Identification of synaptophysin-like immunoreactivity in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa).

Synaptophysin is a membrane protein of synaptic vesicles that serves as an antigenic marker for nervous and endocrine systems in mammals. Monoclonal antisera generated against synaptophysin were used for immunocytochemical staining in tissues of the tentacles of the sea anemone Condylactis gigantea (Cnidaria: Anthozoa). Specific staining, visible at the light and electron microscope levels, was found in the tentacle. Proteins were extracted from the tissues and solubilized. Using SDS-polyacrylamide gel electrophoresis and Western blotting, we identified proteins with apparent molecular weights of 38,000, 78,000, and 114,000. The data suggest the tissues of this anthozoan contain synaptophysin-like proteins with molecular properties similar to those of mammalian neurons.

Neurofilament-like immunoreactivity in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa).

The neuronal cytoskeleton contains neurofilament proteins that serve as markers for nervous tissue in many species across phyla. Antiserum generated to mammalian neurofilaments was used for immunocytochemical staining of tissues in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa). Specific staining, visible at the light and electron microscope levels, was found in the tissues of the tentacle. Proteins were extracted from the tissues and solubilized. SDS-polyacrylamide gel electrophoresis and Western blotting revealed two bands of MWr 156 kD and 74 kD that reacted with antiserum generated to neurofilaments. The protein bands also bound a monoclonal antibody shown to react with a highly conserved epitope in many classes of intermediate filaments. These data suggest that the neurons of this anthozoan contain neurofilament-like proteins with molecular properties similar to those of mammalian neurons.

Investigations of fetal development models for prenatal drug exposure and schizophrenia. Prenatal d-amphetamine effects upon early and late juvenile behavior in the rat.

Recent evidence suggests that mid-pregnancy is a critical period for production of fetal abnormalities that cause behavioral and neuropathological changes in adult offspring. The present experiments provide an animal model of these effects by treating pregnant Sprague-Dawley rats during gestational days 11-14 with d-amphetamine (AM). Offspring were tested for neurological signs, foraging activity, reversal learning, and sensitivity to amphetamine challenge. In the Early Juvenile period, postnatal days (PND) 20-30, female AM offspring initially showed reductions in rearing, holepoking, and midfield activity. On later trials, and as young adults, AM females showed signs of locomotor hyperactivity despite continued poor foraging efficiency, and were also more sensitive to a 1.0 mg/kg d-amphetamine challenge. AM males showed initially slower and more perseverative responding than controls, but then developed excessive response switching. These changes continued during tests for Retention, Reversal, and Extinction in the Late Juvenile/Early Adult stage (PND 50-90), when both AM-exposed sexes showed increased eating time, significantly more perseverative lateral turning preference (right or left), and slower reversal learning than controls. Behavioral data were consistent with aberrations in thalamo-frontal and mesolimbic/nigrostriatal projection systems that have been reported in AM animals and which are also affected by maternal drug abuse and schizophrenia.

The relationship of sweat gland count to electrodermal activity.

This study assessed whether greater skin conductance activity at the distal versus medial site (Scerbo, Freedman, Raine, Dawson, & Venables, 1992) is attributable to a greater number of active (open) sweat glands at the distal site. The number of sweat glands was measured using the Palmar Sweat Index (PSI). Twenty-four subjects were exposed to 10 auditory stimuli. Electrodes were placed on the fore and middle fingers of each hand, using distal sites on one hand and medial sites on the other. The PSI was measured at the medial and distal phalanges adjacent to the electrode placement sites. The distal site contained more open and total sweat glands. Open gland count had the strongest correlations with skin conductance. Multivariate analyses of covariance revealed that site effects for nonspecific and orienting response frequency and trials to habituation were associated with site differences in open glands. Skin conductance measures and the PSI reveal greater electrodermal activity at the distal site. In addition, the number of open glands may be a useful measure related to electrodermal response frequency when polygraph measurement is unavailable.

Purification and characterization of iotrochotin, a novel toxin from the Caribbean sponge Iotrochota birotulata, which selectively permeabilizes synaptosomes.

A protein termed iotrochotin (IOT) was isolated from the exudate of the Caribbean sponge Iotrochota birotulata using as an assay its stimulation of the release of radioactivity from synaptosomes preloaded with [3H]choline. Sephadex G-50 chromatography of the exudate produced one peak, with a mol. wt of approximately 18,000, which was further resolved into two active fractions by anion exchange chromatography. The more tightly bound of the two fractions was characterized and referred to as IOT. The action of IOT was essentially complete by 0.5-1.0 min and was independent of the Ca2+ or Na+ content of the incubation mixture. Released radioactivity included about 50% each of [3H]acetylcholine and [3H]choline. Release of radioactivity increased as a function of IOT concentration and then reached a maximum. Extrapolated asymptotic release was nearly equal to that obtained by lysing the synaptosomes. IOT also released radioactivity from synaptosomes which had been preincubated with other tritiated neurotransmitters or with 2-[3H]deoxy-D-glucose. Lactate dehydrogenase and choline acetyltransferase activities were not released from synaptosomes by treatment with IOT, but were released by digitonin. IOT therefore releases some of the smaller molecular weight components of synaptosomes, but does not permeabilize the synaptosomal membrane in the same way as digitonin treatment.

Effect of duration of haloperidol treatment on DA receptor supersensitization in aging C57BL/6J mice.

Apomorphine-induced behavior, striatal [3H]spiperone binding, and striatal choline acetyltransferase (ChAT) activity were assessed in 6 1/2, 13, and 27-30 month-old male C57BL/6J mice following 0, 30, 60 or 90 days treatment with the dopaminergic (DA) antagonist haloperidol. Both apomorphine-induced behavior and [3H]spiperone binding (Bmax) increased linearly with duration of haloperidol treatment, with no detectable age difference in the degree of supersensitization, although basal receptor density declined with age. Middle- and old-aged mice showed prolonged stereotypic behavior relative to young mice, suggesting slower apomorphine clearance. No differences in ChAT activity were detected with either age or duration of haloperidol treatment. Although the group means of binding and behavior were highly related, the within group correlations were poor. Overall, the results suggest that aged animals are capable of DA receptor supersensitization when given a sufficient stimulus--in this case, relatively long treatment regimes. Previously reported deficits in neuroleptic-induced supersensitization in old mice may be confined to relatively short treatment periods at low doses.

Purification and characterization of beta-leptinotarsin-h, an activator of presynaptic calcium channels.

A new neuroactive protein, beta-leptinotarsin-h, has been purified to near-homogeneity from the hemolymph of the beetle Leptinotarsa haldemani by column chromatography. beta-Leptinotarsin-h has a molecular weight of 57 000. Rat brain synaptosomes incubated with appropriate radioactive precursors release acetylcholine (ACh), norepinephrine, and 4-aminobutyrate when exposed to beta-leptinotarin-h, but do not release lactate dehydrogenase. Release of ACh has been examined in some detail. Release of ACh varies with the concentration of beta-leptinotarsin-h in a rectangular hyperbolic fashion. Half-maximal release is stimulated by a concentration of 50 ng/mL. Altering the ionic composition of the bathing solution affects the release in a manner which suggests that neither Na+ channels nor K+ channels are affected by beta-leptinotarsin-h but that the beta-leptinotarsin-h acts to increase permeability to Ca2+. Varying the concentration of Ba2+, Sr2+, Co2+, and Cd2+ indicates that beta-leptinotarsin-h acts to open the voltage-sensitive presynaptic Ca2+ channel. beta-Leptinotarsin-h may be a useful tool for studying the Ca2+ channel associated with the release of neurotransmitters.

Effect of liposomes containing various divalent cations on the release of acetylcholine from synaptosomes.

The effect of increasing the cytoplasmic levels of various divalent cations on the release of [3H]acetylcholine ([3H]ACh) from synaptosomes was investigated. Synaptosomes prepared from rat brain and prelabeled with [3H]choline were incubated with liposomes containing Mg2+, Ca2+, Mn2+, Co2+, Sr2+, or Ba2+. This treatment allows the transfer of the aqueous contents of the liposomes to the cytoplasm of the synaptosomes. The efflux of radioactivity subsequent to this treatment was measured, and the relative proportions of [3H]ACh and [3H]choline were determined. The release of radioactivity from synaptosomes incubated with liposomes containing Mg2+, Mn2+, or Co2+ was not altered when compared with synaptosomes incubated either without liposomes or with liposomes containing isotonic K+/Na+. Synaptosomes incubated with liposomes containing Ca2+, Sr2+, or Ba2+, however, released significantly more radioactivity than did controls. Moreover, the released radioactivity consisted almost entirely of [3H]ACh. Liposomes containing either Ca2+ or Sr2+ were equally effective in promoting the release of [3H]ACh from synaptosomes, whereas liposomes containing Ba2+ were 2.5 times more effective in promoting the release of [3H]ACh than were liposomes containing either Ca2+ or Sr2+. Since liposomes introduce their aqueous contents into cytoplasm via a mechanism not involving plasma membrane channels, the increased release of [3H]ACh caused by liposomes containing Ca2+, Sr2+, or Ba2+ is attributable to an increase in the intrasynaptosomal concentration of these ions, and not to their passage through calcium channels.

Inhibition by styrylpyridines of purified rat and bovine brain choline acetyltransferase.

Nine styrylpyridine analogs were tested as inhibitors of choline acetyltransferase which had been highly purified from rat cerebrum and bovine caudate nuclei. In general, concentrations required to achieve 50% inhibition (I50 values) were in the micromolar range. For some analogs, I50 values were similar to those obtained previously by other investigators who used less purified enzyme preparations. With certain analogs, however, the measured values of I50 changed as the transferase became more purified, which may indicate the presence in the extract of other molecules which can interact with the enzyme. The methods used in purification of the enzyme suggest that the molecule which modifies the activity of CAT is probably a protein. The mode of inhibition by naphthylvinylpyridinium was found to be uncompetitive with respect to both choline and acetyl coenzyme A for both the rat and bovine transferases.

Fast axonal transport in the presence of high Ca2+: evidence that microtubules are not required.

Microtubules have long been associated with the mechanism of fast axoplasmic transport, although experimental evidence to support an involvement has been equivocal. Electron microscopic studies demonstrated that incubation of the axons of excised rat sciatic nerves in media containing 75 mM Ca2+ caused complete loss of microtubules within 6 hr. To evaluate the role of microtubules in fast anterograde transport, studies of transport in nerves exposed to these conditions were undertaken. Prior to measurement of axoplasmic transport, nerves ligated distal to the dorsal root ganglia were preincubated in vitro in 75 mM Ca2+ for 0-6 hr. Fast axonal transport was subsequently monitored by measuring the amount of trichloroacetic acid-insoluble radioactivity that accumulated at the ligature after incubation for 12-18 hr with L-[3H]proline. Nerves in which microtubules had been depolymerized by preincubation in high Ca2+ maintained control levels of transport. We conclude that intact microtubules are not required for fast anterograde axoplasmic transport.

Leptinotarsin: a presynaptic neurotoxin that stimulates release of acetylcholine.

Leptinotarsin, a toxin found in the hemolymph of the beetle Leptinotarsa haldemani, can stimulate release of acetylcholine from synaptic termini. Leptinotarsin causes an increase in the frequency of miniature end plate potentials (mepps) of the rat phrenic nerve-diaphragm preparation. The increase in the frequency of mepps induced by leptinotarsin is biphasic: about 10% of the total mepps are released in an initial burst that lasts about 90 sec, after which the remaining mepps are released over a period of 10-20 min. Tetrodotoxin has no effect upon the release induced by leptinotarsin, but low-Ca(2+) conditions abolish the first phase. The two phases of release may represent two presynaptic pools of acetylcholine, both of which can be released in quantized form. In a second study, rat brain synaptosomes were incubated with [(3)H]choline and were immobilized on Millipore filters. Leptinotarsin induced release of [(3)H]acetylcholine from this preparation, confirming the release seen by using neurophysiological methods. The ability of leptinotarsin to induce release from either intact nerve terminals or synaptosomes was abolished when the toxin was heated. The releasing activity of leptinotarsin from synaptosomes was also partially dependent upon the presence of Ca(2+) in the perfusing solution. Release from synaptosomes followed first-order kinetics, and was not inhibited by commercial antibodies to black widow spider antigens. The data suggest that leptinotarsin acts as a presynaptic neurotoxin and may be of value as a mechanistic probe in understanding the storage and release of neurotransmitters.

The release of acetylcholine elicited by extracts of black widow spider glands: studies using rat superior cervical ganglia and inhibitors of electrically stimulated release.

An extract of the venom glands of black widow spiders (BWGE) induces the release of acetylcholine (ACh) from the superior cervical ganglia of rats. The release of ACh follows first-order kinetics, which suggests that the venom either lowers the ganglionic store of ACh, or continually reduces the rate of release. Since ganglionic ACh did not decrease in the presence of BWGE, it is likely that the venom continually reduces the rate of release. The rate constant for BWGE-induced release of ACh is depressed about 45% by treatment of the ganglion with either botulinum toxin or a low Ca++/high Mg++Ringer's solution. The rate constant is depressed about 30% by treatment of the ganglion with 8.3 microng/ml of cytochalasin-B. Since these agents inhibit the release of ACh which is elicited by electrical stimulation of the ganglion, it is suggested that one action of BWGE is the stimulation of some step in the physiological mechanism involved in the release of neurotransmitters. Treatment of the ganglion with tetrodotoxin had no effect on the rate constant for release induced by BWGE. The action of BWGE on the ganglion was not reversible after 10 minutes. When treated with BWGE, ganglia whose stores of transmitter had been labeled by electrical stimulation in the presence of [3H]choline released ACh having uniform specific activity. The data suggest the presence of more than one activity in the extracts.

A comparison of rapidly transported proteins in the sensory fibers of the rat and mouse sciatic nerve.

Fast axoplasmic transport through the sensory fibers of the sciatic nerve has been compared in rats and mice. The use of in vitro incubation permits high levels of specific activity to be attained when labeling with [(35)S]L-methionine. The specific activity of the transported proteins was about 10-fold greater in mice than in rats. Proteins labeled with radioactive methionine were examined after separation on polyacrylamide gels. There are no differences between mice and rats when the proteins carried by rapid transport are compared. Similarly, the proteins synthesized by the Schwann cells of these two species are not distinguishable. The dorsal root ganglia of mice, however, yield a band of radioactivity that is not seen in ganglia from rats. This band migrates with an apparent molecular weight of 31,000 daltons.