Inhibitory neurones of the spinal substantia gelatinosa mediate interaction of signals from primary afferents

Abstract

The spinal substantia gelatinosa (SG; lamina II) is a major synaptic zone for unmyelinated (C) primary afferents. Whereas a substantial proportion of intrinsic SG neurones are GABAergic inhibitory, their relationship to afferent activity is unknown. In spinal cord slices from a transgenic mouse in which certain GABAergic lamina II neurones are labelled with green fluorescent protein (GFP), we compared primary afferent input with local efferent connections made by inhibitory SG neurones. Simultaneous whole-cell recordings from characterized neurones establish that inhibitory SG neurones receive monosynaptic input from a subset of unmyelinated primary afferents and connect to other lamina II cells that have input from a different set of afferents, permitting interactions between distinctive afferent messages. Certain lamina II inhibitory cells were found to connect to one another by reciprocal links. Inhibitory lamina II connections appear arranged to modulate activity from different sets of peripheral unmyelinated fibres through neural circuitry that includes disinhibition.

Figure 1

A monosynaptic inhibitory connection between an islet and a PrP-GFP neurone A, eight successive pulses (5 Hz, 10 ms duration) initiated action potentials in the presynaptic islet cell (upper traces) evoking IPSPs in the postsynaptic PrP-GFP cell (lower traces). Shaded portions of the records on the left are on the right expanded in time to show the IPSP latencies. Traces are aligned relative to the peak of the presynaptic action potential. Note the one-to-one generation of fixed latency IPSPs relative to presynaptic action potentials. B, left, averaged traces from 10 successive recordings (0.2 Hz, 10 ms duration). Right, the same protocol was repeated in the presence of glutamate receptor antagonists CNQX (20 μm) and APV (50 μm) to test for multisynaptic linkages.

Figure 2

Reciprocal inhibitory connections between PrP-GFP and islet neurones A, maximum projection confocal images. Upper, biocytin-labelled cells (arrowhead, PrPGFP; arrow, islet). Middle: PrP-GFP (arrowhead). Lower, overlay (arrowhead, PrP-GFP; arrow, islet). Part of the dendritic processes of the islet cell are out of the field of view. B, depolarization by current injection (1 s) in PrP-GFP and islet cells induces tonic firing that generates monosynaptic IPSPs in the other neurone. The IPSPs evoked in each neurone were blocked by 10 μm bicuculline. Resting membrane potential: −60 mV for the presynaptic cell; −50 mV for the postsynaptic cell. Bic: bicuculline.

Figure 3

Differences in threshold and latency of EPSPs evoked by graded DR stimulation in pairs of synaptically connected PrP-GFP and islet neurones A, reciprocally connected PrP-GFP and islet cells. B, unidirectional connection from PrP-GFP to an islet cell. C, unidirectional connection from islet to PrP-GFP cell. Note that the threshold and latency show a consistent difference for the two cells in each connection pattern. All resting membrane potentials were −60 mV.

Figure 4

The effects of TRPM8 and TRPV1 receptor agonists on the frequency of mEPSCs recorded from reciprocally connected PrP-GFP and islet cells A, TRPM8 receptor agonist, icilin (20 μm), increased the frequency of mEPSCs recorded from the PrP-GFP neurone but not from the islet cell. B, TRPV1 receptor agonist, capsaicin (1 μm), increased the frequency of mEPSCs recorded from both cells. TTX (1 μm) was present during all mEPSC recordings. Membrane potential: −70 mV for voltage-clamp recordings.

Figure 5

Schematic illustration of inhibitory neuronal networks in the spinal SG The relative conduction velocities of direct primary afferent input are estimated from comparisons of latencies of DR evoked EPSPs in pairs of linked neurones. Vertical and transient central neurones receive inputs from different populations of primary afferents and represent two distinct excitatory pathways in the SG. DRG: dorsal root ganglia; GFP: PrP-GFP neurone; Ver: vertical neurone; TrC: transient central neurone.