Serotonin receptor subtypes that depolarize guinea pig inferior mesenteric ganglion neurons

Our previous studies indicated that serotonin (5-HT) depolarized a majority of guinea pig inferior mesenteric ganglion (IMG) neurons and may be another transmitter for the noncholinergic late slow excitatory postsynaptic potential (ls-EPSP) in the IMG. However, the subtypes of 5-HT receptor mediating these responses have not yet been identified. Using intracellular recording, we examined the effect of 5-HT receptor antagonists with specificity to various 5-HT receptor subtypes on the 5-HT-mediated depolarization and ls-EPSP in IMG neurons in vitro. Cyproheptadine, a 5-HT(1/2) receptor antagonist, reversibly inhibited the slow, but not the fast, depolarization and ls-EPSP in the 5-HT-sensitive neurons. Both mianserin and spiperone, 5-HT(2) and 5-HT(1A) receptor antagonists, did not significantly alter either the fast or slow depolarizing responses or the ls-EPSP. The 5-HT(3) receptor antagonist MDL 72222 (Bemesetron) completely inhibited the fast depolarization with little diminution of the slow depolarization and ls-EPSP. Superfusion of putative 5-HT(1P) receptor antagonist, BRL 24924 (Renzapride), reversibly attenuated both the depolarization and ls-EPSP. However, 5-HT-insensitive neurons with ls-EPSP were found to be insensitive to both cyproheptadine and BRL 24924. In most 5-HT-sensitive neurons, the 5-HT(3) receptor agonist, 2-methyl-5-HT, and the selective 5-HT(1P) agonist, MCPP or 5-OHIP, evoked a fast and a slow depolarization in 55.6 and 71.4% of the neurons, respectively, without a significant effect on the membrane potential in 85.7 and 100% of the 5-HT-insensitive neurons. In 5-HT-sensitive neurons, MDL 72222 reversibly abolished the fast depolarization induced by 2-methyl-5-HT; BRL 24924 significantly inhibited the slow depolarization induced by MCPP or 5-OHIP, but not by SP. Prolonged superfusion of 5-HT-sensitive neurons with MCPP abolished the evoked ls-EPSP without inhibition of action potential. These results suggest that the fast and slow depolarizations in these neurons are mediated by 5-HT(3) and 5-HT(1P) receptor subtypes, respectively. The latter may also mediate the ls-EPSP in 5-HT-sensitive neurons.     

Ionic currents determining the membrane characteristics of type I spiral ganglion neurons of the guinea pig

Enzymatically isolated type I spiral ganglion neurons of the guinea pig have been investigated in the present study. The identity of the cells was confirmed by using anti-neuron-specific enolase immunostaining. The presence and shredding of the myelin sheath was also documented by employing anti-S100 immunoreaction. The membrane characteristics of the cells were studied by using the whole-cell patch-clamp technique. The whole-cell capacitance of the cells was 9 +/- 2 pF (n = 51), while the resting membrane potential of the cells was -62 +/- 9 mV (n = 19). When suprathreshold depolarizing stimuli were applied, the neurons fired a single action potential at the beginning of the stimulation. It was confirmed in this study that type I spiral ganglion cells possess a hyperpolarization-activated nonspecific cationic current (Ih). The major characteristics of this current component were unaffected by the enzyme treatment. Type I spiral ganglion cells also expressed various depolarization-activated K+ current components. A high-threshold outward current was sensitive to 1-10 mm TEA+ application. The ganglion cells also expressed a relatively small, but nevertheless present, transient outward current component which was less sensitive to TEA+ but could be inhibited by 100 micro m 4-aminopyridine. A DTX-I-sensitive current was responsible for some 30% of the total outward current (at 0 mV), showed rapid activation at membrane potentials positive to -50 mV and demonstrated very little inactivation. However, inhibition of the highly 4-AP- or DTX-I-sensitive component did not alter the rapidly inactivating nature of the firing pattern of the cells.     

Effect of labyrinthectomy on the spike generator of vestibular neurons in the guinea pig

In the guinea pig, in the absence of any stimulation, all the neurons of the vestibular nuclei are tonically firing. After an ipsilateral labyrinthectomy, these neurons first cease to fire but recover their previous discharge in 7 days. Here, we tested whether a modification of the spike generator, the process transforming synaptic currents into spike patterns, could be a factor underlying this restoration. For this purpose, we studied the firing rate responses of neurons of the medial vestibular nucleus in brain stem slices to intracellularly injected currents. We conclude that although labyrinthectomy induces some plastic changes in the excitability of the neurons of the medial vestibular nucleus, these changes do not underlie the restoration of activity which occurs in these neurons when they are deprived of their labyrinthine input.     

Actions of noradrenaline on myenteric neurons in the guinea pig gastric antrum.

We used intracellular electrophysiological recording to study the actions of noradrenaline on myenteric neurons in the guinea pig gastric antrum. Noradrenaline caused a dose-dependent inhibition of the stimulus-evoked cholinergic fast excitatory postsynaptic potentials (EPSPs). Noradrenaline had no effect on the postsynaptic response to acetylcholine, suggesting a presynaptic site of action. The slow EPSP was also presynaptically inhibited by noradrenaline. In only 5% of the neurons, noradrenaline caused a postsynaptic depolarization, accompanied by increased input resistance and enhanced excitability. Studies with adrenergic antagonists and agonists revealed that the presynaptic inhibitory effect was mediated by an alpha 2-receptor, while the postsynaptic excitatory effect seemed to be mediated by an alpha 1 receptor. We conclude that noradrenaline inhibits neurotransmitter release from cholinergic and non-cholinergic nerve terminals in the myenteric plexus of the antrum and that it excites a subpopulation of antral neurons. Both mechanisms may contribute to the neurally mediated inhibitory action of noradrenaline on gastric contractility.     

Electrophysiological properties and cholinergic responses in guinea-pig celiac ganglion neurons in primary culture.

Prevertebral neurons enzymatically dissociated from celiac ganglia of adult guinea-pigs were maintained in long-term primary culture. Cells were plated at a density of 95 +/- 15 cm-2, and intracellular electrical activity was measured between 2 and 7 weeks after dissociation. Neurite outgrowth began within 24 h of enzymatic dissociation. Cell survival dropped below 50% after more than two weeks in culture. The resting potential (-53 mV +/- 0.8), time constant (12 ms +/- 1.3), input resistance (47 M omega +/- 8.6), rheobase (0.33 nA +/- 0.02), degree of accommodation, spike amplitude (70 mV +/- 3.0), after hyperpolarization amplitude (-9.5 mV +/- 0.55), and after hyperpolarization duration (88 ms +/- 7.6) in these cells were not different from those recorded from neurons in intact celiac ganglia. A larger proportion (greater than 90%) of cells exhibited fast accommodation (phasic) in response to depolarizing current pulses. Unevoked (spontaneous) depolarizations and action potentials were observed. The cells responded to pressure ejected acetylcholine. Two types of responses consisted of an early rapid depolarization which was attenuated by hexamethonium and a later slow depolarization which was attenuated by atropine. We conclude that prevertebral neurons from guinea-pigs can be maintained in long-term primary culture, that they retain electrophysiological properties similar to intact ganglia and exhibit complex responsivity to acetylcholine.     

Peripheral nerve pathways to neurons in the guinea pig inferior mesenteric ganglion determined electrophysiologically after chronic nerve section

Peripheral synaptic pathways to neurons in the guinea pig inferior mesenteric ganglion (IMG) were studied. Nerve trunks innervating neurons in the ganglion were surgically sectioned and intracellular electrical responses to nerve stimulation were measured 6-8 days after surgery. In all animals ganglia were decentralized by removal of the lumbar sympathetic chain ganglia L2 through L4 and in addition two peripheral nerves were sectioned leaving the ganglion innervated by only one peripheral nerve. Fast and slow excitatory postsynaptic potential (EPSP) were evoked with electrical stimulation of each of the nerve trunks and with distension of the colon. The thresholds to evoke fast EPSPs and the amplitude of slow EPSPs were compared for each nerve trunk among the different surgical groups including sham-operated controls and completely denervated ganglia. Both fast and slow EPSPs could be evoked electrically from each intact peripheral nerve trunk after the other three nerve trunks had been sectioned, which demonstrates that nerve fibers with cell bodies in the regions innervated by the peripheral nerves make functional synaptic connections with neurons in the inferior mesenteric ganglion. In general, nerve sections increased the threshold for evoking fast EPSPs and decreased the amplitude of electrically-evoked slow EPSPs compared to control ganglia. Synaptic potentials could also be evoked with stimulation of cut nerve trunks, demonstrating that branches of nerve fibers from peripheral nerves enter other nerve trunks. The hypogastric nerve was unique in that branches of axons eliciting fast but not slow synaptic potentials in the ganglion entered this nerve trunk. Distension-induced fast and slow EPSPs were present only if the lumbar colonic nerve was intact and they were not altered by section of the other nerve trunks. In contrast, the slow EPSPs evoked with electrical stimulation of the lumbar colonic nerve were significantly smaller when at least one other nerve trunk was sectioned suggesting that the axon branches from other nerve trunks which enter the lumbar colonic nerve are not activated by distension. These studies demonstrate that neurons eliciting either fast or slow synaptic potentials with cell bodies in regions innervated by the peripheral nerve trunks make functional synaptic connections with neurons of the inferior mesenteric ganglion. The results also suggest that the majority of mechanosensory neurons mediating excitatory synaptic responses to colon distension are neurons with a peripheral cell body.     

Distribution of cholecystokinin-like-immunoreactive neurons in the guinea pig forebrain

The distribution of cholecystokinin (CCK)-immunoreactive nerve fibers and cell bodies was studied in the forebrain of control and colchicine-treated guinea pigs by using an antiserum directed against the carboxyterminus of CCK octapeptide (CCK-8) in the indirect immunoperoxidase technique. Virtually all forebrain areas examined contained immunoreactive nerve fibers. A dense innervation was visualized in; neocortical layers II-III, piriform cortex, the medial amygdala, the medial preoptic area, a circumventricular organ-like structure located at the top of the third ventricle in the preoptic area, the subfornical organ, the posterior bed nucleus of the stria terminalis, the posterior globus pallidus (containing labeled woolly fiber-like profiles), the ventromedial hypothalamus, the median eminence, and the premammillary nucleus. A moderately dense innervation was visualized elsewhere excepted in the septum and thalamus where labeled axons were comparatively few. Immunoreactive perikarya were abundant in: neocortex (especially layers II-III), piriform cortex, amygdala, the median preoptic nucleus, the bed nucleus of the stria terminalis, the hypothalamic paraventricular (parvicellular part), arcuate, and dorsomedial (pars compacta) nuclei, the dorsal and perifornical hypothalamic areas, and throughout the thalamus. Areas also containing a moderate number of labeled cell bodies were the medial preoptic area, the globus pallidus, the caudate-putamen, and the periventromedial area in the hypothalamus. Immunostained perikarya were absent or only occasionally observed in the septum, the suprachiasmatic nucleus, the magnocellular hypothalamoneurohypophyseal nuclei, and the ventral mesencephalon. In the adenohypophysis, corticomelanotrophs were labeled in both males and females, and thyrotrophs were labeled in females only. This distribution pattern of CCK-8 immunoreactivity is compared to those previously recorded in other mammals. This shows that very few features are peculiar to the the guinea pig. It is discussed whether some interspecific differences in immunostaining are real rather than methodological.     

Electrophysiological properties of guinea pig septal neurons in vitro

The electrophysiological properties of septal neurons have been examined in vitro in guinea pig brain slices. These cells display different firing modes when stimulated by transmembrane current pulses depending on the amplitude of the depolarization. With small pulses septal neurons fire repetitive Na spikes but on larger depolarizations they respond with a single full-Na action potential which is followed by a number of spikes of smaller amplitude. A further increase in the amplitude of the pulse evokes powerful Ca spikes possibly generated in the dendrites. These Ca spikes appear with larger amplitude in presumptive intradendritic recordings. In many cells stimulation of the fimbria evoked postsynaptic responses consisting of either a depolarization, a hyperpolarization or a depolarization-hyperpolarization sequence.     

An electron microscopic study on enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs

Enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs were characterized by a high population of large granular vesicles mixed with small clear vesicles. The immunoreactive material is confined to the large granular vesicles. The immunoreactive nerve fibers formed many axo-dendritic as well as axo-somatic synapses and also formed a few synapses with presumed preganglionic axons containing numerous vesicles. The immunoreactive fibers were regarded as presynaptic at these synapses. These findings suggest that enkephalin might play a role as a neurotransmitter or neuromodulator in the ganglionic transmission of this prevertebral ganglion.     

An electron microscopic study on VIP-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs

An immuno-electron microscopic study revealed that VIP-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs were characterized by a conspicuously numerous large granular vesicles mixed with small clear vesicles. The immunoreactive materials were localized in the core of the large granular vesicles and a distinct halo was recognized between the core and the limiting membrane of the vesicles. These fibers made numerous axo-dendritic and a few axo-somatic synapses with the post-ganglionic principal neurons and also formed some synapses with vesicle-containing neuronal profiles which are presumably preganglionic axons. The immunoreactive fibers were presynaptic at all these synaptic sites. In addition, some synaptic contacts were found between two adjacent immunoreactive nerve fibers. These findings strongly suggests that VIP might be involved in the ganglionic transmission of the prevertebral ganglia.     

Localization of substance P-like immunoreactivity in guinea pig vestibular endorgans and the vestibular ganglion.

The immunocytochemical distribution of substance P (SP) in guinea pig vestibular endorgans and the vestibular ganglion was investigated. Two kinds of SP-immunoreactive fibers were distinguished. Most were thick, and found around or beneath sensory hair cells. These SP-immunoreactive fibers were distributed predominantly on the slope of the crista and the peripheral region of the macula. By electron microscopy, we confirmed this type of SP-like immunoreactivity to be restricted within primary afferent neurons. Some vestibular ganglion cells also showed SP-like immunoreactivity, suggesting that SP is present in some primary afferent neurons, and is involved in afferent neurotransmission. The characteristic distribution of SP may indicate functional differences within each endorgan. The other group of immunoreactive nerve fibers, varicous thin fibers, could be found in the stroma of vestibular endorgans, nerve trunk, vestibular ganglion, and along blood vessels of the vestibular ganglion. These fibers may have a different origin, and have an influence on blood flow and certain other functions.     

Possible relation between structure and spike shapes of neurones in guinea pig cochlear ganglion.

A two-component positive single unit spike could be recorded extracellularly from neurones in the acoustic ganglion of the guinea pig cochlea. Light and electron microscopy showed that the bipolar afferent neurones and their processes were myelinated except at nodes of Ranvier on either side of the cell soma, about 40 mum from the point of emergence of the myelinated processes. This anatomical finding and the properties of the two-component spike suggest that impulse conduction in these cells is achieved by rapid successive activation of the low threshold nodes on either side of the cell soma. The possible advantages of such a conduction system are discussed.     

Transmitter diversity in ganglion cells of the guinea pig gallbladder: an immunohistochemical study.

Several neurotransmitters have been reported to exist in the ganglionated plexus of the guinea pig gallbladder. These include substance P, neuropeptide Y (NPY), calcitonin gene-related peptide, vasoactive intestinal peptide (VIP), acetylcholine, norepinephrine, serotonin, and dopamine. To determine which neuropeptides are intrinsic to gallbladder ganglia, we performed immunohistochemistry on colchicine-treated preparations. In separate, single-labeled preparations, a majority of neurons contained substance P-, NPY-, or somatostatin-like immunoreactivity. In double-labeled preparations, a large majority of the neurons that contained substance P-like immunoreactivity also contained NPY-like immunoreactivity and somatostatin-like immunoreactivity. Immunoreactivity for VIP was present in a small percentage of the gallbladder neurons which did not contain substance P-like immunoreactivity. Additional experiments were done to test for the presence of other compounds, known to exist in the neurons of the gut. Although immunoreactivity was found in control preparations of small intestine, the ganglionated plexus of the gallbladder lacked immunoreactivity for galanin, dynorphin, enkephalin, gastrin-releasing peptide, or gamma-aminobutyric acid. We conclude that ganglia of the guinea pig gallbladder contain at least two populations of neurons, based on transmitter phenotype. One of these populations appears to contain substance P, NPY, and somatostatin. Another population, which represents a small contingent of the total population of neurons, contains VIP.     

The effects of pumiliotoxin-B on sodium currents in guinea pig hippocampal neurons

The actions of pumiliotoxin-B, extracted from the skin of the frog Dendrobates pumilio, were examined on hippocampal slices and on acutely dissociated hippocampal neurons from the adult guinea pig. Application of 0.5-1 microM pumiliotoxin-B to hippocampal slices caused spontaneous, repetitive field discharges in the CA3 subfield. In whole-cell patch-clamp recordings of isolated CA1 and CA3 neurons, 1-2 microM pumiliotoxin-B shifted the midpoint of Na+ current activation by -11.4 +/- 1.1 mV. This shift was not dependent upon prior activation of the sodium channel. Pumiliotoxin-B did not block macroscopic Na+ inactivation but did reduce the apparent voltage-dependence of inactivation such that currents decayed faster at membrane potentials more negative than -30 mV. Single-channel recordings of sodium currents from excised membrane patches indicated that pumiliotoxin-B had little or no effect on channel closings due to entry into inactivated state(s) but did increase the rate of channel closings due to reversal of channel opening. The increase in the channel closing rate was consistent with a +8.7 mV shift in voltage sensitivity. Negative shifts in activation and positive shifts in closing rates implied a negative shift in the voltage-dependence of channel opening, suggesting that pumiliotoxin-B increases the rate of Na+ channel opening and closing in cells at rest, which could result in spontaneous activity in the neurons.     

Direct activation of guinea pig vagal afferent neurons by FMRFamide

Vagus nerve comprises two distinct kinds of nerves, nodose and jugular ganglionic nerves. We tested pharmacological difference between two vagal nerves in the responsiveness to FMRFamide. The response probability to FMRFamide was significantly higher in nodose than jugular nerves in intracellular calcium measurement. Nodose nerves also depolarized membrane potential to FMRFamide more than jugular nerves did, in patch-clamp recording, although the probability of action potential discharge was same in both nerves. The inward current induced by FMRFamide was characterized as mixed cations. These results suggest that FMRFamide may act as an activator and modulator of vagal sensory nerves for treating symptoms in visceral diseases.     

Innervation of guinea pig heart by neurons sensitive to capsaicin

To determine the origin of non-vagal afferent fibers innervating the heart of guinea pigs, capsaicin was injected into the ventricular myocardium to induce depletion of substance P (SP). The lower cervical, upper thoracic and lumbar spinal ganglia, as well as the left atrium and base of ventricles, were assayed for SP depletion by using the enzyme-linked immunosorbent assay (ELISA) and immunohistochemical procedures. Capsaicin affected spinal ganglia from the 3 regions differently. The substance P level in lumbar spinal ganglia remained fairly constant, while the level of SP from cervical and thoracic regions declined significantly. At the maximal depletion dosage (173 micrograms of capsaicin/kg), SP concentration decreased 72.3% in cervical spinal ganglia, 45.5% in thoracic ganglia and 56.1% in the atrium. The lack of SP depletion in lumbar ganglia indicates that capsaicin acted locally on cardiac afferents rather than systemically. Data from this study suggest that capsaicin-sensitive neurons of the heart have cell bodies in the lower cervical spinal ganglia as well as in the upper thoracic spinal ganglia.     

Electrophysiological study of neurotropin-induced responses in guinea pig hypothalamic neurons

To investigate the direct actions of neurotropin (NSP, a nonproteinaceous extract from inflamed skin of rabbits which is in therapeutic use), intracellular recordings were made from neurons of the ventromedial hypothalamic nucleus (VMH) and lateral hypothalamic area (LHA) in slices of guinea pig brain. In the VMH, NSP, applied by perfusion (0.1-3.0 NU/ml), caused dose-dependent depolarization in 29 of 48 neurons (60%) tested. No change in membrane resistance was observed during the depolarization, which hypothesized that the NSP-induced depolarization might be mediated through the inactivation of the Na-K pump. The NSP-induced depolarization persisted even after the elimination of synaptic activity by perfusion with Ca(2+)-free and high Mg2+ Ringer solution. NSP hyperpolarized the cell membrane of three neurons (6%) while two neurons (4%) showed biphasic responses; transient depolarization followed by long-lasting hyperpolarization. Membrane potential of the remaining 14 neurons was not changed by application of NSP. Of 14 LHA neurons tested for NSP effects, eight (57%) were depolarized, three (21%) were hyperpolarized, and one showed a biphasic response. The present results suggest that NSP significantly modulates hypothalamic neuron activity, and the central modulation of autonomic functions by NSP might be mediated through hypothalamic neurons.     

Exocytosis and mobility of histamine vesicles exhibit no difference to that of histamine-negative vesicles in guinea pig superior cervical ganglion neurons

Our previous studies have shown that histamine existed widely in the sympathetic nervous system and functioned differentially on the sympathetic nerve activation level. Therefore, in this study, we tried to find out whether it is the special exocytosis/recycling of histamine-containing vesicles that contribute to those differential histamine synaptic effects. By using N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide and histamine immunostaining methods, we confirmed that histamine was stored in small vesicles and found that the histamine-containing vesicles included the recycling pool and the reserve pool. However, we also found for the first time that the release and mobility kinetics of histamine-containing vesicles were identical to that of histamine-negative vesicles. In conclusion, these findings provide further characters of histamine as a sympathetic neurotransmitter.     

An electron microscopic study on substance P-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs

Substance P-immunoreactive nerve fibers in the celiac ganglion of guinea pigs were revealed with the PAP procedures to contain abundant small clear vesicles mixed with a few large granular vesicles. The immunoreactive materials were localized around cytoplasmic components including vesicles and on the inside of the plasma membrane. The immunoreactive fibers directly apposed to unlabelled dendrites of postganglionic neurons and also to preganglionic axons. Morphological features of synapses could be identified at sites of apposition to unlabelled dendrites: clusters of vesicles in the immunoreactive fibers, intercellular spaces of about 20 nm, and an intermediate density on the postjunctional membrane of unlabelled dendrites. On the other hand, no distinct electron density together with accumulations of vesicles was seen underneath the apposed membrane of unlabelled axons. These findings indicate at the ultrastructural level that substance P-fibers form axo-dendritic synapses on the postganglionic neurons and also suggest the presence of the presynaptic interaction between substance P-fibers and some preganglionic axons in this ganglion.     

Allografted fetal dorsal root ganglion neuronal survival in the guinea pig cochlea

Neural grafting is a potential strategy to help restore auditory function following loss of spiral ganglion cells. As a first step towards the reconstruction of a neural pathway from the cochlea to the brainstem, we have examined the survival of fetal dorsal root ganglion (DRG) neurons allografted into the cochlea of adult guinea pigs. In some animals implantation of DRGs was combined with a local infusion of neurotrophic substances whereas in others auditory sensory receptors were chemically destroyed prior to DRG implantation by injection of the ototoxin neomycin into the middle ear. The results show that many transplanted DRG neurons attached close to the cochlear spiral ganglion neurons. The survival of the implant was significantly increased by treatment with neurotrophic factors, but not reduced by the absence of auditory sensory structures. This study shows that implanted sensory neurons can survive heterotopic grafting immediately adjacent to the eighth cranial nerve, thereby providing a basis for further studies of the anatomical and functional influence of neural grafts in the inner ear.