Effects of glycine and GABA on bulbar respiratory neurons of cat

1. Bulbar respiratory neurons of unanesthetized, decerebrate cats were impaled with the center pipette of a compound, coaxial microelectrode. This electrode allowed intracellular recording of membrane potential (MP) through the central pipette and extracellular iontophoresis of glycine or gamma-aminobutyric acid (GABA) from micropipettes encircling the center pipette with their tips recessed 20-40 microns from the tip of the center pipette. 2. Seventy-seven studies were carried out on 32 inspiratory and 28 postinspiratory neurons with the use of brief pulses (0.3-0.5 s) or long pulses (3-10 s) spanning one or more respiratory cycles. In both neuronal types, GABA and glycine decreased spike frequency, synaptic "noise," respiratory fluctuations of MP, and "input" resistance in a dose-related fashion. 3. In most cases, the membrane was hyperpolarized by the amino acid. The reverse response (depolarization) was observed when the membrane had been hyperpolarized by current clamp. This reversal from hyperpolarization to depolarization occurred at a MP of -81 +/- 2.3 mV (mean +/- SE, n = 7) for glycine and -81 +/- 1.6 (n = 6) for GABA. 4. After intracellular iontophoresis of chloride ions, application of GABA and glycine depolarized the membrane. 5. During relatively long (3-10 s) periods of iontophoresis of glycine or GABA, the effects on MP and input resistance waned. In some cases (23%), the amino acid depolarized the membrane at the most hyperpolarizated portion of the MP trajectory. This was never observed with brief iontophoretic pulses. Such effects of long duration iontophoresis may reflect changes in membrane properties secondary to the primary action of the amino acid on the membrane of the impaled neuron or indirect synaptic actions via changes in discharge of neighboring neurons. 6. Extracellular iontophoresis of a GABA uptake inhibitor, nipecotic acid, potentiated the effects of GABA. 7. Extracellular application of tetrodotoxin appeared to act pre- and postsynaptically to reduce respiratory fluctuations in membrane potential and to increase input resistance without altering the effects of iontophoresed glycine and GABA, suggesting that the amino acids act on postsynaptic membrane receptors not linked to fast sodium channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of GABA in medullary raphe-evoked modulation of neuronal activity in the periaqueductal grey matter in the rat

Experiments were carried out in urethane-anaesthetised rats to investigate whether GABA is involved in mediating inhibition of neuronal activity in the dorsal half of the periaqueductal grey matter (PAG) after stimulation of the serotonin-containing projection to the PAG from nucleus raphe obscurus (NRO). Multibarrelled micropipettes were used to make recordings from 42 neurones in the dorsal half of the PAG. Most (n = 36) cells were quiescent. Their firing rate was therefore raised to 10-16 Hz by continuous iontophoretic application of DL-homocysteic acid (DLH) in order to facilitate the study of inhibitory events. Iontophoretic application of GABA (0-10 nA) silenced every neurone tested (n = 42), and the effect was blocked by the GABAA antagonist bicuculline (BIC, 10-20 nA; 15/15 cells). BIC also produced an increase in ongoing activity in 14 of 15 cells, indicating the presence of inhibitory GABAergic tone. Iontophoretically applied serotonin (5-HT; 10-70 nA) also inhibited ongoing activity in 9 of 11 cells. The effect of 5-HT was not blocked by BIC. In six of seven cells, microinjection of 100-200 nl DLH into NRO produced a 72.3 +/- 9.4% decrease in neuronal firing rate which was maximal 112 +/- 18 s after the start of the injection and lasted for a total of 313 +/- 63 s. In five of six cells, the raphe-evoked inhibition was blocked by BIC. It is suggested that activation of the serotonergic projection to the PAG from NRO engages GABA-containing interneurones within the PAG which mediate the inhibitory effects of raphe stimulation.     

Adenosine A2A receptors interact with GABAergic pathways to modulate respiration in neonatal piglets

GABA and adenosine contribute to respiratory inhibition in early postnatal life. In this study the adenosine A2A receptor agonist CGS21680 was used to evaluate adenosine receptor specificity and the interrelation of adenosine and GABA in the inhibition of inspiratory drive. In neonatal piglets (n = 10), CGS21680 was injected into the fourth ventricle resulting in apnea and/or decreased burst area and frequency of phrenic discharge. Phrenic burst area decreased to 58.9 +/- 8.6% (S.E.M.) after CGS21680 injection (control = 91.8 +/- 1.0%). Expiratory time increased 261.0 +/- 59.9% after CGS21680 from control (87.7 +/- 2.7%). When bicuculline was injected locally within the rostral ventrolateral medulla (n = 5), or into the fourth ventricle (n = 5), the CGS21680 induced inhibition of phrenic was abolished. To define expression of A2A receptor at the message level (mRNA), we employed in situ hybridization with a digoxigenin-coupled oligonucleotide. Adenosine A2A receptor mRNA was expressed in regions of the medulla oblongata known to contain GABAergic neurons. We conclude that GABAergic inputs affecting respiratory timing and inspiratory drive are modulated by activation of A2A receptors. These findings offer new insight into the mechanism whereby xanthine therapy diminishes apnea of prematurity.     

Role of GABAA and GABAC receptors in the biphasic GABA responses in neurons of the rat major pelvic ganglia.

The role of gamma-aminobutyric acid-A (GABAA) and GABAC receptors in the GABA-induced biphasic response in neurons of the rat major pelvic ganglia (MPG) were examined in vitro. Application of GABA (100 microM) to MPG neurons produced a biphasic response, an initial depolarization (GABAd) followed by a hyperpolarization (GABAh). The input resistance of the MPG neurons was decreased during the GABAd, whereas it was increased during the GABAh. The GABAd could be further separated into the early component (early GABAd) with a duration of 27 +/- 5 s (mean +/- SE; n = 11) and the late component (late GABAd) with a duration of 109 +/- 11 s (n = 11). The duration of the GABAh was 516 +/- 64 s (n = 11). The effects of GABA (5-500 microM) in producing the depolarization and the hyperpolarization were concentration-dependent. GABA (5-30 microM) induced only late depolarizations. The early component of the depolarization appeared when the concentration of GABA was >50 microM. Muscimol produced only early depolarizing responses. Baclofen (100 microM) had no effect on the membrane potential and input resistance of MPG neurons. Bicuculline (60 microM) blocked the early GABAd but not the late GABAd and the GABAh. Application of picrotoxin (100 microM) with bicuculline (60 microM) blocked both the late GABAd and the GABAh. CGP55845A (3 microM), a selective GABAB receptor antagonist, did not affect the GABA-induced responses. cis-4-Aminocrotonic acid (CACA, 1 mM) and trans-4-aminocrotonic acid (TACA, 1 mM), selective GABAC receptor agonists, produced late biphasic responses in the MPG neurons. The duration of the CACA responses was almost the same as those of the late GABAd and GABAh obtained in the presence of bicuculline. Imidazole-4-acetic acid (I4AA, 100 microM), a GABAC receptor antagonist, depressed the late GABAd and the GABAh but not the early GABAd. I4AA (100 microM) and picrotoxin (100 microM) also suppressed the biphasic response to CACA. The early GABAd and the late GABAd were reversed in polarity at -32 +/- 3 mV (n = 7) and -38 +/- 2 mV (n = 4), respectively, in the Krebs solution. The reversal potential of the GABAh was -34 +/- 2 mV (n = 4) in the Krebs solution. The reversal potentials of the late GABAd and the GABAh shifted to -20 +/- 3 mV (n = 5) and -22 +/- 3 mV (n = 5), respectively, in 85 mM Cl- solution. These results indicate that the late GABA(d) and the GABAh are mediated predominantly by bicuculline-insensitive, picrotoxin-sensitive GABA receptors, GABAC (or GABAAOr) receptors, in neurons of the rat MPG.     

Modulation of human GABArho1 receptors by taurine

A study was made of the effects of taurine on GABArho1 receptors expressed in Xenopus oocytes. The EC(50) and reversal potentials for GABA, taurine and glycine currents were 2.3+/-0.4 microM (-25+/-0.9 mV), 5+/-0.8mM (-27+/-0.4 mV) and 7+/-0.5mM (-22+/-0.6 mV), respectively. Co-application of GABA and taurine, revealed a taurine concentration-dependent biphasic-modulation of the receptor: at 0.3-30 microM taurine potentiated the GABA-currents, whereas at 0.3-30 mM the GABA-currents were reduced. In contrast glycine potentiated the GABA-currents at all concentrations tested. TPMPA, a GABA(C) specific receptor antagonist, also blocked effectively and reversibly the taurine and glycine currents. Finally, lanthanum and zinc modulated the currents generated by the three amino acids. Taurine is abundant in the retina and our observations suggest that taurine may play an important role modulating the retinal GABAergic transmission.     

The GABA(B) receptor antagonist CGP 55845A reduces presynaptic GABA(B) actions in neocortical neurons of the rat in vitro.

Use-dependent depression of inhibitory postsynaptic potentials was investigated with intracellular recordings and the paired-pulse paradigm in rat neocortical neurons in vitro. Pairs of stimuli invariably reduced the second inhibitory postsynaptic potential-A (GABA(A) receptor-mediated inhibitory postsynaptic potential) of a pair; at interstimulus intervals of 500 ms, the amplitude of the second inhibitory postsynaptic potential-A was considerably smaller than the first (36.2 +/- 6.2%, n= 17). Decreasing the interstimulus interval reduced the second inhibitory postsynaptic potential-A further and with interstimulus intervals shorter than 330 ms the compound excitatory postsynaptic potential-inhibitory postsynaptic potential response reversed from a hyperpolarizing to a depolarizing response. The depression of the inhibitory postsynaptic potential-A exhibited a maximum at interstimulus intervals near 150 ms and recovered with a time constant of 282 +/- 96.2 ms. Elimination of excitatory transmission by the application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonovaleric acid yielded an essentially unaltered time-course of paired-pulse depression (maximum depression near 150 ms, time constant of recovery 232 +/- 98 ms). The polarity change of the compound excitatory postsynaptic potential response at shorter interstimulus intervals was abolished in the presence of CNQX and D(- )-2-amino-5-phosphonovaleric acid. CNQX and D(-)-2-amino-5-phosphonovaleric acid also reduced the apparent depolarizing shift of the reversal potential between the first and second inhibitory postsynaptic potential-A from about 6 mV to less than 2 mV. Application of the GABA(B) receptor antagonist CGP 55845A in the presence of CNQX and (-)-2-amino-5-phosphonovaleric acid abolished the inhibitory postsynaptic potential-B and paired-pulse depression. Under these conditions, the amplitude of the second inhibitory postsynaptic potential was, on average, about 90% of the first, i.e. reduced by about 10%. The second inhibitory postsynaptic potential-A was approximately constant at interstimulus intervals between 100 and 500 ms. It is concluded that paired-pulse depression of cortical inhibition is predominantly mediated by presynaptic GABA(B) receptors of GABAergic interneurons. The abolition of net inhibition at interstimulus intervals near 330 ms may facilitate spread of excitation and neuronal synchrony during repetitive cortical activation near 3 Hz. This use-dependent depression of inhibition may contribute to highly synchronized slow electroencephalogram activity during spike-and-wave or delta activity.     

Contribution of GABAergic inhibition to the response characteristics of auditory units in the avian forebrain

1. We tested the contribution of GABAergic inhibition to the response characteristics of 213 neurons in the auditory telencephalon of chronically prepared nonanesthetized chickens. Extracellular recordings were obtained with multibarrel glass electrodes containing a tungsten wire. Auditory stimuli consisted of tones, two-tone combinations, and noise bursts presented either free field or via earphones. 2. Response properties of the neurons were studied both before and during iontophoretic application of GABA, glutamate, bicuculline methiodide (BIC), and acetylcholine. 3. During BIC application excitatory responses were facilitated. With the exception of transient off-responses, which occasionally appeared only in the BIC condition, the temporal response patterns to tone stimuli at the units' best frequency usually were unaltered. In no case was an inhibitory response component to binaurally presented pure tones antagonized by BIC. 4. BIC iontophoresis enlarged the isointensity-response areas of the vast majority of neurons in the structures of the auditory forebrain lying postsynaptic to the thalamorecipient layer L2. This effect was not obtained when neurons were depolarized to perithreshold levels with glutamate. 5. Two-tone stimulation resulted in a suppression of the excitatory response to a neuron's best frequency when the second frequency lay outside the excitatory response area. In lamina L2, the frequency range inducing two-tone suppression was narrow, and the suppressive effect was not antagonized by BIC. In the postsynaptic layers, frequencies up to three octaves from the neurons' best frequency induced two-tone suppression that was sensitive to BIC. In addition, these neurons also displayed a BIC-insensitive suppression similar to the one seen in layer L2. 6. Neurons displaying no or only a poor response to white-noise stimulation strongly responded to this wide-band stimulus during BIC iontophoresis. 7. Neurons without tone responses usually displayed clear response areas to tones during BIC application. Iontophoretic application of acetylcholine, but not glutamate, also induced such tone responses. Two-tone combinations with frequencies lying within the response areas observed in the BIC condition elicited excitatory responses after full recovery from the BIC application. 8. During BIC iontophoresis nonmonotonic intensity-response functions were converted to monotonic functions in most of the neurons studied. 9. A model of GABAergic inhibitory interactions is proposed that is based on two independent GABAergic systems.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterisation of rat superficial superior colliculus neurones: firing properties and sensitivity to GABA

Physiological, pharmacological and morphological properties of superficial superior colliculus neurones (n=93) were characterised using whole-cell patch-clamp recordings in rat brain slices. Six cell types (narrow- and wide-field vertical, horizontal, piriform, marginal and stellate) were identified based on Lucifer Yellow labelling but no cell type-specific spike pattern could be identified. Resting membrane potentials were homogeneous (mean: -67.1 +/- 0.7 mV, n=48), and spike frequencies ranged from 10 to 70 Hz (80 pA current injection). About 66% of the cells displayed regular and sustained spike production, throughout all neuronal categories. Rebound spikes and spontaneous activity were observed frequently in all cell types. Synaptically evoked action potentials appeared as single spikes (mean amplitude: 76.0 +/- 3.2 mV, n=34) followed by a fast after-hyperpolarising potential (mean amplitude: 25.4 +/- 1.4 mV, n=34) and variable late potentials (late after-depolarising and/or -hyperpolarising). Pharmacologically, a characterisation using GABA and its subtype-specific agonists indicated a strong inhibitory influence of this transmitter system on >90% of cells. The GABA(A) receptor agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (100 microM), caused a reversible hyperpolarisation (approximately 9 mV) and spike inhibition of all neurones studied. This was more pronounced for intrinsic than for synaptically evoked spikes. Assessment of the GABA(C) receptor agonist, cis-4-aminocrotonic acid (1 mM), also revealed a hyperpolarisation (approximately 3 mV) and an inhibitory action on firing, but this was not as potent and homogeneous, compared to the GABA(A) receptor agonist. Further, the GABA(B) receptor agonist, baclofen (50-100 microM), had more variable (hyperpolarising, depolarising or no change) effects on the membrane potential. It showed little modulation of current-induced action potentials but fully blocked synaptic spikes. Assessment of GABA receptor antagonist actions revealed the presence of weak tonic and strong phasic GABA(A) receptor-mediated inhibition in the superficial superior colliculus: application of the GABA(A) receptor antagonist, bicuculline (100 microM), led to a generally enhanced excitability and depolarisation (approximately 5 mV). Intrinsic firing was somewhat enhanced, but synaptic spiking was drastically potentiated and prolonged. In contrast, 1,2,5,6-tetrahydro-(pyridin-4-yl) methylphosphinic acid (TPMPA; 100 microM), the GABA(C) receptor antagonist, produced little effect on these physiological parameters. The GABA(B) receptor antagonist, CGP35348 (200 microM), caused a partial inhibition of late after-hyperpolarising potentials (approximately 30%). Uptake of GABA contributes little to endogenous inhibition in the superior colliculus slice preparation, as suggested by the action of GABA uptake inhibitors SKF89976 (50-100 microM) and nipecotic acid (200-500 microM), both had no obvious effect on physiological parameters. However, in the presence of these compounds, sub-maximal inhibitory actions of GABA were potentiated.In conclusion, different cell types in the superficial superior colliculus do not display distinct physiological properties and are subject to strong inhibitory modulation. We therefore suggest that signal processing in this brain region does not require cell type-specific encoding of information. In line with evidence provided by previous in vivo investigations, identification of visual stimuli and orientation responses appears to be realised via the network properties of the receptive fields that form topographic maps.     

Inhibitory synaptic plasticity regulates pyramidal neuron spiking in the rodent hippocampus

Spike-timing modifies the efficacy of both excitatory and inhibitory synapses onto CA1 pyramidal neurons in the rodent hippocampus. Repetitively spiking the presynaptic neuron before the postsynaptic neuron induces inhibitory synaptic plasticity, which results in a depolarization of the reversal potential for GABA (E(GABA)). Our goal was to determine how inhibitory synaptic plasticity regulates CA1 pyramidal neuron spiking in the rat hippocampus. We demonstrate electrophysiologically that depolarizing E(GABA) by 24.7 mV increased the spontaneous action potential firing frequency of cultured hippocampal neurons 254% from 0.12+/-0.07 Hz to 0.44+/-0.13 Hz (n=11; P<0.05). Next we used a single compartment model of a CA1 pyramidal neuron to explore in detail how inhibitory synaptic plasticity of feedforward and feedback inhibition regulates the generation of action potentials, spike latency, and the minimum excitatory conductance required to generate an action potential; plasticity was modeled as a depolarization of E(GABA), which effectively weakens inhibition. Depolarization of E(GABA) at feedforward and feedback inhibitory synapses decreased the latency to the 1st spike by 2.27 ms, which was greater that the sum of the decreases produced by depolarizing E(GABA) at feedforward (0.85 ms) or feedback inhibitory synapses (0.02 ms) alone. In response to a train of synaptic inputs, depolarizing E(GABA) decreased the inter-spike interval and increased the number of output spikes in a frequency dependent manner, improving the reliability of input-output transmission. Moreover, a depolarizing shift in E(GABA) at feedforward and feedback synapses triggered by spike trains recorded from CA1 pyramidal layer neurons during field theta from anesthetized rats, significantly increased spiking on the up- and down-strokes of the first half of the theta rhythm (P<0.05), without changing the preferred phase of firing (P=0.783). This study provides the first explanation of how depolarizing E(GABA) affects pyramidal cell output within the hippocampus.     

Behavioral inspiratory inhibition: inactivated and activated respiratory cells

1. Eleven adult cats were trained to stop inspiration in response to a conditioning stimulus. The conditioning stimuli were presented at the onset of inspiration at intervals of approximately 20-30 s. Intratracheal pressures, diaphragmatic activity, and the extracellular activity of single medullary respiratory neurons were recorded while the animals performed this response. 2. Inactivation of the diaphragm to the conditioning stimuli occurred at latencies that varied from 40 to 110 ms and averaged 74 +/- 32 (SD) ms. 3. The subjects of this report are 38 inspiratory neurons that were inactivated and 19 cells that were activated when inspiration was stopped behaviorally. These cells were located in the region of n. ambiguus and the ventrolateral n. of tractus solitarius. 4. The inspiratory cells that were inactivated behaviorally had the following characteristics: 1) Most had an augmenting inspiratory profile with (n = 14) or without (n = 9) postinspiratory activity. Other types were inspiratory throughout (n = 5), decrementing inspiratory (n = 3), tonic inspiratory (n = 4), early inspiratory (n = 2), and expiratory-inspiratory (n = 1). 2) Their mean discharge rate was 39 +/- 2.7 (SE) Hz. 3) The latency of their inactivation in response to the task averaged 81 +/- 4.9 (SE) ms, and 4) Their activity corresponded closely to breathing not only during the behavioral response but also during eupnea (eta 2 = 0.62 +/- 0.04, mean +/- SE) and respiratory acts such as sneezing, sniffing, meowing, and purring. 5. The cells that were activated when inspiration was stopped behaviorally had the following characteristics. 1) As a group, they had discharge profiles related to every phase of the respiratory cycle. 2) They were recorded in the same region as, and often simultaneously with, respiratory cells that were inactivated. 3) Their activity patterns were highly variable such that the signal strength and consistency of the respiratory component of that activity were weak (eta 2 = 0.27 +/- 0.03, mean +/- SE). 4) The latency of their activation in response to the task averaged 58 +/- 2.7 (SE) ms and was significantly shorter than the latency of inactivation of the high eta 2-valued inspiratory cells. 5) This activation was intense and prolonged. 6. It is hypothesized that the activated cells integrate nonrespiratory and respiratory inputs and act to inhibit other respiratory cells during the behavioral inhibition of inspiration.     

Reduction in the myocardial sodium current by halothane and thiamylal

Effects of two general anesthetics, halothane and thiamylal, on the fast sodium inward current (INa) of enzymatically isolated single rat ventricular cells were studied under current clamp and voltage clamp conditions. A suction pipette technique was used for potential measurement, current injection and internal perfusion of isolated cells. In current clamp experiments, sodium action potential was elicited in a Ca-free Co Krebs solution and the action potential was reduced by 0.5% halothane and 5 X 10(-5) M thiamylal. In voltage clamp experiments, the calcium current was suppressed by replacing Ca with Co and the potassium current was eliminated by replacing K with Cs and adding 4-aminopyridine and tetraethylammonium. Both anesthetics decreased INa, in a dose dependent manner, without changing the shape of the current-voltage curve. Halothane (1%) shifted the steady state inactivation curve in a negative direction along the potential axis by 8.5 +/- 2 mV (mean +/- S.D., n = 4). Thiamylal, 5 X 10(-5) and 10(-4) M, shifted the curve in a negative direction by 4.4 +/- 0.8 mV (n = 5) and 8.6 +/- 3.2 mV (n = 5), respectively. Both agents slightly reduced the maximum sodium conductance (gNa). Halothane (1%) increased half recovery time from inactivation measured at -80 mV from 30 +/- 15 to 80 +/- 25 ms (n = 4). Thiamylal (10(-4) M) prolonged it at - 75 mV from 50 +/- 20 to 110 +/- 15 ms (n = 5). With a test pulse duration of 50 ms, neither drug produced a use-dependent inhibition of INa. Halothane and thiamylal depress the INa of cardiac muscles mainly by shifting the steady state inactivation curve in a negative direction along the potential axis. Relatively small prolongation of half recovery time from inactivation and no sign of use-dependent inhibition suggest a molecular mechanism which differs in some respects from the local anesthetics.     

Inhibitory synaptic transmission differs in mouse type A and B medial vestibular nucleus neurons in vitro

Fast inhibitory synaptic transmission in the medial vestibular nucleus (MVN) is mediated by GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs). To assess their relative contribution to inhibition in the MVN, we recorded miniature inhibitory postsynaptic currents (mIPSCs) in physiologically characterized type A and type B MVN neurons. Transverse brain stem slices were prepared from mice (3-8 wk old), and whole cell patch-clamp recordings were obtained from visualized MVN neurons (CsCl internal; Vm = -70 mV; 23 degrees C). In 81 MVN neurons, 69% received exclusively GABA(A)ergic inputs, 6% exclusively glycinergic inputs, and 25% received both types of mIPSCs. The mean amplitude of GABA(A)R-mediated mIPSCs was smaller than those mediated by GlyRs (22.6 +/- 1.8 vs. 35.3 +/- 5.3 pA). The rise time and decay time constants of GABA(A)R- versus GlyR-mediated mIPSCs were slower (1.3 +/- 0.1 vs. 0.9 +/- 0.1 ms and 10.5 +/- 0.3 vs. 4.7 +/- 0.3 ms, respectively). Comparison of type A (n = 20) and type B (n = 32) neurons showed that type A neurons received almost exclusively GABA(A)ergic inhibitory inputs, whereas type B neurons received GABA(A)ergic inputs, glycinergic inputs, or both. Intracellular labeling in a subset of MVN neurons showed that morphology was not related to a MVN neuron's inhibitory profile (n = 15), or whether it was classified as type A or B (n = 29). Together, these findings indicate that both GABA and glycine contribute to inhibitory synaptic processing in MVN neurons, although GABA dominates and there is a difference in the distribution of GABA(A) and Gly receptors between type A and type B MVN neurons.     

GABAA-Dependent chloride influx modulates GABAB-mediated IPSPs in hippocampal pyramidal cells.

The relationship between postsynaptic inhibitory responses [the fast GABA(A)-mediated inhibitory postsynaptic potential (IPSP) and the slow GABA(B)-mediated IPSP] were investigated in hippocampal CA3 pyramidal cells. Mossy fiber-evoked GABA(B)-mediated IPSPs were, paradoxically, of greater amplitude in cells with resting membrane potential of -62 mV (13.6 +/- 0.5 mV; mean +/- SE) as compared with cells with resting membrane potential of -54 mV (7.0 +/- 0.8 mV). In addition, when a cell's membrane potential was artificially manipulated, GABA(B)-mediated IPSPs were reduced at relatively depolarized levels (-55 mV) and enhanced at relatively hyperpolarized potentials (at least -60 mV). In contrast, the preceding GABA(A)-mediated IPSPs were larger at the more positive membrane potentials and smaller as the cell was hyperpolarized. Similar voltage dependency was obtained when monosynaptic GABA(A)- and GABA(B)-mediated IPSPs were isolated in the presence of glutamatergic receptor antagonists. However, monosynaptic GABA(B)-mediated IPSPs isolated in the presence of glutamatergic and GABA(A) receptor antagonists were not reduced at the more positive membrane potentials, and were significantly larger in amplitude than GABA(B)-mediated IPSPs preceded by a monosynaptic GABA(A)-mediated IPSP. The amplitude of the isolated monosynaptic GABA(B)-mediated IPSPs recorded with potassium chloride-containing microelectrodes was significantly smaller than the comparable potential recorded with potassium acetate microelectrodes without chloride. We conclude that voltage-dependent chloride influx, via GABA(A) receptor-gated channels, modulates postsynaptic GABA(B)-mediated inhibition in hippocampal CA3 pyramidal cells.     

Relative contributions of passive equilibrium and active transport to the distribution of chloride in mammalian cortical neurons

1. Active and passive factors affecting the chloride gradient of cortical neurons were assessed using intracellular recordings from neurons in slices of cingulate cortex maintained in vitro. The chloride equilibrium potential (ECl-) was estimated indirectly from the reversal potentials of responses to perisomatic gamma-aminobutyric acid (GABA) application and the Cl(-)-dependent inhibitory postsynaptic potential (IPSP). Under control conditions the mean resting potential (Vm; -69.7 mV) was not significantly different than the mean IPSP reversal potential (EIPSP; -70.1 mV). 2. Increasing the external potassium concentration ([K+]o) from 1 to 10 mM shifted the mean EIPSP from -80.4 to -61.8 mV. The mean EIPSP was approximately equal to the mean Vm at all [K+]oS. The conditions of Donnan equilibrium are not met in [K+]o less than 10 mM. 3. Polarization of Vm up to 20 mV away from EIPSP for 4 min with maintained current injection had no significant effect on EIPSP. 4. The GABA reversal potential was maintained 37-52 mV less negative than Vm after equilibration in saline in which the external chloride concentration had been reduced from 133 to 5 mM by substitution with isethionate. Vm and input resistance were not significantly different from control values in cells recorded under these conditions. 5. We conclude that Cl- is not passively distributed in cortical neurons, perhaps due to a low resting Cl- permeability. 6. Impalement with electrodes containing 2 M KCl resulted in a rapid 10 mV depolarizing shift in EIPSP that then remained relatively constant. Intracellular iontophoresis of Cl- resulted in a further depolarizing shift of EIPSP of 5-10 mV that returned to control in less than 1 min. The time course of recovery of IPSP amplitude could be fit with a single exponential having a mean time constant of 6.9 +/- 1.5 s and was independent of the amount of Cl- injected or stimulation frequency. 7. Reductions in temperature from 37 to 32 degrees C significantly increased the mean time constant of IPSP recovery from Cl- injection to 11.1 +/- 3.3 s, corresponding to Q10 = 2.6.(ABSTRACT TRUNCATED AT 400 WORDS)     

Orexins cause depolarization via nonselective cationic and K+ channels in isolated locus coeruleus neurons

The locus coeruleus (LC) contains noradrenergic neurons that are innervated by orexin (ORX)-like immunoreactive axons and express both orexin receptor-1 and -2. We studied effects of ORX-A and -B (ORX-A/B) on dissociated LC neurons by using whole-cell patch clamp techniques. In current-clamp mode, LC neurons were depolarized by application of ORX-A (10(-7) M) [53% of neurons tested; 9.0+/-0.2 mV (n=5)], or ORX-B (10(-7) M) [38% of neurons tested; 4.0+/-0.1 mV (n=5)]. Firing frequencies of action potentials increased during application [1.1+/-0.2 Hz (n=5) in ORX-A; 0.8+/-0.2 Hz (n=5) in ORX-B] and returned to the control level [0.2+/-0.1 Hz (n=5)] after removal. The ORX-A/B-induced depolarization was well maintained in the presence of TTX (3x10(-7) M), CNQX (10(-6) M) and AP5 (10(-5) M). In voltage-clamp mode, removal of external Na+ suppressed both ORX-A/B-induced currents and shifted their reversal potentials from approximately -45 mV to -60 mV. In addition, ORX-A/B inhibited sustained K+ currents. These results suggest that ORX-A/B increase the firing frequency of LC neurons through the depolarization probably produced by both augmentation of the nonselective cationic conductance and inhibition of the sustained K+ conductance.     

Membrane potential modulates the activation of GABA-gated channels

1. The activity of single gamma-aminobutyric acid (GABA)-gated Cl- channels (GABA = 0.5-2.0 microM) was recorded in inside-out patches of membrane from cultured chick cerebral neurons. 2. The distribution of open intervals of the GABA channel was described by the sum of two exponentials, which suggests the presence of at least two open states of the channel. The time constants of these two components were 0.39 +/- 0.1 and 2.1 +/- 0.9 ms (+/- SD, n = 9). 3. The distribution of shut intervals was described by the sum of either three (n = 5) or four (n = 3) exponentials. This suggests the presence of at least three or four shut states. 4. At all GABA concentrations examined, the activity of the GABA channel decreased over time. This decline in activity was most likely the result of desensitization of the GABA channels. 5. The distribution of open intervals was unchanged during desensitization of the GABA channel. Thus desensitization is not associated with an alteration in either the mean lifetime of the two open states or the relative number of transitions to these two states. Rather, desensitization results from a decrease in the probability of channel opening. 6. There was an e-fold increase in the probability of finding a GABA channel open for every 80 +/- 43 mV (n = 4) of depolarization. The degree of voltage dependence decreased as the GABA channels desensitized. 7. The depolarization-induced increase in open channel probability was not associated with any change in the distribution of open intervals. Thus depolarization does not affect the mean open time of the channel but rather increases the likelihood that it will open. 8. A simple model with three or four shut and two open states is considered for the gating of the GABA channel by the agonist. Possible sites for the voltage dependence within this proposed model are discussed.     

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Rectal gland tubule (RGT) segments of the spiny dogfish (Squalus acanthias) were perfused in vitro. The effects of inhibitors of known mode of action on transepithelial PD (PDte resistance (Rte), the PD across the basolateral membrane (PDbl), the fractional resistance of this membrane (FRbl), and intracellular activities of NA+, Cl-, K+ (apha cell) were examined. Furosemide (5 x 10(-4) mol x 1(-1)) reduced PDte from -12 +/- 0.7 to -2.3 +/- 0.2 mV (n = 63), hyperpolarized PDbl from -71 +/- 1.3 to -79 +/- 0.9 mV (n = 59), FRbl decreased from 0.2 +/- 0.03 to 0.13 +/- 0.01 (n = 21), alpha cell cl- fell from 38 +/- 4 to 11 +/- 2 mmol x 1(-1) (n = 21), alpha cell Na+ fell from 37 +/- 4 to 17 +/- 2 mmol x 1(-1) (n = 12) and alpha cell K+ was constant [113 +/- 14 vs. 117 +/- 15 mmol x 1(-1) (n = 6)]. Furosemide exerted its effects within some 20-40s. Its action was completely reversible. Analysis of the time courses revealed that the furosemide induced initial fall in alpha cell cl- was approximately twice as rapid when compared to that of alpha cell Na+. Ba2+ 0.5 mmol x 1(-1) (bath) reduced PDte from -7.1 +/- 1.2 to -4.1 +/- 0.6 mV (n = 24), increased Rte from 18 +/- 2 to 22 +/- 2.5, omega cm2 (n = 14). PDbl depolarized from -75 +/- 2 to -48 +/- 2 mV (n = 42), FRbl increased from 0.2 +/- 0.02 to 0.34 +/- 0.04 (n = 14) and alpha cell K+ increased from 143 +/-28 to 188 +/- mmol x 1(-1) (n = 4). Ouabain (50 x 10(-6) mol x 1(-1), bath) reduced PDte from -12 +/-2 to -3 +/- 0.5 mV (n = 9), Rte increased from 18 +/- 3 to 21 +/- 3 omega cm2 (n = 5). PDbl depolarized from -67 +/- 4 to -26 + 3 mV (n = 14), FRbl increased from 0.23 +/- 0.04 to 0.45 +/- 0.05 (n = 6), alpha cell K+ fell only slightly from 135 +/- 15 to 112 +/- 30 mmol x 1(-1) (n = 4), but alpha cell cl- increased from 35 +/- 12 to 111 +/- 37 mmol x 1(-1) (n = 3). These effects of ouabain were slow when compared to those exerted by furosemide or Ba2+. The ouabain effects on PDte and PDbl were completely prevented if furosemide was applied first.(ABSTRACT TRUNCATED AT 400 WORDS)     

GABA-mediated inhibition of glutamate release during ischemia in substantia gelatinosa of the adult rat

An ischemia-induced change in glutamatergic transmission was investigated in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by use of the whole cell patch-clamp technique; the ischemia was simulated by superfusing an oxygen- and glucose-free medium (ISM). Following ISM superfusion, 21 of 37 SG neurons tested produced an outward current (23 +/- 4 pA at a holding potential of -70 mV), which was followed by a slow and subsequent rapid inward current; the remaining neurons had only inward currents. During such a change in holding currents, spontaneous excitatory postsynaptic currents (EPSCs) were remarkably decreased in a frequency with time (half-decay time of the frequency: about 65 s). The frequency of spontaneous EPSCs was reduced to 28 +/- 13% (n = 37) of the control level during the generation of the slow inward current (about 4 min after the beginning of ISM superfusion) without a change in the amplitude of spontaneous EPSCs. When ISM was superfused together with either bicuculline (10 microM) or CGP35348 (20 microM; GABA(A) and GABA(B) receptor antagonists, respectively), spontaneous EPSC frequency reduced by ISM recovered to the control level and then the frequency markedly increased [by 325 +/- 120% (n = 22) and 326 +/- 91% (n = 17), respectively, 4 min after ISM superfusion]; this alteration in the frequency was not accompanied by a change in spontaneous EPSC amplitude. Superfusing TTX (1 microM)-containing ISM resulted in a similar recovery of spontaneous EPSC frequency and following increase (by 328 +/- 26%, n = 12) in the frequency; strychnine (1 microM) did not affect ISM-induced changes in spontaneous EPSC frequency (n = 5). It is concluded that the ischemic simulation inhibits excitatory transmission to SG neurons, whose action is in part mediated by the activation of presynaptic GABA(A) and GABA(B) receptors, probably due to GABA released from interneurons as a result of an ischemia-induced increase in neuronal activities. This action might protect SG neurons from an excessive excitation mediated by L-glutamate during ischemia.     

Electrical properties of phrenic motoneurons in the cat: correlation with inspiratory drive

1. Resting membrane potential (Vmp), input resistance (Rn), rheobase (Irh), and after hyperpolarization duration (AHPdur) and amplitude (AHPamp) were measured in 38 phrenic motoneurons of anesthetized, paralyzed, and artificially ventilated cats during hypocapnic apnea. The mean +/- SD and range of values for these variables were as follows: Vmp, -68 +/- 5mV (range: -60 to -82); Rn, 1.3 +/- 0.6 M omega (0.6-2.4); Irh, 9.7 +/- 5 nA (2-20); AHPdur, 68 +/- 19 ms (37-134); AHPamp, 3.3 +/- 1.8 mV (1.0-8.5). In 31 motoneurons, the membrane potential level at which firing occurred (Vthr) during intracellular current injection was measured. The mean value of Vthr was -58 +/- 3 mV (range: -52 to -64). 2. A histogram of Rn revealed a bimodal distribution. Also a plot of Irh against Rn showed a grouping of the motoneurons into two subpopulations: 1) low-Rn and high-Irh cells, called type L neurons, and 2) high-Rn, low-Irh cells, called type H neurons. The overall negative linear correlation between Irh and Rn (r = -0.85; P less than 0.0001) resulted from this grouping rather than from a strictly linear relation between these two variables. 3. Electrical properties were compared for type L (n = 20) and type H (n = 18) phrenic motoneurons. The following mean values were found for each group, respectively: Rn, 0.8 and 1.8 M omega; Irh, 13.7 and 5.3 nA; AHPdur, 58 and 79 ms; AHPamp, 2.4 and 4.4 mV. All differences were significant (t test, P less than 0.001). Mean Vthr was the same for the two groups. 4. Comparison of these data with those available for lumbosacral motoneurons revealed that almost all investigated electrical properties of type L and type H phrenic motoneurons are similar to the analogous properties of type F (fast twitch) and type S (slow twitch) lumbosacral motoneurons, respectively. The apparent exception is the lower mean value of Irh for type L phrenic motoneurons compared with type F lumbosacral motoneurons. 5. For 13 cells, membrane potential was continuously monitored while spontaneous respiratory activity was restored by increasing CO2. It was found that at approximately the same end-tidal CO2 (about 7%) and a similar end-expiratory mean membrane potential level (approximately -70 mV), mean amplitude of peak inspiratory synaptic depolarization was higher in type H motoneurons (8.8 mV, n = 5) than in type L (2.9 mV, n = 8; P less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)     

Secretion of lung fluid by the developing fetal rat alveolar epithelium in organ culture.

We studied lung explants in submersion organ culture to examine the role of the developing fetal alveolar epithelium in the production of lung fluid. Fourteen-day-gestation fetal rat lungs were grown in a collagen gel matrix supplemented with F-12 media and 10% fetal calf serum. In this model, the lung continues to grow, secrete fluid, and become progressively cystic in morphology. There is gradual thinning of the distal epithelial layer, which is lined by alveolar type II cells and their precursors. After 6 to 8 days in culture, we impaled the cyst walls with a microelectrode and continuously recorded the transepithelial potential (psi t). Stable, baseline transepithelial potentials of -1.1 to -6.2 mV (mean +/- SEM = -3.3 +/- 0.11 mV, lumen negative, n = 34) were measured in bicarbonate-buffered Ringer's solution, suggesting active electrolyte transport. When bumetanide, an inhibitor of chloride secretion in other systems, was added to the bathing solution, psi t decreased from a baseline of -3.5 +/- 0.07 mV (mean +/- SEM) to a value of -2.2 +/- 0.07 mV, suggesting chloride transport contributes to the voltage (n = 18, P less than 0.0005). Isoproterenol hyperpolarized psi t from a baseline of -4.3 +/- 1.0 mV to -6.5 +/- 1.0 mV (n = 7, P less than 0.005). 8-(4-Chlorophenylthio) adenosine 3':5'cyclic monophosphate (CPT-cAMP) plus isobutylmethylxanthine (IBMX) similarly hyperpolarized psi t from a baseline of -4.6 +/- 0.4 mV to -7.3 +/- 0.7 mV (n = 11, P less than 0.005). Addition of bumetanide after stimulation with isoproterenol or CPT-cAMP/IBMX depolarized psi t.(ABSTRACT TRUNCATED AT 250 WORDS)