Retinal ganglion cells (RGCs

The habenula is a dorsal diencephalic structure consisting of medial and lateral subnuclei and a principal output tract, the fasciculus retroflexus, which together form a link between the limbic forebrain and ventral midbrain. Here, we have used microarray and bioinformatic approaches in the mouse to show that the habenula is a distinctive molecular territory of the CNS, with a unique profile of neurotransmitter, ion channel, and regulatory factor expression. Neurons of the medial habenula and part of the lateral habenula express the transcription factor Brn3a/Pou4f1, and Brn3a-expressing habenular neurons project exclusively to the interpeduncular nucleus in the ventral midbrain. In Brn3a mutant embryos, the fasciculus retroflexus is directed appropriately, but habenular neurons fail to innervate their targets. Microarray analysis of Brn3a null embryos shows that this factor regulates an extensive program of habenula-enriched genes, but not generic neural properties. The orphan nuclear receptor Nurr1/Nr4a2 is coexpressed with Brn3a in the developing habenula, is downstream of Brn3a, and mediates expression of a subset of Brn3a-regulated transcripts. Together, these findings begin to define a gene regulatory pathway for habenula development in mammals.

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 GABAergic 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. Using retrograde tract tracing with cholera toxin subunit B, we show that LHb neurons projecting to the hypothalamus, VTA, median raphe, and 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.

The Chrna5 gene encodes the ␣5 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, ␣5 GFP , to examine the cellular phenotype, connectivity, and function of ␣5-expressing neurons. Although the medial habenula (MHb) has been proposed as a site of ␣5 function, ␣5GFP is not detectable in the MHb, and ␣5 mRNA is expressed there only at very low levels. However, ␣5GFP 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 ␣5 GFP neurons in the IP project principally to the MnR/PMnR and DTg/interfascicular dorsal raphe, both areas rich in serotonergic neurons. The ␣5 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 ␣5 GFP IP neurons. Selective inhibitors of both ␣4␤2-and ␣3␤4-containing nicotinic receptors were able to reduce nicotine-evoked inward currents in ␣5 GFP neurons in the IP, suggesting a mixed nicotinic receptor profile in these cells. Together, these findings show that the ␣5-GABAergic interneurons form a link from the MHb to serotonergic brain centers, which is likely to mediate some of the behavioral effects of nicotine.

The habenulopeduncular pathway consists of the medial habenula (MHb), its output tract, the fasciculus retroflexus, and its principal target, the interpeduncular nucleus (IP). Several IP subnuclei have been described, but their specific projections and relationship to habenula inputs are not well understood. Here we have used viral, transgenic, and conventional anterograde and retrograde tract-tracing methods to better define the relationship between the dorsal and ventral MHb, the IP, and the secondary efferent targets of this system. Although prior studies have reported that the IP has ascending projections to ventral forebrain structures, we find that these projections originate almost entirely in the apical subnucleus, which may be more appropriately described as part of the median raphe system. The laterodorsal tegmental nucleus receives inhibitory inputs from the contralateral dorsolateral IP, and mainly excitatory inputs from the ipsilateral rostrolateral IP subnucleus. The midline central gray of the pons and nucleus incertus receive input from the rostral IP, which contains a mix of inhibitory and excitatory neurons, and the dorsomedial IP, which is exclusively inhibitory. The lateral central gray of the pons receives bilateral input from the lateral IP, which in turn receives bilateral input from the dorsal MHb. Taken together with prior studies of IP projections to the raphe, these results form an emerging map of the habenulopeduncular system that has significant implications for the proposed function of the IP in a variety of behaviors, including models of mood disorders and behavioral responses to nicotine.

No abstract

Numerous transcription factors have been identified which have profound effects on developing neurons. A fundamental problem is to identify genes downstream of these factors and order them in developmental pathways. We have previously identified 85 genes with changed expression in the trigeminal ganglia of mice lacking Brn3a, a transcription factor encoded by the Pou4f1 gene. Here we use locus-wide chromatin immunoprecipitation in embryonic trigeminal neurons to show that Brn3a is a direct repressor of two of these downstream genes, NeuroD1 and NeuroD4, and also directly modulates its own expression. Comparison of Brn3a binding to the Pou4f1 locus in vitro and in vivo reveals that not all high affinity sites are occupied, and several Brn3a binding sites identified in the promoters of genes that are silent in sensory ganglia are also not occupied in vivo. Site occupancy by Brn3a can be correlated with evolutionary conservation of the genomic regions containing the recognition sites and also with histone modifications found in regions of chromatin active in transcription and gene regulation, suggesting that Brn3a binding is highly context dependent.

The ability of the central nervous system (CNS) to generate innate immune responses was investigated in an in vitro model of CNS infection. Cultures containing CNS cells were infected with mouse hepatitis virus-JHM, which causes fatal encephalitis in mice. Immunostaining indicated that viral infection had a limited effect on culture characteristics, overall cell survival, or cell morphology at the early postinfection times studied. Results from Affymetrix gene array analysis, assessed on RNA isolated from virally and sham-infected cultures, were compared with parallel protein assays for cytokine, chemokine, and cell surface markers. Of the 126 transcripts found to be differentially expressed between viral and sham infections, the majority were related to immunological responses. Virally induced increases in interleukin-6 and tumor necrosis factor alpha mRNA and protein expression correlated with the genomic induction of acute-phase proteins. Genomic and protein analysis indicated that viral infection resulted in prominent expression of neutrophil and macrophage chemotactic proteins. In addition, mRNA expression of nonclassical class I molecules H2-T10, -T17, -M2, and -Q10, were enhanced three-to fivefold in virus-infected cells compared to sham-infected cells. Thus, upon infection, resident brain cells induced a breadth of innate immune responses that could be vital in directing the outcome of the infection and, in vivo, would provide signals which would summon the peripheral immune system to respond to the infection. Further understanding of how these innate responses participate in immune protection or immunopathology in the CNS will be critical in efforts to intervene in severe encephalitis.