Organelle dynamics in lobster axons: anterograde and retrograde particulate organelles.

Particulate organelles in isolated axons from the walking legs of the lobster were detected with differential interference contrast optics and video microscopic techniques. The motion of the organelles was studied in normal axons, in axons whose surface membrane was rendered permeable with saponin, and in axoplasm extruded from the axons. In normal axons at 20-22 degrees C, organelles moved more rapidly in the anterograde direction than in the retrograde direction (respective mean velocities 1.73 micron/s and 0.63 micron/s). The instantaneous velocities of both sets of organelles were variable: those of the anterograde organelles varied less than those of retrograde organelles. The variation in instantaneous velocity was patterned; all organelles studied had velocities that fluctuated slowly with a major frequency at about 0.1 Hz. Some organelles oscillated about a fixed position at a similar major frequency. In axons with a permeabilized surface membrane there was no organelle motion unless adenosine 5'-triphosphate (ATP) was present in the bathing medium. Organelle motion reactivated with ATP was patterned in a way similar to that in normal intact axons. In extruded axoplasm in the presence of ATP, organelles moved along transport filaments that were assumed to be microtubules. Movement of organelles from one transport filament to another was not accompanied by changes in motion that could explain the normal fluctuation in velocity. The evidence indicates that the variable, or oscillatory, character of organelle motion in lobster axons is caused by an active component of the mechanisms of axonal transport.

Morphology of astroglial cells is controlled by beta-adrenergic receptors.

Astroglial cells in vivo and in vitro respond to hormones, growth factors, and neurotransmitters by changing from an epithelial-like to stellate morphology. We have studied the temporal relationship between receptor activation, second messenger mobilization, and morphological changes using LRM55 astroglial cells. Maintenance of an altered morphology required continuous beta-adrenergic receptor activation. These changes appeared to be mediated by cAMP since they were elicited by its analogue, dibutyryl cAMP, and by forskolin, a direct activator of adenylate cyclase. Changes in cell morphology may require a relatively small increase in intracellular cAMP, since receptor-stimulated changes in cAMP levels were transient and peaked approximately 5 min after receptor activation while changes in morphology took at least 30 min to reach a new steady state. Time-lapse videomicroscopy and high voltage electron microscopy indicated that receptor activation resulted in a sequence of morphological events. Time-lapse observations revealed the development and enlargement of openings through the cytoplasm associated with cytoplasmic withdrawal to the perinuclear region and process formation. Higher resolution high voltage electron microscopy indicated that the transition to a stellate morphology was preceded by the appearance of two distinct cytoplasmic domains. One contained an open network of filaments and organelles. The other was characterized by short broad cytoplasmic filaments. The first domain was similar to cytoplasm in control cells while the second was associated with the development and enlargement of openings through the cytoplasm and regions of obvious cytoplasmic withdrawal.

Organelle dynamics in lobster axons: anterograde, retrograde and stationary mitochondria.

Mitochondria in isolated motor axons from the walking legs of lobster were observed with differential interference contrast optics and video microscopic techniques. Movements of the mitochondria were analyzed in time-lapse videotape records. The mean velocity of transport in the retrograde direction (1.33 +/- 0.64 micron/s) was greater than the mean velocity of transport in the anterograde direction (0.72 +/- 0.26 micron/s). The mean lengths of the mitochondria moving in the retrograde and anterograde directions were only slightly different (6.9 microns and 5.5 microns, respectively). No correlation was found between mitochondrial length and average velocity or reciprocal velocity. The instantaneous velocities of mitochondria were distributed over a range of approximately 3 micron/s; both the anterograde and retrograde distributions contained a small proportion of values whose sign was opposite to the modal value. The variation in instantaneous velocity took place at frequencies close to 0.1 Hz. Some mitochondria displayed longitudinally oriented oscillatory movements of a similar low frequency. While the movement of most mitochondria was parallel to the axis of the axon, transverse deviations and complex circular paths were sometimes observed. Some mitochondria reversed their orientation and continued in the same direction, so that the end which had been the leading end became the trailing end. Many mitochondria immediately beneath the plasma membrane were stationary and adhered strongly to the plasma membrane when the axoplasmic structure was disrupted. In electron micrographs, fine strands connected peripheral mitochondria and the plasma membrane. These strands may anchor the stationary mitochondria to the plasma membrane.

Polarity orientations of microtubules in squid and lobster axons.

Polarity orientations of microtubules in periaxolemmal and internal regions of squid and lobster axons were determined in order to test the hypothesis that regional differences in particle transport are produced by differentially distributed microtubule subclasses. Over 95% of the microtubules in all regions of the axons investigated were oriented with plus ends located distally, pointing away from axonal somata, and there were no significant differences in orientation ratios in periaxolemmal and internal axoplasm. In axonal sheath glial cells of lobsters, microtubules were found to be oriented parallel to axonal microtubules and to have approximately equally mixed polarities. The results for axonal microtubules did not support the possibility of subclasses of axonal microtubules.

Enkephalin and neuropeptide Y: two colocalized neuropeptides are independently regulated in primary cultures of bovine chromaffin cells.

We have found that Neuropeptide Y is colocalized with enkephalin in bovine adrenal chromaffin cells. The two peptides can be found in the same granules in those cells where they coexist. These cells correspond to the adrenergic subpopulation of chromaffin cells since they contain the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase. Despite their coexistence, production of the two peptides is independently regulated. Enkephalin levels are doubled after nicotinic depolarization (which increases enkephalin synthesis) or after treatment with reserpine (which increases enkephalin precursor processing). Neither of these treatments, acting by different mechanisms, has any effect on the levels of Neuropeptide Y.

Slow pulsatile movements of Schwann cells in vitro: a time-lapse cinemicrographic study.

Slow pulsatile movements of Schwann cells in vitro were studied quantitatively by using time-lapse cinemicrography. Schwann cells from peripheral nerves of 3-day-old rats were cultured in serum-free medium. Most Schwann cells showed intermittent episodes of pulsatile movement; each episode consisted of one or several contractile pulses. About half of the episodes consisted of a single pulse, and episodes with more than four pulses were rare. The average episode of activity lasted 2.6 min, while the average duration of a single pulse was 1.5 min. The mean quiescent interval between episodes of activity was 3.7 min. Some cells showed no pulsatile activity. Active cells averaged 6.6 episodes/h. The fraction of time which a Schwann cell spent in pulsatile activity varied widely, with an average of 28%. Behavior of Schwann cells in HEPES-buffered Hanks saline was generally similar to that in the complete medium. Raising K+ to 40 mM or Ca++ to 10 mM did not markedly affect the time course of the pulsatile motility, although the contractions were more vigorous in the high Ca++. Pulsatile movement was reversibly inhibited by cytochalasin B and appeared to be potentiated by drugs that disrupt microtubules.

Nucleotide specificity for reactivation of organelle movements in permeabilized axons.

In a permeabilized axon model, exogenous ATP can reactivate intraaxonal saltatory organelle movements (microscopically visible manifestations of fast axonal transport). We have studied the dependence of the reactivated movements on the ATP concentration and have also examined the nucleotide specificity of the reactivation. Organelle transport was visualized in isolated lobster giant motor axons using Nomarski optics and video microscopy. The axons were permeabilized with saponin, and movement was reactivated with ATP or other nucleotides. Some slight movement was seen with ATP concentrations as low as 10 microM. The velocity and frequency of the reactivated transport increased with increasing ATP concentrations up to about 5 mM. Movement was also reactivated by deoxyadenosine triphosphate, but not by AMP-PNP (a nonhydrolyzable ATP analogue), ADP, or AMP. Although other nucleotides (CTP, GTP, UTP, ITP) could reactivate transport, movement equivalent to that produced by 0.1 mM ATP was only seen with tenfold or greater concentrations of the other nucleotides. This pattern of specificity is consistent with the hypothesis that a dynein-like ATPase, rather than a myosin, is involved in fast axonal transport.

Selective inhibition of retrograde axonal transport by erythro-9-[3-(2-hydroxynonyl)]adenine.

Retrograde transport of microscopically visible organelles in isolated lobster axons was selectively inhibited by erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA), with little or no effect on anterograde transport. The retrograde velocity was reduced both in living axons and in permeabilized axons in which movement was reactivated with exogenous ATP. This is the first report of an agent that selectively inhibits axonal transport in one direction.

Fast axonal transport in permeabilized lobster giant axons is inhibited by vanadate.

We have developed a method for permeabilizing axons and reactivating the fast transport of microscopically visible organelles. Saltatory movements of organelles in motor axons isolated from lobster walking legs were observed using Nomarski optics and time-lapse video microscopy. In the center of the axon most of the particles and mitochondria moved in the retrograde direction, but immediately below the axolemma the majority moved in the anterograde direction. When axons were permeabilized with 0.02% saponin in an adenosine 5'-triphosphate (ATP)-free "internal" medium, all organelle movement ceased. Saltatory movements resembling those in intact axons immediately reappeared upon the addition of MgATP. Very slight movement could be detected with ATP concentrations as low as 10 microM, and movement appeared to be maximal with 1 to 5 mM ATP. Vanadate, which does not affect axonal transport in intact axons, inhibited the reactivated organelle movements in permeabilized axons. Movement was rapidly and reversibly inhibited by 50 to 100 microM sodium orthovanadate. The effects of vanadate, including the time course of inhibition, its reversibility, and its concentration dependence, are consistent with the hypothesis that a dyneinlike like molecule may play a role in the mechanism of fast axonal transport.

Morphologic observations of contact-induced lysis of EBV-infected B lymphocytes by autologous hand mirror T cells.

To study the possible immunologic role of hand mirror lymphocytes (HML) in the control of Epstein-Barr virus (EBV)-infected B lymphocytes in patients with infectious mononucleosis (IM), we studied the in vitro interactions of these cells by time-lapse video light microscopy. Peripheral blood T lymphocytes isolated from three patients in the early recovery phase of IM were mixed with their autologous EBV-infected B cells. Motile lymphocytes with their characteristic hand mirror shape were observed to attach by their uropods to the B cells. The HML remained attached for variable periods ranging from 45-75 min. Following detachment, B cells that were in contact with HML underwent lysis. Mixtures of T cells from healthy donors and EBV-infected cell lines exhibited only rare uropod formation with no attachment or lysis of B cells. The present experiment indicates that contact-induced lysis of EBV-infected B lymphocytes is operative in IM and that this process is mediated by the HML.

Explant cultures of adult amphibian sympathetic ganglia: electrophysiological and pharmacological investigation of neurotransmitter and nucleotide action.

Explant cultures from adult bullfrog sympathetic ganglia can be maintained in vitro for several months and are suitable for electrophysiological recording. The cultured neurons display morphological, electrophysiological and pharmacological characteristics similar, in most respects, to those reported for acutely isolated sympathetic ganglia. Individual cells were visualized by Nomarski optics and impaled with a glass micropipette, which was used for voltage recording and current injection. The average specific membrane properties, calculated from cell dimensions and responses to current injection, were Vm = -46 mV, Rin = 27 M omega, Rm = 1665 omega cm2, tau m = 5 msec, and Cm = 3.2 microF/cm2. Bath perfusion of the cholinergic agonist muscarine depolarized most neurons with an increase in input resistance, while carbachol depolarized neurons with both increases and decreases in input resistance. GABA depolarized all neurons tested with a decreased resistance. High concentrations of catecholamines (2-5 mM) generally hyperpolarized explanted neurons, usually in association with an increased resistance. Extracellularly or intracellularly applied cyclic AMP and two other analogues produced weak and inconsistent hyperpolarizations. In contrast, perfusion or iontophoresis of most non-cyclic purine and pyrimidine nucleotides markedly depolarized most neurons in association with an increased input resistance. UTP and UDP were most potent, revealing threshold concentrations of about 10(-8) to 5 x 10(-8) M. The related nucleosides were largely ineffective. The nucleotide-evoked depolarizations were similar to the muscarine responses but were not blocked by atropine. These results suggest that the purine or pyrimidine nucleotides should be considered for a possible involvement in neurotransmission in sympathetic ganglia.

Batrachotoxin blocks saltatory organelle movement in electrically excitable neuroblastoma cells.

Batrachotoxin has been reported to inhibit fast axonal transport. We have examined the effect of batrachotoxin on saltatory organelle movements in N115 neuroblastoma cells (a model of fast axonal transport) using time-lapse video intensification microscopy. Batrachotoxin (0.1-1.0 microM) inhibits saltatory organelle movement. Contrary to previously published hypotheses, this inhibition of intra-axonal movement depends upon the action of batrachotoxin on action potential Na+ channels. Evidence for this conclusion is: (1) the range of concentrations of batrachotoxin which inhibit saltatory organelle movement is consistent with the dose-response curve for the activation of action potential Na+ channels by batrachotoxin in N18 neuroblastoma cells; (2) tetrodotoxin, which blocks action potential Na+ channels, prevents the inhibition of organelle movements by batrachotoxin; (3) batrachotoxin has no effect on saltatory movement in cells, including some neuroblastoma cell lines, which lack action potential Na+ channels; and (4) in Na+-free or low Na+ media, batrachotoxin does not block organelle movement. We suggest that changes in internal ion concentrations which follow the influx of Na+ are responsible for the inhibition of fast axonal transport by batrachotoxin.

Interaction of fluorescent gonadotropin-releasing hormone with receptors in cultured pituitary cells.

A fluorescent derivative of the gonadotropin-releasing hormone (GnRH) agonist analog, [D-Lys6]GnRH, was synthesized for receptor studies and shown to be biologically active. The rhodamine-derivatized peptide (Rh-GnRH) retained 40% of the receptor binding activity of [D-Lys6]GnRH, and 50% of the luteinizing hormone-releasing activity assayed in cultured pituitary cells. The fluorescent analog was employed to visualize the distribution of GnRH receptors in cultured pituitary cells, using the technique of video-intensified fluorescence microscopy. The binding of Rh-GnRH was confined to the large gonadotrophs which comprised 15% of the cell population. The specificity of the binding was shown by the absence of significant fluorescence in the presence of a 100-fold excess of [D-Lys6]GnRH, or when Rh-GnRH was incubated with choriocarcinoma, neuroblastoma, or 3T3 cell lines devoid of GnRH receptors. The interaction of Rh-GnRH with living pituitary cells was characterized by an initial diffuse distribution, followed by the formation of polar aggregates that later appeared to be internalized. These observations emphasize the value of fluorescent derivatives of GnRH for elucidating the course of the interaction with specific receptors on pituitary gonadotrophs. The initial results indicate that GnRH-receptor complexes undergo aggregation during stimulation of luteinizing hormone release, and are later internalized for subsequent degradation and/ or intracellular actions.

Comparative analysis of rapidly transported axonal proteins in sensory neurons of the frog and rat.

35S-labeled proteins carried by fast axonal transport in sciatic sensory axons of bullfrog and rat were separated electrophoretically on discontinuous polyacrylamide gradient slab gels. In contrast to the previously reported similarity in the electrophoretic profiles of rapidly transported proteins from functionally different neurons, we have found that there is very little correspondence in the profiles of these proteins in functionally similar neurons from two widely studied species. We also found very little correspondence between the two species in the profiles of locally synthesized sciatic nerve protein. The results demonstrate the difficulty inherent in comparing the electrophoretic profiles obtained using these two model systems for the study of rapidly transported axonal proteins. In particular, relationships between the major rapidly transported proteins in the two species could not be analyzed with this technique.