The main aims of this paper are (a) to locate possible dopaminergic neurons in the eyestalk with anti-tyrosine hydroxylase antibodies, (b) to search for the presence of dopamine (DA) in the nervous structures of the eyestalk, (c) to explore its release, and (d) to test the effect of DA on neurosecretory cells in the eyestalk. Experiments were performed in adult crayfishes Procambarus clarkii, in isolated optic peduncle. Immunocytochemistry was made with the antibody against its precursor synthesizing enzyme tyrosine-hydroxylase. The content and release studies of DA were made using high performance liquid chromatography (HPLC). Extracellular and intracellular recordings were conducted with conventional recording techniques. A large number (approximately 2000) of immunopositive somata of different sizes and shapes were identified in various regions of the eyestalk. The majority of somata are of the smallest size (5-25 microm diameter). DA content in the eyestalk was 5.6 +/- 0.1 pmol per structure; the greatest content is in the MT (over 60%). A basal level release of DA was observed. Incubation of eyestalks in solution containing a high K+ concentration increased the DA release (79%). Two effects of DA on the excitability of X-organ neurons were observed; an excitatory effect on neurons of approximately 25 microm somata diameter and another inhibitory effect in the group of approximately 35-microm somata diameter neurons. The excitation occurs with a depolarization and decrement of membrane conductance in the cell soma while the inhibition occurs with a hyperpolarization and increment of membrane conductance in soma. We concluded the following: (1) Dopamine is present in each optic ganglia of the crayfish eyestalk. (2) There is a basal release of DA from the isolated eyestalk. (3) DA release is enhanced threefold by eyestalk incubation in 40 mM [K+] solution. (4) DA selectively excites a population of neurons with low-speed conduction axons, and small somata in the X-organ-sinus gland system, while inhibiting another population characterized by higher axonal conduction speed and large somata. (5) These observations support a role for DA as a neurotransmitter or neuromodulator in the X-organ neurons of the crayfish eyestalk.

The content and regional distribution of 5-hydroxytryptamine (5-HT) in the crayfish eyestalk was determined by high-performance liquid chromatography. Levels of the 5-HT precursors l-tryptophan (L-TRP) and 5-hydroxytryptophan (5-OH-TRP), and of three metabolites, 5-hydroxytryptophol (5-HTPH), N-acetylserotonin (NA-5-HT) and 5-hydroxy-indole-3-acetic acid (5-HIAA), were also determined. The total content of 5-HT in the eyestalk was 95.4+/-49.3 pg mg-1 wet mass (mean +/- s.d., N=55) while the specific content was 9.6+/-4.9 fmol microg-1 protein (mean +/- s.d. N=5). 5-HT was present in all four ganglia of the eyestalk. The highest proportion was found in the medulla terminalis (40.2 %) and the lowest in the retina lamina ganglionaris (9.9 %), which also had the lowest specific content. Conversely, the highest specific content of L-TRP was in the retina lamina ganglionaris. 5-HT biosynthesis and metabolism were explored in isolated eyestalks. The monoamine oxidase blocker pargyline, at concentrations between 0.8 and 10 mmol l-1, elicited a dose-dependent increase in 5-HT content. The biosynthesis of 5-HT in the crayfish eyestalk is suggested by the presence of its immediate precursor (5-OH-TRP) and by the suppression of 5-HT synthesis induced by m-hydroxybenzyl-hydrazine (m-HBH), a blocker of 5-OH-TRP decarboxylase. The presence of immunopositive cell bodies and axons was demonstrated using an anti-5-HT antiserum. 5-HT-like immunopositivity was detected in various regions of the eyestalk. Efferent immunopositive axons were also identified in the optic nerve, and these may have originated in the protocerebral lobe of the supraoesophageal ganglion. The branchings of these axons were profusely distributed in the neuropile of the medulla terminalis. A basal level release of 5-HT was detected in isolated eyestalks. The amount recovered was increased two-to threefold after blocking 5-HT uptake with fluoxetine (1 micromol l-1). Incubation of eyestalks in solutions containing a high K+ concentration (80 mmol l-1) released 5-HT. Electrical stimulation of the optic nerve released 5-HT as a function of the intensity of stimulation. Both the basal and evoked release were suppressed by lowering the Ca2+ concentration in the medium. These observations support a role for 5-HT as a neurotransmitter or neuromodulator in the crayfish eyestalk.