Neurochemical characterisation of lamina II inhibitory interneurons that express GFP in the PrP-GFP mouse

Abstract

Background: Inhibitory interneurons in the superficial dorsal horn play important roles in modulating sensory transmission, and these roles are thought to be performed by distinct functional populations. We have identified 4 non-overlapping classes among the inhibitory interneurons in the rat, defined by the presence of galanin, neuropeptide Y, neuronal nitric oxide synthase (nNOS) and parvalbumin. The somatostatin receptor sst2A is expressed by ~50% of the inhibitory interneurons in this region, and is particularly associated with nNOS- and galanin-expressing cells. The main aim of the present study was to test whether a genetically-defined population of inhibitory interneurons, those expressing green fluorescent protein (GFP) in the PrP-GFP mouse, belonged to one or more of the neurochemical classes identified in the rat.

Results: The expression of sst2A and its relation to other neurochemical markers in the mouse was similar to that in the rat, except that a significant number of cells co-expressed nNOS and galanin. The PrP-GFP cells were entirely contained within the set of inhibitory interneurons that possessed sst2A receptors, and virtually all expressed nNOS and/or galanin. GFP was present in ~3-4% of neurons in the superficial dorsal horn, corresponding to ~16% of the inhibitory interneurons in this region. Consistent with their sst2A-immunoreactivity, all of the GFP cells were hyperpolarised by somatostatin, and this was prevented by administration of a selective sst2 receptor antagonist or a blocker of G-protein-coupled inwardly rectifying K+ channels.

Conclusions: These findings support the view that neurochemistry provides a valuable way of classifying inhibitory interneurons in the superficial laminae. Together with previous evidence that the PrP-GFP cells form a relatively homogeneous population in terms of their physiological properties, they suggest that these neurons have specific roles in processing sensory information in the dorsal horn.

Figure 1

nNOS, GABA and sst_2A in lamina II in the mouse. A single confocal scan through a section that had been reacted to reveal nNOS, GABA and sst_2A. A nNOS⁺/GABA⁺ neuron (double arrow) is sst_2A⁺. Several other GABA⁺ cells that lack nNOS are present in this field. Two of these that are sst_2A⁺ are marked with arrows, while two that are sst_2A^- are indicated with arrowheads. Three nearby GABA-negative cells are labelled with asterisks. Scale bar: 20 μm.

Figure 2

GFP and sst_2A expression among different neurochemical interneuron populations in the PrP-GFP mouse. a-e, a field from lamina II contains three GFP-labelled neurons, all of which are sst_2A⁺. The cells numbered 1 and 3 show weak immunoreactivity for both nNOS and galanin, while the cell numbered 2 is strongly nNOS⁺ and galanin^-. f-j, a nearby region from the same section includes a cell with weak GFP (arrow) that is sst_2A⁺ and shows strong galanin immunoreactivity, but lacks nNOS. k-o, This field from lamina II includes a GFP cell (arrow) that is sst_2A⁺ but does not contain NPY or parvalbumin (PV). Two nearby cells are indicated with arrowheads. The one on the left is PV⁺/NPY^-, while the one on the right is PV^-/NPY⁺. Both of these cells lack sst_2A. Scale bar (in o): 20 μm.

Figure 3

Expression of galanin and nNOS among sst_2A⁺ neurons in the superficial dorsal horn, and their relationship with GFP in the PrP-GFP mouse. The pie chart shows the sizes of different neurochemical populations among the inhibitory interneurons in laminae I-II. We have estimated that 54% of the inhibitory interneurons in this region are sst_2A-immunoreactive in a different mouse strain (C57Bl/6) [4], and the present results indicate that the proportion of these cells that contain only nNOS, only galanin or both nNOS and galanin are 17%, 31% and 13%, respectively. The proportions of each of these populations that are accounted for by GFP⁺ neurons in the PrP-GFP mouse are shown in green, with the corresponding percentages indicated.

Figure 4

Strength of immunostaining for nNOS and galanin among sst_2A⁺ and GFP⁺ neurons in laminae I-II of the PrP-GFP mouse. a, The frequency of cells with different intensities of labelling for nNOS and galanin, graded from 4 (strong) to 1 (very weak), or 0 (negative). Data were pooled from 4 mice (two dorsal horns from each mouse). Although many cells were only immunoreactive for nNOS or galanin, some showed both types of immunoreactivity. b, The equivalent frequency histogram for all of the cells shown in a that were also GFP-labelled.

Figure 5

GFP intensity among galanin and nNOS cells in the superficial dorsal horn of the PrP-GFP mouse and their laminar distribution. The GFP⁺ cells that contained either galanin or nNOS were divided into 3 groups: those that had a higher intensity score for galanin (galanin), those with a higher score for nNOS (nNOS) and those for which the scores were equal (both). a The numbers of cells belonging to each group that were defined as having weak (1), medium (2), strong (3) or very strong (4) labelling for GFP. Each column shows the mean number of cells per mouse and the range across the 4 mice. Note that cells in the galanin group tended to have a low level of GFP, while those in the nNOS group often had high GFP levels. b The laminar location of cells in these 3 groups plotted onto an outline of the superficial dorsal horn. In each drawing the upper line represents the edge of the dorsal horn and the lower line the lamina II-III border. Cells belonging to the nNOS group are shown as open circles, those in the galanin group as filled circles and those defined as both as filled triangles. The lower right drawing shows all 3 groups combined.

Figure 6

The effect of somatostatin in PrP-GFP neurons. a An example trace showing the membrane hyperpolarisation in response to somatostatin application. b The membrane potential before (control) and during somatostatin application. The mean hyperpolarisation was 8.9 ± 2.8 mV, and this was statistically significant (p < 0.05, n = 7). c The membrane potential did not change when somatostatin was applied in the presence of the sst₂ receptor antagonist, CYN 154806 (n = 5). d Currents measured in response to brief voltage steps (100 ms, -70 to -50 mV) in control and somatostatin conditions. e The current–voltage (I-V) relationship for the somatostatin-evoked current (n = 7). The reversal potential of this current is approximately -90 mV. f The membrane potential did not change when somatostatin was applied in the presence of the GIRK channel blocker, tertiapin-Q (n = 5), suggesting that the somatostatin-mediated membrane hyperpolarisation in the PrP-GFP cells involves activation of GIRK channels.