Neuromodulation of rhythmic motor patterns in the blue crab Callinectes sapidus by amines and the peptide proctolin

The blue crab Callinectes sapidus, provides an opportunity to study neuromodulation of three variations of rhythmic behavior produced by the same appendages. These behaviors are sideways swimming, backward swimming, and courtship display (CD). Each behavior has a different context, and despite similarities among them, each is quantifiably distinguishable. CD behavior occurs in males, is stimulated naturally by pheromone, and elements of the behavior are evoked by proctolin and dopamine. Sideways and backward swimming do not share these characteristics. Bath-applied proctolin, combined with either electrical or pheromonal stimulation, was used to search for interneurons influencing motor outflows from the fifth legs. Interneurons were found which, when stimulated electrically, initiated rhythmic behavior. At least one of these neurons responded to pheromonal stimulation. Application of proctolin combined with stimulation of descending trigger cells resulted in changes from a backward swimming motor pattern toward a CD pattern. Dopamine applied with proctolin lowered the concentration threshold for proctolin-evoked changes in motor outflows. Octopamine co-applied with proctolin extinguished the proctolin effect unless dopamine was co-applied. Combinations of modulators appear to play critical roles in shaping patterns of rhythmic motor activity of the fifth legs.Abbreviations CD courtship display - DA dopamine - OA octopamine - PROC proctolin - EMG electromyogram - OC oesophageal connective - VNC ventral nerve cord - PIR proctolin-like immunoreactivity - SNK Student-Neuman-Keuls - PO pericardial organ     

Coordination and neuromuscular control of rhythmic behaviors in the blue crab,Callinectes sapidus

The stereotypical courtship display (CD) behavior of the male blue crab, Callinectes sapidus, includes an unusual component: the rhythmic waving of the swimming appendages above the carapace. This behavior occurs in a unique context but it resembles two other rhythmic behaviors performed using the swimming legs: sideways swimming and backward swimming. As a first step to understanding the mechanisms that allow the expression of apparently different rhythmic motor patterns, we have examined these behaviors using slow motion video analysis and electromyography of the basal muscles of the swimming legs in freely behaving crabs. The results show that these behaviors are distinguished by four parameters: the frequency of leg waving, the phase relationship between the legs, the presence of a stationary pause in basal muscle activity combined with rotation of the distal leg during CD, and an extended range of motion of these legs during CD and backward swimming, relative to sideways swimming. EMG analysis revealed that during sideways swimming, the sequence of muscular activity between the two legs was different. In contrast, during CD and backward swimming the sequence of activity for these legs is identical.Abbreviations CD courtship display - EMGs electromyograms - CD AMP courtship display in crabs with amputated fifth legs - CD1 crabs that voluntarily used one leg to perform courtship display waving - CD 1–3 courtship waving in cycles 1–3 - CD MID courtship waving after cycles 1–3 - M-C meral-carpal joint     

Neuromuscular modulation in Limulus by both octopamine and proctolin

Both octopamine and proctolin potentiate nerve-evoked skeletal muscle contractions in the horseshoe crab, Limulus. The threshold concentration for octopamine was 10^?9 to 10^?8M, while for proctolin it was 3 × 10^?9M. Norepinephrine and dopamine produced effects similar to octopamine but at higher thresholds; tyramine and serotonin were ineffective. Octopamine caused significant increases in amplitudes of excitatory postsynaptic potentials (epsps) of muscle fibers, but had little effect on muscle fiber input resistance or membrane potential. Also, octopamine did not affect depolarization of muscle fibers and subsequent contraction due to the direct action of exogenously applied glutamate. These results suggest that octopamine potentiates nerve-evoked contractions primarily by facilitating release of neuromuscular transmitter. At concentrations above 10^?7M, however, octopamine sometimes caused muscle spikes in response to motoneuron stimulation, a finding that suggests that octopamine may also have some postsynaptic action. Proctolin potentiated the muscle contractions evoked by glutamate but had little effect on glutamate-evoked muscle fiber depolarization, muscle fiber input resistance, or membrane potential. Thus, proctolin appears to act directly on skeletal muscle to enhance contractility. The proctolin-induced potentiations of contraction were sometimes accompanied by modest increases in epsp amplitude, so that unlike lobster skeletal and Limulus cardiac neuromuscular preparations, proctolin may have a secondary direct synaptic effect. Both octopamine and proctolin have been found in Limulus cardiac ganglion. This potential access to the hemolymph and the relatively low threshold concentrations needed for physiological action suggest that octopamine and proctolin could function as hormonal modulators of neuromuscular function in Limulus.     

Aminergic modulation of the myogenic heart in the branchiopod crustacean Triops longicaudatus

Although crustaceans typically have a neurogenic heart, the primitive crustacean Triops longicaudatus has a myogenic heart with the heartbeat arising from the endogenous rhythmic activity of the myocardium. In the present investigation, the effects of six biogenic amines, epinephrine, norepinephrine, dopamine, octopamine, serotonin and histamine, on the myogenic heart of T. longicaudatus were examined. Epinephrine, norepinephrine, dopamine and octopamine accelerated the heartbeat, increasing both the frequency and amplitude of the action potential of the myocardium in a concentration dependent manner. The ability of epinephrine and norepinephrine to produce the acceleratory effects was more potent than that of dopamine and octopamine; the threshold concentrations of epinephrine and norepinephrine were approximately 10(-10) M and those of dopamine and octopamine approximately 10(-7) M. Serotonin weakly inhibited the heartbeat, decreasing both the frequency and amplitude of the myocardial action potential in a concentration dependent manner with a threshold concentration of approximately 10(-6) M. Histamine exhibited no effect on the heartbeat. The results provide the first evidence for direct effects of amines on the crustacean myocardium and suggest neurohormonal regulation of the myogenic heart in T. longicaudatus.     

Elevated temperature alters the ionic dependence of amine-induced pacemaker activity in a conditional burster neuron

Summary The anterior burster neuron of the lobster (Panulirus interruptus) stomatogastric ganglion is a conditional burster that functions as the primary pacemaker for the pyloric motor network. When modulatory inputs to this cell are blocked, it loses its bursting properties and becomes quiescent. Applications of the monoamines, dopamine, octopamine or serotonin restore rhythmic bursting in this cell (Flamm and Harris-Warrick 1986). At 15 °C, serotonin- and octopamine-induced oscillations depend critically upon sodium entry (blocked by low sodium saline or tetrodotoxin); dopamine-induced oscillations depend upon calcium entry (blocked by reduced extracellular calcium; Harris-Warrick and Flamm 1987). We show here that the ionic dependence of amine-induced oscillations in the anterior burster cell differs at 15 and 21 °C. At 21 °C, all amines have the potential to induce rhythmic oscillations in saline containing tetrodotoxin. At the elevated temperature and in tetrodotoxin, both calcium and sodium currents are essential for the maintenance of dopamine-induced oscillaions; serotonin-induced oscillations do not depend upon either calcium or sodium alone; octopamine-induced oscillations do not depend upon calcium and show a variable dependence upon sodium. Thus, multiple ionic mechanisms, which vary with both the modulator and the ambient temperature, can be recruited to support rhythmic activity in a conditional burster neuron.Abbreviations AB anterior burster - PD pyloric dilator - PY pyloric constrictor - DA dopamine - 5HT serotonin - Oct octopamine - STG stomatogastric ganglion - TTX tetrodotoxin - GSP graded synaptic potential     

Modulation of swimming in Tritonia: excitatory and inhibitory effects of serotonin

1. In the mollusc Tritonia escape swimming is produced by a network of central pattern generator (CPG) neurons. The purpose of this study was to determine which neurotransmitters might be involved in the swim system.
2. Injection of serotonin (5HT) into whole animals elicited swimming followed by a long-lasting inhibition of swimming. In isolated brain preparations, bath-applied 5HT elicited a swim pattern at short latency and also caused a long-lasting inhibition of the swim pattern. The activation of swimming by 5HT was associated with a tonic depolarization of cerebral cell 2 (C2) and the dorsal swim interneurons (DSI) which form part of the swim CPG network.
3. In isolated brain preparations, bath applied glycine, histamine, proctolin, and FMFRamide had no effect on the swim motor pattern elicited by electrical stimulation of a peripheral nerve. Aspartate, carbacol, dopamine, glutamate, octopamine, pilocarpine, and small cardioactive peptide-B (SCP_B) inhibited the activation of swimming by nerve stimulation.
4. The 5HT antagonists cyproheptidine, tryptamine, and 7-methyltryptamine had no effect on swimming, but methysergide and fenfluramine inhibited swimming to both normal sensory stimuli and exogenously applied 5HT.
5. Staining with a polyclonal antibody indicated that one class of CPG neurons, the dorsal swim interneurons (DSI), was immunoreactive for 5HT.
6. Taken together, the data suggest that pattern generator interneurons, particularly the DSIs, use 5HT as a neurotransmitter.

     

Neurochemical Bases of Tremor and Other Disorders of Movement

Monkeys inflicted with specific unilateral electrolytic lesions in the upper brain stem developed extrapyramidal disorders. Hypokinesia of the limbs was associated contralaterally with a lesion of the substantia nigra and depletion of striatal dopamine. Choreiform movements were observed in animals that had, contralaterally, a lesion severing the most dorsomedial fibres of the cerebral peduncle and the rubro-tegmentospinal tract, associated with depletion of striatal serotonin. Monkeys showing sustained postural tremor and hypokinesia had lesions affecting these three tracts contralaterally and loss of striatal dopamine and serotonin on the lesion side. Of many drugs tested, only harmaline and harmine affected the dyskinesias. The nigrostriatal fibres appear to be dopaminergic; the cerebral peduncular (dorsomedial) fibres, serotoninergic. The role of striatal dopamine and serotonin in the control of normal movements and posture of the limbs represents the first directly demonstrated function of these amines in the central nervous system.     

The effect of neuropeptides from Limulus on its circadian rhythm in retinal sensitivity

Retinal responses of the Limulus lateral eyes to light are greater at night than during the day. A circadian clock in the brain of the horseshoe crab controls these rhythmic changes of light sensitivity. The increase in sensitivity (as measured by the amplitude of the electroretinogram) is mediated at least in part by octopamine that is released from efferent axons terminating in the visual cells. Earlier studies indicate that certain factors in Limulus hemolymph can act in conjunction with octopamine. More recently, five neuropeptides (LP1-LP4 and Lip-HP) had been isolated from acetone extracts of the Limulus central nervous system using HPLC fractionation and radioimmunoassay with antisera against FMRFamide-like peptides for detection. Presently, we have injected into the Limulus lateral eye these five peptides and observed changes in retinal sensitivity. Injection during daytime had no immediate effect on that daytime electroretinogram but decreased the electroretinogram amplitude for the entire subsequent night (12 h). However, upon injection at night, we observed an immediate but only transitory decrease in electroretinogram amplitude for about 1 h without effect on the subsequent daytime electroretinogram. We suggest that the peptides act antagonistically to octopamine and are highly dependent upon the activity state of the efferent nerve terminals. Accepted: 25 August 1996     

Behavioral and cellular effects of serotonin on locomotion and male mating posture in Ascaris suum (Nematoda)

The site and mode of action of serotonin on locomotion were investigated in the parasitic nematode Ascaris suum. Injection of serotonin into Ascaris immediately caused paralysis in animals that were generating locomotory waveforms. Injected serotonin also increased body length and decreased the number of propagating body waves. Similar injections into the male tail produced a ventral tail curl. Injection of N-acetyl-serotonin had no effect on the generation of locomotory waveforms, but increased the body length and decreased the number of body waves in the waveform. Other biogenic amines were also tested but were much less potent.Serotonin decreased the amplitude of a submaximal acetylcholine-induced muscle contraction and increased the time to attain this contraction. The time course of this effect on the response to ACh was much slower than the action of injected serotonin on locomotory waveforms, suggesting that additional elements are involved in the action of serotonin on locomotory behavior.Serotonin abolished spontaneous slow potentials in VI motor neurons and decreased the frequency of EPSPs in DE2 motor neurons, probably by a pre-synaptic mechanism. In the male tail, serotonin depolarized the male-specific transverse ventral muscle cells, but did not affect either dorsal or ventral longitudinal muscle cells.Abbreviations ACh acetylcholine - cAMP cyclic adenosine monophosphate - DA dopamine - DE dorsal excitatory motor neuron - DI dorsal inhibitory motor neuron - DM dorsal muscle - ELP egg-laying pore - EPSP excitatory post synaptic potential - GABA gamma aminobutyric acid - HRB head restricted behavior - IPSP inhibitory post synaptic potential - 5-HT 5-hydroxytryptamine, serotonin - NA-5-HT N-acetyl-5-hydroxytryptamine - NE norepinephrine - OA octopamine - PBS phosphate buffered saline - PCF pseudocoelomic fluid - tVM malespecific transverse ventral muscle - TRYP tryptamine - VI ventral inhibitory motor neuron     

Modulation of Drosophila heartbeat by neurotransmitters

The heart of Drosophila melanogaster is a simple muscular tube with a posterior pulsatile portion and a thoracic-cranial vessel. The pacemaker, located caudally, is myogenic. Its rate of firing is modulated by neurotransmitters. Serotonin, octopamine, norepineph-rine, dopamine, and acetylcholine accelerate the heart, in that order of potency. Dihydroxyphenylalanine, γ-aminobutyric acid, glutamate, and glycine have no effect. Generally, the regularity of the heartbeat is not adversely affected by treatment with any of these neurotransmitters. We show here that amnesiac, a neurological mutation, and Dihydroxyphenylalanine decarboxylase ^{temperature sensitive}, a mutation that interferes with synthesis of dopamine, norepinephrine, and serotonin, result in slower heart rate and reduced regularity across a normal range of temperatures for these flies. Dopamine-N-acetyltransferase, which is on the catabolic route to dopamine, serotonin, and octopamine, has no effect. hypoactiveC reduces the rate of the heart, but its mechanism of action is unknown. Accepted: 5 August 1996     

Are females of Ilyoplax pusilla (Brachyura: Dotillidae) attracted to groups having more waving males?

Males of the dotillid crab Ilyoplax pusilla wave at approaching females during the breeding season. They also, however, often perform waving that is not directed toward any particular individual. This undirected waving is associated with the presence of male neighbors and may function in male–male competition. It may also, however, act as a long-range female attractant. To test whether undirected waving functions to attract females, we conducted a field experiment that manipulated the abundance of waving males. We found that females preferred to approach groups that had more waving males. This suggests that undirected waving by male I. pusilla functions as a long-range courtship signal.     

Modulatory effects of proctolin on a crab ventilatory muscle

Proctolin enhances nerve-evoked, phasic contractions of a selected respiratory muscle of the shore crab, Carcinus maenas, but has no effect on muscle tonus. Proctolin also increases the work and power output of this muscle. These effects are functionally appropriate in view of previous reports that proctolin stimulates the ventilatory rhythm. They also suggest that proctolin exerts coordinated modulatory control at the central and peripheral levels of the gill ventilatory system. In contrast, serotonin, dopamine and octopamine have no effect on this muscle.     

Putative neurohemal areas in the peripheral nervous system of an insect,Gryllus bimaculatus,revealed by immunocytochemistry

The morphology and position of putative neurohemal areas in the peripheral nervous system (ventral nerve cord and retrocerebral complex) of the cricket Gryllus bimaculatus are described. By using antisera to the amines dopamine, histamine, octopamine, and serotonin, and the neuropeptides crustacean cardioactive peptide, FMRFamide, leucokinin 1, and proctolin, an extensive system of varicose fibers has been detected throughout the nerves of all neuromeres, except for nerve 2 of the prothoracic ganglion. Immunoreactive varicose fibers occur mainly in a superficial position at the neurilemma, indicating neurosecretory storage and release of neuroactive compounds. The varicose fibers are projections from central or peripheral neurons that may extend over more than one segment. The peripheral fiber varicosities show segment-specific arrangements for each of the substances investigated. Immunoreactivity to histamine and octopamine is mainly found in the nerves of abdominal segments, whereas serotonin immunoreactivity is concentrated in subesophageal and terminal ganglion nerves. Immunoreactivity to FMRFamide and crustacean cardioactive peptide is widespread throughout all segments. Structures immunoreactive to leucokinin 1 are present in abdominal nerves, and proctolin immunostaining is found in the terminal ganglion and thoracic nerves. Codistribution of peripheral varicose fiber plexuses is regularly seen for amines and peptides, whereas the colocalization of substances in neurons has not been detected for any of the neuroactive compounds investigated. The varicose fiber system is regarded as complementary to the classical neurohemal organs.     

A Spatially Explicit Model of Synchronization in Fiddler Crab Waving Displays

Fiddler crabs (Uca spp., Decapoda: Ocypodidae) are commonly found forming large aggregations in intertidal zones, where they perform rhythmic waving displays with their greatly enlarged claws. While performing these displays, fiddler crabs often synchronize their behavior with neighboring males, forming the only known synchronized visual courtship displays involving reflected light and moving body parts. Despite being one of the most conspicuous aspects of fiddler crab behavior, little is known about the mechanisms underlying synchronization of male displays. In this study we develop a spatially explicit model of fiddler crab waving displays using coupled logistic map equations. We explored two alternative models in which males either direct their attention at random angles or preferentially toward neighbors. Our results indicate that synchronization is possible over a fairly large region of parameter space. Moreover, our model was capable of generating local synchronization neighborhoods, as commonly observed in fiddler crabs under natural conditions.     

Biogenic amines, caffeine and tonic immobility in Tribolium castaneum

Biogenic amines are physiologically neuroactive substances that affect behavioural and physiological traits in invertebrates. In the present study, the effects of dopamine, octopamine, tyramine and serotonin on tonic immobility, or death-feigning, were investigated in Tribolium castaneum. These amines were injected into the abdomens of beetles artificially selected for long or short duration of tonic immobility. In beetles of the long strains, the durations of tonic immobility were shortened by injection of dopamine, octopamine and tyramine, and the effects of these amines were dose-dependent. On the other hand, serotonin injection did not affect the duration of tonic immobility. In the short-strain beetles that rarely feign death, no significant effects of the amines were found on the duration of tonic immobility. Brain expression levels of octopamine, tyramine and serotonin did not differ between long- and short-strain beetles, in contrast to the higher dopamine levels in short strains previously reported. Caffeine decreased the duration of death-feigning in both oral absorption and injection experiments. It is known that caffeine activates dopamine. Therefore, the present results suggest that the duration of tonic immobility is affected by dopamine via the dopamine receptor in T. castaneum.     

Primary afferent responses of a crustacean mechanoreceptor are modulated by proctolin, octopamine, and serotonin

Modulation of sensory responses recorded intracellularly in primary sensory afferents of a crustacean proprioceptor is described. The neuropeptide proctolin enhances the sensory response, whereas the bioamines octopamine and serotonin depress it. The lobster oval organ of the second maxilla, a simple stretch receptor lacking centrifugal control, provides a useful model for studies on nonsynaptic modulation at peripheral sensory loci. Its three large afferents, X, Y, and Z, were prepared for intracellular recording and tested under five experimental conditions: (1) when fully rested, (2) when adapted to maintained stretch and firing tonically, (3) when showing reduced responses after habituation to repetitive stimulation, (4) not stretched but depolarized with current injections, (5) after TTX blockade. The results, taken together, indicate that conductances contributing to the overall amplitude of the receptor potential are major targets for modulators. Thus proctolin increased receptor potential amplitudes with consequent augmentation of spiking, whereas serotonin and octopamine depressed the receptor potentials, often to subthreshold levels with loss of spiking. Octopamine was a less potent agent than serotonin and failed to act upon fibers under TTX blockade. Fibers Y and Z consistently showed sensitivity to the modulators tested. The largest fiber, X, typically was resistant to proctolin, octopamine, and serotonin. Threshold concentrations of 10(-10)-10(-11) M determined in vitro are well below the circulating levels for serotonin and octopamine found in vivo. Proctolin, however, is usually not detectable in the hemolymph, and it is suggested that a significant site of proctolin release may be the oval organ itself.     

Responses of Heterodera glycines and Meloidogyne incognita to exogenously applied neuromodulators

Biogenic amines regulate important behaviours in nematodes and are associated with pharyngeal activity in plant-parasitic nematodes. A robust behavioural assay based upon nematode body movements was developed to expand the study of these and other neuroregulators in plant-parasitic nematodes. Dopamine, octopamine and serotonin each had significant but differing effects on the behaviour of soybean cyst nematode Heterodera glycines and root-knot nematode Meloidogyne incognita juveniles. Body movement frequency was increased twofold in H. glycines by 5 mM dopamine (P = 0.0001), but decreased by 50 mM dopamine in H. glycines (88%) and M. incognita (53%) (P < 0.0001). Movement frequency in both species was increased by 50-70% (P < 0.0001) by 50 mM octopamine, and 5 mM octopamine increased M. incognita movement frequency more than twofold (P < 0.0001). Movement frequency in each species was reduced by more than 90% by 5 mM serotonin (P < 0.0001). While amplitude of body movement in H. glycines was unaffected by any amine, it was significantly reduced in M. incognita by all amines (P < 0.0006). Stylet pulsing frequencies in either species were unaffected by dopamine or octopamine, but 5 mM serotonin stimulated pulsing in H. glycines by nearly 13-fold (P < 0.0001) and in M. incognita by more than 14-fold (P < 0.0001). The invertebrate neuropeptide FLRFamide (N-Phe-Leu-Arg-Phe) increased M. incognita body movement frequency 45% (P = 0.02) at 1 mM but did not affect stylet activity. Finally, H. glycines egg hatch was completely suppressed by 50 mM serotonin, and partially suppressed by 50 mM dopamine (75%; P < 0.0001) and 50 mM octopamine (55%; P < 0.0001).     

Biogenic amines in cultured neuroblastoma and astrocytoma cells

The presence of biogenic amines in cultured cells of mouse neuroblastoma C-1300 (clone NB-2a) was suggested by fluorescence-microscope histochemistry. Incubation in media containing L-[¹⁴C]tyrosine and L-[¹⁴C]tryptophan for 24 h, followed by high-voltage electrophoresis, radiochromatogram scanning, and scintillation counting, confirmed the presence of [¹⁴C]dopamine, [¹⁴C]norepinephrine, [¹⁴C]epinephrine, [¹⁴C]serotonin, [¹⁴C]tyramine, and [¹⁴C]octopamine. Dopamine, norepinephrine, epinephrine, and serotonin were demonstrated spectrophotofluorometrically in concentrations, expressed as micrograms amine per milligram protein, of 1.19, 0.027, 0.038, and 0.148, respectively, for cells in a stationary growth phase. Fluorescence-microscope histochemistry also suggested the presence of biogenic amines in cultured astrocytoma cells (cell line C6). Spectrophotofluorometric assay of cells in a stationary growth phase demonstrated intracellular dopamine, norepinephrine, epinephrine, and serotonin in concentrations significantly lower than those of neuroblastoma cells.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.