R.E. McCaman

The determination of picomole levels of 5-hydroxytryptamine and dopamine inAplysia, Tritonia and leech nervous tissues

Abstract Methods have been presented for the determination of two biogenic amines (5-HT and DA) in nervous tissues of various invertebrates. The sensitivity of these methods is such that 2 and 4 pmoles of 5-HT and DA, respectively, can be measured quantitatively. Results have been presented on the levels of these amines in individual ganglia and nerve trunks of Aplysia, Tritonia and leech and in isolated Retzius cells of the leech.

Imunocytochemical localization and direct assays of serotonin-containing neurons in Aplysia

Serotonin-containing neurons were localized in the central nervous system of Aplysia californica by the combination of an immunohistochemical procedure for wholemounts of Aplysia ganglia and, in parallel experiments, by the direct measurement of serotonin in individual neurons with a radioenzymatic assay. Paraformaldehyde fixed, desheathed ganglia were incubated in a commercially available antiserum against a conjugate of serotonin and bovine serum albumin. The bound antibody was visualized by an indirect antibody procedure using the horseradish peroxidase catalyzed reduction of the chromogen, diaminobenzidine, and a cobalt-nickel intensification procedure. The specificity of the immunoreaction for serotonin-containing cells was demonstrated by (1) the consistent staining of previously identified serotonin-containing neurons; (2) the absence of staining in identifiable neurons which do not contain measurable serotonin; and (3) the absence of staining in ganglia treated with antiserum which had been absorbed by serotonin and the serotonin conjugate. Previously unidentified serotonin-containing neurons were localized in the cerebral and pedal ganglia by the combination of immunocytochemistry and direct assay for serotonin. Immunoreactive fibers were found surrounding many neuronal somata. In addition, serotonin assays of known cholinergic neurons that were covered by immunoreactive fibers indicated that measurable amounts of serotonin were associated with such neurons, but the concentration of serotonin was an order of magnitude lower than in neurons known to stain with the anti-serotonin serum. These studies have localized more than a hundred neurons that appear to contain serotonin in concentrations (greater than 0.5 mM) in the Aplysia central nervous system. In addition, it appears that the long-held belief that the somata of invertebrate neurons are relatively free of impinging nerve fibers may no longer be tenable. The immunoreactive fibers surrounding many cell bodies may be the source of measurable serotonin associated with neurons known to utilize or to contain transmitter substances other than serotonin. Immunocytochemical techniques applied to wholemounts of molluscan preparations facilitate identification of stained neurons for parallel physiological and chemical experiments.

Identification of additional histaminergic neurons in Aplysia: improvement of single cell isolation techniques for in tandem physiological and chemical studies.

Abstract Identifiable neurons in the marine mollusc, Aplysia californica , were used to improve isolation techniques for in tandem physiological and chemical studies of the same neuron. A previous study demonstrated that the histaminergic neurons, RC-2 and LC-2, which had been subjected to intracellular recording and staining, were particularly sensitive to a freeze-substitution procedure used in isolating neurons for subsequent chemical assay. The present report describes an improvement of the freeze-substitution procedure which permits excellent recovery of histamine in physiologically manipulated C-2 neurons. The in tandem physiological and chemical procedures developed for identified neurons were used to characterize additional histamine-containing neurons which cannot be identified by visual inspection alone. These new histaminergic neurons, designated RC-3 and LC-3, are located on the ventral surface of the cerebral ganglion and are distinguishable from other adjacent histamine-containing neurons by their monosynaptic connections with some of the same follower neurons previously described for RC-2 and LC-2. The results of this study further support the role of histamine as a multiaction neurotransmitter and demonstrate the feasibility of measuring endogenous levels of various neurotransmitters in neuronal somata subsequent to intracellular recording.

The role of serotonin in the control of pedal ciliary activity by identified neurons in Tritonia diomedea

Abstract 1. A pair of identified neurons (Pd 21) in the pedal ganglia of Tritonia diomedea are capable of exciting pedal ciliary beating. 2. These neurons contain serotonin, but not acetylcholine, dopamine, norepinephrine or histamine. Other identifiable serotonergic neurons are also found in the Tritonia brain. 3. Serotonin (10 −7 g/ml) excites ciliary beating in isolated foot strips bathed in a high Mg 2+ -low Ca 2+ solution. 4. Serotonin injected into intact Tritonia prolonged pedal ciliary beating. 5. The Pd 21 neurons probably release serotonin into spaces in the foot, where the serotonin exerts its excitatory effect on the ciliated cells.

Presence of octopamine in firefly photomotor neurons

Various tissues involved in producing luminescence in larval fireflies (Photuris versicolor) were examined for the presence of octopamine. These tissues included the terminal abdominal ganglion (A8) which innervates the paired lantern organs, the cell bodies of the photomotor neurons and the isolated larval lanterns. A previous study has identified the 4 motoneurons arising from A8 which bilaterally innervate the paired larval lanterns through symmetrical axons existing both sides of the ganglion. Individual photomotor neuron somata were isolated, pooled and found to contain about 0.03 pmol/soma giving an effective concentration of 2.8 mM octopamine. Significant amounts of octopamine were also found within the peripheral effector tissue. The presence of octopamine throughout the luminescence-producing pathway further supports the hypothesis that octopamine serves a neurotransmitter function in firefly bioluminescence. In this system, it appears that octopamine serves a more direct role as a neurotransmitter that that postulated for its modulatory and hormonal functions in other arthropod systems. Furthermore, the bioluminescent response of the larval firefly lantern provides a useful dynamic system to study the physiology, pharmacology and biochemistry of octopaminergic transmission.

Dopamine measurements in molluscan ganglia and neurons using a new, sensitive assay

DOPAMINE is the principal catecholamine found in the molluscan CNS, and its role as a neurotransmitter has been studied in various species (see reviews by WELSH, 1972; GERSCHENFELD, 1973; KEHOE & MARDER, 1976). Using histofluorescence techniques, only specific neurons show a green fluorescence characteristic of DA. Biochemical studies have demonstrated the presence both of appropriate biosynthetic enzyme systems and of specific uptake mechanisms. Electrophysiological studies have described different receptors for DA in various neurons which mediate excitatory, inhibitory, or mixed responses. The most convincing evidence for a neurotransmitter role for DA was provided by studies of the giant DA-containing neuron (GDC) and its postsynaptic neurons in the pond snail, Planorbis corneus (see review by BERRY & PENTREATH, 1978). In this communication we have used a new, ultrasensitive radiochemical assay (MCCAMAN et a/., submitted for publication) to measure DA in the ganglia and in individual neurons of several marine and fresh water gastropod molluscs: Aplysia californica, Tritonia diomedia, Lymnaea stagnalis, and Helisoma trioolis. Neurons containing measurable amounts of DA were found only in the pedal ganglia of the fresh water species, Lymnaea and Helisoma and are probably homologous to the G D C described in Planorbis.

5-Hydroxytryptamine Measurements in Molluscan Ganglia and Neurons Using a Modified Radioenzymatic Assay

Abstract: A radioenzymatic procedure for the determination of sub‐picomole amounts of 5‐hydroxytryptamine (5‐HT) is described. It is a modification of the method originally described by Saavedra et al. (1973), in which 5‐HT was measured as the radiolabelled product [3H]5‐ methoxy‐N‐acetyltryptamine, after incubation with [3H]S‐adenosylmethionine, acetyl‐CoA, and the enzymes hydroxyindole‐O‐methyltransferase (EC 2.1.1.4) and N‐ acetyltransferase (EC 2.3.1.5). Ganglia from various gastropod molluscs (Aplysia californica, Tritonia diomedia, Lymnaea stagnalis, and Helisoma trivolvis), as well as individual neuronal somata isolated from these ganglia, were assayed for 5‐HT. Among the homologous giant cerebral cells in these animals, the 5‐HT concentrations were similar. Statistical analysis of the 5‐HT values in paired 5‐HT‐containing neurons demonstrated that the variability was considerably greater in samples obtained from different animals than in those obtained from the same animal. This suggests that experiments aimed at manipulating amine levels in individual neurons may benefit by using a paired‐cell paradigm. The effects of incubating Aplysia ganglia with 5‐HT or with the 5‐HT precursors tryptophan and 5‐hydroxytryptophan (5‐HTP) were studied. High concentrations of 5‐HTP and 5‐HT (100 μM) increased the levels of 5‐HT in ganglia, but only incubation in high concentrations of 5‐HTP resulted in an increase of 5‐HT in the isolated somata of 5‐HT‐containing neurons C1 and P5.

Measurement of endogenous transmitter levels after intracellular recording

Chemical measurements of endogenous acetylcholine (ACh), serotonin (5HT), dopamine, y-aminobutyric acid, and histamine (HA) in individual invertebrate neuron somata have been important in establishing the transmitter function of these substances 6. The neurons used in these studies were identified on the basis of anatomical features (size, location, pigmentation, etc.), and the chemical measurements were carried out in parallel with the physiological and pharmacological experiments necessary to establish transmitter function. However, many neurons are not easily identified by visual inspection alone and require physiological characterization for identification. In this case, as well as in other experimental paradigms, it would be desirable and often necessary to assay the same neuron subsequent to physiological studies which utilize intraceilular recording techniques. We therefore set out to determine whether chemical procedures used in some of the above studies were applicable to the measurement of amines in neurons subsequent to intracellular recording. Identifiable Aplysia neurons known to contain or to utilize ACh (B-4 and B-5 of the buccal ganglia 2,a) HA (C-2s of the cerebral ganglionS,ll,la), and 5HT (C-Is of the cerebral ganglion 4,12 and the P-1 s of the pedal ganglionT), were chosen for this study. Preliminary attempts to measure ACh and HA in the designated neurons subsequent to intracellular recording resulted in significantly lower levels of ACh in B-4 and B-5 and undetectable levels of HA in the C-2s in comparison to intact control neurons (see Table 1). This problem was traced to the procedure used in preparing the ganglia for isolation of the individual neurons, i.e., the freeze-substitution procedure originally described by Giller and Schwartz 5. The freeze-substitution procedure for isolating individual neuronal somata has been extremely useful for measuring different biogenic amines, amino acids, enzyme activities, etc. in a variety of invertebrate preparations. This procedure greatly facilitates removal of the ganglionic sheath without damaging the underlying neurons and permits the isolation of somata virtually free of contamination. We have

In situ hybridization of whole-mounts of Aplysia ganglia using non-radioactive probes

A protocol for carrying out in situ hybridization with non-radioactive, digoxigenin-labelled probes has been developed for whole-mounts of Aplysia ganglia. Whole-mount preparations preserve the anatomical relationships of neurons within intact ganglia and facilitate the precise identification of a particular neuron in live preparations so that functional studies can be correlated with biochemical attributes of an identified neuron. The protocol was developed with the use of probes to messenger RNAs that are abundant in Aplysia neurons. In situ hybridization with a cDNA probe to a neuronal form of beta-actin stained all neurons, including their processes, whereas use of a cDNA probe for the neuropeptide FMRFamide resulted in staining of a select group of Aplysia neurons.

Evidence for genetic influences on neurotransmitter content of identified neurones of Lymnaea stagnalis

Neurotransmitter content was measured in two identified giant neurones in isogenic and wild-type populations of the freshwater pond snail Lymnaea stagnalis. The paired serotonergic cerebral giant neurones (LC1 and RC1) have higher transmitter levels and less variability in inbred animals than in wild-type animals. The transmitter content of the unpaired dopaminergic right pedal giant neurone (RPeD1) does not differ between inbred and wild-type animals in either level or variability. It is proposed that serotonin content of the cerebral giant neurones is under partial genetic control, and that animals of the wild-type population may possess a number of different alleles for the genes influencing serotonin levels. Inbreeding resulted in fixation of an allele promoting high serotonin levels. This particular wild-type population is probably already isogenic for genes influencing dopamine content in the right pedal giant neurone.