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Biomedical subjects

E A Kravitz

Publications and source records attributed to E A Kravitz.

At least 37 records · Page 2Linked to original sources
PubMed1990

Patterns of appearance of serotonin and proctolin immunoreactivities in the developing nervous system of the American lobster.

Serotonin (5-HT) and proctolin, neurohormones widely distributed in the lobster nervous system, have been implicated in a variety of behaviors and also are known to coexist in large pairs of identified neurons in the fifth thoracic (T5) and first abdominal ganglia (A1) of adults (Siwicki, Beltz, and Kravitz, 1987). Earlier studies also have shown that these paired neurons already contain 5-HT in embryos approximately halfway through development, whereas proctolin immunoreactivity does not appear in these cells until near the time of hatching (Beltz and Kravitz, 1987a). In the current studies, the brain and ventral nerve cord have been screened for the appearance of serotonin and proctolin immunoreactivities using immunocytochemical and biochemical methods, in order to determine whether the late appearance of proctolin in the paired T5 and A1 cells is a general feature of development in other neurons as well. In embryos approximately halfway through development, the adult complement of 5-HT-staining cells is already present. In several cases, embryonic serotonin cells are proportionally very large and prominent, suggesting possible developmental roles. In contrast to serotonin, fewer than 10% of the proctolin-staining neurons of juvenile animals are seen in embryos halfway through development. The number of immunoreactive cells gradually increases, but even by the sixth larval stage only half the number of cells that will eventually stain for proctolin are observed. Therefore, the developmental appearance of proctolin in lobster neurons, assayed using immunocytochemical methods, is relatively late and protracted compared to the appearance of serotonin. Quantitative measurements for 5-HT in lobster larvae were performed using high pressure liquid chromatography (HPLC) with dual electrochemical detection and for proctolin using radioimmunoassay. A gradual, probably growth-related increase in the amounts of serotonin and proctolin were seen during larval development. The implications of the biochemical data, in light of the immunocytochemical studies, are discussed.

Animals↗
PubMed1989

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.

1-Methyl-3-isobutylxanthine↗
PubMed1987

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.

Animals↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

Proctolin in identified serotonergic, dopaminergic, and cholinergic neurons in the lobster, Homarus americanus.

In order to explore the functions of the peptide proctolin in the lobster nervous system, 3 classes of neurons showing proctolin-like immunocytochemical staining were selected for study. These neurons were identified on the basis of physiological and/or morphological criteria, isolated by dissections, and analyzed with biochemical methods to determine whether they contained authentic proctolin and which classical neurotransmitters coexisted with the peptide. Pairs of large proctolin-immunoreactive neurons in fifth thoracic and first abdominal ganglia were identified as serotonin-immunoreactive neurons (Beltz and Kravitz, 1983, 1987) by staining serial sections of the ganglia alternately with the 2 antisera. Physiologically identified cells, dissected from the ganglia and analyzed with high-performance liquid chromatography (HPLC), contained approximately 20 microM proctolin and 0.5 mM serotonin. A large proctolin-immunoreactive neuron in the circumesophageal ganglion was identified as the lobster homolog of a dopaminergic neurosecretory cell found in other crustaceans (Cooke and Goldstone, 1970). The large lobster cell stained with antityrosine hydroxylase antiserum, and synthesized 3H-dopamine from 3H-tyrosine. Dissected cell bodies, analyzed by HPLC, contained approximately 25 microM proctolin. Proctolin-immunoreactive sensory neurons were identified as large stained fibers that terminated in sensory dendrites of the oval organ mechanoreceptor in the scaphognathite (Pasztor, 1979; Pasztor and Bush, 1982). The largest sensory fiber was isolated for biochemical studies. It synthesized 3H-acetylcholine from 3H-choline and, by HPLC analysis, was found to contain approximately 3 microM proctolin. Thus, proctolin coexists with different conventional transmitters in several classes of identified lobster neurons. Investigations of the actions of proctolin in these different contexts should contribute to a more complete understanding of the diverse functions of neuropeptides and their roles as cotransmitters.

Acetylcholine↗
PubMed1987

Physiological identification, morphological analysis, and development of identified serotonin-proctolin containing neurons in the lobster ventral nerve cord.

Amines and peptides exert a wide range of physiological actions on both central neurons and peripheral tissues. Among these actions, serotonin and octopamine are known to trigger contrasting postures when injected into freely moving lobsters. Immunocytochemical studies of lobster ganglia have identified presumptive serotonergic neurons, their central and peripheral projections, and their terminal fields of arborization. More than 100 neurons that show serotonin-like immunoreactivity have been found in the lobster nervous system (Beltz and Kravitz, 1983). From immunocytochemical studies it appears that varicosities within peripheral neurosecretory structures and endings in certain central neuropil regions arise from the same 2 pairs of large cells located in the fifth thoracic (T5) and first abdominal (A1) ganglia. Because we believed that such cells could account for the central and peripheral actions of serotonin on the postural system, we chose to study these 2 pairs of neurons in greater detail. In the previous paper, Siwicki et al. (1987) report that these neurons contain the pentapeptide proctolin in addition to serotonin. In this communication, we report that these cells can be identified reliably in living preparations; they have large fields of innervation projecting anteriorly into at least 4 segmental ganglia; these neurons are the origin of the fibers that form the thoracic second root neurosecretory regions; they are generally spontaneously active neurons that have overshooting action potentials in their cell bodies; and the serotonin and proctolin immunoreactivities are first expressed in these cells at widely different times in development.

Animals↗
PubMed1985

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.

Acetylcholine↗
PubMed1984

Proctolin in the lobster: the distribution, release, and chemical characterization of a likely neurohormone.

A radioimmunoassay, immunohistochemical techniques, and high pressure liquid chromatography (HPLC) methods have been developed for the study of the pentapeptide proctolin in the lobster Homarus americanus. Proctolin-like immunoreactivity is present in nearly every portion of the lobster nervous system; immunoreactivity is found in the brain, in each of the ganglia and connectives of the ventral nerve cord, and in many of the nerve roots that emerge from the cord. The greatest amounts are found in the pericardial organs, which are well known neurosecretory structures, and these structures have been selected for more detailed study. The immunoreactive material in the pericardial organs appears to be authentic proctolin. This material co-migrates with synthetic proctolin in two HPLC systems. Furthermore, a peptide that is purified from pericardial organs by HPLC is indistinguishable from synthetic proctolin in high resolution fast atom bombardment mass spectrometry. Cytochemistry reveals that the surface of the pericardial organs is densely covered with immunoreactive varicosities. No cell bodies that stain for proctolin are found in the pericardial organs, and the cells that give rise to the varicosities have not yet been located. The nerve endings in pericardial organs are capable of releasing proctolin-like material when depolarized in the presence of Ca++. These findings suggest that proctolin is a neurohormone in the lobster.

Animals↗
PubMed1984

Serotonin-induced protein phosphorylation in a lobster neuromuscular preparation.

Three substances believed to be neurohormones in the lobster--serotonin, octopamine, and proctolin--can modulate the physiology of the dactyl opener muscle of the lobster walking leg. All three act directly on muscle fibers to cause long-lasting contractures. In addition, serotonin enhances the release of transmitter from the excitatory and inhibitory axons that innervate the muscle. We now report that serotonin, applied to intact nerve-muscle preparations, increases the phosphorylation of a soluble 29,000-dalton protein by as much as 20-fold. In two respects the phosphorylation parallels the physiological effects of serotonin: both have a slow time course, and both are caused by low concentrations of serotonin (thresholds of approximately 10(-8) M). The 29,000-dalton protein is present in many lobster tissues, including thoracic ganglia, gills, and several different muscles. This suggests that the phosphorylation participates in some aspect of metabolism common to many types of cells, rather than in an activity unique to nerve or muscle. The function of the phosphorylated protein remains unknown. Several lines of evidence suggest that the phosphorylation is mediated by a cyclic nucleotide, probably cyclic AMP. (1) Adding cyclic AMP or cyclic GMP to nerve-muscle homogenates leads to the phosphorylation. (2) Adding 8-Br-cyclic AMP or 8-Br-cyclic GMP to intact preparations leads to the phosphorylation. (3) In intact tissue, forskolin, an adenylate cyclase activator, and isobutylmethylxanthine, a phosphodiesterase inhibitor, increase cyclic AMP and cyclic GMP and also cause the phosphorylation. (4) Serotonin increases cyclic AMP in intact preparations. (5) RMI 12,330A, which blocks the serotonin-induced cyclic AMP increase, also blocks the phosphorylation. (6) Serotonin has no detectable effect on cyclic GMP. This suggests that cyclic AMP, and not cyclic GMP, is the second messenger that mediates phosphorylation in vivo. In contrast, calcium is not likely to mediate the phosphorylation. Agents that block calcium channels do not block the serotonin-induced phosphorylation, and agents that increase sarcoplasmic calcium do not cause it. Furthermore, calcium does not induce the phosphorylation in a homogenate. One of the other modulators, proctolin, does not increase cyclic AMP or cause the phosphorylation even at concentrations 10(6)-fold greater than the hormone's threshold for producing contractures. The third modulator, octopamine, causes small increases in cyclic AMP and is a weak agonist of the phosphorylation. Neither proctolin nor octopamine alters cyclic GMP levels.

Animals↗
PubMed1984

Cellular mechanisms for modulation of posture by octopamine and serotonin in the lobster.

Injection of the monoamines octopamine and serotonin into the hemolymph of lobsters causes them to assume opposing static postures: octopamine causes a tonic extension of all extremities, while serotonin induces a tonic flexion. These amines have been shown previously to act as circulating neurohormones and to cause contraction of isolated exoskeletal muscles. The opposing postures elicited by amine injection do not appear to arise from peripheral actions, however, since serotonin and octopamine have similar effects on both flexor and extensor muscles. Instead, the amines are present within the central nervous system where they are probably released synaptically to activate coordinated patterns of tonic motor activity generating the observed postures. These motor patterns have been recorded from abdominal ganglia and involve the synchronized activation and inhibition of multiple excitatory and inhibitory motoneurons innervating the postural flexor and extensor muscles. These effects can be observed in an isolated abdominal ganglion. To begin a search for the cellular targets of amine action within the central nervous system, we recorded from identified postural motoneurons in abdominal ganglia. In two motoneurons that act to produce postural extension, octopamine reduced and serotonin raised the apparent threshold for action potential generation recorded in the somata. The amines do not significantly affect the resting potential or input resistance of the motoneurons measured in the cell bodies. Although the amine effect on motoneuron excitability could be due to actions directly on the motoneurons, our evidence suggests that at least part of the effect results from activation or inhibition of premotor interneurons: (1) an increase in EPSP frequency is observed in the motoneurons during octopamine superfusion, while a small decrease is observed during serotonin superfusion; (2) the amine effects on action potential threshold are abolished by cobalt ion. These results suggest that octopamine and serotonin have dual effects on posture: within the central nervous system, they act specifically to generate coordinated motor patterns for flexion or extension; as circulating neurohormones, they act nonspecifically to enhance the responses of exoskeletal muscles to motoneuronal activation.

Animals↗
PubMed1983

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.

Action Potentials↗
PubMed1983

Mapping of serotonin-like immunoreactivity in the lobster nervous system.

Serotonin exerts a wide range of physiological actions on many different lobster tissues. To begin the examination of the role of serotonin in lobsters at a cellular level, we have used immunohistochemical methods to search for presumptive serotonergic neurons, their central and peripheral projections, and their terminal fields of arborization. Whole mount preparations of the ventral nerve cord and various peripheral nerve structures have been used for these studies. With these tissues, more than 100 cell bodies have been found that show serotonin-like immunoreactivity. Although a few of the cell bodies are located peripherally (near the pericardial organs, a well known crustacean neurohemal organ), the vast majority are located in central ganglia. Every ganglion in the ventral nerve cord contains at least one immunoreactive cell body. The projections of many of the neurons have been traced, and we have constructed a map of the system of serotonin-immunoreactive cell bodies, fibers, and nerve endings. In addition, a dense plexus of nerve endings showing serotonin-like immunoreactivity surrounds each of the thoracic second roots in the vicinity of groups of peripheral neurosecretory neurons. These peripheral nerve plexuses originate from central neurons of the ventral nerve cord. In some cases we have been able to trace processes from particular central cell bodies directly to the peripheral nerve root plexuses; in other cases we have traced ganglionic neuropil regions to these peripheral endings.

5,7-Dihydroxytryptamine↗
PubMed1982

The action of serotonin on excitatory nerve terminals in lobster nerve-muscle preparations.

1. The action of serotonin on excitatory transmission in the opener muscle of the dactyl of the lobster walking leg was examined by intracellular recording techniques. 2. Serotonin, at concentrations as low as 5 x 10(-9) M, caused a sustained increase in the size of the excitatory junctional (synaptic) potential (e.j.p.). When serotonin was washed out of the bath the e.j.p. declined in two steps (T 1/2 approximately equal to 1-2 min; T 1/2 approximately equal to 30 min) to the control size. The increased e.j.p. size was predominantly due to a serotonin-induced increase in the release of quanta of excitatory transmitter with nerve stimulation. 3. The increase in transmitter release did not require nerve stimulation or the presence of Na+ or Ca2+ ions in the bathing medium during the period of serotonin treatment. 4. Three types of experiments suggested that a part of the action of serotonin on excitatory nerve terminals might involve a long-term metabolic change within terminals, possibly involving the buffering or storage of Ca2+ ions. First, serotonin increased the frequency of spontaneous release of transmitter in both normal saline (26 mM-Ca2+) and saline with very low levels of Ca2+ (less than 10(-8) M). Secondly, serotonin greatly potentiated increases in miniature excitatory junctional potential frequency induced by the loading of the nerve terminal with Na+ either by veratridine or by inhibition of the Na+ pump or by the addition of the Na-ionophore monensin in low-Ca2+ salines. Thirdly, in some experiments, serotonin treatment produced a partial restoration of the nerve-evoked release of transmitter in the low-Ca2+ medium (less than 10(-8) M).

Animals↗
PubMed1981

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.

Animals↗
PubMed1981

Biochemistry and ultrastructure of serotonergic nerve endings in the lobster: serotonin and octopamine are contained in different nerve endings.

In this article we report that the distribution of serotonin in the lobster nervous system parallels the distribution of octopamine and that the same tissues that contain endogenous serotonin can synthesize it from tryptophan. Octopamine and serotonin are highly concentrated in a neurosecretory region of the second thoracic roots in association with a group of neurosecretory cells. The roots possess separate high-affinity uptake systems for both serotonin and tryptophan. Radioactive serotonin, accumulated in tissues during incubations with either tritiated serotonin or tritiated tryptophan, can be released, in a calcium-dependent manner, by depolarization with potassium. A detailed morphological examination of the second thoracic roots shows four distinct categories of nerve endings in the vicinity of the neurosecretory cells. Octopamine is synthesized in one of these types of endings and serotonin in another. The high-affinity uptake systems for serotonin and tryptophan are found only in association with the endings that make serotonin. These endings and all the biochemical parameters of serotonin metabolism in the roots are selectively destroyed by previous injection of animals with the neurotoxin 5,7-dihydroxytryptamine.

Animals↗
PubMed1980

A peptide action in a lobster neuromuscular preparation.

The neuropeptide proctolin causes a sustained contraction of the opener muscle of the dactyl of the lobster walking leg. This substance acts directly on the muscle at concentrations as low as 10(-10)M. The contraction is dependent on extracellular calcium. Neither a significant depolarization nor a detectable change in the input resistance accompanies the response. No presynaptic action of proctolin is indicated; excitatory and inhibitory junctional potential sizes and the frequency of spontaneous miniature excitatory junctional potentials are unaffected.

Animals↗
PubMed1980

Amines and a peptide as neurohormones in lobsters: actions on neuromuscular preparations and preliminary behavioural studies.

In this communication we report that four substances, thought to function as neurohormones in Crustacea, all produce long-term changes in the physiological properties of lobster opener muscle preparations. The substances are the amines, octopamine, serotonin and dopamine, and the peptide, proctolin. The actions of these substances are superimposed on the normal synaptic apparatus that utilizes the amino acids GABA and glutamate (probably) as the inhibitory and excitatory neurotransmitter compounds. Serotonin acts on excitatory and inhibitory nerve endings to facilitate transmitter release and directly on muscle fibres to produce a contracture and to induce the appearance of Ca2+ action potentials. The latter two actions of serotonin are shared by proctolin and octopamine as well. Dopamine, on the other hand, relaxes muscle baseline tension. The mechanism of action of these substances at their target site (or sites) has been explored with electrophysiological and biochemical techniques and the results will be presented. In addition preliminary behavioural experiments have been carried out with serotonin and octopamine. These substances produce opposite postures when injected into lobsters. The amines act on central ganglia to produce these effects where they cause a programmed readout of firing of neurones that will produce either a flexed posture (serotonin) or an extended posture (octopamine).

Action Potentials↗