Presynaptic Regulation of Tonic Inhibition by Neuromodulatory Transmitters in the Basal Amygdala.

Tonic inhibition mediated by ambient levels of GABA that activate extrasynaptic GABAA receptors emerges as an essential factor that tunes neuronal network excitability in vitro and shapes behavioral responses in vivo. To address the role of neuromodulatory transmitter systems on this type of inhibition, we employed patch clamp recordings in mouse amygdala slice preparations. Our results show that the current amplitude of tonic inhibition (Itonic) in projection neurons of the basal amygdala (BA) is increased by preincubation with the neurosteroid THDOC, while the benzodiazepine diazepam is ineffective. This suggests involvement of THDOC sensitive δ subunit containing GABAA receptors in mediating tonic inhibition. Moreover, we provide evidence that the neuromodulatory transmitters NE, 5HT, and ACh strongly enhance spontaneous IPSCs as well as Itonic in the BA. As the increase in frequency, amplitude, and charge of sIPSCs by these neuromodulatory transmitters strongly correlated with the amplitude of Itonic, we conclude that spill-over of synaptic GABA leads to activation of Itonic and thereby to dampening of amygdala excitability. Since local injection of THDOC, as a positive modulator of tonic inhibition, into the BA interfered with the expression of contextual fear memory, our results point to a prominent role of Itonic in fear learning.

The Relation Between Long-Term Synaptic Plasticity at Glutamatergic Synapses in the Amygdala and Fear Learning in Adult Heterozygous BDNF-Knockout Mice.

Brain-derived neurotrophic factor (BDNF) heterozygous knockout mice (BDNF+/- mice) show fear learning deficits from 3 months of age onwards. Here, we addressed the question how this learning deficit correlates with altered long-term potentiation (LTP) in the cortical synaptic input to the lateral amygdala (LA) and at downstream intra-amygdala synapses in BDNF+/- mice. Our results reveal that the fear learning deficit in BDNF+/- mice was not paralleled by a loss of LTP, neither at cortical inputs to the LA nor at downstream intra-amygdala glutamatergic synapses. As we did observe early fear memory (30 min after training) in BDNF+/- mice while long-term memory (24 h post-training) was absent, the stable LTP in cortico-LA and downstream synapses is in line with the intact acquisition of fear memories. Ex vivo recordings in acute slices of fear-conditioned wildtype (WT) mice revealed that fear learning induces long-lasting changes at cortico-LA synapses that occluded generation of LTP 4 and 24 h after training. Overall, our data show that the intact LTP in the tested amygdala circuits is consistent with intact acquisition of fear memories in both WT and BDNF+/- mice. In addition, the lack of learning-induced long-term changes at cortico-LA synapses in BDNF+/- mice parallels the observed deficit in fear memory consolidation.

Impaired transmission at corticothalamic excitatory inputs and intrathalamic GABAergic synapses in the ventrobasal thalamus of heterozygous BDNF knockout mice.

Beside its role in development and maturation of synapses, brain-derived neurotrophic factor (BDNF) is suggested to play a critical role in modulation and plasticity of glutamatergic as well as GABAergic synaptic transmission. Here, we used heterozygous BDNF knockout (BDNF(+/-)) mice, which chronically lack approximately 50% of BDNF of wildtype (WT) animals, to investigate the role of BDNF in regulating synaptic transmission in the ventrobasal complex (VB) of the thalamus. Excitatory transmission was characterized at glutamatergic synapses onto relay (TC) neurons of the VB and intrathalamic inhibitory transmission was characterized at GABAergic synapses between neurons of the reticular thalamic nucleus (RTN) and TC neurons. Reduced expression of BDNF in BDNF(+/-) mice did not affect intrinsic membrane properties of TC neurons. Recordings in TC neurons, however, revealed a strong reduction in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in BDNF(+/-) mice, as compared to WT littermates, whereas mEPSC amplitudes were not significantly different between genotypes. A mainly presynaptic impairment of corticothalamic excitatory synapses in BDNF(+/-) mice was also indicated by a decreased paired-pulse ratio and faster synaptic fatigue upon prolonged repetitive stimulation at 40 Hz. For miniature inhibitory postsynaptic currents (mIPSCs) recorded in TC neurons, both, frequency and amplitude showed a significant reduction in knock-out animals, concurrent with a prolonged decay time constant, whereas paired-pulse depression and synaptic fatigue of inhibitory synapses were not significantly different between WT and BDNF(+/-) mice. Spontaneous IPSCs (sIPSCs) recorded in VB neurons of BDNF(+/-) animals showed a significantly reduced frequency. However, the glutamatergic drive onto RTN neurons, as revealed by the percentage reduction in frequency of sIPSCs after application of AMPA and NMDA receptor blockers, was not significantly different. Together, the present findings suggest that a chronically reduced level of BDNF to ∼50% of WT levels in heterozygous knock-out animals, strongly attenuates glutamatergic and GABAergic synaptic transmission in thalamic circuits. We hypothesize that this impairment of excitatory and inhibitory transmission may have profound consequences for the generation of rhythmical activity in the thalamocortical network.

Modulation by extracellular pH of low- and high-voltage-activated calcium currents of rat thalamic relay neurons.

The effects of changes in the extracellular pH (pH(o)) on low-voltage- (LVA) and high-voltage- (HVA) activated calcium currents of acutely isolated relay neurons of the ventrobasal thalamic complex (VB) were examined using the whole cell patch-clamp technique. Modest extracellular alkalinization (pH 7.3 to 7.7) reversibly enlarged LVA calcium currents by 18.6 +/- 3.2% (mean +/- SE, n = 6), whereas extracellular acidification (pH 7.3 to 6.9) decreased the current by 24.8 +/- 3.1% (n = 9). Normalized current amplitudes (I/I(7.3)) fitted as a function of pH(o) revealed an apparent pK(a) of 6.9. Both, half-maximal activation voltage and steady-state inactivation were significantly shifted to more negative voltages by 2-4 mV on extracellular alkalinization and to more positive voltages by 2-3 mV on extracellular acidification, respectively. Recovery from inactivation of LVA calcium currents was not significantly affected by changes in pH(o). In contrast, HVA calcium currents were less sensitive to changes in pH(o). Although extracellular alkalinization increased maximal HVA current by 6.0 +/- 2.0% (n = 7) and extracellular acidification decreased it by 11.9 +/- 0.02% (n = 11), both activation and steady-state inactivation were only marginally affected by the moderate changes in pH(o) used in the present study. The results show that calcium currents of thalamic relay neurons exhibit different pH(o) sensitivity. Therefore activity-related extracellular pH transients might selectively modulate certain aspects of the electrogenic behavior of thalamic relay neurons.

Activity-related changes in intracellular pH in rat thalamic relay neurons.

Activity-related shifts in intracellular pH (pHi) can exert potent neuromodulatory actions. Different states of neuronal activity of thalamocortical neurons were found to differentially modulate pHi. Tonic activity evoked by injection of depolarizing current led to a reversible rise in [H+]i which was nearly abolished in the presence of TTX. Block of voltage-gated calcium channels with I mM Ni2+ reduced the [H+]i transients related to tonic activity. Rhythmic activation of burst discharges caused changes of [H+]i which were decreased by TTX, whereas I mM Ni2+ almost abolished the [H+]i transients. The present results show that different forms of neuronal activity can lead to intracellular acidification caused by different mechanisms, i.e. Na+ and Ca2+ influx through sodium and Ca2+ channels, respectively, and the subsequent activation of a Ca2+/H+ pump. The resulting acidosis is suggested to reduce further Ca2+ influx and prevent excessive neuronal excitation.

Leech giant glial cell: functional role in a simple nervous system.

The giant glial cell in the central nervous system of the leech Hirudo medicinalis has been the subject of a series of studies trying to link its physiological properties with its role in neuron-glia interactions. Isolated ventral cord ganglia of this annelid offer several advantages for these studies. First, single giant glial cells can easily be identified and are quite accessible to electrophysiological and microfluorometric studies. Second, only two giant macroglial cells are located in the neuropil of each ganglion, rendering them well suited for studying neuron-glia interactions. Third, many neurons can be identified and are well known with respect to their physiology and their roles in controlling simple behaviors in the leech. This review briefly outlines the major recent findings gained by studying this preparation and its contributions to our knowledge of the functional role of glia in nervous systems. Emphasis is directed to glial responses during neuronal activity and to the analysis of intracellular Ca(2+) and H(+) transients mediated by neurotransmitter receptors and ion-driven carriers. Among its numerous properties, the leech giant glial cell prominently expresses a large K(+) conductance, voltage-dependent Ca(2+) channels, ionotropic non-NMDA glutamate receptors, and an electrogenic, reversible Na(+)-HCO(3)(-) cotransporter.

Modulation of the hyperpolarization-activated cation current of rat thalamic relay neurones by intracellular pH.

1. Properties of the hyperpolarization-activated cation current (Ih) were investigated in thalamocortical neurones of an in vitro slice preparation of the rat ventrobasal thalamic complex (VB) before and during changes of pipette pH (pHp), intracellular pH (pHi) and bath pH (pHb) using the whole-cell patch-clamp technique and fluorescence ratio imaging of the pH indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). 2. Recording of Ih with predefined pHp revealed significant shifts in the voltage dependence of Ih activation (V ) of 4-5 mV to more positive values for a pHp of 7.5 and 2-3 mV to more negative values for a pHp of 6.7 as compared to control values (pHp = 7.1). 3. Application of the weak acid lactate (20 mM), which produced a slow monophasic intracellular acidification, induced a reversible negative shift of V of up to 3 mV. Application of 20 mM TMA, which caused a distinct intracellular alkalinization, shifted V to 4-5 mV more positive values. 4. In slices bathed in Hepes-buffered saline, no significant pHo dependence of Ih was observed. Changing pHo by altering the extracellular [HCO3-] in the presence of constant pCO2 also revealed no significant pHo dependence of Ih. 5. Rhythmic stimulation of thalamocortical neurones with repetitive depolarizing pulse trains caused an intracellular acidification, which reversibly decreased the amplitude and time course of activation of Ih. 6. The results of the present study indicate that shifts in pHi result in a significant modulation of the gating properties of Ih channels in TC neurones. Through this mechanism activity-dependent shifts in pHi may contribute to the up- and downregulation of Ih.

Upregulation of the hyperpolarization-activated cation current in rat thalamic relay neurones by acetazolamide.

1. The effect of inhibition of brain carbonic anhydrase (CA) on the hyperpolarization-activated cation current (Ih) of thalamocortical (TC) neurones of the rat ventrobasal thalamic complex (VB) was investigated in an in vitro slice preparation using the whole-cell patch-clamp technique and fluorescence ratio imaging of the pH indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). 2. Recording of Ih before and after addition of 0.4-0.8 mM acetazolamide to the bathing fluid revealed a significant shift in the voltage dependence of activation (V ) of 5-7 mV to more positive potentials. 3. Simultaneous recording of Ih and BCECF fluorescence ratio (F420/F495) revealed an increase in Ih amplitude accompanied by an intracellular alkalinization upon application of acetazolamide. The CA inhibitor ethoxyzolamide (EZA, 50 microM) also led to an intracellular alkalinization and a subsequent 4-5 mV positive shift of V of Ih. 4. Acetazolamide and EZA both profoundly slowed the rapid fall of pHi upon switching from Hepes- to CO2/HCO3--buffered superfusate, indicating intracellular CA isoforms in TC neurones. 5. In slices bathed in Hepes-buffered saline, addition of acetazolamide had no effect on the amplitude and time course of activation of Ih, indicating that the action of acetazolamide on Ih was dependent on the presence of HCO3-. 6. Under current-clamp conditions, the neuronal response to hyperpolarizing current pulses in the presence of acetazolamide was decreased as compared to control. This resulted in a strongly reduced ability of TC neurones to produce rebound Ca2+-mediated spikes. 7. The present results implied that in TC neurones acetazolamide led to an intracellular alkalinization which causes, due to its pH sensitivity, an increase in Ih.

Distribution of L-type calcium channels in rat thalamic neurones.

One major pathway for calcium entry into neurones is through voltage-activated calcium channels. The distribution of calcium channels over the membrane surface is important for their contribution to neuronal function. Electrophysiological recordings from thalamic cells in situ and after acute isolation demonstrated the presence of high-voltage activated calcium currents. The use of specific L-type calcium channel agonists and antagonists of the dihydropyridine type revealed an about 40% contribution of L-type channels to the total high-voltage-activated calcium current. In order to localize L-type calcium channels in thalamic neurones, fluorescent dihydropyridines were used. They were combined with the fluorescent dye RH414, which allowed the use of a ratio technique and thereby the determination of channel density. The distribution of L-type channels was analysed in the three main thalamic cell types: thalamocortical relay cells, local interneurones and reticular thalamic neurones. While channel density was highest in the soma and decreased significantly in the dendritic region, channels appeared to be clustered differentially in the three types of cells. In thalamocortical cells, L-type channels were clustered in high density around the base of dendrites, while they were more evenly distributed on the soma of interneurones. Reticular thalamic neurones exhibited high density of L-type channels in more central somatic regions. The differential localization of L-type calcium channels found in this study implies their predominate involvement in the regulation of somatic and proximal dendritic calcium-dependent processes, which may be of importance for specific thalamic functions, such as those mediating the transition from rhythmic burst activity during sleep to single spike activity during wakefulness or regulating the relay of visual information.

Lack of regulation by intracellular Ca2+ of the hyperpolarization-activated cation current in rat thalamic neurones.

1. The regulation of the hyperpolarization-activated cation current, Ih, in thalamocortical neurones by intracellular calcium ions has been implemented in a number of mathematical models on the waxing and waning behaviour of synchronized rhythmic activity in thalamocortical circuits. In the present study, the Ca2+ dependence of Ih in thalamocortical neurones was experimentally investigated by combining Ca2+ imaging and patch-clamp techniques in the ventrobasal thalamic complex (VB) in vitro. 2. Properties of Ih were analysed before and during rhythmic stimulation of Ca2+ entry by trains of depolarizing voltage pulses. Despite a significant increase in intracellular Ca2+ concentration ([Ca2+]i) from resting levels of 74 +/- 23 nM to 251 +/- 78 nM upon rhythmic stimulation, significant differences in the voltage dependence of Ih activation did not occur (half-maximal activation at -86.4 +/- 1.3 mV vs. -85.2 +/- 2.9 mV; slope of the activation curve, 11.2 +/- 2.4 mV vs. 12.5 +/- 2.5 mV). Recording of Ih with predefined values of [Ca2+]i (13.2 nM or 10.01 microM in the patch pipette) revealed no significant differences in the activation curve or the fully activated I-V relationship of Ih. 3. In comparison, stimulation of the intracellular cyclic adenosine monophosphate (cAMP) pathway induced a significantly positive shift in Ih voltage dependence of +5.1 +/- 1.9 mV, with no alteration in the fully activated I-V relationship. 4. These data argue against a direct regulation of Ih by intracellular Ca2+, and particularly do not support a primary role of Ca(2+)-dependent modulation of the Ih channels in the waxing and waning of sleep spindle oscillations in thalamocortical neurones.

Voltage-activated intracellular calcium transients in thalamic relay cells and interneurons.

The dynamics of intracellular calcium concentration ([Ca2+]i) following activation of low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ currents were studied in identified relay neurons and interneurons of the rat dorsal lateral geniculate nucleus (LGNd) in situ using Ca2+ imaging and patch-clamp techniques. In relay neurons, [Ca2+]i transients associated with the LVA Ca2+ current showed a fairly homogeneous somatodendritic distribution, whereas HVA transients significantly decreased to 65% of the somatic value at 60 microns dendritic distance. In interneurons, LVA transients significantly increased to 239% of the somatic value at 60 microns dendritic distance, whereas HVA transients were not significantly different in the soma and dendrites. These results indicate differences in [Ca2+]i dynamics, which may reflect a heterogeneous distribution of Ca2+ channels contributing to subcellular compartmentation in the two types of thalamic neurons.

Intracellular Ca2+, Na+ and H+ transients evoked by kainate in the leech giant glial cells in situ.

The membrane responses to the glutamate receptor agonist kainate and the subsequent changes in intracellular Ca2+, H+ and Na+ concentration were measured in giant glial cells of the leech central nervous system using ion-selective microelectrodes and microfluorimetry of Fura-2. The membrane depolarization or membrane inward current of exposed neuropile glial cells in situ, evoked by 2-20 microM kainate, were reversibly blocked by 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX), (50-100 microM) and by Ni2+ (2 mM), but not by methoxyverapamil (D600, 500 microM), which blocked voltage-gated Ca2+ influx. Local iontophoretic application of kainate on to the somatic membrane of single neuropile glial cells in situ, resulted in CNQX-sensitive depolarization and rises in intraglial Ca2+ concentration similar to those observed with bath-application of the agonist, indicating the presence of non-N-methyl-D-aspartate-type (NMDA) glutamate receptors in the somatic membrane of these cells. In voltage-clamped glial cells bath-application of kainate (5-10 microM) evoked inward currents and an increase in the membrane conductance,. while the intracellular Ca2+ increased (up to 200 nM). This increase in Ca2+i was not affected by substitution of Na+ by Li+, indicating that it is not due to reversed Na+/Ca2 exchange following intracellular Na+ accumulation. The intracellular Na+ concentration increased (up to 40 mM), and the intracellular pH decreased (0.2-0.3 pH units) in voltage-clamped glial cells following bath application of kainate. All these changes of the concentration of intracellular cations were reversibly suppressed by CNQX and Ni2+. The results indicate that Ca2+, Na+ and H+ enter leech neuropile glial cells presumably through non-selective cation channels, activated by the non-NMDA glutamate receptor agonist kainate.

Inhibition of voltage-dependent Ca2+influx by extracellular ATP in salivary cells of the leech Haementeria ghilianii

The effects of extracellular ATP on intracellular free Ca2+ concentration ([Ca2+]i) and depolarization-induced elevations of [Ca2+]i were investigated in salivary cells of the leech Haementeria ghilianii using the fluorescent Ca2+ indicator Fura-2. Simultaneously, the membrane potential was monitored or controlled by voltage-clamp. The cell membrane was depolarized either by transient elevations of the extracellular K+ concentration ([K+]o) to 90 mmol l-1 or by depolarizing steps under voltage-clamp. The resulting transient elevations of [Ca2+]i (Ca2+ transients) could be repeatedly elicited with little variability in amplitude. Ca2+ transients were completely inhibited by 2 mmol l-1 Ni2+ or in Ca2+-free saline. The transients are, therefore, dependent on Ca2+ influx from the external medium through voltage-gated Ca2+ channels. The Ca2+ influx was rapidly and reversibly inhibited by extracellular application of ATP. The effect was dose-dependent with a threshold concentration below 10(-7) mol l-1. A 50 % reduction in the amplitude of Ca2+ transients was obtained by application of 1­2 µmol l-1 ATP or ATP-gamma-S (apparent IC50, 1.6 µmol l-1 ATP) and Ca2+ transients were almost completely inhibited by 30­100 µmol l-1 ATP. Resting [Ca2+]i, the resting membrane potential and membrane potential changes induced by 90 mmol l-1 [K+]o were not affected by ATP. Adenosine (10 µmol l-1) did not affect resting [Ca2+]i, the resting membrane potential or membrane potential changes induced by 90 mmol l-1 [K+]o and had little effect on Ca2+ transients. Suramin, an antagonist of vertebrate P2 receptors, was without effect on the inhibitory actions of ATP. We conclude that activation of a suramin-insensitive purinoceptor by ATP inhibits Ca2+ influx through voltage-gated Ca2+ channels in the salivary cells of Haementeria ghilianii.

Maintenance of Fura-2 fluorescence in glial cells and neurons of the leech central nervous system.

Identified glial cells and neurones of the leech central nervous system (CNS) were injected iontophoretically with the calcium indicator dye Fura-2 to measure intracellular Ca2+, while simultaneously recording the membrane potential using a double-barrelled theta-type microelectrode. Both glial cells and neurones responded with Ni(2+)-sensitive Ca2+ transients upon membrane depolarization, indicating Ca2+ influx through voltage-gated Ca2+ channels. In contrast to neurones, the glial cells showed a rapid loss of fluorescence with a half-time of 6.3 +/- 1.8 min (n = 6) after dye injection. Both kinetics and amplitudes of the stimulus-induced Ca2+ transients were affected by this rapid dye loss. The anion exchange inhibitor probenicid (2 mM) significantly reduced, but did not prevent, the loss of Fura-2 fluorescence, suggesting that some dye left the glial cell via an anion exchanger. In order to compensate this fluorescence loss, we injected Fura-2 throughout the experiment. Under this condition, similar Ca2+ transients could be elicited repeatedly for more than 1 h. In Retzius neurones single injections of Fura-2 yielded enough intracellularly trapped dye to allow measurement of intracellular Ca2+ for up to 30 min after the end of injection without large decrease in absolute fluorescence.

Intracellular chloride activity of leech neurones and glial cells in physiological, low chloride saline.

Leech blood apparently contains considerably less chloride than generally used in physiological experiments. Instead of 85-130 mM Cl- used in experimental salines, leech blood contains around 40 mM Cl- and up to 45 mM organic anions, in particular malate. We have reinvestigated the distribution of Cl- across the cell membrane of identified glial cells and neurones in the central nervous system of the leech Hirudo medicinalis L., using double-barrelled Cl(-)- and pH-selective microelectrodes, in a conventional leech saline, and in a saline with a low Cl- concentration (40 mM), containing 40 mM malate. The interference of anions other than Cl- to the response of the ion-selective microelectrodes was estimated in Cl(-)-free salines (Cl- replaced by malate and/or gluconate). The results show that the absolute intracellular Cl- activities (aCli) in glial cells and neurones, but not the electrochemical gradients of Cl- across the glial and the neuronal cell membranes, are altered in the low Cl-, malate-based saline. In Retzius neurones, aCli is lower than expected from electrochemical equilibrium, while in pressure neurones and in neuropil glial cells, aCli is distributed close to its equilibrium in both salines, respectively. The steady-state intracellular pH values in the glial cells and Retzius neurones are little affected (< or = 0.1 pH units) in the low Cl-, malate-based saline.

Fura-2 signals evoked by kainate in leech glial cells in the presence of different divalent cations.

The glutamate-agonist kainate evokes Ca2+ transients in both neurones and glial cells. Owing to the membrane depolarization elicited by kainate, a Ca2+ influx could occur through voltage-gated Ca2+ channels or through the kainate-gated cation channels directly. We have measured ratio signals of the calcium indicator dye fura-2, injected into giant glial cells of the leech Hirudo medicinalis, as response to kainate (5-20 microM) in the presence of different divalent cations. The responses to kainate increased during the first 2-4 kainate applications, both in unclamped and in voltage-clamped cells. The fura-2 fluorescence ratio (F350/F380) still increased when Ca2+ was replaced by Ba2+ but was suppressed in Ca(2+)-free saline and in the presence of Ni2+ (2 mM). Co2+ and Mn2+ (2 mM) also reduced the kainate-induced fura-2 fluorescence signals, due to entry of these divalent cations into the cells and subsequent quenching the fluorescence of the intracellular dye. It is concluded that Ni2+ blocks the kainate-induced membrane depolarization and Ca2+ transient but apparently does not enter the cells, while Ba2+, Co2+, and Mn2+ appear to permeate the membrane, presumably through the kainate-gated channels.

Intracellular pH of giant salivary gland cells of the leech Haementeria ghilianii: regulation and effects on secretion.

1. Intracellular pH (pHi) and membrane potential (Em) of giant salivary gland cells of the leech, Haementeria ghilianii, were measured with double-barrelled, neutral-carrier, pH-sensitive microelectrodes. 2. Em was -51 +/- 11.2 mV and pHi was 6.98 +/- 0.1 (mean +/- S.D., N = 41) in Hepes-buffered saline (nominally HCO3(-)-free; extracellular pH, pHe = 7.4). pHi was independent of Em. 3. Amiloride (2 mmol l-1) had no effect on resting pHi or on pHi recovery from an acid load (induced by the NH4+ pre-pulse technique). Removal of external Na+ produced a progressive acidification which was blocked by amiloride, and the drug also slowed the recovery of pHi on reintroduction of Na+. The results indicate the presence of an electroneutral Na+/H+ exchanger whose access to amiloride is competitively blocked by Na+. 4. In certain smaller cells of the gland, which probably form a separate population, removal of external Na+ did not affect pHi, and recovery from an acid load was blocked by amiloride. There may, therefore, be two types of Na+/H+ exchanger, differing in reversibility and sensitivity to amiloride. 5. Recovery of pHi from NH4(+)-induced acid loading was not affected by bicarbonate-buffered saline (2% CO2; 11 mmol l-1 HCO3-) or by addition of the anion-exchange blocker SITS (10(-4) mol l-1). This suggests that there is no significant contribution of a HCO3(-)-dependent transport mechanism to pHi regulation in the gland cells. 6. Removal of external Cl- slowly reduced pHi and there was a transient increase (overshoot) in pHi when Cl- was reintroduced. These effects of Cl- are probably explained by changes in the Na+ gradient. Intracellular Na+ and Cl- activities were measured with ion-selective microelectrodes. 7. Acidification with NH4+ was difficult, probably because of the cells' poor permeability to this ion. Attempts to introduce NH4+ via the Na+ pump or Na+/Cl- transporter were not successful. The H+/K+ ionophore nigericin (1 microgram ml-1), however, produced a rapid and reversible acidification. 8. N-methylmaleimide (0.5-1 mmol l-1), which blocks proton-pumping ATPase, produced a prolonged acidification of almost 1 pH unit, well beyond the level expected for simple equilibration with pHe. The results are consistent with the presence of a vesicular proton pump, acidifying the secretory vesicles which pack the cell body. 9. NH4+ (50 mmol l-1) or trimethylamine (50 mmol l-1) increased pHi and stimulated salivary secretion, while propionate (50 mmol l-1) decreased pHi and stopped secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Sodium-bicarbonate cotransport current in identified leech glial cells.

1. The membrane current associated with the cotransport of Na+ and HCO3- was investigated in neuropil glial cells in isolated ganglia of the leech Hirudo medicinalis L. using the two-electrode voltage-clamp technique. 2. The addition of 5% CO2-24 mM HCO3- evoked an outward current, which slowly decayed, and which was dependent upon the presence of external Na+. Removal of CO2-HCO3- elicited a transient inward current. Re-addition of Na+ to Na(+)-free saline in the presence of CO2-HCO3- also produced an outward current. Under these conditions an intracellular alkalinization and a rise in intracellular [Na+] were recorded using triple-barrelled, ion-sensitive microelectrodes. Addition or removal of HCO3-, in the absence of external Na+, caused little or no change in membrane voltage, membrane current and intracellular pH, indicating that the glial membrane has a very low HCO3- conductance. 3. Voltage steps revealed nearly linear current-voltage relationships both in the absence and presence of CO2-HCO3-, with an intersection at the assumed reversal potential of the HCO(3-)-dependent current. These results suggest a cotransport stoichiometry of 2HCO3-: 1 Na+. The HCO(3-)-dependent current could be inhibited by diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). 4. Simultaneous recording of current and intracellular pH showed a correlation of the maximal acid-base flux with the transient HCO(3-)-dependent current during voltage steps in the presence of CO2-HCO3-. The maximum rate of acid-base flux and the HCO(3-)-dependent peak current showed a similar dependence on membrane voltage. Lowering the external pH from 7.4 to 7.0 produced an inward current, which increased twofold in the presence of CO2-HCO3-. This current was largely inhibited by DIDS, indicating outward-going electrogenic Na(+)-HCO3- cotransport during external acidification. 5. When external Na+ was replaced by Li+, a similar outward current and intracellular alkalinization were observed in the presence of CO2-HCO3-. The Li(+)-induced intracellular alkalinization was not inhibited by amiloride, a blocker of Na+(Li+)-H+ exchange, but was sensitive to DIDS. These results suggest that Li+ could, at least partly, substitute for Na+ at the cotransporter site. 6. Our results indicate that the Na(+)-HCO3- cotransport produces a current across the glial cell membrane in both directions with a reversal potential near the membrane resting potential, rendering pHi a function of the glial membrane potential.

Intracellular chloride activity and the effect of 5-hydroxytryptamine on the chloride conductance of leech Retzius neurons.

Intracellular Cl- activity (aCli) and 5-hydroxytryptamine (5-HT)-induced membrane currents of Retzius neurons in the central nervous system of the medicinal leech were measured using Cl- sensitive microelectrodes and a two-microelectrode voltage-clamp technique. At the membrane of Retzius neurons Cl- ions were not passively distributed. Under different conditions the chloride equilibrium potential (ECl, -60.1 mV for isotonic saline and -57.8 mV for a hypertonic saline) was negative with respect to the membrane potential (Em, -55 +/- 3.8 and -47 +/- 3.4 mV respectively). The endogenous neurohormone 5-HT always polarized the membrane of Retzius neurons in the direction of ECl. When voltage-clamping the membrane of Retzius neurons near the resting potential both in situ and in primary culture, application of 5-HT produced an outward current (I5-HT) and an increase in membrane conductance. Current-voltage relationships for I5-HT showed a slight outward rectification and reversal potentials of -61.6 +/- 3.1 mV in situ and -66 +/- 3.1 mV in primary culture, both values being comparable to the ECl of Retzius neurons as measured in situ. The results indicate that 5-HT increases the Cl- conductance of Retzius neurons, thereby hyperpolarizing the cell membrane and affecting both the excitability of the neuron and 5-HT release from it. This could affect the feeding and swimming behaviour of the leech.

Potassium channels in growth cones of leech Retzius cells.

1. Potassium-selective channels were analysed in growth cones of cultured leech Retzius cells. 2. In the cell-attached mode and at physiological bath and pipette solution little channel activity was observed at resting membrane potential. The channel open probability (p(o)) increased with cell depolarization, and the slope conductance of the single K+ channel current was about 60 pS. 3. With symmetrical high KCl solution on both sides of the excised membrane patch three K(+)-selective channels could be discriminated. Two channels exhibited a linear current-voltage relation of about 18 pS and 106 pS, respectively. 4. The most frequently observed K+ channel showed a non-linear current-voltage relation and p(o) increased with increasing free cytoplasmic Ca2+ and during cell hyperpolarization.