Multisegmental A{delta}- and C-fiber input to neurons in lamina I and the lateral spinal nucleus

Abstract

Spinal lamina I and the lateral spinal nucleus (LSN) receive and integrate nociceptive primary afferent inputs to project through diverse ascending pathways. The pattern of the afferent supply of individual lamina I and LSN neurons through different segmental dorsal roots is poorly understood. Therefore, we recorded responses of lamina I and LSN neurons in spinal segments L4 and L3 to stimulation of six ipsilateral dorsal roots (L1-L6). The neurons were viewed through the overlying white matter in the isolated spinal cord preparation using the oblique infrared LED illumination technique. Orientation of myelinated fibers in the white matter was used as a criterion to distinguish between the LSN and lamina I. Both types of neurons received mixed (monosynaptic and polysynaptic) excitatory Adelta- and C-fiber input from up to six dorsal roots, with only less than one-third of it arising from the corresponding segmental root. The largest mixed input arose from the dorsal root of the neighboring caudal segment. Lamina I and LSN neurons could fire spikes upon the stimulation of up to six different dorsal roots. We also found that individual lamina I neurons can receive converging monosynaptic Adelta- and/or C-fiber inputs from up to six segmental roots. This study shows that lamina I and LSN neurons function as intersegmental integrators of primary afferent inputs. We suggest that broad monosynaptic convergence of Adelta- and C-afferents onto a lamina I neuron is important for the somatosensory processing.

Figure 1.

Identification of lamina I and LSN neurons in the isolated spinal cord. A, Preparation of the lumbar spinal cord with unilateral six dorsal roots, L1–L6. The roots were stimulated through suction electrodes. Inset, Lamina I neuron viewed using oblique LED illumination. B, A cross section of the fixed spinal cord (L4, 27-d-old rat) with indications of the LSN and the lamina I region accessible for the recording. Continuous lines show the border of the gray matter and the LSN. C, Schematic drawing of the fiber orientation in the dorsal and dorsolateral white matter used for the identification of the LSN and lamina I. The photographs shown below were taken from the regions (1–4) indicated on the schematic drawing. The focal plane was chosen to show the fibers. Region 1, The LSN: large neuronal cell bodies are surrounded by the parallel rostrocaudal myelinated fibers of the dorsolateral funiculus. Region 2, Transitional zone between the dorsolateral funiculus (left) and lamina I (right). Region 3, Network of the randomly oriented fibers and the scattered cell bodies in lamina I. Region 4, The medial border of accessible part of lamina I (left) and myelinated fibers in the dorsal root entry zone (right). D, Parasagittal sections (100 μm thick) of the spinal cord with a biocytin-labeled LSN neuron (left) and lamina I neuron (right). Locations of the sections are shown in the schematic drawings where asterisks indicate the cell body positions. Left, LSN neuron is found outside the spinal gray matter within the dorsolateral funiculus. Continuous lines show the dorsal borders of the white matter in the top and bottom of the section. Right, Lamina I neuron is seen within the dorsolateral gray matter. Dashed line shows the border between the gray and white matter. Continuous lines indicate the dorsal borders of the white matter in the top and bottom of the section.

Figure 2.

Mixed inputs to an L4 lamina I neuron. Mixed (monosynaptic and polysynaptic) EPSCs were elicited in an L4 lamina I neuron by stimulating L1–L6 roots. A, Each root was stimulated at least 10 times with a 1 ms current pulse at 0.1 Hz to elicit both Aδ- and C-fiber mixed EPSCs. For each root, individual recordings (upper traces) were averaged (lower traces). B, Each dorsal root was stimulated at least 10 times with a 50 μs pulse at 1 Hz to elicit only Aδ-fiber mixed EPSCs. Averaged responses (lower traces) were obtained from individual recordings (upper traces). C, The averaged traces are shown superimposed. Holding potential was −70 mV. Here and in the following figures, the fastest evoked EPSCs (monosynaptic and polysynaptic) appeared with latencies corresponding to afferent conduction velocity below 4.4 m/s (conduction velocity of the fastest Aδ-afferents in dorsal roots, Pinto et al., 2008a). Note that all roots differed in length, and therefore, had different latency criteria for separation of Aδ- and C-fiber EPSCs.

Figure 3.

Integrated mixed inputs to lamina I neurons. A, integrated mixed inputs to an L4 lamina I neuron from L1–L6 roots. For each root, the areas under the corresponding averaged traces (similar to those in Fig. 2C) were calculated by integration. The integrated input is given in pC (1 pC is the electrical charge transferred, for example, by a 100 pA current for 10 ms). B, Distributions of integrated mixed inputs to an L3 lamina I neuron. C, The distribution of the integrated Aδ- and C-inputs, Aδ-inputs, and C-inputs centered on the segment where the neuron was located (0, vertical gray bar). More caudal and more rostral segmental dorsal roots are indicated by positive and negative numbers, respectively. D, Comparison of the integrated mixed inputs to L4 lamina I and lamina II neurons from the strongest root (L5). The data for lamina I neurons (n = 13) are from A, while those for lamina II neurons (n = 19) were obtained by reevaluation of recordings from Pinto et al. (2008b). SG, Substantia gelatinosa (lamina II).

Figure 4.

Efficacy of inputs to a lamina I neuron. A, Voltage- and current-clamp recordings of synaptic inputs to an L4 lamina I neuron. Roots (L1–L6) were stimulated by 1 ms pulses to activate both Aδ- and C-fiber mixed EPSCs and EPSPs. The membrane potential was close to −70 mV. B, The mean number of spikes evoked per stimulation of a root. For each neuron, each root was stimulated at least 10 times and the mean numbers were calculated. Graphs are based on data from 13 L4 lamina I neurons and 11 L3 lamina I neurons. Vertical gray bars indicate the segment of the neuron location. APs, action potentials.

Figure 5.

Converging monosynaptic Aδ- and C-fiber inputs to an L4 lamina I neuron. Recording from the lamina I neuron with the broadest input observed. Left, Recordings of EPSCs elicited by stimulating L1–L6 roots with 1 ms pulses. Holding potential, −70 mV. Monosynaptic C- and Aδ-fiber-mediated EPSCs are indicated by filled and open triangles, respectively. The triangles also show the time moment for which the latency analysis was done (see insets). Middle, The roots were also stimulated with 50 μs pulses. Recordings are shown as a superposition of 10 consecutive traces for roots with monosynaptic inputs (indicated by triangles) and 5 consecutive traces for roots without monosynaptic input. The C-fiber-mediated input from the L6 root (indicated by a horizontal gray bar) was too strong and could not be analyzed. Right, Schematic drawing of the monosynaptic projections of C- and Aδ-afferents originating from the L1–L6 roots to this L4 lamina I neuron. Insets at bottom show two examples of analysis of the monosynaptic inputs. Inset 1, EPSCs elicited by stimulating the L4 root at Aδ-fiber intensity. The faster component (marked by an asterisk) showing failures and the latency variation exceeding 1 ms was not considered as monosynaptic. The slower component (indicated by an open triangle) fulfilled criteria of monosynaptic input. The vertical gray bar has a width of 1 ms (maximum latency variation allowed for the monosynaptic response). Inset 2, The response to the L6 root stimulation consisted of multiple overlapping components of composite EPSC, one of which (indicated by an open triangle) was considered as monosynaptic. The triangle indicates the exact time moment for which the analysis was done. Note that here and in the following figures the triangles indicate the monosynaptic component, and therefore, may not coincide with the beginning of the composite EPSCs.

Figure 6.

Converging monosynaptic Aδ-fiber inputs to an L3 lamina I neuron. A, Left, EPSCs elicited by stimulating L1–L6 roots with 1 ms pulses. Holding potential, −70 mV. Monosynaptic Aδ-fiber EPSCs are indicated by open triangles. Middle, The roots were stimulated at 50 μs. Recordings are shown as a superposition of 10 and 5 consecutive traces for roots with and without monosynaptic inputs, respectively. Inputs from the roots L4 and L5 also show composite EPSCs (indicated by asterisks) consisting of overlapping slow Aδ- and fast C-fiber components. In these composite EPSCs, neither the C-fiber component (analyzed at 1 ms stimulation) nor the-Aδ-fiber component (analyzed at 1 ms and 50 μs stimulations) satisfied our criteria for monosynaptic inputs. Right, Schematic drawing of the monosynaptic Aδ-fibers projecting from the L3, L4, and L5 dorsal roots to this L3 lamina I neuron. B, Biocytin-labeled soma, axon, and dendrites of the lamina I neuron from A. The axon had local collaterals around the cell body and one prominent caudally oriented branch (indicated by an asterisk), which gave rise to faint collaterals. Gray lines indicate the contours of the bottom of the serial sections (some contour lines were omitted).

Figure 7.

Aδ- and C-fiber mixed inputs to an LSN neuron. A, Mixed EPSCs elicited in an L4 LSN neuron by stimulating L1–L6 roots. The stimulation protocol was as in Figure 2. Holding potential, −70 mV. B, Integrated mixed inputs to an L4 LSN neuron. C, Centered mixed inputs to an LSN neuron. Pooled data from L4 LSN neurons (n = 6) and L3 LSN neurons (n = 5).

Figure 8.

Efficacy of inputs to an LSN neuron. A, Voltage- and current-clamp recordings of synaptic inputs to an L4 LSN neuron. The L1–L6 roots were stimulated by 1 ms pulses to activate both Aδ- and C-fiber mixed EPSCs and EPSPs. Stimulation of five roots evoked spikes. The membrane potential was −70 mV. B, Membrane responses of an LSN neuron to an injection of 500 ms current pulses of −5 pA, +15 pA, +35 pA, and +55 pA. C, A fast afterhyperpolarization (fAHP) and a slow afterhyperpolarization (sAHP) observed in the majority of LSN neurons.

Figure 9.

Converging monosynaptic Aδ-fiber inputs to an L3 LSN neuron. A, Left, EPSCs elicited by stimulating L1–L6 roots (1 ms pulses). Holding potential, −80 mV. Monosynaptic Aδ-fiber EPSCs are indicated by open triangles. Middle, The roots were stimulated with short pulses (50 μs). Recordings are shown as a superposition of 10 and 5 consecutive traces for the roots with and without monosynaptic inputs, respectively. Right, Schematic drawing of the monosynaptic Aδ-fibers projecting from the L3 and L4 roots to this L3 LSN neuron. B, Biocytin-labeled soma, axon, and dendrites of the neuron from A. The soma located inside the dorsolateral funiculus gave rise to four major dendrites. The axon gave rostrocaudal and ventral collaterals with numerous en-passant varicosities. The main axon (indicated by an asterisk) had constant diameter and descended ventrally and medially in several sections before crossing the level of the central canal toward the contralateral anterolateral white matter. Gray lines indicate the contours of the bottom of the serial sections (some contour lines were omitted).

Figure 10.

Proposed model of monosynaptic convergence of thin afferents onto a lamina I neuron. A model of inputs to an L4 lamina I neuron from the L1–L6 roots was developed on the basis of our data and studies described in Table 3. Solid and dotted lines indicate the most probable and less probable types of afferents, respectively. Cutaneous afferents (blue) originating from the sciatic nerve provide the major supply of an L4 lamina I neuron from the L3, L4, and L5 roots. The monosynaptic projections to an L4 lamina I neuron arising from the L1 and L2 roots are of noncutaneous somatic (muscles and joints) origin (red). DR, Dorsal root; SN, spinal nerve; DRS, dorsal ramus of the spinal nerve; VRS, ventral ramus of the spinal nerve; SCI, sciatic nerve.