Physiological, neurochemical and morphological properties of a subgroup of GABAergic spinal lamina II neurones identified by expression of green fluorescent protein in mice

Abstract

The processing of sensory, including nociceptive, information in spinal dorsal horn is critically modulated by spinal GABAergic neurones. For example, blockade of spinal GABA A receptors leads to pain evoked by normally innocuous tactile stimulation (tactile allodynia) in rats. GABAergic dorsal horn neurones have been classified neurochemically and morphologically, but little is known about their physiological properties. We used a transgenic mouse strain coexpressing enhanced green fluorescent protein (EGFP) and the GABA-synthesizing enzyme GAD67 to investigate the properties of a subgroup of GABAergic neurones. Immunohistochemistry showed that EGFP-expressing neurones accounted for about one-third of the GABAergic neurones in lamina II of the spinal dorsal horn. They constituted a neurochemically rather heterogeneous group where 27% of the neurones coexpressed glycine, 23% coexpressed parvalbumin and 14% coexpressed neuronal nitric oxide synthase (nNOS). We found almost no expression of protein kinase Cγ (PKCγ) in EGFP-labelled neurones but a high costaining with PKCβII (78%). The whole-cell patch-clamp technique was used to intracellularly label and physiologically characterize EGFP-and non-EGFP-expressing lamina II neurones in spinal cord slices. Sixty-two per cent of the EGFP-labelled neurones were islet cells while the morphology of non-EGFP-labelled neurones was more variable. When stimulated by rectangular current injections, EGFP-expressing neurones typically exhibited an initial bursting firing pattern while non-EGFP-expressing neurones were either of the gap or the delayed firing type. EGFP-expressing neurones received a greater proportion of monosynaptic input from the dorsal root, especially from primary afferent C-fibres. In conclusion, EGFP expression defined a substantial but, with respect to the measured parameters, rather inhomogeneous subgroup of GABAergic neurones in spinal lamina II. These results provide a base to elucidate the functional roles of this subgroup of GABAergic lamina II neurones, e.g. for nociception.

Identification of Early RET+ Deep Dorsal Spinal Cord Interneurons in Gating Pain

“…Early RET+ dDH neurons can be categorized into five groups based on morphology: islet cells (29%), radial cells (22%), vertical cells (16%), inverted stalked cells (8%), and unclassified neurons (25%) ( Figures 2H-I). Islet cells are predominantly inhibitory, while other morphologies have been associated with both excitatory and inhibitory phenotypes (Heinke et al, 2004;Todd, 2010). Taken together, our results reveal complex intrinsic firing properties and morphologies of the early RET+ dDH neurons, which are consistent with their DH inhibitory neuron identity.…”

“…They have complicated morphologies and firing patterns (Heinke et al, 2004;Todd, 2010), which makes the task of defining functional subpopulations of spinal cord inhibitory interneurons challenging. Here, we identified a novel population of DH interneurons located in the deep layers III through V of the spinal cord, which express Ret neonatally.…”

Genetically Defined Inhibitory Neurons in the Mouse Spinal Cord Dorsal Horn: A Possible Source of Rhythmic Inhibition of Motoneurons during Fictive Locomotion

“…We cannot definitively determine whether these boutons arise from the GFPϩ interneurons located in laminae V/VI, dorsal laminae I-II, or descending neurons (Holstege, 1991). However, it is unlikely that inhibitory neurons in the superficial dorsal horn project directly to motoneurons (Heinke et al, 2004). Although axonal projections could not be completely reconstructed in the slice, fibers were observed originating from the region of medial laminae V/VI and coursing toward the motor pools.…”

“…Given the coexpression of GAD isoforms in the spinal cord , and the coexpression of GABA and glycine neurotransmitters (Todd and Sullivan, 1990), it was not surprising that GFP-expressing neurons also contained GAD67 or in rare instances GlyT2 in their terminals. By the same principles, it is possible that either the dorsally located GFPϩ neurons in laminae I-II and/or laminae V/VI seen in these GAD65::GFP animals are the same as those expressing GFP in GAD67:GFP animals (Heinke et al, 2004;Dougherty et al, 2009).…”

“…Given the location of these superficial GABAergic interneurons, it is possible that they are associated with local afferent processing such as inhibitory transmission to substantia gelatinosa neurons (Yoshimura and Nishi, 1995). It has been shown that small inhibitory cells in the superficial dorsal horn are mostly related to processing ascending sensory information by modulating neurotransmitter release from the central terminals of primary afferents (Lao and Marvizon, 2005) and being the substrate for lateral inhibition in ascending sensory systems (Le Bars, 2002;Heinke et al, 2004;Graham et al, 2007). Heinke et al (2004) suggested that GFP-positive cells in laminae I and II in GAD67-GFP transgenic mice are involved in the transmission of nociceptive information to the brain.…”