A quantitative study of neurochemically-defined populations of inhibitory interneurons in the superficial dorsal horn of the mouse spinal cord

Around a quarter of neurons in laminae I-II of the dorsal horn are inhibitory interneurons. These play an important role in modulating somatosensory information, including that perceived as pain or itch. Previous studies in rat identified four largely non-overlapping neurochemical populations among these cells, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin. The galanin cells were subsequently shown to coexpress dynorphin. Several recent studies have used genetically-modified mice to investigate the function of different interneuron populations, and it is therefore important to determine whether the same pattern applies in mouse, and to estimate the relative sizes of these populations. We show that the neurochemical organisation of inhibitory interneurons in mouse superficial dorsal horn is similar to that in the rat, although a larger proportion of these neurons (33%) express NPY. Between them, these four populations account for ∼75% of inhibitory cells in laminae I-II. Since ∼25% of inhibitory interneurons in this region belong to a novel calretinin-expressing type, our results suggest that virtually all inhibitory interneurons in superficial dorsal horn can be assigned to one of these five neurochemical populations. Although our main focus was inhibitory neurons, we also identified a population of excitatory dynorphin-expressing cells in laminae I-II that are largely restricted to the medial part of the mid-lumbar dorsal horn, corresponding to glabrous skin territory. These findings are important for interpretation of studies using molecular-genetic techniques to manipulate the functions of interneuron populations to investigate their roles in somatosensory processing

A Descending Circuit Derived From the Superior Colliculus Modulates Vibrissal Movements

The superior colliculus (SC) is an essential structure for the control of eye movements. In rodents, the SC is also considered to play an important role in whisking behavior, in which animals actively move their vibrissae (mechanosensors) to gather tactile information about the space around them during exploration. We investigated how the SC contributes to vibrissal movement control. We found that when the SC was unilaterally lesioned, the resting position of the vibrissae shifted backward on the side contralateral to the lesion. The unilateral SC lesion also induced an increase in the whisking amplitude on the contralateral side. To explore the anatomical basis for SC involvement in vibrissal movement control, we then quantitatively evaluated axonal projections from the SC to the brainstem using neuronal labeling with a virus vector. Neurons of the SC mainly sent axons to the contralateral side in the lower brainstem. We found that the facial nucleus received input directly from the SC, and that the descending projections from the SC also reached the intermediate reticular formation and pre-Bötzinger complex, which are both considered to contain neural oscillators generating rhythmic movements of the vibrissae. Together, these results indicate the existence of a neural circuit in which the SC modulates vibrissal movements mainly on the contralateral side, via direct connections to motoneurons, and via indirect connections including the central pattern generators

Preprotachykinin A (PPTA) is expressed by a distinct population of excitatory neurons in the mouse superficial spinal dorsal horn including cells that respond to noxious and pruritic stimuli

The superficial dorsal horn, which is the main target for nociceptive and pruritoceptive primary afferents, contains a high density of excitatory interneurons. Our understanding of their roles in somatosensory processing has been restricted by the difficulty of distinguishing functional populations among these cells. We recently defined three non-overlapping populations among the excitatory neurons, based on the expression of neurotensin, neurokinin B (NKB) and gastrin-releasing peptide (GRP). Here we identify and characterise another population: neurons that express the tachykinin peptide substance P. We show with immunocytochemistry that its precursor protein (preprotachykinin A, PPTA) can be detected in ~14% of lamina I-II neurons, and these are concentrated in the outer part of lamina II. Over 80% of the PPTA-positive cells lack the transcription factor Pax2 (which determines an inhibitory phenotype), and these account for ~15% of the excitatory neurons in this region. They are different from the neurotensin, NKB or GRP neurons, although many of them contain somatostatin, which is widely expressed among superficial dorsal horn excitatory interneurons. We show that many of these cells respond to noxious thermal and mechanical stimuli, and to intradermal injection of pruritogens. Finally, we demonstrate that these cells can also be identified in a knock-in Cre mouse line (Tac1Cre), although our findings suggest that there is an additional population of neurons that transiently express PPTA. This population of substance P-expressing excitatory neurons is likely to play an important role in transmission of signals that are perceived as pain and itch

Expression of calretinin among different neurochemical classes of interneuron in the superficial dorsal horn of the mouse spinal cord

Around 75% of neurons in laminae I-II of the mouse dorsal horn are excitatory interneurons, and these are required for normal pain perception. We have shown that four largely non-overlapping excitatory interneuron populations can be defined by expression of the neuropeptides neurotensin, neurokinin B (NKB), gastrin-releasing peptide (GRP) and substance P. In addition, we recently identified a population of excitatory interneurons in glabrous skin territory that express dynorphin. The calcium-binding protein calretinin is present in many excitatory neurons in this region, but we know little about its relation to these neuropeptide markers. Here we show that calretinin is differentially expressed, being present in the majority of substance P-, GRP- and NKB-expressing cells, but not in the neurotensin or dynorphin cells. Calretinin-positive cells have been implicated in detection of noxious mechanical stimuli, but are not required for tactile allodynia after neuropathic pain. Our findings are therefore consistent with the suggestion that neuropathic allodynia involves the neurotensin and/or dynorphin excitatory interneuron populations. Around a quarter of inhibitory interneurons in lamina I-II contain calretinin, and recent transcriptomic studies suggest that these co-express substance P. We confirm this, by showing that inhibitory Cre-expressing cells in a Tac1Cre knock-in mouse are calretinin-immunoreactive. Interestingly, there is evidence that these cells express low levels of peptidylglycine alpha-amidating monooxygenase, an enzyme required for maturation of neuropeptides. This may explain our previous finding that although the substance P precursor preprotachykinin A can be detected in some inhibitory interneurons, very few inhibitory axonal boutons are immunoreactive for substance P