Diverse physiological properties of hypoglossal motoneurons innervating intrinsic and extrinsic tongue muscles.

The mammalian tongue contains eight muscles that collaborate to ensure that suckling, swallowing, and other critical functions are robust and reliable. Seven of the eight tongue muscles are innervated by hypoglossal motoneurons (XIIMNs). A somatotopic organization of the XII motor nucleus, defined in part by the mechanical action of a neuron's target muscle, has been described, but whether or not XIIMNs within a compartment are functionally specialized is unsettled. We hypothesize that developing XIIMNs are assigned unique functional properties that reflect the challenges that their target muscle faces upon the transition from in utero to terrestrial life. To address this, we studied XIIMNs that innervate intrinsic and extrinsic tongue muscles, because intrinsic muscles play a more prominent role in suckling than the extrinsic muscles. We injected dextran-rhodamine into the intrinsic longitudinal muscles (IL) and the extrinsic genioglossus, and physiologically characterized the labeled XIIMNs. Consistent with earlier work, IL XIIMNs (n = 150) were located more dorsally within the nucleus, and GG XIIMNs (n = 55) more ventrally. Whole cell recordings showed that resting membrane potential was, on average, 9 mV more depolarized in IL than in GG XIIMNs (P = 0.0019), and the firing threshold in response to current injection was lower in IL (-31 ± 23 pA) than in GG XIIMNs (225 ± 39 pA; P < 0.0001). We also found that the appearance of net outward currents in GG XIIMNs occurred at more hyperpolarized membrane potentials than IL XIIMNs, consistent with lower excitability in GG XIIMNs. These observations document muscle-specific functional specializations among XIIMNs.NEW & NOTEWORTHY The hypoglossal motor nucleus contains motoneurons responsible for innervating one of seven different muscles with notably different biomechanics and patterns of use. Whether or not motoneurons innervating the different muscles also have unique functional properties (e.g., spiking behavior, synaptic physiology) is poorly understood. In this work we show that neonatal hypoglossal motoneurons innervating muscles that shape the tongue (intrinsic longitudinal muscles) have different electrical properties than those innervating the genioglossus, which controls tongue position.

"Conversational Advice": A mixed-methods analysis of medical residents' experiences co-managing primary care patients with behavioral health providers.

OBJECTIVE: When integrated behavioral health clinicians (IBHCs) and residents co-manage patients, residents may learn new approaches. We aimed to understand the effect of co-management on residents' behavioral health (BH) management learning. METHODS: Residents completed a web-based survey enquiring: whether co-management included a shared visit and/or face-to-face meeting with an IBHC, whether residents received feedback from the IBHC, and what they learned. Qualitative responses were coded thematically using a constant comparative method. RESULTS: Among 117 respondents (overall response rate 72%, 117/163), from five residencies recruited from 40 residencies with BH integration, residents were significantly more likely to receive feedback if they had a shared visit with the patient and an IBHC (yes 69% vs. no 33%; adjusted OR 3.0, 95% CI 1.2-7.6). Residents reported three major learning themes: interpersonal communication skills awareness, BH skills awareness, and newly adopted attitudes toward BH. Residents who received feedback were more likely to report themes of interpersonal communication skills awareness (yes 26.6% vs. no 9.4%). CONCLUSION: BH integration promotes increased feedback for residents practicing face-to-face co-management with IBHCs, and a positive influence regarding residents' attitudes and perceived skills. PRACTICAL IMPLICATIONS: Residency programs can meaningfully improve residents' learning by promoting face-to-face co-management with IBHCs.

Residents' Confidence Providing Primary Care With Behavioral Health Integration.

BACKGROUND AND OBJECTIVES: Behavioral health integration (BHI) entails integrated behavioral health clinicians (IBHCs) providing care-generally for mental health and substance abuse disorders and behavioral comorbidity- within the operational functioning of primary care. Because limited data exist regarding BHI in residency, we studied its impact on resident education by examining whether increased behavioral health (BH) co-management improved residents' perceived ability to treat BH conditions. METHODS: We included residents from internal and family medicine training programs using BHI in residents' continuity clinics and assessed the level of co-management between primary care and IBHCs and the following domains: (1) confidence in managing BH conditions, (2) barriers to BH provision, (3) perception of autonomy when working with IBHCs, (4) satisfaction with the clinic, and (5) perceived educational value of BH learning modes. RESULTS: Altogether, 117 residents participated in our survey (73.1% response rate). Residents who had co-managed ≥ five patients alongside IBHCs reported significantly higher confidence than those who had co-managed < five patients with BH conditions. The association remained significant after adjustment for residents' level of training and specialty. In rating BH learning modes, residents rated most highly active collaboration with IBHCs and observation with feedback from clinic preceptors. CONCLUSIONS: BHI training within residency enhances perceived learning and confidence in providing BH care.

Development of a case report review instrument.

Case reports are valued components of the medical literature. The assessment of case reports by editors of medical journals and peer reviewers is largely subjective. The purpose of this study was to develop a reliable instrument to evaluate the quality of written case reports. Instrument development involved review of the literature and the materials provided to peer reviewers who review manuscripts, communications with journal editors and discussions of the study team. After multiple amendments, the instrument was pilot tested on both published and unpublished case reports. Further revisions resulted in the final 11-item tool. Four independent reviewers evaluated 28 case reports in their original submission format that had been submitted to five medical journals. The reviewers were blinded to the specific journal that the manuscripts had been submitted and to whether the case reports had been accepted for publication. Inter-rater reliability was assessed using multirater kappa. Inter-rater reliability ranged from 0.03 to 0.90. The four variables with the highest agreement between raters were (i) rationale for writing the case report; (ii) implications of the case report; (iii) adequacy of the literature review; and (iv) overall impression about whether to accept or reject the manuscript (kappas of 0.67, 0.67, 0.90 and 0.67, respectively). Six of the instrument's first 10 variables were highly correlated with the reviewers' decision about whether to accept or reject the case report for publication (item 11) (all p < 0.001). No correlation existed between the reviewers' decision to accept or reject the manuscript and the actual decision that had been made by the various journals. The case report review instrument is the first such tool for objectively evaluating case reports and appears to have reasonable reliability. Medical journals may wish to incorporate the use of this instrument into the decision making about a case report's suitability for publication.

Changes in calcium signaling during postembryonic dendritic growth in Manduca sexta.

Activity-dependent Ca(2+) influx plays crucial roles in adult and developing nervous systems through its influence on signal processing, synaptic plasticity, and neuronal differentiation. The responses to internal Ca(2+) elevations vary depending on the spatial distribution of Ca(2+) accumulation in different cell compartments. In this study, the mechanisms and the distribution of Ca(2+) accumulation are addressed by in situ Ca(2+) imaging of an identified insect motoneuron, MN5, at critical stages of postembryonic life. During metamorphosis of Manduca sexta, MN5 undergoes extensive dendritic regression followed by regrowth. The time course, amplitude, and distribution of Ca(2+) accumulation within MN5 change during development. During the initial stage of rapid dendritic growth and branching, dendritic growth cones are present, and voltage-dependent Ca(2+) currents are small. At this stage, activity-induced elevations of internal Ca(2+) are largest in the distal dendrites, suggesting that the density of voltage-gated Ca(2+) channels is highest in these regions. Later phases of dendritic growth are accompanied by the transient occurrence of prominent Ca(2+) spikes. Single Ca(2+) spikes cause robust Ca(2+) influx of similar amplitudes and time courses in all central compartments of MN5. The resting Ca(2+) levels also increase during development. Ca(2+)-induced Ca(2+) release from intracellular stores did not contribute to the elevations measured at either stage, although Ca(2+) stores are present in the dendrites. These developmental changes of the internal Ca(2+) signaling are consistent with a regulatory role for activity-dependent Ca(2+) influx in postembryonic dendritic growth.

Nerve-muscle interactions regulate motor terminal growth and myoblast distribution during muscle development.

Interactions between motoneurons and muscles influence many aspects of neuromuscular development in all animals. These interactions can be readily investigated during adult muscle development in holometabolous insects. In this study, the development of the dorsolongitudinal flight muscle (DLM) and its innervation is investigated in the moth, Manduca sexta, to address the specificity of neuromuscular interactions. The DLM develops from an anlage containing both regressed larval template fibers and imaginal myoblasts. In the adult, each fiber bundle (DLM1-5) is innervated by a single motoneuron (MN1-MN5), with the dorsal-most fiber bundle (DLM5) innervated by a mesothoracic motoneuron (MN5). The DLM failed to develop following complete denervation because myoblasts failed to accumulate in the DLM anlage. After lesioning MN1-4, MN5 retained its specificity for the DLM5 region of the anlage and failed to rescue DLM1-4. Thus specific innervation of the DLM fiber bundles does not depend on interactions among motoneurons. Myoblast accumulation, but not myonuclear proliferation, increased around the MN5 terminals, producing a hypertrophied adult DLM5. Therefore, motoneurons compete for uncommitted myoblasts. MN5 terminals subsequently grew more rapidly over the hypertrophied DLM5 anlage, indicating that motoneuron terminal expansion is regulated by the size of the target muscle anlage.

Remodeling of the femoral chordotonal organ during metamorphosis of the hawkmoth, Manduca sexta.

During metamorphosis of the moth, Manduca sexta, the larval legs degenerate and are replaced by adult legs with a diverse array of new sensory organs. The majority of the larval sensory neurons degenerate but some hair sensilla and chordotonal organ sensory neurons survive metamorphosis (Consoulas [2000] J. Comp. Neurol. 419:154-174). In the present study nerve-tracing techniques, birth-date labeling (5-bromodeoxyuridine), and electrophysiology were used to describe the remodeling of the femoral chordotonal organ (FCO) in the prothoracic legs. The larval FCO is composed of two scoloparia, which are associated with separate apodemes. At the onset of metamorphosis, some of the 13 larval neurons degenerate, together with the larval FCO apodemes. The remaining larval FCO sensory neurons persist in the imaginal leg to become the precursors of the adult femoral and tibial chordotonal organs respectively. Early in the pupal stage, 45 to 60 new sensory neurons are generated de novo and become associated with 6 persistent larval neurons in the imaginal femur to compose the adult FCO. Two clusters of persistent and new neurons are enclosed into two scoloparia with short apodemes that eventually become fused. In both larval and adult stages, the FCO contains units that respond phasically and others that respond tonically to femorotibial movements. Nerve activity from the FCO neurons can be recorded continuously during the remodeling of the organ. A persistent leg flexor motoneuron receives inputs from the FCO sensory neurons in both larval and adult stages, offering the opportunity to investigate the remodeling of the neural circuits underlying the proprioceptive control of the femorotibial joint.

Remodeling of membrane properties and dendritic architecture accompanies the postembryonic conversion of a slow into a fast motoneuron.

The postembryonic acquisition of behavior requires alterations in neuronal circuitry, which ultimately must be understood as specific changes in neuronal structure, membrane properties, and synaptic connectivity. This study addresses this goal by describing the postembryonic remodeling of the excitability and dendritic morphology of an identified motoneuron, MN5, which during the metamorphosis of Manduca sexta (L.) changes from a slow motoneuron that is involved in larval-crawling behavior into a fast adult flight motoneuron. A fivefold lower input resistance, a higher firing threshold, and an increase in voltage-activated K(+) current contribute to a lower excitability of the adult MN5, which is a prerequisite for its newly acquired behavioral role. In addition, the adult MN5 displays larger Ca(2+) currents. The dendrites of MN5 undergo extensive remodeling. Drastic regression of larval dendrites during early pupal stages is followed by rapid growth of new dendrites. Critical changes in excitability take place during the onset of adult dendrite formation. Larval Ca(2+) currents are absent when dendritic remodeling is most dramatic but increase markedly during later development. Changes in Ca(2+) and K(+) currents follow different time courses, allowing the transient occurrence of Ca(2+) spikes during pupal stages when new dendritic branching ceases. The adult MN5 can produce prolonged Ca(2+) spikes after K(+) currents are reduced. We suggest that alterations in Ca(2+) and K(+) currents are necessary for the participation of MN5 in flight behavior and that the transient production of Ca(2+) spikes may influence postembryonic dendritic remodeling.

Postembryonic development of the dorsal longitudinal flight muscle and its innervation in Manduca sexta.

The neuromuscular systems of holometabolous insects must be remodeled during metamorphosis to allow striking behavioral changes, such as the acquisition of flight. The fast contracting dorsal longitudinal flight muscle (DLM) of Manduca arises from an anlage containing both remnants of specific larval dorsal body wall muscles and extrinsic myoblasts. In the mesothorax, the DLM is innervated by five persisting larval motoneurons: one in the mesothoracic and four in the prothoracic ganglion. These motoneurons innervate two slowly contracting body wall muscles in the larva. 2 days before pupation, the DLM template fibers begin to degenerate, whereas other muscles remain intact until pupation. Correspondingly, the motor terminals retract from the template fibers while they remain on other muscle fibers until pupation. Accumulation and proliferation of putative myoblasts also starts 2 days before pupation in close spatial relationship to the retracted motor tufts around the degenerating larval template fibers. Proliferation increases through the early pupal stages, and is detected within the anlage until the ninth day after pupation. 2 days after pupation, the anlage splits into five bundles, each innervated by one motoneuron. Striations occur on the seventh day after pupation when the growing motor axons reach the attachment sites. Subsequently, the muscle grows in volume and higher-order motor branches are formed. Within the central nervous system, there is dramatic regression of larval dendrites followed by growth of new dendrites as the persistent motoneurons assume their new role in flight behavior. Both central and peripheral remodeling follow similar time courses.

Comparison of identified leg motoneuron structure and function between larval and adult Manduca sexta.

Persistent leg motoneurons of the moth Manduca sexta were investigated in larval and adult animals to compare their dendritic structures, intrinsic electrical properties and pattern of target innervation. The study focused on two identified motoneurons of the prothoracic leg. Despite the complete remodeling of leg muscles, the motoneurons innervated pretarsal flexor muscles in both larval and adult legs. Similarly, although the central dendrites regress and regrow, the branching pattern was similar with the exception of a prominent midline branch that was not present in the adult stage. The intrinsic electrical properties of the motoneurons differed between larval and adult stages. Larval motoneurons had significantly higher membrane input resistances and more depolarized resting membrane potentials than did motoneurons in pharate adults or adults. In all stages, one motoneuron had a low maximal firing frequency, whereas the second motoneuron, which innervated the other half of the muscle, had a high maximum firing frequency. Although the two motoneurons continued to innervate the same halves of the target muscle, their relative effects on muscular contraction were reversed during metamorphosis along with concomitant changes in intrinsic properties. Pretarsal flexor motoneurons in pharate adults (just prior to emergence) displayed properties similar to those in emerged adults.

Behavioral transformations during metamorphosis: remodeling of neural and motor systems.

During insect metamorphosis, neural and motor systems are remodeled to accommodate behavioral transformations. Nerve and muscle cells that are required for larval behavior, such as crawling, feeding and ecdysis, must either be replaced or respecified to allow adult emergence, walking, flight, mating and egg-laying. This review describes the types of cellular changes that occur during metamorphosis, as well as recent attempts to understand how they are related to behavioral changes and how they are regulated. Within the periphery, many larval muscles degenerate at the onset of metamorphosis and are replaced by adult muscles, which are derived from myoblasts and, in some cases, remnants of the larval muscle fibers. The terminal processes of many larval motoneurons persist within the periphery and are essential for the formation of adult muscle fibers. Although most adult sensory neurons are born postembryonically, a subset of larval proprioceptive neurons persist to participate in adult behavior. Within the central nervous system, larval neurons that will no longer be necessary die and some adult interneurons are born postembryonically. By contrast, all of the adult motoneurons, as well as some interneurons and modulatory neurons, are persistent larval cells. In accordance with their new behavioral roles, these neurons undergo striking changes in dendritic morphology, intrinsic biophysical properties, and synaptic interactions.

An outbreak of gram-negative bacteremia in hemodialysis patients traced to hemodialysis machine waste drain ports.

OBJECTIVE: To investigate an outbreak of gram-negative bacteremias at a hemodialysis center (December 1, 1996-January 31, 1997). DESIGN: Retrospective cohort study. Reviewed infection control practices and maintenance and disinfection procedures for the water system and dialysis machines. Performed cultures of the water and dialysis machines, including the waste-handling option (WHO), a drain port designed to dispose of saline used to flush the dialyzer before patient use. Compared isolates by pulsed-field gel electrophoresis. SETTING: A hemodialysis center in Maryland. RESULTS: 94 patients received dialysis on 27 machines; 10 (11%) of the patients had gram-negative bacteremias. Pathogens causing these infections were Enterobacter cloacae (n = 6), Pseudomonas aeruginosa (n = 4), and Escherichia coli (n = 2); two patients had polymicrobial bacteremia. Factors associated with development of gram-negative bacteremias were receiving dialysis via a central venous catheter (CVC) rather than via an arterio-venous shunt (all 10 infected patients had CVCs compared to 31 of 84 uninfected patients, relative risk [RR] undefined; P<.001) or dialysis on any of three particular dialysis machines (7 of 10 infected patients were exposed to the three machines compared to 20 of 84 uninfected patients, RR = 5.8; P = .005). E cloacae, P aeruginosa, or both organisms were grown from cultures obtained from several dialysis machines. WHO valves, which prevent backflow from the drain to dialysis bloodlines, were faulty in 8 (31%) of 26 machines, including 2 of 3 machines epidemiologically linked to case-patients. Pulsed-field gel electrophoresis patterns of available dialysis machine and patient E cloacae isolates were identical. CONCLUSIONS: Our study suggests that WHO ports with incompetent valves and resultant backflow were a source of cross-contamination of dialysis bloodlines and patients' CVCs. Replacement of faulty WHO valves and enhanced disinfection of dialysis machines terminated the outbreak.

Peripheral distribution of presynaptic sites of abdominal motor and modulatory neurons in Manduca sexta larvae.

Insect muscle fibers are commonly innervated by multiple motor neurons and efferent unpaired median (UM) neurons. The role of UM neurons in the modulation rather than rapid activation of muscle contraction (Evans and O'Shea [1977] Nature 270:257-259) suggests that their terminal varicosities may differ structurally and functionally from the presynaptic terminals of motor neurons. Furthermore, differences in the characteristics of UM neuron terminal varicosities may be correlated with functional differences among their diverse target muscles. Larval abdominal body wall muscles in the hawkmoth, Manduca sexta, consist of large, elongated fibers that are multiterminally innervated by one and occasionally two motor neurons (Levine and Truman [1985] J. Neurosci. 5:2424-2431). The fibers are also innervated by one of two efferent UM neurons that bifurcate to innervate targets on both sides of the abdomen (Pflüger et al. [1993] J. Comp. Neurol. 335:508-522). In this study, the intracellular tracer biocytin was used to identify the targets of the UM neurons and to distinguish their terminal axonal varicosities on the muscle fibers. An antiserum to the synaptic vesicle protein, synaptotagmin, was used to label synaptic vesicles, and the styryl dye FM1-43 was used to demonstrate release and recycling. Most of the abdominal muscles in a given hemisegment were found to be supplied by one of the two UM neurons. Terminal varicosities of the excitatory motor neurons were large (3-7 pm) and were found in rows of rosettes that extended to every aspect of the muscle fiber; these varicosities were designated as type I terminals. The UM neuron terminal varicosities also occupied every aspect of the fiber but were smaller (1-3 microm) and more separated from each other; these were designated as type II terminals. Both type I and type II terminals are synaptotagmin immunoreactive and, as shown by FM1-43 staining, are sites of synaptic vesicle recycling. The excitatory motor neuron terminals (type I) could easily be loaded and unloaded with FM1-43, which indicates their capacity for repeated vesicular exocytosis and recycling. In contrast, the dye could not as readily be unloaded from UM neuron terminals (type II), which may indicate that they have a slower turnover of synaptic vesicles.

Steroid hormone enhancement of neurite outgrowth in identified insect motor neurons involves specific effects on growth cone form and function.

Dramatic reorganization of dendrites and axonal terminals is a hallmark of neuronal remodeling during metamorphosis in the hawkmoth, Manduca sexta. The dendritic and axonal arbors of leg motor neurons regress in late larval stages, then regrow during adult development. Ecdysteroids, the insect steroids that trigger metamorphosis, control both regression and outgrowth in vivo and stimulate neuritic growth in cultured pupal leg motor neurons. To identify subcellular targets of ecdysteroid action in these neurons, we examined the dynamic and structural features of branching and their modulation by ecdysteroids in vitro. Delayed treatment of pupal leg motor neurons with ecdysteroid led to a robust enhancement of neuritic branch accumulation accompanied by a subtle effect on total neuritic length. Repeated imaging revealed that branch formation occurred almost exclusively at the growth cone; interstitial branching was extremely rare. Ecdysteroid treatment significantly enhanced both the formation and retention of branches at the growth cone. Branches formed via two distinct processes: engorgement (of fine protrusions) and condensation (of lamellae) with the relative contributions of these mechanisms being unaltered by ecdysteroid. Confocal imaging of the cytoskeleton demonstrated that growth cones consisted of microtubule-based domains fringed by actin-based filopodia. Treated growth cones were larger and displayed increased numbers of microtubule-based branches, whereas filopodial density was unaffected. These findings indicate that ecdysteroid enhances neuritic branching by altering growth cone structure and function, and suggest that hormonal modulation of cytoskeletal interactions contributes significantly to neuritic remodeling during metamorphosis.

Ecdysteroid control of ionic current development in Manduca sexta motoneurons.

The steroid hormone 20-hydroxyecdysone (20-HE) regulates several processes during insect metamorphosis. We studied the effects of 20-HE on the development of voltage-sensitive ionic currents of thoracic leg motoneurons of Manduca sexta. The larval leg motoneurons persist throughout metamorphosis but undergo substantial morphological reorganization, which is under the control of 20-HE and accompanied by changes in Ca2+ and K+ current densities. To determine whether 20-HE controls the changes in Ca2+ and K+ current levels during postembryonic development, identified thoracic leg motoneurons isolated from late larval and early pupal stages were taken into primary cell culture. Whole-cell Ca2+ and K+ currents were measured after 1-4 days of steroid hormone incubation. In the presence of 20-HE, peak Ca2+ currents of pupal leg motoneurons increased from day 1 to day 4 in vitro. Thus, at culture day 4 the pupal Ca2+ current levels were larger in 20-HE-treated than in untreated cells. By contrast, 20-HE did not affect the Ca2+ current amplitudes of larval leg motoneurons. Whole-cell K+ currents, measured at 4 days in pupal motoneurons, consisted of a fast-activating transient current and a sustained, slowly inactivating current. 20-HE did not affect the amplitude of the transient or sustained currents after 4 days in vitro. Thus, a direct steroid hormone effect may control the proper maturation of voltage-sensitive Ca2+ currents in leg motoneurons.

The steroid hormone 20-hydroxyecdysone enhances neurite growth of Drosophila mushroom body neurons isolated during metamorphosis.

Mushroom bodies (MBs) are symmetrically paired neuropils in the insect brain that are of critical importance for associative olfactory learning and memory. In Drosophila melanogaster, the MB intrinsic neurons (Kenyon cells) undergo extensive reorganization at the onset of metamorphosis. A phase of rapid axonal degeneration without cell death is followed by axonal regeneration. This re-elaboration occurs as levels of the steroid hormone 20-hydroxyecdysone (20E) are rising during the pupal stage. Based on the known role of 20E in directing many features of CNS remodeling during insect metamorphosis, we hypothesized that the outgrowth of MB axonal processes is promoted by 20E. Using a GAL4 enhancer trap line (201Y) that drives MB-restricted reporter gene expression, we identified Kenyon cells in primary cultures dissociated from early pupal CNS. Paired cultures derived from single brains isolated before the 20E pupal peak were incubated in medium with or without 20E for 2-4 d. Morphometric analysis demonstrated that MB neurons exposed to 20E had significantly greater total neurite length and branch number compared with that of MB neurons grown without hormone. The relationship between branch number and total neurite length remained constant regardless of hormone treatment in vitro, suggesting that 20E enhances the rate of outgrowth from pupal MB neurons in a proportionate manner and does not selectively increase neuritic branching. These results implicate 20E in enhancing axonal outgrowth of Kenyon cells to support MB remodeling during metamorphosis.

Presynaptic function during muscle remodeling in insect metamorphosis.

During metamorphosis the leg neuromuscular system of the moth Manduca sexta undergoes an extensive remodeling as the larval muscles degenerate and are replaced by new muscles in the adult. The terminal processes of persistent leg motoneurons undergo severe regression followed by regrowth (Consoulas et al., 1996), accompanied, as shown here, by the loss and re-establishment of functional presynaptic specializations. Before and shortly after the degeneration of the larval muscle, immunoreactivity for the vesicular protein synaptotagmin was localized to the presynaptic varicosities of the motoneurons. Similarly localized were distinct sites of Ca2+-dependent uptake of the fluorescent dye FM1-43. During myoblast migration and accumulation about the re-expanding motor axons, synaptotagmin immunoreactivity was widely distributed in axons, and specific FM1-43 staining revealed vesicle exocytosis in distal axon branches. During myoblast proliferation and fusion, and myotube formation, synaptotagmin staining remained widely distributed in nerve branches, whereas FM1-43 staining was more localized to subdomains of these nerve branches. These initial presynaptic active sites were transient and were replaced by new sites in more distal nerve processes as the muscle anlage increased in size and additional myotubes formed. After myotube separation, synaptotagmin staining disappeared from primary branches but remained distributed within secondary and high-order nerve branches. FM1-43 staining was detected in high-order branches only. During muscle fiber striation, growth, and maturation, both FM1-43 staining and synaptotagmin immunoreactivity became localized to terminal varicosities. Thus, presynaptic function can persist after the loss of the target and occurs transiently in axon shafts before becoming restricted to terminal domains as the underlying muscle fibers mature.

Segmentally distributed metamorphic changes in neural circuits controlling abdominal bending in the hawk moth Manduca sexta.

During the metamorphosis of Manduca sexta the larval nervous system is reorganized to allow the generation of behaviors that are specific to the pupal and adult stages. In some instances, metamorphic changes in neurons that persist from the larval stage are segment-specific and lead to expression of segment-specific behavior in later stages. At the larval-pupal transition, the larval abdominal bending behavior, which is distributed throughout the abdomen, changes to the pupal gin trap behavior which is restricted to three abdominal segments. This study suggests that the neural circuit that underlies larval bending undergoes segment specific modifications to produce the segmentally restricted gin trap behavior. We show, however, that non-gin trap segments go through a developmental change similar to that seen in gin trap segments. Pupal-specific motor patterns are produced by stimulation of sensory neurons in abdominal segments that do not have gin traps and cannot produce the gin trap behavior. In particular, sensory stimulation in non-gin trap pupal segments evokes a motor response that is faster than the larval response and that displays the triphasic contralateral-ipsilateral-contralateral activity pattern that is typical of the pupal gin trap behavior. Despite the alteration of reflex activity in all segments, developmental changes in sensory neuron morphology are restricted to those segments that form gin traps. In non-gin trap segments, persistent sensory neurons do not expand their terminal arbors, as do sensory neurons in gin trap segments, yet are capable of eliciting gin trap-like motor responses.

Multisegmental motor activity in the segmentally restricted gin trap behavior in Manduca sexta pupae.

Stimulation of sensory neurons innervating hairs in the gin traps on the abdomen of Manduca sexta pupae evokes a rapid bending of the abdomen that is restricted to one or more of the three articulating posterior segments. However, electrical stimulation of the gin trap sensory nerve in an isolated abdominal nerve cord evokes characteristic motor neuron activity in every abdominal segment. To determine if the segmentally distributed motor activity also occurred in intact animals and how it contributed to the segmentally restricted reflex movement, mechanical stimulation of the sensory hairs in intact animals was used to evoke reflex responses that were recorded as electromyograms synchronized with video recordings of the behavior. Motor activity was monitored during movements to determine if there was activity in many segments when the movement was restricted to one segment. Coordinated muscle activity was evoked throughout the abdomen in response to stimulation of any of the three gin traps, even when movement was restricted to one segment. Differences in the timing of ipsilateral and contralateral motor activity among segments allowed the closing of gin traps to be segmentally restricted. These findings suggest that the neural circuit underlying the gin trap reflex is distributed throughout the abdominal nerve cord. This network generates a complex, yet coordinated, motor pattern with muscular activity in many abdominal segments that produces a localized bending reflex.