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γ-Aminobutyric acid type A (GABAA) receptor activation inhibits many primary afferent neurons by depolarization and increased membrane conductance. Deterministic (step and sinusoidal) functions are commonly used as stimuli to test such inhibition. We found that when the VS-3 mechanosensory neurons innervating the spider lyriform slit-sense organ were stimulated by randomly varying white-noise mechanical or electrical signals, their responses to GABAA receptor agonists were more complex than the inhibition observed during deterministic stimulation. Instead, there was rapid excitation, then brief inhibition, followed by long-lasting excitation. During the final excitatory phase, VS-3 neuron sensitivity to high-frequency signals increased selectively and their linear information capacity also increased. Using experimental and simulation approaches we found that the excitatory effect could also be achieved by depolarizing the neurons without GABA application and that excitation could override the inhibitory effect produced by increased membrane conductance (shunting). When the VS-3 neurons were exposed to bumetanide, an antagonist of the Cl− transporter NKCC1, the GABA-induced depolarization decreased without any change in firing rate, suggesting that the effects of GABA can be maintained for a long time without additional Cl− influx. Our results show that the VS-3 neuron's response to GABA depends profoundly on the type of signals the neuron is conveying while the transmitter binds to its receptors.

Afferent neurons entering the central nervous systems of vertebrates and invertebrates receive presynaptic inhibition on their axon terminals. This usually involves an increase in membrane conductance (shunting) and depolarization (primary afferent depolarization, PAD). In arachnids and crustaceans the peripherally located parts of afferent neurons also receive efferent synapses. GABA (γ-aminobutyric acid) plays a major role in both central and peripheral inhibition, activating chloride channels that depolarize the membrane and increase its conductance. Although both central and peripheral inhibition have been widely investigated, debate continues about the mechanisms involved, especially concerning the relative contributions of shunting versus inactivation of sodium channels by depolarization. Sensory neurons innervating spider VS-3 slit sensilla are accessible to intracellular recordings during mechanical or electrical stimulation. These neurons are inhibited by GABA, and both the electrophysiology and pharmacology of this inhibition have been studied previously. Here, we developed a Hodgkin–Huxley style model to simulate VS-3 neuron activity before and after GABA treatment. The model indicates that GABA-activated chloride current can entirely account for action potential suppression, and that either shunting or inactivation are sufficient to produce inhibition. This model also demonstrates that slowing of sodium current contributes to inhibition.

Peripherally located parts of spider mechanosensory neurons are modulated by several neurotransmitters released from apposed efferent fibers. Activities of acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) and ACh degrading enzyme acetylcholine esterase (AChE) were previously found in some efferent fibers. ChAT activity was also present in all the mechanosensory neurons, while AChE activity was only found in some. We show that spider mechanosensory neurons and probably some efferent neurons are immunoreactive to a monoclonal antibody against muscarinic ACh receptors (mAChRs). However, application of muscarinic agonists did not change the physiological responses or membrane potentials of neurons in the lyriform organ VS-3. Similarly, the sensitivities of the neurons of trichobothria (filiform hairs) remained unchanged after application of these agonists. Therefore, activation of mAChRs may only modulate the function of spider mechanosensory neurons indirectly, for example, by affecting the release of other transmitter(s). However, a subgroup of VS-3 neurons was inhibited by ACh, which also depolarized the membrane similar to these neurons' responses to GABA, suggesting that ACh activates anion channels in these neurons. Interestingly, all of the neurons responding to ACh were the rapidly adapting Type A neurons that were previously shown to express AChE activity.

GABAergic inhibition of mechanosensory afferent axon terminals is a widespread phenomenon in vertebrates and invertebrates. Spider mechanoreceptor neurons receive efferent innervation on their peripherally located axons, somata and sensory dendrites, and the dendrites have recently been shown to be excitable. Excitability of the spider sensory neurons is inhibited by muscimol and GABA, agonists of ionotropic GABA receptors. Here we asked where in the neurons this inhibition occurs. We found no evidence for inhibition of action potentials in the sensory dendrites, but axonal action potentials were rapidly suppressed by both agonists. Earlier work showed that metabotropic GABA(B) receptors are located on the dendrites and distal somata of the spider sensory neurons, where they modulate voltage-activated conductances and may provide slower, prolonged inhibition. Therefore, GABA released from single peripheral efferents may activate both ionotropic and metabotropic receptor types, providing rapid suppression of axonal activity followed by slower inhibition that eventually prevents action potential initiation in the distal dendrites.

The mechanosensilla in spider exoskeleton are innervated by bipolar neurons with their cell bodies close to the cuticle and dendrites attached to it. Numerous efferent fibers synapse with peripheral parts of the mechanosensory neurons, with glial cells surrounding the neurons, and with each other. Most of these efferent fibers are immunoreactive to γ-aminobutyric acid (GABA), and the sensory neurons respond to agonists of ionotropic GABA receptors with a rapid and complete inhibition. In contrast, little is known about metabotropic GABABreceptors that may mediate long-term effects. We investigated the distribution of GABABreceptors on spider leg mechanosensilla using specific antibodies against 2 proteins needed to form functional receptors and an antibody that labels the synaptic vesicles on presynaptic sites. Both anti-GABABreceptor antibodies labeled the distal parts of the sensory cell bodies and dendrites but anti-GABABR1 immunoreactivity was also found in the axons and proximal parts of the cell bodies and some glial cells. The fine efferent fibers that branch on top of the sensory neurons did not show GABABreceptor immunoreactivity but were densely labeled with anti-synapsin and indicated synaptic vesicles on presynaptic locations to the GABABreceptors. Intracellular recordings from sensory neurons innervating the slit sensilla of the spider legs revealed that application of GABABreceptor agonists attenuated voltage-activated Ca2+current and enhanced voltage-activated outward K+current, providing 2 possible mechanisms for controlling the neurons' excitability. These findings support the hypothesis that GABABreceptors are present in the spider mechanosensilla where their activation may modulate information transmission.

G-protein-coupled octopamine (OA) receptors mediate their effects by Ca²(+) signaling or adjusting intracellular cAMP levels. Depending on OA concentration and cell type, activation of OA receptors in excitable cells triggers excitatory or inhibitory effects, but the mechanisms by which Ca²(+) or cAMP mediates these effects are not well understood. We investigated signaling mechanisms that are potentially activated by OA, and OA effects on excitability and frequency sensitivity in mechanosensory neurons innervating the VS-3 slit sensilla on the patella of the spider Cupiennius salei. These neurons are directly innervated by octopaminergic efferents, and possess OA receptors that were immunoreactive to an antibody against an OA receptor highly expressed in mushroom bodies. OA application enhanced VS-3 neuron sensitivity, especially at high stimulation frequencies. This enhancement lasted for at least 1 h after OA application. Changes in sensitivity were also detected when the Ca²(+) ionophore ionomycin or the cAMP analog 8-Br-cAMP was applied. However, the cAMP pathway was unlikely to mediate the OA effect, as the protein kinase A inhibitor RP-cAMPS did not diminish this effect. In contrast, the OA-induced sensitivity enhancement was significantly reduced by KN-62, an inhibitor of Ca²(+) /calmodulin-dependent protein kinase II (CaMKII), and by the Ca²(+) chelator BAPTA-AM. OA depolarized the neurons by 3.8 mV from resting potential, well below the threshold for opening of voltage-activated Ca²(+) channels. OA also reduced the amplitudes of voltage-activated K(+) currents. We propose that OA receptors in VS-3 neurons activate inositol 1,4,5-trisphosphate, leading to Ca²(+) release from intracellular stores. The Ca²(+) surge switches on CaMKII, which modulates voltage-activated K(+) channels, resulting in persistent enhancement in excitability.

Activation of ionotropic γ-aminobutyric acid type A (GABAA) receptors depolarizes neurons that have high intracellular [Cl−], causing inhibition or excitation in different cell types. The depolarization often leads to inactivation of voltage-gated Na channels, but additional ionic mechanisms may also be affected. Previously, a simulated model of spider VS-3 mechanosensory neurons suggested that although voltage-activated Na+current is partially inactivated during GABA-induced depolarization, a slowly activating and inactivating component remains and may contribute to the depolarization. Here, we confirmed experimentally, by blocking Na channels prior to GABA application, that Na+current contributes to GABA-induced depolarization in VS-3 neurons. Ratiometric Ca2+imaging experiments combined with intracellular recordings revealed a significant increase in intracellular [Ca2+] when GABAAreceptors were activated, synchronous with the depolarization and probably due to Ca2+influx via low-voltage–activated (LVA) Ca channels. In contrast, GABAB-receptor activation in these neurons was previously shown to inhibit LVA current. Blockade of voltage-gated K channels delayed membrane repolarization, extending GABA-induced depolarization. However, inhibition of Ca channels significantly increased the amplitude of GABA-induced depolarization, indicating that Ca2+-activated K+current has an even stronger repolarizing effect. Regulation of intracellular [Ca2+] is important for many cellular processes and Ca2+control of K+currents may be particularly important for some functions of mechanosensory neurons, such as frequency tuning. These data show that GABAA-receptor activation participates in this regulation.

Most mechanosensory neurons are inhibited by GABAergic efferent neurons. This inhibition is often presynaptic and mediated by ionotropic GABA receptors at the axon terminals. GABA receptor activation opens Cl- channels, leading to membrane depolarization and an increase in membrane conductance. In many invertebrate preparations, efferent neurons that innervate mechanosensory afferents contain glutamate in addition to GABA, suggesting that the sensory neurons are also modulated by glutamate. However, the effects of glutamate on these neurons are not well understood. Peripheral parts of the spider (Cupiennius salei) mechanosensory neurons are surrounded by efferent fibers immunoreactive to antibodies against GABA and glutamate. GABA and its analogue muscimol were shown to effectively inhibit spider mechanosensory neurons innervating lyriform slit sensilla VS-3 that detects cuticular strains in the leg. Here, we show that glutamate also inhibits the VS-3 neurons, but its effects are different from those of GABA or muscimol, suggesting that it acts on a different group of receptors. GABA and muscimol always depolarized these neurons and the inhibitory effect was strongly correlated with the amount of depolarization. In contrast, glutamate inhibited the VS-3 neurons even when it did not depolarize them. In addition, while glutamate inhibited both the axonal action potentials elicited with electrical stimulation and dendritic action potentials produced by mechanical stimulation, muscimol only inhibited the axonal action potentials. Therefore, the inhibitory glutamate receptors in the VS-3 neurons are distinct from and differently distributed than the GABA receptors, providing a subtle control of the neurons' sensitivity in varying behavioural situations.

Spider mechanosensory neurons receive an extensive network of efferent synapses onto their sensory dendrites, somata and distal axonal regions. The function of these synapses is unknown. Peripheral synapses are also found on crustacean stretch-receptor neurons but not on mechanosensory afferents of other species, although inhibitory GABAergic synapses are a common feature of centrally located axon terminals. Here we investigated the effects of GABA receptor agonists and antagonists on one group of spider mechanosensory neurons, the slit sense organ VS-3, which are accessible to current- and voltage-clamp recordings. Bath application of GABA activated an inward current that depolarized the membrane and increased the membrane conductance leading to impulse inhibition. VS-3 neuron GABA receptors were activated by muscimol and inhibited by picrotoxin but not bicuculline, and their dose-response relationship had an EC(50) of 103.4 microm, features typical for insect ionotropic GABA receptors. Voltage- and current-clamp analysis confirmed that, while the Na(+) channel inhibition resulting from depolarization can lead to impulse inhibition, the increase in membrane conductance (i.e. 'shunting') completely inhibited impulse propagation. This result argues against previous findings from other preparations that GABA-mediated inhibition is caused by a depolarization that inactivates Na(+) conductance, and it supports those findings that assign this role to membrane shunting. Our results show that GABA can rapidly and selectively inhibit specific mechanoreceptors in the periphery. This type of peripheral inhibition may provide spiders with a mechanism for distinguishing between signals from potential prey, predators or mates, and responding with appropriate behaviour to each signal.