Characterisation of interneurons in lamina II of the rat spinal cord

Lamina II of the dorsal horn contains numerous small neurons with varying morphologies, most of which have axons that remain within the spinal cord. It can be distinguished from the other laminae by its lack of myelinated fibres and its constituent interneurons that are densely packed. This region is the major termination site for unmyelinated (C) primary afferent fibres, which convey mostly nociceptive information. It also receives inputs from thinly myelinated (Aδ) fibres, some of which are nociceptive. In spite of its importance and several past attempts, little is known of its neuronal circuitry. This is mainly due to the great functional and morphological diversity of lamina II interneurons, which has made characterisation difficult. A comprehensive classification scheme is essential to identify discrete functional populations of lamina II interneurons, and to enable understanding of their roles in the local neuronal circuitry. The present study aims to investigate the physiological, pharmacological and morphological properties of lamina II interneurons recorded in an in vitro slice preparation from adult rat spinal cord. These properties were correlated with the neurotransmitter content of each cell, which was identified by detection of vesicular transporters in axonal boutons, in order to distinguish discrete functional subpopulations of cells in this region. Both inhibitory and excitatory interneurons were identified in lamina II, based on their expression of vesicular GABA transporter (VGAT) or vesicular glutamate transporter (VGLUT2), respectively. None of the cells that had VGAT-immunoreactive axons displayed staining for VGLUT2, and vice-versa. Injection of depolarising current evoked tonic-, transient-, delayed-, gap-, reluctant- and single spike-firing among these cells. Discharge pattern was strongly related to neurotransmitter phenotype, since most excitatory cells, but very few inhibitory cells had firing patterns that could be attributed to A-type potassium (IA) currents (i.e. delayed, gap or reluctant-firing). This suggests that excitatory lamina II interneurons with IA –type firing patterns are involved in plasticity that contributes to pain states. The majority of inhibitory cells displayed tonic-firing pattern in response to depolarisation. There was also an obvious difference in the response of lamina II neurons to hyperpolarisation, since the majority of inhibitory cells showed inward currents while most excitatory cells displayed transient outward currents. Noradrenaline and serotonin hyperpolarised both inhibitory and excitatory neurons, while only inhibitory neurons responded to somatostatin. This is consistent with the findings of a previous study that had shown that the somatostatin 2 receptor (sst2a) is only expressed by inhibitory neurons in lamina II, and suggests that the pro-nociceptive effects of somatostatin are mediated by ‘disinhibition’. The somatodendritic morphology of 61 lamina II interneurons was reconstructed from projected confocal images of Neurobiotin labelling and assessed according to the morphological scheme developed by Grudt and Perl (2002). Although cells in the islet, central, vertical and radial class were identified, a substantial number of cells (19/61) had morphology that was atypical or intermediate between two classes and therefore could not be classified. Certain morphological types were consistently found in the inhibitory or excitatory population: all islet cells were GABAergic, while all radial cells and most vertical cells were glutamatergic. However, the correlation between these properties may be complex, since there was a considerable diversity in the remaining cells. Some glutamatergic interneurons had axons that contained somatostatin and many of these also contained enkephalin. Somatostatin-expressing glutamatergic cells included various morphological types, while enkephalin was detected in the axons of vertical and radial cells. All cells with axons that were somatostatin- and enkephalin-immunoreactive had delayed-firing patterns. Taken together with the pharmacological data from the present study, this suggests that somatostatin released from these glutamatergic neurons would hyperpolarise subsets of inhibitory neurons and causes disinhibition. This could lead to alterations of pain thresholds. The results from this study demonstrate that distinctive populations of inhibitory and excitatory interneurons can be recognised in lamina II, and these cells are most likely to correspond to discrete functional groups. Electrophysiological, neurochemical, morphological and pharmacological properties of neurons can be correlated but this is likely to be very complex. Future investigations that combine various approaches should allow further understanding of the specific roles of lamina II interneurons in nociceptive processing within the spinal cord.