Electrogenesis of the slow inhibitory postsynaptic potential in bullfrog sympathetic ganglia

The ionic mechanisms of the slow surface positive (P)-potential and the slow inhibitory postsynaptic potential (IPSP), an intracellularly recorded P-potential in sympathetic ganglia, were analysed by means of sucrose-gap, intracellular microelectrode techniques, and voltage clamp technique. Both the P-potential and the slow IPSP consist of two different potential components, namely the ouabain-sensitive and the ouabain-insensitive components. The ouabain-sensitive component was enhanced by a moderate conditioning hyperpolarization. This component was most reasonably explained as a potential change generated by an activation of the electrogenic Na+ pump. The ouabain-insensitive potential component of the P-potential and the slow IPSP decreased in the amplitude and finally reversed its polarity by conditioning hyperpolarization. The reversal potential of ouabain-insensitive component of slow IPSP and slow inhibitory postsynaptic current (IPSC) was close to the EK. The amplitude of ouabain-insensitive component of P-potential and slow IPSP was markedly decreased by an elevation of external K+ concentration. The reversal potential of ouabain-insensitive component shifted to a more positive potential level in high K+ Ringer's solution. On the other hand, it was augmented in K+-free Ringer's solution. A reduction of the membrane resistance was observed during the generation of the slow IPSP, when the membrane potential of ganglion cells was held at a membrane potential level more negative than -60 mV. The slow IPSC recorded by voltage-clamp method was associated with an increase in membrane conductance. It was concluded that the ouabain-insensitive component was generated by an activation of K+ conductance.     

Analysis of the slow excitatory postsynaptic potential in bullfrog sympathetic ganglion cells.

The ionic mechanism of the slow excitatory postsynaptic potential (slow EPSP), i.e. the muscarinic action of acetylcholine (ACh), was studied either by stimulating preganglionic nerves or by applying ACh in curarized sympathetic ganglion cells of bullfrogs. There are three different types of cells characterized by the effects of membrane hyperpoliarization on the amplitude of slow EPSP. One group of cells showed an increase in amplitude (type 1 cell) and, in two other groups of cells, it remained unchanged (type 2 cell) or decreased (type 3 cell), when the membrane was hyperpolarized. Under the muscarinic effects of ACh, the slope membrane conductance was increased (type 1 cell), unchanged (type 2 cell) or decreased (type 3 cell) at 10-20 mV hyperpolarized levels, while it was unchanged (type 1 cell) or decreased (types 2 and 3 cells) at resting and depolarized levels. In all cells, the slow ACh potential, corresponding to the slow EPSP, was almost completely suppressed in a high K+, Ca2+-free, Na+-free solution. These results suggest that the slow EPSP is generated by increases in Na+ and Ca2+ conductance and also by a simultaneous decrease in the K+ conductance.     

Phaclofen inhibition of the slow inhibitory postsynaptic potential in hippocampal slice cultures: a possible role for the GABAB-mediated inhibitory postsynaptic potential

The GABAA antagonist bicuculline methiodide and the GABAB antagonist phaclofen were used to examine the function of the fast inhibitory postsynaptic potential and slow inhibitory postsynaptic potential, in hippocampal slice cultures in the rat. These cultures form easily-visualized monolayers of nerve cells which maintain the structure and synaptic organization of transverse hippocampal slices. The present study shows that the cellular and synaptic physiological properties of slice cultures are very similar, but not identical, to those observed in acutely-prepared hippocampal slices. The major difference is a higher incidence of fast excitatory postsynaptic potentials and inhibitory postsynaptic potentials compared to slices, and the appearance of spontaneous slow inhibitory postsynaptic potentials. This increase in synaptic drive has been useful for our investigation of the role of GABA-mediated inhibitory postsynaptic potentials. Bath application of 10 microM bicuculline blocked the fast inhibitory postsynaptic potentials and gave rise to bursts 1-11 s in duration. The presence of the slow inhibitory postsynaptic potentials did not prevent bicuculline-induced burst activity. Phaclofen (1 mM) perfused in the bath reversibly blocked the slow inhibitory postsynaptic potential, but did not result in the formation of large paroxysmal depolarizing shift-like bursts as seen with bicuculline. Rather, block of the slow inhibitory postsynaptic potential resulted in the formation of repetitive "afterdischarge bursts". These afterdischarge potentials typically appeared with a delay of 2-15 min following block of the slow inhibitory postsynaptic potential, during which time there was a gradual increase in non-synchronized excitatory activity. Once established, this cycle of increasing excitatory activity culminating in afterdischarge potentials recurred at 2-4 min intervals while phaclofen was present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin attenuates a slow inhibitory postsynaptic potential in rat hippocampal neurons

Activity of hippocampal neurons was recorded in an in vitro slice preparation. Topical application of serotonin produced hyperpolarization, blockade of a slow afterhyperpolarization which follows a burst discharge and blockade of a slow inhibitory postsynaptic potential. The slow inhibitory postsynaptic potential evoked by stimulation of the apical dendritic region of the hippocampus is more sensitive to serotonin than the membrane potential or conductance. The effects of serotonin on the inhibitory postsynaptic potentials are blocked by the 5-HT1a antagonist spiperone, and not by mianserin, a 5-HT2 antagonist. The attenuation of the inhibitory postsynaptic potentials is not accompanied by a change in postsynaptic reactivity to GABA or baclofen. Serotonin blocks repetitive large inhibitory postsynaptic potentials evoked in hippocampal neurons by topical application of 4-aminopyridine. Putative interneurons are more sensitive to topical application of serotonin than pyramidal neurons. Fenfluramine, a serotonin releaser mimics the effects of topical application of serotonin indicating that synaptically released serotonin can produce the changes in membrane potential and reactivity to afferent stimulation. It is suggested that serotonin attenuates slow inhibitory postsynaptic potentials by inhibiting feed forward inhibitory interneurons which impinge upon the recorded pyramidal neurons.     

Adrenaline inhibits muscarinic transmission in bullfrog sympathetic ganglia

The effect of adrenaline (Ad) on muscarinic transmission was examined in B neurones of bullfrog sympathetic ganglia by using intracellular and voltage-clamp recording methods. Bath-application of Ad (5–500 M) caused a depression of the slow excitatory postsynaptic potential (EPSP) elicited by repetitive stimulations of preganglionic nerve fibres in the presence of curare (30 M). Ad also depressed the muscarinic ACh potential induced by ionophoretic application of ACh directly to curarized sympathetic neurones in a concentration-dependent manner. Isoprenaline mimicked the effect of Ad in producing the inhibition of the muscarinic ACh potential. Propranolol antagonized the inhibitory action of Ad. Dibutyryl adenosine 3,5-monophosphate had no significant effect on the muscarinic ACh potential. Under voltage-clamp conditions, Ad caused an inward current associated with inhibition of the M-current (Brown and Adams 1980). Ad depressed the amplitude of slow postsynaptic currents produced by applications of ACh and muscarine. At a concentration of 100 M, Ad produced a 68±8% (n=12) depression of the amplitude of the muscarinic ACh current. The inhibition of muscarinic transmission induced by Ad is due to a direct suppression of the muscarinic current at the postsynaptic membrane in bullfrog sympathetic ganglia.     

Mechanisms regulating the adrenaline-induced long-term potentiation in bullfrog sympathetic ganglia

Two regulatory mechanisms on the long-term potentiation of transmitter release induced by adrenaline (adr.-l.t.p.) in bullfrog sympathetic ganglia were studied by recording intracellularly the fast excitatory postsynaptic potentials. An increase in exposure time to adrenaline from 10 min to 60 min did not enhance the magnitude of adr.-l.t.p. However, increasing an exposure time to dibutyryl cyclic AMP (1 mM) up to 60 min progressively enhanced the magnitude of the nucleotide-induced potentiation, indicating the desensitization of the -adrenoceptor. The desensitization remained at 20 min after the removal of adrenaline in all the five cells, but disappeared at 60–90 min in four cells out of eight. Under the latter condition, the second l.tp. was summated on the first one. Dibutyryl cyclic GMP (100 M) blocked the generation of the l.t.p. induced by dibutyryl cyclic AMP (1 mM) as well as that of adr.-l.t.p. Muscarine (10 M) or adenosine (1 mM), a possible candidate for raising intraterminal cyclic GMP, did not significantly affect adr.-l.t.p. These results suggest that adr.-l.t.p. is regulated by the desensitization of -adrenoceptor and a process which involves endogenous cyclic GMP acting on a step subsequent to the cyclic AMP production.     

Peptidergic inhibition of cholinergic transmission in bullfrog sympathetic ganglia

Intracellular and voltage-clamp recordings were made from sympathetic B neurons to investigate an interaction between peptidergic and cholinergic responses in bullfrog sympathetic ganglia. Stimulations of both 3rd-5th (0.2 Hz) and 8th (30 Hz) spinal nerves evoked the fast excitatory postsynaptic potential (EPSP) superimposed with the late slow EPSP at the same sympathetic neuron. The amplitude of fast EPSPs was decreased during the course of the late slow EPSP in a majority of sympathetic neurons. The mean depression of the fast EPSP amplitude was 51 +/- 4% (n = 24). The quantal content of the fast EPSP was also depressed by 54 +/- 3% (n = 10) during the late slow EPSP. Acetylcholine-induced depolarization (ACh potential) and current (ACh current) produced by an ionophoretic application of ACh were not reduced during the late slow EPSP. Bath-application of LH-RH (40 nM-4 microM) depressed the fast EPSP in a concentration-dependent manner; at a concentration of 1 microM, it produced a 63 +/- 8% (n = 8) depression of the quantal content of the fast EPSP. LH-RH (1-4 microM) depressed the frequency of the miniature (M) EPSPs by 25 +/- 4% (n = 5) of control. Antagonists for luteinizing hormone-releasing hormone (LH-RH) receptor, [D-Phe2,6, Pro3]-LH-RH and [D-pGlu1, D-Phe2, D-Trp3,6]-LH-RH, prevented the presynaptic inhibition of the fast EPSP induced by LH-RH. These results suggest that the fast EPSP is depressed during the late slow EPSP by decreasing the evoked release of ACh from presynaptic nerve terminals in bullfrog sympathetic ganglia.     

"Cholinergic" postsynaptic membranes of bullfrog sympathetic ganglia: electron microscopy of thin sections and freeze-fracture replicas

"Cholinergic" synapses of the bullfrog sympathetic ganglion cells were investigated with thin sectioning, complementary freeze-fracturing, and deep-etching methods after glutaraldehyde fixation. The protoplasmic (-fracture) face (PF) of the postsynaptic membrane was characterized by intramembranous particles (IMPs), 3,500/micron 2 in density, consisting of larger particles, 10-12 nm in diameter, and smaller ones, 8-9 nm; the complementary exoplasmic (-fracture) face (EF) contained larger and smaller IMPs, 750/micron 2 in density, and numbers of pits. By close inspection of the sections and freeze-fracture replicas at high magnification and with deep-etching in particular, it was concluded that aggregated IMPs might represent transmembranous components and that the particulate entities existing in the postsynaptic active zones might be larger in number than those exposed to view and counted here in the "cholinergic" synapses. An individual IMP often appeared to consist of five or six subunits arranged in a rosette with a central pit. These findings suggest that the aggregated IMPs, particularly the larger ones, may be closely related to the structure of the nicotinic ACh receptor-ion channel complex.     

Characteristics of fast excitatory postsynaptic current in bullfrog sympathetic ganglion cells

The membrane current underlying the fast excitatory postsynaptic potential (EPSC) of bullfrog sympathetic ganglion cells was studied. The relationship between the EPSC amplitude and membrane potential was linear at negative levels of membrane potential, but deviated from the linearity toward a smaller amplitude at positive levels. The falling phase of EPSC almost followed a single exponential decay. The half-decay time (HDT) of EPSC's increased exponentially with an increase in the negativity of membrane potential. The rise time (RT) was also prolonged slightly with membrane hyperpolarization. Lowering of temperature decreased the EPSC amplitude, lengthened markedly the HDT and increased the slope relating the logarithm of the HDT to membrane potential. Neostigmine (1×10^–5 M) prolonged both the RT and HDT. A decrease in Ca²⁺ concentration caused a marked reduction in the EPSC amplitude, and a slight shortening in the RT and HDT. An increase in Ca²⁺ concentration significantly prolonged the RT and HDT without altering the slope of the relationship between the HDT and membrane potential, while the amplitude of EPSC was increased slightly. The HDT was independent of EPSC amplitude. It is suggested that the mechanism responsible for closing the ion channels of the nicotinic receptor at the subsynaptic membrane is regulated by membrane potential. The possible mechanisms of the action of Ca²⁺ on the decay phase of EPSC were discussed.     

A muscarine-resistant M-current in C cells of bullfrog sympathetic ganglia

C cells of bullfrog sympathetic ganglia have an M-current similar to that of the larger B cells. However, the M-current of C cells is much less sensitive to muscarinic agonists. The M-current of C cells is blocked by barium and by LHRH (mammalian luteinizing hormone-releasing hormone). LHRH sometimes also decreases a different conductance, with an extrapolated reversal potential near 0 mV. These results demonstrate that the coupling of receptors to ion channels can vary between cell types, even within a single ganglion.     

Luteinizing hormone-releasing hormone inhibits nicotinic transmission in bullfrog sympathetic ganglia

Intracellular and voltage-clamp recordings were made from neurons in bullfrog sympathetic ganglia to investigate the effects of luteinizing hormone-releasing hormone (LH-RH) on nicotinic transmission. LH-RH (50 nM-4 microM) decreased the amplitude of the fast excitatory postsynaptic potential (fast EPSP) in a dose-dependent manner. LH-RH (1-4 microM) reduced the quantal content of the fast EPSP by 60-85%. LH-RH did not change the frequency of the miniature (m) EPSP, but it slightly depressed the mEPSP amplitude. LH-RH (1-4 microM) caused a 22-32% decrease in the amplitude of the acetylcholine-induced synaptic responses due to the iontophoretic application of acetylcholine (ACh) to neurons in the presence of atropine (1 microM). These results suggested that LH-RH decreased nicotinic transmission in the bullfrog sympathetic ganglion, primarily by reducing the release of ACh from the preganglionic nerve terminals.     

Presynaptic inhibition of cholinergic transmission by peptidergic neurons in bullfrog sympathetic ganglia

Intracellular recordings were made from sympathetic B neurons to investigate an interaction between peptidergic and cholinergic responses in bullfrog sympathetic ganglia. Simultaneous stimulations of 3rd-5th and 8th spinal nerves evoked the fast excitatory postsynaptic potential (EPSP) superimposed with the late slow EPSP at the same sympathetic neuron. The amplitude of fast EPSPs was reduced during the course of the late slow EPSP in a majority of sympathetic neurons. A nicotinic depolarization produced by an ionophoretic application of ACh (ACH potential) was not significantly affected during the late slow EPSP. The quantal content of the fast EPSP calculated by the variance method was depressed during the late slow EPSP. Luteinizing hormone-releasing hormone (LH-RH), a putative transmitter for the late slow EPSP decreased the amplitude and the quantal content of the fast EPSP. [D-Phe^2,6, Pro³]-LH-RH, and [D-_DGlu¹, D-Phe², D-Trp^3,6]-LH-RH, antagonists for LH-RH receptors prevented the inhibition of the fast EPSP induced by the late slow EPSP and LH-RH. These results suggest that cholinergic nicotinic transmission is inhibited during the late slow EPSP by a decreased ACh-release from nerve terminals in bullfrog sympathetic ganglia.     

The slow inhibitory postsynaptic potential in rat hippocampal CA1 neurones is blocked by intracellular injection of QX-314

Intracellular recordings were made from CA1 pyramidal neurones in the rat hippocampus slice preparation. The recording electrodes contained potassium acetate (4 M) with or without the quaternary lidocaine derivative, QX-314 (50 mM). Both fast (f) and slow (s) inhibitory postsynaptic potentials (IPSP) were evoked by low-frequency orthodromic stimulation. The s-IPSP was rapidly reduced by QX-314 injection. It decreased along a similar time course to the dV/dt of the action potential (AP). The f-IPSP and excitatory postsynaptic potential were not significantly reduced in size at a time when the s-IPSP was virtually abolished by QX-314. It is concluded that conductance through the K⁺ channels which are coupled to GABA_B receptors is readily blocked by QX-314, while the Cl⁻ channels which are coupled to GABA_A receptors and the cation channels coupled to the glutamate receptors are relatively resistant to the local anaesthetic.     

Voltage-clamp studies of a slow inward current in bullfrog sympathetic ganglion cells

Voltage-dependent inward membrane currents of bullfrog sympathetic ganglion cells were analyzed with the voltage clamp method. Two distinct inward currents, a tetrodotoxin (TTX)-sensitive fast inward current (INa) and a TTX-insensitive slow inward current (Isi), were recorded in Ringer solution. The Isi was markedly depressed by removal of external Ca2+ and by the addition of Mn2+, Co2+ or D-600. This suggests that the Isi is, almost exclusively, due to Ca2+. These results indicate that under physiological conditions a Ca2+ influx is induced during initiation of action potentials in these ganglion cells.     

Pathologic alterations in pre- and postsynaptic elements in aged mouse sympathetic ganglia

Summary Dysfunction of the sympathetic autonomic nervous system is an increasingly recognized, although poorly understood, complication of increasing age in experimental animals and man. In this study of young adult (4–6 months old) and aged (12– 24 months old) mice we have examined the ultrastructural appearance of perikarya, dendritic processes, preterminal axons, and synapses in selected sympathetic ganglia as well as the three-dimensional structure of the dendritic arborizations of principal sympathetic neurons using intracellular injections of Lucifer Yellow. Ultrastructural examination demonstrated numerous markedly enlarged presynaptic terminal axons and synapses which distorted the contours of perikarya and dendrites of neurons within the prevertebral celiac/superior mesenteric and paravertebral superior cervical and stellate sympathetic ganglia of aged mice. Dilated preterminal axons had the distinctive ultrastructural appearance of neuroaxonal dystrophy, a pathologic process described in a wide variety of clinical and experimental entities. Dystrophic axons were identical in ultrastructural appearance in young and old animals, differing only in frequency. A distinctive type of ultrastructural alteration, characterized by markedly distended neurites containing numerous vacuoles, was confined to the superior cervical ganglia and also increased in frequency with aging. Although many intraganglionic vacuolated processes disappeared with surgical interruption of the cervical sympathetic trunk, which contains the preganglionic axons innervating the superior cervical ganglia, others persisted. In addition, the presence in some processes of admixed ribosomes, lipofuscin, or continuity with the cell body indicated that numerous neuritic alterations within aged sympathetic ganglia were likely of dendritic origin. Intracellular injections of Lucifer Yellow into principal sympathetic neurons demonstrated that the dendritic arborizations of the celiac/superior mesenteric ganglia neurons of young adult mice were significantly more complex and extensive than those of the superior cervical ganglia. Sympathetic neurons of aged superior cervical ganglia, but not superior mesenteric ganglia, appeared significantly smaller with regard to total dendritic length, extent, and branching when compared to those of young animals. In the aged superior cervical ganglia, short, stunted dendritic processes also exhibited large, focal, often multiple, swellings, a phenomenon infrequently observed in the superior cervical ganglia of young animals. The celiac/ superior mesenteric ganglia of aged or young adult mouse failed to exhibit comparable dendritic swellings. These observations may provide a neuropathological basis for understanding age-related changes in autonomic function observed in animals and man, and provide a model system in which age-related pathogenetic mechanisms resulting in neuroaxonal dystrophy and dendritic alterations can be studied.     

“Cholinergic” postsynaptic membranes of bullfrog sympathetic ganglia: Electron microscopy of thin sections and freeze-fracture replicas

“Cholinergic” synapses of the bullfrog sympathetic ganglion cells were investigated with thin sectioning, complementary freeze-fracturing, and deepetching methods after glutaraldehyde fixation. The protoplasmic (-fracture) face (PF) of the postsynaptic membrane was characterized by intramembranous particles (IMPs), 3,500/μm ² in density, consisting of larger particles, 10–12 nm in diameter, and smaller ones, 8–9 nm; the complementary exoplasmic (-fracture) face (EF) contained larger and smaller IMPs, 750/μm ² in density, and numbers of pits. By close inspection of the sections and freeze-fracture replicas at high magnification and with deep-etching in particular, it was concluded that aggregated IMPs might represent transmembranous components and that the particulate entities existing in the postsynaptic active zones might be larger in number than those exposed to view and counted here in the “cholinergic” synapses. An individual IMP often appeared to consist of five or six subunits arranged in a rosette with a central pit. These findings suggest that the aggregated IMPs, particularly the larger ones, may be closely related to the structure of the nicotinic ACh receptor-ion channel complex.