Immunocytochemical 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.

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.

Long-term regulation of synaptic acetylcholine release and nicotinic transmission: the role of cyclic AMP.

1. Using the rat superior cervical ganglion in vitro, the relative efficacy of nicotinic synaptic transmission was estimated by recording the postganglionic compound action potential and the amount of endogenous acetylcholine (ACh) released. These two parameters were correlated in individual ganglia by sampling the bathing medium for the assay of ACh while simultaneously recording the postganglionic response. 2. The beta-adrenoceptor agonist isoprenaline potentiated both the evoked release of ACh and the postganglionic response by about 20% during preganglionic stimulation at 0.2 Hz. 3. The adenosine receptor agonist 2-chloroadenosine inhibited ACh release and the postganglionic response by about 35%. 4. Tetanic preganglionic stimulation for a few seconds induced a long-term potentiation of nicotinic responses and of ACh release. Both of these potentiations were dependent upon extracellular Ca2+ during the tetani. 5. Forskolin and analogues of cyclic AMP also caused a long-lasting potentiation of both the evoked release of ACh and the postganglionic response, indicating that cyclic AMP may regulate transmission by a presynaptic mechanism. The specificity of the cyclic AMP analogues was tested using various butyryl- and bromo-purine nucleotides. 6. The effects of forskolin and 8-bromo-cyclic AMP did not appear to be dependent upon extracellular Ca2+. 7. The potentiation caused by forskolin was consistently augmented by three phosphodiesterase inhibitors--AH 21-132, papaverine and SQ 20-006. However, the effect of forskolin was not consistently enhanced by theophylline, nor was it reduced by the adenylate cyclase inhibitor SQ 22-536. 8. The neurogenic long-term potentiation was augmented by two of the phosphodiesterase inhibitors that also augmented the forskolin-induced potentiation--papaverine and SQ 20-006. 9. It was concluded that cyclic AMP can enhance nicotinic transmission, and can do so by increasing the evoked release of ACh. However, it was not possible to prove that cyclic AMP mediates the long-term potentiation induced by tetanic preganglionic stimulation.

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.

Single RNA species injected in Xenopus oocyte directs the synthesis of active choline acetyltransferase.

In vitro synthesized complementary RNA (cRNA) transcribed from a non-full length Drosophila choline acetyltransferase (ChAT) cDNA clone will direct the synthesis of enzymatically active and immunologically recognizable protein when injected into Xenopus oocytes. The levels of ChAT activity expressed in injected oocytes are proportional, over 4 orders of magnitude difference, to the concentration of injected 'sense' orientation cRNA. GpppG capping of the in vitro synthesized cRNA is not necessary for expression of active ChAT but inclusion of the capping compound during in vitro transcription results in higher levels of enzyme expression at lower levels of cRNA injection. In addition the capped cRNA results in increasing ChAT expression by the oocytes up to 7 days after injection while uncapped cRNA results in maximum enzyme activity after a single day and decreasing levels of activity during subsequent days. A single immunologically detectable protein is produced by oocytes injected with 'sense' cRNA which has a molecular size of 75 kDa and is indistinguishable from the major form of ChAT present in Drosophila. Oocytes making enzymatically active ChAT also accumulate significant levels of acetylcholine. We conclude from these results that our our non-full length Drosophila ChAT cDNA clone contains all the necessary coding information to make a functional protein which appears to have the same size and activity as native Drosophila ChAT.

Choline acetyltransferase and acetylcholine levels in Drosophila melanogaster: a study using two temperature-sensitive mutants.

Choline acetyltransferase (ChAT, acetyl-CoA-choline-O-acetyltransferase, EC 2.3.1.6) activity was measured in homogenates prepared from wild type (Canton S) and two temperature-sensitive presumed ChAT structural gene mutants (Chats1 and Chats2; originally described by Greenspan, R. (1980) J. Comp. Physiol. 137: 83-92) of Drosophila melanogaster. Wild type flies grown at 32 degrees C for 12 or 24 hr showed increased ChAT activity, whereas Chats1 and Chats2 flies showed a progressive decrease in enzyme activity at 32 degrees C (restrictive temperature) when compared to flies reared at 18 degrees C (permissive temperature). Acetylcholine (ACh) and choline levels were determined in formic acid-acetone extracts of individual fly heads, and the ACh levels showed the same variation with time at 32 degrees C as did the ChAT activity. In contrast, choline levels did not vary in any regular pattern. Acetylcholinesterase (EC 3.1.1.7) activity did not vary during heat treatment (except for Chatts2 flies held at 32 degrees C for 24 hr, where a decrease was observed) indicating that this treatment may be specific for ChAT. We conclude that ChAT activity is strongly correlated with ACh levels in Drosophila heads and may thus have an important regulatory role in determining the levels of ACh available for physiological function. We also report on the preliminary characterization of ChAT in both Chats mutants and compare the biochemical properties to those of wild type enzyme. Isoelectric focusing profiles of ChAT from both Chats mutants revealed enzymes with altered patterns compared to wild type, indicating that the mutations are most probably in the structural gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Histaminergic synaptic transmission in the cerebral ganglion of Aplysia.

Standard intracellular stimulating and recording techniques including voltage-clamp were used to analyze the synaptic responses mediated by two identifiable histamine-containing neurons (HCNs), designated C2 neurons, located in bilaterally symmetric clusters of the isolated cerebral ganglion of Aplysia california. Activation of each C2 induced unitary chemically mediated synaptic potentials in over 15 identified ipsilateral follower neurons. Several additional followers were connected to the HCNs by nonrectifying electrical synapses. Most of the follower neurons examined received only chemical synapses from the C2s. Some of the followers were reciprocally connected with each other through nonrectifying electrical synapses. A single C2 action potential can evoke six distinctive types of chemically mediated postsynaptic potentials (PSPs) in different follower neurons. Most of the PSPs have been shown to be multicomponent, i.e., they are comprised of various combinations of individual fast (less than or equal to 150 ms), slow (1-2 s), and very slow (greater than or equal to 4 s) depolarizing and hyperpolarizing components. The combination of these components produces PSPs of varying complexity, from simple monophasic responses such as the frequently observed slow excitatory PSPs and slow inhibitory PSPs to responses consisting of two to three components such as fast excitatory, slow inhibitory PSPs or fast inhibitory, slow excitatory PSPs. All of the multicomponent PSPs appear to be mediated through monosynaptic connections from the C2, as determined by various electrophysiological criteria. The slow and very slow synaptic components of the multicomponent PSPs were markedly potentiated in amplitude and duration after repetitive C2 activation. This property of the slow components permits the slower PSPs to exert a major influence on the excitability and integrative properties of the follower neurons.

On the nature of histamine-mediated slow hyperpolarizing synaptic potentials in identified molluscan neurones.

1. Standard intracellular stimulating and recording techniques were used to test the correspondence between monosynaptic post-synaptic potentials (p.s.p.s) evoked by histamine-containing C-2 neurones and responses to focally applied histamine recorded from two classes of identified post-synaptic neurones in the cerebral ganglion of Aplysia californica.2. Two types of p.s.p.s were examined: (1) a monophasic slow hyperpolarizing potential (I(s)p.s.p.) lasting 1-2 sec; and (2) a biphasic p.s.p. consisting of a fast depolarizing component <0.5 sec in duration (E(f)p.s.p.) plus a slow hyperpolarizing potential (I(s)p.s.p.) designated the E(f)I(s)p.s.p.3. Ionophoretic or pressure applied histamine mimicked both p.s.p.s and produced conductance increases in the post-synaptic neurones similar to those associated with the evoked p.s.p.s.4. The reversal potentials (E(rev)) for the I(s)p.s.p. and E(f)I(s)p.s.p., estimated by extrapolation, were -85+/-5.3, -35+/-5.5, and -83+/-8.1 mV (mean+/-S.D.), respectively. The I(s)p.s.p.s were produced by an increase in potassium conductance because their E(rev)s were shifted about 16 mV by doubling or halving the concentration of extracellular potassium and they could be eliminated by cooling or by intracellular injection of TEA ions.5. The average E(rev) values for the slow hyperpolarizing histamine responses were similar to those for the I(s)p.s.p.s; about -83 and -86 mV in neurones receiving the monophasic I(s)p.s.p.s and biphasic E(f)I(s)p.s.p., respectively.6. Cimetidine, an antihistamine drug that selectively blocks histamine receptors associated with potassium conductances in Aplysia, reversibly abolished the I(s)p.s.p.s and slow hyperpolarizing responses to focally applied histamine. In similar concentrations, cimetidine had no discernible effects on the E(f)p.s.p. and depolarizing response to histamine or on several different types of p.s.p.s mediated by the C-2 neurones.7. It is proposed that the I(s)p.s.p.s are mediated by histamine released from the C-2 neurones.

Long-term potentiation of synaptic acetylcholine release in the superior cervical ganglion of the rat.

The release of endogenous acetylcholine (ACh) from the in vitro rat superior cervical ganglion was measured by assaying the bathing medium. Simultaneously, synaptic transmission in the ganglion was assessed by recording post-ganglionic compound action potentials. A brief period of tetanic preganglionic stimulation (20 Hz for 20 s) induced a long-term potentiation of the post-ganglionic compound action potential. The same tetanic stimulation also consistently induced a long-term potentiation of stimulated ACh release. Spontaneous (non-stimulated) ACh release was not enhanced after tetanic stimulation. The content of ACh in the ganglion was not measurably increased after tetanic stimulation. These results suggest that the long-term increase in synaptic efficacy is due, at least in part, to an increase in the amount of ACh released by the afferent impulse.

5-hydroxytryptamine measurements in molluscan ganglia and neurons using a modified radioenzymatic assay.

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 microM) 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.