Effects of nocodazole and taxol on glycine evoked currents on rat spinal cord neurones in culture

We examined the effect of altering the cytoskeleton polymerization state by treatment with nocodazole and taxol on glycine-evoked currents in patch-clamp recordings from cultured spinal cord neurones. Adding ATP and GTP to the pipette solution did not prevent the rundown of the peak current. In the absence or in the presence of ATP, the proportion of the non-desensitizing part of the glycine evoked-current declined with time. Adding intracellular GTP and ATP stabilized glycine-evoked responses although the proportion of non-inactivating current was reduced. Nocodazole reduced by itself the proportion of the non-inactivating current whereas taxol (with ATP and GTP) had an opposite effect. These results suggest that the polymerization state of microtubules has functional consequences on glycine receptors.     

Evidence for a non-GABAergic action of quaternary salts of bicuculline on dopaminergic neurones

Intracellular recordings were made from neurones, presumed to be dopaminergic, in the rat midbrain slice preparation. Bicuculline methiodide (BMI) and methochloride (BMC) reversibly blocked the slow, apamin-sensitive component of the afterhyperpolarization in these cells. The IC50 for this effect was about 26 microM. In comparison, BMC antagonized the input resistance decrease evoked by muscimol (3 microM) with an IC50 of 7 microM. The base of bicuculline was less potent in blocking the slow afterhyperpolarization. SR95531 (2-[carboxy-3'-propyl]-3-amino-6-paramethoxy-phenyl-pyridaziniu m bromide), another competitive GABA(A) antagonist, and picrotoxin, a non-competitive GABA(A) antagonist, also blocked the action of muscimol (IC50s: 2 and 12 microM respectively), but had no effect on the afterhyperpolarization at a concentration of up to 100 microM. Moreover, 100 microM SR95531 did not affect the blockade of the afterhyperpolarization by BMC. This blockade persisted in the presence of tetrodotoxin and was apparently not due to a reduction of calcium entry, suggesting that it involved a direct action on the channels which mediate this afterhyperpolarization. These results strongly suggest that quaternary salts of bicuculline act on more than one target in the central nervous system. Thus, the involvement of GABA(A) receptors in a given effect cannot be proven solely on the basis of its blockade by these agents.     

Distribution of GABA, glycine, and glutamate immunoreactivities in the vestibular nuclear complex of the frog

This study describes the localization of gamma-aminobutyric acid (GABA), glycine, and glutamate immunoreactive neurons, fibers, and terminal-like structures in the vestibular nuclear complex (VNC) of the frog by using a postembedding procedure with consecutive semithin sections at the light microscopic level. For purposes of this study, the VNC was divided into a medial and lateral region. Immunoreactive cells were observed in all parts of the VNC. GABA-positive neurons, generally small in size, were predominantly located in the medial part of the VNC. Glycine-positive cells, more heterogeneous in size than GABA-positive cells, were scattered throughout the VNC. A quantitative analysis of the spatial distribution of GABA glycine immunoreactive cells revealed a complementary relation between the density of GABA and glycine immunoreactive neurons along the rostrocaudal extent of the VNC. In about 10% of the immunolabeled neurons, GABA and glycine were colocalized. Almost all vestibular neurons were, to a variable degree, glutamate immunoreactive, and colocalization of glutamate with GABA and/or glycine was typical. GABA, glycine, or glutamate immunoreactive puncta were found in close contact to somata and main dendrites of vestibular neurons. A quantitative analysis revealed a predominance of glutamate-positive terminal-like structures compared to glycine or GABA containing profiles. A small proportion of terminal-like structures expressed colocalization of GABA and glycine or glycine and glutamate. The results are compared with data from mammals and discussed in relation to vestibuloocular and vestibulo-spinal projection neurons, and vestibular interneurons. GABA and glycine are the major inhibitory transmitters of these neurons in frogs as well as in mammals. The differential distribution of GABA and glycine might reflect a compartmentalization of neurons that is preserved to some extent from the early embryogenetic segmentation of the hindbrain.     

The effects of GABA and glycine on horizontal cells of the rabbit retina

Intracellular and patch-clamp recordings have been used to characterize GABA-activated channels in axonless horizontal cells (ALHC) of the rabbit retina. In our intracellular recordings on an everted eyecup preparation, GABA depolarized the horizontal cells (HC), diminished their light response amplitude and slowed the response rise time. Glycine showed similar effects on the HC light responses. In our whole cell patch-clamp recordings on dissociated ALHC, all HCs responded to 3 microM GABA but none to glycine, even at 100 microM. Dose-response relationship for GABA gave EC50 values around 10 microM and Hill slopes of 1.3. Whole-cell current-voltage (I-V) relationships of GABA-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with isothetionate shifted the GABA reversal potential to a more negative value. Muscimol (30 microM), a GABAA agonist mimicked the effect of GABA, but baclofen (30 microM), a GABAB agonist and cis-aminocaprionic acid (30 microM), a GABAC agonist did not elicit any effect on ALHC. Responses to GABA were blocked by the GABAA receptor antagonist bicuculline (10 microM) and picrotoxin (100 microM). According to our results, we conclude that ALHC express GABA receptors coupled to ion channels, and they correspond to GABAA receptor subtypes.     

Calretinin in the cat retina: colocalizations with other calcium-binding proteins, GABA and glycine

Regeneration of skeletal muscle was studied in the sea bream Sparus aurata, in which extensive post-larval muscle hyperplasia contributes to its large adult size, and in the zebrafish Brachydanio rerio, which shows little post-larval hyperplasia and reaches only a small adult size. Small mechanical lesions of body wall muscle were made under general anaesthesia, and the progress of subsequent regeneration was assessed at various intervals by histology and electron microscopy (for general morphology), by immunostaining for desmin and myosin isoforms (to identify the phenotype of new fibres), and by 5'-bromo-2'-deoxyuridine (BrdU) incorporation (to identify proliferating cells). Despite the difference in normal growth-related hyperplasia in these fish, a vigorous regeneration occurred in both species, giving rise to new fibres with an initial myosin composition that differed from that in mature fast-white fibres. However, species differences in myosin expression in these fibres suggest that they may have derived from different myoblast populations. In sea bream, myosin expression in regenerating fibres resembled that seen in new fibres produced in post-larval white muscle, whereas in the zebrafish it resembled that of the primitive monolayer fibres formed during embryonic development. Subsequently, most regenerating fibres gradually transformed into the mature fast-white phenotype in both species.     

Inhibitory action of CGS 21680 on cerebral cortical neurons is antagonized by bicuculline and picrotoxin-is GABA involved?

The possibility of an involvement of endogenously released GABA in the inhibitory actions of A1 and A2a adenosine receptor agonists on rat cerebral cortical neurons discharges was examined using the GABAA antagonists bicuculline and picrotoxin. The A1 agonist N6-cyclopentyladenosine (CPA), the A2a agonist CGS 21680 and the non-selective receptor agonist, adenosine, depressed neuronal firing. Applications of bicuculline or picrotoxin enhanced the spontaneous firing rate of cortical neurons, indicating the presence of ongoing GABA-ergic inhibition. Antagonism of GABAA receptors blocked the depressant effects of CGS 21680 on neuronal firing; was without effect on CPA-evoked inhibitions and tended to reduce the duration of adenosine-evoked inhibitions. These results suggest that the depressant effects of A2a receptor activation are due to an increase in GABA-ergic inhibition, likely as a consequence of increased GABA release. GABA does not appear to be involved in adenosine A1 receptor-mediated inhibition of neuronal firing.     

Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.     

Immunocytochemical evidence for an axonal localization of GABA in the optic nerves of rabbits, rats, and cats

We have examined, by light-microscopic immunocytochemistry, the distribution of GABA in the optic nerves of adult rabbits, rats, and cats. Within the optic nerves, immunoreactivity for GABA was restricted to a small subset of axons; some axons were strongly labelled, others weakly labelled, whilst most axons were unlabelled. Glia and other non-neuronal elements were always unlabelled. Our ability to detect GABA in optic nerve axons of adult mammals contrasts with previous reports that indicate a lack of GABA immunoreactivity in such axons. We suggest that this discrepancy may be due to the sensitivity of our immunocytochemical techniques which enable us to detect low concentrations of GABA.     

Effects of bicuculline and baclofen on paired-pulse depression in the dentate gyrus of epileptic patients

Neurofibromatosis 2 (NF2) is an uncommon, autosomal dominant disorder in which patients are predisposed to neoplastic and dysplastic lesions of Schwann cells (schwannomas and schwannosis), meningeal cells (meningiomas and meningioangiomatosis) and glial cells (gliomas and glial hamartomas). Clinical and genetic criteria that distinguish NF2 from neurofibromatosis 1 have allowed more accurate assignment of specific pathological features to NF2. The NF2 tumor suppressor gene on chromosome 22q12 encodes a widely expressed protein, named merlin, which may link the cytoskeleton and cell membrane. Germline NF2 mutations in NF2 patients and somatic NF2 mutations in sporadic schwannomas and meningiomas have different mutational spectra, but most NF2 alterations result in a truncated, inactivated merlin protein. In NF2 patients, specific mutations do not necessarily correlate with phenotypic severity, although grossly truncating alterations may result in a more severe phenotype. In schwannomas, NF2 mutations are common and may be necessary for tumorigenesis. In meningiomas, NF2 mutations occur more commonly in fibroblastic than meningothelial subtypes, and may cluster in the first half of the gene. In addition, in meningiomas, a second, non-NF2 meningioma locus is probably also involved. Future efforts in NF2 research will be directed toward elucidating the role of merlin in the normal cell and the sequelae of its inactivation in human tumors.     

Effects of quarantine on cats and their owners

The effects of quarantine on 16 cats and their owners were assessed by means of four questionnaires completed by the owners at the beginning of their cat's stay in quarantine, three months later, and two weeks and three months after the cats left quarantine. Changes in body condition were evident in two-thirds of the cats during and at the end of quarantine but not three months later. Mid-way through quarantine, the owners considered their cats were less attached to them, less relaxed, more excitable, more aggressive, more nervous and less playful than before quarantine. When they left quarantine the cats were friendlier, more affectionate and more timid, and three months later they were more affectionate, more nervous and more vocal than before quarantine. When they left quarantine and three months later the cats spent more time with their owners than before quarantine. Most owners visited their cats once or twice a month; the location of the cattery and the limited opening hours restricted the number of visits they made.     

Immunocytochemical localization of GABA and glycine in amacrine and displaced amacrine cells of macaque monkey retina

Recent studies have varied widely in the percentages of GABA- and glycine-immunoreactive (GABA+, GLY+) amacrines reported for primate retina. We compared the distributions of GABA+ and GLY+ amacrines and displaced amacrines at seven locations along the horizontal meridian of macaque retina using postembedding immunogold labeling with silver intensification. The percentage of GABA+ amacrine profiles was higher in central retina (50-55%) than peripheral retina (30-40%), whereas the percentage of GLY+ amacrine profiles did not vary much with eccentricity (52-57%). GABA and glycine were colocalized in 5-20% of amacrines, depending on the eccentricity, whereas 5-30% of amacrines were not immunoreactive for either neurotransmitter. GABA+ amacrines were slightly larger than GLY+ amacrines or Müller cells. In the ganglion cell layer, 5-20% of neurons were labeled for either GABA or glycine and were identified as displaced amacrines. Of these, 53% were GABA+ only, 11% were GLY+ only, and 37% were double-labeled. A few large, very lightly labeled GABA+ cells were identified as ganglion cells. Other features that varied with eccentricity included the linear density of GABA+ and GLY+ amacrines, and the ratio of amacrines to Müller cells.     

Inhibition of glycine currents by dextromethorphan in neurones dissociated from the guinea-pig nucleus tractus solitarii

1. The effect of dextromethorphan (DM) on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea-pigs was studied by use of the whole-cell patch clamp technique. The effect of DM on gamma-aminobutyric acid (GABA)-induced currents (IGABA) was also examined. 2. DM inhibited 30 microM glycine-induced current (IGly), without affecting the current caused by 30 microM GABA. The action of DM was concentration-dependent, with a maximum effect at 100 microM, and reversible. The half-maximum inhibitory concentration (IC50) of DM was 3.3 microM, about 85 times higher than that of strychnine. 3. DM 3 microM shifted the concentration-response curve for glycine to the right without affecting the maximum value. DM 10 microM shifted the curve even more to the right, although it was not a parallel shift. Strychnine at a concentration of 0.1 microM shifted the curve for glycine in a nearly parallel fashion. 4. The effect of 10 microM DM was slightly weak voltage-dependency, but the lower concentration of DM, 3 microM, inhibited IGly equally at -50 mV and +50 mV. The effect of 3 microM DM on IGly showed no use-dependence. Blockade by strychnine 0.1 microM showed no voltage- or use-dependence. 5. The results indicate that DM inhibits IGly in single neurones of NTS, and further suggest that DM at a low concentration may act on the glycine receptor-ionophore complex, but not on the Cl channel of the complex. However, a relatively high concentration of DM may at least partly affect the Cl- channel of the complex.     

Differential intracellular regulation of cortical GABA(A) and spinal glycine receptors in cultured neurons

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.     

Effects of arachidonic, linoleic, linolenic and oleic acid on experimental arrhythmias in cats, rabbits and guinea-pigs

Antiarrhythmic effects of the Prostaglandin (PG) precursors arachidonic and Linoleic acid were demonstrated on three models of experimental arrhythmias, whereas the fatty acids linolenic and oleic acid proved to be ineffective in these models. In ouabain-induced arrhythmias infusions of arachidonic acid (1, 0 mg/kg/min) caused a strong antiarrhythmic effect in 80 percent of the animals. On the same model linoleic acid showed a maximum effct in 40 percent of the animals. BaCl2-induced arrhythmias were abolished by arachidonic and linoleic acid in 60 percent and 66 percent of the rabbits, respectively. Pretreatment by indomethacin reduced the antiarrhythmic effects of linoleic acid from 40 percent to 9 percent on ouabain-induced arrhythmias in cats. The results suggest a participation of PG synthesis in the antiarrhythmic effect of PG precursors.     

Effects of ketamine on somatosympathetic reflex discharges in cats

Effects of ketamine on somatosympathetic reflex discharges induced from sympathetic trunk with electrical stimulation of superficial peroneal nerve were investigated in 51 cats under anesthesia with urethane and alpha chloralose. These reflex discharges through spinal cord and medulla oblongata consist of two components, A and C reflexes, which are derived from somatic myelinated and unmyelinated afferent fiber respectively. Amplitudes of both A and C reflex potentials were depressed significantly after intravenous injection of ketamine 10 mg.kg-1. The maximum depression was observed 5 min after administration. In decerebrated cats with surgical transection at the midbrain, both A and C reflexes were also depressed after administration of the same dosages, and the maximum level of the depression was more profound than that in brain intact cats. After intrathecal injection of ketamine 1 mg.kg-1 to the lumbar spinal region, a slight depression of C reflex was found, but, dosages of 10 mg.kg-1 significantly depressed both A and C reflexes to the similar levels as those in iv injection to brain intact cats. The maximum depression was observed 30 min after administration. The depressive effects on both reflexes of intravenous ketamine 10 mg.kg-1 were not antagonized by naloxone 0.06 mg.kg-1 in brain intact cats. These results suggest that the suppressive effects of ketamine on somatosympathetic reflexes are caused by direct inhibition of medulla oblongata and spinal cord, whereas supra-midbrain regions may be activated by ketamine, and the effect of ketamine is predominant on medulla oblongata in this situation rather than on the spinal cord.     

Currents evoked by GABA and glycine in acutely dissociated neurons from the rat medial preoptic nucleus

The responses of acutely dissociated medial preoptic neurons to application of GABA, and glycine were studied using the perforated-patch whole-cell recording technique under voltage-clamp conditions. GABA, at a concentration of 1 mM, evoked outward currents in all cells (n = 33) when studied at potentials positive to -80 mV. The I-V relation was roughly linear. The currents evoked by GABA were partially blocked by 25-75 microM picrotoxin and were also partially or completely blocked by 100-200 microM bicuculline. Glycine, at a concentration of 1 mM, did also evoke outward currents in all cells (n = 12) when studied at potentials positive to -75 mV. The I-V relation was roughly linear. The currents evoked by glycine were largely blocked by 1 microM strychnine. In conclusion, the present work demonstrates that neurons from the medial preoptic nucleus of rat directly respond to the inhibitory transmitters GABA and glycine with currents that can be attributed to GABAA receptors and glycine receptors respectively.     

Activities of expiratory neurones of the B?tzinger complex during vocalization in decerebrate cats

Repetitive electrical stimulation of the midbrain peri-aqueductal grey (PAG) terminates quiet breathing and initiates inspiration that precedes vocalization. To understand the neuronal mechanisms underlying this phenomenon, activities of expiratory neurones (n = 39) of the B?tzinger complex (BOT) were examined in decerebrate cats. Most augmenting expiratory (E-aug) neurones (20/22) of the BOT, including 15 bulbospinal neurones, decreased their activities (9/20) or ceased to discharge (11/20) after the onset of stimulation of the PAG. This suggests that suppression of E-aug neurones of the BOT, which project to phrenic motoneurones, results in disinhibition of these neurones, and, in turn, terminates expiration and initiates inspiration preceding vocalization.     

Effects of GABA on horizontal cells in the tiger salamander retina

Application of gamma-aminobutyric acid (GABA) slows down the horizontal cell response time course (HCRRT) and induces membrane depolarization in horizontal cells (HCs) after synaptic inputs are blocked by Co2+. We present evidence that suggests both effects are probably mediated by GABAA receptors which open chloride channels in the HC membrane. In any given concentration of GABA, ranged from 0 to 100 microM, the HC membrane potential (VHC) in saturating light and in the presence of 100 microM Co2+ are identical. This result suggests that GABA in both light and 100 microM Co2+ opens the same amount of chloride channels (same gCl) so that VHC determined by chloride and leak conductances has the same value. Higher concentrations of Co2+ (> 300 microM) not only blocks synaptic transmission from photoreceptors to HCs, but also acts as an antagonist that suppresses the GABA-mediated depolarization in HCs.