Neurotrophic agents prevent motoneuron death following sciatic nerve section in the neonatal mouse

We have examined the ability of different neurotrophic and growth factors to prevent axotomy-induced motoneuron cell death in the developing mouse spinal cord. After postnatal unilateral section of the mouse sciatic nerve, most motoneuron (MN) loss occurs in the lateral motor column of the fourth lumbar segment (L4). Significant axotomy-induced cell death occurred after surgery performed on or before postnatal day (PN) 5. In contrast, no significant cell loss was found when axotomy was performed after PN10. Axotomy on PN2 or PN5 resulted in a 44% loss of L4 motoneurons by 7 days, and a 66% loss of motoneurons by 10 days postsurgery. Implantation of gelfoam presoaked in various neurotrophic factors at the lesion site rescued axotomized motoneurons. Nerve growth factor (NGF), nedurotrophin-4/5 (NT-4/5) and ciliary neurotrophic factor (CNTF) rescued 20%–30% of motoneurons, whereas brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and insulin-like growth factor 1 (IGF-1) rescued virtually all motoneurons from axotomy-induced death. By contrast, platelet-derived growth factor (PDGF)-AA, PDGF-AB, basic fibroblast growth factor (bFGF), and interleukin (IL-6) were ineffective on motoneuron survival following axotomy. NGF, BDNF, NT-3, IGF-1, and CNTF also prevented axotomy-induced atrophy of surviving motoneurons. These data show that mouse lumbar motoneurons continue to be vulnerable to axotomy up to about 1 week after birth and that a number of trophic agents, including the neurotrophins, CNTF, and IGF-1, can prevent the death of these neurons following axotomy. Our studies confirm and extend previous reports on the time course of axotomy-induced mouse motoneuron death and the survival promoting effects of neurotrophic factors. 1994 John Wiley & Sons, Inc.     

Biological studies of a putative avian muscle-derived neurotrophic factor that prevents naturally occurring motoneuron death in vivo

A series of in vivo studies have been carried out using the chick embryo to address several critical questions concerning the biological, and to a lesser extent, the biochemical characteristics of a putative avian muscle-derived trophic agent that promotes motoneuron survival in vivo. A partially purified fraction of muscle extract was shown to be heat and trypsin sensitive and rescued motoneurons from naturally occurring cell death in a dose-dependent fashion. Muscle extract had no effect on mitotic activity in the spinal cord and did not alter cell number when administered either before or after the normal cell death period. The survival promoting activity in the muscle extract appears to be developmentally regulated. Treatment with muscle extract during the cell death period did not permanently rescue motoneurons. The motoneuron survival-promoting activity found in skeletal muscle was not present in extracts from a variety of other tissues, including liver, kidney, lung, heart, and smooth muscle. Survival activity was also found in extracts from fetal mouse, rat, and human skeletal muscle. Conditioned medium derived from avian myotube cultures also prevented motoneuron death when administered in vivo to chick embryos. Treatment of embryos in ovo with muscle extract had no effect on several properties of developing muscles. With the exception of cranial motoneurons, treatment with muscle extract did not promote the survival of several other populations of neurons in the central and peripheral nervous system that also exhibit naturally occurring cell death. Initial biochemical characterization suggests that the activity in skeletal muscle is an acidic protein between 10 and 30 kD. Examination of a number of previously characterized growth and trophic agents in our in vivo assay have identified several molecules that promote motoneuron survival to one degree or another. These include S100β, brain-derived neurotrophic factor (BDNF), neurotrophin 4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), transforming growth factor β (TGFβ), platelet-derived growth factor-AB (PDGF-AB), leukemia inhibitory factor (CDF/LIF), and insulin-like growth factors I and II (IGF). By contrast, the following agents were ineffective: nerve growth factor (NGF), neurotrophin-3 (NT3), epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF, bFGF), and the heparin-binding growth-associated molecule (HB-GAM). Of those agents that were effective, CDF/LIF, IGF-1 and -2, BDNF, and TGF are reported to be expressed in developing or adult muscle. Studies are underway to determine whether the survival activity found in avian muscle extract can be accounted for by one or more of these growth factors. Of all the tissue extracts and purified proteins tested here, only the neurotrophins—NGF, NT-3, and BDNF (but not NT-4/5)—rescured sensory neurons from naturally occurring cell death. © 1993 John Wiley & Sons, Inc.     

Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult.

Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-beta and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L-Cre; Stat3(flox/KO) mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.     

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.     

Prevention of thrombin‐induced motoneuron degeneration with different neurotrophic factors in highly enriched cultures

Previous reports have shown that neuronal and glial cells express functionally active thrombin receptors. The thrombin receptor (PAR‐1), a member of a growing family of protease activated receptors (PARs), requires cleavage of the extracellular amino‐terminus domain by thrombin to induce signal transduction. Studies from our laboratory have shown that PAR‐1 activation following the addition of thrombin or a synthetic thrombin receptor activating peptide (TRAP) induces motoneuron cell death both in vitro and in vivo. In addition to increasing motoneuron cell death, PAR‐1 activation leads to decreases in the mean neurite length and side branching in highly enriched motoneuron cultures. It has been suggested that motoneuron survival depends on access to sufficient target‐derived neurotrophic factors through axonal branching and synaptic contacts. However, whether the thrombin‐induced effects on motoneurons can be prevented by neurotrophic factors is still unknown. Using highly enriched avian motoneuron cultures, we show here that alone, soluble chick skeletal muscle extracts (CMX), brain‐derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial cell line–derived neurotrophic factor (GDNF) significantly increased motoneuron survival compared to controls, whereas nerve growth factor (NGF) did not have a significant effect on motoneuron survival. Furthermore, cotreatment with muscle‐derived agents (i.e., CMX, BDNF, GDNF) significantly prevented the death of motoneurons induced by α‐thrombin. Yet, non–muscle‐derived agents (CNTF and NGF) had little or no significant effect in reversing thrombin‐induced motoneuron death. CMX and CNTF significantly increased the mean length of neurites, whereas NGF, BDNF, and GDNF failed to enhance neurite outgrowth compared to controls. Furthermore, CMX and CNTF significantly prevented thrombin‐induced inhibition of neurite outgrowth, whereas BDNF and GDNF only partially reversed thrombin‐induced inhibition of neurite outgrowth. These findings show differential effects of neurotrophic factors on thrombin‐induced motoneuron degeneration and suggest specific overlaps between the trophic and stress pathways activated by some neurotrophic agents and thrombin, respectively. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 571–580, 1999     

The response of motoneurons to neurotrophins

The ongoing search for neurotrophic factors for motoneurons has led to the identification of a number of molecules which regulate motoneuron survival and function. Among these factors, the neurotrophins brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5 but not nerve growth factor (NGF), can prevent embryonic and postnatal motoneuron cell death in a variety of experimental paradigms. Analysis of expression of p75, trkB and trkC—components of the neurotrophin receptors—supports a potential physiological role for these factors as muscle- and glial-derived trophic factors for motoneurons. However, the survival of motoneurons during embryonic development is not reduced in the absence of BDNF, NT-3 or NT-4, as revealed by gene knockout experiments. This points to the involvement of additional trophic factors in the regulation of embryonic and postnatal motoneuron survival. The purpose of this review is to bring together the often prophetic observations from earlier studies—prior to the identification and characterization of these neurotrophins—with more recent results. Special issue dedicated to Dr. Hans Thoenen.     

Prevention of thrombin-induced motoneuron degeneration with different neurotrophic factors in highly enriched cultures

Previous reports have shown that neuronal and glial cells express functionally active thrombin receptors. The thrombin receptor (PAR-1), a member of a growing family of protease activated receptors (PARs), requires cleavage of the extracellular amino-terminus domain by thrombin to induce signal transduction. Studies from our laboratory have shown that PAR-1 activation following the addition of thrombin or a synthetic thrombin receptor activating peptide (TRAP) induces motoneuron cell death both in vitro and in vivo. In addition to increasing motoneuron cell death, PAR- 1 activation leads to decreases in the mean neurite length and side branching in highly enriched motoneuron cultures. It has been suggested that motoneuron survival depends on access to sufficient target-derived neurotrophic factors through axonal branching and synaptic contacts. However, whether the thrombininduced effects on motoneurons can be prevented by neurotrophic factors is still unknown. Using highly enriched avian motoneuron cultures, we show here that alone, soluble chick skeletal muscle extracts (CMX), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF) significantly increased motoneuron survival compared to controls, whereas nerve growth factor (NGF) did not have a significant effect on motoneuron survival. Furthermore, cotreatment with muscle-derived agents (i.e., CMX, BDNF, GDNF) significantly prevented the death of motoneurons induced by alpha-thrombin. Yet, non-muscle-derived agents (CNTF and NGF) had little or no significant effect in reversing thrombin-induced motoneuron death. CMX and CNTF significantly increased the mean length of neurites, whereas NGF, BDNF, and GDNF failed to enhance neurite outgrowth compared to controls. Furthermore, CMX and CNTF significantly prevented thrombin-induced inhibition of neurite outgrowth, whereas BDNF and GDNF only partially reversed thrombin-induced inhibition of neurite outgrowth. These findings show differential effects of neurotrophic factors on thrombin-induced motoneuron degeneration and suggest specific overlaps between the trophic and stress pathways activated by some neurotrophic agents and thrombin, respectively.     

Simultaneous GDNF and BDNF application leads to increased motoneuron survival and improved functional outcome in an experimental model for obstetric brachial plexus lesions

Motoneurons of the neonate rat respond to proximal axonal injury with morphologic and functional changes and ultimately with neuronal death. Recent studies showed that both glial cell-line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) reduce induced degeneration of motoneurons after axotomy and avulsion. Whether rescued motoneurons are functionally intact has been argued. In the present investigation, the authors have used a proximal crush lesion of the brachial plexus in neonatal rats as the experimental model of neuronal injury. This allowed the authors to study the effects of trophic factor administration on injured motoneurons and the relationship between motoneuron survival and extremity function. Trophic factors were locally released by small polymer implants in a low-dose slow-release mode. Six groups of 10 animals were prepared: BDNF, GDNF, GDNF/BDNF, control, sham, and normals. The number of surviving motoneurons was determined by retrograde tracer techniques using Fluorogold and Fastblue. Extremity function was quantitatively evaluated with functional muscle testing at day 56. The results of this study demonstrate that trophic factors applied separately had no effect, whereas combined trophic factor application (GDNF/BDNF group) had a dramatic rescue effect on motoneuron survival as compared with the control groups, which also effected significantly greater strength. The authors conclude that a combination of trophic factors leads to enhanced motoneuron survival, with improved voluntary function as the animal enters adulthood so that exogenous trophic support of motoneurons might have a role in the treatment of all types of severe neonatal plexopathies, maintaining the viability of motoneurons until reconstructive surgery provides them with a pathway for regeneration and endogenous trophic support.     

Chronic application of curare does not increase the level of motoneuron survival-promoting activity in limb muscle extracts during the naturally occurring motoneuron cell death period

Naturally occurring motoneuron cell death during development is a well-described phenomenon and the existence of a survival factor provided by target muscles has been postulated. Blockade of activity by chronic application of a neuromuscular junction blocker rescues almost all motoneurons from cell death. The present study was conducted in order to examine the possibility that the motoneuron survival-promoting activity in muscles increases following activity blockade. Cell culture was used to assess the degree of motoneuron survival-promoting activity present in muscle extracts. Embryonic chick motoneurons were labeled by injecting the water-insoluble fluorescent dye, DiI (Molecular Probes, Inc.) into the spinal nerves both before and during the cell death period. The labeled cells extending long neurites were counted after 2 days of culture as viable motoneurons in low-density heterogeneous cell cultures. The culture medium, Ham F12/DMEM (1:1 mixture) supplemented with 10% horse serum, 5% chick serum, and 5% fetal calf serum, was employed as a basic culture medium for assessing motoneuron survival factor, since it supported the survival of a significantly higher number of motoneurons derived from embryos before cell death than those during the cell death period, thus representing the motoneuron's requirement for survival factor in vivo. The number of surviving motoneurons clearly increased in proportion to the amount of muscle extract added to the culture medium. In comparison with control chick embryos, the dose-response relation between the number of surviving motoneurons and the amount of muscle extract added did not change when embryos were used after chronic application of curare. These results therefore indicate that survival factor derived from target muscle is crucial to the in vitro motoneurons during the cell death period, but do not support the idea that inactive muscle contains a higher amount of the survival factor.     

Interactions between spinal cord stimulation and activity blockade in the regulation of synaptogenesis and motoneuron survival in the chick embryo

The present study investigated the effects of spinal cord stimulation, neuromuscular blockade, or a combination of the two on neuromuscular development both during and after the period of naturally occurring motoneuron death in the chick embryo. Electrical stimulation of the spinal cord was without effect on motoneuron survival, synaptogenesis, or muscle properties. By contrast, activity blockade rescued motoneurons from cell death and altered synaptogenesis. A combination of spinal cord stimulation and activity blockade resulted in a marked increase in motoneuron death, and also altered synaptogenesis similar to that seen with activity blockade alone. Perturbation of normal nerve–muscle interactions by activity blockade may increase the vulnerability of developing motoneurons to excessive excitatory afferent input (spinal cord stimulation) resulting in excitotoxic-induced cell death. © 1993 John Wiley & Sons, Inc.     

Cell death of motoneurons in the chick embryo spinal cord. XI. Acetylcholine receptors and synaptogenesis in skeletal muscle following the reduction of motoneuron death by neuromuscular blockade

Treatment of chick embryos with neuromuscular blocking agents such as curare during periods of naturally occurring motoneuron death results in a striking reduction of this normal cell loss. Inactivity-induced changes in motoneuron survival were found to be associated with increased levels of AChRs and AChR-clusters in skeletal muscle and with increased focal sites of AChE that are innervated ('synaptic sites'). Treatment of embryos with curare after the normal cell death period (E12-E15) resulted in no change in motoneuron survival. Although AChR-clusters and focal sites of AChE were increased in these embryos on E16, many of these sites were uninnervated. Treatment of embryos with nicotine or decamethonium (E6-E10) also reduced neuromuscular activity but did not alter motoneuron survival nor did such treatment alter AChRs. The different effects of curare vs nicotine and decamethoniam on motoneuron survival and AChRs may be related to the fact that the former is a competitive blocker whereas the latter two drugs are depolarizing blockers. Finally, treatment of embryos (E6-9) with doses of curare (1 mg daily) that allow for the almost complete recovery of neuromuscular activity a few days following treatment (by E16) resulted in the gradual loss of the excess motoneurons that were present on E10, and by E16 the number of remaining AChR clusters and focal sites of AChE were also decreased to levels comparable to control values. Inactivity-induced changes in AChRs or AChR-clusters may be an important factor in the reduced motoneuron death that accompanies neuromuscular blockade during critical stages of development. These receptor changes very likely reflect increased synaptogenesis in the muscles of paralyzed embryos which in turn may act to reduce motoneuron death by providing increased access to muscle-derived neurotrophic molecules.     

Developmental motoneuron cell death and neurotrophic factors

During the development of higher vertebrates, motoneurons are generated in excess. In the lumbar spinal cord of the developing rat, about 6000 motoneurons are present at embryonic day 14. These neurons grow out axons which make contact with their target tissue, the skeletal muscle, and about 50% of the motoneurons are lost during a critical period from embryonic day 14 until postnatal day 3. This process, which is called physiological motoneuron cell death, has been the focus of research aiming to identify neurotrophic factors which regulate motoneuron survival during this developmental period. Motoneuron cell death can also be observed in vitro when the motoneurons are isolated from the embryonic avian or rodent spinal cord. These isolated motoneurons and other types of primary neurons have been a useful tool for studying basic mechanisms underlying neuronal degeneration during development and under pathophysiological conditions in neurodegenerative disorders. Accumulating evidence from such studies suggests that some specific requirements of motoneurons for survival and proper function may change during development. The focus of this review is a synopsis of recent data on such specific mechanisms.     

Insulin-like growth factors: Putative muscle-derived trophic agents that promote motoneuron survival

Treatment of chick embryos in ovo with IGF-I during the period of normal, developmentally regulated neuronal death (embryonic days 5–10) resulted in a dose-dependent rescue of a significant number of lumbar motoneurons from degeneration and death. IGF-II and two variants of IGF-I with reduced affinity for IGF binding proteins, des(1-3) IGF-I and long R³ IGF-I, also elicited enhanced survival of motoneurons equal to that seen in IGF-I-treated embryos. IGF-I did not enhance mitogenic activity in motoneuronal populations when applied to embryos during the period of normal neuronal proliferation (E2-5). Treatment of embryos with IGF-I also reduced two types of injury-induced neuronal death. Following either deafferentation or axotomy, treatment of embryos with IGF-I rescued approximately 75% and 50%, respectively, of the motoneurons that die in control embryos as a result of these procedures. Consistent with the survival-promoting activity on motoneurons in ovo, IGF-I, -II, and des(1-3) IGF-I elevated choline acetyltransferase activity in embryonic rat spinal cord cultures, with des (1-3) IGF-I demonstrating 2.5 times greater potency than did IGF-I. A single addition of IGF-I at culture initiation resulted in the maintenance of 80% of the initial ChAT activity for up to 5 days, during which time ChAT activity in untreated control cultures fell to 9%. In summary, these results demonstrate clear motoneuronal trophic activity for the IGFs. These findings, together with previous reports that IGFs are synthesized in muscle and may participate in motoneuron axonal regeneration and sprouting, indicate that these growth factors may have an important role in motoneuron development, maintenance, and recovery from injury. © 1993 John Wiley & Sons, Inc.     

Identified nonspiking interneurons in leg reflexes and during walking in the stick insect

In the stick insect Carausius morosus identified nonspiking interneurons (type E4) were investigated in the mesothoracic ganglion during intraand intersegmental reflexes and during searching and walking.In the standing and in the actively moving animal interneurons of type E4 drive the excitatory extensor tibiae motoneurons, up to four excitatory protractor coxae motoneurons, and the common inhibitor 1 motoneuron (Figs. 1–4).In the standing animal a depolarization of this type of interneuron is induced by tactile stimuli to the tarsi of the ipsilateral front, middle and hind legs (Fig. 5). This response precedes and accompanies the observed activation of the affected middle leg motoneurons. The same is true when compensatory leg placement reflexes are elicited by tactile stimuli given to the tarsi of the legs (Fig. 6).During forward walking the membrane potential of interneurons of type E4 is strongly modulated in the step-cycle (Figs.8–10). The peak depolarization occurs at the transition from stance to swing. The oscillations in membrane potential are correlated with the activity profile of the extensor motoneurons and the common inhibitor 1 (Fig. 9).The described properties of interneuron type E4 in the actively behaving animal show that these interneurons are involved in the organization and coordination of the motor output of the proximal leg joints during reflex movements and during walking.Abbreviations CLP reflex, compensatory leg placement reflex - CI1 common inhibitor I motoneuron - fCO femoral chordotonal organ - FETi fast extensor tibiae motoneuron - FT femur-tibia - SETi slow extensor tibiae motoneuron     

Rescue of motoneurons from cell death by a purified skeletal muscle polypeptide: effects of the ChAT development factor, CDF

Rat skeletal muscle contains a 22 kd polypeptide that increases the level of choline acetyltransferase (ChAT) activity in cultures of embryonic rat spinal cord neurons and has been purified to homogeneity. The application of this factor, ChAT development factor or CDF, to developing chick embryos during the period of naturally occurring motoneuron cell death significantly increased the survival of motoneurons but did not affect the survival of dorsal root ganglion neurons or sympathetic preganglionic neurons (column of Terni). These results provide the first demonstration that an isolated, skeletal muscle-derived molecule can selectively enhance the survival of motoneurons in vivo and suggest that CDF may function in vivo to regulate the survival and development of motoneurons.     

Cell death of motoneurons in the chick embryo spinal cord. X. Synapse formation on motoneurons following the reduction of cell death by neuromuscular blockade

Chronic treatment of chick embryos with neuromuscular blocking agents, such as curare, rescues motoneurons from naturally occurring cell death. In the present study, embryos treated with curare from E6 to E9 had 35% more motoneurons than controls on E10 and 42% more than controls on E16. Previous studies have shown that several aspects of motoneuron differentiation occur normally in curare-treated embryos. We report here that dendrite growth and arborization is also unaltered on E10 and E16 following curare treatment. A quantitative analysis of afferent synapses on motoneurons shows that the packing density of both axosomatic and axodendritic synapses is also normal on E10 in curare-treated embryos, despite the greater number of motoneurons present. This indicates that the interneurons that provide presynaptic input to motoneurons are able to compensate for the increased number of synaptic sites made available by curare treatment. However, by E16 the packing density of synapses is reduced by about half. Because motoneurons and their dendrites continue to grow between E10 and E16, the further increase in synaptic sites made available in curare-treated embryos apparently exceeds the compensatory capacity of presynaptic interneurons on E16. One can conclude from these results that the increased survival of motoneurons in curare-treated embryos is not owing to an increase in afferent synapses. Motoneurons in these embryos continue to survive in the face of either no change (E10) or a reduction (E16) in the number of axodendritic and axosomatic synapses. Therefore, increased motoneuron survival in this situation is very likely regulated primarily by motoneuron-target interactions.     

The role of the frontal ganglion in foregut movements of the moth,Manduca sexta

Two types of rhythmic foregut movements are described in fifth instar larvae of the moth, Manduca sexta. These consist of posteriorly-directed waves of peristalsis which move food toward the midgut, and synchronous constrictions of the esophageal region, which appear to retain food within the crop. We describe these movements and the muscles of the foregut that generate them.The firing patterns of a subset of these muscles, including a constrictor and dilator pair from both the esophageal and buccal regions of the foregut, are described for both types of foregut movement.The motor patterns for the foregut muscles require innervation by the frontal ganglion (FG), which lies anterior to the brain and contains about 35 neurons. Eliminating the ventral nerve cord, leaving the brain and FG intact, did not affect the muscle firing patterns in most cases. Eliminating both the brain and the ventral nerve cord, leaving only the FG to innervate the foregut, generally resulted in an increased period for both gut movements and muscle bursts. This manipulation also produced increases in burst durations for most muscles, and had variable effects on the phasing of muscle activity. Despite these changes, the foregut muscles still maintained a rhythmic firing pattern when innervated by the FG alone.Two nerves exit the FG to innervate the foregut musculature: the anteriorly-projecting frontal nerve, and the posteriorly-directed recurrent nerve. Cutting the frontal nerve immediately and irreversibly stopped all muscle activity in the buccal region, while cutting the recurrent nerve immediately stopped all muscle activity in the pharyngeal and esophageal regions. Recordings from the cut nerves leaving the FG showed that the ganglion was spontaneously active, with rhythmic activity continuing within the nerves. These observations indicate that all of the foregut muscle motoneurons are located within the FG, and the FG in isolation produces a rhythmic firing pattern in the motoneurons. We have identified several motoneurons within the FG, by cobalt backfills and/or simultaneous intracellular recordings and fills from putative motoneurons and their muscles.Abbreviations BC Buccal Constrictor - BC1 buccal constrictor motoneuron 1 - BC2 buccal constrictor motoneuron 2 - BD Buccal Dilator - BD1 buccal dilator motoneuron 1 - EC Esophageal Dilator - EC1 esophageal dilator motoneuron 1 - EC2 esophageal dilator motoneuron 2 - EC3 esophageal dilator motoneuron 3 - ejp excitatory junction potential - FG frontal ganglion - psp postsynaptic potential