyperpolarizing potentials were increased in amplitude. In the remaining cell, large EPSPs followed by IPSPs were produced by cortical stimulation. Synaptic potentials motoneurons
in
reinnervating
hypoglossal
Synaptic potentials in the tongue protruder motoneurons (P-Mns) 126 days after their cut axons were reunited to the tongue retractor muscle, the styloglossus muscle, were explored and results are shown in Fig. 3. As illustrated in Fig. 3A,,B,, a motoneuron was identified from the antidromic spike evoked by stimulation of the previously sutured axons in the 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. 3A was stimulated with depolarizing current (Fig. 3A,,) unitary muscle activity in the styloglossus muscle followed the cell spike one-for-one with constant latency (Fig. 3A,,), indicating that this P-Mn reinnervated the tongue retractor muscles, the styloglossus muscle. In this innervating P-Mn, stimulation of the inferior alveolar nerve produced an IPSP, illustrated in
788
M. TAKATAet al.
Fig. 3. PSPs in P-Mns after cut axons were reunited to the foreign muscle. (A) Reinnervating P-Mn. The record a shows an antidromic spike. Unitary muscle activity in the styloglossus muscle (b,) followed an induced spike in a P-Mn (b,). An inferior alveolar-induced IPSP is shown in c, The record c1 shows field potentials. The records d show the effect of membrane polarization on an inferior alveolar-induced IPSP. (B) Non-reinnervating P-Mn. No muscle responses were produced in the styloglossus muscle (b,) by an induced spike in a P-Mn (b2). Record a shows an antidromic spike. An inferior alveolar-induced IPSP is shown in c, The record cj shows field potentials.
Fig. 3A,, The relation between the amplitude of inferior alveolar-induced hyperpolarizing synaptic potentials and the membrane potential displacements was examined and results are shown in Fig. 3A,. 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 to 4.5 ms (n = 23). In nine reinnervating cells, synaptic potentials produced by inferior alveolar nerve 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. 3B, no muscle responses were produced (Fig. 3B,,) by an induced spike of the cell (Fig. 3B,,), indicating that this cell failed to reinnervate the styloglossus muscle. In this cell stimulation of the inferior alveolar nerve produced an IPSP (Fig. 3B,,). In 31 out of 54 explored cells no muscle responses were produced by an induced spike of the cell. In I8 non-reinnervating cells synaptic potentials produced by stimulation of the inferior alveolar nerve 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 produced in reinnervating and non-reinnervating P-Mns by inferior alveolar nerve stimulation. Repetitive firing
in reinnervating
motoneurons
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. 4. In a P-Mn illustrated in Fig. 4A, unitary muscle activity in the styloglossus muscle (Fig. 4A,,) followed the cell spike (Fig. 4A,,) one-for-one, indicating that this P-Mn reinnervated the styloglossus muscle. In this reinnervating P-Mn, the relationship between the firing frequency (impulses/s) and the amount of injected depolarizing current (nanoamperes) was examined. Repetitive firing of this cell responding to increasing amounts of injected depolarizing current is illustrated in Fig. 4A,. 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 (k 2.5, n = 10) and 45 (k 5.5, n = 10) imp/s per nA, illustrated by open circles in Fig. 4C. In a P-Mn illustrated in Fig. 4B, no muscle responses were produced (Fig. 4B,,) by an induced spike of the cell (Fig. 4B,,). As illustrated in Fig. 4B,,, this non-reinnervating P-Mn shows sustained firing to maintain pulses of intracellularly injected depolarizing currents. The rheobase of this cell was 2 nA (2-6 nA, n = 10). The f-1 relation
789
Postsynaptic potentials in reinnervating motoneurons C imp/s
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)2ooJN 300-
b
I
Smrrc
_ Smrec
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)20mV
0
250-
l-
B 0
0
100
50
0,
1 0
5
10 nA
Fig. 4. Repetitive firing of P-Mns after cut axons were reunited to the foreign muscle. (A) Reinnervating P-Mn. Unitary muscle activity in the styloglossus muscle (a,) followed an induced spike in a P-Mn (az). Repetitive firing caused by injecting current is shown in b. (B) Non-reinnervating P-Mn. No muscle responses were produced in the styloglossus muscle (a,) by an induced spike in a P-Mn (a,). Repetitive firing caused by injecting current is shown in b. (C)f-I relation. Results obtained from an reinnervating and non-reinnervating P-Mn are illustrated by open and filled circles.
plotted from the first interspike intervals of a non-reinnervating P-Mn is represented by filled
circles in Fig. 4C. In a non-reinnervating P-Mn, a distinct breakpoint in the f-1 plots was missing, as has already been found in axotomized spinal and trigeminal motoneurons.‘3’38
In a Mass.Mn 146 days after the self-union operation, the f-1 relation was examined and results are shown in Fig. 5. As illustrated in Fig. SA, the reinnervating Mass.Mn shows sustained firing to maintained pulses of intracellularly injected depolarizing currents. Unitary muscle activity in the deep masseter muscle (the upper in each record) followed the cell spike one-for-one with constant latency. In this Mass.Mn, the f-I relation plotted from the first interspike intervals revealed two distinct slopes, illustrated in Fig. 5B. The slope of the primary and the secondary range was 15 ( f 2.5, n = 7) and 45 (&-4.5, n = 7) imp/s per nA, respectively.
DISCUSSION
5
10
15 nA
Fig. 5. Repetitive firing of a Mass.Mn after the self-union operation. (A) Repetitive firing of an reinnervating Mass.Mn to maintained pulses of intracellularly injected depolarizing currents. Unitary muscle activity in the deep masseter muscle (the upper in each record) followed by a spike in a Mass.Mn. (B) f-l relation plotted from the first interspike intervals.
In two-thirds of explored Mass.Mns 146 days after the self-union operation, an induced spike of the cell produced unitary muscle activity with a large amplitude in the masseter muscle. In 23 out of 54 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 31 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. In this respect, it was reported
790
M. TAKATA~~
that cut nerves often failed to regenerate to the muscle by mechanical stresses8s9 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.” Furthermore, Ip and Vrbora14 showed that in leg muscles either soleus’ own nerve or a foreign nerve was eventually able to innervate soleus muscle, indicating that motoneurons show little or no preference for their former muscle fibers. 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. ‘“~‘7~‘8~23 It was also reported that full recovery of motoneuron properties and synaptic contacts onto axotomized motoneurons occurs after muscle reinnervation.3’ In axotomized MassMns, it was 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 greatly reduced while that of excitatory synapses unaffected.38 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 and the excitatory influence was masked by IPSPS’~.*~.‘~ and that excitation of the inferior alveolar nerve evoked multiphasic responses, the IPSP-EPSP-IPSP sequence.3 In the present experiments, it was found that in both reinnervating and non-reinnervating Mass.Mns, stimulation of either the lingual nerve or the inferior alveolar nerve produced large IPSPs, suggesting that the recovery of the synaptic efficacy of inhibitory synapses is time-dependent rather than muscle reinnervation. In the Mass.Mns 146 days after the self-union operation, cortical stimulation produced large IPSPs in 36 out of 37 explored cells, suggesting that the recovery of synaptic contacts made by the corticobulbar fibers is also time-dependent. It was demonstrated that the majority of the total sample of explored P-Mns 40 days after axotomy showed an EPSP-IPSP sequence when the peripheral nerves were stimulated.” In the present experiments, in P-Mns 126 days after their cut axons were reunited to the styloglossus muscle, it was found that in non-reinnervating cells, when a single shock was delivered to the inferior alveolar nerve, IPSPs were evoked in two-thirds of explored cells, as demonstrated in normal PMns,34.40 suggesting that the recovery of the synaptic efficacy of inhibitory synapses in hypoglossal motoneurons is also time-dependent rather than muscle reinnervation. In superior cervical ganglion cells, a disjunction of presynaptic contacts on axotomized neurons was diminished if exogenous nerve growth factor
al.
was supplied to the ganglion cells, suggesting that one of the roles of the target tissue is to supply a trophic factor.26 Moreover, in sympathetic ganglion cells, it was suggested that a trophic 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.2’ In invertebrates, it was demonstrated that inhibitory chemical synapses on buccal motoneurons of Helisoma show a transient decrease of efficacy during organ culture, and motoneurons exhibit recovery of their chemical synapses without target innervation.5 In trigeminal and hypoglossal motoneurons, it is probably safe to assume that the synapse recovery is not dependent on retrograde trophic messages from muscle. In sympathetic ganglion cells and spinal motoneurons, it was revealed that axotomy causes loss of functional inputs22,24,28and the death of many injured neurons, 27~30 and revealed that there is an increase in the number of synaptic inputs to the surviving neurons.4’.42 However, in the present studies, there were no differences in the patterns of PSPs produced in reinnervating and non-reinnervating motoneurons by peripheral nerve stimulation. In rat hypoglossal nucleus, cell loss did not exceed 25% and was not detectable until more than 28 days had elapsed after operation.’ However, Aldskogius and Thomander2 studied the organization of the facial motor nucleus and reported that after transection of the facial nerve at the adult stage, no significant loss of motoneurons occurred. Recently, Kashihara et al.” reported that in rats four days after birth the majority of axotomized motoneurons die if target contact is prevented, whereas, in rats four weeks after birth, section of the sciatic nerve is known to cause no loss of the motoneurons.” In reinnervating MassMns and P-Mns fired in sustained manner by injecting depolarizing currents into the cell, the f-Z relation revealed two distinct slopes (the primary and the secondary range). In contrast, a distinct breakpoint in the f-Z relation was missing in non-reinnervating motoneurons as demonstrated in axotomized motoneurons. Therefore, the recovery of processes that control rhythmical firing of motoneurons is probably dependent on muscle reinnervation, as suggested by Kuno and Llinas” and Czeh et al.’ that AHP duration is regulated by retrograde trophic messages from muscle. Recently, of motor units Foehring et al.” studied properties following cross-reinnervation of cat lateral gastrocnemius and soleus muscles by the medial gastrocnemius nerve and conclude that the innervated muscle may influence the expression of motoneuron electrical properties. Acknowledgement-This study was supported in part by a grant-in-aid for scientific research 01304045 from the Japanese Ministry of Education, Science and Culture.
Postsynaptic potentials in reinnervating motoneurons
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21. Mahler- J. and Nja A. (l-984) Rearrangement of synapses’ on guinea-pig sympathetic ganglion cells after partial interruption of the preganglionic nerve. J. Physiol., Lond. 348, 43-56. 22. 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. Physjoi,, Land. 245, 91-13.5. 23. Mendell L. M. (1984) Modifiability of spinal synapses. Physiol. Rev. 64, 260-324. 24. Mendell L. RI., Munson J. B. and Scott J. G. (1976) Alterations of synapses on axotomized motoneurons. Lond. 256. 67-79.
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25. Nakamura 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. 26. Nja A. and Purves D. (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. 27. Purves D. (1975) Functional and structural changes in mammalian sympathetic neurons following interruption of their axons. J. Physiol., Land. 252, 429-463. 28. Purves D. (1976) Functional and structural changes in mammalian sympathetic neurons following colchicine application to post-ganglionic nerves. J. Physiol., Lond. 259, 159-175. 29. Schmalbruch H. (1984) Motoneuron death after sciatic nerve section in newborn rats. J. camp. Neurol. 224, 252-2.58. 30. Smolen A. J. (1983) Retrograde transneuronal regulation of the afferent innervation to the rat superior cervical sympathetic ganglion. 1. Neurocytol. 12, 27-45. 31. Sumner B. E. H. (1975) A quantitative analysis of boutons with different types of synapses in normal and injured hypo~lo~al nuclei. Exol Neural. 49. 406418. 32. Sumner B. E. H. and Sutherland F.‘I. (1973) Q~ntitative electron microscopy on the injured hypoglossal nucleus in the tat. J. Neurocytoi. 2, 315328. 33. Takata M. (1981) Lingually induced inhibitory postsynaptic potentials in hypoglossal motoneurons after axotomy. Brain Res. 224, 165-169. 34. Takata M. (1982) Inhibitory postsynaptic potentials evoked in hypoglossal motoneurons by lingual nerve stimulation. Expl Neural. 75, 103-I 1I. 35. Takata M. and Fujita S. (1979) The properties of lingually induced IPSPs in the masseteric motoneurons. Brain Res. 168, 64865 1. 36. Takata M., Fujita S. and Kanamori N. (1982) Repetitive firing in trigeminal mesencephahc tract neurons and trigeminal motoneurons. J. Neurophysiol. 47, 23-30.
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37. Takata M. and Nagahama T. (1983) Synaptic efficacy of inhibitory synapses in hypoglossal motoneurons after transection of the hypoglossal nerves. Neuroscience 10, 23-29. 38. Takata M. and Nagahama T. (1986) Cortically and lingually induced postsynaptic potentials in trigeminal motoneurons after axotomy. Expl Brain Res. 61, 2722279. 39. Takata M., Shohara E. and Fujita S. (1980) The excitability of hypoglossal motoneurons undergoing chromatolysis. Neuroscience 5, 413419. 40. Takata M. and Tomomune N. (1986) Properties of postsynaptic potentials evoked in hypoglossal motoneurons by inferior alveolar nerve stimulation. Expl Neural. 93, 117-127. 41. Womble M. D. and Poper S. (1987) Retrograde effects of target atrophy on submandibular ganglion neurons. J. Neurophysiol. 58, 276-286. 42. Womble M. D. and Poper S. (1987) Neonatal synapse elimination in the rat submandibular ganglion: effects of retarded target growth. J. Neurophysiol. 58, 288-299. 43. Yarom Y., Sugimori M. and Llinas R. (1985) Ionic currents and firing patterns of mammalian vagal motoneurons in oitro. Neuroscience 16, 719-737. (Accepted 15 November
1989)
0306-4522/90$3.00+ 0.00 Pergamon Press plc 0 1990IBRO
1990
Printed in Great Britain
SYNAPTIC EFFICACY OF INHIBITORY SYNAPSES AND REPETITIVE FIRING IN THE REINNERVATING TRIGEMINAL AND HYPOGLOSSAL MOTONEURONS M. TAKATA,* N. TOMOMUNE,T. NAGAHAMA, S. TOMIOKAand N. NAKAJO Department of Physiology, School of Dentistry, Tokushima University, Kuramoto-cho, Japan
Tokushima 770,
Abstract-The synaptic efficacy and repetitive firing in masseteric motoneurons after the self-union operation and in tongue protruder motoneurons after their cut axons were reunited to tongue retractor muscles, the styloglossus muscle, were studied in cats. To ensure the correct identification of reinnervating motoneurons, the muscle response produced by an induced spike in a motoneuron by intracellularly injected depolarizing current was recorded. In both masseteric and tongue protruder motoneurons there were no differences on the patterns of postsynaptic potentials produced in reinnervating and non-reinnervating motoneurons by peripheral nerve stimulation, suggesting that the recovery of the synaptic efficacy of inhibitory synapses is time-dependent rather than muscle reinnervation. However, the present study demonstrated that the recovery of processes that control rhythmical firing of motoneurons is probably dependent on muscle reinnervation.
reveal two distinct slopes, the primary and the secIn axotomized motoneurons it ondary range. 4,6,36*39 was found that a distinct breakpoint in they-l plots the was missing,‘3,38 and that in spinal motoneurons slopes of the f-Z curves were steeper following axotomy, and these steeper slopes were well correlated with the decreased AHP conductance.12 Thus, it is expected that axotomy will result in marked changes in the firing of motoneurons. 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.20~43 The purpose of the present paper is to present the synaptic efficacy in Mass.Mns after the self-union operation and in hypoglossal motoneurons after their cut axons were reunited to the foreign muscles.
In adult cats, sectioning the masseter and the hypoglossal nerves causes a disjunction of presynaptic contacts on axotomized masseteric and hypoglossal motoneurons.33,37,38 In hypoglossal motoneurons, morphological studies have revealed that by one week after axotomy there is a loss of about 50% of the synapses.3’q32 Sumner and Sutherland32 and Sumner” have also observed a reduced number of dendritic synapses accompanied with detachment of synapses from the soma membrane. In masseteric motoneurons (Mass.Mns), it has been demonstrated that axotomy is followed by the decline of synaptic efficacy of inhibitory rather than of excitatory underlying the synapses. 38 The precise mechanism loss of efficacy at these chemical synapses is unclear, and it is not known whether muscle reinnervation itself is necessary for synapse recovery. Another effect of axotomy that occurs in motoneurons is a reduction in the amplitude and duration of the afterhyperpolarization (AHP), which follows an action potential.i2,‘9 These changes may be indicative of a transient response to axotomy. However, the maintenance of certain intrinsic properties of motoneurons (resting potential and input resistance) is unchanged, indicating that these membrane properties are independent of target innervation.5 As is already known for cat spinal, hypoglossal and trigeminal motoneurons, the frequency-current (f-Z) functions plotted for the first interspike intervals
EXPERIMENTAL PROCEDURES All experiments were performed on 15 young, adult cats (1.75-2.1 kg). In the self-union operation, the central stump of the right masseter nerves was cut and immediately reunited with its own peripheral stump by suturing the nerve sheath with a 9/10 silk thread under sodium pentobarbital anesthesia (Nembutal, 30 mg/kg, i.p.). The left masseter nerves were left intact and animals were allowed to survive for 146 days. The hypoglossal nerve of the cat divides into the medial and the lateral branches. The fibers of the medial branch innervate the tongue protruder muscles, and those of the lateral branch the tongue retractor muscles. In experiments, the medial and the lateral branch of the right hypoglossal nerve were cut and cut medial branches were reunited with a 9/10 silk thread to the tongue retractor muscle, the styloglossus muscle, aseptically under sodium pentobarbital anesthesia (Nembutal, 30 mg/kg, i.p.). The proximal stump of the lateral branch was ligated with silk thread and the left hypoglossal nerves were left intact. In some cases regeneration was prevented by inserting the cut end of the lateral
*To whom correspondece should be addressed. AHP, afterhyperpolarization; EPSP, excitatory postsynaptic potential; f-I,frequency-current; IPSP, inhibitory postsynaptic potential; Mass.Mn, masseteric motoneuron; P-Mn, tongue protruder motoneuron; PSP, postsynaptic potential.
Abbreviations:
785
786
M. TAKATAC~
branch into polyethylene tube with a blind distal end. Animals were then allowed to survive for 126 days. In the terminal experiments, the cerebellum was sucked out under sodium pentobarbital anesthesia (Nembutal, 30mg/kg, i.p.) in order to enable penetration into the trigeminal and the hypoglossal nucleus by a micropipette. Unitary muscle activity was recorded by a wire electrode (diameter 120 pm, interelectrode distance 3 mm, insulation removed for 3 mm) implanting the masseter and the styloglossus muscle when a motoneuron was stimulated by injecting a depolarizing current. In the masseter muscle, a wire electrode was implanted into the superficial masseter muscle, the anterior and the posterior deep masseter muscle. In the animals used for the experiments on the effect of cortical stimulation on Mass.Mns a pneumothorax was made, and cats were immobilized by intravenous injection of gallamine triethiodide, and respiration was maintained artificially. The left eye was enucleated and the orbitofrontal area of the cerebral cortex was exposed. Before the administration of gallamine triethiodide, the dura was opened and a spring-mounted silver ball electrode (diameter I mm) was placed on the cortical surface and was fixed at the optimum point for inducing jaw opening-closing movements by repetitive stimuli at 50/s, the reference electrode being inserted in temporal muscles. Single shock, surface anodal stimuli (02O.S ms, below 2 mA) were applied at this site. Body temperature was maintained by means of an electric blanket thermostatically controlled from a rectal thermister. A sleeve elecrode was used to stimulate the previously sutured axons and the ipsilateral lingual nerve. Two screw electrodes implanted into the mandible were used to stimulate the ipsilateral inferior alveolar nerve. In the present experiments both the lingual nerve and the inferior alveolar nerve were stimulated at an intensity of five times the nerve threshold. In intracellular recordings. a glass micropipette
A
1
al
filled with 2 M potassium citrate ing and for current injection. between 15 and 25 MR.
was used both for recordElectrode resistance was
RESULTS
Synaptic potentials mo toneurons
in
reinnervating
masseteric
Postsynaptic potentials (PSPs) in the Mass.Mns 146 days after the central stump of the cut masseter nerve was reunited with its own peripheral stump (self-union) were explored. As illustrated in Fig. 1, the degree of reinnervation was estimated by the ratio of the maximum jaw-closure produced by stimulation of the masseter nerve in intact side (A*) to that produced by stimulation of sutured axons in operated side (A, ). From this test, the degree of reinnervation was estimated as 70% in this sample 146 days after the self-union operation. As illustrated in Fig. lB,, a Mass.Mn was identified from the antidromic spike evoked by stimulation of the previously sutured axons. To ensure the correct identification of reinnervating Mass.Mns, the following study was performed. The muscle response in the masseter muscle produced by an induced spike in a Mass.Mn by intracellularly injected depolarizing current was recorded and illustrated in Fig. lB,. When a spike was elicited in a Mass.Mn by passing brief depolarizing currents across the membrane
2 -Il-
llmm
B
2
.
w I
5mV
20mZ Fig. 1. PSPs in a reinnervating Mass.Mn. (A) Degree of reinnervation. The jaw-closure produced by stimulation of the masseter nerve in intact (2) and operated side (1) is shown. (B) Reinnervating MassMn. The record a shows an antidromic spike. Unitary muscle activity in the anterior deep masseter muscle (b,) followed an induced spike in a Mass.Mn (b,). (C) PSPs. The records a, and b, show a lingually induced and an inferior alveolar-induced PSP. The records a2 and b, show filed potentials. (D) Effect of membrane polarization. The records a and b show a lingually induced and an inferior alveolar-induced PSP.
Postsynaptic potentials in reinnervating motoneurons unitary muscle activity was evoked in the anterior deep masseter muscle (Fig. lBb,) and followed an induced spike one-for-one with a constant latency. No muscle responses were produced in the superficial masseter and the posterior deep masseter muscle by an induced spike of this MassMn, indicating that this Mass.Mn reinnervated the anterior deep masseter muscle (reinnervating motoneurons). In 18 out of 30 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 2.5 to 4.5 ms (n = 18). As illustrated in Fig. 1C in the reinnervating Mass.Mn stimulation of the lingual nerve (Fig. lC,,) or the inferior alveolar nerve (Fig. lC,,) produced large inhibitory postsynaptic potentials (IPSPs). The latency of an inferior alveolar-induced IPSP was about 3.0 ms from stimulus artifact (mean + S.D., 3.5 + 0.5 ms, n = 18). When a single shock was delivered to the lingual nerve, a small excitatory postsynaptic potential (EPSP) was seen before the generation of large IPSPs. The relation between the amplitude of lingually induced or inferior alveolarinduced hyperpolarizing potentials and the membrane potential displacements was examined and results are shown in Fig. 1D,, D, Numerals denote the amount of injected current in nanoamperes. In both cases, by displacing the membrane potential towards hyperpolarization, a hyperpolarizing synaptic potential was decreased in amplitude and reversed to a depolarizing potential. By displacement of the membrane potential to depolarization, a hyperpolarizing synaptic potential was increased in amplitude. In 12 out of 30 explored cells, no muscle responses were produced by an induced spike of the cell. A sample record is shown in Fig. 2. The antidromic spike evoked by stimulation of the previously sutured axons is shown in Fig. 2A,. Simultaneous recordings of an induced spike (Fig. 2A,,,) and muscle responses of the anterior deep masseter muscle (Fig. 2A,,) are shown in Fig. 2A,. No muscle responses were produced in the superlicial masseter, the anterior deep and the posterior deep masseter muscle by an induced spike of this Mass.Mn, indicating that this cell failed to reinnervate the masseter muscle (non-reinnervating motoneurons). In this cell stimulation of the lingual nerve or the inferior alveolar nerve produced a large IPSP (illustrated in Fig. 2B,, ,B,, ). By lingual nerve stimulation, a small EPSP followed by predominant IPSPs was produced. Finally, we found that there were no differences on the patterns of PSPs produced in reinnervating and non-reinnervating Mass.Mn by lingual or inferior alveolar nerve stimulation. In the following studies, the effect of cortical stimulation on MassMns 146 days after the selfunion operation was explored. In 36 out of 37 explored MassMns stimulation of the cerebral cortex (Fig. lB,,),
Fig. 2. PSPs in a non-reinnervating Mass.Mn. (A) Nonreinnervating Mass.Mn. The record a shows an antidromic spike. No muscle reponses were produced in the masseter muscle (b,) by an induced spike in a Mass.Mn (b,). (B) PSPs. The PSPs produced by stimulation of the lingual nerve (a,) or the inferior alveolar nerve (b,) are shown. The records a2 and b, show field potentials. (C) Effect of membrane polarization on cortically induced PSPs in a cell 146 days after the self-union operation.
produced large IPSPs. A sample record is shown in Fig. 2C. At the resting membrane level (0 nA), stimulation of the cerebral cortex produced large IPSPs. By displacing the membrane potential towards hyperpolarization, cortically induced hyperpolarizing synaptic potentials were decreased in amplitude and reversed to a depolarizing potential. By displacement of the membrane potential to depolarization, hyperpolarizing potentials were increased in amplitude. In the remaining cell, large EPSPs followed by IPSPs were produced by cortical stimulation. Synaptic potentials motoneurons
in
reinnervating
hypoglossal
Synaptic potentials in the tongue protruder motoneurons (P-Mns) 126 days after their cut axons were reunited to the tongue retractor muscle, the styloglossus muscle, were explored and results are shown in Fig. 3. As illustrated in Fig. 3A,,B,, a motoneuron was identified from the antidromic spike evoked by stimulation of the previously sutured axons in the 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. 3A was stimulated with depolarizing current (Fig. 3A,,) unitary muscle activity in the styloglossus muscle followed the cell spike one-for-one with constant latency (Fig. 3A,,), indicating that this P-Mn reinnervated the tongue retractor muscles, the styloglossus muscle. In this innervating P-Mn, stimulation of the inferior alveolar nerve produced an IPSP, illustrated in
788
M. TAKATAet al.
Fig. 3. PSPs in P-Mns after cut axons were reunited to the foreign muscle. (A) Reinnervating P-Mn. The record a shows an antidromic spike. Unitary muscle activity in the styloglossus muscle (b,) followed an induced spike in a P-Mn (b,). An inferior alveolar-induced IPSP is shown in c, The record c1 shows field potentials. The records d show the effect of membrane polarization on an inferior alveolar-induced IPSP. (B) Non-reinnervating P-Mn. No muscle responses were produced in the styloglossus muscle (b,) by an induced spike in a P-Mn (b2). Record a shows an antidromic spike. An inferior alveolar-induced IPSP is shown in c, The record cj shows field potentials.
Fig. 3A,, The relation between the amplitude of inferior alveolar-induced hyperpolarizing synaptic potentials and the membrane potential displacements was examined and results are shown in Fig. 3A,. 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 to 4.5 ms (n = 23). In nine reinnervating cells, synaptic potentials produced by inferior alveolar nerve 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. 3B, no muscle responses were produced (Fig. 3B,,) by an induced spike of the cell (Fig. 3B,,), indicating that this cell failed to reinnervate the styloglossus muscle. In this cell stimulation of the inferior alveolar nerve produced an IPSP (Fig. 3B,,). In 31 out of 54 explored cells no muscle responses were produced by an induced spike of the cell. In I8 non-reinnervating cells synaptic potentials produced by stimulation of the inferior alveolar nerve 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 produced in reinnervating and non-reinnervating P-Mns by inferior alveolar nerve stimulation. Repetitive firing
in reinnervating
motoneurons
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. 4. In a P-Mn illustrated in Fig. 4A, unitary muscle activity in the styloglossus muscle (Fig. 4A,,) followed the cell spike (Fig. 4A,,) one-for-one, indicating that this P-Mn reinnervated the styloglossus muscle. In this reinnervating P-Mn, the relationship between the firing frequency (impulses/s) and the amount of injected depolarizing current (nanoamperes) was examined. Repetitive firing of this cell responding to increasing amounts of injected depolarizing current is illustrated in Fig. 4A,. 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 (k 2.5, n = 10) and 45 (k 5.5, n = 10) imp/s per nA, illustrated by open circles in Fig. 4C. In a P-Mn illustrated in Fig. 4B, no muscle responses were produced (Fig. 4B,,) by an induced spike of the cell (Fig. 4B,,). As illustrated in Fig. 4B,,, this non-reinnervating P-Mn shows sustained firing to maintain pulses of intracellularly injected depolarizing currents. The rheobase of this cell was 2 nA (2-6 nA, n = 10). The f-1 relation
789
Postsynaptic potentials in reinnervating motoneurons C imp/s
3!!;::: ;I?
)2ooJN 300-
b
I
Smrrc
_ Smrec
I
)20mV
0
250-
l-
B 0
0
100
50
0,
1 0
5
10 nA
Fig. 4. Repetitive firing of P-Mns after cut axons were reunited to the foreign muscle. (A) Reinnervating P-Mn. Unitary muscle activity in the styloglossus muscle (a,) followed an induced spike in a P-Mn (az). Repetitive firing caused by injecting current is shown in b. (B) Non-reinnervating P-Mn. No muscle responses were produced in the styloglossus muscle (a,) by an induced spike in a P-Mn (a,). Repetitive firing caused by injecting current is shown in b. (C)f-I relation. Results obtained from an reinnervating and non-reinnervating P-Mn are illustrated by open and filled circles.
plotted from the first interspike intervals of a non-reinnervating P-Mn is represented by filled
circles in Fig. 4C. In a non-reinnervating P-Mn, a distinct breakpoint in the f-1 plots was missing, as has already been found in axotomized spinal and trigeminal motoneurons.‘3’38
In a Mass.Mn 146 days after the self-union operation, the f-1 relation was examined and results are shown in Fig. 5. As illustrated in Fig. SA, the reinnervating Mass.Mn shows sustained firing to maintained pulses of intracellularly injected depolarizing currents. Unitary muscle activity in the deep masseter muscle (the upper in each record) followed the cell spike one-for-one with constant latency. In this Mass.Mn, the f-I relation plotted from the first interspike intervals revealed two distinct slopes, illustrated in Fig. 5B. The slope of the primary and the secondary range was 15 ( f 2.5, n = 7) and 45 (&-4.5, n = 7) imp/s per nA, respectively.
DISCUSSION
5
10
15 nA
Fig. 5. Repetitive firing of a Mass.Mn after the self-union operation. (A) Repetitive firing of an reinnervating Mass.Mn to maintained pulses of intracellularly injected depolarizing currents. Unitary muscle activity in the deep masseter muscle (the upper in each record) followed by a spike in a Mass.Mn. (B) f-l relation plotted from the first interspike intervals.
In two-thirds of explored Mass.Mns 146 days after the self-union operation, an induced spike of the cell produced unitary muscle activity with a large amplitude in the masseter muscle. In 23 out of 54 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 31 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. In this respect, it was reported
790
M. TAKATA~~
that cut nerves often failed to regenerate to the muscle by mechanical stresses8s9 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.” Furthermore, Ip and Vrbora14 showed that in leg muscles either soleus’ own nerve or a foreign nerve was eventually able to innervate soleus muscle, indicating that motoneurons show little or no preference for their former muscle fibers. 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. ‘“~‘7~‘8~23 It was also reported that full recovery of motoneuron properties and synaptic contacts onto axotomized motoneurons occurs after muscle reinnervation.3’ In axotomized MassMns, it was 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 greatly reduced while that of excitatory synapses unaffected.38 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 and the excitatory influence was masked by IPSPS’~.*~.‘~ and that excitation of the inferior alveolar nerve evoked multiphasic responses, the IPSP-EPSP-IPSP sequence.3 In the present experiments, it was found that in both reinnervating and non-reinnervating Mass.Mns, stimulation of either the lingual nerve or the inferior alveolar nerve produced large IPSPs, suggesting that the recovery of the synaptic efficacy of inhibitory synapses is time-dependent rather than muscle reinnervation. In the Mass.Mns 146 days after the self-union operation, cortical stimulation produced large IPSPs in 36 out of 37 explored cells, suggesting that the recovery of synaptic contacts made by the corticobulbar fibers is also time-dependent. It was demonstrated that the majority of the total sample of explored P-Mns 40 days after axotomy showed an EPSP-IPSP sequence when the peripheral nerves were stimulated.” In the present experiments, in P-Mns 126 days after their cut axons were reunited to the styloglossus muscle, it was found that in non-reinnervating cells, when a single shock was delivered to the inferior alveolar nerve, IPSPs were evoked in two-thirds of explored cells, as demonstrated in normal PMns,34.40 suggesting that the recovery of the synaptic efficacy of inhibitory synapses in hypoglossal motoneurons is also time-dependent rather than muscle reinnervation. In superior cervical ganglion cells, a disjunction of presynaptic contacts on axotomized neurons was diminished if exogenous nerve growth factor
al.
was supplied to the ganglion cells, suggesting that one of the roles of the target tissue is to supply a trophic factor.26 Moreover, in sympathetic ganglion cells, it was suggested that a trophic 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.2’ In invertebrates, it was demonstrated that inhibitory chemical synapses on buccal motoneurons of Helisoma show a transient decrease of efficacy during organ culture, and motoneurons exhibit recovery of their chemical synapses without target innervation.5 In trigeminal and hypoglossal motoneurons, it is probably safe to assume that the synapse recovery is not dependent on retrograde trophic messages from muscle. In sympathetic ganglion cells and spinal motoneurons, it was revealed that axotomy causes loss of functional inputs22,24,28and the death of many injured neurons, 27~30 and revealed that there is an increase in the number of synaptic inputs to the surviving neurons.4’.42 However, in the present studies, there were no differences in the patterns of PSPs produced in reinnervating and non-reinnervating motoneurons by peripheral nerve stimulation. In rat hypoglossal nucleus, cell loss did not exceed 25% and was not detectable until more than 28 days had elapsed after operation.’ However, Aldskogius and Thomander2 studied the organization of the facial motor nucleus and reported that after transection of the facial nerve at the adult stage, no significant loss of motoneurons occurred. Recently, Kashihara et al.” reported that in rats four days after birth the majority of axotomized motoneurons die if target contact is prevented, whereas, in rats four weeks after birth, section of the sciatic nerve is known to cause no loss of the motoneurons.” In reinnervating MassMns and P-Mns fired in sustained manner by injecting depolarizing currents into the cell, the f-Z relation revealed two distinct slopes (the primary and the secondary range). In contrast, a distinct breakpoint in the f-Z relation was missing in non-reinnervating motoneurons as demonstrated in axotomized motoneurons. Therefore, the recovery of processes that control rhythmical firing of motoneurons is probably dependent on muscle reinnervation, as suggested by Kuno and Llinas” and Czeh et al.’ that AHP duration is regulated by retrograde trophic messages from muscle. Recently, of motor units Foehring et al.” studied properties following cross-reinnervation of cat lateral gastrocnemius and soleus muscles by the medial gastrocnemius nerve and conclude that the innervated muscle may influence the expression of motoneuron electrical properties. Acknowledgement-This study was supported in part by a grant-in-aid for scientific research 01304045 from the Japanese Ministry of Education, Science and Culture.
Postsynaptic potentials in reinnervating motoneurons
791
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13. Heyer C. B. and Llinis R. (1977) Control of rhythmic firing in normal and axotomized cat spinal motoneurons. J. Neurophysiol. 40, 480-488. 14. Ip M. C. and Vrbora G. (1983) Reinnervation
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