Synaptic Transmission and Modulation in Submandibular Ganglia: Aspects of a Current-Clamp Study

Bull. Tokyo dent. Coll., Vol. 41, No. 4, pp.149ϳ167, November, 2000 149

Review Article

SYNAPTIC TRANSMISSION AND MODULATION IN SUBMANDIBULAR GANGLIA: ASPECTS OF A CURRENT-CLAMP STUDY

TAKASHI SUZUKI

Department of Physiology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan

Received 8 September, 2000/Accepted for Publication 10 October, 2000

Abstract The superior salivatory nucleus in the medulla oblongata is the parasympathetic center of the sublingual and the submandibular (SM) glands. The preganglionic axons originating in this parasympathetic center connect postganglionic neurons in the submandibular ganglia. In spite of an earlier electrophysiological study by Langley in 189013), intracellular electrical studies on SM ganglion neurons had not been done because of the technical difficulties in impaling neurons. It was in only 1972 that the authors begun the intracellular electrical studies of SM ganglia in adult rats and hamsters. In this review, we describe the membrane properties of neurons, spontaneous activities of neurons, types of connection between pre- and post-ganglionic neurons, synaptic potentials including fast EPSP, slow IPSP, slow EPSP and slow hyperpolarizing synaptic potential, characteristics of the reflex spike discharges and modulation of synaptic transmission by biogenic substances in SM ganglion neurons. Transfer of information within the ganglia is more complex than simple nicotinic-cholinergic relay, and seems to be specialized for compatibility with the salivary glands. These data reflect the specific characteristics of synaptic transmission in the SM ganglia. Key words: Membrane properties—Spontaneous membrane activities— Postsynaptic potentials—Biogenic substances—Reflex spike discharges

INTRODUCTION humans, it causes release of small amounts of saliva with rich organic constituents from The salivary glands5,13) are supplied with major salivary glands. both sympathetic and parasympathetic nerves. Since the end of the 1970s, studies using Both autonomic nerves are secretory nerves. the method of tracing the retrograde axonal Stimulation of the parasympathetic nerve transport of horseradish peroxidase have con- causes copious secretion of watery saliva with ﬁrmed the results of the early histological a relatively low content of organic material, studies utilizing the neuronal degeneration while the effect of stimulation of the sympa- method16). The superior and the inferior thetic nerve varies from species to species. In salivatory nuclei consist of parvocellular neu-

149 150 T. SUZUKI rons sparsely scattered in the lateral reticular formation of the medulla at the level of the facial nucleus. The superior subdivision is situated rostral to the inferior subdivision, but there is no anatomical boundary. The rostral part of the parasympathetic center, the superior salivatory nucleus, connects with the submandibular and the sublingual glands. It is also known that some of the preganglionic fibers diverge to more than one postganglionic neuron14). The postganglionic parasympathetic nerve fibers are known to con- verge at the glandular cell level8,15). The importance of nerves for salivary secretion was revealed in 1850 by Ludwig8), who found that electrical stimulation of the lingual nerve in dogs caused secretion of saliva from the submandibular gland. Professor Langley13) is a noted pioneer in Fig. 1 Compound action potentials of the secretory nerve bundles electrophysiological study of submandibular Stimulus strength: A: 2V, B: 5.8V, C: 7.8V and D: 26 V ganglia. The so-called submandibular (SM) Duration: 0.1msec. Conducting distance: 7.5mm (see ganglia with the smaller cluster in the chordo- ref. 19) lingual triangle and the small ganglion near the hilus of the SM gland are scattered on the course of nerve fibers to the sublingual (SL) count the number of the unmyelinated and the SM glands. His study concluded that fibers19). The myelinated and the unmyeli- secretory fibers of the chorda tympani in dogs nated fibers of less than 3␮m were presumed are paralysed by nicotine in the applied gan- to be the parasympathetic fibers involved glion more readily than the peripheral vasodi- in secretion and vasodilation. Some of the lator nerve. Therefore, Langley’s findings unmyelinated fibers are probably visceral suggested that the SM ganglion included sensory fibers. both secretory and vasodilatory neurons. The major and minor diameters of 604 In 1972, intracellular electrophysiological cells in the same preparation which permitted studies on the SM ganglion cells were con- measurement were 13.2–42.9␮m and 9.9– ducted in the small ganglion near the SM 33.0␮m. The peaks of histograms were at gland of adult rats19). 23.1–26.4␮m and 16.5–19.8␮m, respectively19).

2. Electrophysiological investigation of the LIGHTMICROSCOPIC OBSERVATIONS secretory nerve The compound action potentials of the 1. The size of preganglionic fibers and secretory nerve bundles were recorded imme- ganglion neurons diately before a small ganglion in the hilus of The approximately 10 secretory nerve bun- the SM gland (shown in Fig. 1) and the con- dles near the origin which branched out from duction velocities were estimated from the the lingual nerve contained 156 fibers. Thirty- latency of each action potential and the con- eight fibers were less than 1.5␮m; 100 fibers, ducting distance. The fastest conduction 1.5–3␮m; 11 fibers, 3–4.5␮m; 3 fibers, 4.5– velocities, 6–12 m/sec, were considered to be 6␮m; 2 fibers, 6–7.5␮m; and 2 fibers, 7.5– those of B fibers or the somatosensory nerve 9␮m in diameter. However, it was difficult to fibers. The conduction velocities of 3.0 m/sec CURRENT-CLAMP STUDY IN SUBMANDIBULAR GANGLIA 151

(in B), 1–1.6 m/sec (in D), 0.32–0.9 m/sec and its mean duration is 250 mV at the level of

(in C), 0.21–0.3 m/sec (in D), and 0.17– the mean Em. The values of active membrane 0.21 m/sec (in C) were presumed to be those properties in vitro are similar to those of in of preganglionic B and C fibers. In the ham- vivo20). SM ganglion neurons usually discharge ster SM ganglion, the conduction velocity of a single spike only at the onset of depolarizing some preganglionic fibers was slightly faster current pulses. In bladder ganglion, three than 1m/sec. The mean velocity of the fibers types of neurons can be distinguished on the S.D.) m/sec. The post gangli- basis of the nature of the spike discharge) 0.17עwas 0.24 onic fibers had a conduction velocity of evoked by intrasomatic injection of depolariz- .(m/sec20). ing current pulses9 0.11ע0.24 Hyperpolarizing after-potentials are associated with the spikes in all of autonomic gan- MEMBRANE PROPERTIES OF glion neurons. The after-hyperpolarizing po- SUBMANDIBULAR GANGLION NEURONS tentials summate and are prolonged when spikes are evoked repetitively; their durations Most intracellular electrophysiological stud- are less than 1sec. The ionic mechanisms for ies had been done on ganglia in vitro because the postspike hyperpolarization is an increase of the technical difficulties of impaling the in calcium-activated gK that is triggered by an neurons with microelectrodes in situ. The influx of calcium during the rising phase of work of Suzuki and Kusano20) was exceptional the action potential20). The functional signifi- in that they compared the electrophysiologi- cance of this mechanism in all of the neurons cal properties of hamster SM ganglion neu- is to restrict the frequency of spike discharge. rons in vitro and in vivo. The results indicate Electrophysiological findings in parasym- that the properties of the neurons do not pathetic ganglia indicate that the neurophysi- change in the in vitro situation. ology is more complex than simple nicotinic- cholinergic relay and divergence of pregan- 1. The passive properties glionic information to autonomic effector sys- The resting membrane potentials of SM tems. The transfer function of these ganglia mV is, no doubt, specialized for compatibility with 51מ ganglion neurons in rats are mean mV in vitro19). The mean the effector system they control, and it is 77מ–40מ and range resting Ems of hamster SM ganglion neurons evident that this specialization is associated mV, with heterogenous synaptic interactions and 8.0ע53מ S.D.) mV and) 7.2ע53מ are -mV electrophysiological properties that are sug 70מ–40מ respectively, and the range is in the in vitro and in vivo conditions20). Mean gestive of integrative and modulatory func- membrane input resistances in hamster SM tions within ganglia41). M⍀ and 17.2עganglion neurons are 42.4 M⍀ in the in vitro and on in vivo 26.2ע40.4 conditions, respectively. The range of the SPONTANEOUS ACTIVITY IN membrane time constant is 5–10 msec both in SUBMANDIBULAR GANGLION NEURONS vitro and in vivo20). Spontaneously occurring hyperpolarizing 2. The active properties potentials occur in submandibular ganglion The mean amplitude of the antidromic neurons20) and in the cardiac ganglion neu- S.D.) mV in hamster SM rons of Necturus10). These potentials reflect) 12.4עspike is 71.5 ganglion neurons in the in vivo condition. periodic increases in calcium-dependent gK The mean amplitude of the directly evoked and are similar to the spontaneous IPSPs that mV. The critical level of occur in myenteric neurons40). However, they 9.8עspike is 69.0 mV. The mean ampli- are not synaptic potentials in SM ganglion 5.4עspike firing is 14.4 tude of spike after-hyperpolarization is 13 mV, neurons. Spontaneous transient hyperpolar- 152 T. S UZUKI

Fig. 2 Spontaneous hyperpolarizing potentials and effects of caffeine on membrane potential A. In this neuron, the spontaneous HPs occurred with various amplitudes and durations at irregular intervals in normal Krebs solution. B. Application of 2.5mM caffeine induced the HPs at a regular interval. Amplitudes of HPs increased, and spikes fired at the ends of HPs as an off-response. C. Five minutes after caffeine application, repetitive spiking begun. .(ceased the repetitive spike firings, but the rhythmic HPs continued (see ref. 20 (7מD. TTX(10 free induced membrane-םizations (HPs) occur in some neurons at at times before K irregular intervals (shown in Fig. 2). HPs are depolarization occurred. The amplitude of -free saline with con-םinsensitive to TTX but are suppressed by SOMP decreased in Ca2 Caffeine induces low frequency rhyth- comitant depolarization; conversely, in saline .םMn2 ,concentration was doubled םmic HPs in many neurons, often alternating in which the Ca2 with periods of repetitive spiking. This mech- the membrane potential (Em) was stable near ם2 ם2 anism contributes to the regulation of low the EK level. Ca -free, Ba -substituted saline frequency repetitive firing in SM neurons. prevented SOMPs. A persistent application of We termed another type of rhythmic poten- 5 mM caffeine hyperpolarized a different cell מ מ tial in hamster SM neurons slow oscillations to a stable Em level of 76– 77 mV, and pre- of membrane potential; SOMP23,24). SOMPs vented the SOMP. Caffeine rhythmic HPs occurred spontaneously, roughly in sinusoi- were still induced at a frequency of 17.5/min. dal forms between the subthreshold range Replacement with caffeine-free control saline and the potassium equilibrium potential (EK, recovered the SOMP activity of this cell. The ם2 מ approximately 85 mV) (shown in Fig. 3). activation of slow gCa(V) for Ca entry from the

The most positive and negative Em levels of external medium may occur during the rising .S.D.) mV and phase near the peak of SOMPעmean) 3.3ע58.0מ SOMP were mV. One cycle of SOMP was A possible functional implication of these 2.8ע87.6מ ע 3.7 2.9 min. The interval did not depend on oscillations of the Em is a modulation of cellu- ם2 the resting Em. In physostigmine-treated pre- lar excitability. Activation of the Ca -activated parations, the SOMP was triggered by pregan- gK increase by preganglionic impulses could glionic repetitive stimulation. Bethanechol, a act as a long-lasting inhibitory postsynaptic muscarinic agonist, also can induce SOMP potential. Spontaneous impulse discharges (shown in Fig. 4). It is generated by slow peri- may occur in a postsynaptic origin under cer- odic activation of gK(Ca) brought by changes in tain circumstances; they are patterned by ם2 [Ca ]i. The membrane input resistance was these Em oscillations and affect submandibu- higher at the crest than at the trough (Fig. 3). lar gland activity. Such cellular activities may concentration be signiﬁcant for the long-term modulation םReduction of extracellular K increased the amplitude of SOMP without of neuronal excitability in autonomic ganglia changing its frequency. This effect was noted where information transfer is processed slowly CURRENT-CLAMP STUDY IN SUBMANDIBULAR GANGLIA 153

Fig. 3 Slow oscillation of membrane potential (SOMP) in a hamster SM ganglion neuron M physostigmine-treated 4מsolutions on synaptically enhanced SOMPs in 10 םfree and low Ca2-םEffects of K מס ganglion are shown. Initial resting Em 80mV. The preganglionic stimulation in a, b and c was applied at 10Hz for 20 sec. The traces are continuous. Changes in the membrane input resistance (Rm) were A, 100msec pulses at 10sec intervals. The traces were recorded for total 9מ10ן3מ monitored by applying 2.4hr recording using a 3M KCl-ﬁlled electrode (see ref. 24).

Fig. 4 SOMPs initiated by bethanechol (BCh) and enhanced by BCh-hyperpolarization מס Initial resting Em 40mV. The traces were obtained from a neuron, but were not continuous in this 9מ Figure. Changes in Rm were monitored by 10 A, 100msec pulses (see Suzuki, Proc. Int. Union Physiol. Sci. XVI 109, 1986). 154 T. S UZUKI and sustained for relatively long periods41).

SYNAPTIC TRANSMISSION IN SUBMANDIBULAR GANGLIA

Transfer of information within SM ganglia seems to be specialized for compatibility with the function of the salivary glands; this is reflected in the specific characteristics of synaptic transmission in the SM ganglia. Morphological studies indicate that axosomatic, axoaxonal, and axodendritic synapses are all present in the parasympathetic ganglia; however there is considerable vari- ability between different ganglia and between the same ganglia in different species with respect to such elements as the length of den- drites and synaptic structure41). This is impor- tant for understanding the synaptic circuitry of these ganglia, because intracellular micro- electrode studies of synaptic transmission have been limited to axosomatic synapses located on the axon hillock near the soma. Intrasomatic recording from SM ganglia shows fast and slow synaptic potentials. The Fig. 5 Two types of SM ganglion neurons innervated by a single preganglionic and by multiple pregangli- fast synaptic potentials are excitatory. The onic fibers slow synaptic potentials are both excitatory Left-hand trace: An orthodromic action potential evoked and inhibitory. by a single EPSP. Right-hand trace: Steps of 3–5 EPSPs for a single action potential generation by graded preganglionic stimulation (see ref. 19). FAST-EXCITATORY POSTSYNAPTIC POTENTIALS IN SUBMANDIBULAR GANGLION NEURONS excitatory postsynaptic potential (f-EPSP) of the rat SM ganglion neurons with single Fast synaptic transmission between pregan- preganglionic innervation has the following glionic and postganglionic parasympathetic parameters: the mean amplitude is 20.4 mV neurons is mediated by nicotinic-cholinergic and the range is 15–31 mV; the mean time to receptors in all the ganglia. The behaviour peak is 4.0 msec and the range is 2–11 msec; of the nicotinic synapses is not the same in the mean time constant of decay is 12.7 msec all the ganglia. Both single preganglionic and the range is 7–25 msec; and the mean and multiple preganglionic innervations are duration is 39 msec and the range is 20– found in SM ganglion neurons19–21). Approxi- 73 msec. The f-EPSP of neurons with single mately 75% of the SM ganglion neurons are preganglionic innervation responds com- innervated by a single preganglionic fiber14,19). pletely to the preganglionic stimulation up to The remaining percentages are multiplely 100 Hz for 1sec (shown in Fig. 6). Potentia- innervated neurons, in which two or three tion of the amplitude (approximately 30% fibers, and sometimes more than three, are of the control) occurs for approximately preganglionic14,19) (shown in Fig. 5). The fast 250 msec from the beginning of the repetitive CURRENT-CLAMP STUDY IN SUBMANDIBULAR GANGLIA 155

Fig. 6 Response of f-EPSP in a SM ganglion neuron with single preganglionic innervation to repetitive preganglionic stimulation The f-EPSP of the neuron with single preganglionic innervation responded completely to the repetitive preganglionic stimulation up to 100Hz for 1 sec. Potentiation of f-EPSP amplitude occurred up to approximately 250msec from the initiation of stimulation. Stimulus frequency: A: 10Hz, B: 20Hz, C: 40Hz, D: 80Hz and E: 100 Hz (see ref. 19).

Fig. 7 Responses of action potential to repetitive stimulation in two SM ganglion neurons Series Trace 1: The responses of action potential in a neuron with single preganglionic innervation. Stimulus frequency: A: 20Hz, B: 40Hz, C: 50Hz, D: 60 Hz, and E: 80Hz (see ref. 19). Series trace 2: The responses of a neuron which evoked two action potentials to single preganglionic stimulation. Stimulus frequency: A: 20Hz, B: 30Hz (see ref. 19). 156 T. S UZUKI

Fig. 8 Two orthodromic action potentials in a SM Fig. 9 Two orthodromic action potentials in a SM ganglion neuron evoked by graded pregangli- ganglion neuron by graded preganglionic onic stimulation stimulation The first orthodromic action potential is evoked by Disappearing of the initial spike was preceded by a graded steps of EPSP. decrease in stimulus strength. The chemical intrinsic The spike interval: 33msec (see ref. 19). connections may be present among neurons in the intact ganglia (see ref. 19). preganglionic stimulation. The ganglionic subtypes. Two or three steps of the EPSP gating property is accounted for by the tem- elicite a single spike in a ganglion neuron poral facilitation of the amplitude of the by graded stimulation of the preganglionic f-EPSP that results from increased release of nerve (Fig. 5). The other two subtypes of gan- acetylcholine from the presynaptic terminals glion neurons evoked two spikes with differ- when they are activated repetitively. The gan- ent latencies. In the first subtype of ganglion glionic neurons innervated by a single pre- neurons, both spikes were evoked according ganglionic B fiber respond with spikes of up to all or none law by graded stimulation of to 30–60 Hz to the repetitive preganglionic the preganglionic nerve. In the other subtype stimulation, while the neurons multiplely of ganglion neurons, either of two spikes is innervated by preganglionic C fibers respond evoked with graded steps of EPSP (shown in until 20 Hz19) (shown in Fig. 7). Therefore, Fig. 8). The latencies of all the orthodromic f-EPSP with a high safety factor for evoking spikes recorded successively are within range spikes occurs in the SM ganglia21). The SM of 3–38 msec. The highest and the lowest con- ganglion neurons with convergence of the duction velocities estimated from these laten- preganglionic fibers are divided into three cies are 3.3–0.26 m/sec. The exceptional low CURRENT-CLAMP STUDY IN SUBMANDIBULAR GANGLIA 157 conduction velocities are 0.24, 0.23 and 0.18 m/sec. In some neurons, the latencies of the second spike fluctuate. Kawa and Roper11) demonstrated the existence of electrical and chemical intrinsic synaptic connections in the rat SM ganglia after preganglionic denervation. Electrical connections are demonstrated in the intact ganglia. However, it is unclear whether the chemical intrinsic synapses are present in the intact ganglia or whether they are induced only after preganglionic denervation. The second spikes evoked by the graded preganglionic stimulation must be induced by the chemical intrinsic synaptic transmission because of their low threshold, the long latencies (shown in Fig. 9), and their fluctu- ation in some neurons19). Chronic preganglionic denervation of the hamster SM ganglion neuron did not change the nicotinic receptor sensitivities for 3–43 days after denervation25). There are no interneurons in the rat SM gan- glion14). The reversal potential of f-EPSP in the rat SM ganglion neurons was estimated using the superimposing method, in which the f-EPSP was evoked at various potential levels on the falling phase of the antidromic spike. The 8mV19) (shown inמ–6מ reversal potential is Fig. 10). The reversal potential of fast-excitatory Fig. 10 Estimation of f-EPSP reversal potential postsynaptic currents (f-EPSCs) in rat SM The 1st action potential: Antidromic action potential. .The 2nd action potential: Orthodromic action potential (17 מ ganglion neurons is approximately 10 mV ; A–E: The orthodromic response was superimposed at This was measured using the single microelec- various potential levels of the falling phase of the antidromic spike. D–E: The rising phase of f-EPSP disappeared at 8mV levels on the falling phase of the antidromicמ–6מ trode voltage-clamp technique. In hamster SMG neurons, the reversal potential of f- spike (see ref. 19). EPSCs was also measured using the same style of voltage-clamp technique, and was found to -mV26). The decay phase of (slow excitatory postsynaptic potential), s 9.7מ be at mean the hamster ganglionic f-EPSC was composed IPSP (slow inhibitory postsynaptic potential) of fast and slow components similar to those and s-HSP (slow hyperpolarizing synaptic po- of f-EPSC in rat SM ganglion neurons17,26). tential) are evoked by repetitive pregangli- Thus hamster SM ganglion neurons seem onic stimulation, for several seconds at a fre- to possess two types of ACh-operated ionic quency of 5–40 Hz. Both s-IPSP and s-EPSP in channels26). the SM ganglion are induced by direct activation of muscarinic receptors and are associ-

ated with changes in gK (shown in Fig. 11). SLOW-POSTSYNAPTIC POTENTIALS IN Approximately 50% of ganglion neurons SUBMANDIBULAR GANGLION NEURONS respond with f-EPSP and s-IPSP to the repetitive preganglionic stimulation. The remain- In hamster SM ganglion neurons, s-EPSP ing neurons respond with f-EPSP and s-EPSP. 158 T. SUZUKI

Fig. 11 Diagrammatic representation of synaptic connection between preganglionic ﬁbers and a postganglionic neuron, and postsynaptic potentials

N: nicotinic receptor, M1: muscarinic M1-receptor, M2: muscarinic M2-receptor, P1: P1 purinoceptor, slow HSP: slow hyperpolarizing synaptic potential, AF-DX 116: muscarinic M -receptor antagonist, ␤ 2 dTC: d-tubocurarine chloride, dHE: dihydro- -erythroidine, C6: hexamethonium, caffeine: P1 purinoceptor antagonist, Left-hand number of each trace in slow EPSP and slow HSP shows the

resting Em (see ref. 19, 33 and 36).

1. Functional significance of the slow inhibited during s-IPSP, indicating an inhibi- postsynaptic potentials tion of neuronal excitability. Therefore, the The s-PSPs serve as a physiological signifi- s-EPSP and the s-EPSP function in the SM cant modulator of submandibular ganglionic ganglion neurons as excitatory and inhibitory transmission. The s-EPSP enhances an excit- modulators of cholinergic excitatory pathway ability in SM ganglion neurons and has a in the SM ganglia. facilitatory action. The spontaneous spike firings are enhanced during the s-EPSP. On the 2. Slow excitatory postsynaptic potential other hand, the spontaneous firings are s-EPSPs are evoked by 40–100 pregangli- CURRENT-CLAMP STUDY IN SUBMANDIBULAR GANGLIA 159 onic stimuli at 10–25 Hz in the presence of which respond with f-EPSP and s-IPSP to 0.3–0.5 mM hexamethonium33). The s-EPSPs repetitive preganglionic stimulation. The BCh- induced by 40 preganglionic stimuli at 25 Hz hyperpolarizations were also blocked by AF- are mean 4 mV in amplitude and mean 11 sec DX 116. However, BCh-hyperpolarization is (21 מ in duration at mean resting Em of 60 mV, associated with an increase in Rm . The s- and are accompanied by a decrease in gK. The IPSPs in mudpuppy cardiac and cat vesicle s-EPSPs are blocked by 100 nM pirenzepine, pelvic ganglia are induced by direct monosyn- a muscarinic M1-receptor antagonist. The aptic activation of muscarinic receptors and 7,10) muscarinic agonists, bethnechol (BCh) and accompanied by an increase in gK . The acetyl-␤-methylcholine, induce depolariza- electrogenesis mechanism of s-IPSP in the tions associated with an increase in input hamster SM ganglion neurons is not yet well resistance (Rm) in some SM ganglion neu- understood at the level of ionic currents. rons21). BCh-depolarizations induced by 10 ejections (100 mM BCh) using a pressure 4. Slow hyperpolarizing synaptic potential ejection system are mean 4.4 mV in amplitude Hamster SM ganglion neurons induce s- and mean 5.9 min in duration at mean resting HSPs in the presence of hexamethonium and מ Em of 46 mV; they are accompanied with AF-DX 116 in response to repetitive pregan- spike firing at 4–5 Hz in the rising phase. The glionic stimulation, which is 40 stimuli at BCh-depolarization is induced in the neu- 25 Hz. The s-HSP is mean 4 mV in amplitude rons which respond with f-EPSP and s-EPSP and mean 38.7 sec in duration at mean resting מ to repetitive preganglionic stimulation. The Em of 68.7 mV. Rm decreased during the s- membrane input resistance (Rm) during BCh- HSP in some cells. In the remainings, Rm depolarization increases by approximately hardly changed. However, the reversal poten- 30% of the control value at a maximum. BCh- tial of s-HSP is estimated at approximately -mV. The s-HSP is blocked by 1mM caf 90מ depolarization is also blocked by pirenzepine 33) (300 nM) . Recently, we proved the existence feine, a P1 purinoceptor antagonist. Adenos- of M currents in the hamster SM ganglion ine causes hyperpolarizations accompanied neurons using the whole-cell patch clamp by a decrease in Rm in the same neurons which technique39). induce the s-HSP. 2-chloroadenosine (30mM) was applied to the neurons using a pressure 3. Slow inhibitory postsynaptic potential ejection system. The adenosine hyperpolar- s-IPSP is evoked by 40–50 stimuli at 25 Hz ization is also blocked by caffeine. Both the s- in the presence of 0.3–0.5 mM hexametho- HSP and adenosine hyperpolarization in the nium33), which is mean 7 mV in amplitude hamster SM ganglion neurons are mediated and mean 10 sec in duration at mean Em of by P1 purinoceptors and are caused by an (36 מ 53 mV. The membrane input resistance increase in gK . Adenosine is established

(Rm) during s-IPSP decreased by 32% of the as the neurotransmitter mediating the slow control value at a maximum. The reversal HSP in vesical pelvic ganglia. The slow HSP -mV, and the adenosine hyperpolarization are asso 90מ potential of s-IPSP is approximately suggesting that the s-IPSP is associated with an ciated with an increase in the potassium 1) increase in gK. AF-DX 116, a muscarinic M2- conductance . receptor antagonist (500 nM), blocked the s- IPSP. After-hyperpolarizations following sum- mated f-EPSPs insensitive to the M2 receptor REFLEX SPIKE DISCHARGES antagonist remained. BCh-hyperpolarizations FROM THE SUBMANDIBULAR induced by 10 ejections (100 mM BCh) using GANGLION NEURONS a pressure ejection system are mean 11 mV in amplitude, and mean 5.6 min in duration at We studied reﬂex spike discharges from rat מ the mean resting Em of 44 mV in neurons, SM ganglion neurons in order to analyze 160 T. S UZUKI

Fig. 12 Heat and gustatory reflex responses obtained from two type 1 neurons Upper row, Trace A: Reflex spike discharges obtained after exposing a rat to 38°C heat stimulus. Lower row, Trace A: Reflex spike discharges after exposure to 36.5°C, B: After lowering the ambient temperature to room temperature (20°C), C: Reflex spike discharges evoked by the application of 0.2M acetic acid to the tongue, E: After washing with water. Average spike frequencies in upper row trace A, and lower row trace A–C: 10.8Hz, 2.5Hz, 1.3Hz and 5.8Hz, respectively (see ref. 22). parasympathetic information in situ for saliva- for a period of stimulation (shown in Fig. 12). tion22). Salivation is induced reflexly by apply- The maximal spike frequencies 100 min after ing gustatory stimulation to the tongue. High exposing the rats to the ambient temperature ambient temperature is another and different of 38°C were 10–12 Hz. When the ambient salivatory stimulation for rodents, because temperature was lowered to 20°C, the spike they secrete a large amount of saliva to regu- frequencies decreased to 1–2 Hz after 20 min. late their body temperature in such an envi- The frequency of gustatory reflex discharges ronment. In the intracellular recording from was 9–10 Hz. The applied gustatory stimuli the rat SM ganglion neuron in situ, the rats were 0.2 M acetic acid, 0.5 M NaCl, and 0.4 M were anesthetized by chloralose and ure- sucrose. After a latency of several seconds, thane. The resting membrane potentials of applications of the acetic acid and NaCl solu- SM neurons with single preganglionic (type tions increased the spike frequency to 9–10 Hz. 1) and multiple preganglionic (type 2) inner- The sucrose solution elicited reflex spikes .mV. only in the initial phase of the application 50מ to 40מ vations were in the range of Under resting conditions, the frequencies of spontaneous spikes were approximately 0.6 Hz 2. Reflex responses from type 2 neurons in type 1 neurons and 0.1Hz in type 2 neurons. The maximal frequency of the reflex spikes in type 2 was approximately 4 Hz, 90 min after 1. Reflex responses from type 1 neurons exposing the rat to an ambient temperature The discharge pattern of type 1 neurons is of 36.5°C. The reflex spikes were accompa- characterized by a persistent burst of spikes nied by a number of subthreshold EPSPs. CURRENT-CLAMP STUDY IN SUBMANDIBULAR GANGLIA 161

Fig. 13 Reflex spike responses obtained from a type 2 neuron Trace A: Reflex spike discharges of a type 2 neuron to 36.5°C heat stimulus. Trace B: Reflex spike discharges to 0.5NaCl. Average spike frequencies: 0.9Hz in A and 1Hz in B (see ref. 22).

Table 1 Receptors of biogenic substances and their responses in the SM ganglion

Receptor Ions, Permeability Potential Increase f-EPSP ,םK ,םNicotinic receptor Na

ם Muscarinic M1-receptor K , Decrease s-EPSP

ם Muscarinic M2-receptor K , Increase s-IPSP

ם P1 purinoceptor K , Increase s-HSP ם ␣ 1-adrenergic receptor K , Decrease Excitatory potential Increase Inhibitory potential ,םK ␣ 2-adrenergic receptor Decrease? (ref. 30) Inhibitory potential ,מor Cl

ם2 ם ם 5-HT3 receptor Na , K , Ca , Increase Excitatory potential

ם ם H1 receptor Na , K , Increase Excitatory potential Increase Excitatory potential ,מCl

GABAA receptor Decrease Inhibitory after-potential ,מCl

ם GABAB receptor K , Increase Inhibitory potential

ם D2 receptor K , Increase Inhibitory potential Increase Preceding inhibitory potential ,םK

BK2 receptor Decrease Following excitatory potential ,םK

P2 purinoceptor Non-selective cation, Increase Excitatory potential

However, type 2 neurons are almost com- The parasympathetic preganglionic nerve pletely taste-insensitive (shown in Fig. 13). to the SM gland of the rabbit contains taste- When a number of subthreshold EPSPs with sensitive and taste-insensitive ﬁbers12). These different amplitudes were evoked, the sum- correspond the type 1 and type 2 neurons of mated EPSPs generated spikes. Among the the rat SM ganglion neurons. When SM gan- three gustatory stimuli, the maximal spike glion neurons were impaled 60–80 min after frequency was approximately 1Hz in the exposing the animal to 38°C and 90–100 min response to 0.5M NaCl. after exposing it to 36–37°C, the spike fre- 162 T. SUZUKI quencies recorded from most of the type 1 depolarizations are also accompanied by a de- ם neurons were similar to those in the chorda crease in gK. Classical K channel blockers do tymani reported by Elmer and Ohlin4). The not signiﬁcantly affect them. These pharma- maximal 10–12 Hz to taste stimuli is also cological characteristics are similar to those of the optimal preganglionic stimulus frequency cat vesical pelvic ganglion1). accompanying the maximal secretory response Hyperpolarizations in hamster SM gan- in the SM gland6). It is known that subman- glion neurons are induced by clonidine, ␣ dibular ganglion neurons innervate secretory an 2-adrenoceptor agonist, and NE- and cells, myoepithelial cells, and blood vessels6). clonidine-hyperpolarizations are blocked by ␣ 27,28,30) Type 1 neurons probably innervate secretory yohimbine, 2-adrenoceptor antagonist . cells, thereby controlling the saliva secretory Therefore, the NE-hyperpolarizations in ham- ␣ rate, whereas type 2 neurons seem to inner- ster SM ganglion neurons are mediated by 2- vate the blood vessels of the SM gland and adrenoceptors. The receptors responsible for play a role in vasodilation. NE-hyperpolarization in hamster SM ganglion neurons are the same as those of cat vesical pelvic ganglion neurons1). However, in

BIOGENIC SUBSTANCES hamster SM ganglion neurons, Rm during NE- MODULATING SYNAPTIC TRANSMISSION hyperpolarization increases by mean 12.6% IN SUBMANDIBULAR GANGLIA of the control value at a maximum, and the mV28). These 10מ reversal potential is close to We have been studying various biogenic two characteristics differ from the NE-hyper- substances modulating synaptic transmission polarization in the vesical pelvic ganglion in hamster SM ganglion neurons. Receptors neurons. of these biogenetic substances and their responses in the SM ganglion neurons are 2. 5-Hydroxytriptamine (5-HT) summarized in Table 1. Examples of responses Application of 5-HT (serotonin) by pres- to biogenic substances in the SM ganglion sure pulses induces a fast depolarization in neurons are shown in Fig. 14. almost all hamster SM ganglion neurons31). The sensitivity to 5-HT is distinct between

1. Norepinephrine neurons. Rm decreases by mean 13% of the Norepinephrine (NE) either depolarized control value during 5-HT-depolarization at a or directly hyperpolarized the SM ganglion maximum. The reversal potential of 5-HT- ,mV. Multiple ions 20ם–neurons. In the hamster SM ganglion, NE depolarization is 0 are involved ,מand Cl םK ,םCa2 ,םcaused a blockade of transmission at synapses including Na when the ganglion neurons were either depo- in the electrogenesis mechanism. ICS 205- larized or hyperpolarized21). In the vesical pel- 930, a 5-HT M-receptor antagonist, inhibits vic ganglia, the activation of ␣-adrenergic the 5-HT-depolarization, whereas methyser- receptors has been suggested to inhibit nico- gide, a 5-HT D-receptor antagonist, has no tinic transmission by reducing the release effect. Therefore, 5-HT-depolarization is medi- 1) of ACh from preganglionic terminals . The ated by an activation of M (5-HT3)-receptors. responses to NE in hamster SM ganglion neu- 5-HT enhances the excitability of hamster SM rons are mediated by ␣-adrenergic receptors21). ganglion neurons. 5-HT-like immunoreactivi- Depolarizations are induced by phenyl- ties exists in neurons and varicoses of ﬁbers in ␣ ephrine, an 1-adrenoreceptor agonist, and rabbit vesical pelvic ganglia and mudpuppy NE- and phenylephrine-depolarizations are cardiac ganglia1). ␣ blocked by prazosin, an 1-adrenoreceptor antagonist in hamster SM ganglion neu- 3. ␥-Aminobutyric acid (GABA) rons27–29). Therefore, the NE-depolarizations ␥-Aminobutyric acid (GABA) produces a ␣ are mediated by 1-adrenoceptors. The NE- biphasic response, consisting of an initial fast- CURRENT-CLAMP STUDY IN SUBMANDIBULAR GANGLIA 163

Fig. 14 Responses to biogenic substances in SM ganglion neurons Biogenic substances were applied to a neuron from a pipette by air pressure pulse. Depolarizations (upward deﬂection) exert an excitatory effect on the neuron, and hyperpolarizations exert an inhibitory effect. GABA produces a biphasic response. Right-hand graph: The relationship between

resting Em and the amplitudes of GABA-depolarization and -afterhyperpolarization. The reversal potentials of GABA-depolarization and -afterhyperpolarization were estimated by extrapolation. The 22.2mV when the amplitude wasמ reversal potential of this GABA-depolarization was estimated as מ at resting Em of 40mV (shown by a broken line). The value was omitted. The SM ganglion neuron possessed GABA - and GABA -receptors. Baclofen is a GABA -receptor agonist. Dopamine-hyper- B מ A B polarizations were blocked by ( )-sulpirid, a D2 receptor antagonist (the lower trace). Histamine-

depolarizations were blocked by pyrilamine, an H1 receptor antagonist (in B). The left-hand number

of each response shows the resting Em (see ref. 29, 28, 31, 34, 38, 35, 37 and 32).

depolarization associated with an increase in gCl GABA-depolarization, Rm decreases by mean followed by a delayed slow-hyperpolarization 29.5% of the control value. The GABA-after- (GABA-after-hyperpolarization), associated with hyperpolarization is mean 18.4 mV in ampli- Ϫ a decrease in gCl and partly with an increase tude and mean 7.2 min in duration. Rm 34) in gK . GABA was applied to a neuron by a increased by 26.5% of the control value. single pulse of pressure ejection (100 mM, The reversal potential of GABA-depolarization mV), and the 25.6מ–9.6מ mV (range 16מ msec in duration). GABA-depolarization is 10 was mean 19 mV and mean 2.8 min in dura- reversal potential of GABA-after-hyperpolar- מ מ מ tion at mean resting Em of 60 mV. During ization ranges from 10 to 24 mV. The 164 T. SUZUKI

effects of muscimol, a GABAA-receptor agonist, 1–3 pulses) induced hyperpolarizations in all 37) picrotoxin, a GABAA-antagonist, and bicu- tested neurons . The DA-hyperpolarization culine, a selective GABAA-antagonist, on was mean 3 mV in amplitude and mean

GABA-depolarization indicate that it is medi- 37 sec in duration at mean resting Em of מ ated by GABAA-receptors. Baclofen, a GABAB- 65 mV. The Rm does not indicate obvious receptor agonist, induces a slow hyperpolar- changes during DA-hyperpolarization. The ization in hamster SM ganglion neurons. The reversal potential of DA-hyperpolarization is מ increase in gK is probably produced by open- close to 90 mV. However, their amplitudes free Krebs solution-םchannels. Therefore, the hamster SM and durations in K םing of K ganglion neurons possess both GABAA- and increase markedly in comparison with those

GABAB-receptors. The GABA-depolarization in the normal solution. Rm during the DA- enhances the excitability of the neuron, hyperpolarization decreased by 36% of the and, conversely, the GABA-after-hyperpolar- control value. The reversal potential of DA- -(מ) .mV 177.2מ ization plays an inhibitory role in synaptic hyperpolarization shifted to transmission. Sulpiride, a D2 receptor antagonist, blocks the DA-hyperpolarization. Applied DA directly

4. Adenosine triphosphate (ATP) activates D2 receptors on neurons of the ham-

In the vesical pelvic ganglion, the presence ster SM ganglion to increase the gK. DA of a noncholinergic and nonadrenergic released from SIF (small intensely ﬂuores- nerve, that releases ATP, and supplies the uri- cent) cells activates D1 receptors on the supe- nary bladder has been postulated and termed rior cervical ganglion neurons of the rabbit. purinergic3). Purinoceptors have been into two types; P1 and P2 for adenosine and ATP. 6. Bradykinin In the hamster SM ganglion, pressure appli- Lysylbradykinin is formed in the activated cation of ATP (30 mM, 1–5 pulses) induced a salivary gland by release of a enzyme, tissue fast depolarization in 75% of the neurons, kallikrein, from the gland cells and is involved and this fast depolarization was followed by in maintaining blood ﬂow for the activated a hyperpolarization in 11% of them35). The salivary gland. Lysylbradykinin may stimulate remaining neurons (14%) responded with axon terminals of the SM ganglion neurons. persistent hyperpolarizations alone. The fast Rodent neuroblastoma hybrid cells (NG 108- depolarization is mean 14 mV in amplitude 15 hybrid cells), descendents of tumor cells and mean 33 sec in duration at mean resting probably originating in the sympathetic gan- מ Em of 62 mV. Rm decreased by 32% of the glion produced a hyperpolarization followed control value during the fast depolarization. by a depolarization when bradykinin (BK) The reversal potential of the fast depolariza- was applied to the cells2). The BK-biphasic ם2 מ tion is 11mV, and is probably associated response is caused by an increase in [Ca ]i with an increase in non-selective cation con- and an activation of protein kinase C, which ductance. Caffeine (1mM), a P1 purinoceptor are induced after breakdown of phosphatidy- antagonist, has no effect on this fast depolar- linositol 4,5-biphosphate via BK2-receptors. ization. Therefore, it is mediated by the P2 In the hamster SM ganglion neurons, pres- purinoceptors, and the persistent hyperpolar- sure applications of BK (500␮M, 3–5 pulses) ization is probably mediated by the P1 purino- caused responses in most neurons (23/29). ceptors36). In the vesical ganglia, the relative The remaining neurons did not respond38). order of potency for the P1 purinoceptors is Fifteen neurons responded with a depolariza- 2-chloroadenosineϾAMPϾϾADPϾATP 18). tion, with mean 4 mV in amplitude and mean

6.5 min in duration at mean resting Em of מ 5. Dopamine 58 mV. The Rm increased by 24% of the In the hamster SM ganglion neurons, pres- control value during BK-depolarization. sure application of dopamine (DA) (100 mM, These depolarizations caused spike ﬁrings, CURRENT-CLAMP STUDY IN SUBMANDIBULAR GANGLIA 165 that continued for 30 min at a maximum. induced by release of ACh from pregangli- The BK-depolarization often was evoked onic terminals. Pyrilamine (100␮M) and ␮ מ even at an Em negative level to 70 mV. The diphenhydramine (100 M), H1 receptor responses of four neurons was biphasic; the antagonists, decreased the amplitude of hista- BK-depolarization was preceded by a BK- mine-depolarization by 71–73% and 23–30%, hyperpolarization, with mean 3 mV in ampli- respectively. Cimetidine (100␮M–1mM), an tude and mean 2.5 min in duration at mean H2 receptor antagonist, had no effect. The מ resting Em of 74 mV. The Rm decreased by histamine-depolarization is mediated by an

17% of the control value during the BK- activation of H1 receptors on the neuron hyperpolarization. The reversal potential was soma. Increases in cation conductances seem -mV. Four different neurons to be involved in the electrogenesis mecha 90מ close to responded with a hyperpolarization alone. nism in the hamster SM ganglion neurons.

The Rm decreased by more than 30% of the control value. NEUROPEPTIDES MODULATING םThe concentrations of cytosolic free Ca2 were measured in primary cultured SM gan- SYNAPTIC TRANSMISSION glion neurons using fura-2 microﬂuorometry. IN SUBMANDIBULAR GANGLIA ם2 BK increased [Ca ]i in the hamster SM ganglion neurons38). The ineffectiveness of the Modulating effects of neuropeptides, such

BK1 receptor agonist and antagonist suggest as vasoactive intestinal peptide (VIP), en- that the BK responses in hamster SM gan- kephalin, substance P (SP), neurokinin A ␣ ␣ glion neurons are mediated by BK2 receptors, (NK), -calcitonin gene-peptide ( -CGRP), although it is indirect evidence. In NG 108-15 angiotensin II (Angio II) and pituitary adeny- hybrid cells, the BK-biphasic response is late cyclase activating peptide (PACAP) on slowly acti- the submandibular ganglia will be described-םcaused by an activation of Ca2 .channels and an inhibition of M in the next paper in this series םvated K channels. The dual responses are mediated 2) by BK2 receptors . These ﬁndings in SM ganglion neurons suggest that the preceding hyperpolarization is caused by an activation of REFERENCES slow gK(Ca), whereas, in the electrogenesis mechanism of the BK-depolarization in ham- 1) Akasu, T. (1992). Synaptic transmission and ster SM neurons, a type of cation channel dif- modulation in parasympathetic ganglia. Jpn J ferent from M channels seems to be involved, Physiol 42, 839–864. because it is evoked even at an Em negative to 2) Brown, D.A. and Higashida, H. (1988). Mem- mV. brane current responses of NG 108-15 mouse 70מ rat glioma hybrid cells toןneuroblastoma bradykinin. J Physiol (Lond) 397, 167–184. 7. Histamine 3) Burnstock, G.A. (1978). Basis for distinguish- In the hamster SM ganglion neurons, pres- ing two types of purinergic receptor. In Cell sure application of histamine (100 mM, 10–20 Membrane Receptors for Drugs and Hormones: A pulses) induced a fast depolarization associ- Multidisciplinary Approach (L. Bolis and R.W. 32) Staub eds.), pp.107–108, Raven Press, New ated with a decrease in Rm (range 0–23%) . York. This histamine-depolarization was mean 4) Elmer, M. and Ohlin, P. (1971). Salivary secre- 9.7 mV in amplitude and mean 1.6 min at tion in the rat in a hot environment. Acta מ mean resting Em of 63 mV. The reversal Physiol Scand 83, 174–178. potential of histamine-depolarization was at 5) Emmelin, N. (1967). Nervous control of salivary glands. In Handbook of Physiology. (C.F. an Em of 0–5 mV. Histamine-depolarizations -Code and W. Heidel eds.), section 6: Alimen ם2 are also induced in Ca -free Krebs solution. tary Canal, vol.II, pp.595–632, American Therefore, the effect of histamine is not Physiological Society, Washington. 166 T. S UZUKI

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33) Suzuki, T., Fukuda, S. and Sakada. S. (1989a). sponses in hamster submandibular ganglion Slow postsynaptic potentials recorded from cells. Bull Tokyo dent Coll 33, 25–28. hamster submandibular ganglion cells. Bull 39) Suzuki, T., Hayashi, K. and Ikegami, H. (2000). Tokyo dent Coll 30, 89–92. Angiotensin II-induced depolarizations in sub- 34) Suzuki, T., Hada, R. and Sakada, S. (1989b). mandibular ganglion neurons. Jpn J Physiol 50, GABA-induced biphasic response in the sub- Suppl. (in press) mandibular ganglion cell. Bull Tokyo dent Coll 40) Wood, J.D. and Mayer, C.J. (1978). Intracellu- 30, 85–88. lar study of electrical activity of Auerbach’s 35) Suzuki, T., Aida, H., Aeba, A. and Sakada, S. plexus in guinea pig small intestine. Pﬂugers (1990). Responses of hamster submandibu- Arch 374, 265–275. lar ganglion cells to ATP. Bull Tokyo dent Coll 31, 41) Wood, D.J. (1983). Neurophysiology of para- 63–65. sympathetic and enteric ganglia. In Autonomic 36) Suzuki, T., Fukuda, S. and Sakada, S. (1990). Ganglia. (L.G. Elfvin ed.), pp.367–398, John Adenosine hyperpolarization and slow hyper- Wiley and Sons, New York. polarizing synaptic potential in the hamster submandibular ganglion cell. Bull Tokyo dent Coll 31, 67–70. Reprint requests to: 37) Suzuki, T. (1992a). Hyperpolarization in ham- Dr. Takashi Suzuki ster submandibular ganglion cell mediated by Department of Physiology,

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