Physiological properties of hypothalamic MCH neurons identified with selective expression of reporter gene after recombinant virus infection.

Neurons that synthesize melanin-concentrating hormone (MCH) may modulate arousal and energy homeostasis. The scattered MCH neurons have been difficult to study, as they have no defining morphological characteristics. We have developed a viral approach with AAV for selective long-term reporter gene (GFP) expression in MCH neurons, allowing the study of their cellular physiology in hypothalamic slices. MCH neurons showed distinct membrane properties compared to other neurons infected with the same virus with a cytomegalovirus promoter. Transmitters of extrahypothalamic arousal systems, including norepinephrine, serotonin, and the acetylcholine agonist muscarine, evoked direct inhibitory actions. Orexigenic neuropeptide Y was inhibitory by pre- and postsynaptic mechanisms; an anorexigenic melanocortin agonist had no effect. In contrast, the hypothalamic arousal peptide hypocretin/orexin evoked a direct inward current and increased excitatory synaptic activity and spike frequency in the normally silent MCH neurons. Together, these data support the view that MCH neurons may integrate information within the arousal system in favor of energy conservation.

p120 nucleolar-proliferating antigen is a direct target of G-CSF signaling during myeloid differentiation.

Granulocyte-colony stimulating factor (G-CSF) is an essential cytokine, which contributes to proliferation and differentiation of granulocyte precursor cells in the bone marrow. Despite recent progress in understanding G-CSF signaling events, the mechanisms that underlie the distinct spectrum of biological functions attributed to G-CSF-mediated gene expression remain unclear. Previous studies have identified a number of genes, which are up-regulated in G-CSF-stimulated myeloid precursor cells. In this study, we sought to identify additional target genes of G-CSF-mediated proliferation and/or differentiation. cDNA representational difference analysis was used with the 32Dcl3 cell line as a model system to isolate genes, which are up-regulated in an immediate-early manner upon G-CSF stimualtion. We isolated p120 nucleolar-proliferation antigen (NOL1), a highly conserved, nucleolar-specific, RNA-binding protein of unknown function, and confirmed its expression by Northern blot analysis in 4-h, G-CSF-induced 32Dcl3 cells. Isolation of a mouse p120 genomic clone revealed the presence of a signal tranducer and activator of transcription (STAT)-binding site in the first intron of the gene. We demonstrate the importance of STAT3 and STAT5 in mediating the G-CSF response with respect to p120 expression by transient transfection analysis, oligonucleotide pull-down assays, and the loss of p120 expression in the bone marrow of mice lacking normal STAT3 signaling. In addition, overexpression of p120 in G-CSF-induced 32D cells revealed normal, morphologic maturation and growth characteristics but loss of lactoferrin expression, a marker of normal neutrophil maturation, suggesting that inappropriate expression of the p120 gene can result in aberrant neutrophil maturation.

Cytomegalovirus induces interferon-stimulated gene expression and is attenuated by interferon in the developing brain.

Cytomegalovirus (CMV) is considered the most common infectious agent causing permanent neurological dysfunction in the developing brain. We have previously shown that CMV infects developing brain cells more easily than it infects mature brain cells and that this preference is independent of the host B- and T-cell responses. In the present study, we examined the innate antiviral defenses against mouse (m) and human (h) CMVs in developing and mature brain and brain cells. mCMV infection induced interferon (IFN)-stimulated gene expression by 10- to 100-fold in both glia- and neuron-enriched cultures. Treatment of primary brain cultures with IFN-alpha, -beta, and -gamma or a synthetic RNA, poly(I:C), reduced the number of mCMV-infected cells, both in older cells and in fresh cultures from embryonic mouse brains. When a viral dose that killed almost all unprotected cells was used, IFN-protected cells had a natural appearance, and when they were tested with whole-cell patch clamp recording, they appeared physiologically normal with typical resting membrane potentials and action potentials. mCMV infection increased expression of representative IFN-stimulated genes (IFIT3, OAS, LMP2, TGTP, and USP18) in both neonatal and adult brains to similarly large degrees. The robust upregulation of gene expression in the neonatal brain was associated with a much higher degree of viral replication at this stage of development. In contrast to the case for downstream gene induction, CMV upregulated IFN-alpha/beta expression to a greater degree in the adult brain than in the neonatal brain. Similar to the case with cultured brain cells, IFN treatment of the developing brain in vivo depressed mCMV replication. In parallel work with cultured primary human brain cells, IFN and poly(I:C) treatment reduced hCMV infection and prevented virus-mediated cell death. These results suggest that coupling IFN administration with current treatments may reduce CMV infections in the developing brain.

Neurons synthesizing melanin-concentrating hormone identified by selective reporter gene expression after transfection in vitro: transmitter responses.

Neurons from the lateral hypothalamus that synthesize melanin-concentrating hormone (MCH) play an important role in the regulation of energy homeostasis. Relatively little is known of the cellular physiology and transmitter responses of these neurons, in part because of the difficulty in identifying live MCH cells. Here we use a novel approach of transfection of specific gene constructs with the MCH promoter driving green fluorescent protein (GFP) or red fluorescent protein (dsRed2) in CNS cultures to identify live rat MCH neurons; all neurons expressing the reporter gene showed MCH immunoreactivity, indicating selective expression. MCH neurons had a resting membrane potential of -57.5 +/- 0.6 mV, a linear current-voltage relation and a mean input resistance of 1,013 MOmega. Long depolarizing pulses revealed significant spike frequency adaptation. Functional glutamate and GABA receptors were expressed by MCH neurons. MCH neurons were hyperpolarized by norepinephrine in the presence or absence of tetrodotoxin, suggesting direct inhibition. Orexigenic peptides neuropeptide Y (NPY) and MCH showed no direct effect on membrane potential, input resistance, action potential width, or afterhyperpolarization potential, but inhibited voltage-dependent calcium channels, indicating that MCH neurons expressed both MCH and NPY receptors.

Hypocretin (orexin) enhances neuron activity and cell synchrony in developing mouse GFP-expressing locus coeruleus.

The noradrenergic neurons of the locus coeruleus (LC) play an important role in modulating arousal and selective attention. A similar function has been attributed to the hypocretin neurons of the hypothalamus which maintain a strong synaptic projection to the LC. As the LC can be difficult to detect in the embryonic and neonatal mouse brain, we used a new transgenic mouse with strong GFP expression in the LC under the regulation of a mouse prion promoter. GFP colocalized with immunoreactive tyrosine hydroxylase in sections and dispersed cultures of the LC, allowing visualization and whole cell or single-unit recording from the LC in early stages of cellular development. GFP expression in the LC had no apparent effect on cellular physiology, including resting membrane potential, input resistance, spike threshold, depolarization-induced spike frequency increase, current-voltage relations, or hypocretin responses. In slices of the mature mouse and rat LC, hypocretin-1 and -2 increased spike frequency, with hypocretin-1 being an order of magnitude more potent. In the postnatal day (P) 0-2 developing mouse slice during a developmental period when spikes could be elicited in some cells, other developing LC neurons showed rhythmic, subthreshold oscillations (approximately 1 Hz) in membrane potential (2.9-7.4 mV amplitude); others were arrhythmic. Hypocretin-1 depolarized the membrane potential, resulting in the appearance of spikes in developing LC cells that showed no spikes under control conditions. In the presence of TTX and glutamate receptor antagonists, hypocretin-1-mediated inward currents were blocked by substitution of choline-Cl for NaCl, suggesting an excitatory mechanism based on an inward cation current. Hypocretin-1 initiated strong regular membrane voltage oscillations in arrhythmic immature neurons. Hypocretin increased the temporal synchrony of action potentials studied with dual-cell recording in P1-P5 mouse LC slices, consistent with the view that synchrony of LC output, associated with improved cognitive performance, may be increased by hypocretin. Together these data suggest that the hypothalamus, via hypocretin projections, may therefore be in a position to enhance arousal and modulate plasticity in higher brain centres through the developing LC.