The deep dorsal horn is a poorly characterized spinal cord region implicated in processing low-threshold mechanoreceptor (LTMR) information. We report an array of mouse genetic tools for defining neuronal components and functions of the dorsal horn LTMR-recipient zone (LTMR-RZ), a role for LTMR-RZ processing in tactile perception, and the basic logic of LTMR-RZ organization. We found an unexpectedly high degree of neuronal diversity in the LTMR-RZ: seven excitatory and four inhibitory subtypes of interneurons exhibiting unique morphological, physiological, and synaptic properties. Remarkably, LTMRs form synapses on between four and 11 LTMR-RZ interneuron subtypes, while each LTMR-RZ interneuron subtype samples inputs from at least one to three LTMR classes, as well as spinal cord interneurons and corticospinal neurons. Thus, the LTMR-RZ is a somatosensory processing region endowed with a neuronal complexity that rivals the retina and functions to pattern the activity of ascending touch pathways that underlie tactile perception.

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 (I(A)) 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 I(A) 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.

GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABA(A) 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 nai;ve 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 nai;ve 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.

Key points: Perception of normal bodily sensations relies on the precise regulation of sensory information entering the dorsal horn of the spinal cord. Inhibitory, axoaxonic, synapses provide a mechanism for this regulation, but the source of these important inhibitory connections remains to be elucidated. This study shows that a subpopulation of spinal interneurons that expresses parvalbumin and have specific morphological, connectivity and functional characteristics are a likely source of the inhibitory inputs that selectivity regulate non‐noxious tactile input in the spinal cord. Our findings suggest that a loss of normal function in parvalbumin positive dorsal horn neurons may result in the development of tactile allodynia, where non‐painful stimuli gain the capacity to evoke the sensation of pain. Abstract  Axo‐axonic synapses on the central terminals of primary afferent fibres modulate sensory input and are the anatomical correlate of presynaptic inhibition. Although several classes of primary afferents are under such inhibitory control, the origin of these presynaptic inputs in the dorsal horn is unknown. Here, we characterize the neurochemical, anatomical and electrophysiological properties of parvalbumin (PV)‐expressing cells in wild‐type and transgenic mice where enhanced green fluorescent protein (eGFP) is expressed under the PV promoter. We show that most PV cells have either islet or central cell‐like morphology, receive inputs from myelinated primary afferent fibres and are concentrated in laminae II inner and III. We also show that inhibitory PV terminals in lamina II inner selectively target the central terminals of myelinated afferents (∼80% of 935 PVeGFP boutons) and form axo‐axonic synapses (∼75% of 71 synapses from PV boutons). Targeted whole‐cell patch‐clamp recordings from PVeGFP positive cells in laminae II and III showed action potential discharge was restricted to the tonic firing and initial bursting patterns (67% and 33% respectively; n= 18), and virtually all express I_h subthreshold voltage‐gated currents (94%; n= 18). These neurons show higher rheobase current than non‐eGFP cells but respond with high frequency action potential discharge upon activation. Together, our findings show that PV neurons in laminae II and III are a likely source of inhibitory presynaptic input on to myelinated primary afferents. Consequently PV cells are ideally placed to play an important role in the development of central sensitization and tactile allodynia.