Interactions ofγ-aminobutyric acid (GABA), pentobarbital,and homopantothenic acid (HOPA) on internally perfused frog sensory neurons

Augmentatory actions among Cl- currents (ICl) induced by gamma-aminobutyric acid (GABA), pentobarbital (PB), and homopantothenic acid (HOPA) were investigated in isolated frog sensory neurons after suppression of Na+, K+, and Ca2+ currents using a suction pipette technique which combines internal perfusion with voltage clamp. GABA-sensitive neurons responded to both PB and HOPA, and the responses behaved as a simple Cl- electrode and reversed at the Cl- equilibrium potential (ECl). The dose-response curve for GABA-induced Cl- conductance was sigmoidal with the GABA concentration producing a half-maximum response (4.2 X 10(-5) M). Both GABA and HOPA dose-response curves shifted to the left in the presence of PB, though the facilitatory action of PB on GABA- and HOPA-induced ICl was more effective in the former. There was a significant facilitatory interaction between GABA- and HOPA-induced ICl. It is concluded that HOPA affects the GABA-GABA or PB-PB receptor interactions.     

Role of uptake inγ-aminobutyric acid (GABA)-mediated responses in guinea pig hippocampal neurons

Intracellular recordings were obtained from hippocampal pyramidal neurons maintained in vitro. Measurements were made of the conductance change induced by iontophoretically applied gamma-aminobutyric acid (GABA) and, using voltage-clamp techniques, of inhibitory postsynaptic currents resulting from activation of inhibitory pathways. Analysis of GABA iontophoretic charge-response curves indicated that there was considerable variation among neurons with respect to the slope of this relation. The placement of the GABA-containing pipette did not appear to be responsible for the observed variation, since vertical repositioning of the pipette did not alter the slope of the charge-response relationship. Steady iontophoresis of GABA from one barrel of a double-barreled pipette markedly affected the charge-response relation obtained when short pulses were applied to the other barrel. The curve was shifted to the left, and the slope was decreased. Concomitantly, the enhanced GABA-induced responses were prolonged. Similar alterations in GABA responsiveness were observed when the uptake blocker, nipecotic acid, was iontophoretically applied. Furthermore, bath application of saline containing a reduced sodium concentration (25% of control) also produced a prolongation of GABA-mediated responses. Under voltage clamp, inhibitory postsynaptic currents were observed to have biphasic decays. The initial, fast decay was prolonged by an average of 18% by nipecotic acid, whereas the later, slow phase was prolonged by 23%. The results of these studies support the hypothesis that a saturable GABA uptake system is responsible for the observed variation in the charge-response curves and, in turn, underlies the apparent sensitizing effect of excess GABA application. The results also suggest that a reduction of transmitter uptake affects the time course of inhibitory postsynaptic currents in the hippocampus.     

Effects ofγ-irradiation on embryonic development and nucleic acids in trigeminal neurons

Summary Pregnant rats were exposed to absorbed doses of 0.5, 1, 2, and 4 Gy of⁶⁰Co- rays either on day 8 or on day 12 p.c.. The embryos were collected on day 18 p.c.. Irradiations both on day 8 and 12 p.c. result in a dose dependent decrement of weight. The effect is larger for irradiation on day 8 p.c.. Caryometric studies of the neurons in the trigeminal ganglion show, on the other hand, that the reduction of nuclear sizes in this tissue is more substantial for an irradiation on day 12 p.c. when the ganglion is in a stage of formation. Cytophotometric determinations of the Feulgen-DNA content and determination of the RNA content by staining with chromic gallocyanin in combination with ribonuclease digestion lead to the conclusion that the irradiation induces no significant hyperploidy and that the irradiated neurons have the nuclear RNA content that is normal at this stage of gestation. This applies both to the irradiations on day 8 and on day 12 p.c.     

Excitatory action of γ-aminobutyric acid (GABA) on crustacean neurosecretory cells

Summary 1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of -aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd²⁺ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a –50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10^–6 M [Ca²⁺]_i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from–25 to–10 mV. At 10^–9 M [Ca²⁺]_i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.     

Differential expression of sodium channel β subunits in dorsal root ganglion sensory neurons

The small-diameter (<25 μm) and large-diameter (>30 μm) sensory neurons of the dorsal root ganglion (DRG) express distinct combinations of tetrodotoxin sensitive and tetrodotoxin-resistant Na(+) channels that underlie the unique electrical properties of these neurons. In vivo, these Na(+) channels are formed as complexes of pore-forming α and auxiliary β subunits. The goal of this study was to investigate the expression of β subunits in DRG sensory neurons. Quantitative single-cell RT-PCR revealed that β subunit mRNA is differentially expressed in small (β(2) and β(3)) and large (β(1) and β(2)) DRG neurons. This raises the possibility that β subunit availability and Na(+) channel composition and functional regulation may differ in these subpopulations of sensory neurons. To further explore these possibilities, we quantitatively compared the mRNA expression of the β subunit with that of Na(v)1.7, a TTX-sensitive Na(+) channel widely expressed in both small and large DRG neurons. Na(v)1.7 and β subunit mRNAs were significantly correlated in small (β(2) and β(3)) and large (β(1) and β(2)) DRG neurons, indicating that these subunits are coexpressed in the same populations. Co-immunoprecipitation and immunocytochemistry indicated that Na(v)1.7 formed stable complexes with the β(1)-β(3) subunits in vivo and that Na(v)1.7 and β(3) co-localized within the plasma membranes of small DRG neurons. Heterologous expression studies showed that β(3) induced a hyperpolarizing shift in Na(v)1.7 activation, whereas β(1) produced a depolarizing shift in inactivation and faster recovery. The data indicate that β(3) and β(1) subunits are preferentially expressed in small and large DRG neurons, respectively, and that these auxiliary subunits differentially regulate the gating properties of Na(v)1.7 channels.     

Does angiotensin II have a significant tonic action on cardiovascular neurons in the rostral and caudal VLM?

The peptidic ANG II receptor antagonists [Sar(1),Ile(8)]ANG II (sarile) or [Sar(1),Thr(8)]ANG II (sarthran) are known to decrease arterial pressure and sympathetic activity when injected into the rostral part of the ventrolateral medulla (VLM). In anesthetized rabbits and rats, the profound depressor and sympathoinhibitory response after bilateral microinjections of sarile or sarthran into the rostral VLM was unchanged after prior selective blockade of angiotensin type 1 (AT(1)) and ANG-(1---7) receptors, although this abolished the effects of exogenous ANG II. Unlike the neuroinhibitory compounds muscimol or lignocaine, microinjections of sarile in the rostral VLM did not affect respiratory activity. Sarile or sarthran in the caudal VLM resulted in a large pressor and sympathoexcitatory response, which was also unaffected by prior blockade of AT(1) and ANG-(1---7) receptors. The results indicate that the peptidic ANG receptor antagonists profoundly inhibit the tonic activity of cardiovascular but not respiratory neurons in the VLM and that these effects are independent of ANG II or ANG-(1---7) receptors.     

Inhibitory effect of tumor necrosis factor α on gluconeogenesis in perfused rat liver

Tumor necrosis factor α (TNFα) is a cytokine involved in many metabolic responses in both normal and pathological states. Considering that the effects of TNFα on hepatic gluconeogenesis are inconclusive, we investigated the influence of this cytokine in gluconeogenesis from various glucose precursors. TNFα (10 μg/kg) was intravenously injected in rats; 6 h later, gluconeogenesis from alanine, lactate, glutamine, glycerol, and several related metabolic parameters were evaluated in situ perfused liver. TNFα reduced the hepatic glucose production (p < 0.001), increased the pyruvate production (p < 0.01), and had no effect on the lactate and urea production from alanine. TNFα also reduced the glucose production (p < 0.01), but had no effect on the pyruvate production from lactate. In addition, TNFα did not alter the hepatic glucose production from glutamine nor from glycerol. It can be concluded that the TNFα inhibited hepatic gluconeogenesis from alanine and lactate, which enter in gluconeogenic pathway before the pyruvate carboxylase step, but not from glutamine and glycerol, which enter in this pathway after the pyruvate carboxylase step, suggesting an important role of this metabolic step in the changes mediated by TNFα.     

Effects of dragon’s blood resin and its component loureirin B on tetrodotoxinsensitive voltage-gated sodium currents in rat dorsal root ganglion neurons

Using whole-cell patch clamp technique on the membrane of freshly isolated dorsal root ganglion (DRG) neurons, the effects of dragon’s blood resin and its important component loureirin B on tetrodotoxin-sensitive (TTX-S) voltage-gated sodium currents were observed. The results show that both blood resin and loureirin B could suppress TTX-S voltage-gated sodium currents in a dose-dependent way. The peak current amplitudes and the steady-state activation and inactivation curves are also made to shift by 0.05% blood resin and 0.2 mmol/L loureirin B. These results demonstrate that the effects of blood resin on TTX-S sodium current may contribute to loureirin B in blood resin. Perhaps the analgesic effect of blood resin is caused partly by loureirin B directly interfering with the nociceptive transmission of primary sensory neurons.     

Modulation of dragon’s blood on tetrodotoxin-resistant sodium currents in dorsal root ganglion neurons and identification of its material basis for efficacy

To clarify the modulation of dragon's blood on the tetrodotoxin-resistant (TTX-R) sodium currents in dorsal root ganglion (DRG) neurons and explore its corresponding material basis for the efficacy, using whole-cell patch clamp technique, the effects of dragon's blood and the combined effects of three components (cochinchinenin A, cochinchinenin B, and loureirin B) extracted from dragon's blood on the TTX-R sodium currents in acute-isolated DRG neurons of rats were observed. According to the operational definition of material basis for the efficacy of TCM established, the material basis of the modulation on the TTX-R sodium currents in DRG neurons of dragon's blood was judged from the experimental results. The drug interaction equation of Greco et al. was used to assess the interaction of the three components extracted from dragon's blood. This investigation demonstrated that dragon's blood suppressed the peak TTX-R sodium currents in a dose-dependent way and affected the activations of TTX-R sodium currents. The effects of the combination of cochinchinenin A, cochinchinenin B, and loureirin B were in good agreement with those of dragon's blood. Although the three components used alone could modulate TTX-R sodium currents, the concentrations of the three components used alone were respectively higher than those used in combination when the inhibition rates on the TTX-R sodium currents of them used alone and in combination were the same. The combined effects of the three components were synergistic. These results suggested that the interference with pain messages caused by the modulation of dragon's blood on TTX-R sodium currents in DRG neurons may explain some of the analgesic effect of dragon's blood and the corresponding material basis for the efficacy is the combination of cochinchinenin A, cochinchinenin B, and loureirin B.     

Do neurons in the vertebrate CNS migrate on laminin?

In adult rat brain the extracellular matrix glycoprotein, laminin, is found only in basement membranes, but is transiently expressed by astrocytes after brain injury. Here, I show that laminin also appears in immature brain cells during CNS development, and that its presence coincides with phases of neuronal migration. In early embryos, laminin is seen throughout the whole thickness of the forming brain, and is apparently synthesized by the cells, as judged by its intracytoplasmic localization. As development proceeds, intracellular laminin becomes restricted to the periventricular regions while punctate deposits of laminin follow the course of vimentin-positive radial glial fibers. In most brain regions, the adult pattern of laminin expression is achieved by birth. In the post-natal rat cerebellum, however, laminin is detected in external granule cells, in Purkinje cells, and in punctate deposits along the radial Bergmann glial fibers. By day 24 after birth, when the migration of external granule cells is complete, all laminin immunoreactivity disappears from these structures. The transient expression of laminin in regions where neurons are migrating raises the possibility that laminin plays a role in neuronal migration during CNS development.     

Effects of dragon’s blood resin and its component loureirin B on tetrodotoxin-sensitive voltage-gated sodium currents in rat dorsal root ganglion neurons

Copyright by Science in China Press 2004 Dragons blood resin is one of famous precious Traditional Chinese Medicine (TCM), which has been widely applied in clinical treatment of cardiovascular disease, cervical spondylosis, gynecological disease, etc., due to its actions of dissipating blood stasis, eas-ing pain, arresting bleeding, promoting tissue regen-eration and wound healing[1]. At present, the investi-gation on the pharmacological mechanism of blood resin is concentrated on promoting…     

Effects of dragon’s blood resin and its component loureirin B on tetrodotoxin-sensitive voltage-gated sodium currents in rat dorsal root ganglion neurons

Using whole-cell patch clamp technique on the membrane of freshly isolated dorsal root ganglion (DRG) neurons, the effects of dragon’s blood resin and its important component loureirin B on tetrodotoxin-sensitive (TTX-S) voltage-gated sodium currents were observed. The results show that both blood resin and loureirin B could suppress TTX-S voltage-gated sodium currents in a dose-dependent way. The peak current amplitudes and the steady-state activation and inactivation curves are also made to shift by 0.05% blood resin and 0.2 mmol/L loureirin B. These results demonstrate that the effects of blood resin on TTX-S sodium current may contribute to loureirin B in blood resin. Perhaps the analgesic effect of blood resin is caused partly by loureirin B directly interfering with the nociceptive transmission of primary sensory neurons.     

Multiple mechanisms of block by the anesthetic isoflurane of a γ-aminobutyric acid activated chloride channel in crayfish

Outside-out patches were excised from the membrane of the deep extensor abdominal muscle (DEAM), containing γ-aminobutyric acid (GABA)- activated chloride channels, in the crayfish Astacus astacus. GABA and isoflurane (iso) were applied in pulses by a liquid filament switch, and their effects on the GABA-elicited chloride currents were investigated. Application of iso alone elicited no current responses and pre-application of iso prior to GABA had no effects on the GABA-elicited current. Co-application of GABA and iso resulted in a reduction of the initial chloride current and subsequent decline of the current to a steady state, indicating that iso binds to the receptor after GABA has bound. Recovery currents at the end of the co-application pulse, their amplitudes decreasing with pulse duration, confirmed this suggestion. Open-time distributions of the blocked channel showed a shift of the long open-time towards a new time constant, indicating a second block mechanism via the long open state A₅O_s of the channel. Removal of GABA and iso after reaching the equilibrium state of the block resulted in recovery currents containing exclusively openings from the long open state A₅O_s, confirming the suggestion of an open channel block only at one of the open states. Accepted: 24 June 1997     

γ-aminobutyric acid in the central nervous system of metamorphosing and mature Manduca sexta

We have begun to examine the factors controlling the accumulation of the neurotransmitter γ-aminobutyric acid (GABA) in the central nervous system (CNS) of the sphinx moth Manduca sexta. Analysis of soluble amino acids in CNS structures from mature moths outlines the regional distribution of GABA. Analysis of amino acids in the antennal lobes (the primary olfactory centres) of Manduca during metamorphosis reveals that GABA accumulates gradually and continuously through most of adult development until eclosion; within 18 hr after eclosion, levels of GABA abruptly increase 27–50%. The activity of the biosynthetic enzyme glutamic acid decarboxylase (EC 4.1.1.15), assayed in extracts of antennal lobes from developing moths, does not change after eclosion. Extracts of hemolymph from mature moths contain low levels of glutamate ( <0.2 mM) and higher levels of certain other amino acids such as serine, glutamine and proline. The concentration of proline in hemolymph increases up to 2-fold after eclosion. Glutamate, glutamine and proline are interconvertible in the CNS, and each can serve as precursor for GABA synthesis both in vivo and in vitro. The efficiency of the precursor role in vitro is similar for each amino acid, as estimated from the ratio of the specific radioactivities of GABA and glutamic acid in the ganglion derived from each precursor. Exogenous proline and glutamine can equilibrate rapidly with the ganglionic pools of the same amino acids while glutamic acid is relatively excluded. Taken together, the findings of this study show that proline and glutamine may contribute substantially to synthesis of GABA in the CNS of M. sexta.     

Mechanism of action of snake venom α-neurotoxins on nicotinic acetylcholine receptors of rabbit sympathetic ganglion neurons

Currents evoked by iontophoretic applications of acetylcholine and postsynaptic currents evoked by single stimulation of the cervical sympathetic nerve were recorded in neurons of the isolated rabbit superior cervical ganglion with membrane voltage clamped and muscarinic acetylcholine receptors blocked by atropine (10^?6 M). The α-neurotoxins from snake venom (α-bungarotoxin and α-cobratoxin) in a concentration of 10^?6 M caused an increase in amplitude (potentiation) of the acetylcholine current, inhibition of that current, or initial potentiation followed by inhibition, in different neurons. Spectral analysis of the fluctuations of this current showed that α-neurotoxins affect neither the current through a single channel nor the duration of the open state of long-living channels (evidently extrasynaptic), but they approximately double the duration of the open state of long-living channels. This last effect in all probability causes potentiation of the acetylcholine current. The α-neurotoxins also depressed the amplitude of the postsynaptic current evoked by sympathetic nerve stimulation (on average by 44%) and lengthened its decline (on average by 24%). It is postulated that α-neurotoxins may both block and modify activity of the receptor-channel complex in the neurons tested, lengthening the duration of its open state. This latter mechanism of action of α-neurotoxins is exhibited only in long-living channels, evidence that the phamacological properties of the two populations of channels connected with nicotinic acetylcholine receptors are not identical.     

Differential blocking actions of 4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) and γ-hexachlorocyclohexane (γ-HCH) on γ-aminobutyric acid- and glutamate-induced responses of American cockroach neurons

4′-Ethynyl-4-n-propylbicycloorthobenzoate (EBOB) has been employed extensively as a radioligand in binding assays to evaluate the pharmacology of γ-aminobutyric acid (GABA)-gated Cl⁻ channels (GABARs) of insects and mammals, and γ-hexachlorocyclohexane (γ-HCH) was used as an insecticide targeting insect GABARs. Since recent studies have shown that not only GABARs but also glutamate-gated chloride channels (GluCls) are blocked by picrotoxinin, dieldrin and fipronil, the actions of EBOB and γ-HCH on native GABARs and GluCls of terminal abdominal ganglion neurons in American cockroach (Periplaneta americana) were tested using patch-clamp electrophysiology. A marked run-down of the GABA- and glutamate-induced responses of the cockroach neurons occurred, when a standard pipette solution was employed, but addition of pyruvate to the solution permitted stable recordings of these responses. With this solution, EBOB and γ-HCH were found to block not only the GABA- but also glutamate-gated responses, with the actions augmented by repeated co-application with the agonists. It was also found that prolonged pre-application of EBOB and γ-HCH prior to co-application with GABA and glutamate resulted in enhanced blocking actions, indicating resting-state actions of the blockers. The blocking actions of EBOB and γ-HCH on the GABA- and glutamate-induced responses were compared by determining IC₅₀ values under steady state condition. The IC₅₀ values for the actions of EBOB on GABAR and GluCls differed less than those of γ-HCH.