c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy.

Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-MafEX) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-MafEX neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-MafEX neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-MafEX neuron activation. Our study identifies c-MafEX neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control.

Does toe clipping for genotyping interfere with later-in-life nociception in mice?

INTRODUCTION: Genetically modified mice are widely used in studies on human and animal physiology and pharmacology, including pain research. The experimental design usually includes comparisons of genetically modified mice with wild-type littermates, requiring biopsy material for genotyping and methods for unequivocal identification of individual mice. Ethical standards and, in some countries, legislation require that both needs are reached with a single procedure. Clipping of the most distal phalanx of up to two toes per paw (toe clipping) is the favored procedure in most research fields, but it may be problematic in sensory physiology and pain research. OBJECTIVES: To systematically investigate whether toe-clipping influences later-in-life nociceptive sensitivity or the susceptibility to neuropathic or inflammatory hyperalgesia. METHODS: We tested in male mice whether the clipping of 2 toes of a hind paw influences nociceptive sensitivities to noxious heat or cold, or to mechanical stimulation under baseline conditions, after peripheral nerve injury (chronic constriction of the sciatic nerve) or during peripheral inflammation induced by subcutaneous zymosan A injection. We tested not only for the presence of significant differences but also specifically addressed bioequivalence using the 2 one-sided t test procedure. We chose a threshold of 25% variation of the control value for nonequivalence, which is usually taken as a threshold for biological relevance in pain tests. RESULTS: Using this value, we found that for all conditions (non-neuropathic and non-inflamed, neuropathic and inflamed), nociceptive sensitivities significantly fell within the equivalence bounds of the non-toe-clipped control mice. CONCLUSIONS: These results suggest that toe clipping does not have long-term effects on nociceptive sensitivities and does not alter the susceptibility of male mice to neuropathic or inflammatory hyperalgesia.

Spinal nociceptive circuit analysis with recombinant adeno-associated viruses: the impact of serotypes and promoters.

Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer into genetically defined neuron subtypes has become a powerful tool to study the neuroanatomy of neuronal circuits in the brain and to unravel their functions. More recently, this methodology has also become popular for the analysis of spinal cord circuits. To date, a variety of naturally occurring AAV serotypes and genetically modified capsid variants are available but transduction efficiency in spinal neurons, target selectivity, and the ability for retrograde tracing are only incompletely characterized. Here, we have compared the transduction efficiency of seven commonly used AAV serotypes after intraspinal injection. We specifically analyzed local transduction of different types of dorsal horn neurons, and retrograde transduction of dorsal root ganglia (DRG) neurons and of neurons in the rostral ventromedial medulla (RVM) and the somatosensory cortex (S1). Our results show that most of the tested rAAV vectors have similar transduction efficiency in spinal neurons. All serotypes analyzed were also able to transduce DRG neurons and descending RVM and S1 neurons via their spinal axon terminals. When comparing the commonly used rAAV serotypes to the recently developed serotype 2 capsid variant rAAV2retro, a > 20-fold increase in transduction efficiency of descending supraspinal neurons was observed. Conversely, transgene expression in retrogradely transduced neurons was strongly reduced when the human synapsin 1 (hSyn1) promoter was used instead of the strong ubiquitous hybrid cytomegalovirus enhancer/chicken ß-actin promoter (CAG) or cytomegalovirus (CMV) promoter fragments. We conclude that the use of AAV2retro greatly increases transduction of neurons connected to the spinal cord via their axon terminals, while the hSyn1 promoter can be used to minimize transgene expression in retrogradely connected neurons of the DRG or brainstem. Cover Image for this issue: doi. 10.1111/jnc.13813.