Correlations between neuronal morphology and electrophysiological features in the rodent superficial dorsal horn

Abstract

Relationships between the morphology of individual neurones of the spinal superficial dorsal horn (SDH), laminae I and II, and their electrophysiological properties were studied in spinal cord slices prepared from anaesthetized, free-ranging hamsters. Tight-seal, whole-cell recordings were made with pipette microelectrodes filled with biocytin to establish electrophysiological characteristics and to label the studied neurones. Neurones were categorized according to location and size of the somata, the dendritic and axonal pattern of arborization, spontaneous synaptic potentials, evoked postsynaptic currents, pattern of discharge to depolarizing pulses and current-voltage relationships. Data were obtained for 170 neurones; 13 of these had somata in lamina I and 157 in lamina II. Stimulation of the segmental dorsal root evoked a prompt excitatory response in almost every neurone sampled (161/166) with nearly 3/4 displaying putative monosynaptic EPSCs. The majority of neurones (133/170) fitted one of several distinctive morphological categories. To a considerable extent, neurones with a common morphological configuration and neurite disposition shared electrophysiological characteristics. Five of the 13 lamina I neurones were relatively large with extensive dendritic arborization in the horizontal dimension and a prominent axon directed ventrally and contralaterally. These presumptive ventrolateral projection neurones differed structurally and electrophysiologically from the other lamina I neurones, which had ipsilateral, locally arborizing axons and/or branches entering the dorsal lateral funiculus. One hundred and twenty lamina II neurones fitted one of five morphological categories: islet, central, medial-lateral, radial or vertical. Central cells were further divided into three groups on functional features. We conclude that the spinal SDH comprises many types of neurones whose morphological characteristics are associated with specific functional features implying diversity in functional organization of the SDH and in its role as a major synaptic termination for thin primary afferent fibres.

Figure 1. Locations of the recorded SDH (laminae I, II_o and II_i) neurones

Each neurone studied was plotted as a circle on a schematic drawing of the lower lumbar dorsal horn. The position of each neurone was established using both darkfield microscopy, in which the myelin-poor SDH appears dark, and bright field cytoarchitectonic markers of the laminae.

Figure 2. Micrographs of biocytin-stained lamina I neurones

Two examples are shown of each category as captured from 60 μm parasagittal histological sections. Dorsal is shown up. A, lamina I projection cells. Note the thick ventrally directed axon in A1, which in adjacent sections extended ventral to the central canal and turned toward the contralateral side of the spinal cord. B, lamina I non-projection cells. Axons of non-projection lamina I neurones, identified as uniform thin profiles originating from the soma (or on occasion a proximal dendrite) arborized locally.

Figure 4. Micrographs of unclassified lamina II cells

About 25 % of lamina II cells in the sample did not fit the criteria for any of the categorized groups. A, a lamina II neurone that had a smaller and less dense dendritic tree than islet cells and longer, less compact dendritic arborization than central cells (Fig. 3C). B, a lamina II neurone with dendrites spreading in several directions with a less dense arbor than the radial category (Fig. 3B). C, a lamina II neurone with vertically directed dendrites with a much denser arborization than the vertical category (Fig. 3E). In addition to morphological distinctions from the most similar of the categorized class, unclassified neurones exhibited functional properties that differed from those of classified groups.

Figure 3. Micrographs of biocytin-stained lamina II neurones exemplifying classified categories

Two examples of each category are shown. Images were captured from 60 μm parasagittal histological sections except D2, which was taken from a 60 μm transverse section. Dorsal is shown up. See text for additional details. A, islet cells. The dendritic and axonal arbors of this class extended further rostro-caudally than any other type of lamina II neurone. Although the soma was occasionally located in II₀, most of the dendritic and axonal arborizations were in lamina II_i. B, radial cells. The soma of these neurones was usually located near the border between lamina II_o and II_i. C, central cells. This class had a general configuration similar to islet cells, but with a much smaller dendritic expansion in the rostro-caudal direction. D, medial-lateral cells. A group of three neurones notable for being the sole lamina II neurones with dendrites that extended substantially in the medio-lateral dimension. Dendrites of the cell shown in D1 were present in five adjacent 60 μm parasagittal sections. The image illustrated in D2 is from a 60 μm transverse section. E, vertical cells. A partially heterogeneous group which had in common a dendritic arbor expanding more prominently dorso-ventrally than other lamina II neurones.

Figure 5. Whole-cell, voltage clamp recording of spontaneous synaptic currents from a SDH neurone

Membrane potential was held at −50 mV. Spontaneous inward (downward) and outward (upward) postsynaptic currents are prominent.

Figure 10. Electrophysiological properties of lamina II vertical neurones

The displayed data are from the neurones for which morphology is shown in Fig. 3E1 and E2. See the legend for Fig. 6 and Methods for details of the experimental procedures.

Figure 6. Electrophysiological properties of lamina I neurones

Two examples are shown for each neuronal category. The responses are from the cells whose morphology is illustrated in Figure 2. A1 and 2, projection cells. B1 and 2, non-projection cells. From above down: a, current clamp recordings of action potential firing patterns in response to a depolarizing step from −60 mV; b, voltage clamp recordings of the synaptic response elicited by stimulating a segmental dorsal root at a holding potential of −50 mV; c, current-voltage relationship obtained by holding the cell at −60 mV in voltage clamp and stepping in 10 mV increments initially from −60 to −50 mV and then stepwise to −120 mV.

Figure 7. Electophysiological properties of lamina II islet (A1 and 2) and radial (B1 and 2) neurones

Recordings were obtained from neurones for which morphology is shown in Fig. 3A1 and 2 (islet cells) and Fig. 3B1 and 2 (radial cells). See the legend for Fig. 6 and Methods for description of the experimental procedures.

Figure 8. Electrophysiological properties of lamina II transient central I_A (A1 and 2) and medial-lateral (B1 and 2) neurones

The data for the medial-lateral neurones are from neurones for which morphology is displayed in Fig. 3D1 and 2. See the legend for Fig. 6 and Methods for description of the experimental procedures. Note the difference in the horizontal axis (time) in A1c, A2c and B1c from that for B2c. The holding potential in A1c, A2c and B1c was −50 mV; for B2c it was −60 mV. Although both some transient central and the medial-lateral categories exhibit a transient outward I_A-like current, they differ substantially in other physiological as well as in morphological characteristics (see Fig. 3).

Figure 9. Electrophysiological properties of lamina II transient central (non- I_A) (A1 and 2) and tonic central (B1 and 2) neurones

The data were obtained from the transient central (non-I_A) neurones for which morphology is shown in Fig. 3C1 and 2. See the legend for Fig. 6 and Methods for details of the experimental procedures.