Softworms: the design and control of non-pneumatic, 3D-printed, deformable robots.

Robots that can easily interact with humans and move through natural environments are becoming increasingly essential as assistive devices in the home, office and hospital. These machines need to be safe, effective, and easy to control. One strategy towards accomplishing these goals is to build the robots using soft and flexible materials to make them much more approachable and less likely to damage their environment. A major challenge is that comparatively little is known about how best to design, fabricate and control deformable machines. Here we describe the design, fabrication and control of a novel soft robotic platform (Softworms) as a modular device for research, education and public outreach. These robots are inspired by recent neuromechanical studies of crawling and climbing by larval moths and butterflies (Lepidoptera, caterpillars). Unlike most soft robots currently under development, the Softworms do not rely on pneumatic or fluidic actuators but are electrically powered and actuated using either shape-memory alloy microcoils or motor tendons, and they can be modified to accept other muscle-like actuators such as electroactive polymers. The technology is extremely versatile, and different designs can be quickly and cheaply fabricated by casting elastomeric polymers or by direct 3D printing. Softworms can crawl, inch or roll, and they are steerable and even climb steep inclines. Softworms can be made in any shape but here we describe modular and monolithic designs requiring little assembly. These modules can be combined to make multi-limbed devices. We also describe two approaches for controlling such highly deformable structures using either model-free state transition-reward matrices or distributed, mechanically coupled oscillators. In addition to their value as a research platform, these robots can be developed for use in environmental, medical and space applications where cheap, lightweight and shape-changing deformable robots will provide new performance capabilities.

Self-assembled insect muscle bioactuators with long term function under a range of environmental conditions.

The use of mammalian muscles as device actuators is severely limited by their sensitivity to environmental conditions and short lifetime. To overcome these limitations insect muscle stem cells were used to generate organized 3D muscle constructs with significant enhancements in environmental tolerance and long term function. These tissues self-assembled, self-repaired, survived for months in culture without media replenishment and produced stresses of up to 2 kPa, all under ambient conditions. The muscle tissues continued to function for days even under biologically extreme temperature and pH. Furthermore, the dimensions and geometry of these tissues can be easily scaled to MEMS or meso-scale devices. The versatility, environmental hardiness and long term function provide a new path forward for biological actuators for device needs.

Bone-free: soft mechanics for adaptive locomotion.

Muscular hydrostats (such as mollusks), and fluid-filled animals (such as annelids), can exploit their constant-volume tissues to transfer forces and displacements in predictable ways, much as articulated animals use hinges and levers. Although larval insects contain pressurized fluids, they also have internal air tubes that are compressible and, as a result, they have more uncontrolled degrees of freedom. Therefore, the mechanisms by which larval insects control their movements are expected to reveal useful strategies for designing soft biomimetic robots. Using caterpillars as a tractable model system, it is now possible to identify the biomechanical and neural strategies for controlling movements in such highly deformable animals. For example, the tobacco hornworm, Manduca sexta, can stiffen its body by increasing muscular tension (and therefore body pressure) but the internal cavity (hemocoel) is not iso-barometric, nor is pressure used to directly control the movements of its limbs. Instead, fluid and tissues flow within the hemocoel and the body is soft and flexible to conform to the substrate. Even the gut contributes to the biomechanics of locomotion; it is decoupled from the movements of the body wall and slides forward within the body cavity at the start of each step. During crawling the body is kept in tension for part of the stride and compressive forces are exerted on the substrate along the axis of the caterpillar, thereby using the environment as a skeleton. The timing of muscular activity suggests that crawling is coordinated by proleg-retractor motoneurons and that the large segmental muscles produce anterograde waves of lifting that do not require precise timing. This strategy produces a robust form of locomotion in which the kinematics changes little with orientation. In different species of caterpillar, the presence of prolegs on particular body segments is related to alternative kinematics such as "inching." This suggests a mechanism for the evolution of different gaits through changes in the usage of prolegs, rather than, through extensive alterations in the motor program controlling the body wall. Some of these findings are being used to design and test novel control-strategies for highly deformable robots. These "softworm" devices are providing new insights into the challenges faced by any soft animal navigating in a terrestrial environment.

Locomotion in caterpillars.

Most species of caterpillar move around by inching or crawling. Their ability to navigate in branching three-dimensional structures makes them particularly interesting biomechanical subjects. The mechanism of inching has not been investigated in detail, but crawling is now well understood from studies on caterpillar neural activity, dynamics and structural mechanics. Early papers describe caterpillar crawling as legged peristalsis, but recent work suggests that caterpillars use a tension-based mechanism that helps them to exploit arboreal niches. Caterpillars are not obligate hydrostats but instead use their strong grip to the substrate to transmit forces, in effect using their environment as a skeleton. In addition, the gut which accounts for a substantial part of the caterpillar's weight, moves independently of the body wall during locomotion and may contribute to crawling dynamics. Work-loop analysis of caterpillar muscles shows that they are likely to act both as actuators and energy dissipaters during crawling. Because caterpillar tissues are pseudo-elastic, and locomotion involves large body deformations, moving is energetically inefficient. Possession of a soft body benefits caterpillars by allowing them to grow quickly and to access remote food sources safely.

A constitutive model for muscle properties in a soft-bodied arthropod.

In this paper, we examine the mechanical properties of muscles in a soft-bodied arthropod under both passive and stimulated conditions. In particular, we examine the ventral interior lateral muscle of the tobacco hornworm caterpillar, Manduca sexta, and show that its response is qualitatively similar to the behaviour of particle-reinforced rubber. Both materials are capable of large nonlinear elastic deformations, show a hysteretic behaviour and display stress softening during the first few cycles of repeated loading. The Manduca muscle can therefore be considered as different elastic materials during loading and unloading and is best described using the theory of pseudo-elasticity. We summarize the basic equations for transversely isotropic pseudo-elastic materials, first for general deformations and then for the appropriate uniaxial specialization. The constitutive relation proposed is in good agreement with the experimental data for both the passive and the stimulated conditions.

Expression and function of two nicotinic subunits in insect neurons.

Nicotinic acetylcholine receptors (nAChRs) in insects are neuron-specific oligomeric proteins essential for the central transmission of sensory information. Little is known about their subunit composition because it is difficult to express functional insect nAChRs in heterologous systems. As an alternative approach we have examined the native expression of two subunits in neurons of the nicotinic-resistant, tobacco-feeding insect Manduca sexta. Both the alpha-subunit MARA1 and the beta-subunit MARB can be detected by in situ hybridization in the majority of cultured neurons with an overlapping, but not identical, distribution. Changes in intracellular Ca(2+) evoked by nicotinic stimulation are more strongly correlated to the expression of MARA1 than MARB and are independent of cell size. Unlike the previously reported critical role of MARA1 in mediating nicotinic Ca(2+) responses, down-regulation of MARB by RNA interference (RNAi) did not reduce the number of responding neurons or the size of evoked responses, suggesting that additional subunits remain to be identified in Manduca.

Combined imaging and chemical sensing of L-glutamate release from the foregut plexus of the lepidopteran, Manduca sexta.

A new combined imaging and chemical detection sensor for the measurement of localized L-glutamate release at the insect neuromuscular junction (NMJ) is presented. The sensor is comprised of an L-glutamate-sensitive fluorescent gel, spin-coated onto the tip of an optical imaging fiber. The gel is composed of L-glutamate oxidase (GLOD); a pH-sensitive fluorescent dye, SNAFL; and poly(acrylamide-co-N-acryloxysuccinimide) (PAN). NH(3) is liberated from the interaction of L-glutamate with GLOD, which reversibly reduces the emitted fluorescence signal from SNAFL. This sensor has a spatial resolution of 3-4 micro m, and an L-glutamate detection limit of between 10 and 100 micro M. L-glutamate release and re-uptake from the foregut plexus of Manduca sexta was detected by the sensor in the presence of the L-glutamate re-uptake blocker dihydrokainate, and the post-synaptic L-glutamate receptor antagonist CNQX.

The nicotinic alpha subunit MARA1 is necessary for cholinergic evoked calcium transients in Manduca neurons.

The functional contribution of cloned subunits to insect nicotinic acetylcholine (ACh) receptors has been difficult to determine using heterologous expression. Instead, in this study we explore the subunit composition of naturally expressed functional receptors in an insect using RNA interference. The nicotinic alpha subunit, Manduca ACh Receptor Alpha 1 (MARA1) can be detected in neuronal cultures isolated from the ventral nerve cord of fifth instar larvae of Manduca sexta by in situ hybridization. It's presence correlates with large ACh induced, nicotinic Ca2+ responses. The expression of MARA1 is downregulated by treatment with dsRNA which significantly reduced both the number of responding cells and the amplitude of remaining Ca2+ responses. These results suggest that MARA1 is part of a nicotinic receptor functionally coupled to Ca2+ entry.

Nitric oxide and the control of firefly flashing.

Bioluminescent flashing is essential for firefly reproduction, yet the specific molecular mechanisms that control light production are not well understood. We report that light production by fireflies can be stimulated by nitric oxide (NO) gas in the presence of oxygen and that NO scavengers block bioluminescence induced by the neurotransmitter octopamine. NO synthase is robustly expressed in the firefly lantern in cells interposed between nerve endings and the light-producing photocytes. These results suggest that NO synthesis is a key determinant of flash control in fireflies.

Neurons involved in nitric oxide-mediated cGMP signaling in the tobacco hornworm, Manduca sexta.

Recently, both nitric oxide synthase (NOS), and nitric oxide (NO)-sensitive guanylyl cyclase were cloned in Manduca sexta and implicated in several cellular, developmental, and behavioral processes (Nighorn et al. [1998] J Neurosci 18:7244-7255). However, NO is a highly diffusive gas, and little is known about the range and specificity of its actions on neurons. To begin examining the role of NO as a neurotransmitter in the central nervous system (CNS) of larval Manduca, we have mapped potential NO-producing neurons using fixation-resistant NADPH-diaphorase staining and antisera that recognize a NOS-specific epitope. In addition, to detect NO-responsive neurons, we treated the CNS with NO donors and used antibodies that recognize elevated levels of cyclic 3;,5;-guanosine monophosphate (cGMP). Many potential NO-producing neurons were mapped, including the ventral unpaired median cells and three pairs of lateral cells in each abdominal ganglion. Additional neurons in the dorsal midline of ganglia A5-7 (PM2) appear to express NOS in a segment-specific manner. At the larval-to-pupal transition, this staining pattern changes; the PM2 neurons stain weakly or are undetectable and there is novel expression of NOS in cell 27. In response to NO donors, a small number of neurons produce detectable cGMP accumulation in a segment-specific pattern. These include a pair of posteriodorsally positioned interneurons (IN505) in all the abdominal ganglia, PM2 neurons in A5, and PM1 and PM2 neurons in A7. Hence, PM2 neurons in A5 and A7 are potentially capable of producing and responding to NO. These identified NO-producing and responding neurons provide a tractable model system for studying the dynamics and specificity of NO signaling in the CNS.

Combined kinematic and electromyographic analyses of proleg function during crawling by the caterpillar Manduca sexta.

The planta retractor muscles in the prolegs of Manduca sexta caterpillars are a frequently-used model system for investigating a number of problems in neurobiology. We have combined kinematic and electromyogram analysis of proleg movements during crawling to examine the roles of these muscles during normal behavior. We found that retractor muscle activity is highly stereotyped, and that the primary function of these muscles is to disengage the crochets at the tip of the proleg for the swing phase of crawling. The duration of activity of the muscles was tightly coupled to the phasing of crawling behavior. The stepping patterns of animals changed to accommodate variations in the substrate, but the relative timing of retractor muscle activity was unaffected. There were no clear correlations between the various properties of motoneuronal input to the muscle (duration of activity, number of spikes, peak frequency of spikes) and the resulting muscle length change. Perhaps because it functions partially as a hydrostat, this may represent a neuromuscular system in which a significant part of the control algorithm is embedded in its morphology.

Context dependency of a limb withdrawal reflex in the caterpillar Manduca sexta.

The proleg withdrawal reflex in the caterpillar Manduca sexta is a robust, well-characterized system for investigating the integration of sensory information with centrally patterned behavior. The reflex is evoked by stimulating mechanosensory hairs--planta hairs--located at the tip of each proleg. We studied the expression of this reflex by combining video recordings and electromyographic recordings from the main retractor muscles of the proleg, the principal and accessory planta retractor muscles. In intact animals, the nature of the response depended on the motor context of the animal. Animals which were standing quietly showed great variability in both the kinematic properties of proleg withdrawal, and the corresponding muscle electrical activity. Animals which were hanging upside down from a wooden dowel exhibited a much faster reflex, with retraction of the proleg occurring slightly faster than in standing animals, but re-extension of the proleg to the substrate being considerably faster. In crawling animals, expression of the reflex depended on the phase of the crawling cycle during which stimulation occurred. The reflex in a given proleg was suppressed during stance phase of that proleg. During swing phase, however, planta hair stimulation evoked proleg withdrawal, resulting in an assistance reflex. In contrast. isolated abdomens showed much less variability in the reflex. A comparison of the relationship between retractor muscle activity and the resulting proleg movement showed significant correlations between both the duration of activity and the number of muscle spikes, and the size of the associated proleg withdrawal. This is a promising system in which to investigate how central neuronal circuits accomplish context-dependency of motor behavior.

The role of inositol 1,4,5-trisphosphate 5-phosphatase in inositol signaling in the CNS of larval Manduca sexta.

Production of inositol 1,4,5-trisphosphate (IP3) in cells results in the mobilization of intracellular calcium. Therefore, the dynamics of IP3 metabolism is important for calcium dependent processes in cells. This report investigates the coupling of mAChRs to the inositol lipid pathway in the CNS of the larval Manduca sexta. Stimulation of intact abdominal ganglia prelabeled with [3H]-inositol using a muscarinic agonist, oxotremorine-M (oxo-M), increased total inositol phosphate levels in a dose dependent manner (EC50 = 4.23 microM). These inositol phosphates consisted primarily of inositol 1,4-bisphosphate (IP2) and inositol monophosphate (IP1). Similarly, when nerve cord homogenates were provided with [3H]-phosphatidylinositol 4,5-bisphosphate ([3H]-PIP2) (10-13 microM) the predominant products were IP2 and IP1. In contrast, incubation of purified membranes with 1 mM oxo-M in the presence of 100 microM GTP gamma S and [3H]-PIP2 increased IP3 levels, suggesting that the direct activation of phospholipase C (PLC) by mAChRs occurs in a membrane delimited process. Together, these results suggest that in the intact nerve cord and in crude homogenates, a cytosolic 5-phosphatase quickly metabolizes IP3 to produce to IP2 and IP1. This enzyme was kinetically characterized using IP3 (Km = 43.7 microM, Vmax = 864 pmoles/min/mg) and IP4 (Km = 0.93 microM; Vmax = 300pmoles/min/mg) as substrates. The enzyme activity can be potently inhibited by two IP thiol compounds; IP3S3 (1,4,6) and IP3S3 (2,3,5), that show complex binding kinetics (Hill numbers < 1) and can distinguish different forms of the 5-phosphatase in purified membranes. These two inhibitors could be very useful tools to determine the role of the inositol lipid pathway in neuroexcitability.

The role of nitric oxide in motoneuron spike activity and muscarinic-evoked changes in cGMP in the CNS of larval Manduca sexta.

Nitric oxide and muscarinic agonists both stimulate motoneuron spike activity and cGMP production in the central nervous system of larval Manduca sexta. The possible role of nitric oxide in mediating muscarinic changes in excitability was examined by measuring cGMP accumulation and proleg motoneuron activity while blocking or mimicking the production of nitric oxide. All the muscarinic-induced changes in cGMP are blocked by the nitric oxide-synthase inhibitor, nitro-l-arginine, an effect that is partially prevented by co-incubation with arginine. Action potential blockage with tetrodotoxin revealed that muscarinic increases in cGMP production have both spike-dependent and spike-independent mechanisms. Furthermore, nitric oxide donors can increase proleg motoneuron activity and this stimulation is blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one suggesting that it is mediated by a nitric oxide-sensitive guanylyl cyclase. In contrast, nitro-l-arginine and a variety of other nitric oxide-synthase inhibitors and nitric oxide scavengers have no significant effect on muscarinic stimulation of motoneuron activity. Therefore, although a nitric oxide sensitive guanylyl cyclase is capable of elevating spike activity and muscarinic agonists can increase cGMP, this mechanism is not necessary for the normal muscarinic increase in excitability. It is concluded that muscarinic receptors are coupled to nitric oxide and cGMP production in neurons other than those controlling the prolegs.

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.

Characterization of muscarinic binding sites in the central nervous system of larval Manduca sexta.

Putative muscarinic receptors (mAChRs) were characterized in the CNS of larval Manduca sexta through the binding of 3H-quinuclidyl benzilate ([3H]-QNB). Specific binding isotherms revealed a high affinity binding site in both crude homogenates (Ka = 3.22 +/- 0.62 nM-1 (Kd 0.311 nM), Bmax = 65.4 +/- 9.8 fmoles/mg protein) and in purified membrane preparations (Ka = 7.61 +/- 1.78 nM-1 (Kd 0.130 nM), Bmax = 22.8 +/- 2.15 fmoles/mg protein). In purified membrane preparations the binding was complex, consisting of at least two sites (Hill coefficient = 0.514 +/- 0.041). Because of the high proportion of non-specific binding at QNB concentrations greater than 10 nM, the binding parameters for the additional low affinity site could not be determined accurately but the Kd was estimated to be greater than 8 nM. Complex binding was also exhibited in the kinetics of [3H]-QNB association and dissociation in purified membranes. Analysis resolved two high affinity sites (dissociation rate constants = 80.4 and 1.2 min, Kds = 0.272 and 0.909 nM, consisting of 65.9 +/- 3.7 and 26 +/- 4.9% of the sites respectively). Presumably, the high affinity site identified in saturation studies consists of these two components. The primary high affinity site was isolated using a dissociative method and its pharmacology determined in competition studies. [3H]-QNB binding at this site was displaced by pirenzepine and methoctramine (Kis = 248 and 707 nM) closely matching the pharmacology of the cloned Drosophila mAChR. Competition data for 4-DAMP were better fitted by a two site model (Kis = 37 and 547 nM). These results demonstrate unexpected complexity in muscarinic ligand binding and are consistent with mAChR heterogeneity in the CNS of Manduca.

Modulation of second messengers in the nervous system of larval Manduca sexta by muscarinic receptors.

Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta. Cyclic AMP levels were increased by treatment with 3-isobutyl-1-methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine-M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine-M and by oxotremorine-M in the presence of 3-isobutyl-1-methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine-M. The primary inositol metabolite generated during a 5-min exposure to oxotremorine-M coeluted from ion-exchange columns with inositol-1-monophosphate, although other more polar metabolites were also detected. This agonist-evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell-free preparation of nerve cord membranes labeled with [3H]inositol. Incubation with oxotremorine-M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell-free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.

Current excitement from insect muscarinic receptors.

Recent electrophysiological, pharmacological and molecular studies suggest that muscarinic ACh receptors (mAChRs) in insects are related to, but distinct from, their mammalian counterparts. Insect mAChRs perform two primary roles that are distinguished by their locations. Presynaptic mAChRs, present on sensory terminals, inhibit transmitter release, thereby reducing the effectiveness of specific afferent inputs. In contrast, postsynaptic mAChRs depolarize and increase the excitability of motoneurons and interneurons, thereby acting as dynamic-gain controls. This postsynaptic modulation is achieved in different ways in specific neurons but generally results from the activation of persistent inward and outward currents. At the level of neural processing, these distinct roles enable insect mAChRs to regulate the transfer of sensory information, and modulate the contributions of central neurons to central pattern generators and reflexes. Because these phenomena can be studied in identified neurons, a combination of physiological and molecular studies of mAChRs in insects should help to elucidate some of their behavioral roles. Furthermore, such studies could lead to the identification of general mechanisms of functional plasticity in neuronal networks.

Characterization of a muscarinic current that regulates excitability of an identified insect motoneuron.

1. Application of the muscarinic agonist oxotremorine-M (oxo-M) to isolated abdominal ganglia of larval Manduca sexta excited an identified proleg retractor motoneuron called PPR. This excitation consisted of a persistent depolarization and an increased tendency to generate action potentials. Previous work has established that the action of oxo-M is probably mediated by muscarinic acetylcholine receptors (mAChRs) on PPR and that oxo-M mimics an afferent-induced long-lasting depolarization called the slow excitatory postsynaptic potential (sEPSP). 2. Action potentials in the ganglion could be blocked by applying tetrodotoxin (TTX) in the bath saline. Under these conditions all excitatory postsynaptic potentials in PPR were also blocked, but the depolarizing action of oxo-M was unaffected. In the absence of background activity PPR could be voltage clamped using a single-electrode switching clamp to study the currents underlying the response to oxo-M. 3. At a membrane potential of -50 mV, application of oxo-M to the ganglion in the bath saline (3-6 x 10(-7) M) or by brief (20-40 ms) pulses from a micropipette into the neuropil (1 x 10(-5) M) evoked an apparently inward current called Iox. The mean peak current change in response to pulses was -0.80 +/- 0.04 nA (n = 48 preparations). 4. The voltage dependence of Iox was determined by subtracting the current-voltage relationship for PPR in control saline from that during a response to oxo-M. Iox was maximal near the resting potential of PPR (-45 to -40 mV), decreasing slightly with hyperpolarization and strongly with depolarization. 5. Peak Iox was directly dependent on the bath Na+ concentration. Complete replacement of Na+ with N-methyl-D-glucamine in the saline blocked Iox. Changes in the bath K+ concentration (extracellular K+ concentration, [K+]o) had only a small effect on Iox. Reducing [Cl-]o from 140 to 74.5 mM had no significant effect on Iox during a 15-min exposure. Intracellular injections of Cl- from a KCl-containing electrode also had no measurable effect on Iox. 6. Changes in the bath Ca2+ concentration above or below 2 mM inhibited Iox. Furthermore, the divalent cations Ni2+, Co2+, Mg2+, and Ba2+ at millimolar concentrations and the Ca2+ channel blocking agents nifedipine and Cd2+ at micromolar concentrations inhibited Iox. 7. These results suggest that mAChRs on PPR activate an inward current that is persistent, TTX insensitive, voltage dependent and carried predominantly by Na+. However, the results cannot eliminate the possibility that changes in K+ or Cl- conductances might also be involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic acetylcholine receptors modulate the excitability of an identified insect motoneuron.

1. With the use of an isolated, perfused ganglion preparation from the tobacco hornworm, Manduca sexta, we have examined the responses of an identified proleg retractor motoneuron (designated PPR) to trains of stimuli delivered to sensory branches of the ventral nerve (VN). 2. Trains of stimuli (50 Hz, 100 ms to 5 s) delivered to the proleg sensory nerve, VNA, caused PPR to depolarize and initiate a bout of spiking activity that outlasted the stimulus. A fast component of this response was due to monosynaptic input from planta hair sensory neurons, acting on nicotinic acetylcholine receptors (nAChRs). The fast response to VNA stimulation was abolished when the ganglion was treated with the nicotinic antagonist, mecamylamine, leaving a slow, long-lasting depolarization of PPR, which we have called the slow excitatory postsynaptic potential (sEPSP). 3. A sEPSP could be evoked by stimulation of all the major subbranches of VNA ipsilateral to PPR's cell body. Small sEPSPs were also evoked by stimulation of all but one of the major contralateral subbranches of VNA. 4. During a sEPSP the spike threshold of PPR became more negative. This increase in excitability was not correlated with changes in membrane potential measured at the cell body, and there was no detectable change in input resistance. We conclude that the spike-threshold change reflects either a depolarization electrically remote from the cell body, or a change in PPR's spike initiation properties that are not reflected in the membrane potential. 5. Both the sEPSP and the associated change in PPR's spike threshold were blocked by several muscarinic antagonists. Scopolamine was effective at concentrations > 2 x 10(-7) M, atropine at concentrations > 1 x 10(-6) M, and pirenzepine at 5 x 10(-5) M. 4-Diphenylacetoxy-N-methylpiperidine (4-DAMP), methoctramine, hexahydrosiladifenidol, and AF-DX 116 were all poor antagonists. 6. Bath application of the muscarinic agonist oxotremorine-M (oxo-M) at concentrations > 3 x 10(-7) M increased the spontaneous spiking activity of PPR and other proleg motoneurons. In PPR, this increased activity was accompanied by a small depolarization and a more negative spike threshold, both of which were inhibited by 1 x 10(-7) M scopolamine. 7. At concentrations > 6 x 10(-8) M, bath-applied oxo-M depolarized PPR even when spike activity in the ganglion was blocked with tetrodotoxin. During such spike blockage, pressure ejection of brief puffs of oxo-M into the neuropil evoked a long-lasting depolarization of PPR that resembled the sEPSP.(ABSTRACT TRUNCATED AT 400 WORDS)