Bicuculline-sensitive primary afferent depolarization remains after greatly restricting synaptic transmission in the mammalian spinal cord

Abstract

Primary afferent neurotransmission is the fundamental first step in the central processing of sensory stimuli. A major mechanism producing afferent presynaptic inhibition is via a channel-mediated depolarization of their intraspinal terminals which can be recorded extracellularly as a dorsal root potential (DRP). Based on measures of DRP latency it has been inferred that this primary afferent depolarization (PAD) of low-threshold afferents is mediated by minimally trisynaptic pathways with GABAergic interneurons forming last-order axoaxonic synapses onto afferent terminals. We used an in vitro rat spinal cord preparation under conditions that restrict synaptic transmission to test whether more direct low-threshold pathways can produce PAD. Mephenesin or high divalent cation solutions were used to limit oligosynaptic transmission. Recordings of synaptic currents in dorsal horn neurons and population synaptic potentials in ventral roots provided evidence that conventional transmission was chiefly restricted to monosynaptic actions. Under these conditions, DRP amplitude was largely unchanged but with faster time to peak and reduced duration. Similar results were obtained following stimulation of peripheral nerves. Even following near complete block of transmission with high Mg(2+)/low Ca(2+)-containing solution, the evoked DRP was reduced but not blocked. In comparison, in nominally Ca(2+)-free or EGTA-containing solution, the DRP was completely blocked confirming that Ca(2+) entry mediated synaptic transmission is required for DRP genesis. Overall these results demonstrate that PAD of low-threshold primary afferents can occur by more direct synaptic mechanisms, including the possibility of direct negative-feedback or nonspiking dendroaxonic pathways.

Figure 1.

A, Experimental setup (see Materials and Methods for details). B–D, Stimulation is via the L5 dorsal root at 100 μA, 100 μs. Bi, A single stimulus produces a DRP that is almost entirely blocked by bicuculline. The remaining potential is TTX-sensitive. Bii, Low-threshold activation is confirmed by measuring the afferent volley produced by this stimulation and its TTX sensitivity. C, Picrotoxin completely blocks DRP after synaptic isolation with mephenesin. D, Similarly, gabazine blocks the DRP after preincubation in mephenesin. E, The DRP requires chemical synaptic transmission. Evoked responses are shown before and after bath exchange to a nominally 0 Ca²⁺-containing saline. Identical results were seen in another animal after application of the Ca²⁺ chelator EGTA (100 μm). Left column is a single shock and right column is five pulses at 20 Hz. Traces were low-pass filtered at 300 Hz.

Figure 2.

A, Relation between afferent volleys and evoked field potentials. Stimuli were delivered at 10T to the L5 dorsal root. Afferent volleys were recorded from the same dorsal root. Extracellular field potentials (EFPs) were recorded in the L5 deep dorsal horn. Ai, Stimulation recruits 2 separable afferent volleys, each leading to a monosynaptic field potential. These responses are unaffected by the presence of mephenesin. Aii, In the presence of a high divalent cation solution, the low-threshold afferent volley waveforms and corresponding evoked field potentials are slowed. Aiii, Relation of monosynaptic EFPs to the evoked DRP and VRPs. The evoked DRP waveform shape is largely unchanged in the presence of the high divalent cation solution whereas VRP duration is greatly shortened. Bi, Bii, EPSCs in 2 neurons measured at a holding potential of −80 mV. Identity of synaptic current was verified as excitatory at a holding potential of −40 mV. Under both conditions (high Mg²⁺/high Ca²⁺ and mephenesin) the longer-latency synaptic response (at arrow) is blocked while the monosynaptic component is not significantly affected. Ci, Cii, High divalent cation solution and mephenesin both block longer latency DRPs in the same animal (arrows on difference trace identifying inflection of longer-latency component). D, Similarly, most of the DRP remains after treatment with mephenesin following stimulation of the tibial or deep peroneal nerve at 4T (Di) or 10T (Dii). Traces were low-pass filtered at 3 kHz and at 300 Hz in C.

Figure 3.

A, Greatly reducing synaptic transmission does not completely block the DRP. Evoked responses are shown before and after bath exchange to a high Mg²⁺/low Ca²⁺ saline (6.5 mm Mg²⁺/0.85 mm Ca²⁺). Stimulation is via the L5 dorsal root at 100 μA, 100 μs. Ai, Left, Single shock; right, five pulses at 20 Hz. In high Mg²⁺/low Ca²⁺ saline, the DRP is facilitated following repetitive stimulation. Aii, Under the same conditions in another animal, the high Mg²⁺/low Ca²⁺ saline virtually abolished monosynaptic EPSCs, but transmission is increased by repetitive stimulation (upper row). Traces were low-pass filtered at 300 Hz. B, Putative circuits for low-threshold afferent-evoked PAD not requiring trisynaptic pathways. As mephenesin and high divalent cation solutions block part of the DRP, a conventional disynaptic pathway may serve PAD of some primary afferents. The remaining DRP may arise from direct monosynaptic actions of transmitter onto afferent terminals or via a disynaptic nonspiking dendroaxonic microcircuit. For details see Discussion.