Localization and quantification of 5-hydroxytryptophan and serotonin in the central nervous systems of Tritonia and Aplysia.

Serotonin (5-hydroxytryptamine, 5-HT) plays a central role in several behaviors in marine molluscs and other species. In an effort to better understand the regulation of 5-HT synthesis, we used high performance liquid chromatography (HPLC) with electrochemical detection and immunohistochemistry to measure and map the distribution of the immediate precursor of 5-HT, 5-hydroxytryptophan (5-HTP), in two model opisthobranch molluscs, the nudibranch Tritonia diomedea and the anaspid Aplysia californica. HPLC measurements showed that 5-HTP is present at approximately the same level as the 5-HT metabolite, 5-hydroxyindolacetic acid (5-HIAA) but is more than 100 times lower in concentration than either 5-HT or dopamine in the same tissue. Specific 5-HTP immunoreactivity was colocalized with serotonin in both species. The overall intensity of 5-HTP immunoreactivity in individual ganglia agreed with HPLC measurements for those ganglia. The intensity of 5-HTP immunolabeling varied between cell types and was correlated with the intensity of 5-HT immunolabeling. In particular, differences in staining intensity were consistently seen among the three dorsal swim interneurons of the Tritonia swim central pattern generator circuit. Some nonserotonergic neurons also displayed low levels of 5-HTP immunolabeling that were above background levels. Together, these results support the notion that production of 5-HTP is a rate-limiting step in serotonin synthesis and suggest that there may be additional regulation that allows 5-HTP to accumulate to varying levels.

Paradoxical actions of the serotonin precursor 5-hydroxytryptophan on the activity of identified serotonergic neurons in a simple motor circuit.

Neurotransmitter synthesis is regulated by a variety of factors, yet the effect of altering transmitter content on the operation of neuronal circuits has been relatively unexplored. We used electrophysiological, electrochemical, and immunohistochemical techniques to investigate the effects of augmenting the serotonin (5-HT) content of identified serotonergic neurons embedded in a simple motor circuit. The dorsal swim interneurons (DSIs) are serotonergic neurons intrinsic to the central pattern generator (CPG) for swimming in the mollusc Tritonia diomedea. As expected, treatment with the serotonin precursor 5-hydroxytryptophan (5-HTP) increased the intensity of serotonin immunolabeling and enhanced the potency of synaptic and modulatory actions elicited by the DSIs. It also greatly enhanced the ability of the DSIs to evoke rhythmic CPG activity. After 5-HTP treatment, microvoltammetric measurements indicated an increase in a putative 5-HT electrochemical signal during swim CPG activation. Paradoxically, the spiking activity of the serotonergic neurons decreased to a single burst at the onset of the rhythmic motor program, whereas the overall duration of the episode remained about the same. 5-HTP treatment gradually reduced the rhythmicity of the CPG output. Thus, more serotonin did not result in a more robust swim motor program, suggesting that serotonin synthesis must be kept within certain limits for the circuit to function correctly and indicating that altering neurotransmitter synthesis can have serious consequences for the output of neural networks.

Upregulation of calcium homeostatic mechanisms in chronically depolarized rat myenteric neurons.

Perturbations of intracellular Ca2+ ion concentration ([Ca2+]i) have important effects on numerous neuronal processes and influence development and survival. Neuronal [Ca2+]i is, in large part, dependent on activity, and changes in activity levels can alter how neurons handle calcium (Ca). To investigate the ability of neuronal Ca homeostatic mechanisms to adapt to the persistent elevation of [Ca2+]i, we used optical and electrophysiological recording techniques to measure [Ca2+]i transients in neurons from the rat myenteric plexus that had been chronically depolarized by growth in culture medium containing elevated (25 mM) KCl. When studied in normal saline, neurons that had previously been chronically depolarized for 3-5 days had briefer action potentials than control neurons, their action potentials produced smaller, more rapidly decaying increases in [Ca2+]i, and voltage-clamp pulses with action potential waveforms evoked smaller Ca currents than in control neurons. Simultaneous voltage-clamp measurements and calcium imaging revealed that increases in the Ca handling capacities of the chronically depolarized neurons permitted them to limit the amplitudes of action potential-evoked [Ca2+]i transients and to restore [Ca2+]i to basal levels more rapidly than control neurons. Release of Ca from endoplasmic reticulum-based Ca stores made smaller contributions to action potential-evoked [Ca2+]i transients in chronically depolarized neurons even though those neurons had larger caffeine-releasable Ca stores. Endoplasmic reticulum-based Ca sequestration mechanisms appeared to contribute to the faster decay of [Ca2+]i transients in chronically depolarized neurons. These results demonstrate that when neurons experience prolonged perturbations of [Ca2+]i, they can adjust multiple components of their Ca homeostatic machinery. Appropriate utilization of this adaptive capability should help neurons resist potentially lethal metabolic and environmental insults.

Long-term regulation of neuronal calcium currents by prolonged changes of membrane potential.

Although rapid-onset, short-term regulation of neuronal Ca currents by neurotransmitters and second messengers is well documented, little is known about conditions that can cause longer-lasting changes in Ca channel function. We report here that persistent depolarization is accompanied by slowly developing long-term reduction of neuronal Ca currents. Rat myenteric neurons grown in cell culture for 1-7 d were studied with the tight-seal whole-cell recording technique. Macroscopic Ca-channel currents had decaying and sustained components at all days studied. When the neurons were grown in medium containing 25 mM KCl, which depolarized them to -40 mV and caused significant elevation of intracellular Ca, the densities of both components of Ca-channel current decreased by 40-80%. Several results suggest that different mechanisms underlie the downregulation of the two components. (1) The density of the decaying component decreased approximately four times faster than did that of the sustained component. (2) When neurons were returned to control medium, which contained 5 mM KCl, the density of the sustained component returned to control levels within 24 hr, while that of the decaying component did not recover significantly. (3) Inhibitors of RNA and protein synthesis reduced or prevented downregulation of the sustained but not of the decaying component. (4) The dihydropyridine antagonist nitrendipine, which prevented the sustained elevation of intracellular Ca in neurons grown in 25 mM KCl, prevented downregulation of the sustained component but had no effect on downregulation of the decaying component. We suggest that these forms of regulation of Ca current density could help neurons adapt to altered levels of electrical activity and may contribute to changes in synaptic strength that occur during periods of increased or decreased electrical activity.