Activin tunes GABAergic neurotransmission and modulates anxiety-like behavior.

Activin, a member of the transforming growth factor-beta superfamily, affords neuroprotection in acute brain injury, but its physiological functions in normal adult brain are largely unknown. Using transgenic (tg) mice expressing a dominant-negative activin receptor mutant under the control of the CaMKIIalpha promoter in forebrain neurons, we identified activin as a key regulator of gamma-aminobutyric acid (GABA)ergic synapses and anxiety-like behavior. In the open field, wild-type (wt) and tg mice did not differ in spontaneous locomotion and exploration behavior. However, tg mice visited inner fields significantly more often than wt mice. In the light-dark exploration test, tg mice made more exits, spent significantly more time on a well-lit elevated bar and went farther away from the dark box as compared to wt mice. In addition, the anxiolytic effect of diazepam was abrogated in tg mice. Thus the disruption of activin receptor signaling produced a low-anxiety phenotype that failed to respond to benzodiazepines. In whole-cell recordings from hippocampal pyramidal cells, enhanced spontaneous GABA release, increased GABA tonus, reduced benzodiazepine sensitivity and augmented GABA(B) receptor function emerged as likely substrates of the low-anxiety phenotype. These data provide strong evidence that activin influences pre- and postsynaptic components of GABAergic synapses in a highly synergistic fashion. Given the crucial role of GABAergic neurotransmission in emotional states, anxiety and depression, dysfunctions of activin receptor signaling could be involved in affective disorders: and drugs affecting this pathway might show promise for psychopharmacological treatment.

Distinct roles of Galpha(q) and Galpha11 for Purkinje cell signaling and motor behavior.

G-protein-coupled metabotropic glutamate group I receptors (mGluR1s) mediate synaptic transmission and plasticity in Purkinje cells and, therefore, critically determine cerebellar motor control and learning. Purkinje cells express two members of the G-protein G(q) family, namely G(q) and G11. Although in vitro coexpression of mGluR1 with either Galpha11 or Galpha(q) produces equally well functioning signaling cascades, Galpha(q)- and Galpha11-deficient mice exhibit distinct alterations in motor coordination. By using whole-cell recordings and Ca2+ imaging in Purkinje cells, we show that Galpha(q) is required for mGluR-dependent synaptic transmission and for long-term depression (LTD). Galpha11 has no detectable contribution for synaptic transmission but also contributes to LTD. Quantitative single-cell RT-PCR analyses in Purkinje cells demonstrate a more than 10-fold stronger expression of Galpha(q) versus Galpha11. Our findings suggest an expression level-dependent action of Galpha(q) and Galpha11 for Purkinje cell signaling and assign specific roles of these two G(q) isoforms for motor coordination.

P2 purinoceptor-mediated intracellular Ca2+ transients in human sural nerve.

Segments of biopsied human sural nerve were stained with the Ca(2+)-sensitive fluorescent dyes Calcium Green-1 and Fura Red. The emission ratio was used to follow changes in the intracellular free Ca2+ concentration ([Ca2+]i). Application of ATP and analogues in concentrations between 0.3 and 300 microM via the bathing solution resulted in a transient rise in [Ca2+]i. The rank order of agonist potency, 2-methylthioATP > ATP > UTP, and the failure of adenosine, alpha,beta-MeATP and beta,gamma-MeATP to evoke rises in [Ca2+]i indicate the presence of P2Y/U subtypes of purinoceptors in this preparation. ATP-induced Ca2+ transients in biopsied human nerve preparations might serve as a diagnostic tool in neuropathies.

Activation kinetics and single channel properties of recombinant alpha1beta2gamma2L GABA(A) receptor channels.

Recombinant alpha1beta2gamma2L GABA(A) receptor channels, transiently expressed in HEK 293 cells, were investigated using the patch-clamp technique in combination with a device for ultra-fast solution exchange. The dose-response relationship revealed an EC50 of 11.6 +/- 0.9 microM and saturated with 3 mM GABA. The slope between 0.001 and 0.01 mM GABA was 2.2 +/- 0.4, indicating at least three binding sites for GABA. The rise time decreased from about 120 ms at 0.001 mM GABA to about 0.8 ms at 10 mM GABA. Single channel openings were grouped in bursts with an average duration of 10.3 +/- 3.0 ms. More than 95% of the current was represented by a single channel slope conductance of about 29 pS.

Immunohistochemical and electrophysiological evidence for omega-conotoxin-sensitive calcium channels in unmyelinated C-fibres of biopsied human sural nerve.

In vitro electrophysiological measurements of Ca2+ potentials in human sural nerve fascicles revealed that Ca2+ conductances might be present on unmyelinated C-fibres. Furthermore, these Ca2+ potentials were partially blocked by omega-conotoxin, a calcium antagonist for the N-type Ca2+ channels. Therefore, immunohistochemical staining with indirect immunofluorescent omega-conotoxin GVIA was used to localize N-type Ca2+ channels in intact and in enzymatically dissociated human sural nerve fascicles. Densities of toxin binding sites were highly heterogeneous throughout the different nerve fascicles investigated and putative N-type Ca2+ channels were localized in about 20% of the unmyelinated C-fibres. Myelinating Schwann cells as well as enzymatically demyelinated axons displayed no specific binding indicating the absence of N-type Ca2+ channels.

Activation of rat recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptor by the insecticide ivermectin.

In the present study, the activation of rat recombinant alpha(1)beta(2)gamma(2S) gamma-aminobutyric acid (GABA)-ergic Cl(-) channel expressed in human embryonic kidney (HEK) 293 cells by ivermectin was investigated. Maximal activation of the channel occurred with GABA concentrations of 10 mM or 20 microM ivermectin both achieving about the same current amplitudes. With those saturating concentrations, the currents rose with GABA within 1 ms to the maximal values, whereas the rise time for ivermectin was about 500 times longer. In contrast to activation with GABA, no desensitisation in the presence of the agonist was observed with ivermectin. With both agonists, two different open states were detected. On simultaneous application of GABA and ivermectin the current amplitudes and the kinetics were determined by the agonist applied in the concentration eliciting the higher open probability. It is concluded that GABA and ivermectin activated the channel independently resulting in different kinetic properties.

Modulatory effects of gamma-hydroxybutyric acid on a GABA(A) receptor from crayfish muscle.

The effects of gamma-hydroxybutyric acid (GHB) were evaluated with a gamma-aminobutyric acid (GABA) activated Cl- channel on crayfish deep extensor abdominal muscle. GABA and GHB were applied to outside-out patches using a fast application system. Application of GHB up to 10 mM did not result in detectable activation of the channel. After coapplication of GABA and GHB, a dose-dependent potentiation of the GABA-elicited current by GHB was observed. The maximal effect was obtained with 0.5-1 mM GHB, with which the amplitude was enhanced by about 50% with 0.4 or 1 mM GABA. Simultaneously with the potentiating effect, a decrease of the rise times was seen. Preapplication of GHB, prior to the GABA pulses, resulted in a reduction of the current amplitude elicited by GABA. This block was persistent throughout the application time of GABA. Therefore, two contrasting effects of GHB on this chloride channel, a potentiating one and a blocking one, seemed to occur simultaneously.

The chemotherapeutic oxaliplatin alters voltage-gated Na(+) channel kinetics on rat sensory neurons.

The chemotherapeutic oxaliplatin causes a sensory-motor neuropathy with predominantly hyperpathic symptoms. The mechanism underlying this hyperexcitability was investigated using rat sensory nerve preparations, dorsal root ganglia and hippocampal neurons. Oxaliplatin resulted in an increase of the amplitude and duration of compound action potentials. It lengthened the refractory period of peripheral nerves suggesting an interaction with voltage-gated Na(+) channels. Application of oxaliplatin to dorsal root ganglion neurons resulted in an increase of the Na(+) current, a block of the maximal amplitude and a shift of the voltage-response relationship towards more negative membrane potentials. The effect was detectable on 13 of 18 tested cells. This observation, together with the absence of any effect on Na(+) currents of hippocampal neurons, suggests that the interaction of oxaliplatin is restricted to one or more channel subtypes. The effect of oxaliplatin could be antagonised by the Na(+) channel blocker carbamazepine which could be used to reduce side effects of oxaliplatin therapy in patients.

A patch clamp study of a glutamatergic chloride channel on pharyngeal muscle of the nematode Ascaris suum.

Glutamatergic chloride channels on the pharyngeal muscle of Ascaris suum could be activated with glutamate and ivermectin and reversibly blocked with picrotoxin using the patch clamp technique. No activation was observed with GABA, glycine and acetylcholine. Most of the current was carried by the main subconductance state of 21 pS. Two smaller subconductance states occurred rarely. Open time histograms could be best fitted by two time constants of tau(o1) = 0.33 ms and tau(o2) = 9.8 ms present at all glutamate concentrations applied. The results suggest that some properties of the channel investigated here are different from other glutamatergic chloride channels reported from various animals.

Evoked transmitter release at neuromuscular junctions in wild type and cysteine string protein null mutant larvae of Drosophila.

Cysteine string proteins (CSPs) are synaptic vesicle proteins thought to be involved in neurotransmitter release. To obtain more information about the function of these proteins motor nerve terminals of wild type and CSP null mutant Drosophila larvae were depolarized and excitatory postsynaptic currents (EPSCs) were recorded with an extracellular electrode at 16-18 degrees C. At this temperature the amplitude of average EPSCs was reduced and the time constant of the exponential fit of the current decay was increased in CSP null mutant compared to wild type larvae. The number of quanta released per pulse was not different but the time course of release was distributed differently in CSP null mutant and wild type larvae. In measurements of the latency of quantal EPSCs the probability of release after a pulse reached a lower peak value and the decay after the peak was delayed in CSP null mutant compared to wild type larvae. In addition facilitation in response to twin-pulse stimulation was slightly increased at low levels of release in CSP null mutant larvae. It is concluded that CSPs are involved in neurotransmitter release and help to synchronise evoked release at nerve terminals.

The amplitude of quantal currents is reduced during short-term depression at neuromuscular synapses in Drosophila.

Focal extracellular excitatory postsynaptic currents were recorded to investigate short-term depression at glutamatergic Drosophila neuromuscular synapses. The amplitudes of quantal excitatory postsynaptic currents (qEPSCs) elicited before and after depolarizations eliciting large release were compared. Depression reduced the amplitude of the qEPSCs to 0.65 +/- 0.14 of control. Recovery from depression and of the receptor channels from desensitization follow a similar time course. Thus receptor desensitization seems to be involved in short-term depression at Drosophila neuromuscular junctions.

Desensitization characteristics of rat recombinant GABA(A) receptors consisting of alpha1beta2gamma2S and alpha1beta2 subunits expressed in HEK293 cells.

Desensitization kinetics of rat recombinant typeA GABAergic receptors consisting of the subunits alpha1beta2gamma2S or alpha1beta2 was investigated on application of 10-0.001 mM GABA to whole cell patches using a piezo driven liquid filament switch for fast application and deapplication. At high GABA concentrations desensitization was triphasic showing increasing time constants and a decreasing extent of desensitization on lowering the GABA concentration. Below agonist concentrations of 1 mM for the trimeric receptor and 0.1 mM for the dimeric one desensitization was biphasic switching to monophasic kinetics at GABA concentrations < or = 0.01 mM for the alpha1beta2gamma2S-type and < or = 0.003 mM for the alpha1beta2-type, respectively. Comparison with former studies performed with GABAergic receptors consisting of different subunits revealed differences in the desensitization kinetics.

A patch-clamp study on a novel gamma-aminobutyric acid activated chloride channel of crayfish deep extensor abdominal muscle.

The patch-clamp technique was used to study the effect of rapid pulses of gamma-aminobutyric acid (GABA) on excised membrane-patches in the outside-out configuration of the deep extensor abdominal muscle (DEAM) of crayfish. Channel currents reversed at the equilibrium potential of Cl- and were blocked by picrotoxin. Rare channel openings were elicited by 0.1 mM GABA, and a saturating open probability of 0.9 was reached with 10 nM GABA. The investigated channel was only sensitive to GABA and is different from a previously described GABAergic channel in crayfish that is in addition sensitive to acetylcholine and glutamate and shows three subconductance states.

Isoflurane slows inactivation kinetics of rat recombinant alpha1beta2gamma2L GABA(A) receptors: enhancement of GABAergic transmission despite an open-channel block.

Recombinant alpha1beta2gamma2L gamma-aminobutyric acid (A) receptor (GABA(A)R) channels expressed in human embryonic kidney (HEK293) cells were used for patch-clamp experiments. The currents activated by brief pulses of GABA (10(-4) M) applied with a device for fast solution exchange to cells clamped in the whole-cell configuration mimicked GABA(A)R-mediated inhibitory postsynaptic currents. Isoflurane (ISO) at clinically relevant concentrations (0.6 mM) decreased the amplitude and prolonged the decay of the GABA-evoked response. To further detail the mechanism underlying the prolonged decay time, we made simulations based on these measurements. These simulations suggest that ISO slows the rate of GABA unbinding from the receptor. Under these conditions, ISO increases the GABA-induced charge transfer and, thus, could enhance GABAergic inhibition despite the concomitant open-channel block causing the decrease in the current amplitude.

Neuromuscular glutamatergic and GABAergic channels.

Our laboratory has worked extensively on glutamatergic and GABA-ergic channels, predominantly in crayfish, but also in locust, Drosophila and recently Ascaris. Channel currents were recorded in the different modes of the patch-clamp technique (Hamill et al., 1981). The opening kinetics of the channels were derived from open and closed time histograms obtained from single channel recordings. From these, channel conductances could also be evaluated. The most relevant data were obtained by very rapidly rising and falling pulses (time of change about 0.1 ms) of agonists applied to outside-out patches containing the respective channels (Franke et al., 1987). From such recordings we constructed dose-response curves for peak and steady-state currents, for the rise times of the currents and for the time constants of desensitization. In double-pulse experiments we measured recovery from desensitization and predesensitization due to low agonist concentrations. For most of the channel types, we succeeded in constructing a reaction scheme which in computer simulations mimicked channel behaviour to a good approximation.

Block by picrotoxin of a GABAergic chloride channel expressed on crayfish muscle after axotomy.

Outside-out patches containing a gamma-aminobutyric acid (GABA)-activated chloride channel expressed after axotomy on crayfish deep extensor abdominal muscle were excised. GABA and the blocker picrotoxin (PTX) were applied to the patches using a liquid filament switch to study the effects of picrotoxin on the GABA-elicited currents. Coapplication of GABA and PTX resulted in a reduction of the current amplitude compared with that elicited by the same GABA concentration alone. This reduction of the amplitude was dependent on both the GABA and PTX concentrations. The rise time of the current decreased after coapplication of GABA and PTX. Evaluation of the single channel currents and off-currents in the presence of GABA and PTX showed a dramatic shortening of the burst duration of the channel. The open time distributions were not altered, whereas in the closed time distributions a new closed time was apparent in presence of PTX. Preincubation with PTX prior to the GABA pulse resulted in an increase of the rise time. This effect was dependent on the PTX concentration only. Possible mechanisms are discussed to explain the effects of PTX and are implemented into the existing molecular reaction scheme of the channel.

A molecular scheme for the reaction between gamma-aminobutyric acid and the most abundant chloride channel on crayfish deep extensor abdominal muscle.

Single-channel measurements were performed with the aim of constructing a detailed molecular scheme for the reaction between gamma-aminobutyric acid (GABA) and a chloride channel of crayfish deep extensor abdominal muscle (DEAM). GABA was applied in pulses to outside-out patches of muscle membrane, and, based on the dose-response of the peak currents and of their rise times, a linear model with five binding steps has been proposed. Evaluation of the single-channel kinetics indicated at least three open states. Two of them originate most probably from the fully liganded receptor state and are grouped in mixed bursts due to their different life times. The third one appears independently, outside the bursts, and originates from a lower liganded receptor state. Simulations of the dose-responses and the open time distributions with this model led to a set of rate constants which generated relatively optimal fits.

Characterization and molecular reaction scheme of a chloride channel expressed after axotomy in crayfish.

The nerve to the deep extensor abdominal muscle (DEAM) in crayfish species Astacus astacus, containing four excitatory and one inhibitory motor axons, was cut in the third segment on one side of the animal. The distal axon stump was not subject to phagocytosis but was present for months after the axotomy. The two lateral bundles of the DEAM were prepared 4-6 weeks after the axotomy. The gamma-aminobutyric-acid-(GABA-) activated chloride channel of these bundles was characterized by applying pulses of GABA to outside-out patches of the muscle membrane and measuring the responses. Based on the dose/response relationship of the peak current and of the rise time as well as on single-channel kinetics, a detailed molecular scheme for the reaction of the channel with GABA was derived. This scheme contains four binding steps of the agonist to the receptor and two open states. Simulations of the dose/response relationships with this model resulted in a set of rate constants which generate proper fits. In comparison to the channels present in innervated muscles, the channels of denervated muscles have a higher affinity for GABA, a lower single-channel conductance, four versus five binding steps, and non-cooperative binding. The first three of these adaptations of denervated muscles correspond to similar changes in denervated vertebrate muscles.

Affinity purification of cellulose-binding enzymes of Clostridium stercorarium.

Cellulose-affinity chromatography proved to be a fast and efficient purification procedure for exoenzymes of the thermophilic anaerobe Clostridium stercorarium, suitable for the preparation of large amounts of enzymes for technical applications. The cellulose-binding enzymes could be identified as the C. stercorarium; cellulolytic enzymes Avicelase I and Avicelase II characterized previously as endo-1,4-beta-glucanase and exo-1,4-beta-glucanase. A third protein was identified as xylanase A, the major endo-1,4-beta-xylanase of this organism.

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

Outside-out patches were excised from the membrane of the deep extensor abdominal muscle (DEAM), containing gamma-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 A5Os 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 A5Os, confirming the suggestion of an open channel block only at one of the open states.