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Key Points: GABA is an essential molecule for sensory information processing. It is usually assumed to be released by neurons. Here we show that in the dorsal horn of the spinal cord, astrocytes respond to glutamate by releasing GABA. Our findings suggest a novel role for astrocytes in somatosensory information processing., Abstract: Astrocytes participate in neuronal signalling by releasing gliotransmitters in response to neurotransmitters. We investigated if astrocytes from the dorsal horn of the spinal cord of adult red-eared turtles (Trachemys scripta elegans) release GABA in response to glutamatergic receptor activation. For this, we developed a GABA sensor consisting of HEK cells expressing GABA A receptors. By positioning the sensor recorded in the whole-cell patch-clamp configuration within the dorsal horn of a spinal cord slice, we could detect GABA in the extracellular space. Puff application of glutamate induced GABA release events with time courses that exceeded the duration of inhibitory postsynaptic currents by one order of magnitude. Because the events were neither affected by extracellular addition of nickel, cadmium and tetrodotoxin nor by removal of Ca 2+ , we concluded that they originated from non-neuronal cells. Immunohistochemical staining allowed the detection of GABA in a fraction of dorsal horn astrocytes. The selective stimulation of A∂ and C fibres in a dorsal root filament induced a Ca 2+ increase in astrocytes loaded with Oregon Green BAPTA. Finally, chelating Ca 2+ in a single astrocyte was sufficient to prevent the GABA release evoked by glutamate. Our results indicate that glutamate triggers the release of GABA from dorsal horn astrocytes with a time course compatible with the integration of sensory inputs., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

This report considers serotonergic (5-HT) effects on spinal motoneurons, reviewing previous data and presenting a new study showing distinct effects of two 5-HT receptor subtypes. We previously investigated the effects of 5-HT on motoneurons in a slice preparation from the spinal cord of the adult turtle. In agreement with previous studies, we had found that 5-HT applied to the extracellular medium promoted a voltage sensitive plateau potential. However, we also reported that this effect was only observed in half of the motoneurons; 5-HT inhibited the firing of the other half of the motoneurons recorded from. To investigate the reasons for this, we applied 5-HT focally by means of the microiontophoresis technique. Facilitation of plateau potentials was observed when 5-HT was released at sites throughout the somatodendritic region. However, motoneurons were inhibited by 5-HT when selectively applied in the perisomatic region. These two effects could be induced in the same motoneuron. With pharmacological tools, we demonstrate here that the facilitation of plateau potentials is mediated by 5-HT(2) receptors and the inhibitory effect is due to the activation of 5-HT(1A/7) receptors.

The details of behaviour are determined by the interplay of synaptic connectivity within neuronal circuitry and the intrinsic membrane properties of individual neurones. One particularly dramatic intrinsic property displayed by neurones in many regions of the nervous system is membrane potential bistability, in which transient excitation of a neurone results in a persistent depolarization outlasting the initial excitation. Here we characterize the contribution of such intrinsic bistability, also referred to as plateau properties and mediated by persistent inward currents (PICs), in spinal motor neurones to the production of withdrawal behaviours in the frog. We performed experiments on the isolated frog spinal cord with attached hindlimb. This preparation allowed the simultaneous monitoring of muscle activations during motor behaviour and intracellular neuronal recordings. We found that PICs, following their potentiation by serotonin (5-HT), are recruited and contribute to the production of withdrawal behaviours. These properties conferred a voltage-dependent prolongation to the duration of motor neuronal activity. Consistent with this potentiation of motor neuronal PICs, 5-HT also increased the duration of evoked muscle activations. This behavioural potentiation, as well as the expression of PICs in individual neurones, was reduced following antagonism of L-type Ca(2+) channels. These results demonstrate that PICs in motor neurones can be recruited during the production of behaviour and play a role in specifying the temporal details of motor output.

The modulatory effects of serotonin mediated by 5-HT1A receptors in adult spinal motoneurons were investigated by intracellular recordings in a slice preparation from the turtle. In current-clamp mode, activation of 5-HT1A receptors by 8-OH-DPAT led to depolarization and an increase in input resistance in most motoneurons but caused hyperpolarization and a decrease in input resistance in the remaining smaller fraction of cells. When slices were preincubated in medium containing the 5-HT1A receptor antagonist WAY-100635, 8-OH-DPAT had no effect. In voltage-clamp mode, with 1 mM CsCl in the bathing medium, 8-OH-DPAT consistently inhibited a leak current that was sensitive to extracellular acidification and anandamide, a TASK-1 channel blocker. In medium with a low pH, as in the presence of anandamide, 8-OH-DPAT had no effect. Our results show that activation of 5-HT1A receptors contributes to the excitatory effect of serotonin on spinal motoneurons by inhibition of a TASK-1 potassium channel leading to depolarization and increased input resistance.

The excitatory action of muscarine on spinal motoneurones was investigated with intracellular recordings in a slice preparation from adult turtles. In these cells muscarine is known to facilitate a persistent inward current mediated by L-type Ca(2+) channels. When this effect was blocked by nifedipine, muscarine still increased the excitability. In voltage clamp, a slowly activating outward current, generated during depolarizing voltage commands and deactivating as a tail current on return to the holding voltage, was reduced by muscarine. This outward current was activated when the voltage was stepped to potentials positive to -60 mV, was voltage sensitive and had a deactivation time constant of approximately 80 ms. These findings are compatible with an M-current. This possibility was also supported by the finding that the current was reduced by XE-991 - a selective blocker of the KCNQ potassium channels underlying M-currents in other cell types. Our findings suggest that an M-like current, mediated by a KCNQ channel, contributes to the intrinsic response properties of motoneurones in the adult spinal cord by increasing adaptation of repetitive firing and decreasing the slope of the frequency-current relation.

1. The involvement of intracellular calcium and calmodulin in the modulation of plateau potentials in motoneurones was investigated using intracellular recordings from a spinal cord slice preparation. 2. Chelation of intracellular calcium with BAPTA-AM or inactivation of calmodulin with W-7 or trifluoperazine reduced the amplitude of depolarization-induced plateau potentials. Inactivation of calmodulin also inhibited facilitation of plateau potentials by activation of group I metabotropic glutamate receptors or muscarinic receptors. 3. In low-sodium medium and in the presence of tetraethylammonium and tetrodotoxin, calcium action potentials evoked by depolarization were followed by a short hyperpolarization ascribed to the calcium-activated non-selective cationic current (ICAN) and by a dihydropyridine-sensitive afterdepolarization. The amplitude of the afterdepolarization depended on the number of calcium spikes and was mediated by L-type calcium channels. 4. The dihydropyridine-sensitive afterdepolarization induced by calcium spikes was reduced by blockade of calmodulin. 5. It is proposed that plateau potentials in spinal motoneurones are facilitated by activation of a calcium-calmodulin-dependent pathway.

1. The spatial distribution of synaptic facilitation of plateau potentials in dendrites of motoneurones was investigated in transverse sections of the spinal cord of the turtle using differential polarization by applied electric fields. 2. The excitability of motoneurones in response to depolarizing current pulses was increased following brief activation of either the dorsolateral funiculus (DLF) or the medial funiculus (MF) even when synaptic potentials were eliminated by antagonists of ionotropic receptors. 3. The medial and lateral compartments of motoneurones were differentially polarized by the electric field generated by passing current between two electrodes on either side of the preparation. In one direction of the field lateral dendrites were depolarized while the cell body and medial dendrites were hyperpolarized (S- configuration). With current in the opposite direction the cell body and medial dendrites were depolarized while lateral dendrites were hyperpolarized (S + configuration). 4. Following brief activation of the DLF the excitability and the generation of plateau potentials were facilitated during differential depolarization of the lateral dendrites but not during differential depolarization of the cell body and medial dendrites. Following brief activation of the MF the excitability and generation of plateau potentials were facilitated during differential depolarization of the cell body and medial dendrites but not during differential depolarization of the lateral dendrites. 5. It is concluded that the synaptic facilitation of the dihydropyridine-sensitive response to depolarization is compartmentalized in turtle motoneurones.

1. The effect of a brief train of electric stimuli in the dorsolateral funiculus on the intrinsic response properties of turtle motoneurones was investigated in transverse sections of the spinal cord in vitro. 2. Even when glutamatergic, GABAergic and glycinergic ionotropic synaptic transmission was blocked by antagonists of AMPA, NMDA, glycine and GABA receptors, dorsolateral funiculus (DLF) stimulation induced a facilitation of plateau potentials during current clamp and the underlying inward current in voltage clamp. This facilitation lasted more than 10 s. 3. The plateau potential and the facilitation by DLF stimulation was absent in the presence of 10 microM nifedipine. The DLF-induced facilitation was reduced by antagonists of 5-HT1A, group 1 metabotropic glutamate receptors and muscarine receptors. 4. These findings suggest that the intrinsic properties of spinal motoneurones are dynamically regulated by afferent synaptic activity. These afferents can be of spinal and extraspinal origin. Continuous regulation of intrinsic response properties could be a mechanism for motor flexibility.

1. Clarke's column neurons of the dorsal spinocerebellar tract (DSCT) were recorded intracellularly in anaesthetized cats during weak sustained contractions of triceps surae (TS) produced by direct electrical stimulation of the muscle. 2. Of 145 DSCT neurons, 77 (53%) were contraction sensitive suggesting that information about weak contraction of a limited number of muscles is widely distributed among DSCT neurons. Four types of effects were observed in individual neurons during TS contractions. 3. In the first group of 11 DSCT neurons (14% of the contraction-sensitive cells), the effect was excitation persisting throughout the duration of contractions. These responses were ascribed to actions of afferents from contraction-activated tendon organs. 4. In a second group of 15 neurons (20% of the contraction-sensitive cells), quickly declining excitatory potentials were recorded during sustained TS contractions. By analogy with previous observations of contraction-induced effects in motoneurons, the decline of excitation might be explained by contraction-induced presynaptic inhibition of group I afferents in Clarke's column. 5. Declining inhibitions, resembling those previously observed in homonymous and synergic motoneurons, were recorded in 49% of contraction-sensitive DSCT neurons. This appears in keeping with the fact that interneurons mediating Ib inhibition to motoneurons project axon collaterals to DSCT neurons. Presynaptic inhibition of Ib fibres might therefore cause parallel reductions of inhibitory potentials in motoneurons and in DSCT neurons. 6. In a final group of 13 neurons, mixed excitatory and inhibitory effects were observed during TS contractions. Such DSCT neurons might monitor the excitability of Ib interneurons by integration of information about input to and output from these neurons. 7. The non-uniform patterns of DSCT responses to TS contractions suggest complex processing of information on ankle extensor activity in cerebellum. Phasic signalling of contraction onset is observed in many DSCT neurons while others carry messages about duration and strength of contraction.