Bhlhb5::flpo allele uncovers a requirement for Bhlhb5 for the development of the dorsal cochlear nucleus.

Auditory information is initially processed in the cochlear nuclei before being relayed to the brain. The cochlear nuclei are subdivided into dorsal, anterior ventral, and posterior ventral domains, each containing several subtypes of neurons that are thought to play discrete roles in the processing of sound. However, the ontogeny of these neurons is poorly understood, and this gap in knowledge hampers efforts to understand the basic neural circuitry of this nucleus. Here, we reveal that Bhlhb5 is expressed in both excitatory (unipolar brush cells) and inhibitory neurons (cartwheel cells) of the DCN during development. To gain genetic access to Bhlhb5-expressing neurons in the DCN, we generated a Bhlhb5::flpo knockin allele. Using an intersectional genetic strategy, we labeled cartwheel cells, thereby providing proof of concept that subpopulations of Bhlhb5-expressing neurons can be genetically targeted. Moreover, fate-mapping experiments using this allele revealed that Bhlhb5 is required for the proper development of the DCN, since mice lacking Bhlhb5 showed a dramatically diminished number of neurons, including unipolar brush and cartwheel cells. Intriguingly, the Bhlhb5::flpo allele also genetically labels numerous other regions of the nervous system that process sensory input, including the dorsal horn, the retina, and the nucleus of the lateral olfactory tract, hinting at a more general role for Bhlhb5 in the development of neurons that mediate sensory integration.

Generation of a NK1R-CreER knockin mouse strain to study cells involved in Neurokinin 1 Receptor signaling.

The Neurokinin 1 Receptor (NK1R), which binds Substance P, is expressed in discrete populations of neurons throughout the nervous system, where it has numerous roles including the modulation of pain and affective behaviors. Here, we report the generation of a NK1R-CreER knockin allele, in which CreERT2 replaces the coding sequence of the TACR1 gene (encoding NK1R) in order to gain genetic access to these cells. We find that the NK1R-CreER allele mediates recombination in many regions of the nervous system that are important in pain and anxiety including the amygdala, hypothalamus, frontal cortex, raphe nucleus, and dorsal horn of the spinal cord. Other cell types that are labeled by this allele include amacrine cells in the retina and fibroblasts in the skin. Thus, the NK1R-CreER mouse line is a valuable new tool for conditional gene manipulation enabling the visualization and manipulation of cells that express NK1R.

Generation of a KOR-Cre knockin mouse strain to study cells involved in kappa opioid signaling.

The kappa opioid receptor (KOR) has numerous important roles in the nervous system including the modulation of mood, reward, pain, and itch. In addition, KOR is expressed in many non-neuronal tissues. However, the specific cell types that express KOR are poorly characterized. Here, we report the development of a KOR-Cre knockin allele, which provides genetic access to cells that express KOR. In this mouse, Cre recombinase (Cre) replaces the initial coding sequence of the Opkr1 gene (encoding the kappa opioid receptor). We demonstrate that the KOR-Cre allele mediates recombination by embryonic day 14.5 (E14.5). Within the brain, KOR-Cre shows expression in numerous areas including the cerebral cortex, nucleus accumbens and striatum. In addition, this allele is expressed in epithelium and throughout many regions of the body including the heart, lung, and liver. Finally, we reveal that KOR-Cre mediates recombination of a subset of bipolar and amacrine cells in the retina. Thus, the KOR-Cre mouse line is a valuable new tool for conditional gene manipulation to enable the study of KOR.

The class 4 semaphorin Sema4D promotes the rapid assembly of GABAergic synapses in rodent hippocampus.

Proper circuit function in the mammalian nervous system depends on the precise assembly and development of excitatory and inhibitory synaptic connections between neurons. Through a loss-of-function genetic screen in cultured hippocampal neurons, we previously identified the class 4 Semaphorin Sema4D as being required for proper GABAergic synapse development. Here we demonstrate that Sema4D is sufficient to promote GABAergic synapse formation in rodent hippocampus and investigate the kinetics of this activity. We find that Sema4D treatment of rat hippocampal neurons increases the density of GABAergic synapses as detected by immunocytochemistry within 30 min, much more rapidly than has been previously described for a prosynaptogenic molecule, and show that this effect is dependent on the Sema4D receptor PlexinB1 using PlxnB1(-/-) mice. Live imaging studies reveal that Sema4D elicits a rapid enhancement (within 10 min) in the rate of addition of synaptic proteins. Therefore, we demonstrate that Sema4D, via PlexinB1, acts to initiate synapse formation by recruiting molecules to both the presynaptic and the postsynaptic terminals; these nascent synapses subsequently become fully functional by 2 h after Sema4D treatment. In addition, acute treatment of an organotypic hippocampal slice epilepsy model with Sema4D reveals that Sema4D rapidly and dramatically alters epileptiform activity, which is consistent with a Sema4D-mediated shift in the balance of excitation and inhibition within the circuit. These data demonstrate an ability to quickly assemble GABAergic synapses in response to an appropriate signal and suggest a potential area of exploration for the development of novel antiepileptic drugs.

Organic small hairpin RNAs (OshR): a do-it-yourself platform for transgene-based gene silencing.

The RNA interference (RNAi) pathway in animal cells can be harnessed to silence gene expression with artificial small interfering RNAs (siRNAs) or transgenes that express small hairpin RNAs (shRNAs). The transgene-expressing shRNA approach has been adapted into large-scale resources for genome-wide loss-of-function screens, whereas focused studies on a narrow set of genes can be achieved by using individual shRNA constructs from these resources. Although current shRNA repositories generally work, they might fail in certain situations and therefore necessitate other alternatives. We detail here a new highly-accessible and rational design of custom shRNAs that utilizes a refined backbone configuration termed the 'organic' shRNA (OshR) platform. The OshR platform is 'organic' because it conforms more naturally to the endogenous vertebrate miRNAs by maintaining specific bulges and incorporating strategic mismatches to insure the desired guide strand is produced while reducing the accumulation of passenger strands that might contribute to off-target effects. We also demonstrate that the reliability of the OshR platform for gene silencing is increased when sequences target the 3' UnTranslated Region (3'UTR) of a gene. We further compare the OshR platform with the current and emerging shRNA designs, and propose that the OshR platform is a novel approach that can allow investigators to generate custom and effective shRNAs for individual gene functional studies.

An Unexpected Role for TRPV4 in Serotonin-Mediated Itch.

Previous studies have revealed that TRPV1 and TRPA1 function downstream of many itch receptors, where they mediate inward current to trigger action potentials in primary afferents. Although other TRP channels, such as TRPV4, are expressed in primary afferents, whether or not they play an analogous role in itch was previously unknown. Now, Akiyama et al. provide evidence that TRPV4 is a key mediator of serotonin-induced itch. This finding is important because it uncovers an unanticipated role for TRPV4 in itch, thereby identifying a novel therapeutic target.

Emerging themes in GABAergic synapse development.

Glutamatergic synapse development has been rigorously investigated for the past two decades at both the molecular and cell biological level yet a comparable intensity of investigation into the cellular and molecular mechanisms of GABAergic synapse development has been lacking until relatively recently. This review will provide a detailed overview of the current understanding of GABAergic synapse development with a particular emphasis on assembly of synaptic components, molecular mechanisms of synaptic development, and a subset of human disorders which manifest when GABAergic synapse development is disrupted. An unexpected and emerging theme from these studies is that glutamatergic and GABAergic synapse development share a number of overlapping molecular and cell biological mechanisms that will be emphasized in this review.