Andrew J. Todd

Neuronal circuitry for pain processing in the dorsal horn

Neurons in the spinal dorsal horn process sensory information, which is then transmitted to several brain regions, including those responsible for pain perception. The dorsal horn provides numerous potential targets for the development of novel analgesics and is thought to undergo changes that contribute to the exaggerated pain felt after nerve injury and inflammation. Despite its obvious importance, we still know little about the neuronal circuits that process sensory information, mainly because of the heterogeneity of the various neuronal components that make up these circuits. Recent studies have begun to shed light on the neuronal organization and circuitry of this complex region.

The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid‐lumbar spinal cord of the rat. The great majority (94–100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2‐immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin‐ and enkephalin‐containing boutons (including somatostatin‐immunoreactive boutons that lacked calcitonin gene‐related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present.

GABA-immunoreactive neurons in the dorsal horn of the rat spinal cord

An antiserum to GABA was used on semithin resin-embedded sections of rat dorsal horn. Immunoreactive neurons were evenly distributed throughout laminae I-III and constituted between 24 and 33% of the total neuronal population within three laminae. Fifty Golgi-stained cells in lamina II were tested with the antiserum. Most of the islet cells examined were immunoreactive, although some small islet cells were not. None of the 14 stalked cells tested was immunoreactive. These results provide further evidence that the stalked and islet cells of lamina II form two distinct functional classes and suggest that the islet cells function as inhibitory interneurons.

Neurokinin 1 receptor expression by neurons in laminae I, III and IV of the rat spinal dorsal horn that project to the brainstem.

Large neurons in laminae III and IV of the spinal cord which express the neurokinin 1 receptor and have dendrites that enter the superficial laminae are a major target for substance P (SP)‐containing (nociceptive) primary afferents. Although some of these neurons project to the thalamus, we know little about other possible projection targets. The main aim of this study was to determine whether all cells of this type are projection neurons and to provide information about brainstem sites to which they project. Injections of cholera toxin B subunit were made into four brainstem areas that receive input from the spinal cord, and the proportion of cells of this type in the L4 spinal segment that were retrogradely labelled was determined in each case. The results suggest that most of these cells (>90%) project to the contralateral lateral reticular nucleus (or to a nearby region), while many (>60%) send axons to the lateral parabrachial area and some to the dorsal part of the caudal medulla. However, few of these cells project to the periaqueductal grey matter. As lamina I neurons with the neurokinin 1 receptor appear to be important in the generation of hyperalgesia, we also examined projection neurons in this lamina and found that for each injection site the great majority possessed the receptor. These results demonstrate that dorsal horn neurons which express the neurokinin 1 receptor contribute to several ascending pathways that are thought to be important in pain mechanisms.

Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion

To effect movement, motoneurons must respond appropriately to motor commands. Their responsiveness to these inputs, or excitability, is regulated by neuromodulators. Possible sources of modulation include the abundant cholinergic “C boutons” that surround motoneuron somata. In the present study, recordings from motoneurons in spinal cord slices demonstrated that cholinergic activation of m2-type muscarinic receptors increases excitability by reducing the action potential afterhyperpolarization. Analyses of isolated spinal cord preparations in which fictive locomotion was elicited demonstrated that endogenous cholinergic inputs increase motoneuron excitability during locomotion. Anatomical data indicate that C boutons originate from a discrete group of interneurons lateral to the central canal, the medial partition neurons. These results highlight a unique component of spinal motor networks that is critical in ensuring that sufficient output is generated by motoneurons to drive motor behavior.

Selective loss of spinal GABAergic or glycinergic neurons is not necessary for development of thermal hyperalgesia in the chronic constriction injury model of neuropathic pain.

&NA; GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABAA and glycine receptor antagonists produces behavioural signs of allodynia, suggesting that these transmitters have an important role in spinal pain mechanisms. Several studies have described a substantial loss of GABA‐immunoreactive neurons from the dorsal horn in nerve injury models, and it has been suggested that this may be associated with a loss of inhibition, which contributes to the behavioural signs of neuropathic pain. We have carried out a quantitative stereological analysis of the proportions of neurons in laminae I, II and III of the rat dorsal horn that show GABA‐ and/or glycine‐immunoreactivity 2 weeks after nerve ligation in the chronic constriction injury (CCI) model, as well as in sham‐operated and naïve animals. At this time, rats that had undergone CCI showed a significant reduction in the latency of withdrawal of the ipsilateral hindpaw to a radiant heat stimulus, suggesting that thermal hyperalgesia had developed. However, we did not observe any change in the proportion of neurons in laminae I–III of the ipsilateral dorsal horn that showed GABA‐ or glycine‐immunoreactivity compared to the contralateral side in these animals, and these proportions did not differ significantly from those seen in sham‐operated or naïve animals. In addition, we did not see any evidence for alterations of GABA‐ or glycine‐immunostaining in the neuropil of laminae I–III in the animals that had undergone CCI. Our results suggest that significant loss of GABAergic or glycinergic neurons is not necessary for the development of thermal hyperalgesia in the CCI model of neuropathic pain.

Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat

In order to determine whether spinothalamic neurons in the lumbar spinal cord of the rat process neurokinin-1 (substance P) receptors, we injected cholera toxin B subunit into the thalamus and carried out dual-labelling immunocytochemistry to search for neurons that were immunoreactive with antibodies to cholera toxin and neurokinin-1 receptor. We examined 356 spinothalamic neurons in transverse sections and found that 35% of these were neurokinin-1 receptor-immunoreactive. Double-labelled cells made up the majority of the spinothalamic population in lamina I and the lateral spinal nucleus, and were also present in laminae III-V and the area around the central canal. On the side contralateral to the injection site, 77% of spinothalamic neurons in lamina I also showed neurokinin-1 receptor immunoreactivity, while 33% of those in laminae III-V and 14% of the ventromedial group possessed the receptor. Several of the double-labelled neurons with cell bodies in laminae III and IV had dendrites which could be followed dorsally into the superficial dorsal horn. These results indicate that substance P released from nociceptive primary afferents into the superficial dorsal horn is likely to act on spinothalamic tract neurons in lamina I, and also on those with cells bodies in laminae III-IV and long dorsal dendrites.

Conditional Rhythmicity of Ventral Spinal Interneurons Defined by Expression of the Hb9 Homeodomain Protein

The properties of mammalian spinal interneurons that underlie rhythmic locomotor networks remain poorly described. Using postnatal transgenic mice in which expression of green fluorescent protein is driven by the promoter for the homeodomain transcription factor Hb9, as well as Hb9-lacZ knock-in mice, we describe a novel population of glutamatergic interneurons located adjacent to the ventral commissure from cervical to midlumbar spinal cord levels. Hb9+ interneurons exhibit strong postinhibitory rebound and demonstrate pronounced membrane potential oscillations in response to chemical stimuli that induce locomotor activity. These data provide a molecular and physiological delineation of a small population of ventral spinal interneurons that exhibit homogeneous electrophysiological features, the properties of which suggest that they are candidate locomotor rhythm-generating interneurons.

Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach.

&NA; Lamina II contains a large number of interneurons involved in modulation and transmission of somatosensory (including nociceptive) information. However, its neuronal circuitry is poorly understood due to the difficulty of identifying functional populations of interneurons. This information is important for understanding nociceptive processing and for identifying changes that underlie chronic pain. In this study, we compared morphology, neurotransmitter content, electrophysiological and pharmacological properties for 61 lamina II neurons recorded in slices from adult rat spinal cord. Morphology was related to transmitter content, since islet cells were GABAergic, while radial and most vertical cells were glutamatergic. However, there was considerable diversity among the remaining cells, some of which could not be classified morphologically. Transmitter phenotype was related to firing pattern, since most (18/22) excitatory cells, but few (2/23) inhibitory cells had delayed, gap or reluctant patterns, which are associated with A‐type potassium (IA) currents. Somatostatin was identified in axons of 14/24 excitatory neurons. These had variable morphology, but most of those tested showed delayed‐firing. Excitatory interneurons are therefore likely to contribute to pain states associated with synaptic plasticity involving IA currents. Although noradrenaline and serotonin evoked outward currents in both inhibitory and excitatory cells, somatostatin produced these currents only in inhibitory neurons, suggesting that its pro‐nociceptive effects are mediated by disinhibition. Our results demonstrate that certain distinctive populations of inhibitory and excitatory interneuron can be recognised in lamina II. Combining this approach with identification of other neurochemical markers should allow further clarification of neuronal circuitry in the superficial dorsal horn.

The types of neuron which contain protein kinase C gamma in rat spinal cord

Protein kinase C (PKC) is thought to have a role in sensitization of dorsal horn neurons in certain pain states, and a recent study has reported that mice which lack the gamma isoform (PKCgamma) show reduced neuropathic pain after peripheral nerve injury. Although PKCgamma is present at high levels in the ventral part of lamina II we have limited information concerning the types of neuron in which it is located. In this study we have used immunocytochemistry to characterise the neurons which contain PKCgamma. Immunoreactive neurons were concentrated in ventral lamina II, but were also present in lamina III. Some weakly-immunoreactive neurons were located in the dorsal part of lamina II and in lamina I. The great majority (92%) of cells with PKCgamma were not GABA-immunoreactive, and these cells are likely to be excitatory interneurons. Dual-immunofluorescence labelling showed that PKCgamma was not randomly distributed amongst non-GABAergic neurons, since it was present in 76% of cells with neurotensin and 45% of those with somatostatin, but only 5% of those with the mu-opioid receptor (MOR-1). Cells with the neurokinin 1 receptor are found in lamina I and lamina III, and PKCgamma was present in 22% and 37% of these populations, respectively. These results suggest that excitatory interneurons in laminae II and III which lack the micro-opioid receptor may have a significant role in generating neuropathic pain.