The effects of ethanol on pre-synaptic components of synaptic transmission in a model glutamatergic synapse: the crayfish neuromuscular junction

We have elucidated some of the mechanisms by which ethanol (EtOH) reduces synaptic efficacy at model glutamatergic synapses. The crayfish phasic and tonic neuromuscular junctions are superb models for directly assessing the effects of EtOH on pre-synaptic components of synaptic transmission. The ability to perform quantal analysis of synaptic transmission has allowed us to assess pre-synaptic alterations of release. Using this system, we report that the application of EtOH, within a range observed in intoxicated humans (44 and 88 mM), resulted in a diminution of excitatory post-synaptic potentials (EPSP) amplitudes. Additionally, using focal macro-patch recordings, quantal synaptic currents were recorded to assess the pre-synaptic component as potential target sites for EtOH's action. At the tonic neuromuscular junctions, EtOH (88 mM) reduced the probability of release (p), and in some cases, reduced the number of the release sites (n), but did not alter facilitation index nor did it affect the latency of vesicular release. At the phasic neuromuscular junction, a reduction in synaptic charge occurred during the presence of EtOH. Thus, the observed decrease in synaptic strength is at least partially attributable to a pre-synaptic alteration, specifically the release of fewer vesicles.     

The abdominal motor system of the crayfish, Cherax destructor. I. Morphology and physiology of the superficial extensor motor neurons

Using extracellular and intracellular stimulation, recording and dye-filling, we identified and studied the superficial extensor motor neurons of the crayfish, Cherax destructor. Functional associations of each neuron were characterised by recording its responses to sensory and abdominal cord inputs, its extensor muscle innervation pattern and its relationships with other neurons. Two clear associations were found among the six neurons of each segment. A medium-sized excitor (no. 3), that innervates a substantial percentage of extensor muscle fibres, and the largest excitor (no. 6), recruited during peak, excitation, were inhibited by input from unknown interneurons that excited the common inhibitor (no. 5). Likewise, these excitors received excitatory input when the inhibitor was silent. Another medium-sized neuron (no. 4) that innervates many muscle fibres was co-active with one of the small excitors (no. 2). The two medium-sized neurons were never active at the same time, and these two groupings may be determined by pre-motor interneurons. The implications of these findings for our understanding of motor control in this system are discussed. Accepted: 21 June 1998     

The abdominal motor system of the crayfish, Cherax destructor. II. Morphology and physiology of the deep extensor motor neurons

Two opposing muscle systems underlie abdominal contractions during escape swimming in crayfish. In this study we used extracellular and intracellular stimulation, recording and dye-filling to systematically identify each of the five deep extensor excitors and single inhibitor of the crayfish, Cherax destructor. Functional associations of each neuron were characterised by recording its responses to sensory and abdominal cord inputs, its extensor muscle innervation pattern, and its relationships with other neurons. Each excitor receives excitatory input from the tonic abdominal stretch receptors and the largest neuron also receives input from the phasic stretch receptor. The two largest excitors innervate the muscle bundle containing the fastest fibres and may be electronically coupled. The smaller neurons may also be electronically coupled and innervate the remaining deep extensor fibres which display dynamic characteristics from fast to medium-fast. The inhibitor does not receive input from the stretch receptors, but is strongly excited by tactile afferents. The implications of these findings for the current models of the control of abdominal tailflips and swimming are discussed. Accepted: 21 June 1998     

Innervation and motor patterns of the abdominal superficial flexor muscles in larval lobsters

The pattern of innervation and motor program of the abdominal superficial flexor muscle was investigated electrophysiologically in larval lobsters (Homarus americanus). The muscle receives both excitatory and inhibitory innervation in the larval as well as in the embryonic stages. Individual muscle fibers receive a single inhibitory neuron (f5) and a maximum of three excitors. Based on spike heights these axons belong to either the small (f1 or f2) or large (f3, f4) motoneurons. While the small axons preferentially innervate the medial muscle fibers the large axons innervate medial as well as lateral fibers. This larval pattern of innervation resembles the pattern in the adult lobster. The resemblance extends to the firing patterns as well with both large and small excitors firing spontaneously. Furthermore, evoked activity in the larvae produces reciprocal (and occasionally cyclical) bursts of excitor and inhibitor neurons denoting abdominal extension and flexion and resembling the firing patterns in adults. Consequently motor programs employed in steering the pelagic larvae are reminiscent of the programs for maintaining posture in the benthic adult lobsters.     

The ultrastructure of giant fibre and serial synapses in crayfish

Summary The ultrastructure of synapses between the cord giant fibres (lateral and medial) and the motor giant fibres in crayfish, Astacus pallipes, third abdominal ganglia have been examined. These electrotonic synapses are asymmetrical, they have synaptic vesicles only in the presynaptic fibre, and they have synaptic cleft widths normally of about 100 Å but narrowed to about 50 Å in restricted areas. Localized increases in density of the synaptic cleft and adjacent membranes also occur within a synapse, and synaptic vesicles are most tightly grouped at the membrane in such areas. Tight or gap junctions with 30 Å or narrower widths have not been found, but the junctions probably function in a similar way to gap junctions.Three small nerves are closely associated with the synapses between the giant fibres. One of these small nerves has round synaptic vesicles and is thought to be excitatory on morphological grounds; one has flattened vesicles and is thought to be inhibitory; and one is postsynaptic to the lateral giant and the two small presynaptic nerves. It is proposed that these small nerves modulate activity in the much larger giant fibre synapse.     

Synaptic interactions among neurons that coordinate swimmeret and abdominal movements in the crayfish

1. Many interneurons in the crayfish (Procambarus clarkii) abdominal nervous system influence two behaviors, abdominal positioning and swimmeret movements. Such neurons are referred to as dual output cells. Other neurons which influence either one behavior or the other are single output cells. 2. Extensive synaptic interactions were observed between both dual and single output neurons involved in the control of abdominal positioning and swimmeret movements. Over 60% of all neuron pairs examined displayed interactions. Pairs of agonist neurons displayed excitatory interactions, while pairs of antagonists had inhibitory interactions. This pattern of interaction was observed in about 75% of interactive neuron pairs whether abdominal positioning or swimmeret outputs were considered. 3. Evidence for both serial and parallel connectivity, as well as, reciprocal or looping connections was observed. Looping connections can be found both between the abdominal positioning and swimmeret systems and within each system. 4. Most (28/34) single output neurons were not presynaptic to dual output neurons. No single output neurons were found to excite dual output neurons to spiking, although inhibitory interactions and weak excitations were observed. 5. Abdominal positioning inhibitors displayed properties consistent with a role in mediating some of the coordination between the swimmeret and abdominal positioning systems. 6. None of the dual output neurons examined influenced the swimmeret motoneurons directly.     

Sensitivity of transformed (phasic to tonic) motor neurons to the neuromodulator 5-HT

Long-term adaptation resulting in a 'tonic-like' state can be induced in phasic motor neurons of the crayfish, Procambarus clarkii, by daily low-frequency stimulation [Lnenicka, G.A., Atwood, H.L., 1985b. Long-term facilitation and long-term adaptation at synapses of a crayfish phasic motoneuron. J. Neurobiol. 16, 97-110]. To test the hypothesis that motor neurons undergoing adaptation show increased responses to the neuromodulator serotonin (5-HT), phasic motor neurons innervating the deep abdominal extensor muscles of crayfish were stimulated at 2.5 Hz, 2 h/day, for 7 days. One day after cessation of conditioning, contralateral control and conditioned motor neurons of the same segment were stimulated at 1 Hz and the induced excitatory post-synaptic potentials (EPSPs) were recorded from DEL(1) muscle fibers innervated by each motor neuron type. Recordings were made in saline without and with 100 nM 5-HT. EPSP amplitudes were increased by 5-HT exposure in all cases. Conditioned muscles exposed to 5-HT showed a 2-fold higher percentage of increase in EPSP amplitude than did control muscles. Thus, the conditioned motor neurons behaved like intrinsically tonic motoneurons in their response to 5-HT. While these results show that long-term adaptation (LTA) extends to 5-HT neuromodulation, no phenotype switch could be detected in the postsynaptic muscle. Protein isoform profiles, including the myosin heavy chains, do not change after 1 week of conditioning their innervating motor neurons.     

Effects of Lead Salts on the Uptake, Release, and Binding of γ-Aminobutyric Acid: The Importance of Buffer Composition

The effects of lead on the uptake and release of gamma-[3H]aminobutyric acid [( 3H]GABA) from rat brain slices were examined in solutions buffered with Tris-HCl, sodium phosphate, and sodium bicarbonate. Lead acetate (10-250 microM) inhibited uptake and potassium-stimulated release and facilitated spontaneous efflux only in solutions buffered with Tris-HCl. Calcium-independent binding of [3H]GABA was unaffected by lead acetate (1-100 microM) in Tris-citrate buffer but was significantly inhibited by 3 microM lead acetate in Tris-HCl solution. At the rat soleus neuromuscular junction, lead caused a dose-dependent reduction of end-plate potential amplitude at concentrations of 10-100 microM lead acetate in HEPES-buffered solution but had no effect at these concentrations in phosphate-buffered solution. Stability constants of lead complexes indicate that buffers containing carbonate and phosphate are unlikely to contain a significant concentration of Pb2+, as complexing by these anions would reduce the availability of free Pb2+. This study indicates that the choice of buffer is important when investigating the effects of lead on biological systems and that negative findings may result from the use of inappropriate buffers. It also has important clinical implications suggesting that some effects of lead poisoning may result from its ability to affect neurotransmitter systems directly and that local changes in pH and complexing anion concentrations in the CNS may influence its biological availability and, hence, variable biological responses.     

Behavioral effects of serotonin and serotonin agonists in two crayfish species, Procambarus clarkii and Orconectes rusticus

Exogenous serotonin elicits several behaviors in Procambarus clarkii, including a flexed, elevated posture, reduced locomotion, and changes in aggressive behavior. We conducted experiments to determine if several serotonin agonists mimicked the behavioral effects of serotonin in two crayfish species, P. clarkii and Orconectes rusticus. Drugs tested were 1-(3-Chlorophenyl)-piperazine dihydrochloride (mCPP), Oxymetazoline, 2,5-dimethoxy-4-iodoamphetamine (DOI), CGS-12066A, and (+/-)-8-hydroxy-2-(di-n-dipropylamino) tetralin (8-OH-DPAT). In P. clarkii, mCPP most closely mimicked the effects of serotonin, significantly increasing the performance of the flexed, elevated posture and reducing locomotion; 8-OH-DPAT significantly reduced locomotion as well. Both of these drugs produced significant increases in elevated posture and decreases in locomotion in O. rusticus, and in this species, the drugs at test concentrations were more effective in eliciting these effects than serotonin. The effects of the drugs on behaviors performed during fighting bouts were variable. In both species, only 8-OH-DPAT significantly reduced several agonistic behaviors, and no agonist or 5-HT itself produced significant increases in agonistic behavior.     

FGF binding proteins (FGFBPs): Modulators of FGF signaling in the developing,adult, and stressed nervous system

Members of the fibroblast growth factor (FGF) family are involved in a variety of cellular processes. In the nervous system, they affect the differentiation and migration of neurons, the formation and maturation of synapses, and the repair of neuronal circuits following insults. Because of the varied yet critical functions of FGF ligands, their availability and activity must be tightly regulated for the nervous system, as well as other tissues, to properly develop and function in adulthood. In this regard, FGF binding proteins (FGFBPs) have emerged as strong candidates for modulating the actions of secreted FGFs in neural and non-neural tissues. Here, we will review the roles of FGFBPs in the peripheral and central nervous systems.     

The result of evolutionary limb loss on the complement of motor neurons in a crayfish limb nerve revealed by serial homology

Many macruran decapod crustaceans show sexual dimorphism of abdominal appendages adapted for use as secondary reproductive organs. Not only does the Australian crayfish, Cherax destructor, show no external, abdominal dimorphism, but both males and females have lost the pleopods of the first abdominal segment entirely. The first nerves of the abdominal ganglia of crayfish and lobsters carry the axons of the pleopod motor neurons. We used intracellular cobalt infusion into the first nerves of the first and second abdominal ganglia to reveal the motor neuron complement of these ganglia in males and females. The first nerves of the second abdominal ganglia of both males and females have approximately 37 motor neurons associated with them. The homologous nerves in the first abdominal segment, where there are no pleopods, have only 8 or 9 motor neurons associated with them. The evolutionary implications of this difference are discussed.     

Physiological changes of premotor nonspiking interneurons in the central compensation of eyestalk posture following unilateral sensory ablation in crayfish

We investigated how the physiological characteristics and synaptic activities of nonspiking giant interneurons (NGIs), which integrate sensory inputs in the brain and send synaptic outputs to oculomotor neurons innervating eyestalk muscles, changed after unilateral ablation of the statocyst in order to clarify neuronal mechanisms underlying the central compensation process in crayfish. The input resistance and membrane time constant in recovered animals that restored the original symmetrical eyestalk posture 2 weeks after operation were significantly greater than those immediately after operation on the operated side whereas in non-recovered animals only the membrane time constant showed a significant increase. On the intact side, both recovered and non-recovered animals showed no difference. The frequency of synaptic activity showed a complex pattern of change on both sides depending on the polarity of the synaptic potential. The synaptic activity returned to the bilaterally symmetrical level in recovered animals while bilateral asymmetry remained in non-recovered ones. These results suggest that the central compensation of eyestalk posture following unilateral impairment of the statocyst is subserved by not only changes in the physiological characteristics of the NGI membrane but also the activity of neuronal circuits presynaptic to NGIs.     

Characteristics of interaction between central neurons and abdominal stretch receptors in the crayfish

Spontaneous activity of slow- and fast-adapted abdominal stretch receptor (SAR and FAR) of the crayfish and their activity evoked by adequate stimulation were investigated in the presence of efferent regulation. Activity of the receptors was shown to be under effective inhibitory control of two central neurons, principal and accessory; activity of these neurons, in turn, is determined by the current receptor activity. The closest interaction is found between SAR and the principal inhibitory neuron. Two types of efferent regulatory action of this neuron were discovered: grouped and continuous. Its powerful discharges (up to 361 spikes) arising in response to only one SAR afferent impulse are described. The character of synaptic connections between the peripheral and central neurons is discussed.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Institute of Zoology, Academy of Sciences of the Moldavian SSR, Kishinev. Translated from Neirofiziologiya, Vol. 4, No. 3, pp. 317–327, May–June, 1972.     

Mechanisms of interaction between central neurons and abdominal stretch receptors in the crayfish

Mechanisms of interaction between central and receptor neurons of the crayfish (the principal inhibitory neuron — PIN — and the slow-adapting stretch receptor — SAR) when functioning under different conditions were investigated: during regular spontaneous activity of SAR, grouped discharges of PIN, and regular spontaneous activity of PIN. A close connection was found between the various parameters of the PIN and SAR responses. Adaptation of SAR to the action of adequate, regular repetitive stimulation takes place faster in the presence of stationary background activity of PIN. The appearance and disappearance of SAR spike activity are determined by the ratio between the firing rates of SAR and PIN: at the moment of changeover the neuron with the higher firing rate is predominant.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Institute of Zoology, Academy of Sciences of the Moldavian SSR, Kishinev. Translated from Neirofiziologiya, Vol. 4, No. 4, pp. 429–438, July–August, 1972.     

Evaluation of anti-depressant effects of phthalazinone-based triple-acting small molecules against 5-HT2A, 5-HT2C,and the serotonin transporter

Development of highly effective, safe, and fast-acting anti-depressants is urgently required for the treatment of major depressive disorder. It has been suggested that targeting 5-HT_2A and 5-HT_2C in addition to inhibition of serotonin reuptake may be beneficial in generating anti-depressant agents with better pharmacology and less adverse effects. We have developed phthalazinone-based compounds that potently bind to 5-HT_2A, 5-HT_2C, and the serotonin transporter. The representative compounds 11j and 11l displayed strong binding affinities against these targets, and showed favorable toxicity profiles as determined by hERG binding and CYP inhibition assays. Furthermore, these compounds presented promising anti-depressant effects comparable to fluoxetine and also synergistic effects with fluoxetine in forced swimming test, which implicates these compounds can be developed to help the treatment of major depressive disorder.     

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.     

Histochemical heterogeneity of fibers in the abdominal superficial flexor muscles of the Norway lobster, Nephrops norvegicus (L.).

The superficial flexor muscle in the abdomen of the Norway lobster Nephrops norvegicus (L.), comprises medial and lateral bundles with distinct fiber type composition. Fibers of the medial bundle have long sarcomeres (> 9 microns) and a thick fringe of subsarcolemmal mitochondria. In histochemical tests they have a low total myofibrillar ATPase activity, a pH-stable isoform of myosin ATPase, and a high level of oxidative enzyme activity. A few fibers of the lateral bundle also display these morphological and histochemical properties. However, the majority of lateral fibers have shorter sarcomeres (< 8 microns), no subsarcolemmal mitochondria, but a well-developed tubular system. They also have a higher total myofibrillar ATPase activity, a pH-labile isoform of myosin ATPase, and a low level of oxidative enzyme activity. The heterogeneous pattern of different fiber types in the lateral bundle of this muscle is similar but not identical in the different abdominal segments and in different individuals.     

The effects of nitrite on chloride regulation in the crayfish Pacifastacus leniusculus Dana (Crustacea: Decapoda)

Summary Nitrite in the external freshwater medium was found to be toxic to Pacifastacus leniusculus Dana (48 h LC₅₀0.7 mM NO ₂ ^– ). It produced significant changes in haemolymph ionic concentration and acid-base status. Exposure to 1.0 mM NO ₂ ^– resulted in a rapid, active accumulation of nitrite in the haemolymph (to 25 mM NO ₂ ^– after 24 h) and caused the partial inhibition of Cl^– uptake. Some reduction in Cl^– efflux rate was seen. In 1.0 mM NO ₂ ^– a rapid depletion of haemolymph [Cl^–] was observed (50 mM decrease in 27 h). Nitrite competitively inhibited active Cl^– uptake (K_m increased from 0.42 to 1.22 mM; K_i=0.45 mM). To achieve Cl^– balance in this medium, depleted crayfish would require a two-fold increase in external [Cl^–]. A lesser decrease in haemolymph [Na⁺] was found while osmotic pressure was relatively unaffected. Haemolymph [HCO ₃ ^– ] showed a significant increase and was accompanied, unexpectedly, by an acidosis. Possible sources of the excess HCO ₃ ^– , perhaps by inhibition of normal Cl^–/HCO ₃ ^– branchial exchange or release from CaCO₃ stores, are discussed. Haemolymph clearance of NO ₂ ^– was slower than uptake as was the restoration of [Cl^–] on recovery in nitrite-free medium.Abbreviations AFWM artificial freshwater medium - BOD biochemical oxygen demand - J _out ^Cl chloride efflux - J _in ^Cl chloride influx - J _in ^Cl chloride influx - J _net ^base net base flux - J _net ^base net base flux - J _in(p) ^Cl passive chloride influx - J _out efflux - LC ₅₀ median lethal concentration - NEDE N-1-Naphthylethylenediamine - SEM standard error of mean - TEP transepithelial potential difference - V _in ^Cl active chloride uptake     

Types and arrangements of neurons in the crayfish optic lamina

Cell and Tissue Research - The neural arrangements in the optic lamina of the crayfish Pacifastacus leniusculus Dana have been studied by light microscopy by means of silver impregnation...     

The anatomy and motor nerve distribution of the eye muscles in the crayfish

Summary The gross anatomy of the muscles in the crayfish compound eye and the distribution of brain oculomotor neurons were studied by a variety of anatomical and physiological techniques. There are 11 major muscles in each eye. These vary considerably in size and influence upon eye movements and in their source of motor innervation. Muscles that cause defensive eyestalk withdrawal are controlled by axons of a giant motor neuron cluster. Muscles that move the eyecup in vertical planes are innervated by cells of an anterior motor cluster, as well as by cells in the medulla terminalis. Muscles which move the eyecup horizontally are supplied by neurons of the lateral motor cluster. The separation of the oculomotor system into different neuronal groups that supply different sets of muscles thus reflects functional specializations of the component divisions.I am grateful to Mr. Gene Lorton for his technical assistance with some phases of this work. I also thank Sharon Greene for executing the illustration in Figure 1 and Susan Suarez for illustrating Figure 2. This work was supported by USPHS research grant NS04989.