Probes for CRE

Gastrin-releasing peptide (GRP) functions as a neurotransmitter for non-histaminergic itch, but its site of action (sensory neurons vs spinal cord) remains controversial. To determine the role of GRP in sensory neurons, we generated a floxed Grp mouse line. We found that conditional knockout of Grp in sensory neurons results in attenuated non-histaminergic itch, without impairing histamine-induced itch. Using a Grp-Cre knock-in mouse line, we show that the upper epidermis of the skin is exclusively innervated by GRP fibers, whose activation via optogeneics and chemogenetics in the skin evokes itch- but not pain-related scratching or wiping behaviors. In contrast, intersectional genetic ablation of spinal Grp neurons does not affect itch nor pain transmission, demonstrating that spinal Grp neurons are dispensable for itch transmission. These data indicate that GRP is a neuropeptide in sensory neurons for non-histaminergic itch, and GRP sensory neurons are dedicated to itch transmission

Diverse neurons in the parabrachial nucleus (PB) communicate with widespread brain regions. Despite evidence linking them to a variety of homeostatic functions, it remains difficult to determine which PB neurons influence which functions because their subpopulations intermingle extensively. An improved framework for identifying these intermingled subpopulations would help advance our understanding of neural circuit functions linked to this region. Here, we present the foundation of a developmental-genetic ontology that classifies PB neurons based on their intrinsic, molecular features. By combining transcription factor labeling with Cre fate-mapping, we find that the PB is a blend of two, developmentally distinct macropopulations of glutamatergic neurons. Neurons in the first macropopulation express Lmx1b (and, to a lesser extent, Lmx1a) and are mutually exclusive with those in a second macropopulation, which derive from precursors expressing Atoh1. This second, Atoh1-derived macropopulation includes many Foxp2-expressing neurons, but Foxp2 also identifies a subset of Lmx1b-expressing neurons in the Kölliker-Fuse nucleus (KF) and a population of GABAergic neurons ventrolateral to the PB ("caudal KF"). Immediately ventral to the PB, Phox2b-expressing glutamatergic neurons (some coexpressing Lmx1b) occupy the KF, supratrigeminal nucleus, and reticular formation. We show that this molecular framework organizes subsidiary patterns of adult gene expression (including Satb2, Calca, Grp, and Pdyn) and predicts output projections to the amygdala (Lmx1b), hypothalamus (Atoh1), and hindbrain (Phox2b/Lmx1b). Using this molecular ontology to organize, interpret, and communicate PB-related information could accelerate the translation of experimental findings from animal models to human patients.

The potassium channel Kv1.6 has recently been implicated as a major modulatory channel subunit expressed in primary nociceptors. Furthermore, its expression at juxtaparanodes (JXP) of myelinated primary afferents is induced following traumatic nerve injury as part of an endogenous mechanism to reduce hyperexcitability and pain-related hypersensitivity. In this study we compared two mouse models of constitutive Kv1.6 knock-out achieved by different methods: traditional gene trap via homologous recombination, and CRISPR-mediated excision. Both Kv1.6 knock-out mouse lines exhibited an unexpected reduction in sensitivity to noxious heat stimuli, to differing extents: the Kv1.6 mice produced via gene trap had a far more significant hyposensitivity. These mice (Kcna6lacZ ) expressed the bacterial reporter enzyme LacZ in place of Kv1.6 as a result of the gene trap mechanism and we found that their central primary afferent presynaptic terminals developed a striking neurodegenerative phenotype involving accumulation of lipid species, development of 'meganeurites' and impaired transmission to dorsal horn wide dynamic range (WDR) neurons. The anatomical defects were absent in CRISPR-mediated Kv1.6 knock-out mice (Kcna6 -/-) but were present in a third mouse model expressing exogenous LacZ in nociceptors under the control of a Nav1.8-promoted Cre recombinase. LacZ reporter enzymes are thus intrinsically neurotoxic to sensory neurons and may induce pathological defects in transgenic mice, which has confounding implications for the interpretation of gene knock-outs using lacZ Nonetheless, in Kcna6 -/- mice not affected by LacZ, we demonstrated a significant role for Kv1.6 regulating acute noxious thermal sensitivity, and both mechanical and thermal pain-related hypersensitivity after nerve injury.SIGNIFICANCE STATEMENTIn recent decades the expansion of technologies to experimentally manipulate the rodent genome has contributed significantly to the field of neuroscience. While introduction of enzymatic or fluorescent reporter proteins to label neuronal populations is now commonplace, often potential toxicity effects are not fully considered. We show a role of Kv1.6 in acute and neuropathic pain states through analysis of two mouse models lacking Kv1.6 potassium channels, one with additional expression of LacZ and one without. We show that LacZ reporter enzymes induce unintended defects in sensory neurons, with an impact on behavioural data outcomes. To summarise we highlight the importance of: Kv1.6 in recovery of normal sensory function following nerve injury, and careful interpretation of data from LacZ reporter models.