Efferent pathways of the mouse lateral habenula.

The lateral habenula (LHb) is part of the habenula complex of the dorsal thalamus. Recent studies of the LHb have focused on its projections to the ventral tegmental area (VTA) and rostromedial tegmental nucleus (RMTg), which contain γ-aminobutyric acid (GABA)ergic neurons that mediate reward prediction error via inhibition of dopaminergic activity. However, older studies in the rat have also identified LHb outputs to the lateral and posterior hypothalamus, median raphe, dorsal raphe, and dorsal tegmentum. Although these studies have shown that the medial and lateral divisions of the LHb have somewhat distinct projections, the topographic specificity of LHb efferents is not completely understood, and the relative extent of these projections to brainstem targets is unknown. Here we have used anterograde tracing with adeno-associated virus-mediated expression of green fluorescent protein, combined with serial two-photon tomography, to map the efferents of the LHb on a standard coordinate system for the entire mouse brain, and reconstruct the efferent pathways of the LHb in three dimensions. Using automated quantitation of fiber density, we show that in addition to the RMTg, the median raphe, caudal dorsal raphe, and pontine central gray are major recipients of LHb efferents. By using retrograde tract tracing with cholera toxin subunit B, we show that LHb neurons projecting to the hypothalamus, VTA, median raphe, caudal dorsal raphe, and pontine central gray reside in characteristic, but sometimes overlapping regions of the LHb. Together these results provide the anatomical basis for systematic studies of LHb function in neural circuits and behavior in mice. J. Comp. Neurol. 523:32-60, 2015. © 2014 Wiley Periodicals, Inc.

Medial habenula output circuit mediated by α5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus.

The Chrna5 gene encodes the α5 nicotinic acetylcholine receptor subunit, an "accessory" subunit of pentameric nicotinic receptors, that has been shown to play a role in nicotine-related behaviors in rodents and is genetically linked to smoking behavior in humans. Here we have used a BAC transgenic mouse line, α5(GFP), to examine the cellular phenotype, connectivity, and function of α5-expressing neurons. Although the medial habenula (MHb) has been proposed as a site of α5 function, α5(GFP) is not detectable in the MHb, and α5 mRNA is expressed there only at very low levels. However, α5(GFP) is strongly expressed in a subset of neurons in the interpeduncular nucleus (IP), median raphe/paramedian raphe (MnR/PMnR), and dorsal tegmental area (DTg). Double-label fluorescence in situ hybridization reveals that these neurons are exclusively GABAergic. Transgenic and conventional tract tracing show that α5(GFP) neurons in the IP project principally to the MnR/PMnR and DTg/interfascicular dorsal raphe, both areas rich in serotonergic neurons. The α5(GFP) neurons in the IP are located in a region that receives cholinergic fiber inputs from the ventral MHb, and optogenetically assisted circuit mapping demonstrates a monosynaptic connection between these cholinergic neurons and α5(GFP) IP neurons. Selective inhibitors of both α4ß2- and α3ß4-containing nicotinic receptors were able to reduce nicotine-evoked inward currents in α5(GFP) neurons in the IP, suggesting a mixed nicotinic receptor profile in these cells. Together, these findings show that the α5-GABAergic interneurons form a link from the MHb to serotonergic brain centers, which is likely to mediate some of the behavioral effects of nicotine.

Hmx1 is required for the normal development of somatosensory neurons in the geniculate ganglion.

Hmx1 is a variant homeodomain transcription factor expressed in the developing sensory nervous system, retina, and craniofacial mesenchyme. Recently, mutations at the Hmx1 locus have been linked to craniofacial defects in humans, rats, and mice, but its role in nervous system development is largely unknown. Here we show that Hmx1 is expressed in a subset of sensory neurons in the cranial and dorsal root ganglia which does not correspond to any specific sensory modality. Sensory neurons in the dorsal root and trigeminal ganglia of Hmx1dm/dm mouse embryos have no detectable Hmx1 protein, yet they undergo neurogenesis and express sensory subtype markers normally, demonstrating that Hmx1 is not globally required for the specification of sensory neurons from neural crest precursors. Loss of Hmx1 expression has no obvious effect on the early development of the trigeminal (V), superior (IX/X), or dorsal root ganglia neurons in which it is expressed, but results in marked defects in the geniculate (VII) ganglion. Hmx1dm/dm mouse embryos possess only a vestigial posterior auricular nerve, and general somatosensory neurons in the geniculate ganglion are greatly reduced by mid-gestation. Although Hmx1 is expressed in geniculate neurons prior to cell cycle exit, it does not appear to be required for neurogenesis, and the loss of geniculate neurons is likely to be the result of increased cell death. Fate mapping of neural crest-derived tissues indicates that Hmx1-expressing somatosensory neurons at different axial levels may be derived from either the neural crest or the neurogenic placodes.

Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation.

The combinatorial expression of transcription factors frequently marks cellular identity in the nervous system, yet how these factors interact to determine specific neuronal phenotypes is not well understood. Sensory neurons of the trigeminal ganglion (TG) and dorsal root ganglia (DRG) coexpress the homeodomain transcription factors Brn3a and Islet1, and past work has revealed partially overlapping programs of gene expression downstream of these factors. Here we examine sensory development in Brn3a/Islet1 double knock-out (DKO) mice. Sensory neurogenesis and the formation of the TG and DRG occur in DKO embryos, but the DRG are dorsally displaced, and the peripheral projections of the ganglia are markedly disturbed. Sensory neurons in DKO embryos show a profound loss of all early markers of sensory subtypes, including the Ntrk neurotrophin receptors, and the runt-family transcription factors Runx1 and Runx3. Examination of global gene expression in the E12.5 DRG of single and double mutant embryos shows that Brn3a and Islet1 are together required for nearly all aspects of sensory-specific gene expression, including several newly identified sensory markers. On a majority of targets, Brn3a and Islet1 exhibit negative epistasis, in which the effects of the individual knock-out alleles are less than additive in the DKO. Smaller subsets of targets exhibit positive epistasis, or are regulated exclusively by one factor. Brn3a/Islet1 double mutants also fail to developmentally repress neurogenic bHLH genes, and in vivo chromatin immunoprecipitation shows that Islet1 binds to a known Brn3a-regulated enhancer in the neurod4 gene, suggesting a mechanism of interaction between these genes.

TDP-43 and ubiquitinated cytoplasmic aggregates in sporadic ALS are low frequency and widely distributed in the lower motor neuron columns independent of disease spread.

Ubiqitinated and TDP-43 immunoreactive cytoplasmic aggregates are hallmark features of ALS molecular pathology. Since clinically most ALS begins focally and advances contiguously, it is important to characterize their distribution. Our objective was to determine the extent and distribution of TDP-43 immunoreactive aggregates in the lower motor neuron columns as a function of disease onset, and to correlate ubiquitinated with TDP-43 aggregates in the lumbar region. We examined TDP-43 cytoplasmic aggregates at four separate neuraxis levels - hypoglossal nucleus and cervical, thoracic, and lumbar anterior horns - in five controls and 20 sporadic ALS nervous systems from patients whose disease began in various sites, i.e. five bulbar, five arm, five trunk, and five leg onsets. We correlated ubiquitinated to TDP-43 aggregates on adjacent histological sections for the lumbar regions. We found that TDP-43 cytoplasmic aggregates are seen in about 8% of motor neurons but there is marked variability between nervous systems, ranging from 0.4% to 20.6%. The aggregates are uniformly distributed within individual nervous systems. There is no obvious correlation between site of disease onset and rate of spread. Almost all ubiquitinated aggregates correlate to TDP-43 aggregates. Thus, TDP-43 immunoreactive cytoplasmic aggregates have a low overall average frequency that does not correlate with either disease course or clinical spread and is the prime ubiquitinated protein.