Dual personality of GABA/glycine-mediated depolarizations in immature spinal cord

Abstract

The inhibitory action of glycine and GABA in adult neurons consists of both shunting incoming excitations and moving the membrane potential away from the action potential (AP) threshold. By contrast, in immature neurons, inhibitory postsynaptic potentials (IPSPs) are depolarizing; it is generally accepted that, despite their depolarizing action, these IPSPs are inhibitory because of the shunting action of the Cl(-) conductance increase. Here we investigated the integration of depolarizing IPSPs (dIPSPs) with excitatory inputs in the neonatal rodent spinal cord by means of both intracellular recordings from lumbar motoneurons and a simulation using the compartment model program "Neuron." We show that the ability of IPSPs to suppress suprathreshold excitatory events depends on E(Cl) and the location of inhibitory synapses. The depolarization outlasts the conductance changes and spreads electrotonically in the somatodendritic tree, whereas the shunting effect is restricted and local. As a consequence, dIPSPs facilitated AP generation by subthreshold excitatory events in the late phase of the response. The window of facilitation became wider as E(Cl) was more depolarized and started earlier as inhibitory synapses were moved away from the excitatory input. GAD65/67 immunohistochemistry demonstrated the existence of distal inhibitory synapses on motoneurons in the neonatal rodent spinal cord. This study demonstrates that small dIPSPs can either inhibit or facilitate excitatory inputs depending on timing and location. Our results raise the possibility that inhibitory synapses exert a facilitatory action on distant excitatory inputs and slight changes of E(Cl) may have important consequences for network processing.

Fig. 1.

E_IPSP and location of synapses affect the inhibitory action of IPSPs. (A1 and A2) Responses evoked in L4 MNs by VF stimulation (vertical dotted line) in the absence (Top) or presence (Middle) of suprathreshold current pulses (Bottom). V_REST in both cases, −67 mV. Red, time window during which VF stimulation prevented the cells from firing. (A3) Frequency distributions of APs elicited by current pulses (black and gray traces, MNs shown in A1 and A2, respectively). Same time scale as in A1 and A2. (B) Duration of inhibition and E_IPSP are negatively correlated (r = −0.57; n = 23 MNs; P < 0.01, Pearson). (C) Compartment model used in simulations. Excitatory inputs were set on the soma, whereas inhibitory inputs were moved along the dendrites. (D) Depolarizing (Top) and hyperpolarizing (Middle) IPSPs generated on the soma (Left) and at 100 μm from the soma (Right). APs are truncated. V_REST, −75 mV. (Bottom) Changes in input resistance measured at the soma level. (E) Duration of functional inhibition (D, red) generated at the soma level by inhibitory synapses at different locations along dendrites, plotted against E_Cl.

Fig. 2.

Electrotonic spread of dIPSPs. (A) Model. (B) Effect on trains of somatic subthreshold EPSPs of IPSPs generated on the soma and at 100 μm on dendrites. (C) Time course of the IPSP-induced reduction of the somatic EPSP for different locations of inhibitory synapses (same colors as in A). E_Cl was set at V_REST to prevent any change in membrane potential. (D) Time course of membrane potential changes at the soma level induced by dIPSPs generated at different loci. (E and F) Effects of IPSPs on both the somatic membrane potential and reduction of somatic EPSPs (“shunt”), plotted against the location of inhibitory synapses. Both parameters are normalized relative to the maximal effect observed when inhibitory synapses are set to the soma (black trace at time t = e in C and D). E and F correspond to the values obtained at times t = e and t = f, respectively, in C and D.

Fig. 3.

Excitatory actions of dIPSPs. (A) VF stimulation-evoked response in an L4 MN in the absence (Top) or presence of current pulses (suprathreshold: Middle, 50 sweeps; subthreshold: Bottom, single sweep). V_REST = −68 mV. Histogram represents the frequency distribution of APs elicited by current pulses of the same magnitude, which were most often (98%) subthreshold when delivered before VF stimulation (t = 0). (B) Inhibition (at 25 μm from soma; E_Cl = −55 mV) and subthreshold excitation (at the soma) presented independently (Top and Middle) or concurrently (Bottom) in the compartment model. (C) Time windows of the dIPSP-evoked facilitation of cell firing at different values of E_Cl. Colors correspond to different loci for inhibitory synapses. (D) Subthreshold EPSPs trigger APs during the whole duration of the IPSP evoked at 100 μm from soma. Note the hyperpolarized value of E_Cl (−65 mV). (E) Global pictures of inhibitory (inhibition of suprathreshold EPSPs) and excitatory (facilitation of subthreshold EPSPs) effects depending on both E_Cl and the location of inhibitory inputs.

Fig. 4.

Presence of GAD65/67 immunoreactive synaptic boutons on the soma and dendrites of neonatal MNs. (A) A 2D reconstruction from confocal images acquired in the z axis of a P3 MN filled intracellularly with Alexa 568 Hydrazide. (B1, C1, and D1) GAD65/67 immunoreactivity scanned at a single optical plane (thickness, 0.39 μm). (B2, C2, and D2) Single optical planes (as in B1, C1, and D1) showing superimposed GAD65/67 immunoreactivity and the various aspects of MN morphology (red). (B3, C3, and D3) Projection images from several optical planes demonstrating multiple sites of contact (arrows). Total thickness: B3, 2.3 μm; C3, 2.7 μm; D3, 3.5 μm. The white double arrowheads in D3 show putative contact points on dendritic spines. (B1) GAD65/67. (B 2 and 3) GAD65/67 plus Alexa 568 Hydrazide. (C) Dendritic extent: 50–100 μm. (D) Dendritic extent: 100–200 μm. (E) Density measurements of GAD65/67-positive boutons on the different loci of MNs per 25 μm. Numbers of MNs (Upper) and dendrites (Lower) analyzed are indicated.