Localization of the sites of gamma-aminobutyric acid (GABA) uptake in lobster nerve-muscle preparations.

The principal sites of gamma-aminobutyric acid (GABA) uptake in lobster nerve-muscle preparations have been determined with radioautographic techniques after binding of the amino acid to proteins by aldehyde fixation. Semiquantitative studies showed that about 30% of the radioactive GABA taken into the tissue was bound to protein by fixation. Both light and electron micrographs showed dense accumulations of label over Schwann and connective tissue cell cytoplasm; muscle was lightly labeled, but axons and terminals were almost devoid of label. The possible role of Schwann and connective tissue cells in the inactivation of GABA released from inhibitory axons is discussed.

The metabolism of gamma aminobutyric acid in the lobster nervous system. Enzymes in single excitatory and inhibitory axons.

gamma-aminobutyric acid (GABA) is the inhibitory transmitter compound at the lobster neuromuscular junction. This paper presents a comparison of the enzymes of GABA metabolism in single identified inhibitory and excitatory axons from lobster walking legs. Inhibitory axons contain more than 100 times as much glutamic decarboxylase activity as do excitatory axons. GABA-glutamic transaminase is found in both excitatory and inhibitory axons, but about 50% more enzyme is present in inhibitory axons. The kinetic and electrophoretic behavior of the transaminase activity in excitatory and inhibitory axons is similar. Succinic semialdehyde dehydrogenase is found in both axon types, as is an unknown enzyme which converts a contaminant in radioactive glutamic acid to GABA. In lobster inhibitory neurons, therefore, the ability to accumulate GABA ultimately rests on the ability of the neuron to accumulate the enzyme glutamic decarboxylase.

Hormonal control of behavior: amines and the biasing of behavioral output in lobsters.

Hormones and neurohormones act on the nervous system to produce important changes in behavior. Amine actions in the lobster nervous system and their possible relations to aggressive behavior in lobsters were studied in order to explore how such changes might come about.

Neuronal geometry: determination with a technique of intracellular dye injection.

In a study of the specificity of neuronal connections in lobster abdominal ganglia, the dye Procion Yellow M4RS was electrophoretically injected into identified cell bodies. This dye spreads into fine branches of cells, survives fixation and routine histological procedures, and permits the reconstruction of cell shapes through examination of serial sections of ganglia. Certain cells were found to have an internal bilateral symmetry. Repeated injection of the same cells in ganglia from different animals showed that cells have characteristic shapes and that the neuropil is highly structured. This method of dye injection should have general applicability in studies where a knowledge of the geometry of specific cells is important.

Serotonin and octopamine produce opposite postures in lobsters.

Serotonin and octopamine, injected into the circulation of freely moving lobsters and crayfish, produce opposite behavioral effects. Octopamine injection produces sustained extension of the limbs and abdomen; serotonin injection produces sustained flexion. Neurophysiological analyses show that these postures can be accounted for by opposing, coordinated effects of these amines on patterns of motoneuron activity recorded from the ventral nerve cord.

Adrenocorticotropic hormone causes long-lasting potentiation of transmitter release from frog motor nerve terminals.

Exposure of frog neuromuscular preparations to adrenocorticotropic hormone for several minutes increased both nerve-evoked and spontaneous transmitter release for several hours. No changes in postsynaptic sensitivity to transmitter were detected. The long-lasting potentiation shows little sensitivity to changes in extracellular calcium concentration and seems to be entirely presynaptic in origin.

Serotonin, social status and aggression.

Serotonin, social status and aggression appear to be linked in many animal species, including humans. The linkages are complex, and, for the most part, details relating the amine to the behavior remain obscure. During the past year, important advances have been made in a crustacean model system relating serotonin and aggression. The findings include the demonstration that serotonin injections will cause transient reversals in the unwillingness of subordinate animals to engage in agonistic encounters, and that at specific synaptic sites involved in activation of escape behavior, the direction of the modulation by serotonin depends on the social status of the animal.

Purification and characterization of FMRFamidelike immunoreactive substances from the lobster nervous system: isolation and sequence analysis of two closely related peptides.

In the preceding paper (Kobierski et al: J. Comp. Neurol. 266:1-15, '87) FMRFamidelike immunoreactivity (FLI) was localized to specific cells and processes in the nervous system of the lobster Homarus americanus. In an effort to establish a role for this material we have purified and characterized a variety of immunoreactive peptides that can be extracted from the secretory pericardial organs. By using gel-filtration chromatography and three different HPLC systems, it has been established that little or no authentic FMRFamide is present. Of the major immunoreactive components two peptides were purified in sufficient quantity for microsequence analysis and have been tentatively identified as the octapeptides Ser-Asp-Arg-Asn-Phe-Leu-Arg-Phe-amide (FLI 3) and Thr-Asn-Arg-Asn-Phe-Leu-Arg-Phe-amide (FLI 4). Both of these are novel neuropeptides with some sequence homology to the previously described FMRFamide family. The pericardial organs release FLI when depolarized with 100 mM K+ in the presence of calcium. Between 75 and 80% of this release is accounted for by FLI 3 and FLI 4. One of these peptides (FLI 4) has been synthesized and shown to cochromatograph with the endogenous immunoreactive material. Preliminary studies show that this peptide can act as a modulator of exoskeletal and cardiac neuromuscular junctions.

Developmental expression of the octopamine phenotype in lobsters, Homarus americanus.

We have used immunocytochemical methods to examine the sequence of appearance of octopamine-immunoreactive neurons during development, and to try to correlate that appearance with the emergence of behavioral or physiological capabilities. The first octopamine neurons express their transmitter phenotype at approximately 43% of embryonic development. The last cells show immunostaining at the 3rd larval stage. In the wild, therefore, immunoreactivity in cells appears over a 9-12 month period. In contrast, serotonin-immunoreactive neurons stain early in embryonic development and the last serotonin-immunoreactive cells appear at about the same time the first octopamine-immunoreactive neurons show staining. The pattern of appearance of octopamine-immunoreactive cells is cell type-specific. A pair of brain cells and the descending interneurons stain first. Additional brain cell staining is seen throughout embryonic development. The ascending interneurons appear next, and a general anterior-posterior gradient typifies their emergence over a relatively short portion of embryonic life (E 48-62%). The neurosecretory cell staining appears last, is segment-specific, begins at about 62% development, and continues to the 3rd larval stage. The emergence of immunostaining for amine neurotransmitters within groups of identified neurons at precise times in development may specify possible functional units. With at least one group of cells, this possibility seems plausible: the three pairs of claw octopamine neurosecretory cells show immunostaining as a unit.

Mapping of octopamine-immunoreactive neurons in the central nervous system of the lobster.

It has been suggested that serotonin and octopamine serve important roles in behavioral regulation in lobsters. In this paper the locations of octopamine-immunoreactive neurons were mapped in wholemount preparations of the ventral nerve cord of 4th stage lobster (Homarus americanus) larvae. Approximately 86 neurons were found, distributed as follows: brain, 12; circumesophageal ganglia, 2; subesophageal ganglion, 38; thoracic ganglia, 6 each; and 4th and 5th abdominal ganglia, 2 each. All the octopamine-immunoreactive neurons are paired and located along the midline. Of the 86 neurons, 28 were identified as neurosecretory, and 26 as intersegmental ascending thoracic, ascending abdominal, or descending interneurons. The neurosecretory system is arranged segmentally and located entirely within the thoracic and subesophageal neuromeres with extensive terminal fields of endings along 2nd thoracic and subesophageal nerve roots. This set of neurons shares the features of central and peripheral endings with 2 pairs of large serotonin-containing neurosecretory neurons found in the fifth thoracic and first abdominal ganglia. The intersegmental neurons include: (1) two cells in the brain and 2 pairs of cells in the 3rd and 4th neuromeres of the subesophageal ganglion, which project to the 6th abdominal ganglion; (2) a segmentally organized group of ascending interneurons found in the subesophageal and in all thoracic ganglia; and (3) pairs of ascending interneurons found in the 4th and 5th ganglia in the abdominal nerve cord. By means of a biochemical assay, the cell bodies of octopamine-immunoreactive neurosecretory cells in the thoracic segment of the nerve cord were found to contain 40-100 fmol of octopamine, while control neurons had none.

FMRFamidelike peptides of Homarus americanus: distribution, immunocytochemical mapping, and ultrastructural localization in terminal varicosities.

The distribution of FMRFamidelike peptides was studied in the nervous system of the lobster Homarus americanus by using immunocytochemical and radioimmunological techniques. By radioimmunoassay FMRFamidelike immunoreactivity (FLI) was found in low levels (ca. 1 pmol/mg protein) throughout the ventral nerve cord and in much higher amounts (60-100 pmol/mg protein) in the neurosecretory pericardial organs. Immunocytochemical studies showed FLI in approximately 300-350 cell bodies, and in distinct neuropil regions, neuronal fiber tracts, and varicose endings. Specificity of the immunostaining was tested by preabsorbing the antiserum with FMRFamide, with peptides having similar carboxyl termini to FMRFamide (Met-enkephalin-Arg-Phe, Phe-Met-Arg-Tyr-amide), with several amidated peptides (alpha-melanocyte-stimulating hormone, substance P, oxytocin), and with proctolin, a peptide found widely distributed in the lobster nervous system. Of these substances, only FMRFamide blocked the staining. In addition to the pericardial organs, significant levels of FLI were found in neurosecretory regions associated with thoracic second roots and in the connective tissue sheath that surrounds the ventral nerve cord. In all three regions, immunocytochemical studies showed the FLI to be localized to fine fibers and associated terminal varicosities lying close to the surface of the tissue, with no obvious target in their immediate vicinity. When examined at the ultrastructural level, the immunoreactive varicosities of the thoracic second roots and of the ventral nerve cord sheaths were found a few microns from the surface of the tissue and contained electron-dense granules. In the immunoreactive nerve cord sheath endings, in addition to the large, dense granules, small, clear vesicles were found. The appearance and location of these terminals suggest a neurohormonal role for FMRFamidelike peptides in lobsters. The observation that low levels of FLI are found in the hemolymph supports this suggestion. In addition, the localization of FLI to particular neuronal somata, fiber tracts, and neuropil regions suggests possible functional roles for these peptides in (1) integration of visual and olfactory information, (2) function of the anterior and posterior gut, and (3) the control of exoskeletal muscles.

Crustacean hyperglycemic hormone in the lobster nervous system: localization and release from cells in the subesophageal ganglion and thoracic second roots.

Crustacean hyperglycemic hormones (CHHs) are neuropeptides involved in the regulation of hemolymph glucose. The primary source of CHHs has been identified as the neurosecretory neurons of the eyestalk X-organ and its associated neurohemal organ, the sinus gland. We have identified another source of CHH-like peptides in the nervous system. With the use of immunocytochemistry, cells in the second roots of the thoracic ganglia have been observed to stain positively for CHH-reactive material. We also identified a pair of cells in the subesophageal ganglion that contain large amounts of CHH-reactive material. Depolarization of these cells with elevated potassium mediates a calcium-dependent release of CHH-like material from the ganglion as quantified with an enzyme-linked immunosorbent assay (ELISA).

Proctolin in the lobster nervous system.

The pentapeptide proctolin (Arg-Tyr-Leu-Pro-Thr) is present in high concentrations in neurosecretory organs of the lobster, Homarus americanus. The central nervous system contains ca. 1400 proctolin-immunoreactive neurons, which appear to serve a variety of different functions. Some of these neurons have been specifically identified and analyzed biochemically to determine which classical neurotransmitters coexist with the peptide. These include: serotonin-proctolin cell pairs in the fifth thoracic and first abdominal ganglia; a large dopamine-proctolin neuron in the circumesophageal ganglion; and cholinergic-proctolin sensory neurons which innervate a mechanoreceptor in the scaphognathite. With these identified neurons we have begun to investigate the physiological actions of proctolin, the interactions between cotransmitters, and the development of multiple transmitter phenotypes in individual neurons.

Presynaptic action of the neurosteroid pregnenolone sulfate on inhibitory transmitter release in cultured hippocampal neurons.

The effects of the neurosteroid pregnenolone sulfate (PS) were studied in 3- to 9-week-old hippocampal cultures from neonatal rats. Cells were voltage clamped using CsCl filled electrodes, while action potentials and excitatory glutamatergic currents were abolished by superfusing with a combination of tetrodotoxin, 6-cyano-7-nitroquinoxaline (CNQX) and 2-amino-5-phosphonopentanoic acid (AP-5). Under these conditions spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were seen as inward currents at a holding potential of -70 mV. Their amplitude distributions were skewed without clearly detectable peaks. PS at 1-50 microM concentrations decreased the frequency of sIPSCs, with 1 microM being the most effective concentration. The effect appeared after 10-15 min of steroid application and the magnitude of the reduction increased during the early wash period. No recovery of sIPSC frequency was found after 30 min of washing with steroid-free medium. sIPSC amplitudes were not significantly changed at the time the effect of PS on sIPSC frequency was observed. The slow onset of this effect and its duration suggest a novel presynaptic action of the neurosteroid PS on GABAergic inhibition in the mammalian brain.

Noradrenaline augments tetanic potentiation of transmitter release by a calcium dependent process.

Noradrenaline (25 microM-50 microM) causes an increase in tetanic potentiation and in the augmentation phase of posttetanic potentiation of miniature and plate potential frequency. These effects were observed at both the frog and the rat neuromuscular junctions. The action of noradrenaline on quantal transmitter release depends on the presence of calcium ions in the extracellular medium.

Serotonin and aggression: insights gained from a lobster model system and speculations on the role of amine neurons in a complex behavior.

The amine serotonin has been suggested to play a key role in aggression in many species of animals, including man. Precisely how the amine functions, however, has remained a mystery. As with other important physiological questions, with their large uniquely identifiable neurons, invertebrate systems offer special advantages for the study of behavior. In this article we illustrate that principal with a description of our studies of the role of serotonin in aggression in a lobster model system. Aggression is a quantifiable behavior in crustaceans, the amine neuron systems believed to be important in that behavior have been completely mapped, and key physiological properties of an important subset of these netirons have been defined. These results are summarized here, including descriptions of the "gain-setter" role and "autoinhibition" shown by these neurons. Results of other investigations showing socially modulated changes in amine responsiveness at particular synaptic sites also are described. In addition, speculations are offered about how important developmental roles served by amines like serotonin, which have been well described by other investigators, may be related to the behaviors we are examining. These speculations draw heavily from the organizational/activational roles proposed for steroid hormones by Phoenix et al. (1959).

Cyclic AMP only partially mediates the actions of serotonin at lobster neuromuscular junctions.

Serotonin (5-HT) has multiple physiological actions at lobster neuromuscular junctions, including facilitation of transmitter release from nerve terminals and an increase in the tone and excitability of muscle fibers. These physiological effects of 5-HT are accompanied by a rise in intracellular levels of cAMP. We have used combined biochemical and physiological approaches to investigate whether cAMP directly mediates the physiological actions of the hormone. Based on the following lines of evidence, we conclude that the postsynaptic increase in muscle tone occurs independently of cAMP and that while the cyclic nucleotide does play a role in the facilitation of transmitter release by 5-HT, there is also a cAMP-independent component to this facilitation. (1) Agents that mimic the action of 5-HT on cAMP levels (forskolin, IBMX, SQ20009, 8-bromo cAMP) fail to mimic the postsynaptic actions of the amine. These agents do facilitate transmitter release, although none of them has as large an effect as does 5-HT. (2) When 5-HT is removed, presynaptic facilitation decays as the sum of 2 exponentials with very different time courses. The rate of the slower process is similar to the rate of breakdown of cAMP, while the faster process and the postsynaptic response decay significantly more rapidly. (3) IBMX retards the breakdown of cAMP and simultaneously retards the decay of the slower presynaptic process, with little or no effect on the other responses. (4) IBMX and forskolin potentiate the effect of 5-HT on cAMP levels and selectively enhance the slowly decaying presynaptic component with little or no effect on the other responses.