Effect of GABAergic substances on firing rate and thermal coefficient of hypothalamic neurons in the juvenile chicken

The goal of the study is to investigate the GABAergic action on firing rate (FR) and temperature coefficient (TC) on hypothalamic neurons in the juvenile chicken. Extracellular recordings were obtained from 37 warm-sensitive, 32 cold-sensitive and 56 temperature-insensitive neurons in brain slices to determine the effect of GABA(A)-receptor agonist muscimol, GABA(A)-receptor antagonist bicuculline, GABA(B)-receptor agonist baclofen and GABA(B)-receptor antagonist CGP 35348. Muscimol and baclofen in equimolar concentrations (1 microM) significantly inhibited FR of the neurons, regardless of their type of thermosensitivity. In contrast, bicuculline, as well as CGP 35348 (10 microM) increased FR of the majority of the neurons. The TC of most chick hypothalamic neurons could not be estimated during muscimol application because FR was completely inhibited. GABA(B)-receptor agonist specifically increased TC. This effect was restricted to cold-sensitive neurons, which were determined in a high number. The TC was significantly increased (p<0.05) by baclofen and significantly decreased (p<0.05) by CGP 35348. The effects of muscimol and baclofen on FR and TC were prevented by co-perfusion of the appropriate antagonists bicuculline and CGP 35348. The results suggest that the fundamental mechanisms of GABAergic influence on temperature sensitive and insensitive neurons in the chicken PO/AH are conserved during evolution of amniotes.     

γ-氨基丁酸对离体大鼠延髓头端腹外侧区神经元单位放电的抑制作用

在７３张脑片上观察了γ－氨基丁酸（ＧＡＢＡ）对１０６个延髓头端腹外侧区（ＲＶＬＭ）神经元单位放电的影响。外源性的ＧＡＢＡ（０．１￣３．０ｍｍｏｌ／Ｌ）抑制了１０６神经元中的８４个神经元的电活动,这些抑制效应呈剂量－反应关系。ＧＡＢＡ的抑制效应大部分可被ＧＡＢＡＡ受体选择性拮抗剂荷苞牡丹碱甲基碘化物（ＢＭＩ）和Ｃｌ＾－通道阻断剂印防己毒素（ＰＴＸ）所阻断,而单独灌流ＢＭＩ和ＰＴＸ对ＲＶＬＭ神经元主要     

Golgi Cell-Mediated Activation of Postsynaptic GABA(B) Receptors Induces Disinhibition of the Golgi Cell-Granule Cell Synapse in Rat Cerebellum

In the cerebellar glomerulus, GABAergic synapses formed by Golgi cells regulate excitatory transmission from mossy fibers to granule cells through feed-forward and feedback mechanisms. In acute cerebellar slices, we found that stimulating Golgi cell axons with a train of 10 impulses at 100 Hz transiently inhibited both the phasic and the tonic components of inhibitory responses recorded in granule cells. This effect was blocked by the GABA(B) receptor blocker CGP35348, and could be mimicked by bath-application of baclofen (30 μM). This depression of IPSCs was prevented when granule cells were dialyzed with GDPβS. Furthermore, when synaptic transmission was blocked, GABA(A) currents induced in granule cells by localized muscimol application were inhibited by the GABA(B) receptor agonist baclofen. These findings indicate that postsynaptic GABA(B) receptors are primarily responsible for the depression of IPSCs. This inhibition of inhibitory events results in an unexpected excitatory action by Golgi cells on granule cell targets. The reduction of Golgi cell-mediated inhibition in the cerebellar glomerulus may represent a regulatory mechanism to shift the balance between excitation and inhibition in the glomerulus during cerebellar information processing.     

Adrenergic innervation of the gut musculature in vertebrates

Summary The adrenergic innervation of the gut musculature has been compared in various vertebrates (two teleost fish, an amphibian, a reptile and a mammal) by the fluorescent histochemical localization of certain monoamines. Very few, if any, adrenergic nerves occur within the longitudinal gut muscle of any of these animals, except for the taenia coli of the guinea-pig caecum. In contrast, the circular smooth muscle coat is supplied by varicose adrenergic nerves. These nerve fibres are particularly numerous in the toad large intestine, guinea-pig caecum, and throughout the eel gut, but are generally sparse or absent from the musculature of the stomach and small intestine of the trout, toad, lizard and guinea-pig. The extent of adrenergic innervation of the muscle has been discussed in relation to the physiology of the different muscle coats and to the general structure of the enteric plexuses in the vertebrate gut.     

Selective gamma-hydroxybutyric acid receptor ligands increase extracellular glutamate in the hippocampus, but fail to activate G protein and to produce the sedative/hypnotic effect of gamma-hydroxybutyric acid

Two gamma-hydroxybutyric acid (GHB) analogues, trans-gamma-hydroxycrotonic acid (t-HCA) and gamma-(p-methoxybenzyl)-gamma-hydroxybutyric acid (NCS-435) displaced [3H]GHB from GHB receptors with the same affinity as GHB but, unlike GHB, failed to displace [3H]baclofen from GABAB receptors. The effect of the GHB analogues, GHB and baclofen, on G protein activity and hippocampal extracellular glutamate levels was compared. While GHB and baclofen stimulated 5'-O-(3-[35S]thiotriphospate) [35S]GTPgammaS binding both in cortex homogenate and cortical slices, t-HCA and NCS-435 were ineffective up to 1 mm concentration. GHB and baclofen effect was suppressed by the GABAB antagonist CGP 35348 but not by the GHB receptor antagonist NCS-382. Perfused into rat hippocampus, 500 nm and 1 mm GHB increased and decreased extracellular glutamate levels, respectively. GHB stimulation was suppressed by NCS-382, while GHB inhibition by CGP 35348. t-HCA and NCS-435 (0.1-1000 microm) locally perfused into hippocampus increased extracellular glutamate; this effect was inhibited by NCS-382 (10 microm) but not by CGP 35348 (500 microm). The results indicate that GHB-induced G protein activation and reduction of glutamate levels are GABAB-mediated effects, while the increase of glutamate levels is a GHB-mediated effect. Neither t-HCA nor NCS-435 reproduced GHB sedative/hypnotic effect in mice, confirming that this effect is GABAB-mediated. The GHB analogues constitute important tools for understanding the physiological role of endogenous GHB and its receptor.     

Synthesis of [D-Ala2,4'-125I-Phe3,Glu4]deltorphin and characterization of its delta opioid receptor binding properties.

Both [D-Ala2,Glu4]Deltorphin and [D-Ala2,4'-I-Phe3,Glu4]Deltorphin are highly selective ligands for delta, relative to mu, opioid receptors. Radiolabeled [D-Ala2, 4'-125I-Phe3,Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [D-Ala2, 4'-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for mu, delta, and kappa opioid receptors is consistent with binding by the radioligand to a single site having the properties of a delta opioid receptor. The results of these studies are in good agreement with those obtained by structurally different delta opioid receptor ligands. The similarity between the delta receptor site labeled by [125I]Deltorphin and those labeled by other delta receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed.     

Short-term desensitization of phosphatidylinositol turnover via muscarinic acetylcholine receptors and histamine H1-receptors in smooth muscle

Smooth muscle of guinea-pig taenia caecum was desensitized by treatment with 10(-4)M carbachol or 10(-4)M histamine for 30 min in Ca-free solution containing 2mM EGTA. Phosphatidylinositol turnover stimulated by carbachol was not reduced by desensitization with either carbachol or histamine, while the turnover stimulated by histamine was reduced by desensitization with histamine, but not with carbachol. These results are consistent with our previous report (1) that heterologous desensitization induced by carbachol occurs at intracellular Ca stores and homologous desensitization by histamine occurs at H1 receptors.     

Effect of GABA(B) receptor agonist on distension-sensitive pelvic nerve afferent fibers innervating rat colon

Spinal afferents innervating the gastrointestinal tract are the major pathways for visceral nociception. Many centrally acting analgesic drugs attenuate responses of visceral primary afferent fibers by acting at the peripheral site. Gamma-amino butyric acid (GABA), a major inhibitory neurotransmitter, acts via metobotropic GABA(B) and ionotropic GABA(A)/GABA(C) receptors. The aim of this study was to test the peripheral effect of selective GABA(B) receptor agonist baclofen on responses of the pelvic nerve afferent fibers innervating the colon of the rat. Distension-sensitive pelvic nerve afferent fibers were recorded from the S(1) sacral dorsal root in anesthetized rats. The effect of baclofen (1-300 micromol/kg) was tested on responses of these fibers to colorectal distension (CRD; 60 mmHg, 30 s). A total of 21 pelvic nerve afferent fibers was recorded. Mechanosensitive properties of four fibers were also recorded before and after bilateral transections of T(12)-S(3) ventral roots (VR). Effect of baclofen was tested on 15 fibers (7 in intact rats, 4 in rats with transected VR, and 4 in rats pretreated with CGP 54626). In nine fibers (5/7 in intact and 4/4 in VR transected rats), baclofen produced dose-dependent inhibition of response to CRD. Pretreatment with selective GABA(B) receptor antagonist CGP 54626 (1 micromol/kg) reversed the inhibitory effect of baclofen. Results suggest a peripheral role of GABA(B) receptors in the inhibition of mechanotransduction property of distension-sensitive pelvic nerve afferent fibers.     

GABA(B) receptors on vagal afferent pathways: peripheral and central inhibition

To investigate GABA(B) receptors along vagal afferent pathways, we recorded from vagal afferents, medullary neurons, and vagal efferents in ferrets. Baclofen (7-14 micromol/kg i.v.) reduced gastric tension receptor and nucleus tractus solitarii neuronal responses to gastric distension but not gastroduodenal mucosal receptor responses to cholecystokinin (CCK). GABA(B) antagonists CGP-35348 or CGP-62349 reversed effects of baclofen. Vagal efferents showed excitatory and inhibitory responses to distension and CCK. Baclofen (3 nmol i.c.v. or 7-14 micromol/kg i.v.) reduced both distension response types but reduced only inhibitory responses to CCK. CGP-35348 (100 nmol i.c.v. or 100 micromol/kg i.v.) reversed baclofen's effect on distension responses, but inhibitory responses to CCK remained attenuated. They were, however, reversed by CGP-62349 (0.4 nmol i.c.v.). In conclusion, GABA(B) receptors inhibit mechanosensitivity, not chemosensitivity, of vagal afferents peripherally. Mechanosensory input to brain stem neurons is also reduced centrally by GABA(B) receptors, but excitatory chemosensory input is unaffected. Inhibitory mechano- and chemosensory inputs to brain stem neurons (via inhibitory interneurons) are both reduced, but the pathway taken by chemosensory input involves GABA(B) receptors that are insensitive to CGP-35348.     

Gamma-aminobutyric acid modulation of acetylcholine-induced contractions of a smooth muscle from an echinoderm (Sclerodactyla briareus)

This study provides pharmacological evidence for the presence of GABAergic neurons innervating the longitudinal muscle of the body wall (LMBW) of holothurians. Gamma-aminobutyric acid (GABA) A and B receptor subtypes were both present in this system and regulated spontaneous contractions as well as responses to acetylcholine (ACh) that stimulated contraction of the LMBW. GABA dose-dependently relaxed the resting tone of the LMBW. GABA (10(-5) M) inhibited ACh-induced (10(-4) M) contractions by 20%. The GABA B agonist, baclofen, relaxed the LMBW, an effect potentiated by GABA. Pretreatment with baclofen (10(-4) M) inhibited ACh (10(-4) M) contractions of the LMBW by 50%. Phaclofen, a GABA receptor B antagonist, caused a dose-dependent increase in resting tension. Phaclofen-induced (10(-5) M) contractions were reversed by the addition of GABA or baclofen (10(-4) M) and potentiated by the addition of another GABA B receptor antagonist, 2-hydroxy-saclofen (10(-5) M). Pretreatment with phaclofen (10(-5) M) caused a marked potentiation of ACh-induced (10(-4) M) contractions by 101%. 2-Hydroxy-saclofen (10(-5) M) had a toxic effect on the LMBW, rendering it completely unresponsive either to ACh or to a second exposure to GABA, and so exhibiting cross-desensitization. Muscimol, a GABA A receptor agonist, had no effect on the resting tension of the LMBW. Curiously, pretreatment of the muscle with muscimol (10(-5) M) potentiated ACh-evoked (10(-4) M) contractions by nearly 20%. Bicuculline (10(-5) M), a GABA A receptor antagonist, generated large, sustained contractions and partially blocked GABA-induced (10(-4) M) relaxation. Like 2-hydroxy-saclofen, bicuculline (10(-5) M) had a profound cross-desensitizing effect on the LMBW to subsequent exposures to GABA and ACh. ACh was unable to potentiate the sustained contractions induced by bicuculline.     

Role of protein kinase C in regulating smooth muscle electrical and contractile activity: the effect of phorbol ester

The effects of protein kinase C activation by 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) on the functions of guinea-pig smooth muscle taenia coli have been studied, using double-sucrose-gap method. A 15-20-min treatment of the muscle with 2 X 10(-8) M TPA caused a progressing inhibition of spontaneous electrical activity and mechanical tension, suppression of post-hyperpolarizing electrical and contractile "off-responses", a decrease in the number of action potentials during superthreshold membrane depolarization, depression of electrical and mechanical responses induced by acetylcholine, histamine, bradykinin mediators. The treatment of pre-depolarized (140 mM kappa+) muscle with 2.10(-8) TPA has led to a considerable reduction in contractile responses induced by the above mediators. The results obtained indicate that protein kinase C is capable of regulating both voltage-sensitive and receptor-operated ionic channels in smooth muscle cells.     

The role of nitric oxide in mediating non-adrenergic non-cholinergic relaxation in longitudinal muscle of human taenia coli.

Electrical field stimulation (EFS) of isolated longitudinal muscle of human taenia coli at 4Hz produced relaxation which was abolished by tetrodotoxin but not adrenergic and cholinergic blockade (NANC-relaxation). NG-nitro L-arginine (L-NOARG; 1-100 microM), an NO synthesis inhibitor, produced a concentration-dependent partial inhibition of the NANC response; 10 microM L-NOARG inhibited EFS-induced relaxation by 48.6 +/- 5.20% and 100 microM L-NOARG by 54.2 +/- 10.1%. L-Arginine (1mM), but not D-arginine (1mM) partially reversed the inhibitory effect and this was inversely proportional to the concentration of L-NOARG used. Cumulative administration of NO (acidified sodium nitrite solution; 1-100 microM) produced a concentration-dependent relaxation of the strips. L-NOARG (1 mM) did not affect either NO or isoprenaline-induced relaxations. These results provide the first preliminary evidence that NO is partially responsible for the NANC inhibitory transmission in the longitudinal muscle of the taenia coli of human colon.     

Inhibition of nitric oxide synthesis blocks the inhibitory response to capsaicin in intestinal circular muscle preparations from different species

Moderate concentrations of the sensory stimulant drug capsaicin caused relaxation in human and animal intestinal circular muscle preparations (guinea-pig proximal, mouse distal colon, human small intestine and appendix) in vitro. With the exception of the guinea-pig colon, the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 10(-4) M) strongly inhibited the relaxant effect of capsaicin. Tetrodotoxin, an inhibitor of voltage-sensitive Na+ channels failed to significantly reduce the inhibitory effect of capsaicin in the guinea-pig colon, human ileum and appendix; it caused an approximately 50% reduction in the mouse colon. The relaxant effect of capsaicin was strongly reduced in colonic preparations from transient receptor potential vanilloid type (TRPV1) receptor knockout mice as compared to their wildtype controls. It is concluded that nitric oxide, possibly of sensory origin, is involved in the relaxant action of capsaicin in the circular muscle of the mouse and human intestine.     

Use of biological methods in the analysis of the purinergic activity of etimizol

To analyse etimizol purinergic activity, methods of biological analysis were applied using isolated smooth muscle strips of the guinea-pig caecum and developing embryo cultures of the sea urchin (Strongylocentrotus intermedius). Stimulating etimizol action in 10(-8)-10(-12) M concentration was observed on embryonal development in the sea urchin to be eliminated by co-action of etimizol and P1-purinoceptor antagonist theophylline. The analogous action of the preparations was established using isolated smooth muscle strips of the guinea-pig caecum.     

Electrophysiological analysis of the action of kavinton on the smooth muscles

Cavinton at a concentration of 10(-7)-10(-5) M was found to have a dose-dependent relaxing effect on bovine cerebral artery smooth muscles, without changing the resting potential and membrane resistance. Smooth muscles of the rabbit portal vein and guinea-pig taenia coli were insensitive to low cavinton concentrations. The results are consistent with the hypothesis that relaxing action of cavinton is due to the blocking of Ca2+ ions influx into the cells of cerebral artery through receptor-operated calcium channels. At higher concentrations (exceeding 10(-5) M) cavinton exerts nonspecific influence on the smooth muscles under study, inhibiting their excitability and decreasing membrane resistance resulting in the attenuation of tetanic contractions in the smooth muscles of the portal vein and taenia coli.     

Sensitivity of embryonic spontaneous motility to the GABA agonists baclofen and muscimol

The development of the sensitivity of spontaneous motor activity to the GABA agonists baclofen (10 mg/kg egg weight, systemic administration) and muscimol (0.8 mg/kg e.w., systemic administration) was tested in 11-day to 19-day-old chick embryos. 1) Baclofen already significantly depressed the frequency of spontaneous movements in 11-day embryos; its effect attained the maximum (85% depression of spontaneous motility) in 13-day embryos. After the 15th day of incubation, it reduced spontaneous motor activity by 50-60%. In spinal embryos, baclofen had the same, but a quantitatively more pronounced effect, demonstrated from its direct action on the spinal cord uninfluenced by supraspinal modulation, which began to be manifested after the 15th day of incubation. 2) Muscimol did not begin to inhibit spontaneous motility significantly until the 13th day of incubation. Subsequently, the latent period of its effect shortened, its duration lengthened and, lastly, its quantitative result also increased. 3) A comparison of the effect of GABA (Sedlácek 1978), muscimol and baclofen in 17-day chick embryos showed that the depressive effect increased in the sequence baclofen less than GABA less than muscimol, but that GABA took effect faster than the others. The results testify that the maturation of the individual elements of the GABA-ergic central inhibition system is a complex process.     

Relationship between taenia coli electric activity and circular fibers. Study of the human transverse colon

First results of the comparison between the electrical activity of taenia coli and circular muscle are described. Experimental techniques consisted in recording electrical activity for 83 h in 12 patients during the post-operative period after cholecystectomy. Bipolar wire electrodes were surgically inserted in each muscle of the transverse colon's median portion. Statistical treatment leads to the principal conclusion as follows: Whatever type of muscles we have investigated, always could be found slow rhythmic components (called electro-control activity, ECA) in frequency ranges of 1-5 c/min and 9-12 c/min. Major part of taenia coli ECA is in frequency range between 1-5 c/min. ECA in this frequency range can be found in 21% of circular ECA, 23% of taenia coli ECA and 9% of common ECA (% means percents of total recording time). Higher frequencies are found in 7% of circular ECA, 4% of taenia coli ECA and 3% of common ECA. Using long time constant (more than 2 s) the bursts of spikes (10-50 microV) meaning active responses are always accompanied with slower components of important amplitude (greater than or equal to 100 microV): In the discrete mode of activity (DERA) short bursts (during 1-2.5 s) always follow the ECA, whereas in the continuous mode of activity (CERA) mostly longlasting bursts (up to 30 s) could be found. The bursts represent 14% of total time, namely in 8% of longitudinal ECA, 4% of circular ECA and 2% of common ECA. ECA of taenia coli in the case of common ECA is always prolonged. Typical slow waves of greater amplitude (greater than or equal to 500 microV) different from ECA are frequently recorded probably due the intensive contractile process.     

Inhibition of cyclooxygenase-2 and EP1 receptor antagonism reduces human colonic longitudinal muscle contractility in vitro

We investigated the contribution of cyclo-oxygenase enzyme inhibition and prostamide agonism on human colonic contractility in vitro. The effects of the non-specific COX inhibitor diclofenac were compared against selective COX-2 inhibition via nimesulide, the prostanoid EP₁ receptor antagonist SC19220 or the prostaglandin prodrug/prostamide receptor agonist bimatoprost, on potency of contraction to acetylcholine in human colonic circular and longitudinal muscle strips. Pre-treatment with either nimesulide (10^?5 M) or diclofenac (10^?6 M) caused a significant decrease in the potency of acetylcholine-evoked longitudinal muscle contraction, but did not inhibit acetylcholine-evoked circular muscle contraction. Pre-treatment with the EP₁ receptor antagonist SC19220 (10^?5 M) similarly decreased cholinergic potency in longitudinal muscle, without influence on circular muscle contraction. The prostamide agonist bimatoprost (10^?6 M) increased basal circular and longitudinal muscle tone, but did not alter cholinergic potency in either muscle layer. In conclusion, colonic longitudinal muscle contraction is augmented by COX-2 activity, most likely via PGE₂ acting at EP₁ receptors. While colonic contraction is tonically modulated by bimatoprost, it does not share the same functional properties attributed to other endogenous COX-2 metabolites on colonic contractile function.