The nucleotide sequence of SV40 DNA was determined, and the sequence was correlated with known genes of the virus and with the structure of viral messenger RNA's. There is a limited overlap of the coding regions for structural proteins and a complex pattern of leader sequences at the 5' end of late messenger RNA. The sequence of the early region is consistent with recent proposals that the large early polypeptide of SV40 is encoded in noncontinguous segments of DNA.

No abstract

Qu et al., 1996), and blockade of the MCH receptor New Haven, Connecticut 06520 with antagonists reduces body weight (Borowsky et al., 2 Columbus Children's Research Institute 2002). Food deprivation and leptin alter expression of Ohio State University MCH and c-fos mRNA in MCH neurons (Qu et al., 1996; Columbus, Ohio 43205 Huang et al., 1999). MCH peptide (Shimada et al., 1998) and receptor (Marsh et al., 2002) knockout mice eat less, have reduced body weights, and show increased Summary activity. It has been suggested that another orexigenic peptide, neuropeptide Y (NPY), which also stimulates Neurons that synthesize melanin-concentrating horfood intake, acts by stimulating the MCH neurons mone (MCH) may modulate arousal and energy ho-(Schwartz et al., 2000). MCH neurons may also play a meostasis. The scattered MCH neurons have been role in memory and depression (Borowsky et al., 2002). difficult to study, as they have no defining morphologi-As MCH neurons are scattered within the hypothalacal characteristics. We have developed a viral apmus and have no distinct morphological attributes, it proach with AAV for selective long-term reporter gene has been difficult to identify the live neurons in order (GFP) expression in MCH neurons, allowing the study to record from them. We have solved this problem by of their cellular physiology in hypothalamic slices. designing a recombinant adeno-associated virus (AAV) MCH neurons showed distinct membrane properties that expresses a reporter gene, GFP, only in MCH neucompared to other neurons infected with the same rons and appears to have no adverse effect on neuron virus with a cytomegalovirus promoter. Transmitters health. Although many cells may be infected by the virus, of extrahypothalamic arousal systems, including noronly those cells that contain immunoreactivity for the epinephrine, serotonin, and the acetylcholine agonist MCH peptide show reporter gene expression. The virus muscarine, evoked direct inhibitory actions. Orexishould work to identify MCH neurons in a wide variety genic neuropeptide Y was inhibitory by pre-and post-of species, as suggested by experiments here in both synaptic mechanisms; an anorexigenic melanocortin rats and mice. This approach has allowed us to study agonist had no effect. In contrast, the hypothalamic the membrane properties, synaptic activity, and mechaarousal peptide hypocretin/orexin evoked a direct innisms of transmitter responses in hypothalamic slices ward current and increased excitatory synaptic activfrom rodents injected in vivo with the recombinant virus, ity and spike frequency in the normally silent MCH and we have corroborated these findings with parallel neurons. Together, these data support the view that slice experiments from MCH-GFP transgenic mice. We MCH neurons may integrate information within the tested the responses of MCH neurons to two groups of arousal system in favor of energy conservation.neuromodulators that are found in axons in the LH: those that play a role in arousal, such as norepinephrine (NE),

In situ hybridization and Northern blots were used to study the ionotropic subtypes of the glutamate receptor in the rat hypothalamus. Widespread expression of AMPA, kainate, and NMDA receptor RNA was found in the hypothalamus with the transcripts the same size and number as found in other regions of the brain. Most of the glutamate-receptor subunits studied were expressed in greater amounts in hippocampus than in hypothalamus; GluR5, on the other hand, showed a greater expression in hypothalamus than in hippocampus. On the basis of Northern blot analysis, all regions of the brain examined, including hypothalamus, cerebral cortex, cerebellum, olfactory bulb, and hippocampus, expressed all eight of the subunits studied. Each subunit showed different relative expressions in the different regions. In the hypothalamus, GluR1 and GluR2 were among the most widely expressed of the non-NMDA ionotropic receptors. Other AMPA-preferring receptors, GluR3 and -R4, were also found, but to a lesser extent. Scattered cells expressed the kainate-preferring receptors GluR5, -R6, and -R7. The NMDA receptor NMDAR1 was detected throughout the hypothalamus. In many regions of the hypothalamus, only scattered cells showed detectable expression of the glutamate-receptor mRNA as detected by autoradiographic silver grains over neurons; unlabeled cells were mixed among labeled cells. Every region of the hypothalamus had several different glutamate receptors. The expression of many different types of ionotropic glutamate receptors throughout the hypothalamus suggests that multiple modes of ion channel regulation by glutamate probably operate here and provides further support for the importance of the excitatory transmitter glutamate in hypothalamic regulation.

The paraventricular thalamic nucleus (PVT) receives one of the most dense innervations by hypothalamic hypocretin/orexin (Hcrt) neurons, which play important roles in sleep-wakefulness, attention, and autonomic function. The PVT projects to several loci, including the medial prefrontal cortex (mPFC), a cortical region involved in associative function and attention. To study the effect of Hcrt on excitatory PVT neurons that project to the mPFC, we used a new line of transgenic mice expressing green fluorescent protein (GFP) under the control of the vesicular glutamate-transporter-2 promoter. These neurons were retrogradely labeled with cholera toxin subunit B that had been microinjected into the mPFC. Membrane characteristics and responses to hypocretin-1 and -2 (Hcrt-1 and -2) were studied using whole cell recording (n > 300). PVT neurons showed distinct membrane properties including inward rectification, H-type potassium currents, low threshold spikes, and spike frequency adaptation. Cortically projecting neurons were depolarized and excited by Hcrt-2. Hcrt-2 actions were stronger than those of Hcrt-1, and the action persisted in TTX and in low calcium/high magnesium artificial cerebrospinal fluid, consistent with direct actions mediated by Hcrt receptor-2. Two mechanisms of Hcrt excitation were found: an increase in input resistance caused by closure of potassium channels and activation of nonselective cation channels. The robust excitation evoked by Hcrt-2 on cortically projecting glutamate PVT neurons could generate substantial excitation in multiple layers of the mPFC, adding to the more selective direct excitatory actions of Hcrt in the mPFC and potentially increasing cortical arousal and attention to limbic or visceral states.

In order to investigate the 5' terminal structural heterogeneity of the 16S size class of SV40 late RNA, we have bound an SV40 DNA fragment labeled at its 5' termini with P32 to the .939-.945 map unit region of late lytic cytoplasmic polyadenylated RNA, used reverse transcriptase to prepare cDNA copies of the 5' termini of this RNA, separated the cDNA products on an 8% polyacrylamide-7 M urea gel and subjected these products to nucleic acid sequence analysis. A number of discrete cDNAs were obtained. Analysis of these cDNAs has suggested the presence of three categories of 16S species all containing the same body extending from residues 1381-2592 (.939-.170 m.u.) but differring in the structure of their leader segments. Members of the first category contain leaders which are colinear with SV40 DNA, have a common 3' terminus at residue 444 and extend varying distances in a 5' direction. The most abundant 16S species contains a leader of 203 nucleotides and is a member of this group. RNAs of the second category contain leaders with an internal gap between residues 211-352. The single RNA comprising the third category contains a leader with a tandem repetition of nucleotides 351-443 at the 3' terminus of its leader.

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.

The 5' termini of the principal early mRNAs produced in cells transformed by wild-type simian virus 40 lie 21-25