A specific inhibitory pathway between substantia gelatinosa neurons receiving direct C-fiber input

Abstract

The spinal substantia gelatinosa (SG) is a major termination region for unmyelinated (C) primary afferent fibers; however, how the input it receives from these sensory fibers is processed by SG neurons remains primarily a matter of conjecture. To gain insight on connections and functional interactions between intrinsic SG neurons, simultaneous tight-seal, whole-cell recordings were made from pairs of neurons in rat spinal cord slices to examine whether impulses in one cell generated synaptic activity in the other. Most SG neuron pairs sampled lacked synaptic interaction. Those showing a linkage included a recurring pattern consisting of a monosynaptic, bicuculline-sensitive inhibitory connection from an islet cell to a transient central neuron, each of which received direct excitatory input from different afferent C-fibers. This newly defined inhibitory circuit is postulated to represent a SG neural module by which a nociceptive C-fiber input to transient central cells is modified by other C-fiber messages.

Figure 1.

Diagram of the typical experimental arrangement. Sagittal spinal slices (400-600 μm thick, 3-5 mm long) with an attached dorsal root (8-10 mm long) were cut from the lumber spinal cord of adult Sprague Dawley rats and maintained in a recording chamber superfused with ACSF. Separate recording electrodes were used to make simultaneous tight-seal, whole-cell recordings from two SG neurons. A suction electrode was used to stimulate a segmental dorsal root to evoke volleys of impulses in primary afferent fibers. C, Caudal; D, dorsal; R, rostral; V, ventral.

Figure 2.

Simultaneous whole-cell recordings from two pairs of synaptically coupled SG neurons. a, Top trace, Current-clamp recording from the presynaptic (Pre) neuron of one pair (membrane potential, -60 mV). An action potential (AP) was initiated by brief depolarizing current injection (10 msec, 1 nA) through the recording electrode. Bottom trace, Current-clamp recording from the postsynaptic (Post) neuron (membrane potential, -60 mV) showing an excitatory postsynaptic potential (Post EPSP) evoked by the presynaptic AP and its block by bath-applied CNQX (20 μm). b, Top trace, Current-clamp recording from the presynaptic neuron of another pair of neurons (membrane potential, -60 mV) showing an AP evoked by a brief current injection (10 msec, 1 nA) through the recording electrode. Bottom trace, Current-clamp recording from the postsynaptic neuron of this pair (membrane potential, -50 mV) showing an inhibitory postsynaptic potential (Post IPSP) evoked by the presynaptic AP and its suppression by bath-applied bicuculline (10 μm), a GABA_A receptor antagonist. All traces are the average of 40-50 individual records.

Figure 3.

Examples of a monosynaptic inhibitory connection between SG neurons. Top traces, Six superimposed traces recorded from the presynaptic neuron showing action potential (AP) initiated by depolarizing pulses. Holding potential: -60 mV for presynaptic neuron, -50 mV for postsynaptic neuron. APs were evoked in the presynaptic cell by single pulses at 0.2 Hz (a) and by three successive pulses (b). Middle traces, Six traces of recordings from the postsynaptic neuron (Post) showing evoked IPSPs. Bottom traces, Average of 50 successive IPSPs. The latencies from the peak of the presynaptic APs to onset of the IPSPs (2.1 msec) remain relatively constant at 0.2 Hz (a) and for triple repetitions at 100 msec intervals (b).

Figure 4.

Characterization of an inhibitory connection. Data are from the same pair of cells whose features are also illustrated in Figures 3 and 5a. a, Action potential (AP) evoked in the presynaptic (Pre) neuron by a depolarizing pulse. b, IPSP in the postsynaptic (Post) cell under control conditions and in the presence of bath-applied bicuculline (10 μm). c, IPSPs evoked by presynaptic APs at different membrane holding potentials. d, Dependence of the IPSPs on postsynaptic membrane potentials. The reversal potential of IPSP was approximately -70 mV. Traces in a-c are averages of 50 trials.

Figure 5.

Morphology and discharge properties of presynaptic and postsynaptic neurons for two examples of the inhibitory pathway. a, b, Top left, Photomicrographs of biocytin-labeled connected cells. Bottom left, Reconstructions of labeled neurons and their neurite arborization; the presynaptic cell is shown in blue, and the postsynaptic cell is shown in red. Dotted lines indicate approximate borders between lamina IIo and lamina IIi. Right, Action potential firing pattern of the inhibitory connected neurons: presynaptic (top of each pair) and postsynaptic (bottom of each pair). C, Caudal; D, dorsal; R, rostral; V, ventral.

Figure 6.

Responses evoked by graded dorsal root stimulation in pairs of inhibitory connected SG neurons. Pre, Presynaptic neuron; Post, postsynaptic neuron. Data are from the same pair of cells as for Figures 3, 4, 5a. a, Voltage-clamp recordings (holding potential, -60 mV) of individual responses elicited by DR stimulation. a1, Responses (EPSC) appeared in only the presynaptic cell at a relatively weak DR stimulus intensity (4.2 V, 0.5 msec, 0.1 Hz). a2, Increasing the DR stimulus (6 V, 0.5 msec, 0.1 Hz) also evoked an EPSC in the postsynaptic cell. Four trials were superimposed in a1 and a2. b, Current-clamp recordings (holding potentials: presynaptic, -60 mV; postsynaptic, -50 mV). b1, EPSPs evoked by dorsal root stimulation (6 V, 0.5 msec, 0.1 Hz). Note that the latency for EPSP in the postsynaptic cell is longer than that for the presynaptic neuron. b2, The holding potential of the presynaptic neuron was changed from -60 to -50 mV, and at the intensity of DR stimulation for b1, an action potential (AP) was initiated on the rising phase of the DR evoked EPSP. This AP leads to an IPSP at the time of the rising phase of the DR evoked EPSP in the postsynaptic neuron, suppressing most of the latter. Traces in b1 and b2 are averages of 20 trials.

Figure 7.

Schematic summarizing the C-fiber-related inhibitory circuit between islet and transient central SG neurons. The presynaptic islet cell has a GABA (bicuculline-sensitive) projection to the transient central neuron. The islet and the transient central neurons receive monosynaptic input from different primary afferent C-fibers via a glutamate AMPA receptor-mediated connection. The input to the presynaptic islet cell is from larger-diameter, more rapidly conducting C-fibers than those projecting to the postsynaptic transient central neurons. The latter is consistent with the concept that the input to the islet neurons is from C-fibers signaling different (innocuous?) events than those exciting the transient central cells (noxious?). DRG, Dorsal root ganglion.