Increase In Membrane Input Resistance Research Articles

Myelination defects and neuronal hyperexcitability in the neocortex of connexin 32-deficient mice.

Morphological and electrophysiological studies were performed on neocortices of adult Connexin 32 (Cx32)-deficient mice and wild-type mice to investigate the consequences of a lack of the gap junction subunit Cx32 on neocortical structure and function. Morphometrical analysis revealed a reduced volume fraction of myelin within the neuropil and a decreased thickness of the axonal myelin sheaths in the neocortex of Cx32-deficient mice. Intracellular recordings from neurons in neocortical slice preparations provided evidence for an increased membrane input resistance in neurons of Cx32-null mutant mice as compared to neurons of wild-type mice. Consequently, neurons of Cx32-deficient mice displayed an enhanced intrinsic excitability. In addition, approximately 50% of the neurons investigated in slices of Cx32-deficient mice responded to afferent stimulation with delayed and large glutamatergic excitatory postsynaptic potentials resembling paroxysmal depolarizations. GABAergic inhibition sufficient to efficiently control synaptic excitability was virtually absent in these cells. The changes in intrinsic membrane properties observed in neurons of Cx32-null mutant mice were independent of the alterations in synaptic function, since increased membrane resistances were observed also in neurons with normal synaptic response pattern. Thus, in the neocortex, lack of Cx32 correlates with myelination defects, alterations in intrinsic membrane properties and dysfunction of inhibitory synaptic transmission.

Angiotensin II-induced depolarizations in hamster submandibular ganglion neurons.

Angiotensin II acts as an excitatory neurotransmitter of the sympathetic ganglia. In the parasympathetic neurons, however, angiotensin II (angio II)-induced responses have not been recorded. In this study, we investigated the effects of angio II in the hamster submandibular ganglion (SMG) neurons using a intracellular recording technique. Approximately 70% of these ganglion cells responded with persistent depolarization (3-6 mV, 5-9 min) at a concentration of 10 microM. The angio II-induced depolarizations were caused by the combination of an increase in Na+ conductance and a decrease in K+ conductance. An involvement of K+ channels such as M channels and SK channels in the electrogenesis mechanism was suggested by an inhibitory effect of Ba2+ ion on the component of the increased membrane input resistance during the angio II-induced depolarization. In addition, the angio II-induced depolarizations were mediated through angio II type I (AT1) receptors. Thus angio II is probably a neurotransmitter that increases their excitability in most of the neurons in parasympathetic ganglia.

Functional alterations in jejunal myenteric neurons during inflammation in nematode-infected guinea pigs.

Intracellular recordings of jejunal myenteric neurons with an afterspike hyperpolarization (AH) from Trichinella spiralis-infected animals showed enhanced excitability on days 3, 6, and 10 postinfection (PI) compared with uninfected animals. Lower membrane potential, increased membrane input resistance, decreased threshold for action potential discharge, decreased AH amplitude and duration, and increased fast excitatory postsynaptic potential amplitude and duration were characteristic of neuronal recordings from infected animals. Concurrent with electrophysiological changes during T. spiralis infection, increased cytochrome oxidase activity, a marker of neuronal metabolic activity, and the expression of nuclear c-Fos immunoreactivity, an indicator of transcriptional-translational activity, were also observed in myenteric ganglion cells. Double-labeling for calbindin-immunoreactive myenteric neurons revealed that approximately 50% of these neurons also expressed increased c-Fos immunoreactivity during T. spiralis infection. Myeloperoxidase activity was significantly higher in the jejunum of T. spiralis-infected guinea pigs on days 3, 6, and 10 PI vs. uninfected counterparts. The expression of c-Fos in calbindin-immunoreactive neurons together with enhanced neuronal electrical and metabolic activity during nematode-induced intestinal inflammation suggests the onset of excitation-transcription coupled changes in enteric neural microcircuits.

Characterization of carbachol-induced rhythmic bursting discharges in neurons from guinea pig lateral septum slices.

A brain slice preparation from guinea pigs and intracellular recording techniques were used to examine the effects of carbachol application on the three classes (A, B, and C) of neurons (n = 68, 40 of class A, 12 of class B, 16 of class C) within the mediolateral part of the lateral septum (LSml). Bath application of carbachol elicited a sustained depolarization associated with an increase in membrane input resistance, action-potential firing and triggered rhythmic bursting discharges in 59% of recorded neurons. According to the configuration of these bursts, LSml neurons were classified into type I, II, and III neurons with reference to their response to carbachol. The frequency of spontaneous bursts was increased by depolarization caused by applied DC current in the three types of neurons. Bursts in type II and III neurons were voltage and dose dependent. These dependences were responsible for a continuum of variation in carbachol responses in these two types of neurons. As the neuron depolarized in the presence of carbachol, spontaneous action potentials increased in frequency and slow afterdepolarizing potentials (sADPs) appeared and preceded the occurrence of the first burst. These sADPs from adjacent action potentials appeared to progressively increase to initiate a burst. In the presence of carbachol, sADPs and bursts were also observable after action potentials evoked by depolarizing current pulses at the resting membrane potential (RMP) in LSml neurons. Evoked sADPs and bursts were associated with an apparent increase in input conductance. Application of low Na+ medium blocked both the sADP and bursts. Application of zero Ca2+ medium either 1) blocked completely the generation of sADPs and bursts (n = 16), or 2) did not block bursts (n = 14). Evoked sADPs and bursts were blocked by tetraethylammonium but were resistant to external Cs+. The results indicate that the activation of cholinergic receptors does not differentially affect the three classes of LSml neurons. The responses to carbachol in type II and III neurons form a continuum of variation, whereas these of type I neurons constitute a discrete entity. The selective cholinergic induction of a sADP, and the progressive activation of these sADPs in LSml neurons are thought to be responsible for the onset of the three types of rhythmic bursting discharges. We propose that sADPs and bursts induced by carbachol are generated by a cationic conductance largely permeable to Na+. In a subpopulation of LSml neurons (n = 16), the bursts are dependent on the presence of Ca2+ in the medium.

GABAC receptors mediate slow membrane potentials in neurons of the rat major pelvic ganglia.

Effects of gamma-aminobutyric acid (GABA) on the neuronal membrane of the rat major pelvic ganglia (MPG) were studied using intracellular recording techniques, in vitro. Application of GABA (100 microM) to MPG neurons induced a depolarization (GABAd) associated with a decreased membrane input resistance and a slow hyperpolarization (s-GABAh) associated with an increased membrane input resistance. The GABA depolarization had two phases, a fast depolarization (f-GABAd) and a subsequent slow depolarization (s-GABAd). Bicuculline (60 microM) blocked the f-GABAd but not the s-GABAd and s-GABAh. Picrotoxin (100 microM) blocked all the GABA responses. Imidazole-4-acetic acid (I4AA, 100 microM), a GABAc receptor antagonist, depressed the s-GABAd and s-GABAh, but did not block the f-GABAd. Cis-4-aminocrotonic acid (CACA), a GABAc receptor agonist, produced a depolarization followed by a hyperpolarization in MPG neurons. I4AA (100 microM) depressed the CACA-induced responses. It was concluded that GABAA receptors mediate the f-GABAd and that GABAc receptors mediate the s-GABAd and s-GABAh, in neurons of the rat MPG.

Myenteric neurons of the rat descending colon: electrophysiological and correlated morphological properties.

Conventional intracellular electrophysiological recordings were made from 502 myenteric neurons of the rat descending colon. Myenteric neurons could be classified into three groups on the basis of distinct electrophysiological properties. The first group of neurons (51% of all neurons) fired tetrodotoxin-sensitive action potentials in response to direct somal depolarization and the majority (98%) of this group generated fast cholinergic excitatory synaptic potentials in response to focal stimulation and were therefore designated S/Type 1 neurons. The second group (40%) of neurons fired tetrodotoxin-insensitive action potentials which were followed by long-lasting membrane afterhyperpolarizations, hence were termed AH neurons. These neurons did not receive fast cholinergic synaptic inputs but ionophoretic application of acetylcholine induced rapid nicotinic cholinoceptor-mediated depolarizations. The final group of neurons (9%), named Type 3 neurons, received fast cholinergic synaptic inputs but could never be made to fire action potentials. Rundown in amplitude of successive fast excitatory synaptic potentials evoked by a short train of presynaptic nerve stimuli was observed in only a small proportion of neurons (8/37; 22%) with the majority of neurons (29/37; 78%) showing no such decrease in amplitude, even at frequencies of stimulation as high as 10 Hz. Superfusion of 5-hydroxytryptamine could induce both an inhibition and a facilitation of cholinergic fast synaptic transmission. Evidence was adduced that these presynaptic inhibitory and facilitatory actions appeared to be mediated via 5-hydroxytryptamine 1A and 5-hydroxytryptamine 4 receptors, respectively. Muscarinic slow excitatory synaptic potentials were not detected (9/9 neurons tested) and non-cholinergic slow excitatory synaptic potentials following repetitive focal presynaptic nerve stimulation were observed in only 39/502 (8%) of all neurons. In those neurons in which a demonstrable change in membrane input resistance was detectable, slow excitatory potentials were accompanied by an increased input resistance. In addition, in a small subset (4%) of S/Type 1 neurons, slow membrane hyperpolarizations accompanied by an increased membrane input resistance were observed following tetanic presynaptic nerve stimulation. Superfusion of 5-hydroxytryptamine induced both membrane depolarizations and hyperpolarizations. Membrane depolarizations were observed in 40% of all neuronal types (34% of S/Type 1 neurons, 58% of AH neurons and 11% of Type 3 neurons) and were accompanied by an increased membrane input resistance and occasionally, in S/Type 1 and AH neurons, by anodal break excitation or spontaneous action potential firing. Membrane hyperpolarizations were observed in S/Type 1 neurons (5%) only and were accompanied, unexpectedly, by an increased membrane input resistance. In those neurons that responded both to application of 5-hydroxytryptamine and tetanic presynaptic nerve stimulation, 5-hydroxytryptamine always mimicked the slow synaptic response indicating that 5-hydroxytryptamine may function as a slow synaptic mediator in some myenteric neurons. Myenteric neurons identified by intracellular injection of the neuronal marker Neurobiotin TM were found to conform to the morphological classification schemes proposed for myenteric neurons of the guinea-pig and porcine intestine, that is, Dogiel Types I and II and Stach Type IV neurons were present. Simultaneous electrophysiological recording and intracellular staining techniques revealed that a correlation existed between the electrophysiological and morphological properties of myenteric neurons of the rat colon, with electrophysiological classified S/Type 1 neurons having Dogiel Type I morphologies (95/108 neurons; 88%) and electrophysiological classified AH neurons having Dogiel Type II morphologies (87/94 neurons; 93%)...

Facilitating effect of CCK on nicotinic neurotransmission in cat pancreatic ganglion.

Previous studies have demonstrated the presence of cholecystokinin (CCK)-like peptides in nerve terminals surrounding ganglion neurons of the cat pancreas. The present study was undertaken to determine the effect of cholecystokinin octapeptide (CCK-8) on ganglionic transmission. Recordings were made intracellularly in vitro from ganglion neurons in isolated pieces of the pancreas. Sulfated CCK-8 (S-CCK-8) and nonsulfated CCK-8 initiated or increased ongoing fast excitatory postsynaptic potential (fEPSP) activity, an effect antagonized by hexamethonium. Superfusion of S-CCK-8 in concentrations ranging from 10(-11) to 10(-8) M significantly augmented the amplitude of nerve-evoked subthreshold fEPSPs without a significant change in either membrane potential or membrane input resistance. S-CCK-8 (10(-8)M) also increased the quantal content and quantal size of nerve-evoked fEPSPs and increased the response to exogenously applied acetylcholine (ACh). Concentrations of S-CCK-8 higher than 10(-8)M caused depolarization and an increase in membrane input resistance, an effect unaltered by a low-Ca+, high-Mg2+ solution. It was concluded that S-CCK-8 potentiated nicotinic transmission by facilitating release of ACh from preganglionic nerve terminals and by increasing the postsynaptic membrane sensitivity to ACh.

Effects of tachykinins on identified dorsal vagal neurons: an electrophysiological study in vitro.

Intracellular current-clamp recordings were performed using in vitro brainstem slice preparations to compare the actions of substance P, neurokinin A, neurokinin B and their agonists on rat dorsal vagal nucleus neurons with or without antagonists of neurokinin 1 and 2 receptors. The agonists used were either [Sar9,Met(O2)11]substance P or septide for neurokinin 1 and [Nle10]neurokinin A(4-10) for neurokinin 2 receptors. The antagonists were spantide, SR 140333 or RP 67580 for neurokinin 1 receptors and SR 48968 for neurokinin 2 receptors. Identification of vagal neurons was achieved electrophysiologically by testing antidromic responses and confirmed morphologically by an intracellular injection of biocytin. Of the 70 neurons tested, substance P led to depolarization in 36, hyperpolarization in six and no effect in 28. Depolarization was concentration dependent and generally associated with an increase of the membrane input resistance. Addition of tetrodotoxin (1 microM) to the medium had no effect on depolarization. RP 67580 (1 microM) blocked depolarization, but spantide and SR 140333 (microM to 50 microM) did not. Hyperpolarization was never observed using agonists. Neurokinin A and neurokinin 2 agonist induced concentration-dependent depolarization associated with an increase in membrane input resistance in eight of 14 neurons and in four of nine neurons, respectively. Depolarization was only partially abolished by the neurokinin 2 antagonist SR 48968. Neurokinin B had no effect in any of the eight neurons tested. These data prove that vagal neurons have neurokinin 1 and 2 receptors and that tachykinin could produce either depolarization or hyperpolarization. Since membrane potential variations were associated with an increase (during depolarization) or decrease (during hyperpolarization) in the membrane input resistance and since the reversal potential was close to the potassium equilibrium potential, we speculate that these effects are mediated by modulation of potassium conductance.

Cholinergic nucleus basalis neurons are excited by histamine in vitro

Considerable evidence has shown that both cholinergic and histaminergic neurons in the brain may act to facilitate processes of cortical activation that occur during wakefulness. In the present study, the potential influence of histaminergic neurons upon cholinergic neurons of the basal forebrain was investigated in guinea-pig basal forebrain slices. We found that electrophysiologically identified and immunohistochemically verified cholinergic neurons of the nucleus basalis were depolarized and excited by histamine, as manifested by an increase in tonic firing. The depolarization was associated with an increase in membrane input resistance. The effect of histamine persisted in the presence of either tetrodotoxin or a high-magnesium/low-calcium solution, indicating that it is postsynaptic. By a process of elimination, the participation in this response of the three described histamine receptors was examined. Involvement of H3 receptors was excluded on the basis that the H3 agonist (R)-alpha-methyl-histamine had no direct effect, and the H3 antagonist, thioperamide, did not block the effect of histamine. In contrast, the presence of a small response to impromidine, a selective agonist of H2 receptors, and the partial block of the response to histamine by the H2 receptor antagonist, cimetidine, indicated the participation of H2 receptors. Finally, the complete elimination of histamine's effect occurred when low doses of the H1 antagonist, mepyramine, were added to the H2 antagonist, cimetidine, indicating the involvement and predominance of H1 receptors in the response. Our data thus suggest that histamine excites nucleus basalis cholinergic neurons by a concomitant activation of H1 and H2 receptors. Histaminergic tuberomammillary neurons may accordingly facilitate tonic firing of cholinergic neurons during wakefulness. Cholinergic basalis neurons could thus act in tandem with histaminergic neurons during periods of arousal to collectively promote widespread cortical activation.

Noradrenergic Modulation of Cholinergic Nucleus Basalis Neurons Demonstrated by in vitro Pharmacological and Immunohistochemical Evidence in the Guinea‐pig Brain

The effects of noradrenalin were tested upon electrophysiologically characterized cholinergic nucleus basalis neurons in guinea-pig brain slices. According to their previously established intrinsic membrane properties, the cholinergic cells were distinguished by the presence of low-threshold Ca2+ spikes and transient outward rectification that endowed them with the capacity to fire in low-threshold bursts in addition to a slow tonic discharge. A subset of the electrophysiologically identified cholinergic cells that responded to noradrenalin had been filled with biocytin (or biotinamide) and documented in previously published reports as choline acetyltransferase (ChAT)-immunoreactive. The noradrenalin-responsive, biocytin-filled/ChAT+cells were mapped in the present study and shown to be distributed within the substantia innominata amongst a large population of ChAT+ cells. Slices from another subset of noradrenalin-responsive, electrophysiologically identified cholinergic cells were stained for dopamine-beta-hydroxylase to visualize the innervation of the biocytin-filled neurons by noradrenergic fibres. These biocytin-filled neurons were surrounded by a moderate plexus of varicose noradrenergic fibres and were ostensibly contacted by a small to moderate number of noradrenergic boutons abutting their soma and dendrites. Applied in the bath, noradrenalin produced membrane depolarization and a prolonged tonic spike discharge. This excitatory action was associated with an increase in membrane input resistance, suggesting that it occurred through reduction of a K+ conductance. These effects persisted when synaptic transmission was eliminated (by tetrodotoxin or low Ca2+/high Mg2+) and were therefore clearly postsynaptic. The excitatory effect of noradrenalin was blocked by the alpha 1-adrenergic receptor antagonist prazosin and not by the alpha 2-antagonist yohimbine, and it was mimicked by the alpha 1-agonist L-phenylephrine but not by the alpha 2-agonists clonidine and UK14.304, indicating mediation by an alpha 1-adrenergic receptor. There was also evidence for a contribution by a beta-adrenergic receptor to the effect, since the beta-antagonist propranolol partially attenuated the effect of noradrenalin, and the beta-agonist isoproterenol produced, like noradrenalin, alone or when applied in the presence of the alpha 1-antagonist prazosin, membrane depolarization and an increase in tonic spike discharge. These results indicate that through a predominant action upon alpha 1-adrenergic receptors, but with the additional participation of beta-adrenergic receptors, noradrenalin depolarizes and excites cholinergic neurons. This action would tend to drive the cholinergic cells into a tonic mode of firing and to stimulate or increase the rate of repetitive spike discharge for prolonged periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbachol-induced synchronized rhythmic bursts in CA3 neurons of guinea pig hippocampus in vitro

1. Carbachol effects on CA3 hippocampal cells were studied in the absence of ionotropic glutamatergic and GABAergic transmission with intracellular and extracellular recordings from guinea pig septohippocampal slices. 2. In all experiments the perfusing solution contained ionotropic glutamate and gamma-aminobutyric acid (GABA) receptor blockers [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-20 microM), 3-((+/-)-2-carboxypiperazin-4-il)propyl-1-phosphonic acid (CPP, 10-20 microM), and picrotoxin (50 microM), respectively]. Under these conditions, the excitatory and early inhibitory postsynaptic potentials, evoked in CA3 pyramidal cells by mossy fiber stimulation before the addition of the blockers, were completely suppressed. 3. Carbachol (50 microM) introduced via bath perfusion or pulse application elicited a series of rhythmic bursts with overriding action potentials. Each rhythmic burst lasted up to 30 s and repeated at intervals of 0.7-6 min. Rhythmic bursts were blocked by atropine (1 microM). 4. At membrane potentials more depolarized than -70 mV, carbachol also elicited a sustained depolarization associated with an increase in membrane input resistance and action-potential firing. This response was blocked by atropine (1 microM). 5. Carbachol can induce both rhythmic bursts and sustained depolarizations in the same cell. Rhythmic bursts were elicited when the membrane potential of the cell was more hyperpolarized than -70 mV; sustained depolarizing responses were activated by carbachol when the cell membrane potential was more depolarized than -70 mV. 6. Extracellular field potential responses in the CA3 region occurred simultaneously with rhythmic bursts, indicating the synchronization of the event in the CA3 field. Dual intracellular recordings confirmed that rhythmic bursts occurred simultaneously in CA3 hippocampal pyramidal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Hippocampal CA1 neuronal properties in genetically epilepsyprone rats: Evidence for increased excitation

The genetically epilepsy prone rats (GEPRs) are abnormally susceptible to seizures with a variety of treatments and can be used as a model to study generalized seizure predisposition involving the brainstem and forebrain structures. We investigated the basic membrane and synaptic properties of hippocampal CA1 cells in Sprague-Dawley (SD) rats and GEPRs. Several differences in cellular properties were observed in the GEPRs. These include an increase in membrane input resistance and reduced spike frequency adaptation in the majority of GEPR cells. A decrease in the amount of current required to elicit a 5-mV EPSP was observed in the GEPR. A marked increase in excitability with paired pulse stimulation was also observed in GEPRs both in extracellular population spikes and intracellular EPSPs. Applying bicuculline, a GABAA antagonist, markedly increased paired pulse facilitation of the population spike in SD rats but in GEPRs produced only a minimal effect on facilitation. This difference suggests reduced GABA A-mediated inhibition in GEPR hippocampus with paired pulse stimulation. Several factors could interact or act independently to produce these effects because the epileptic phenotype in GEPRs is regulated by multiple genes.

Bidirectional synaptic transmission in Necturus taste buds

Pairs of taste cells were impaled with intracellular recording microelectrodes in intact taste buds in slices of Necturus lingual epithelium. Applying short pulses of 140 mM KCl or 200 mM CaCl2 solutions to the apical pore elicited receptor potentials in taste receptor cells. Chemostimulation of receptor cells elicited postsynaptic responses in basal cells in the taste bud. Postsynaptic responses in basal cells had a threshold for activation and did not saturate with increasing doses of chemical stimulus applied to the receptor cells. We directly depolarized individual receptor cells and tested whether this would evoke postsynaptic responses in basal cells. Depolarizing receptor cells to approximately 0 mV evoked small depolarizing responses in basal cells in 16% of the experiments. The properties of these responses were consistent with their being mediated by a chemical synapse. A comparison of the responses in basal cells evoked by depolarizing single receptor cells, with responses evoked by stimulating the entire receptor cell population with KCl suggests that there is extensive synaptic convergence from receptor cells onto each basal cell. We also tested whether electrical excitation of basal cells would elicit (retrograde) synaptic responses in receptor cells. Single depolarizing pulses (up to 1 sec duration) applied to basal cells through the intracellular recording microelectrode never evoked synaptic responses in receptor cells. However, when repetitive electrical stimuli were applied to basal cells (four to six 1 sec depolarizations to approximately 0 mV every 12 sec) we observed prolonged effects on receptor cells in 11 of 23 experiments. These effects included an increase in the amplitude of receptor potentials elicited by KCI (mean +/- SD = +19 +/- 5%), an increase in membrane input resistance of receptor cells (+27 +/- 11%), and a hyperpolarization of receptor cells (3-10 mV). In control experiments, repetitive stimulation of one receptor cell never elicited such effects in another receptor cell. We investigated the possibility that serotonin (5-HT), released from basal cells, mediated the above modulatory effects on receptor cells. Bath-applied 5-HT (100 microM) mimicked the effects produced by repetitive basal cell stimulation (KCI responses increased by 23 +/- 12%; input resistance increased by 24 +/- 11%; hyperpolarization of 5-15 mV; N = 14). We conclude that basal cells release 5-HT onto adjacent taste receptor cells and that this enhances the electrotonic propagation of receptor potentials from the apical (chemosensitive) tip to the basal (synaptic) processes of receptor cells. The net effect is that activation of basal cells effectively increases the chemosensitivity of taste receptor cells.

Dissection of a model for membrane potential oscillations in bursting neuron of snail, Helix pomatia

A slow-wave model for bursting activity based on the hyperpolarization-activated outward current has been analysed by simulation of the well-known experimental data. It is shown that the slow-wave model demonstrates an increase of membrane input resistance during the development of the depolarizing phase; responds adequately to stationary de- or hyperpolarizing current; demonstrates a phenomenon of induced hyperpolarization and contingency. Change of definite model parameters selectively affects burst or interburst interval, and the period of slow-wave oscillation. It is found that the investigated model, when using the slowest process (1.5 sec) can generate persistent oscillations with a period of more than 100 sec. Introduction of variable parameters in the model simulates an initiation of slow-wave oscillation in Helix bursting neuron evoked by stimulation of identified peptidergic interneuron. The limitation of the presented model of slow-wave oscillation is discussed.

Adenosine reduces the potassium conductance of guinea pig submucosal plexus neurons by activating protein kinase A.

Intracellular recordings were made from S neurons of the submucosal plexus isolated from the guinea pig ileum. Adenosine or its analog 2-chloroadenosine (CADO) depolarized about 80% of neurons; previous work has shown that this results from activation of an A2 receptor. The depolarization was associated with an increase in membrane input resistance, became smaller with membrane hyperpolarization, reversed polarity at the potassium equilibrium potential and was mimicked and occluded by calcium-free solutions or by cadmium, suggesting that it is due to a reduction in a calcium-dependent potassium conductance. Both forskolin (though not 1,9-dideoxyforskolin) and phorbol 12,13-dibutyrate (PDBu) mimicked and occluded the action of CADO. Staurosporine (a nonspecific inhibitor of protein kinases) blocked the depolarization induced by the phorbol ester within 5 min, and blocked the effects of forskolin and CADO in 15-35 min. The depolarization caused by CADO was inhibited by the specific inhibitor of protein kinase A KT5720 [(8R*,9S*,11S*)-(-)-9-hydroxy-9-n-hexylester-8-methyl-2,3,9,10-tet rahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]c ycloocta[cd e]-trin-den-1-one], whereas this inhibitor did not affect the depolarization induced by PDBu. The results are consistent with the control of this potassium conductance by protein kinase C, protein kinase A and intracellular calcium, and they indicate that adenosine reduces the conductance by activating protein kinase A.

Guanosine 3',5'-cyclic monophosphate regulates calcium channels in neurones of rabbit vesical pelvic ganglia.

1. The effects of dibutyryl guanosine 3',5'-cyclic monophosphate (db-cyclic GMP) were studied in vitro on calcium channels of neurones in rabbit vesical parasympathetic ganglia, using intracellular and single-electrode voltage-clamp recordings. 2. Db-cyclic GMP (100 microM) caused membrane depolarization associated with a decrease in membrane input resistance and an after-hyperpolarization associated with an increase in membrane input resistance. 3. Db-cyclic GMP (0.01-1 mM) caused a concentration-dependent, transient inward current followed by a long-lasting outward current. Membrane conductance was increased and decreased during the inward and outward currents, respectively. 4. The db-cyclic GMP-induced inward current was depressed in nominally calcium-free solutions, by cobalt (1 mM) and nicardipine (10 microM). The mean reversal potentials of the inward current were +42 and -20 mV in the presence and absence of calcium in the external solution, respectively. 5. The db-cyclic GMP-induced inward current was not altered by lowering the external sodium concentration, raising external potassium concentration or by intracellular injection of caesium. 6. A calcium-insensitive component of the db-cyclic GMP-induced current was increased by lowering the external chloride concentration and blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, a chloride channel blocker. 7. Voltage-dependent, high-threshold calcium currents were depressed during the db-cyclic GMP-induced inward current and facilitated during the outward current. 8. Cyclic GMP was less potent than db-cyclic GMP in causing both inward and outward currents or modulation of calcium currents. GTP, GDP, GMP, guanosine, 8-bromoadenosine 3',5'-cyclic monophosphate and forskolin did not alter the holding current or voltage-dependent calcium currents. 9. It is concluded that intracellular cyclic GMP causes not only activation of resting calcium and chloride channels but also a transient depression followed by long-lasting facilitation of voltage-dependent calcium currents in neurones of vesical parasympathetic ganglia.

Some effects of octopamine, proctolin and serotonin on dorsal unpaired median neurones of cockroach ( Periplaneta americana) thoracic ganglia

Effects of neuromodulators, two monoamines, octopamine and serotonin, and the pentapeptide, proctolin, known to be present in the cockroach central nervous system, on the excitability of dorsal unpaired median neurones in the thoracic ganglia of the cockroach, Periplaneta americana, were investigated using intracellular recording technique. Octopamine had a marked excitatory effect on these neurones. Bath application of octopamine increased the frequency of spontaneous spikes in a dose-dependent manner associated with an increase in membrane input resistance. Serotonin increased the frequency and the resistance, but much less than octopamine. Proctolin also had an excitatory effect on the dorsal unpaired median somata, but the effect was somewhat equivocal. No appreciable change in the membrane input resistance occurred in the presence of proctolin. Among these neuromodulators tested, octopamine may alter the intrinsic properties of the above somata, resulting in the modulation of neural networks.

Adenosine promotes burst activity in guinea-pig geniculocortical neurones through two different ionic mechanisms.

1. The mechanisms of action of adenosine were examined in relay neurones of the dorsal lateral geniculate nucleus (LGND) using in vitro intracellular recording techniques in guinea-pig thalamic slices. 2. Adenosine hyperpolarized LGND relay neurones due to an increase in membrane potassium conductance. The K+ currents generated by near maximal stimulation of adenosine and GABAB receptors were non-additive. 3. Blockage of membrane K+ conductances by barium unmasked a second response to adenosine; an outward shift of the current versus voltage relationship negative to -65 mV associated with an increase in membrane input resistance. The beta-adrenoceptor agonist isoprenaline elicited an inward current in the same voltage range, which was inhibited and replaced by an outward current during activation of adenosine receptors. The effects of adenosine were due to a decrease in amplitude and rate of rise of the hyperpolarization-activated cation current, Ih. Maximal reduction by 66% of Ih amplitude occurred near the range of half-activation. 4. Both responses to adenosine were mimicked by the selective A1 receptor agonists N6-cyclopentyladenosine or N6-cyclohexyladenosine, and reversibly blocked by the selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 5. The decrease in Ih by adenosine may be mediated by an inhibition of adenylyl cyclase activity and hence a decrease in the intracellular level of cyclic AMP, since local application of the adenylyl cyclase inhibitor 2',3'-dideoxyadenosine imitated the decrease in Ih. Local application of the adenylyl cyclase stimulant forskolin or 8-bromo-cyclic AMP resulted in an enhancement in Ih, and forskolin inhibited the action on Ih evoked by N6-cyclopentyladenosine. 6. The adenosine-induced effects interacted with the intrinsic electrophysiological properties of LGND neurones in that (i) the hyperpolarization due to an increase in K+ conductance inhibited single spike firing and promoted calcium-mediated burst discharges, and (ii) the decrease in Ih inhibited the dampening effect on Ca(2+)-mediated rebound activity of beta-adrenergic receptor stimulation. 7. It is suggested that during increased levels of extracellular adenosine the response of LGND relay neurones to activating brainstem influences will be depressed, and a pattern of Ca(2+)-mediated burst firing will be favoured.

Endothelin causes prolonged inhibition of nicotinic transmission in feline colonic parasympathetic ganglia.

The action of endothelin (0.03-1 microM) on neurons in colonic parasympathetic ganglia of cats was studied in vitro, using intracellular microelectrode recording techniques. Electrical stimulation of the pelvic nerve evoked excitatory postsynaptic potentials (EPSPs) and orthodromic action potentials that were reversibly blocked by (+)-tubocurarine, hexamethonium, or external solutions containing nominal zero calcium and elevated magnesium. Endothelin blocked orthodromic action potentials and caused a concentration-dependent prolonged reversible depression of fast EPSPs. Endothelin had minimal effects on nicotinic depolarizations evoked by pressure application of acetylcholine. Endothelin also caused membrane depolarization (2-12 mV) followed by membrane hyperpolarization (1-8 mV). The depolarization and hyperpolarization were associated with a decrease and increase in membrane input resistance, respectively. The actions of endothelin were not altered by superfusion of the ganglia with external solutions containing atropine (300 nM), yohimbine (300 nM), naloxone (1 microM), or substance P (3 microM). We conclude that endothelin modulates synaptic transmission by slow membrane depolarization, membrane hyperpolarization, and prolonged depression of fast EPSPs. We suggest that the blockade of orthodromic action potentials and the depression of fast EPSPs is primarily due to inhibition of release of acetylcholine from presynaptic terminals.

The low KM-phosphodiesterase inhibitor denbufylline enhances neuronal excitability in guinea pig hippocampus in vitro

The actions of the phosphodiesterase inhibitor denbufylline on the excitability of hippocampal neurons were investigated by means of extracellular and intracellular recordings. Denbufylline, which has been shown to selectively inhibit a low KM, Ca2+/calmodulin-independent phosphodiesterase isozyme, concentration-dependently increased the amplitude of the extracellularly recorded CA1 population spike evoked by electrical stimulation of the Schaffer collateral/commissural pathway. Concentration-response-curves yielded an EC50 for denbufylline of 0.76 microM. In comparison, the non-selective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) also produced an increase in the amplitude of the population spike. From the concentration-response-curve, which was steeper than that of denbufylline, an EC50 for IBMX of 1.04 microM was obtained. However, despite their similar EC50 values, denbufylline was found to be significantly more potent at lower concentrations (less than or equal to 300 nM) than IBMX. Intracellular recordings from CA1 pyramidal cells revealed postsynaptic actions of denbufylline (300 nM) as indicated by a small drug-induced depolarization (2-5 mV) associated with an increase in membrane input resistance by 10-20%. In addition, denbufylline blocked the accommodation of trains of action potentials evoked by the injection of depolarizing current pulses. The results suggest i) that accumulation of adenosine-3',5'-monophosphate (cAMP) in the postsynaptic cell and/or in the presynaptic terminal produced by blockade of phosphodiesterases leads to enhanced synaptic transmission in the CA1 area of the hippocampus and ii) that a low KM, Ca2+/calmodulin-independent cAMP-phosphodiesterase is an important component involved in the regulation of the intracellular cAMP level at synapses of central nervous system neurons.