Monosynaptic convergence of somatic and visceral C-fiber afferents on projection and local circuit neurons in lamina I: a substrate for referred pain

Abstract

Referred pain is a phenomenon of feeling pain at a site other than the site of the painful stimulus origin. It arises from a pathological mixing of nociceptive processing pathways for visceral and somatic inputs. Despite numerous studies based on unit recordings from spinal and supraspinal neurons, the exact mechanism and site of this mixing within the central nervous system are not known. Here, we selectively recorded from lamina I neurons, using a visually guided patch-clamp technique, in thoracic spinal cord preparation with preserved intercostal (somatic) and splanchnic (visceral) nerves. We show that somatic and visceral C fibers converge monosynaptically onto a group of lamina I neurons, which includes both projection and local circuit neurons. Other groups of lamina I neurons received inputs from either somatic or visceral afferents. We have also identified a population of lamina I local circuit neurons showing overall inhibitory responses upon stimulation of both nerves. Thus, the present data allow us to draw two major conclusions. First, lamina I of the spinal cord is the first site in the central nervous system where somatic and visceral pathways directly converge onto individual projection and local circuit neurons. Second, the mechanism of somatovisceral convergence is complex and based on functional integration of monosynaptic and polysynaptic excitatory as well as inhibitory inputs in specific groups of neurons. This complex pattern of convergence provides a substrate for alterations in the balance between visceral and somatic inputs causing referred pain.

Figure 1

Spinal cord preparation to study somatovisceral convergence. (A) Preparation of the thoracic spinal cord with attached intercostal T9 and splanchnic nerves. The nerves were stimulated using suction electrodes. A lamina I neuron was viewed using oblique infrared LED illumination. (B1) Low magnification photograph of a projection neuron. This is a flattened neuron with extensive dendrites, one of which gives rise to a single axon (arrow). The axon descends towards deeper laminae after a short loop (asterisks) without further branching. Scale bar, 100 μm. (B2) High magnification photograph of the initiation point (arrow) of the projection axon (asterisk). Scale bar, 25 μm. (C1) Low magnification photograph of a local circuit neuron, with a fusiform somatodendritic morphology. The soma is located on the surface, while the dendrites (out of focus) protrude ventrally into lamina II. Scale bar, 100 μm. (C2) High magnification photomicrograph of the typical local circuit neuron axon. The main axon (asterisk) is thicker with regular swellings, while frequent side branches are thin and possess numerous varicosities, some of which have a diameter >1.5 μm. Scale bar, 50 μm. Recordings from the neurons shown in (B1) and (B2) and (C1) and (C2) are given in Figs. 5A and B, respectively.

Figure 2

Somatovisceral lamina I neurons. (A) Recording from a lamina I projection neuron receiving suprathreshold inputs from the intercostal (blue traces) and splanchnic nerves (red traces). Left, Intrinsic firing pattern of this projection neuron; injected currents were (from top to bottom) +20, +10, and −10 pA. Dashed line in current-clamp indicates 0 mV. Middle, Current-clamp recording of excitatory postsynaptic potentials and spikes activated by stimulating intercostal and splanchnic nerves (pulse, 1 ms, 100 μA). Each panel shows 5 consecutive traces. Arrows indicate the time of nerve stimulation. Right, Voltage-clamp recordings of excitatory postsynaptic currents (EPSCs) evoked by stimulation of intercostal and splanchnic nerves (holding potential, −70 mV). Each panel shows EPSCs from 5 consecutive stimulations. Arrowheads indicate monosynaptic components. (B) Suppression of evoked EPSCs in an unidentified lamina I neuron by 10 μM CNQX. The nerves were stimulated by a 150 μA pulse (1 ms duration).

Figure 3

Neurons with dominating visceral inputs. (A) A lamina I projection neuron with suprathreshold visceral C-afferent input. The monosynaptic excitatory postsynaptic currents (EPSCs) are indicated by arrowheads (voltage-clamp, −70 mV). For monosynaptic inputs, 5 consecutive traces are superimposed. Intrinsic firing properties are shown for the injected currents of +70, +40, and −20 pA. (B) A nonidentified lamina I neuron with suprathreshold visceral and subthreshold somatic inputs. Monosynaptic EPSCs (indicated by arrowheads) were mediated by somatic and visceral C afferents (holding potential, −70 mV). Five consecutive traces are superimposed for current- and voltage-clamp. Intrinsic firing properties, injected currents are +100, +50, and −20 pA. C, A local circuit neuron with suprathreshold visceral and inhibitory somatic inputs. Note that although intercostal nerve stimulation evoked monosynaptic Aδ- (filled arrowhead) and C-fiber (not indicated) EPSCs (voltage-clamp, −70 mV), the overall response was inhibitory (current-clamp). The short-latency inhibitory postsynaptic current was disynaptic (open arrowhead, voltage-clamp) and Aδ-fiber-mediated. The splanchnic nerve stimulation activated a time-locked first spike (triggered by the monosynaptic C-fiber excitatory postsynaptic potential) followed by a repetitive discharge caused by polysynaptic excitatory postsynaptic potentials. For intrinsic firing properties, injected currents were +30, +20, and −20 pA. Schematic drawing shows possible organization of synaptic inputs to this local circuit neuron.

Figure 4

Neurons with dominating somatic inputs. Recordings from a projection neuron receiving suprathreshold somatic but no visceral input. Somatic excitatory postsynaptic potentials and excitatory postsynaptic currents are C-fiber-mediated. The monosynaptic excitatory postsynaptic current is indicated by an arrowhead. For the intercostal nerve, 5 consecutive traces are shown superimposed in current and voltage-clamp (holding potential, −70 mV). Intrinsic firing properties are shown for the injected currents of +80, +30, and −10 pA.

Figure 5

Neurons with inhibitory inputs. (A) Current- and voltage-clamp recordings of responses evoked in a projection neuron after stimulation of intercostal and splanchnic nerves. The neuron showed a tonic pattern of intrinsic firing (injected currents, +20 and −10 pA). Arrowheads in current-clamp indicate a potential of −70 mV. In voltage-clamp, the holding potential was −70 mV. (B1-B5) Recordings from a rhythmically firing local circuit neuron receiving a complex pattern of synaptic inputs from both nerves. Current-clamp, Recording of rhythmic firing in control (B1) and after stimulation (indicated by an arrow) of intercostal (B2) and splanchnic (B3) nerves. Note, the stimulation evoked one or several extra spikes (shown in the insets) followed by a prolonged inhibition of the discharge. Voltage-clamp, Recordings of excitatory postsynaptic currents and inhibitory postsynaptic currents activated by stimulating intercostal (B4) and splanchnic (B5) nerves. Monosynaptic excitatory postsynaptic currents are indicated by arrowheads in the insets. Schematic drawing illustrates possible organization of synaptic inputs to this local circuit neuron.

Figure 6

Proposed model of somatovisceral convergence of thin afferents onto lamina I neurons. Somatic (blue) and visceral (red) afferents converge directly onto a lamina I projection neuron (PN) and a lamina I local circuit neuron (LCN), where somatic and visceral processing pathways merge together. Lamina I LCNs can be both inhibitory and excitatory and can directly synapse on PNs. Intercalated excitatory (+) and inhibitory (−) neurons are shown by smaller circuits; their laminar location is not known. The excitatory intercalated neurons may amplify the primary afferent-driven input to a PN. The inhibitory intercalated neurons may play diverse roles, eg, disinhibit a PN by supressing activity in a rhythmic inhibitory LCN, or induce reciprocal inhibition of somatic or visceral inputs. At the same time, other PNs receive somatic- or visceral-specific inputs.