- Professor of Neuroscience (Neurosciences and Clinical Psychology)
Neurochemistry and Synaptic Connections in the Mammalian Spinal Cord
My main research interest is the organisation of neuronal circuits in the spinal dorsal horn that underlie the perception of pain and itch. We use immunocytochemistry and confocal microscopy combined with other techniques, such as neuronal tract-tracing, patch-clamp recording from spinal cord slices, and electron microscopy. I have collaborations with: Dr Marco Beato (UCL); Dr Toshi Yasaka (Saga University, Japan), Dr Sarah Ross (University of Pittsburgh, USA); Dr Peter Szucs and Prof Boris Safronov (University of Porto, Portugal); Dr Masahiko Watanabe (Hokkaido University, Japan). My work is funded by the Wellcome Trust and the BBSRC.
Organisation of neuronal circuits in the dorsal horn
Although the dorsal horn plays a major role in transmitting and modifying incoming sensory information, we know little about the nerve circuits responsible for these functions, largely due to the difficulty in defining functional populations of neurons.
Projection neurons, with axons that travel directly to the brain, are concentrated in lamina I, and most of these express the neurokinin 1 receptor (NK1r). There are ~400 lamina I projection neurons per side in the rat L4 segment, corresponding to ~5% of all neurons in this lamina. Many project to more than one brain region. There are also NK1r-expressing projection neurons in lamina III, with similar supraspinal targets, and we have identified a population of giant lamina I projection cells that lack the NK1r.
Most neurons in laminae I-III have axons that remain in the spinal cord and are defined as interneurons. They can be divided into two main groups: excitatory and inhibitory. We have shown that among the inhibitory interneurons 4 populations can be defined by the expression of neuropeptide Y (NPY), galanin/dynorphin, neuronal nitric oxide synthase (nNOS) and parvalbumin. These account for over half the inhibitory interneurons in laminae I-II.
We have shown that they differ in their postsynaptic targets, e.g. NPY/GABA axons preferentially innervate NK1r+ lamina III projection cells, while nNOS/GABA axons innervate lamina I giant cells. Some of the neuronal circuits that have been identified in laminae I-III are summarised below.
Changes in dorsal horn following nerve injury
Injury to peripheral nerves often leads to neuropathic pain, but the underlying mechanisms are poorly understood. There is evidence that changes in the dorsal horn contribute to certain types of neuropathic pain, and several possible mechanisms have been proposed. These include: (1) sprouting of Aβ low-threshold mechanoreceptive afferents into the superficial laminae, where they would have access to nociceptive-specific projection neurons; (2) a phenotypic switch, such that these afferents start to synthesise substance P, which could activate NK1rs on nociceptive projection neurons; (3) loss of inhibitory interneurons from the superficial laminae.
We have provided evidence against each of these suggestions. We found that both before and after nerve injury, the central projections of Aβ afferents extend into, but not beyond the inner half of lamina II. We also showed that the central terminals of these afferents did not express or release substance P after nerve injury. We found that in rat models of neuropathic pain, there was no change in the total number of neurons in laminae I-III, nor in the proportion that were GABA-immunoreactive. There is known to be apoptotic cell death in the dorsal horn in these models, as shown by staining with the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method. However, we showed that this was restricted to microglia (labelled with Iba1 antibody), and that TUNEL positive cells were not neurons.
Interneurons in the ventral horn
Although most of our work is on the dorsal horn, we have also identified two important populations of ventral horn interneurons: those giving rise to the "P" and "C" boutons that are associated with motoneurons.
P boutons are GABAergic terminals that form axoaxonic synapses on Ia muscle spindle afferents and regulate proprioceptive input to motoneurons. We showed that P boutons were unique in expressing the 65kDa molecular weight form of glutamic acid decarboxylase (GAD65) This enabled us to identify their cells of origin, which were in the deep medial dorsal horn, and showed that P boutons are likely to represent the only output for these cells.
The C boutons are cholinergic axons that regulate motoneuronexcitability through m2 muscarinic receptors. In collaboration with Drs Rob Brownstone and Gareth Miles (Dalhousie University, Canada) we used a neurochemical/developmental approach to demonstrate that these boutons originate from a group of cholinergic cells located lateral to the central canal (cc) – the so-called "medial partition cells". This was based on the finding that the C boutons, which express choline acetyltransferase (ChAT), lack neuronal nitric oxide synthase (nNOS) but transiently express the transcription factor Dbx1, which was captured with yellow fluorescent protein (YFP).