Seasonal differences in the physiology and morphology of crayfish motor terminals

The physiology and morphology of identified crayfish motor terminals were compared at different seasons. We examined initial excitatory postsynaptic potential (EPSP) amplitudes, synaptic fatigue, and the frequency of synaptic varicosities along the motor terminals of an identified phasic motoneuron in animals collected over a period of 5 years. The physiology and morphology of identified crayfish motor terminals are different for animals collected in summer and winter. In winter animals, phasic axon motor terminals in the claw closer muscle produce large EPSPs initially, but show dramatic synaptic fatigue during repetitive stimulation. In summer animals, these terminals produce smaller initial EPSPs, but are more fatigue resistant. Due to their greater fatigue resistance, synaptic terminals have a greater over-all capacity for transmitter release in summer animals than do those of winter animals. Morphologically, terminals in summer animals have more synaptic varicosities, this result supports earlier studies that have shown that fatigue-resistant motor terminals have more synaptic varicosities. Experiments in which the electrical activity of the motoneuron was experimentally altered suggest that these differences in motor terminals may be due to seasonal differences in activity.     

Sexual differentiation of identified motor terminals in Drosophila larvae

In Drosophila, we have found that some of the motor terminals in wandering third-instar larvae are sexually differentiated. In three out of the four body-wall muscle fibers that we examined, we found female terminals that produced a larger synaptic response than their male counterparts. The single motor terminal that innervates muscle fiber 5 produces an EPSP that is 69% larger in females than in males. This is due to greater release of transmitter from female than male synaptic terminals because the amplitude of spontaneous miniature EPSPs was similar in male and female muscle fibers. This sexual difference exists throughout the third-instar: it is seen in both early (foraging) and late (wandering) third-instar larvae. The sexual differentiation appears to be neuron specific and not muscle specific because the same axon produces Is terminals on muscle fibers 2 and 4, and both terminals produce larger EPSCs in females than males. Whereas, the Ib terminals innervating muscle fibers 2 and 4 are not sexually differentiated. The differences in transmitter release are not due to differences in the size of the motor terminals. For the terminal on muscle fiber 5 and the Is terminal on muscle fiber 4, there were no differences in terminal length, the number of branches, or the number of synaptic boutons in males compared to females. These sexual differences in neuromuscular synaptic physiology may be related to male-female differences in locomotion.     

Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.     

Sympatric Drosophila simulans flies with distinct mtDNA show age related differences in mitochondrial metabolism

The primary causes of age-related changes in mitochondrial metabolism are not known. The goal of this study is to document the influence of naturally occurring mtDNA variation on age dependent changes in mitochondrial respiration, hydrogen peroxide (H(2)O(2)) generation and antioxidant defenses in the fly Drosophila simulans. Possible changes include an increase in rates of reactive oxygen species production with age and/or an age dependent decrease in antioxidant response. For this study we have used flies harboring distinct siII and siIII mtDNA types. Previously we have shown that males harboring siII mtDNA had higher rates of mitochondrial H(2)O(2) production from complex III at 11d compared to males with the siIII mtDNA type. Here, we corroborate those results and show that Drosophila harboring the siII and siIII mtDNA types exhibit significantly different patterns of pro-oxidant and antioxidant activities as they age. Flies harboring siII mtDNA had higher rates of mitochondrial H(2)O(2) production and manganese superoxide dismutase activity at 11 and 18d of age than siIII mtDNA harboring flies. Copper-zinc superoxide dismutase activity increased from 11 to 25d in siII flies while the accumulation of oxidized glutathione did not change between 11 and 25d. In contrast, siIII harboring flies showed an age dependent increase in H(2)O(2) production, reaching higher production rates on day 25 than that observed in siII flies. Copper-zinc superoxide dismutase activities did not change between 11 and 25d while the oxidized glutathione accumulation increased with age. The results show antioxidant levels correlate with pro-oxidant levels in siII but not siIII flies. These results demonstrate our ability to correlate mtDNA variation with differences in whole mitochondrial physiology and individual complex biochemistry.     

Effect of reduced impulse activity on the development of identified motor terminals in Drosophila larvae

In Drosophila larvae, motoneurons show distinctive differences in the size of their synaptic boutons; that is, axon 1 has type Ib ("big" boutons) terminals and axon 2 has type Is ("small" boutons) terminals on muscle fibers 6 and 7. To determine whether axon 1 develops large boutons due to its high impulse activity, we reduced impulse activity and examined the motor terminals formed by axon 1. The number of functional Na(+) channels was reduced either with the nap(ts) mutation or by adding tetrodotoxin (TTX) to the media (0.1 microg/g). In both cases, the rate of locomotion was decreased by approximately 40%, presumably reflecting a decrease in impulse activity. Locomotor activity was restored to above wild-type (Canton-S) levels when nap(ts) was combined with a duplication of para, the Na(+)-channel gene. Lucifer yellow was injected into the axon 1 motor terminals, and we measured motor terminal area, length, the number of branches, and the number and width of synaptic boutons. Although all parameters were smaller in nap(ts) and TTX-treated larvae compared to wild-type, most of these differences were not significant when the differences in muscle fiber size were factored out. Only bouton width was significantly smaller in both different nap(ts) and TTX-treated larvae: boutons were about 20% smaller in nap(ts) and TTX-treated larvae, and 20% larger in nap(ts); Dp para(+) compared to wild-type. In addition, terminal area was significantly smaller in nap(ts) compared to wild-type. Bouton size at Ib terminals with reduced impulse activity was similar to that normally seen at Is terminals. Thus, differences in impulse activity play a major role in the differentiation of bouton size at Drosophila motor terminals.     

Synaptic ultrastructure in nerve terminals of Drosophila larvae overexpressing the learning gene dunce

We investigated synaptic ultrastructure of individual nerve ending varicosities at the Drosophila larval neuromuscular junction in transgenic larvae overexpressing the learning gene dunce (dnc) in the nervous system. It was previously shown that cAMP is reduced to one-third normal in these larvae and that they have fewer nerve terminal varicosities and smaller junction potentials, although transmitter release from individual nerve ending varicosities is not significantly altered. We tested the hypothesis that synaptic ultrastructure is modified to compensate for possible reduced efficacy of synaptic transmission resulting from lower than normal cAMP. Synaptic size and number of presynaptic dense bodies (active zone structures) per synapse are modestly enhanced in transgenic larvae overexpressing the dnc gene product and in rutabaga (rut(1)) mutant larvae, which have reduced adenylyl cyclase activity and reduced neural cAMP. The incidence of complex synapses (possessing 2 or more presynaptic dense bodies) was not consistently different in experimental larvae compared to controls. The observations suggest that chronic reduction of cAMP levels in the nervous system of Drosophila larvae, although leading to a modest compensatory change in synaptic structure, does not markedly alter several synaptic ultrastructural parameters which are thought to influence the strength of transmitter release; thus, homeostatic mechanisms do not act to maintain normal-sized junction potentials by altering synaptic structure.     

Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae

The innervation of ventral longitudinal abdominal muscles (muscles 6, 7, 12, and 13) of third-instar Drosophila larvae was investigated with Nomarski, confocal, and electron microscopy to define the ultrastructural features of synapse-bearing terminals. As shown by previous workers, muscles 6 and 7 receive in most abdominal segments “Type I” endings, which are restricted in distribution and possess relatively prominent periodic terminal enlargements (“boutons”); whereas muscles 12 and 13 have in addition “Type II” terminals, which are more widely distributed and have smaller “boutons.” Serial sectioning of the Type I innervation of muscles 6 and 7 showed that two axons with distinctive endings contribute to it. One axon (termed Axon 1) has somewhat larger boutons, containing numerous synapses and presynaptic dense bodies (putative active zones for transmitter release). This axon also has more numerous intraterminal mitochondria, and a profuse subsynaptic reticulum around or under the synaptic boutons. The second axon (Axon 2) provides somewhat smaller boutons, with fewer synapses and dense bodies per bouton, fewer intraterminal mitochondria, and less-developed subsynaptic reticulum. Both axons contain clear synaptic vesicles, with occasional large dense vesicles. Approximately 800 synapses are provided by Axon 1 to muscles 6 and 7, and approximately 250 synapses are provided by Axon 2. In muscles 12 and 13, endings with predominantly clear synaptic vesicles, generally similar to the Type I endings of muscles 6 and 7, were found, along with another type of ending containing predominantly dense-cored vesicles, with small clusters of clear synaptic vesicles. This second type of ending was found most frequently in muscle 12, and probably corresponds to a subset of the “Type II” endings seen in the light microscope. Type I endings are thought to generate the ?fast’? and ?slow’? junctional potentials seen in electrophysiological recordings, whereas the physiological actions of Type II endings are presently not known. © 1993 John Wiley & Sons, Inc.     

Crayfish sensory terminals and motor neurones exhibit two distinct types of GABA receptors

Motor neurones of the crayfish walking system display inhibitory responses evoked either by γ-amino butyric acid (GABA) or glutamate, possibly involving the same receptor (Pearlstein et al. 1994). In order to test if this sensibility to both GABA and glutamate was a specific property of crayfish GABA receptors, pharmacological characteristics of GABA-evoked responses in both sensory terminals from CB chordotonal organ and motor neurones of the walking system have been compared. Both receptors are GABA-gated Cl⁻ channels activated by specific GABA_A (muscimol, isoguvacine), GABA_B (3-aminopropyl phosphinic acid), and GABA_C (cis-4-amino crotonic acid) agonists, and blocked by competitive (β-guanidino propionic acid) and non-competitive (picrotoxin) antagonists. They were insensitive to specific GABA_A (bicuculline, SR-95531) and GABA_B (phaclofen) antagonists. Furthermore, in both cases, nipecotic acid and the modulatory drug diazepam had no effect. However, our results demonstrate that GABA receptors of sensory terminals are different from those of motor neurones. GABA-induced desensitisation only occurred in sensory terminals. Moreover, glutamate was shown to activate GABA-gated Cl⁻ channels in motor neurones, but not in sensory terminals. Therefore, GABA is likely to be the endogenous neurotransmitter of presynaptic inhibition in sensory terminals, whereas inhibition between antagonistic motor neurones would be achieved by glutamate. Accepted: 10 July 1996     

Topological differences along mammalian motor nerve terminals for spontaneous and alpha-bungarotoxin-induced sprouting

Spontaneous sproutings can be observed in end plates from normal adult vertebrate muscles and motor end plates develop increased growth signs and sprouts when target muscle cells become less active or paralysed. Nevertheless, very little is known about where in the motor nerve terminal arborization spontaneous and experimentally induced sprouts originate, their similarities and differences and also about their final maturation or elimination. In this study we investigate the topological properties of both spontaneous and alpha-bungarotoxin-induced sprouts (during different periods of intoxication and after recovery) along the motor nerve terminal branches of the Levator auris longus muscle of Swiss mice (between 48-169 day old). Muscles were processed for immunocytochemistry to simultaneously detect postsynaptic AChRs and axons. This procedure permits us to make an accurate identification of the fine sprouts and a morphometric study of the presynaptic branching pattern profile in control muscles, during the toxin action and after recovery from paralysis. The results show that in normal muscles, the initial and trunk segments (those between branch points) of the terminal arborization sprouted proportionally more branches when taking their relative lengths into account than the distal free-end segments. In contrast, every micrometer of alpha-bungarotoxin-treated muscles throughout the full terminal arborization have the same probability of generating a sprout. Moreover, the toxin-induced sprouts can consolidate as new branches once recovered from the paralysis without changing the total length of the nerve terminal arborization.     

Sympatric Drosophila simulans flies with distinct mtDNA show difference in mitochondrial respiration and electron transport

The role of mitochondrial DNA (mtDNA) in mitochondrial metabolism is understudied yet humans harboring specific mtDNA types age at dissimilar rates, are unequally susceptible to various diseases, and differentially adapt to various environmental conditions. This study compares mitochondrial respiration, proton leak and electron transport of Drosophila simulans males with distinct mtDNA haplogroups (siII and -III) that were collected in sympatry in Kenya. Despite the large divergence among haplogroups there is very low intrahaplogroup variation and no correlated variation in the nuclear genome has been detected. We show that repeatable bioenergetic differences exist between 11d old males harboring siII and siIII mtDNA. Males with siIII mtDNA showed higher (i) state 3 respiration rates from isolated mitochondria for both complex I and complex III based substrates, and (ii) complex IV (cytochrome c oxidase) activity. Males harboring siIII mtDNA had lower (i) hydrogen peroxide formation by both complexes I and III, (ii) proton leak from isolated mitochondria, (iii) mitochondrial ATPase activity, and (iv) mitochondrial cytochrome content. In combination, the results suggest that mitochondria isolated from siIII mtDNA harboring males have more efficient metabolism than siII mtDNA harboring males.     

Flight initiations in Drosophila melanogaster are mediated by several distinct motor patterns

We have monitored the patterns of activation of five muscles during flight initiation of Drosophila melanogaster: the tergotrochanteral muscle (a mesothoracic leg extensor), dorsal longitudinal muscles #3, #4 and #6 (wing depressors), and dorsal ventral muscle #Ic (a wing elevator). Stimulation of a pair of large descending interneurons, the giant fibers, activates these muscles in a stereotypic pattern and is thought to evoke escape flight initiation. To investigate the role of the giant fibers in coordinating flight initiation, we have compared the patterns of muscle activation evoked by giant fiber stimulation with those during flight initiations executed voluntarily and evoked by visual and olfactory stimuli. Visually elicited flight initiations exhibit patterns of muscle activation indistinguishable from those evoked by giant fiber stimulation. Olfactory-induced flight initiations exhibit patterns of muscle activation similar to those during voluntary flight initiations. Yet only some benzaldehyde-induced and voluntary flight initiations exhibit patterns of muscle activation similar to those evoked by giant fiber stimulation. These results indicate that visually elicited flight initiations are coordinated by the giant fiber circuit. By contrast, the giant fiber circuit alone cannot account for the patterns of muscle activation observed during the majority of olfactory-induced and voluntary flight initiations.Abbreviations DLM/DLMn dorsal longitudinal muscle/motor neuron - DVM/DVMn dorsal ventral muscle/motor neuron - GF(s) giant fiber interneuron (s) - PSI peripherally synapsing interneuron - TTM/TTMn tergotrochanteral muscle/motor neuron     

The morphology,physiology and function of suboesophageal neck motor neurons in the honeybee

We report some of the neural and muscular circuitry that allows honeybees to control head movements. We studied neck motor neurons with cell bodies in the suboesophageal ganglion, axons in the first cervical nerve (IK1) and terminals in neck muscles 44 and 51 (muscle classification: Snodgrass in Smithsonian Misc Coll 103:1-120, 1942). We show that muscle 44 actually comprises five separate bundles of muscle fibres (subunits), while muscle 51 is split into two subunits. Eight motor neurons innervate muscles 44 and 51. Two motor neurons have cell bodies in the ventral-median cell body group (one innervates a subunit in muscle 44, the other a subunit in muscle 51). One motor neuron has a ventrally located contralateral cell body (innervating a subunit in muscle 44) and five have laterally located ipsilateral cell bodies. Of the five lateral cells, one innervates a subunit in muscle 51, three selectively innervate subunits in muscle 44 and one co-innervates a subunit in muscle 44 with the contralateral cell. Extracellular recordings revealed three types of visually driven, direction-selective cell-types in each IK1 tuned for leftward, rightward and downward motion over the eyes. The spatiotemporal tuning of the units is similar to that of other visual interneurons in the bee brain.     

Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae

During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, we address the question of which neurons attribute these values to odor stimuli. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. We report the use of transgenically expressed channelrhodopsin-2, a light-activated cation channel, as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. We demonstrate that distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning.     

Local inhibitor of the crayfish telson-flexor motor giant neurons: morphology and physiology

The motor circuits that control telson flexion in the crayfish (Procambarus clarkii) include a curiously arranged sub-circuit: a premotor 'command' neuron excites a motor neuron via a trisynaptic pathway, but also inhibits (and prevents firing of) the motor neuron via a shorter latency pathway (Kramer et al. 1981 a). The premotor and motor neurons in this circuit have been previously identified (Kramer et al. 1981 a; Dumont and Wine 1985a, b; see Fig. 1). We have now identified a local interneuron that inhibits the motor neurons. The cell we studied is called the 'C' cell because of its distinctive structure (Figs. 2, 3). A single pair of bilaterally homologous C-cells was found in the last (6th) abdominal ganglion. The C-cells are invariably dye coupled to one another following injections of lucifer yellow into either one of them, and are frequently dye coupled to smaller axons in the 2nd, 3rd, and 6th nerves. In addition, some of the extensive branches of the C-cell extend out into the 6th nerve, where they are in close proximity to the axons of the motor neurons they inhibit (Fig. 3). Two kinds of evidence established that the C-cell directly inhibits the motor neurons. First, when simultaneous recordings were made from the C-cell and the motor neurons, spikes in the C-cell, no matter how evoked, were invariably followed, within 1.5 ms, by depolarizing IPSPs in the motor neuron (Fig. 6). Second, when the C-cell was hyperpolarized so that it could not fire, that same IPSP in the motor neuron was abolished (Fig. 6). The inhibitory pathway to the motor neurons must be fired at short latency in order to prevent firing caused by the trisynaptic excitatory input (Fig. 1). The C-cells were fired at short latency (less than 3 ms) by impulses in either of the escape command cells (Fig. 4), and at even shorter latency by impulses in the Segmental Giant of the 6th ganglion (SG6) (Fig. 5). It has been established elsewhere that the SGs are a major output pathway of the escape command cells; our results suggest that they may be the pathway for command-evoked firing of the C-cell. The C-cells are also excited by two descending, non-giant, flexion premotor neurons, called I2 and I3 (Fig. 5). The EPSPs from a single I2 or I3 impulse were subthreshold, but temporal and spatial summation of EPSPs from the non-giant pathway sometimes fired the C-cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The morphology, physiology, and neural projections of supernumerary compound eyes in Drosophila melanogaster

Supernumerary compound eyes in Drosophila melanogaster produced by the extra eye (ee) mutation were analyzed with regard to their morphology, physiology, and neural projections. Electron and light microscopy revealed that large extra eyes often possess the normal complement of compound-eye cell types and that these cells usually have standard fine structure. In addition, the array of photoreceptor cell rhabdomeres within individual supernumerary ommatidia is standardly trapezoidal, and ommatidial subpopulations having mirror-image configurations of their rhabdomeric trapezoids are separated by an equator in extra eyes. Light stimulation of supernumerary eyes can elicit photoreceptor depolarization potentials as evidenced by electroretinographic recordings from them. In addition, extra-eye photoreceptor cells have a functional pupillary response to light stimulation. Although the supernumerary eyes can be functionally and anatomically standard, examination of serial, silver-stained sections of extra-eye heads has shown that their photoreceptor axons seldom innervate the brain. This situation obtains even in a case in which the normal, ipsilateral compound eye was removed by the eyeless mutation. In contrast, rare supernumerary antennae occasionally found in ee stocks have receptor cells whose axons innervate ventral brain. In addition to duplications of cuticular epithelia, extra glial cells, muscle fibers, and ocellar interneurons are sometimes found in extra-eye bearing flies. Discussion of these results focuses on a polarity guidance hypothesis which models the growth of adult photoreceptor axons into the brain during normal development.     

Dendrites of distinct classes of Drosophila sensory neurons show different capacities for homotypic repulsion

BACKGROUND: Understanding how dendrites establish their territory is central to elucidating how neuronal circuits are built. Signaling between dendrites is thought to be important for defining their territories; however, the strategies by which different types of dendrites communicate are poorly understood. We have shown previously that two classes of Drosophila peripheral da sensory neurons, the class III and class IV neurons, provide complete and independent tiling of the body wall. By contrast, dendrites of class I and class II neurons do not completely tile the body wall, but they nevertheless occupy nonoverlapping territories. RESULTS: By developing reagents to permit high-resolution studies of dendritic tiling in living animals, we demonstrate that isoneuronal and heteroneuronal class IV dendrites engage in persistent repulsive interactions. In contrast to the extensive dendritic exclusion shown by class IV neurons, duplicated class III neurons showed repulsion only at their dendritic terminals. Supernumerary class I and class II neurons innervated completely overlapping regions of the body wall, and this finding suggests a lack of like-repels-like behavior. CONCLUSIONS: These data suggest that repulsive interactions operate between morphologically alike dendritic arbors in Drosophila. Further, Drosophila da sensory neurons appear to exhibit at least three different types of class-specific dendrite-dendrite interactions: persistent repulsion by all branches, repulsion only by terminal dendrites, and no repulsion.