Prabhat K. Ghosh

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.

Heterogeneity and 5′-terminal structures of the late RNAs of simian virus 40

We have investigated the 5′-terminal structures of the late lytic RNAs of simian virus 40 (SV40) by binding the plus strand of small DNA fragments labeled in the 5′-terminal position with 32P to specific regions of cytoplasmic polyadenylated late RNA, extending these “primer” fragments in a 3′ direction with reverse transcriptase, fractionating the extended products on denaturing polyacrylamide gels and performing DNA sequence analyses on the extended products. Extension of a primer bound to the 5′ terminus of the body of 19 S RNA revealed a multiplicity of extended products. RNAs from which these products are derived fall into four classes, the first containing sequences colinear with SV40 DNA and the latter three containing splices which fuse the 5′ terminus of the 19 S body at residue 476 (0.765 map units) to residues 444, 291 and 212, respectively. Of 15 extended products we have analyzed, eight have 3′ termini with the sequence T-A(A). These termini lie at positions 243, 182, 110, 55, and 5189 on the SV40 genome. A number of lines of evidence, including nucleotide sequence analysis of late RNA labeled in vivo and the fact that the principal sequence in SV40 late mRNAs adjacent to capped structures is A-U(U), suggest that the termini at positions 243 and 182 correspond to the 5′ termini of discrete in vivo 19 S RNAs and that stops at the shorter positions may also correspond to the 5′ termini of specific in vivo species of 19 S RNA. An additional extended product was observed with a sequence colinear with SV40 DNA and a 3′ terminus of T-A-A at residue 548 (0.779 map units). It contains sequences complementary to the VP3 but not the VP2 initiation codons of 19 S RNA. For each of the three different gaps we have found in the late 19 S RNAs, as well as four additional gaps we have analyzed in the late 16 S and early 19 S RNAs of SV40, identical di-, tri-or tetranucleotide sequences lie on the ungapped precursor at sites which undergo splicing; these sequences may be involved in determining the specificity of the splicing reaction.

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 (≈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.

Lateral hypothalamus: Early developmental expression and response to hypocretin (orexin)

Hypocretin is a recently discovered peptide that is synthesized by neurons in the lateral hypothalamic area (LH) and is believed to play a role in sleep regulation, arousal, endocrine control, and food intake. These functions are critical for the development of independent survival. We investigated the developmental profile of the hypocretin system in rats. Northern blot analysis showed that the expression of hypocretin mRNA increased from postnatal day 1 to adulthood. Both of the identified hypocretin receptor mRNAs were strongly expressed very early in hypothalamic development, and expression subsequently decreased in the mature brain. Immunocytochemistry revealed hypocretin‐2 peptide expression in the cell bodies of LH neurons and in axons in the brain and spinal cord as early as embryonic day 19. Whole‐cell patch clamp recordings from postnatal P1‐P14 LH slices demonstrated a robust increase in synaptic activity in all LH neurons tested (n = 20) with a 383% increase in the frequency of spontaneous activity upon hypocretin‐2 (1.5 μM) application. A similar increase in activity was found with hypocretin‐1 application to LH slices. Hypocretin‐2 evoked a robust increase in synaptic activity even on the earliest day tested, the day of birth. Furthermore, voltage‐clamp recordings and calcium digital imaging experiments using cultured LH cells revealed that both hypocretin‐1 and ‐2 induced enhancement of neuronal activity occurred as early as synaptic activity was detected. Thus, as in the adult central nervous system, hypocretin exerts a profound excitatory influence on neuronal activity early in development, which might contribute to the development of arousal, sleep regulation, feeding, and endocrine control. J. Comp. Neurol. 433:349–363, 2001.

Developmentally regulated gene expression of all eight metabotropic glutamate receptors in hypothalamic suprachiasmatic and arcuate nuclei – a PCR analysis

Previous studies have demonstrated the critical role glutamate plays in the hypothalamus, both in the developing and adult brain. The expression of metabotropic glutamate receptor (mGluR) mRNA (mGluR1-8) was studied in the suprachiasmatic (SCN) and arcuate (ARC) nuclei. Using reverse Northern blots and cDNA-PCR, we found that all eight cloned mGluRs were expressed in these brain regions. Most had not previously been detected here. Surprisingly, this included mGluRs that had previously been thought to be restricted to the retina, such as mGluR6. We also detected, cloned, and sequenced a splice variant of mGluR7 (mGluR7b). Developmentally, the age of maximal expression of mGluRs was dependent on the region. For instance, mGluR5 was more strongly expressed in neonatal ARC than in adult, whereas the opposite was true in the SCN. Compared with P10 neonates, mGluR1, R3, R6, R7a, R7b, and R8 showed a greater expression in adult SCN and ARC.

Abnormally spliced messenger RNA in erythroid cells from patients with β+ thalassemia and monkey cells expressing a cloned β+-thalassemic gene

Abstract The reduced β-globin synthesis characterizing the β + thalassemia phenotype has been shown to be caused by anomalous processing within the small Intervening sequence (IVS1) of the β-globin mRNA precursor. The β-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3′ junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3′-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with β + thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned β + -thalassemic gene preferentially generated the anomalously spliced RNA when expressed In monkey kidney cells. The anomalously spliced RNA constituted approximately 80%–90%, and normal β RNA approximately 10%–20%, of the total β mRNA. In contrast, the small amount of β mRNA present in reticulocytes from such patients consisted predominantly of normal β mRNA. These results suggest that the reduced amount of normally functioning β mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent Instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.

Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor Golgi-like expression.

Viruses have substantial value as vehicles for transporting transgenes into neurons. Each virus has its own set of attributes for addressing neuroscience‐related questions. Here we review some of the advantages and limitations of herpes, pseudorabies, rabies, adeno‐associated, lentivirus, and others to study the brain. We then explore a novel recombinant vesicular stomatitis virus (dG‐VSV) with the G‐gene deleted and transgenes engineered into the first position of the RNA genome, which replicates only in the first brain cell infected, as corroborated with ultrastructural analysis, eliminating spread of virus. Because of its ability to replicate rapidly and to express multiple mRNA copies and additional templates for more copies, reporter gene expression is amplified substantially, over 500‐fold in 6 hours, allowing detailed imaging of dendrites, dendritic spines, axons, and axon terminal fields within a few hours to a few days after inoculation. Green fluorescent protein (GFP) expression is first detected within 1 hour of inoculation. The virus generates a Golgi‐like appearance in all neurons or glia of regions of the brain tested. Whole‐cell patch‐clamp electrophysiology, calcium digital imaging with fura‐2, and time‐lapse digital imaging showed that neurons appeared physiologically normal after expressing viral transgenes. The virus has a wide range of species applicability, including mouse, rat, hamster, human, and Drosophila cells. By using dG‐VSV, we show efferent projections from the suprachiasmatic nucleus terminating in the periventricular region immediately dorsal to the nucleus. DG‐VSVs with genes coding for different color reporters allow multicolor visualization of neurons wherever applied. J. Comp. Neurol. 516:456–481, 2009.

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

ABSTRACT 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-α, -β, and -γ 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-α/β 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.

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.