NudC associates with Lis1 and the dynein motor at the leading pole of neurons.

NUDC is a highly conserved protein important for nuclear migration and viability in Aspergillus nidulans. Mammalian NudC interacts with Lis1, a neuronal migration protein important during neocorticogenesis, suggesting a conserved mechanism of nuclear movement in A. nidulans and neuronal migration in the developing mammalian brain (S. M. Morris et al., 1998). To further investigate this possibility, we show for the first time that NudC, Lis1, and cytoplasmic dynein intermediate chain (CDIC) colocalize at the microtubule organizing center (MTOC) around the nucleus in a polarized manner facing the leading pole of cerebellar granule cells with a migratory morphology. In neurons with stationary morphology, NudC is distributed throughout the soma and colocalizes with CDIC and tubulin in neurites as well as at the MTOC. At the subcellular level, NudC, CDIC, and p150 dynactin colocalize to the interphase microtubule array and the MTOC in fibroblasts. The observed colocalization is confirmed biochemically by coimmunoprecipitation of NudC with CDIC and cytoplasmic dynein heavy chain (CDHC) from mouse brain extracts. Consistent with its expression in individual neurons, a high level of NudC is detected in regions of the embryonic neocortex undergoing extensive neurogenesis as well as neuronal migration. These data suggest a biochemical and functional interaction of NudC with Lis1 and the dynein motor complex during neuronal migration in vivo.

Neuronal platelet-activating factor receptor signal transduction involves a pertussis toxin-sensitive G-protein.

In most nonneural systems, platelet-activating factor (PAF) receptor effects are mediated by G-proteins that are often pertussis toxin-sensitive. The activation of pertussis toxin-sensitive G-proteins linked to PAF receptors results in the mobilization of intracellular calcium, at least in part, through the second messenger inositol triphosphate. We have sought to determine if a pertussis toxin-sensitive G-protein is involved in the PAF receptor-mediated phenomena of growth cone collapse and of synaptic enhancement in primary neuronal culture. Using infrared differential interference contrast microscopy and patch-clamp recording techniques, pertussis toxin, but not the inactive B oligomer of the toxin, was found to block both the growth cone collapse and the enhanced synaptic release of excitatory transmitter induced by a nonhydrolyzable PAF receptor agonist, making it likely that Go, Gq, or Gi is the G-protein transducer of PAF receptors in primary neurons. We believe that PAF acts directly on neuronal receptors, which are linked to pertussis toxin-sensitive G-proteins, on the tips of developing neurites, and on presynaptic nerve terminals, leading to growth cone collapse and enhanced synaptic release of transmitter.

Opioid modulation of calcium current in cultured sensory neurons: mu-modulation of baroreceptor input.

We used the whole cell open-patch or perforated-patch technique to characterize mu-opioid modulation of Ca(2+) current (I(Ca)) in nodose sensory neurons and in a specific subpopulation of nodose cells, aortic baroreceptor neurons. The mu-opiate receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol enkephalin (DAGO) inhibited I(Ca) in 95% of neonatal [postnatal day (P)1-P3] nodose neurons. To the contrary, only 64% of juvenile cells (P20-P35) and 61% of adult cells (P60-P110) responded to DAGO. DAGO-mediated inhibition of I(Ca) was naloxone sensitive, irreversible in the presence of guanosine 5'-O-(3-thiotriphosphate), absent with guanosine 5'-O-(2-thiodiphosphate), and eliminated with pertussis toxin; DAGO's inhibition of I(Ca) was G protein mediated. Incubation of neurons with omega-conotoxin GVIA eliminated the effect of DAGO in neonatal but not in juvenile cells. In the latter, DAGO reduced 37% of the current remaining in the presence of omega-conotoxin. In the subset of nodose neurons, aortic baroafferents, the effect of DAGO was concentration dependent, with an IC(50) of 1.82 x 10(-8) M. DAGO slowed activation of I(Ca), but activation curves constructed from tail currents were the same with and without DAGO (100 nM). In summary, mu-opiate modulation of I(Ca) in nodose neurons was demonstrated in three age groups, including specifically labeled baroafferents. The demonstration of a mechanism of action of mu-opioids on baroreceptor afferents provides a basis for the attenuation of the baroreflex that occurs at the level of the nucleus tractus solitarii.

Neuronal cytoskeletal alterations evoked by a platelet-activating factor (PAF) analogue.

Platelet-activating factor (PAF), a phospholipid signaling molecule found in brain, modulates several neural functions and is implicated in the human developmental brain disorder Miller-Dieker Lissencephaly (MDL). Exposure to PAF, and a non-hydrolyzable analogue, methyl carbamyl PAF (mc-PAF), produces the following rapid, reversible effects upon cultured hippocampal neurites: growth cone collapse, neurite retraction, and neurite varicosity formation. In this study, the cytoskeletal alterations that mediate these shape changes were investigated by comparing the effects of mc-PAF with other cytoskeletal-altering drugs, through the fluorescent labeling of cytoskeletal proteins and mitochondria, and by electron microscopy. Results indicate that rearrangements of microtubules (MTs), F-actin, and mitochondria underlie the neurite shape changes produced by mc-PAF. Evidence for MT alteration was obtained by comparing the effects of mc-PAF with nocodozole and taxol. Exposure to nocodazole, a MT-depolymerizing agent, produced growth cone collapse and neurite varicosity formation similar to mc-PAF, whereas pre-incubation of neurites in taxol, a MT-stabilizing drug, was effective in blocking mc-PAF-induced neurite effects. Immunofluorescent labeling and EM revealed MT splaying and unbundling within neurite varicosities following mc-PAF treatment. Immunofluorescent labeling also revealed that F-actin shifted from concentration in the growth cone to a diffuse distribution along the neurite shaft following mc-PAF exposure. Fluorescent labeling and EM also revealed retrograde movement and morphological alterations of mitochondria following mc-PAF exposure, resulting in mitochondrial aggregates within neurite varicosities. These cytoskeletal rearrangements may provide insights into the mechanisms by which PAF influences neuronal activity, and could have important implications for the impairment of neuronal motility observed in MDL.

Accentuated antagonism in canine subendocardium is not altered by chronic exercise.

Acetylcholine often affects cardiac action potential repolarization only during augmented adrenergic tone, i.e., the phenomenon of accentuated antagonism. Since chronic exercise involves repeated changes in autonomic outflow, we determined whether it also influenced adrenergic/cholinergic interactions in isolated canine cardiac tissue. Using standard micro-electrode techniques in thin ventricular subendocardial slices isolated from exercised (EX: 8-10 wk daily exercise) and sedentary (SED): 8-10 wk cage rest) dogs, we examined transmembrane potential responses to isoproterenol (ISO: 10(-8), 10(-7), 10(-6) M) and to ISO in the presence of ACH (10(-5) M). Control transmembrane characteristics at BCL = 500 ms were similar for EX (N = 8 dogs) and SED (N = 9 dogs). ISO (10(-6) M) decreased action potential duration at 50% repolarization (APD50): EX = -29 +/- 15 ms; SED = 11 ms and at 90% repolarization (APD90): EX = -37 +/- 17 ms; and SED = -24 +/- 14 ms (P > 0.05, EX vs SED). ACH alone did not alter APD. With ACH (10(-5) M), delta APD50 with ISO (10(-6) M) was -5 +/- ms and 0 +/- 5 ms for EX and SED, respectively; delta APD90 was -8 +/- 4 ms and -8 +/- 7 ms for EX and SED, respectively (P > 0.05, EX vs SED). Thus, ACH antagonized ISO-mediated acceleration of repolarization equally in both groups. Chronic daily exercise does not influence adrenergic/cholinergic interactions at the cellular level.

Platelet-activating factor produces neuronal growth cone collapse.

It is generally believed that neuronal growth cone collapsing factors are proteins that interact with membrane-bound receptors. Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) - a phospholipid autocoid, also interacts with a membrane-bound neuronal receptor which is similar in nature to collapsing factor receptors. We report that PAF and the nonhydrolyzable PAF agonist, methyl carbamyl PAF (1-O-hexadecyl-2N-methylcarbamyl-sn-glycero-3-phosphocholine, mc-PAF), evoke a dose-dependent neuronal growth cone collapse. This collapse is specifically attenuated by the PAF receptor antagonist BN-52021. These data point to a PAF receptor-mediated growth cone collapse. Therefore, PAF must be added to the list of collapsing factors which potentially guide axons to their proper targets in the developing nervous system.

Cardiac adrenergic responses and electrophysiology during ischemia: effect of exercise.

Whether exercise protects the myocardium from arrhythmias during ischemia (ISC) or alters electrophysiology is controversial. We used microelectrode techniques in isolated cardiac fibers from exercise-trained (ET: 8-10 wk daily exercise) and sedentary (SED: 8-10 wk cage-rest) dogs to examine the effect of exercise on cellular electrophysiology during simulated ISC. We superfused fibers first with normal Tyrode's, then "ischemic Tyrode's" ([K+]o = 10 mM, pH = 6.7, pO2 < 25 mm Hg), and then again with normal Tyrode's. In automatic fibers, maximum diastolic potential in normal Tyrode's was -98 +/- 1 mV (ET, N = 22) and -97 +/- 1 mV (SED, N = 23); rates were 20 +/- 2 and 18 +/- 3 bpm for ET and SED, respectively. All fibers depolarized to -61 +/- 2 mV with ISC. Abnormal rhythms (abnormal automaticity with or without delayed afterdepolarizations) during ISC alone were seen in 0% of ET and 33% of SED; during ISC with alpha-adrenergic stimulation with 5 x 10(-8) M phenylephrine the incidence was 25% of ET and 0% of SED; during ISC with isoproterenol it was 75% for ET (P < 0.05 vs control) and 38% for SED. Transmembrane potentials in paced subendocardial fibers were similar for ET and SED during control, ISC, and reperfusion. Exercise did not alter cellular electrophysiology but did influence ectopic rhythms seen with beta-stimulation during ISC.