Carole Torsney

Presynaptic NMDA Receptors Modulate Glutamate Release from Primary Sensory Neurons in Rat Spinal Cord Dorsal Horn

NMDA receptors have the potential to produce complex activity-dependent regulation of transmitter release when localized presynaptically. In the somatosensory system, NMDA receptors have been immunocytochemically detected on presynaptic terminals of primary afferents, and these have been proposed to drive release of substance P from central terminals of a subset of nociceptors in the spinal cord dorsal horn. Here we report that functional NMDA receptors are indeed present at or near the central terminals of primary afferent fibers. Furthermore, we show that activation of these presynaptic receptors results in an inhibition of glutamate release from the terminals. Some of these NMDA receptors may be expressed in the preterminal axon and regulate the extent to which action potentials invade the extensive central arborizations of primary sensory neurons.

The postnatal reorganization of primary afferent input and dorsal horn cell receptive fields in the rat spinal cord is an activity-dependent process

The dorsal horn of the spinal cord in the newborn rat is characterized by large cutaneous mechanoreceptive fields, a predominance of A‐fibre synaptic inputs and diffuse primary afferent A‐fibre projections, all of which are gradually reduced and refined over the first postnatal weeks. This may be partly responsible for the reduction in cutaneous flexion reflex sensitivity of rats over the postnatal period. Here we show that chronic, local exposure of the dorsal horn of the lumbar spinal cord to the NMDA antagonist MK801 from birth prevents the normal functional and structural reorganization of A‐fibre connections. Dorsal horn cells in spinal MK801‐treated animals, investigated at eight weeks of age by in vivo electrophysiological recording, had significantly larger cutaneous mechanoreceptive fields and greater A‐fibre evoked responses than vehicle controls. C‐fibre evoked responses were unaffected. Chronic MK801 also prevented the normal structural reorganization of A‐fibre terminals in the spinal cord. The postnatal withdrawal of superficially projecting A‐fibre primary afferents to deeper laminae did not occur in treated animals although C‐fibre afferent terminals and cell density in the dorsal horn were apparently unaffected. Spinal MK801‐treated animals also had significantly reduced behavioural reflex thresholds to mechanical stimulation of the hindpaw compared to naïve and vehicle‐treated animals, whereas noxious heat thresholds remained unaffected. The results indicate that the normal postnatal structural and functional development of A‐fibre sensory connectivity within the spinal cord is an activity‐dependent process requiring NMDA receptor activation.

Localization and function of ATP and GABAA receptors expressed by nociceptors and other postnatal sensory neurons in rat.

The role of endogenous GABA and ATP in regulating transmitter release from primary afferent terminals in the superficial dorsal horn of the spinal cord is still controversial. ATP is co‐released with GABA from some inhibitory dorsal horn neurons raising the possibility that ATP could act in concert with GABA to regulate transmitter release from primary afferent terminals if receptors to both transmitters are expressed there. Using electrophysiology together with immunocytochemistry, we have investigated the expression of ATP‐gated P2X and GABAA receptors by identified subpopulations of dorsal root ganglion (DRG) neurons known to project primarily to the superficial dorsal horn. Expression of the heat‐sensitive vanilloid receptor 1 (VR1) and sensitivity to capsaicin were used to characterize DRG neurons sensitive to noxious heat. Both P2X and GABAA receptors were expressed on the majority of DRG neurons examined. Recording compound action potentials (CAPs) from dorsal roots in the presence of muscimol, α,β‐methylene‐ATP (α,β‐meATP) or capsaicin resulted in depression of CAP in the slow and medium conducting fibres, indicating cognate receptor expression on the small diameter axons. Dorsal root‐evoked dorsal root potentials (DR‐DRPs), reflecting depolarization of primary afferent terminals by endogenously released substances, were depressed by the GABAA receptor antagonist SR95531 and α,β‐meATP. These results suggest that GABAA and P2X receptors are expressed on DRG cell bodies and slow fibre axons, many of which are heat‐nociceptive. These fibres project to the superficial lamina of the dorsal horn where the receptors may function to modulate transmitter release near their central terminals.

Spinal dorsal horn cell receptive field size is increased in adult rats following neonatal hindpaw skin injury

Local tissue damage in newborn rats can lead to changes in skin sensitivity that last into adulthood and this is likely to be due to plasticity of developing peripheral and central sensory connections. This study examines the functional connections of dorsal horn neurons in young and adult rats that have undergone local skin damage at birth. Newborn rat pups were halothane anaesthetised and received either a unilateral subcutaneous plantar injection of 1 % λ‐carrageenan or a unilateral plantar foot injury made by removal of 2 mm × 2 mm of skin. At 3 weeks, (postnatal day (P) 19–23) and 6 weeks (P40–44) in vivo extracellular recordings of single dorsal horn cells with plantar cutaneous receptive fields were made under urethane anaesthesia (2 g kg−1) and responses to mechanical and electrical stimulation of the skin were assessed. Following neonatal carrageenan inflammation, dorsal horn neuron properties and receptive field sizes at 3 weeks were the same as those of controls. In contrast, following neonatal skin injury, dorsal horn cell receptive field sizes were significantly greater than those of controls at 3 weeks (2.5‐fold) and at 6 weeks (2.2‐fold). Mechanical thresholds, mechanical response magnitudes and evoked responses to single and repeated A and C fibre stimulation remained unaffected. These results show that early skin injury can cause prolonged changes in central sensory connections that persist into adult life, long after the skin has healed. Enlarged dorsal horn neuron receptive field sizes provide a physiological mechanism for the persistent behavioural hypersensitivity that follows neonatal skin injury in rats and for the prolonged sensory changes reported in human infants after early pain and injury.

Modelling the prolonged effects of neonatal pain.

Publisher Summary This chapter discusses the prolonged effects of neonatal pain. A substantial inflammatory response lasting for 2 weeks follows from a single injection of 2% carageenan into one hindpaw within 24 h of birth. This causes no lasting effect on behavioral sensory thresholds or inflammatory pain responses in adulthood. It is important to emphasize, however, that electrophysiological and neuroanatomical analysis may reveal changes in sensory connections that are not evident in reflex behavioral tests and that more research is required in this area. In addition, the reapplication of 2% carageenan or complete Freunds adjuvant (CFA) in rats, when they had reached maturity, caused normal inflammatory, hyperalgesic, and allodynic responses that did not differ from controls. Repetitive painful experiences, prolonged tissue or nerve damage in newborn rats can lead to long-lasting neurobehavioral sequelae not observed when the same stimuli are applied to adults.

Inflammatory Pain Unmasks Heterosynaptic Facilitation in Lamina I Neurokinin 1 Receptor-Expressing Neurons in Rat Spinal Cord

Central sensitization in inflammatory pain conditions results in behavioral mechanical hypersensitivity. Specifically, C-fiber-driven spinal hyperexcitability enables A fibers to gain access to specific spinal circuitry, via heterosynaptic facilitatory mechanisms, to mediate mechanical hypersensitivity. However, the precise circuitry engaged is not known. Lamina I neurokinin 1 (NK1) receptor expressing (NK1R+) dorsal horn neurons, many of which are projection neurons, are required for the development of this hypersensitivity and are therefore likely to be a component of this circuitry. To investigate, whole-cell patch-clamp recordings were made from lamina I NK1R+ neurons in the spinal cord slice preparation with attached dorsal root, obtained from rats with or without complete Freunds adjuvant (CFA) hindpaw inflammation. EPSCs were recorded in response to electrical stimulation of the dorsal root. Control neurons predominantly received monosynaptic C-fiber input (69%) with a smaller proportion receiving monosynaptic Aδ-fiber input (28%). In contrast, CFA inflammation significantly increased the incidence (by twofold) and magnitude (by 75% in a subset) of monosynaptic Aδ-fiber but not monosynaptic C-fiber-evoked responses. Aβ-fiber input to lamina I NK1R+ neurons was minimal, polysynaptic in nature, and unaltered by CFA inflammation. Additional examination of control neurons revealed that a proportion received silent monosynaptic Aδ-fiber input, suggesting that these may provide the substrate for the novel Aδ inputs observed in CFA inflammation. This inflammation induced unmasking and strengthening of monosynaptic Aδ drive to lamina I NK1R+ neurons may contribute to the heterosynaptic facilitatory mechanisms underlying mechanical hyperalgesia in inflammatory pain.

Neonatal capsaicin treatment prevents the normal postnatal withdrawal of A fibres from lamina II without affecting fos responses to innocuous peripheral stimulation

The development of spinal cord sensory pathways has been investigated in postnatal day (P) 21 rat pups following neonatal capsaicin treatment. Capsaicin-induced destruction of C fibres was confirmed by 62% loss of Isolectin B4 (IB4)-binding and an 86% loss of calcitonin gene-related peptide (CGRP)-immunoreactive small diameter dorsal root ganglion cells. Neonatal capsaicin treatment prevented the normal withdrawal of choleragenoid-horseradish peroxidase (B-HRP)-labelled A fibres from lamina II (substantia gelatinosa) to deeper laminae postnatally. A fibre terminals projected more dorsally, extending into 43% of lamina II compared to vehicle-treated littermates. A small cell loss in, and/or shrinkage of, substantia gelatinosa cannot account for this. These support the concept of a competitive interaction between A and C fibre afferents to establish final terminal fields. However the continued exuberant A fibre termination in capsaicin-treated rats did not lead to continued c-fos induction in the superficial dorsal horn by innocuous stimulation. In normal development, exuberant A fibre terminals coincide with c-fos activation in lamina II by innocuous skin stimulation [23]. Despite the continued presence of exuberant A fibre terminals, c-fos was not induced by innocuous peripheral stimulation in P21 capsaicin-treated rats implying that these superficial terminals do not activate lamina II neurons in the same way as in the neonate.

Aberrant dendritic branching and sensory inputs in the superficial dorsal horn of mice lacking CaMKIIα autophosphorylation

Superficial dorsal horn neurones undergo marked structural and functional activity-dependent development during the early postnatal period, but little is known about the molecular mechanisms underlying these changes. Calcium signalling, through activation and autophosphorylation of CaMKII, has been shown to play a major role in the maturation of neuronal morphology and connectivity in the cortex. Here, we show that the normal structural and functional development of superficial dorsal horn neurones requires CaMKII autophosphorylation at the Thr286 residue. The dendritic branching of neurones from mice containing a point mutation at this site (T286A) was significantly increased compared with wild-type littermates. This was accompanied by significant increases in receptive field size, recorded from intact preparations. Whole-cell patch clamp recordings of superficial dorsal horn slices revealed a selective deficit in low-threshold A fibre-evoked synaptic input. These results show that CaMKII autophosphorylation is required for the normal development of spinal sensory circuits.

Neuroscience: A painful factor

Peripheral nerve injury activates cells in the spinal cord called microglia. But how do such cells cause the ensuing chronic pain? It seems that they release a small protein that disrupts normal inhibition of pain signalling.GABA is a painNeuropathic pain, one of the most debilitating of all pain states, often arises from injury to a peripheral nerve that depends on activation of a specific cell type known as microglia. This prompts the question, how do the microglia signal to spinal pain neurons? Coull et al. have now identified the biophysical mechanism by which microglia, activated by ATP, cause hyperexcitability of spinal neurons. The microglia release brain-derived neurotrophic factor, which alters chloride ion distribution across the plasma membrane of neurons in lamina I of the spinal cord. This results in the neurotransmitter, GABA, activating (rather than inhibiting) these cells that form part of a major pathway that signals pain. A collection of recent reprints on neuropathic pain, taken from Nature Publishing Group journals is, now available online via tinyurl.com/dzw86.

Characterization of sensory neuron subpopulations selectively expressing green fluorescent protein in phosphodiesterase 1C BAC transgenic mice

BackgroundThe complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord.ResultsUsing double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X3. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn.ConclusionThe expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.