3-Alpha-chloro-imperialine, a potent blocker of cholinergic presynaptic modulation of glutamatergic afferents in the rat neostriatum

Cortico-thalamic glutamatergic afferents control neuronal activity in the neostriatum. Cholinergic interneurons modulate the activity of medium spiny neurons through both pre- and post-synaptic actions via the activation of muscarinic receptors. The muscarinic pre-synaptic modulation was analyzed electrophysiologically. The transmitter release, induced by 4-AP, was studied and the block of paired pulse facilitation (PPF) by different muscarinic receptor antagonists was analyzed. The GABA(A) antagonist bicuculline isolated the glutamatergic transmission. Muscarinic agonists decreased the frequency of random synaptic potentials induced by 4-AP in about 60% of the cases without changes in input resistance (RN) of the post-synaptic neuron or in the mean amplitude of the synaptic events; indicating a presynaptic action. The administration of both 1 microM carbachol or 20 nM muscarine increased PPF. Muscarinic receptor antagonists blocked this action with a potency order: 3-alpha-chloroimperialine > 4-DAMP>AFDX-116 > or = gallamine > pirenzepine. The IC50's for the first three antagonists were (nM): 0.65, 1.1, and 3.0. Their respective Hill coefficients were: 1.9, 1.4, and 1.3. 3-alpha-Chloroimperialine reduced the PPF almost completely. The M3 and the M2 muscarinic receptor antagonists 4-DAMP and AFDX-116, given at saturating concentrations, consistently blocked only a part of the PPF but had additive effects when given together. These data are consistent with the existence of both M2 and M3 muscarinic receptors in striatal glutamatergic afferents.     

A reductant-induced oxidation mechanism for complex I

We examined whether baroreceptor activation causes a release of acetylcholine (ACh) in the rostral ventrolateral medulla (RVLM) of the rat, in order to investigate a possible connection between RVLM cholinergic systems and cardiovascular baroreflexes. Male Wistar rats were anesthetized, paralyzed and artificially ventilated. Either electrical stimulation of aortic nerve or baroreceptor activation by intravenous phenylephrine produced an increase of the release of ACh in the RVLM, whereas baroreceptor denervation and tetrodotoxin (TTX) microinfusion in the RVLM inhibited the increase in ACh release induced by phenylephrine. TTX injected in the caudal ventrolateral medulla (CVLM) inhibited the phenylephrine-induced increase of ACh release. The excitatory amino acid L-glutamate microinfused in the CVLM produced an release in ACh release in the RVLM. These results suggest that there is a connection between RVLM cholinergic systems and cardiovascular baroreflexes. It is probable that neurons in the CVLM are involved in mediating the release of ACh in the RVLM.     

Dopamine inhibition: enhancement of GABA activity and potassium channel activation in hypothalamic and arcuate nucleus neurons

Dopamine (DA) decreases activity in many hypothalamic neurons. To determine the mechanisms of DA's inhibitory effect, whole cell voltage- and current-clamp recordings were made from primary cultures of rat hypothalamic and arcuate nucleus neurons (n = 186; 15-39 days in vitro). In normal buffer, DA (usually 10 microM; n = 23) decreased activity in 56% of current-clamped cells and enhanced activity in 22% of the neurons. In neurons tested in the presence of glutamate receptor antagonists D,L-2-amino-5-phosphonovalerate (AP5; 100 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM), DA application (10 microM) revealed heterogeneous effects on electrical activity of cells, either hyperpolarization and decrease in activity (53% of 125) or depolarization and increase in spontaneous activity (22% of 125). The DA-mediated hyperpolarization of membrane potential was associated with a decrease in the input resistance. The reversal potential for the DA-mediated hyperpolarization was -97 mV, and it shifted in a positive direction when the concentration of K+ in the incubating medium was increased, suggesting DA activation of K+ channels. Because DA did not have a significant effect on the amplitude of voltage-dependent K+ currents, activation of voltage-independent K+ currents may account for most of the hyperpolarizing actions of DA. DA-mediated hyperpolarization and depolarization of neurons were found during application of the Na+ channel blocker tetrodotoxin (1 microM). The hyperpolarization was blocked by the application of DA D2 receptor antagonist eticlopride (1-20 microM; n = 7). In the presence of AP5 and CNQX, DA (10 microM) increased (by 250%) the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) in 11 of 19 neurons and evoked IPSCs in 7 of 9 cells that had not previously shown any IPSCs. DA also increased the regularity and the amplitude (by 240%) of spontaneous IPSCs in 9 and 4 of 19 cells, respectively. Spontaneous and DA-evoked IPSCs and inhibitory postsynaptic potentials were blocked by the gamma-aminobutyrate A (GABA(A)) antagonist bicuculline (50 microM), verifying their GABAergic origin. Pertussis toxin pretreatment (200 ng/ml; n = 15) blocked the DA-mediated hyperpolarizations, but did not prevent depolarizations (n = 3 of 15) or increases in IPSCs (n = 6 of 10) elicited by DA. Intracellular neurobiotin injections (n = 21) revealed no morphological differences between cells that showed depolarizing or hyperpolarizing responses to DA. Immunolabeling neurobiotin-filled neurons that responded to DA (n = 13) showed that GABA immunoreactive neurons (n = 4) showed depolarizing responses to DA, whereas nonimmunoreactive neurons (n = 9) showed both hyperpolarizing (n = 6) and depolarizing (n = 3) responses. DA-mediated hyperpolarization, depolarization, and increases in frequency of postsynaptic activity could be detected in embryonic hypothalamic or arcuate nucleus neurons after only 5 days in vitro, suggesting that DA could play a modulatory role in early development. These findings suggest that DA inhibition in hypothalamic and arcuate nucleus neurons is achieved in part through the direct inhibition of excitatory neurons, probably via DA D2 receptors acting through a Gi/Go protein on K+ channels, and in part through the enhancement of GABAergic neurotransmission.     

Glutamatergic N2v cells are central pattern generator interneurons of the lymnaea feeding system: new model for rhythm generation

Electrophysiological and pharmacological methods were used to examine the role of glutamate in mediating the excitatory and inhibitory responses produced by the N2v rasp phase neurons on postsynaptic cells of the Lymnaea feeding network. The N2v --> B3 motor neuron excitatory synaptic response could be mimicked by focal or bath application of -glutamate at concentrations of >/=10(-3) M. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were potent agonists for the B3 excitatory glutamate receptor (10(-3) M), whereas kainate only produced very weak responses at the same concentration. This suggested that non-N-methyl--aspartate (NMDA), AMPA/quisqualate receptors were present on the B3 cell. The specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10(-5) M) blocked 85% of the excitatory effects on the B3 cell produced by focal application of glutamate (10(-3) M), confirming the presence of non-NMDA receptors. CNQX also blocked the major part of the excitatory postsynaptic potentials on the B3 cell produced by spontaneous or current-evoked bursts of spikes in the N2v cell. As with focal application of glutamate, a small delayed component remained that was CNQX insensitive. This provided direct evidence that glutamate acting via receptors of the non-NMDA, AMPA/quisqualate type were responsible for mediating the main N2v --> B3 cell excitatory response. NMDA at 10(-2) M also excited the B3 cell, but the effects were much more variable in size and absent in one-third of the 25 B3 cells tested. NMDA effects on B3 cells were not enhanced by bath application of glycine at 10(-4) M or reduction of Mg2+ concentration in the saline to zero, suggesting the absence of typical NMDA receptors. The variability of the B3 cell responses to NMDA suggested these receptors were unlikely to be the main receptor type involved with N2v --> B3 excitation. Quisqualate and AMPA at 10(-3) M also mimicked N2v inhibitory effects on the B7 and B8 feeding motor neurons and the modulatory slow oscillator (SO) interneuron, providing further evidence for the role of AMPA/quisqualate receptors. Similar effects were seen with glutamate at the same concentration. However, CNQX could not block either glutamate or N2v inhibitory postsynaptic responses on the B7, B8, or SO cells, suggesting a different glutamate receptor subtype for inhibitory responses compared with those responsible for N2v --> B3 excitation. We conclude that glutamate is a strong candidate transmitter for the N2v cells and that AMPA/quisquate receptors of different subtypes are likely to be responsible for the excitatory and inhibitory postsynaptic responses.     

Identification of C1 presympathetic neurons in rat rostral ventrolateral medulla by juxtacellular labeling in vivo

The rostral ventrolateral medulla (RVLM) contains barosensitive, bulbospinal neurons that provide the main supraspinal excitatory input to sympathetic vasomotor preganglionic neurons. However, the phenotype of the critical RVLM cells has not been conclusively determined. The goal of the current study was to identify the proportion of electrophysiologically defined, putative, presympathetic RVLM neurons that are C1 cells. We used a juxtacellular labeling technique to individually fill spontaneously active, barosensitive, bulbospinal RVLM neurons with biotinamide following electrophysiological characterization in chloralose-anesthetized rats. To determine whether these neurons could be classified as C1 cells, the biotinamide-labeled cells were processed for detection of tyrosine hydroxylase. The majority of barosensitive bulbospinal RVLM neurons were tyrosine hydroxylase immunoreactive (TH-ir; 28 of 39). All of the barosensitive bulbospinal RVLM neurons with axonal conduction velocities in the C fiber range (<1 m/second) were TH-ir (n = 16), whereas faster conducting cells (1 to 7 m/second) were either lightly TH-ir (n = 12) or not detectably TH-ir (n = 11). Adjacent respiratory-related RVLM units labeled with biotinamide were not detectably TH-ir (n = 10). To verify that TH-ir cells were indeed adrenergic, a subset of barosensitive bulbospinal cells labeled with biotinamide were examined for phenylethanolamine N-methyltransferase immunoreactivity (PNMT-ir). Three slowly conducting cells had detectable PNMT-ir, and two fast-conducting cells had no detectable PNMT-ir. These results indicate that the majority of bulbospinal RVLM neurons with putative sympathoexcitatory function are C1 cells.     

Effect of testosterone and its metabolites upon the level of vasopressin messenger ribonucleic acid in the hypothalamus of the hyperosmotically stimulated male rat

We examined whether the selective 5-hydroxytryptamine 1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) injected systemically can act directly on sympathoexcitatory neurons located in the rostral ventrolateral medulla (RVLM) to cause the hypotensive effect of this agent in rats. Microinjections of 8-OH-DPAT and buspirone into the RVLM produced a dose-dependent decrease in blood pressure. Microinjections of spiperone and pindolol, 5-HT1A antagonists, into the RVLM inhibited the depressor response to 8-OH-DPAT intravenously injected or injected into the RVLM. Microiontophoretic application of 8-OH-DPAT onto RVLM sympathoexcitatory neurons inhibited the firing of RVLM sympathoexcitatory neurons and the inhibition of unit activity by 8-OH-DPAT was blocked by microiontophoretic spiperone. Intravenous administration of 8-OH-DPAT also inhibited the firing of these neurons. Microiontophoretic application of spiperone onto the RVLM sympathoexcitatory neurons reversed the inhibitory response to intravenous 8-OH-DPAT. These results are consistent with the hypothesis that 8-OH-DPAT may exert a portion of its hypotensive effect through a direct inhibition of RVLM sympathoexcitatory neurons in rats. The receptor involved is probably the 5-HT1A type.     

Imidazoline receptor proteins in brains of patients with Alzheimer''s disease

Experiments have been carried out to investigate the chemical substrate in the rostral ventrolateral medulla (RVLM) underlying the depressor responses induced by activation of the greater splanchnic nerve (GSPL) afferent fibres of the rat. In anaesthetised rats with urethane and alpha-chloralose, microinjection of bicuculline, a GABA(A) receptor antagonist, into the RVLM, attenuated largely the depressor responses elicited by electrical stimulation of the GSPL afferent fibres, while strychnine or saline had no effect. In 18 RVLM neurons (including seven identified cardiovascular neurons), iontophoresis of bicuculline also significantly blocked the inhibition evoked by stimulation of the GSPL afferent inputs. We suggest that the depressor responses induced by stimulation of the GSPL afferent fibres involve a GABA(A)-receptor-mediated mechanism in the RVLM in rats.     

No effect of kinins on DNA synthesis in LNCaP prostate cancer cells

1. Prostate has kininogenase activity and expresses members of the tissue kallikrein gene family. The present study examined the effect of exogenous and endogenous kinins on growth of LNCaP prostate adenocarcinoma cells. 2. Rate of DNA synthesis was measured by incorporation over 4 h of [3H]-thymidine into a TCA insoluble fraction of LNCaP cells that had been cultured for 24 h. 3. Increased [3H]-thymidine incorporation was seen in response to 10 nmol/L testosterone (+103 +/- 5 s.e.%), dihydrotestosterone (+113 +/- 14%) and R1881 (+64 +/- 10%) (P < or = 0.001; n = 4). 4. In contrast 0.05, 5 and 1000 nmol/L lysyl-bradykinin had no effect (15 +/- 4, 10 +/- 9 and 5 +/- 3 s.e.%, respectively; n = 7). Des-Arg9[Leu8]-bradykinin (a B1 receptor antagonist) and/or D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin (a B2 receptor antagonist), 1 nmol/L, and indomethacin, 5 mumol/L, also had little or no effect. 5. In conclusion, kallidin and endogenous kinins and prostaglandins have little or no effect on DNA synthesis and therefore on the growth of LNCaP cells in comparison to the two-fold stimulation produced by androgens.     

Bioethical concepts of health in medicine

The effects of both activation and blockade of dopamine (DA) D1 receptors on long-term depression (LTD) of synaptic transmission were examined in CA1 neurons of rat hippocampal slices. Low frequency stimulation (LFS) consisting of 450 pulses at 1 Hz induced LTD (-14.3%, mean, n = 10) in the slope of the field excitatory postsynaptic potential. SKF-38393 (3-10 microM), an agonist of DA D1 receptors, significantly enhanced LFS-induced LTD (-31.1%, n = 11). SCH-23390 (2 microM), an antagonist of DA D1 receptors, blocked the induction of LTD by LFS (2.5%, n = 6). These results indicate that DA D1 receptors play an important role in the modulation of LFS-induced LTD in rat hippocampal CA1 neurons.     

Modulation by endogenous ATP of the light-evoked release of ACh from retinal cholinergic neurones

The retina is an area of the central nervous system that possesses intrinsic cholinergic neurones which release acetylcholine (ACh) in response to stimulation with flickering light. Using an eye-cup preparation in anaesthetized rabbits we found that when the retina was exposed to the P2-purinoceptor antagonist, PPADS, the light-evoked release of ACh was strikingly increased (by over 40%). In contrast, ATP reduced the light-evoked release of ACh by 20%. The inhibitory effect of ATP was not due to its catabolism to adenosine because it was not affected by the A1-adenosine receptor antagonist, DPCPX, in combination with adenosine deaminase. The actions of both ATP and PPADS were completely blocked by strychnine. We conclude that during physiological stimulation of the retina with light, ATP is co-released with ACh and partially inhibits ACh release by activating (with ACh) an inhibitory glycinergic feedback loop.     

Apomorphine-induced hypoattention in rats and reversal of the choice performance impairment by aniracetam

Intense electrical stimulation of meridian points in the rat inhibits the nociceptive tail withdrawal reflex. The objective of the present study was to determine whether spinal opioid receptors mediate this inhibition. Electrical stimulation was applied with 2 ms square pulses, at 4 Hz for 20 min at 20 times the threshold, to previously defined meridian points in the hindlimb. Threshold was the minimum current required to elicit muscle twitch. In lightly anaesthetized intact rats (n = 8) stimulation inhibited tail withdrawal during and for greater than one hour after the end of stimulation. In unanaesthetized spinal rats (n = 12) this inhibition was less and the post-stimulation effect lasted for 15 min. In control anaesthetized intact (n = 28) and unanaesthetized spinal rats (n = 14) placement of electrodes without stimulation had no effect. In spinal rats, preadministration of naloxone (25 mg/kg, i.p.) blocked the evoked inhibition (n = 11). In intact animals both naloxone (n = 8) and the mu-opioid receptor antagonist, beta-funaltrexamine (10 nmol; n = 9), given via a chronic intrathecal catheter, attenuated inhibitions during and after the end of stimulation by 50-60%. The delta-opioid receptor antagonist H-Tyr-tic psi[CH2NH]Phe-Phe-OH (TIPP[psi]; 10 nmol; n = 7) and the kappa-opioid receptor antagonist nor-binaltorphimine (10 nmol; n = 13) given by lumbar puncture attenuated the inhibition during the stimulation by 30% and 56%, respectively; both antagonists blocked the post-stimulation effect and even facilitated the withdrawal. The data suggest that spinal mu-, delta- and kappa-opioid receptors each contribute to the evoked inhibition.     

A trial of the synthetic malaria vaccine SPf66 in Tanzania: rationale and design

By using acetyl strophanthidin (AS) 0.2 mumol/L, the delayed after-depolarization (DAD) was induced in sheep cardiac Purkinje fibers and recorded with intra-cellular microelectrode. When beta-adrenoceptor was blocked by propranolol 1.0 mumol/L, phenylephrine 1.0 mumol/L increased the amplitude of DAD from 8.1 +/- 2.2 mV to 9.5 +/- 2.8 mV, prolonged the duration of DAD from 240 +/- 47 ms to 273 +/- 47 ms (n = 13, P < 0.01) and increased the up rising velocity of DAD from 0.039 +/- 0.023 V/s to 0.051 +/- 0.026 V/s (n = 13, P < 0.05). The DAD occurred earlier for 30 +/- 47 ms to preceding action potential (n = 13, P < 0.05). When triggered action potentials were induced by norepinephrine 1.0 mumol/L on the basis of DAD, propranolol 1.0 mumol/L could suppress the triggered beats while phentolamine 1.8 mumol/L showed little effect. The above results indicate that excitation of alpha-receptor had only slight augmentation effect on DAD. However, for the triggered activity induced by DAD, the inhibitory effect of beta-blockers are stronger than that of alpha-blockers.     

Pharmacology of the spinal adenosine receptor which mediates the antiallodynic action of intrathecal adenosine agonists

The effects of intrathecally delivered adenosine agonists on allodynia induced by L5/L6 spinal nerve ligation in rats with lumbar intrathecal catheters were examined. Tactile allodynia was assessed by measuring the threshold for evoking withdrawal of the lesioned hind paw with calibrated von Frey hairs. Intrathecal administration of the A1 adenosine selective agonist, N6-(2-phenylisopropyl)-adenosine R-(-)isomer (R-PIA), produced a dose-dependent (0.3-3 nmol; ED50 = 0.6 nmol) antiallodynic action and evoked a delayed motor weakness at a dosage of 30 nmol. Intrathecal administration of the A2 adenosine selective agonist, CGS 21680 {2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride}, also produced a dose-dependent reduction in allodynia (2-40 nmol; ED50 = 15 nmol), but this effect was associated at 40 nmol after a short interval with prominent hind limb weakness. Intrathecal pretreatment with A1/A2 adenosine antagonists, caffeine (20 mumol) and 8-sulfophenyltheophylline (60 nmol), blocked antiallodynic actions of R-PIA (1 nmol) and CGS 21680 (40 nmol). Intrathecal pretreatment with the A1 adenosine-selective antagonist, 8-cyclopentyl-1,3-dimethylxanthine (3 nmol), blocked the antiallodynic effect of R-PIA (1 nmol), but even a dose as high as 10 nmol did not block the effect of CGS 21680 (40 nmol). The A2 adenosine-selective antagonist, 3, 7-dimethyl-1-propargylxanthine (3 nmol), prevented the antiallodynic effects of R-PIA (1 nmol) and CGS 21680 (40 nmol). Pretreatment with caffeine (20 mumol), 8-sulfophenyltheophylline (60 nmol) and 3,7-dimethyl-1-propargylxanthine (3 nmol) prevented the motor dysfunction induced by R-PIA (30 nmol) and CGS 21680 (40 nmol), but 8-cyclopentyl-1,3-dimethylxanthine (3 or 10 nmol) did not. Based on these effects, we hypothesize that the antiallodynic effects are mediated through the activation of spinal A1 adenosine receptors and motor dysfunction effects are mediated through A2 adenosine receptors.     

Sympathoexcitation from the rostral ventrolateral medulla is mediated by spinal NMDA receptors

These studies examined the role of spinal N-methyl-D-aspartic acid (NMDA) receptors in mediating sympathoexcitation evoked by stimulation of neurons in the rostral ventrolateral medulla (RVLM). In urethane-anesthetized rats, blood pressure, heart rate, and splanchnic sympathetic nerve activity (SNA) were recorded. The NMDA receptor antagonist D-2-amino-7-phosphonoheptanoic acid (D-AP7) was administered to the spinal cord via intrathecal (IT) catheter. Blockade of spinal NMDA receptors reduced arterial blood pressure, heart rate, and SNA. Spinal administration of D-AP7 markedly attenuated the pressor and sympathoexcitatory responses evoked by L-glutamate stimulation of the RVLM. The small increases in heart rate evoked by stimulation of the RVLM were not affected by IT administration of D-AP7. These results indicate that NMDA receptors in the spinal cord mediate the pressor and sympathoexcitatory responses evoked by activation of a bulbospinal pathway originating from the RVLM. Moreover, these data suggest that excitatory amino acid neurotransmitters and NMDA receptors in the spinal cord play an important role in the maintenance and regulation of SNA and cardiovascular function.     

Noradrenaline modifies the spontaneous spiking activity of red nucleus neurons in the rat by activation of alpha 2- and beta-adrenoceptors

We have investigated the effects of noradrenaline (NA) on the spontaneous firing activity of red nucleus (RN) neurons recorded extracellularly in anesthetized rats by using an in vivo electrophysiological technique. Microiontophoretic applications of NA (5-100 nA for 30 s) modified the background firing rate in 99 out of 124 neurons and three different patterns of response were observed in distinct cells. In 61% of the responding neurons NA decreased the mean firing rate, whereas 22% of the neurons responded to NA application with an increase of their spiking activity; in a smaller group of cells (17%) NA exerted a biphasic inhibitory/excitatory effect on the spontaneous firing rate. The effects of NA were reversible and dose-dependent. From histological examination, the neurons responding to NA with a purely inhibitory effect were scattered throughout the RN. On the other hand, the neurons responding to NA with an excitation were found to be more numerous in the dorso-medial part of the RN, whereas the neurons in which NA induced biphasic effects appeared to be segregated in the outer lateral portion of the RN. The alpha 2-adrenoceptor antagonist yohimbine completely blocked the inhibitory effect of NA but was unable to antagonize the excitatory response. In addition, the inhibitory effect of NA was mimicked by clonidine, a selective agonist of alpha 2-adrenoceptors; clonidine had no effect on those cells which responded to NA with an increase of the mean firing rate. The excitatory effect of NA was mimicked by the beta-receptor agonist isoprenaline and was antagonized by timolol, a selective antagonist of beta-adrenoceptors. Isoprenaline was ineffective in those cells in which NA exerted inhibitory responses. Taken together, our results indicate that the inhibitory effect of NA on the firing activity of rat RN neurons were mediated by alpha 2-adrenoceptors, whereas beta-adrenoceptors were responsible for the excitatory effects.     

Intrathecally administered baclofen for treatment of children with spasticity of cerebral origin

To determine the relative importance of CCK-A, CCK-B, and opioid receptors in mediating the antinociceptive actions of cholecystokinin, we evaluated the actions of selective agonists and antagonists in the mouse hot plate assay. The agonists used were CCK (1-30 nmol i.c.v.), a CCK-A receptor agonist (SNF9019; 0.3-10 nmol i.c.v.), and a CCK-B receptor agonist (SNF9007; 0.3-10 nmol i.c.v.). The antagonists used were the CCK-A receptor antagonist, L364,718 (12.5 nmol i.c.v.), CCK-B receptor antagonist, L365,260 (2.5-25 nmol i.c.v.), and the nonselective opioid receptor antagonist naloxone (1 mg/kg s.c.). CCK and its receptor-selective analogues, SNF9019 and SNF9007, resulted in antinociception that was blocked by naloxone, but was not antagonized by L364,718 or L365,260. In contrast, in positive control experiments, the inhibitory effects of CCK, SNF9019, and SNF9007 on gastrointestinal propulsion in mice were antagonized by identical i.c.v. doses of L364,718 and L365,260. We conclude that centrally administered CCK produces antinociception in the mouse hot plate assay via opioid receptors, but independent of CCK-A or CCK-B receptors. It is necessary to speculate that other CCK receptors, not antagonized by currently available selective antagonists, may exist.     

Excitatory amino-acid receptors contribute to carotid sinus and vagus nerve evoked excitation of neurons in the nucleus of the tractus solitarius

To determine the role of excitatory amino-acid (EAA) receptors in afferent evoked excitation of neurons in the nucleus of the solitarius (NTS), responses of NTS neurons to activation of visceral afferent inputs were examined before and during iontophoretic application of the broad spectrum EAA receptor antagonist kynurenate (KYN). Iontophoretic application of KYN, at doses which attenuated glutamate but not substance P or acetylcholine evoked discharge, inhibited carotid sinus nerve (CSN) and vagus nerve evoked discharge. KYN attenuation of evoked responses was similar whether the evoked input was monosynaptic (CSN evoked discharge reduced by 50 +/- 6% (mean +/- SE; n = 5); vagus nerve evoked discharge reduced by 45 +/- 4%, n = 6) or polysynaptic (CSN evoked discharge reduced by 48 +/- 6%, n = 6; vagus nerve evoked discharge reduced by 43 +/- 3%, n = 8). Spontaneous action potential discharge rate was reduced during KYN iontophoresis in 6 cells (1.8 +/- 0.4 spikes/s vs. 0.7 +/- 0.2 spikes/s). Iontophoretic application of a structural analogue of KYN which has no EAA receptor antagonist properties, xanthurenic acid, had no effect on glutamate, CSN or vagus nerve evoked discharge. Iontophoretic application of KYN reduced the action potential discharge evoked by activation of the carotid body chemoreceptors by 52 +/- 2% in 5 cells tested. The results demonstrate that excitatory amino-acid receptors are involved in visceral afferent evoked activation of NTS neurons. Furthermore, since both mono- and poly-synaptic inputs were attenuated, these receptors appear to be utilized at multiple levels of afferent integration within NTS.     

Post-synaptic activity evoked in the rostral ventrolateral medullary neurones by stimulation of the defence areas of hypothalamus and midbrain in the rat

Neurones in the rostral ventrolateral medulla (RVLM) of the rat were recorded intracellularly (n = 30) and extracellularly (n = 91) in vivo. 91% of them had spontaneous activity with frequencies of 1.1-29.9 Hz. Onset latencies of the excitation induced by the stimulation of the hypothalamic and midbrain defence areas ranged from 1.5 to 44 ms and 2 to 60 ms respectively. There was no statistical difference between two groups. Excitation followed by inhibition or inhibition followed by excitation was observed in these processes. Excitatory post-synaptic potentials (EPSPs) were summated by simultaneous stimulation of both sites and the onset latency was changed with the change of stimulus intensity. It is concluded that projections from the defence areas of hypothalamus and midbrain are relayed to RVLM neurones forming excitatory and inhibitory synapses; one mechanism of the effect summation caused by both sites is via EPSPs.     

Apoptosis in the male gonad

1. Previous work has shown that enalaprilat, an inhibitor of angiotensin-converting enzyme (ACE), potentiated the actions of alpha 1-adrenoceptor antagonists; it was hypothesized that angiotensin II (AngII) modulated the activity of alpha 1-adrenoceptors. This hypothesis was tested in Sprague-Dawley rat isolated perfused tail arteries using the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD123319. 2. Losartan had no alpha 1-adrenoceptor antagonist effects at concentrations below 1 mumol/L. Similarly, losartan (0.1 mumol/L) had no effect on the alpha 1-adrenoceptor antagonist action of doxazosin (1, 10 nmol/L) nor on the potentiation of doxazosin by enalaprilat (1 mumol/L). 3. PD123319 (0.1 mumol/L) had no alpha 1-adrenoceptor antagonist effect but altered the mode of action of the alpha 1-adrenoceptor antagonist doxazosin: PD123319 changed doxazosin from a competitive to a non-competitive antagonist, as evidenced by the reduced slope of the dose-response curve for the alpha 1-adrenoceptor agonist phenylephrine. 4. These results suggest that AngII can modulate alpha 1-adrenoceptor function in rat tail arteries via an indirect action at AT2 receptors. However, the present results do not rule out the involvement of bradykinin, endothelin or prostaglandin in the modulation of alpha 1-adrenoceptor function by angiotensin II.     

Electrophysiological characterization of 5-HT receptors on rat petrosal neurons in dissociated cell culture

The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT). Here, we combined rapid 5-HT application with patch clamp, whole-cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), cultured from 7-12 day-old rat pups. In responsive cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in approximately 43% of the neurons (53/123); however, in a minority population ( approximately 6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease. In the former group, 5-HT produced a transient inward current (I5-HT) in neurons voltage-clamped near the resting potential ( approximately -60 mV); the effect was mimicked by the 5-HT3 receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT3 receptors. Further, I5-HT was selectively inhibited by the 5-HT3 receptor-specific antagonist MDL72222 (1-10 microM), but was unaffected by either 5-HT1/5-HT2 receptor antagonist, spiperone, or by 5-HT2 receptor-specific antagonist, ketanserin (50-100 microM). I5-HT displayed moderate inward rectification and had a mean reversal potential (+/-S.E.M.) of -4.3+/-6.6 mV (n=6). Application of 5-HT (dose range: 0.1-100 microM) produced a dose-response curve that was fitted by the Hill equation with EC50= approximately 3.4 microM and Hill coefficient= approximately 1.6 (n=8). The activation phase of I5-HT (10 microM 5-HT at -60 mV) was well fitted by a single exponential with mean (+/-S.E.M.) time constant of 45+/-30 ms (n=6). The desensitization phase of I5-HT was best fitted by a single exponential with mean (+/-S.E.M.) time constant of 660+/-167 ms (n=6). Fluctuation analysis yielded an apparent mean single-channel conductance (+/-S.E.M) of 2.7+/-1.5 pS (n=4) at -60 mV. In the minority ( approximately 6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT2 receptor antagonist, ketanserin (50 microM). Taken together, these results suggest that 5-HT3 receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets.