Mouse embryonic stem (ES) cells cultured as aggregates and exposed to retinoic acid are induced to express multiple phenotypes normally associated with neurons. A large percentage of treated aggregates produce a rich neuritic outgrowth. Dissociating the induced aggregates with trypsin and plating the cells as a monolayer results in cultures in which a sizable percentage of the cells have a neuronal appearance. These neuron-like cells express class III beta-tubulin and the neurofilament M subunit. Induced cultures express transcripts for neural-associated genes including the neurofilament L subunit, glutamate receptor subunits, the transcription factor Brn-3, and GFAP. Levels of neurofilament L and GAD67 and GAD65 transcripts rise dramatically upon induction. Physiological studies show that the neuron-like cells generate action potentials and express TTX-sensitive sodium channels, as well as voltage-gated potassium channels and calcium channels. We conclude that a complex system of neuronal gene expression can be activated in cultured ES cells. This system should be favorable for investigating some of the mechanisms that regulate neuronal differentiation.

Whole-cell and single-channel recording techniques were used to study the action of the anticonvulsant drug MK-801 {( + )-5-methyl-10,11-dihydro-5H-dibenzo [a,

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Circadian rhythms are modeled as reliable and self-sustained oscillations generated by single cells. The mammalian suprachiasmatic nucleus (SCN) keeps near 24-h time in vivo and in vitro, but the identity of the individual cellular pacemakers is unknown. We tested the hypothesis that circadian cycling is intrinsic to a unique class of SCN neurons by measuring firing rate or Period2 gene expression in single neurons. We found that fully isolated SCN neurons can sustain circadian cycling for at least 1 week. Plating SCN neurons at <100 cells/mm 2 eliminated synaptic inputs and revealed circadian neurons that contained arginine vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) or neither. Surprisingly, arrhythmic neurons (nearly 80% of recorded neurons) also expressed these neuropeptides. Furthermore, neurons were observed to lose or gain circadian rhythmicity in these dispersed cell cultures, both spontaneously and in response to forskolin stimulation. In SCN explants treated with tetrodotoxin to block spike-dependent signaling, neurons gained or lost circadian cycling over many days. The rate of PERIOD2 protein accumulation on the previous cycle reliably predicted the spontaneous onset of arrhythmicity. We conclude that individual SCN neurons can generate circadian oscillations; however, there is no evidence for a specialized or anatomically localized class of cell-autonomous pacemakers. Instead, these results indicate that AVP, VIP, and other SCN neurons are intrinsic but unstable circadian oscillators that rely on network interactions to stabilize their otherwise noisy cycling.luciferase ͉ pacemaker ͉ Period gene ͉ suprachiasmatic nucleus ͉ vasoactive intestinal polypeptide C ircadian pacemakers are schematized as intracellular transcription-translation negative feedback loops (1). In mammals, transcription factors including CLOCK and BMAL1 promote the expression of clock genes, including Period 1 (Per1) and 2 (Per2). The protein products of these genes return to the nucleus after a delay of many hours to repress their own transcription. Genetic deletion of these repressors abolishes circadian rhythms in behavior and physiology (2). The strongest evidence for cell-autonomous, circadian rhythm generation in mammals comes from transcriptional rhythms measured from primary and immortalized fibroblasts (3, 4).The mammalian suprachiasmatic nucleus (SCN) of the anterior hypothalamus coordinates daily rhythms including sleep-wake and hormone release (5). Multielectrode array (MEA) recordings of neuronal firing and luciferase-based reporters of Per1 and Per2 expression showed dissociated SCN neurons in the same culture with different circadian periods (6, 7). Furthermore, Na ϩ -dependent action potentials, vasoactive intestinal polypeptide (VIP), and its receptor, VPAC2, are required for cellular synchrony and maintaining daily oscillations across the SCN (8, 9). Taken together, these results suggest that single SCN neurons are competent circadian oscillators. However, which, if any, SCN neurons are capable ...

The RGS7 (R7) family of RGS proteins bound to the divergent Gβ subunit Gβ5 is a crucial regulator of G protein–coupled receptor (GPCR) signaling in the visual and nervous systems. Here, we identify R7BP, a novel neuronally expressed protein that binds R7–Gβ5 complexes and shuttles them between the plasma membrane and nucleus. Regional expression of R7BP, Gβ5, and R7 isoforms in brain is highly coincident. R7BP is palmitoylated near its COOH terminus, which targets the protein to the plasma membrane. Depalmitoylation of R7BP translocates R7BP–R7–Gβ5 complexes from the plasma membrane to the nucleus. Compared with nonpalmitoylated R7BP, palmitoylated R7BP greatly augments the ability of RGS7 to attenuate GPCR-mediated G protein–regulated inward rectifying potassium channel activation. Thus, by controlling plasma membrane nuclear–shuttling of R7BP–R7–Gβ5 complexes, reversible palmitoylation of R7BP provides a novel mechanism that regulates GPCR signaling and potentially transduces signals directly from the plasma membrane to the nucleus.

Glutamate, the major excitatory neurotransmitter in the central nervous system, activates three different receptors that directly gate ion channels, namely receptors for AMPA (alpha-amino-3-hydroxy-5-methyl isoxozole propionic acid), NMDA (N-methyl-D-aspartate), and kainate, a structural analogue of glutamate. The contribution of AMPA and NMDA receptors to synaptic transmission and plasticity is well established. Recent work on the physiological function of kainate receptors has focused on the hippocampus, where repetitive activation of the mossy-fibre pathway generates a slow, kainate-receptor-mediated excitatory postsynaptic current (EPSC). Here we show that high-intensity single-shock stimulation (of duration 200 microseconds) of primary afferent sensory fibres produces a fast, kainate-receptor-mediated EPSC in the superficial dorsal horn of the spinal cord. Activation of low-threshold afferent fibres generates typical AMPA-receptor-mediated EPSCs only, indicating that kainate receptors may be restricted to synapses formed by high-threshold nociceptive (pain-sensing) and thermoreceptive primary afferent fibres. Consistent with this possibility, kainate-receptor-mediated EPSCs are blocked by the analgesic mu-opiate-receptor agonist Damgo and spinal blockade of both kainate and AMPA receptors produces antinociception. Thus, spinal kainate receptors contribute to transmission of somatosensory inputs from the periphery to the brain.

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Neurons in the mammalian suprachiasmatic nuclei (SCN) generate daily rhythms in physiology and behavior, but it is unclear how they maintain and synchronize these rhythms in vivo. We hypothesized that parallel signaling pathways in the SCN are required to synchronize rhythms in these neurons for coherent output. We recorded firing and clock-gene expression patterns while blocking candidate signaling pathways for at least 8 days. GABA A and GABA B antagonism increased circadian peak firing rates and rhythm precision of cultured SCN neurons, but G i/o did not impair synchrony or rhythmicity. In contrast, inhibiting G i/o with pertussis toxin abolished rhythms in most neurons and desynchronized the population, phenocopying the loss of vasoactive intestinal polypeptide (VIP). Daily VIP receptor agonist treatment restored synchrony and rhythmicity to VIP ؊/؊ SCN cultures during continuous GABA receptor antagonism but not during G i/o blockade. Pertussis toxin did not affect circadian cycling of the liver, suggesting that G i/o plays a specialized role in maintaining SCN rhythmicity. We conclude that endogenous GABA controls the amplitude of SCN neuronal rhythms by reducing daytime firing, whereas G i/o signaling suppresses nighttime firing, and it is necessary for synchrony among SCN neurons. We propose that G i/o, not GABA activity, converges with VIP signaling to maintain and coordinate rhythms among SCN neurons.luciferase ͉ multielectrode array ͉ Period gene ͉ suprachiasmatic nucleus ͉ vasoactive intestinal polypeptide T he suprachiasmatic nuclei (SCN) of the mammalian hypothalamus serve as a master circadian pacemaker, mediating daily rhythms in behavior and physiology. SCN pacemaker function depends on near-24-h oscillations in expression of ''clock genes'' and in firing rate (1). Previous studies have shown that of the nearly 20,000 neurons in the bilateral SCN, one subset comprises cell-autonomous circadian clocks, and another subset requires vasoactive intestinal polypeptide (VIP) signaling to maintain daily rhythms (2).For the SCN to coordinate coherent behavioral rhythms, SCN neurons must synchronize to one another in vivo. Blocking action potentials desynchronizes circadian rhythms among SCN neurons (3, 4). Neurotransmitters likely mediating synchrony within the SCN include VIP and GABA (2). VIP is necessary for synchrony between SCN neurons in vitro (5, 6) and for coherent behavioral rhythmicity in vivo (7,8). GABA has been implicated because most (if not all) SCN neurons express GABA and its receptors (9-11), GABA is released in a daily rhythm within the SCN (12), and daily application of exogenous GABA synchronizes firing-rate rhythms of SCN neurons (13). How VIP mediates circadian synchrony and the necessity of GABA in this process have not been tested.To examine the roles of endogenous GABA and G protein signaling, we recorded Period1::luciferase (Per1::luc) expression from SCN slices and firing rate and PERIOD2::luciferase (PER2::LUC) expression from individual SCN neurons. We find that long-term ant...