Probes for PARVALBUMIN

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.

Relaxin-3 is a highly conserved neuropeptide abundantly expressed in neurons of the nucleus incertus (NI), which project to nodes of the septohippocampal system (SHS) including the medial septum/diagonal band of Broca (MS/DB) and dorsal hippocampus, as well as to limbic circuits. High densities of the Gi/o-protein-coupled receptor for relaxin-3, known as relaxin-family peptide-3 receptor (RXFP3) are expressed throughout the SHS, further suggesting a role for relaxin-3/RXFP3 signaling in modulating learning and memory processes that occur within these networks. Therefore, this study sought to gain further anatomical and functional insights into relaxin-3/RXFP3 signaling in the mouse MS/DB. Using Cre/LoxP recombination methods, we assessed locomotion, exploratory behavior, and spatial learning and long-term reference memory in adult C57BL/6J Rxfp3 (loxP/loxP) mice with targeted depletion of Rxfp3 in the MS/DB. Following prior injection of an AAV((1/2))-Cre-IRES-eGFP vector into the MS/DB to delete/deplete Rxfp3 mRNA/RXFP3 protein, mice tested in a Morris water maze (MWM) displayed an impairment in allocentric spatial learning during acquisition, as well as an impairment in long-term reference memory on probe day. However, RXFP3-depleted and control mice displayed similar motor activity in a locomotor cell and exploratory behavior in a large open-field (LOF) test. A quantitative characterization using multiplex, fluorescent in situ hybridization (ISH) identified a high level of co-localization of Rxfp3 mRNA and vesicular GABA transporter (vGAT) mRNA in MS and DB neurons (~87% and ~95% co-expression, respectively). Rxfp3 mRNA was also detected, to a correspondingly lesser extent, in vesicular glutamate transporter 2 (vGlut2) mRNA-containing neurons in MS and DB (~13% and ~5% co-expression, respectively). Similarly, a qualitative assessment of the MS/DB region, identified Rxfp3 mRNA in neurons that expressed parvalbumin (PV) mRNA (reflecting hippocampally-projecting GABA neurons), whereas choline acetyltransferase mRNA-positive (acetylcholine) neurons lacked Rxfp3 mRNA. These data are consistent with a qualitative immunohistochemical analysis that revealed relaxin-3-immunoreactive nerve fibers in close apposition with PV-immunoreactive neurons in the MS/DB. Together these studies suggest relaxin-3/RXFP3 signaling in the MS/DB plays a role in modulating specific learning and long-term memory associated behaviors in adult mice via effects on GABAergic neuron populations known for their involvement in modulating hippocampal theta rhythm and associated cognitive processes.

Cortical parvalbumin (Pvalb)-expressing neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. This class of inhibitory neurons undergoes extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. While several transcription factors, such as Nkx2-1, Lhx6, and Sox6, are known to be necessary for the differentiation of progenitors into Pvalb+ neurons, which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons' innervation and synaptic function remains largely unknown. Because Sox6 is continuously expressed in Pvalb+ neurons until adulthood, we utilized conditional knockout strategies to investigate its putative role in the postnatal maturation and synaptic function of cortical Pvalb+ neurons in mice of both sexes. We found that early postnatal loss of Sox6 in Pvalb+ neurons leads to failure of synaptic bouton growth, whereas later removal in mature Pvalb+ neurons in the adult causes shrinkage of already established synaptic boutons. Paired recordings between Pvalb+ neurons and pyramidal neurons revealed reduced release probability and increased failure rate of Pvalb+ neurons' synaptic output. Furthermore, Pvalb+ neurons lacking Sox6 display reduced expression of full-length tropomyosin-receptor kinase B (TrkB), a key modulator of GABAergic transmission. Once re-expressed in neurons lacking Sox6, TrkB was sufficient to rescue the morphological synaptic phenotype. Finally, we showed that Sox6 mRNA levels were increased by motor training. Our data thus suggest a constitutive role for Sox6 in the maintenance of synaptic output from Pvalb+ neurons into adulthood.Significance statement:Cortical parvalbumin-expressing (Pvalb+) inhibitory neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. These inhibitory neurons undergo extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. However, it remains largely unknown which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons. Here we show that the transcription factor Sox6 cell-autonomously regulates the synaptic maintenance and output of Pvalb+ neurons until adulthood, leaving unaffected other maturational features of this neuronal population.