CCKB receptor stimulation mediates [Ca2+]i increase but no PKC activation in Jurkat T-cells.

We have investigated the effect of cholecystokinin-octapeptide (CCK-8) on [Ca2+]i and protein kinase C (PKC) activity in Jurkat T-cells. CCK-8 produced a transient [Ca2+]i increase in the presence of extracellular Ca2+. While CCKB receptor antagonist L-365,260 abolished the elevation of [Ca2+]i, CCKA receptor antagonist L-364,718 was without effect. Moreover, the dihydropyridine calcium channel blocker nitrendipine was shown to block the observed calcium response. Results suggest that the calcium effect is caused by an interaction of CCK-8 with CCKB binding sites and an influx of external Ca2+ via dihydropyridine sensitive calcium channels might serve as a source for the increased [Ca2+]i. Because CCK-8 induced no PKC activation CCKB receptor mediated rise of intracellular calcium seems not to include activation of phospholipase C.     

Rat hippocampal slices 'in vitro' display spontaneous epileptiform activity following long-term organotypic culture

Organotypic cultures of rat hippocampal slices were maintained for periods of up to 12 weeks in vitro. Cultures adopted a two-dimensional architecture whilst retaining the subfields characteristic of intact hippocampal slices. Coventional intracellular onset of spontaneous long-lasting epileptiform activity. Epileptiform activity characteristic of both interictal and ictal events (paroxysmal depolarising shifts, tonic/clonic phases and afterdischarges) was observed in the absence of pharmacological manipulation or of orthodromic stimulation. Epileptiform activity was abolished in the presence of high Mg2+ concentration or tetrodotoxin, agents known to block synaptic transmission. In addition, the frequency of epileptiform events was independent of membrane potential and the amplitude of the paroxysmal depolarising shift (PDS) displayed a near linear relationship with membrane potential. The PDS could be reversed at potentials approaching synaptic equilibrium potential. The N-methyl-D-aspartate (NMDA)-receptor antagonist DL-2-amino-5-phosphonovalerate (DL-APV) dose-dependently reduced both the amplitude and duration of the spontaneous paroxysmal shift, having no effect on the initiation of the event or the resting membrane parameters of the neurone. DL-APV also attenuated a late component of the synaptically evoked excitatory postsynaptic potentials (epsp) not observed in non-epileptiform neurones. Application of GABAA receptor antagonists bicuculline or picrotoxin converted interictal events to ictus. In the presence of these agents, ictal events were up to 90 s in duration. These results suggest that long-term culturing of hippocampal explants leads to an alteration in the balance of excitatory and inhibitory synaptic activity. This allows the expression of an excitatory amino acid depolarisation acting through NMDA receptors which contributes to the generation and maintenance of spontaneous epileptiform activity which is synaptic in origin.     

Investigation of behavioral and electrophysiological responses induced by selective stimulation of CCKB receptors by using a new highly potent CCK analog, BC 264

The new CCKB analog, Boc-Tyr (SO3H)-gNle-mGly-Trp-(NMe)-Nle-Asp-PheNH2 (BC 264) exhibited a high affinity (KI = 0.39 +/- 0.15 nM) and selectivity for central (B) versus peripheral (A) receptors (KI CCKA/KI CCKB = 910) in the rat. In agreement with these binding studies, BC 264 was at least 50 times more potent than CCK8 in stimulating the firing of rat CA hippocampal neurones. Furthermore stereotaxic injection of BC 264 or CCK8 in the VTA of rats resulted in potentiation of the dopamine-induced hypolocomotion. These two types of CCK8 responses have been previously shown in involve CCKB receptors. In contrast, after administration into the postero-median nucleus accumbens, the hypoexploration, the increase of emotionality of rats, or the potentiation of dopamine-induced hyperlocomotion were obtained after injection of CCK8 but not of BC 264, supporting the involvement of peripheral CCKA receptors in these CCK8 responses. Owing to its resistance to peptidases, BC 264 appears to be of great interest in the investigation of the still uncertain functional roles of CCK in the central nervous system.     

Cholecystokinin inhibits evoked inhibitory postsynaptic currents in the rat nucleus accumbens indirectly through gamma-aminobutyric acid and gamma-aminobutyric acid type B receptors

We recently reported that cholecystokinin (CCK) excited nucleus accumbens (NAc) cells and depressed excitatory synaptic transmission indirectly through gamma-aminobutyric acid (GABA), acting on presynaptic GABAB receptors (Kombian et al. [2004] J. Physiol. 555:71-84). The present study tested the hypothesis that CCK modulates inhibitory synaptic transmission in the NAc. Using in vitro forebrain slices containing the NAc and whole-cell patch recording, we examined the effects of CCK on evoked inhibitory postsynaptic currents (IPSCs) recorded at a holding potential of -80 mV throughout CCK-8S caused a reversible inward current accompanied by a concentration-dependent decrease in evoked IPSC amplitude. Maximum IPSC depression was approximately 25% at 10 microM, with an estimated EC50 of 0.1 microM. At 1 microM, CCK-8S induced an inward current of 28.3 +/- 4.8 pA (n=6) accompanied by an IPSC depression of -18.8% +/- 1.6% (n=6). This CCK-induced IPSC depression was blocked by pretreatment with proglumide (100 microM; -3.7% +/- 6.9%; n=4) and by LY225910 (100 nM), a selective CCKB receptor antagonist (4.4% +/- 2.6%; n=4). It was not blocked by SCH23390 (10 microM; -23.5% +/- 1.3%; P < 0.05; n=7) or sulpiride (10 microM; -21.8% +/- 5.1%; P <0.05; n=4), dopamine receptor antagonists. By contrast, it was blocked by CGP55845 (1 microM; -0.4% +/- 3.4%; n=5) a potent GABAB receptor antagonist, and by forskolin (50 microM; 9.9% +/- 5.2%; n=4), an adenylyl cyclase activator, and H-89 (1 microM; 6.9% +/- 3.9%; n=4), a protein kinase A (PKA) inhibitor. These results indicate that CCK acts on CCKB receptors to increase extracellular levels of GABA, which then acts on GABAB receptors to decrease IPSC amplitude.     

Interaction of cholecystokinin and glutamate agonists within the dLGN, the dentate gyrus, and the hippocampus

The interaction of sulfated cholecystokinin (CCK-8S) with excitatory amino acids (EAA) was studied on single units of the dorsal lateral geniculate nucleus (dlGN), the dentate gyrus, and the hippocampal CA3 region in rats anaesthetized with urethane. Iontophoretic co-administration of small, individually ineffective currents of CCK-8S and kainic acid or N-methyl-D-aspartate repeatedly elicited an increase of the discharge rate in nearly all geniculate and half of the dentate neurons but not in those of the CA3 region. The effect could be reduced by the CCKB receptor antagonist PD 135, 158 more often than by the CCKA antagonist KL 1001. The increased firing due to co-administration of CCK and kainate could also be suppressed by the non-NMDA antagonist CNQX but not by the NMDA antagonists CPP or AP-5, which were otherwise able to prevent the neuron from responding to co-administration of CCK and NMDA. It is suggested that in distinct brain regions the effectivity of the “low level” EAA transmission may be enhanced by small amounts of CCK-8S. This is thought to be mediated by a coactivation of CCK and EAA receptors.     

Modulation of GABA-mediated Synaptic Potentials by Glutamatergic Agonists in Neonatal CA3 Rat Hippocampal Neurons

Intracellular recordings were made from slices of adult and neonatal hippocampal neurons. During the first 2 weeks of life the majority of pyramidal cells exhibited spontaneous gamma-aminobutyric acid (GABA)-mediated synaptic potentials, which were depolarizing at birth and became hyperpolarizing by the end of the first postnatal week. These synaptic potentials were reduced in frequency or blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist d(-)2-amino-5-phosphonovalerate (AP-5, 50 microM) (13/15 cells). The non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 - 10 microM) abolished the GABA-mediated synaptic potentials in all the cells tested (n=12), Superfusion of l-glutamate (up to 100 microM) increased the frequency of both depolarizing and hyperpolarizing GABA-mediated synaptic potentials. This effect was reduced by AP-5 or dl-2-amino-7-phosphonoheptanoate (AP-7, 50 microM) and fully blocked by concomitant application of AP-5 (50 microM) and CNQX (5 - 10 microM). NMDA (0.5 - 2 microM) increased the frequency of the GABA-mediated synaptic potentials. These effects were blocked by AP-5 (50 microM) and by bicuculline (10 microM). Quisqualate (100 - 300 nM), (RS)-alpha-amino-3-hydroxy-5-methyl-4-izopropionate (AMPA, 100 - 300 nM) and kainate (100 nM) also increased the frequency of the GABA-mediated synaptic potentials. These effects were blocked by CNQX (5 - 10 microM) and by bicuculline (10 microM) but not by AP-5 (50 microM). In the presence of tetrodotoxin (TTX, 1 microM), quisqualate (up to 300 nM), AMPA (up to 500 nM) and kainate (100 nM) had no effect on membrane potential or input resistance. In conclusion, our experiments suggest that, in early postnatal life, NMDA and non-NMDA receptors located on GABAergic interneurons modulate GABAergic synaptic potentials.     

Autoradiographic Localization of Cholecystokinin A and B Receptors in Rat Brain Using [125I]d-Tyr25 (Nle28,31)-CCK 25 - 33S

The distribution of receptors for the sulphated octapeptide cholecystokinin 26 - 33 (CCK - 8S) in rat brain was investigated by radioligand binding in conjunction with autoradiography using the novel iodinable, non-oxidizable, amino- and thiolendopeptidase-resistant CCK analogue, d-Tyr25(Nle28,31)-CCK 25 - 33S. Labelling of the peptide was achieved by synthesis utilizing Na125I and Chloramine-T. [125I]d-Tyr25(Nle28,31)-CCK 25 - 33S (100 pM) bound rapidly and reversibly to a single population of sites on slide-mounted coronal sections of rat forebrain with a dissociation constant of 34 pM. Specific binding was fully inhibited by CCK-8S, CCK-8, CCK-4, L-365,260 and L-364,718, with inhibition constants 2.7, 9.8, 35, 7.0 and 130 nM, respectively. These inhibition data may indicate that the [125I] ligand binds preferentially to a CCKB subtype of receptor, but may also reflect the relative paucity of CCKA receptors in the rat forebrain. Optimum conditions for autoradiography combined the preincubation of brain sections in unlabelled 10 pM d-Tyr25(Nle28,31)-CCK 25 - 33S with a 60-min wash after incubation with the [125I] ligand. Analyses of the autoradiograms obtained from the use of coronal and horizontal brain sections were aided by the high levels of specific binding (80 - 90%), and revealed that CCK receptors were topographically distributed through the neuroaxis. High densities of receptor-associated silver grains were found in the olfactory bulb (internal plexiform layer), neocortex (layer III), nucleus accumbens, parasubiculum, subbrachial nucleus, parabigeminal nucleus, dorsal vagal complex, area postrema and the A2 region. Moderate labelling was observed in many telencephalic and diencephalic nuclei. The majority of these receptors were of the CCKB subtype, as shown by the use of subtype-selective antagonists, although CCKA receptors were present in moderate to high densities in the A2 area, area postrema and nucleus tractus solitarii, and at low density in the interpeduncular nucleus and central amygdala. These findings provide further evidence for the widespread, topographic distribution of CCK receptors and indicate that [125I]d-Tyr25(Nle28,31)-CCK 25 - 33S is very suitable for autoradiographic investigations because of its low non-specific binding.     

Excitatory synaptic inputs on myenteric Dogiel type II neurones of the pig ileum

The synaptic input on myenteric Dogiel type II neurones (n = 63) obtained from the ileum of 17 pigs was studied by intracellular recording. In 77% of the neurones, electrical stimulation of a fibre tract evoked fast excitatory postsynaptic potentials (fEPSPs) with an amplitude of 6 +/- 5 mV (mean +/- S.D.) and lasting 49 +/- 29 ms. The nicotinic nature of the fEPSPs was demonstrated by superfusing hexamethonium (20 microM). High-frequency stimulation (up to 20 Hz, 3 seconds) did not result in a rundown of the fEPSPs, and did not evoke slow excitatory or inhibitory postsynaptic potentials. The effects of neurotransmitters, possibly involved in these excitatory responses, were investigated. Pressure microejection of acetylcholine (10 mM in pipette) resulted in a fast nicotinic depolarisation in 67%(18/27) of the neurones (13 +/- 9 mV, duration 7.0 +/- 7.2 seconds) as did 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) application (10 mM; 14 +/- 10 mV, duration 4.1 +/- 2.8 seconds) in 76% of the cells. The fast nicotinic response to acetylcholine was sometimes (6/27) followed by a slow muscarinic depolarisation (8 +/- 4 mV; duration 38.7 +/- 10.8 seconds). Immunostaining revealed 5-hydroxytryptamine hydrochloride (5-HT)- and calcitonin gene-related peptide (CGRP)-positive neuronal baskets distributed around and in close vicinity to Dogiel type II neuronal cell bodies. Microejection of 5-HT (10 mM) resulted in a fast nicotinic-like depolarisation (12 +/- 6 mV, duration 3.0 +/- 1.3 seconds) in 4 of 8 neurones tested, whereas microejection of CGRP (20 mM) gave rise to a slow muscarinic-like depolarisation (6 +/- 2 mV, duration 56.0 +/- 27.5 seconds) in 8 of 12 neurones tested. In conclusion, myenteric Dogiel type II neurones in the porcine ileum receive diverse synaptic input. Mainly with regard to the prominent presence of nicotinic responses, these neurones behave contrary to their guinea pig counterparts.     

Spontaneous action potential activity and synaptic currents in the embryonic turtle cerebral cortex.

We used loose-patch and whole-cell recording techniques to study the development of spontaneous action potential activity and spontaneous excitatory and inhibitory synaptic currents in embryonic neurons in the cerebral hemispheres of turtles. Sporadic action potential activity appeared early in development at stage 17, soon after morphologically identifiable pyramidal and nonpyramidal neurons were first observed in the cortex. As the cortical plate matured in midembryonic stages, action potential activity became more regular and fell into one of two distinct patterns, tonic and intermittent high-frequency firing. Spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) appeared at developmental stages 18 and 20, respectively, after action potential activity was established. EPSCs and IPSCs exhibited characteristic ionic dependence and pharmacology throughout development. EPSCs reversed in direction at the equilibrium potential for cations and were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of the non-NMDA type of glutamate receptor. IPSCs reversed at the equilibrium potential for chloride and were sensitive to bicuculline methiodide, a GABAA receptor antagonist. Spontaneous synaptic currents differed in their time course of development and in waveform parameters. Spontaneous synaptic currents differed in their time course of development and in waveform parameters. Spontaneous EPSCs appeared at stage 18 and increased progressively in frequency, from 0.2 +/- 0.1 Hz at stage 20 to 3.2 +/- 2.0 Hz at stage 26 (hatching), while spontaneous IPSCs appeared at stage 20 and surpassed EPSCs in frequency, increasing to 7.1 +/- 1.6 Hz at stage 26. EPSCs exhibited stable amplitudes during development, with a mean conductance of 126 +/- 20 pS at stage 26, while IPSCs increased in mean amplitude, from 180 +/- 12 pS at stage 18 to 260 +/- 44 pS at stage 26. The rise time to peak conductance of both types of synaptic currents increased with developmental time, for EPSCs increasing from 1.5 +/- 0.5 msec at stage 20 to 2.7 +/- 0.6 msec at stage 26 and for IPSCs increasing from 2.9 +/- 0.2 msec at stage 18 to 6.2 +/- 0.8 msec at stage 26. While the decay time constants increased for EPSCs, from 3.9 +/- 1.2 msec at stage 20 to 8.7 +/- 2.3 msec at stage 26, decay time constants for IPSCs showed a decreasing trend from 24.0 +/- 5.2 msec at stage 18 to 18.4 +/- 5.3 msec at stage 26. The excitatory and inhibitory synaptic currents were sensitive to the sodium channel blocker TTX and were thus dependent, in part, on spontaneous action potential activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of amino acid antagonists on spontaneous dorsal root activity and evoked dorsal horn field potentials in an isolated preparation of rat spinal cord

Fast and slow dorsal horn field potentials and spontaneous dorsal root activity were recorded from 19-23-day-old rat isolated spinal cord preparations. The effects of GABA, glycine, and glutamate antagonists were tested on these recordings. CNQX, an AMPA/kainate antagonist, reduced all 3 components of the dorsal horn field potential whereas MK801, an NMDA ion channel antagonist, reduced the fast S2 component and the slow wave. Both reduced spontaneous dorsal root activity. NMDA antagonists, D-AP5, 7-chlorokynurenic acid and arcaine, and the metabotropic glutamate antagonists L-AP3 and ethylglutamic acid, while having little effect on the fast components of the field potential, all reduced the slow component. The GABA antagonist, bicuculline, and the glycine antagonist, strychnine, while having no effect on the fast S1 and slow components of the field potential, reduced both the fast S2 component of the field potential and spontaneous dorsal root activity. These results suggest that non-NMDA glutamate receptors are involved in low and high threshold transmission to dorsal horn neurones while NMDA and metabotropic glutamate receptors are primarily involved in high threshold transmission and both GABA and glycine have roles in the transmission or modulation of sensory information within the dorsal horn of the cord.     

Direct inhibition by opioid peptides of neurones located in the ventromedial nucleus of the guinea pig hypothalamus

In slices of guinea pig brain, intracellular recordings were obtained from neurones of the ventromedial nucleus of the hypothalamus (VMH). [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO), an agonist selective for mu-opioid receptors, caused an inhibition of spontaneous firing activity and a membrane hyperpolarization. This effect was reversible, concentration-dependent and could be blocked by naloxone. DAGO directly inhibited VMH neurones since its effect persisted when the slice was perifused with a solution that blocks synaptic transmission. The hyperpolarization induced by DAGO was associated with a marked decrease in membrane input resistance and it was reversed in polarity at membrane potentials 30-40 mV more negative than the resting potential. A chloride current did not contribute to the hyperpolarization brought about by DAGO. We conclude that DAGO inhibits VMH neurones, probably by opening membrane potassium channels.     

Pharmacology of the vestibular hair cell-afferent fiber synapse in the frog

The isolated, intact, membranous labyrinth of the frog (Rana temporaria) has been investigated electrophysiologically in vitro to determine the nature of the transmitter substance at the synapse between the vestibular hair cells and afferent fibers. Spontaneous synaptic activity can be monitored with intra-axonal recordings from the afferents. Increased K+ in the bath results in an increase in frequency of presynaptic release, as indicated by an increased frequency of spontaneous synaptic potentials. Adding Mg2+ and lowering Ca2+ results in a decrease in synaptic potential frequency (often to zero) with no change in their mean amplitude, indicating pre-synaptic blockade. Extracellular recordings from individual vestibular afferents indicate that bath-applied glutamate and related acidic amino acids consistently increase the firing rates of these afferents in a dose-dependent manner with no evidence of desensitization. In the presence of presynaptic blockade (high Mg2+/low Ca2+), bath application of glutamate and its agonists results in a reversible depolarization of vestibular afferents, suggesting a postsynaptic action of these substances. 2-Amino-5-phosphonovaleric acid, kynurenic acid, and other acidic amino acid antagonists reversibly decrease the amplitudes of spontaneously occurring synaptic potentials without affecting their frequency, indicating subsynaptic blockade. These antagonists also block the postsynaptic depolarizations due to glutamate and its agonists. GABA and its agonists and antagonists have no consistent effect upon afferent activity. These findings suggest that glutamate, aspartate, or a related compound is the transmitter at this synapse. However, the antagonists used, or the receptors themselves, are not selective enough to discriminate adequately between the agonists. Therefore, which of these glutamate agonists are actually involved in synaptic transmission remains to be determined.     

Electrophysiological characteristic of corticoaccumbens synapses in rat mesolimbic system reconstructed using organotypic slice cultures

The nucleus accumbens (NAc) is a component of the mesolimbic system involved in drug dependence. Activity of nucleus accumbens neurons is modulated by glutamatergic afferents from the prefrontal cortex and by dopaminergic afferents from the ventral tegmental area (VTA). In the present study, we reconstructed the mesolimbic system using organotypic slice cultures and examined the effects of dopaminergic agents on synaptic activity in the prefrontal cortex-nucleus accumbens synapses. A slice of each of the prefrontal cortex, nucleus accumbens and ventral tegmental area in newborn rat, was arranged on a multi-electrode dish (MED) filled with culture medium so that they contacted each other, termed a 'triple culture'. Extracellular recording using microelectrodes on the multi-electrode dish showed that a single electrical stimulation of the prefrontal cortex slice evoked field excitatory postsynaptic potential, and that population spikes occurred spontaneously in the nucleus accumbens area of the triple culture. The amplitude of evoked field excitatory postsynaptic potentials and the frequency of spontaneous population spikes were decreased by glutamatergic antagonists, D(-)-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. The D1-like receptor agonist SKF38393, but not the D2-like receptor agonist quinpirole, reduced both the amplitude of field excitatory postsynaptic potential and frequency of spontaneous population spikes. Cocaine depressed field excitatory postsynaptic potential and this depression was reversed by D1-like receptor antagonist SCH23390, but not by D2-like receptor antagonist sulpiride. These results suggest that evoked field excitatory postsynaptic potentials and spontaneous population spikes were driven by glutamatergic neurons and were subject to exogenous and endogenous dopaminergic modulation in the triple culture that was similar to that shown in in vivo.     

Cannabinoids modulate synaptic activity in the rat supraoptic nucleus

In the present study, we investigated the effects of the cannabinoid receptor agonist CP55,940 on excitatory and inhibitory synaptic transmission in the rat supraoptic nucleus. Whole-cell patch clamp recordings were performed on supraoptic neurones in in vitro brain slice preparations. CP55,940 significantly reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents in a concentration-dependent manner. These changes were potently reversed by the CB1 receptor antagonist AM251. The results indicate that cannabinoids modulate the activity of magnocellular neurosecretory neurones by presynaptic inhibition of both excitatory and inhibitory synaptic transmission.     

Relationship between colonic motility and cholinergic mechanosensory afferent synaptic input to mouse superior mesenteric ganglion

Abstract Abdominal prevertebral ganglion neurones receive excitatory synaptic input from intestinofugal neurones. To better understand the physiological significance of this input, we examined the relationship between synaptic input to mouse superior mesenteric ganglion (SMG) neurones and intracolonic pressure and volume changes that accompany spontaneous colonic contractions in vitro. Electrical activity was recorded intracellularly from SMG neurones in ganglia attached to a segment of distal colon. The majority of neurones examined received ongoing fast excitatory potentials (F-EPSPs). F-EPSP frequency increased when the colon was distended with fluid and during spontaneous increases in colonic volume that accompanied colonic relaxation. In contrast, F-EPSP frequency in SMG neurones decreased when the colon emptied, and remained at a reduced frequency until the colon refilled and volume increased. Nicotinic blockade of the colon abolished spontaneous colonic contractions and reduced or abolished synaptic input to SMG neurones, suggesting that most of the synaptic input arose from second or higher order neurones. Retrograde labelling identified cell bodies of intestinofugal neurones in myenteric ganglia. Most had short, club-like dendritic processes and appeared uni-axonal. These results show that mechanosensory intestinofugal afferent nerves monitor intracolonic volume changes.     

Nicotinic transmission at functionally distinct synapses in descending reflex pathways of the rat colon

We examined descending reflex pathways in the rat colon using intracellular recording techniques. Inhibitory junction potentials (IJPs) were recorded from circular smooth muscle when descending pathways were excited by combined mucosal compression and distension. IJPs were reduced to 71% of control when synaptic transmission was blocked in the oral stimulation chamber of a divided organ bath suggesting that two reflex pathways exist, the one involving descending sensory neurones and the other involving descending interneurones. Hexamethonium (200 micromol L(-1)) in the recording chamber abolished reflexly evoked IJPs, while in the stimulation chamber, it was as effective as synaptic blockade. When hexamethonium was added to a chamber lying between the stimulation and recording chambers, it again sharply depressed IJPs to 27% of control; an extent similar to synaptic blockade. A P2 receptor antagonist did not reveal any purinergic neurotransmission. Either granisetron (5-HT3 receptor antagonist, 1 micromol L(-1)) or SB204070 (5-HT4 receptor antagonist, 1 micromol L(-1)) in the stimulation chamber significantly decreased IJPs; these decreases were not additive. We conclude that some sensory neurones and interneurones in rat colon have long anally projecting axons and that acetylcholine, acting via nicotinic receptors, is the primary neurotransmitter from sensory neurones, to inhibitory motor neurones and between interneurones.     

Cholinergic excitatory synaptic potentials of neurones in mammalian lumbar paravertebral ganglia

Synaptic potentials and the electrophysiological properties of 201 cells in the 4th lumbar paravertebral ganglia of the rabbit were studied in vitro using intracellular electrophysiological recording techniques. Cells had a mean transmembrane potential of 55.1 +/- 0.8 mV, a mean input resistance of 37.0 +/- 6.6 M omega (range 29.9-61.1) and a mean membrane time constant of 6.0 +/- 0.6 ms. Synaptic potentials in ganglionic neurones were evoked by electrical stimulation of the rami communicantes, inferior lumbar splanchnic nerves and the paravertebral chain from segments both above and below the L4 ganglion. Synaptic responses consisted of a fast, hexamethonium-sensitive component and, following short periods of higher frequency stimulation, a slow, long lasting, pirenzepine and atropine-sensitive depolarization (slow-EPSP). No phenomenon corresponding to a late slow-EPSP was observed and, under our recording conditions no cells exhibited non-cholinergic slow excitatory or slow inhibitory postsynaptic potentials. It is concluded that fast excitatory synaptic events were mediated by nicotinic receptors whereas slow excitatory synaptic events were mediated by muscarinic m1 receptors. McNeil-A-343, a muscarinic agonist, produced membrane depolarization, a decrease in membrane input conductance and in some cells a repetitive discharge of action potentials. In 60% of cells tested substance P produced a depolarization of the membrane potential with an associated decrease in membrane input conductance.     

Antiallodynic effects of NMDA glycine(B) antagonists in neuropathic pain: possible peripheral mechanisms

NMDA receptors are implicated in central sensitisation underlying chronic pain, and NMDA antagonists have a potential for the treatment of neuropathic pain. Functional NMDA receptors are also present on primary afferents, where they may play a role in pro-nociceptive plasticity. The importance of this mechanism in neuropathic pain remains unclear. In the present work, we have compared in models of chronic pain the effects of NMDA antagonists at the glycine(B) site with different central access. L-701,324 (the centrally active antagonist) and 5,7-dichlorokynurenic acid (5,7-DCK, known to have limited central access) were tested after systemic administration in rats in the formalin test and in two models of neuropathic pain. The ability of these compounds to exert central actions (sedation, ataxia) was tested in the open field locomotion test; central NMDA antagonism in vivo was tested in anaesthetised rats on responses of spinal cord neurones to iontophoretic NMDA. Both L-701,324 (2.15-21.5 mg/kg i.p.) and 5,7-DCK (10-46.4 mg/kg i.v.) dose-dependently inhibited Phase II of formalin-evoked behaviour. Likewise, both compounds reversed cold allodynia in the chronic constriction injury model and tactile allodynia in animals with spinal nerve ligation. However, only L-701,324 was able to inhibit neuronal responses to NMDA in the antihyperalgesic dose range; 5,7-DCK was inactive on NMDA responses up to 46.4 mg/kg i.v. or 68.1 mg/kg i.p. Consistent with the lack of inhibition of central NMDA-evoked activity, 5,7-DCK did not alter spontaneous behaviour in the open field test, whereas it was significantly inhibited by L-701,324. Thus, peripheral NMDA receptors may substantially contribute to the efficacy of NMDA antagonists in neuropathic pain.     

Developmental changes in the susceptibility to long-term potentiation of neurones in rat visual cortex slices.

We investigated with intracellular recordings from rat visual cortex slices whether the susceptibility to undergo long-term potentiation (LTP) is age-dependent and whether it is correlated with the expression of synaptic responses mediated by N-methyl-D-aspartate (NMDA) receptors. Test and tetanic stimuli were applied to the white matter and post-tetanic modifications of the amplitude of postsynaptic potentials (PSPs) were assessed in regular spiking cells of supragranular layers. At 2 weeks of age, the amplitudes of early (8-10 ms post-stimulus) and late (20 ms post-stimulus) PSP-components increased after tetanic stimulation to 137.1 +/- 13.4% and 141.3 +/- 12.1% of the pretetanic controls, respectively. At 3 weeks, potentiation of both PSP-components was less pronounced but still significant, the late component being on average more potentiated than the early one. At 4 weeks, PSPs were no longer potentiated. Bath application of 25 microM DL-2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, blocked LTP induction both at 2 and at 3 weeks. We also studied developmental changes of two synaptic responses known to influence the susceptibility of cortical neurones to LTP, the NMDA receptor-mediated excitatory PSP (EPSP) and the initial inhibitory PSP (iIPSP). The amplitude of the APV-sensitive EPSP decreased with age and reached adult values in 4-week-old animals. The iIPSPs were pronounced already at 2 weeks and showed no marked change during further development. The results suggest a close correlation between the susceptibility to undergo LTP and the extent to which NMDA receptor-gated conductances contribute to the synaptic response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological and electrophysiological properties of neurones in the dorsal vagal complex of the rat activated by arterial baroreceptors

This study physiologically identifies and anatomically describes arterial baroreceptive neurones in the nucleus tractus solitarii of the rat. Neurones were recorded using neurobiotin-containing whole cell patch electrodes in a working heart-brainstem preparation and characterized physiologically as arterial baroreceptive in response to stimulation of the aortic arch and/or ipsilateral carotid sinus. Fifteen of 84 neurones tested were arterial baroreceptive, 7 of 8 were morphologically identified as located in the solitary tract nucleus (NTS), and 1 of 8 was located in the dorsal vagal nucleus. The seven NTS neurones had a resting membrane potential of -52 +/- 3.6 mV and a membrane input resistance of 233 +/- 38 M omega. Action potential height was 62 +/- 4.2 mV, width at half amplitude 1.46 +/- 0.38 ms, and duration of after-hyperpolarization 1.7 +/- 2.33 ms. In six of eight neurones labelled there was an invariant excitatory synaptic input (latency 3.95 +/- 0.3 ms) to stimulation of the solitary tract. Labelled somata were dorsomedial or medial to the solitary tract from -0.3 mm to +1.5 mm with regard to obex. Neurones had three to eight primary dendrites, and branches often entered the solitary tract and also extended across the ipsilateral NTS. Axons, which were mostly unmyelinated with boutons of the en passant variety, could ramify within the NTS while the main axon exited the NTS (n = 4/6), in the direction of the ipsilateral ventral medulla (n = 5/6). This is the first morphological and localisation data of physiologically characterised arterial baroreceptive NTS neurones in the rat. By comparing labelled cells with each other as well as with other unidentified cells, we conclude that NTS arterially baroreceptive neurones are morphologically and physiologically heterogenous.