Modulation of the input-output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons

Abstract

The neuronal input-output function depends on recruitment threshold and gain of the firing frequency-current (f-I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input-output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 μm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f-I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 μm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f-I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation.

Figure 1. Relationship between cell size, input resistance and rheobase in MNs of the rat oculomotor nucleus

A and B, cell bodies and main dendritic trees of two MNs with input resistances of 355 MΩ (A) and 134 MΩ (B). Insets in (A) and (B) show the membrane potential responses to negative current steps (20 pA) of labelled MNs. C and D, three-dimensional reconstruction of the MNs shown in (A) and (B), respectively. To obtain the somatic surface values the Imaris software established a separation by a plane between the cell body and main dendrites (one of these planes is shown for each MN). E, relationship between somatic membrane surface area and input resistance (n = 33). F, relationship between input resistance and rheobase (n = 73). Linear correlations are illustrated. MNs, motoneurons.

Figure 2. GABA inputs and currents in MNs of the rat oculomotor nucleus

A–D, vesicular transporter of GABA (VGAT, green) within the boundaries of the rat oculomotor nucleus (delineated by dashed lines, A), in the neuropil (B), and in close association with MN cell bodies (C) and (D). MNs were identified by their positive immunoreactivity against choline acetyltransferase (red). E, inward current evoked by application of 100 μm GABA. This current (107 pA) was mediated by GABA_A receptors as it was blocked by 20 μm gabazine. F, gabazine injection (20 μm) blocked the spontaneous inhibitory postsynaptic currents and revealed a tonic current of 16 pA in the illustrated MN. This current can also be seen in (E) after gabazine application (22 pA). MNs, motoneurons.

Figure 3. Effects of GABA (100 μm) or gabazine (20 μm) on the membrane potential and input resistance of MNs from the rat oculomotor nucleus

A, bath application of 100 μm GABA yielded a membrane potential hyperpolarization and a diminution in the membrane response (see arrows) to negative current steps of 50 pA. B, gabazine produced an increase in the membrane response (see arrows) to negative current steps of 50 pA, while the RMP remained largely unaltered. C, relationship between RMP and membrane potential change evoked by 100 μm GABA. Note that the amplitude of membrane potential change diminished when rest values were close to −67 mV. D, relationship between input resistance and membrane potential change. MNs, motoneurons; RMP, resting membrane potential.

Figure 4. Effects of GABA (100 μm) or gabazine (20 μm) on input resistance of MNs from the rat oculomotor nucleus

A and D, membrane potential responses to current steps of 20 pA in high- and low-input resistance MNs under control conditions and during exposition to GABA (A) or gabazine (D). B and E, plots illustrating the input resistance value of each MN under control conditions and during exposure to GABA (B) or gabazine (E). C and F, relationship between input resistance under control condition and input resistance change in response to exposure to GABA (C) or gabazine (F). Note that the input resistance changed linearly with input resistance in response to gabazine exposure, but not with GABA application. MNs, motoneurons.

Figure 5. Effects of GABA (100 μm) or gabazine (20 μm) on the membrane time constant in motoneurons from the rat oculomotor nucleus

A and B, time constant value for each motoneuron under control condition and following exposure to GABA (A) or gabazine (B), with results expressed as a function of the input resistance under control conditions. C, representative recordings of postsynaptic excitatory potentials evoked by the stimulation of the medial longitudinal fasciculus under control conditions and during GABA application. st, stimulation.

Figure 6. Effects of GABA (100 μm) or gabazine (20 μm) on the rheobase of MNs from the rat oculomotor nucleus

A and D, minimum current (rheobase) required to evoke an action potential under control conditions and in response to GABA (A) or gabazine (D) in two different MNs. B and E, cumulative normalized plots of rheobase under control conditions and during exposure to GABA (B) or gabazine (E). The dashed lines show the rheobase (I₅₀) values at which 50% of MNs fired off an action potential. C and F, relationship between input resistance under control conditions and rheobase change in response to exposure to GABA (C) or gabazine (F). MNs, motoneurons.

Figure 7. Effects of GABA (100 μm) or gabazine (20 μm) on f–I relationships in MNs from the rat oculomotor nucleus

A and C, firing of the same MN in response to two current steps of different intensities under control conditions and in response to GABA (A) or gabazine (C). B and D, f–I plots for two representative MN with low- and high-input resistance under control conditions and during exposure to GABA (B) or gabazine (D). MNs, motoneurons.

Figure 8. An orchestrated system mediated by glutamate- and GABA-synaptic inputs drives firing properties in ocular MNs

A, showing EN and IN that provide glutamatergic and GABAergic inputs, respectively, to the MN. The firing frequency of the MN elicits the pattern of contraction of the extraocular muscle. B, FRs of IN and EN leading to discharge of the MN and altered EP. The bar corresponds to periods of time (in arbitrary units, a.u.) in which: (1) EN does not discharge, whereas IN is firing maximally; (2.1, 2.2, 2.3) EN and IN increase and decrease their FRs, respectively, in successive steps in the MN on-direction; (3,4) EN increases its FR in successive steps while IN does not fire. C, IN, EN and MN FRs during eye fixations represented in (B). D–G, release of GABA and glutamate from IN and EN, respectively. Receptors are in the MN surface membrane. These drawings correspond to the conditions indicated in scenarios 1–4 of (B) in this figure. The gradient from yellow to blue represents the levels of the two neurotransmitters in the synaptic cleft and non-synaptic space. H, reduction in the concentration of extracellular GABA leads to a decrease in the tonic inhibition and an increase in input resistance. I, increase in glutamate concentration leads to a reduction of the recruitment threshold current by reducing the action potential voltage threshold. J, concurrent effects on recruitment threshold and gain of a reduction in the GABA_A-mediated tonic current and activation of metabotropic glutamate receptors. EN, excitatory neurons; EP, eye position; FR, firing rate; IN, inhibitory neurons; MNs, motoneurons.