Janis C. Weeks

A reflex behavior mediated by monosynaptic connections between hair afferents and motoneurons in the larval tobacco hornworm, Manduca sexta.

Summary1.In the tobacco hornworm caterpillar, tactile stimulation of sensory hairs located on the tip of a proleg (the planta) evokes ipsilateral or bilateral retraction of the prolegs in that segment (Figs. 1,2). We have used electrophysiological and anatomical methods to investigate the excitatory neural pathways linking the planta hair afferents and the proleg retractor motoneurons (MNs). An important technical innovation was the development of an isolated proleg and desheathed ganglion preparation that permits rapid and reversible ionic manipulations and drug applications (Fig. 3).2.Action potentials (spikes) in individual planta hair afferents produce time-locked excitatory postsynaptic potentials (EPSPs) in ipsilateral proleg MNs (Fig. 3) which appear to be chemically-mediated and monosynaptic: the EPSPs have a short and constant latency, they follow afferent spikes without failure, they are reversibly abolished in elevated Mg++ saline (Fig. 7), and they persist in saline with elevated Mg++ and Ca++ levels (Fig. 8). Planta hair afferents also excite ipsilateral MNs by polysynaptic pathways, and their excitation of contralateral proleg MNs is exclusively polysynaptic.3.Cobalt-staining of the proleg MNs and planta hair afferents (Fig. 6) shows that the afferents terminate in ventral neuropil, and the proleg MNs have an unusual ventral projection into this region. The ventral projection is on the ipsilateral side, which is consistent with the electrophysiological finding that time-locked EPSPs are found only from ipsilateral hairs.4.Two factors that contribute to the strong monosynaptic excitation of proleg MNs by ipsilateral planta hairs are the convergence of many hair afferents onto each MN (Fig. 5), and the facilitation shown at each afferent-MN synapse (Fig. 9). At least 6 afferents converge on each MN, and at short interspike intervals the afferent-evoked EPSPs are enhanced by as much as 400% by homosynaptic facilitation.5.The EPSP is abolished reversibly by the cholinergic antagonists curare and atropine (Fig. 10), suggesting that the neurotransmitter at the synapse is acetylcholine (ACh). This is of particular interest because the ACh receptors of tobacco-feedingManduca larvae are reported to be less nicotinesensitive than those of other insects.

A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans

This paper describes the fabrication and use of a microfluidic device for performing whole-animal chemical screens using non-invasive electrophysiological readouts of neuromuscular function in the nematode worm, C. elegans. The device consists of an array of microchannels to which electrodes are attached to form recording modules capable of detecting the electrical activity of the pharynx, a heart-like neuromuscular organ involved in feeding. The array is coupled to a tree-like arrangement of distribution channels that automatically delivers one nematode to each recording module. The same channels are then used to perfuse the recording modules with test solutions while recording the electropharyngeogram (EPG) from each worm with sufficient sensitivity to detect each pharyngeal contraction. The device accurately reported the acute effects of known anthelmintics (anti-nematode drugs) and also correctly distinguished a specific drug-resistant mutant strain of C. elegans from wild type. The approach described here is readily adaptable to parasitic species for the identification of novel anthelmintics. It is also applicable in toxicology and drug discovery programs for human metabolic and degenerative diseases for which C. elegans is used as a model.

Hormonal regulation and segmental specificity of motoneuron phenotype during metamorphosis of the tobacco hornworm, Manduca sexta.

The abdominal prolegs of Manduca sexta larvae are eliminated at the onset of metamorphosis. Previous work showed that the prepupal peak of ecdysteroids in the hemolymph causes the dendritic arbors of proleg motoneurons to regress and a stereotyped subset of the motoneurons to die. In the present study we investigated the parameters of ecdysteroid exposure that are important for eliciting these responses by directly infusing 20-hydroxyecdysone (20-HE) into the hemolymph of insects deprived of their own endocrine glands. Doses of 20-HE that were near threshold for evoking regression or death were consistently more effective when infused over a longer duration. Theoretical calculations of hemolymph hormone profiles produced by the infusions support a model of ecdysteroid action in which the hormone concentration must remain above a threshold level for a critical duration of time to be physiologically effective. We further found that segmental location can influence both the metamorphic fate and the hormonal sensitivity of Manduca motoneurons.

Cell Culture Approaches to Understanding the Actions of Steroid Hormones on the Insect Nervous System

During metamorphosis of the hawkmoth, Manduca sexta, ecdysteroids regulate the dendritic remodeling and programmed death of identified motoneurons. These changes contribute to the dramatic reorganization of behavior that accompanies metamorphosis. As a step toward elucidating cellular and molecular mechanisms by which ecdysteroids affect neuronal phenotype, we have investigated the responses of Manduca motoneurons to ecdysteroids in vitro. Following dendritic regression at the end of larval life, thoracic leg motoneurons placed in culture respond to ecdysteroids by an increase in branching complexity, similar to events in vivo. Growth cone structure is affected markedly by ecdysteroids. At pupation, a rise in ecdysteroids triggers the segment-specific death of proleg motoneurons: the same segmental pattern of death is observed when motoneurons from different segments are removed from the nervous system and exposed to ecdysteroids in vitro. These studies provide strong evidence that Manduca motoneurons are direct targets of steroid action and set the stage for further studies of the specific mechanisms involved.

Steroid-triggered programmed cell death of a motoneuron is autophagic and involves structural changes in mitochondria

Neuronal death occurs during normal development and disease and can be regulated by steroid hormones. In the hawkmoth, Manduca sexta, individual accessory planta retractor (APR) motoneurons undergo a segment‐specific pattern of programmed cell death (PCD) at pupation that is triggered directly and cell autonomously by the steroid hormone 20‐hydroxyecdysone (20E). APRs from abdominal segment six [APR(6)s] die by 48 hours after pupal ecdysis (PE; entry into the pupal stage), whereas APR(4)s survive until adulthood. Cell culture experiments showed previously that 20E acts directly on APRs to trigger PCD, with intrinsic segmental identity determining which APRs die. The APR(6) death pathway includes caspase activation and loss of mitochondrial function. We used transmission electron microscopy to investigate the ultrastructure of APR somata before and during PCD. APR(4)s showed normal ultrastructure at all stages examined, as did APR(6)s until approximately stage PE. During APR(6) death, there was massive accumulation of autophagic bodies and vacuoles, mitochondria became ultracondensed and aggregated into compact clusters, and ribosomes aggregated in large blocks. Nuclear ultrastructure remained normal, without chromatin condensation, until the nuclear envelope fragmented late in the death process. Light microscopic immunocytochemistry showed that dying APR(6)s were TUNEL‐positive, which is diagnostic of fragmented DNA. These observations indicate that the steroid‐induced, caspase‐dependent, cell‐autonomous PCD of APR(6)s is autophagic, not apoptotic, and support an early role for mitochondrial alterations during PCD. This system permits the study of neuronal death in response to its bona fide developmental signal, the rise in a steroid hormone. J. Comp. Neurol. 457:384–403, 2003.

Steroid hormone effects on neurons subserving behavior

Recent advances in understanding effects of steroid hormones at the level of individual neurons have been achieved using model systems. Steroid hormone effects on dendritic morphology, synaptic function and ionic conductances have been implicated in the regulation of behavior in both vertebrates and invertebrates. Particularly exciting are studies demonstrating steroid hormone effects on specific synaptic connections and ionic currents. There also has been important progress in understanding the diversity of sites and mechanisms of hormone action, encompassing both genomic and non-genomic effects of steroids on neuronal properties.

Neural Mechanisms of Behavioral Plasticity: Metamorphosis and Learning in Manduca sexta

This review summarizes our current understanding of the neural circuit underlying the larval proleg withdrawal reflex (PWR) of Manduca sexta and describes how PWR function changes in two contexts: metamorphosis and learning. The first form of PWR plasticity occurs during the larval-pupal transformation, when the reflex is lost. One mechanism that contributes to this loss is the weakening of monosynaptic excitatory connection from proleg sensory neurons to proleg retractor motor neurons. This change is associated with the hormonally-mediated regression of proleg motor neuron dendrites, which may break synaptic contacts between the sensory and motor neurons. After pupation, some of the proleg motor neurons die in a segment-specific pattern that persists even after individual motor neurons are isolated from the nervous system and exposed to hormones in vitro. The second form of PWR plasticity involves short-term, activity-dependent changes in neural function during the larval stage. The nicotinic cholinergic connections from proleg sensory neurons to motor neurons exhibit several forms of plasticity including facilitation, depression, post-tetanic potentiation and two types of muscarinic modulation. Larval PWR behavior exhibits two simple forms of learning-habituation and dishabituation-which involve alterations in the central PWR circuit. These studies of a simple circuit illustrate neural mechanisms by which behaviors undergo both short- and long-term modifications.

STEROID HORMONES, DENDRITIC REMODELING AND NEURONAL DEATH : INSIGHTS FROM INSECT METAMORPHOSIS

Steroid hormones influence neuronal structure and function throughout the animal kingdom, via highly conserved receptor proteins. Insights into steroid effects on neurons and behavior have come from a range of vertebrate species including reptiles, amphibians, fish, birds, rodents and primates. In many instances, steroid hormones regulate the volume of particular regions of the nervous system by affecting both the number of constituent neurons and their size. A major determinant of neuronal number is the process of programmed cell death (PCD), which involves molecular machinery that is conserved across species. This article reviews steroid-mediated PCD and dendritic remodeling during metamorphosis of the hawkmoth, Manduca sexta. Metamorphosis is driven by a class of steroid hormones, the ecdysteroids. During the transformation from larva to pupa to adult moth, accessory planta retractor (APR) motoneurons of Manduca undergo dendritic regression and regrowth, and segment-specific PCD, in response to specific ecdysteroid cues. Experiments utilizing APRs in primary cell culture show that PCD is a direct response to ecdysteroids, regulated by the intrinsic segmental identity of individual APRs. As in other systems, activation of caspases (cysteine proteases) is involved in the execution phase of PCD. Other experiments demonstrate that the ecdysteroid-mediated regression of APRs’ dendrites at pupation causes weakening of monosynaptic excitatory inputs from sensory neurons that trigger a larval withdrawal reflex. Thus, the steroid-mediated reduction in dendritic extent is linked to a specific electrophysiological and behavioral change during metamorphosis. The comparative approach, taking advantage of a variety of vertebrate and invertebrate species, holds the most promise for elucidating the full spectrum of steroid effects on neurons and behavior.

Programmed cell death of an identified motoneuron in vitro: Temporal requirements for steroid exposure and protein synthesis

Ecdysteroid hormones trigger the programmed cell death (PCD) of a segmental subset of accessory planta retractor (APR) motoneurons at pupation in the moth, Manduca sexta. APRs from abdominal segment four [APR (4)s] survive through the pupal stage, whereas homologous APR(6)s die 24-48 h after pupal ecdysis (PE) (the shedding of the larval cuticle), in response to the prepupal peak of ecdysteroids. Following retrograde labeling with the vital fluorescent dye, DiI, the morphology of APR(4)s and APR(6)s in vivo was examined at PE and 24-48 h later. During this period, APR(4) somata remained large and ovoid while APR(6)s somata became shrunken and rounded. Similar phenotypes were observed when DiI-labeled APRs were cultured at PE and examined 24 h to 1 week later. During initial shrinkage and rounding of APR(6)s, the plasma membrane remained intact but DNA condensation occurred and mitochondrial activity was lost. The requirements for ecdysteroids and new protein synthesis for APR(6) death were tested by culturing cells with ecdysteroids and cycloheximide (CHX). When cultured at PE, the death of APR(6)s was independent of further exposure to ecdysteroids and could not be blocked by CHX. In contrast, APR(6)s cultured 24 h earlier required additional exposure to ecdysteroids to die and their death was inhibited by CHX. Thus, the final 24 h of larval life represents an important transition period in the commitment of APR(6)s to undergo PCD, and is of interest for pursuing underlying mechanisms of steroid-induced PCD.

Activity-dependent induction of facilitation, depression, and post-tetanic potentiation at an insect central synapse

SummaryIn Manduca sexta larvae, sensory neurons innervating planta hairs on the tips of the prolegs make monosynaptic excitatory connections with motoneurons innervating proleg retractor muscles. Tactile stimulation of the hairs evokes reflex retraction of the proleg. In this study we examined activity-dependent changes in the amplitude of the excitatory postsynaptic potentials (EPSPs) evoked in a proleg motoneuron by stimulation of individual planta hair sensory neurons. Deflection of a planta hair caused a phasic-tonic response in the sensory neuron, with a mean peak instantaneous firing frequency of >300 Hz, and a tonic firing rate of 10–20 Hz. Direct electrical stimulation was used to activate individual sensory neurons to fire at a range of frequencies including those observed during natural stimulation of the hair. At relatively low firing rates (e.g., 1 Hz), EPSP amplitude was stable indefinitely. At higher instantaneous firing frequencies (>10 Hz), EPSPs were initially facilitated, but continuous stimulation led rapidly to synaptic depression. High-frequency activation of a sensory neuron could also produce post-tetanic potentiation, in which EPSP amplitude remained elevated for several min following a stimulus train. Facilitation, depression, and post-tetanic potentiation all appeared to be presynaptic phenomena. These activity-dependent changes in sensory transmission may contribute to the behavioral plasticity of the proleg withdrawal reflex observed in intact insects.