Choline Acetyltransferase Gene Research Articles

126th International Workshop: Congenital Myasthenic Syndromes, 24–26 September 2004, Naarden, The Netherlands

The ENMC hosted a group of 18 experts on Congenital Myasthenic Syndromes (CMS). CMS are inherited disorders in which the safety margin of the neuromuscular transmission is compromised by one or more specific mechanism(s). CMS are caused by various genetic defects. The objectives of the workshop included progress in deciphering the molecular basis of CMS (sessions 1–4) and clinical conclusions for epidemiology, diagnosis and therapy (sessions 5–7). To date, genes known to cause CMS if mutated are the presynaptic choline acetyltransferase gene CHAT, the gene COLQ encoding the synaptic protein ColQ, the genes encoding the different subunits of the postsynaptic acetylcholine receptor (CHRNA1, CHRNB1, CHRND, CHRNE), the genes for the postsynaptic proteins rapsyn (RAPSN), muscle-specific receptor tyrosine kinase (MUSK) and the postsynaptic sodium channel (SCN4A). Since the last ENMC workshop on CMS in October 1999, four novel CMS genes have been identified, namely CHAT, RAPSN, SCN4A and MUSK. As a consequence, several new patients presenting with varying phenotypes of CMS have been described worldwide. In particular, mutations in RAPSN and CHAT turned out to be of high clinical relevance, on one hand because of their apparent worldwide frequency, on the other hand because of their specific clinical phenotype with the occurrence of sudden apneic episodes. Furthermore, considerable progress has been made using a variety of

Expression of high affinity choline transporter during mouse development in vivo and its upregulation by NGF and BMP-4 in vitro

An important feature of cholinergic neurons is high-affinity choline transport, which allows them to reuse choline for the synthesis of ACh needed to support cholinergic neurotransmission. The choline transporter, designated CHT, was recently cloned. We applied RT/PCR to monitor the expression of CHT in the developing mouse CNS from embryonic day 14 (E14) to postnatal day 30 (P30). We found that CHT was expressed early in development, predominantly in the regions containing cholinergic neurons. In the spinal cord, CHT mRNA was present at close to adult levels at the earliest time point examined (E14) and showed almost no changes after birth. In the striatum and the septum, CHT mRNA increased steadily during embryonic stages and leveled off after birth. Surprisingly, CHT mRNA expression was also detected in other brain regions, notably in the cerebellum, where it peaked on E19, and then rapidly declined during postnatal development. CHT protein was detected by Western blotting as a band of apparent molecular weight of 70 kDa. The accumulation of this protein during development lagged behind mRNA accumulation in all tissues. We also examined the effects of NGF and BMP-4, the potent inducers of choline acetyltransferase and vesicular acetylcholine transporter genes, on CHT expression. Both factors increased CHT mRNA accumulation in primary septal cultures. The effect of NGF was dependent on the PI3K signaling, as it was abolished by the PI3K inhibitor LY294002. This result indicates that some of the signals regulating other cholinergic-specific genes also control CHT expression.

Rat choline acetyltransferase of the peripheral type differs from that of the common type in intracellular translocation

Choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of ChAT mRNA, which lacks exons 6–9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type (pChAT), because of its preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type (cChAT) using green fluorescent protein (GFP) in living human embryonic kidney cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT.

Clinical variability of CMS-EA (congenital myasthenic syndrome with episodic apnea) due to identical CHAT mutations in two infants

Congenital myasthenic syndromes (CMS) result from mutations in various synapse-associated genes. Mutations in the choline acetyltransferase (CHAT) gene cause a presynaptic CMS associated with episodic apnea (CMS-EA). We present two unrelated Croatian children affected by CMS-EA. Beside other clinical findings characteristic for CMS, both patients manifested intermittent apneas since early infancy. Whereas the course of disease is mild in the female patient (patient 2), the male patient (patient 1) experienced recurrent and severe episodes of apnea despite adequate treatment with AChE-inhibitors and shows a global developmental delay with delayed myelination and signs of hypoxic-ischemic injury in brain imaging. Interestingly, sequencing of the CHAT gene revealed identical, compound heterozygous mutations S694C and T354M in both children. These findings are in line with a remarkable clinical heterogeneity observed in patients with CHAT mutations and emphasize the potential role of apneic crises for the development of secondary hypoxic brain damage and psychomotor retardation.

Regulation of the cholinergic gene locus by the repressor element-1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF)

The cholinergic gene locus is comprised of two genes, the choline acetyltransferase gene and the vesicular acetylcholine transporter gene. The vesicular acetylcholine transporter gene is located within the first intron of the choline acetyltransferase gene. This arrangement permits coordinate regulation of the locus. Protein kinase A regulates expression of the cholinergic gene locus in PC12 cells. This regulation was found to be dependent on the presence of a 21-bp DNA sequence known as the repressor element-1 (RE-1)/neuron-restrictive silencer element (NRSE). Repressor element-1 silencing transcription factor (REST)/ neuron-restrictive silencer factor (NRSF), which binds to the RE-1/NRSE, is a zinc finger containing transcriptional repressor that blocks the expression of many neuronal RE-1/NRSE containing genes in nonneuronal cells. However, REST/NRSF expression has also been observed in neurons as well as the PC12 cell line used in these studies. REST/NRSF truncated isoforms were expressed in neuronal cells, suggesting they also function in regulating neuronal gene expression. A study of REST4, one of the REST/NRSF isoforms, suggests that it regulates transcription of the cholinergic gene locus by blocking the repressor activity of REST/NRSF. Protein kinase A regulation of the cholinergic gene locus in PC12 cells can thus be attributed, at least in part, to increased synthesis of REST4, which in turn derepresses the repressor activity of REST/NRSF.

Evaluation of the neurorestorative effects of the murine β-nerve growth factor infusions in old rat with cognitive deficit

The nerve growth factor (NGF) is known to participate in the regulation of the expression levels and activity of the choline acetyltransferase (ChAT) in the nervous system. This enzyme is sensitive to the degenerative changes found in Alzheimer’s disease (AD). We compared the effectiveness of intraparenchymal (ip) and intracerebroventricular (icv) administration of the murine β-NGF (β-NGFm) produced in our laboratories, through the determination of the expression levels and activity of the ChAT, and the evaluation of behavioral recovery in aged rat with cognitive deficit. Our results indicated that icv infusion of β-NGFm stimulates the expression levels of ChAT gene in the striatum of old rats. Remarkable losses in the ChAT activity were observed in the septum and striatum of old rats. Exogenous administration of β-NGFm produced a significant increase of ChAT activity in these brain regions differentially according to the administration pathway. The behavioral studies demonstrated that the administration pathway is an important factor in order to obtain the best results for a neurorestorative treatment.

Lack of association between a single nucleotide polymorphism within the choline acetyltransferase gene and patients with Alzheimer's disease

Alterations of the cholinergic system may account for typical clinical and pathophysiological disturbances of Alzheimer's disease (AD). In particular, a marked decline of choline acetyltransferase activity (CHAT) and as a consequence of acetylcholine during the course of the disease has been described. Due to the chromosomal localization of CHAT at 10q11.23 and its possible role in the pathophysiology of AD, CHAT may represent an appropriate candidate gene conferring risk to AD. In fact, a recent study identified a functional single nucleotide polymorphism (SNP) within the first common exon of CHAT, which was associated with AD giving an odds ratio of 3.8 (Neurosci. Lett. 333 (2002) 9). Because of the potential importance of this finding we analyzed this SNP and another functional SNP within exon 9 (rs868749) of the CHAT gene using a German case control sample consisting of 242 patients with AD and 143 cognitively healthy controls. No statistically significant differences were obtained for the previously described polymorphism. In addition, the exon 9 SNP (rs868749) was not polymorphic in the studied population. We conclude that the previously identified polymorphism is not associated with AD.

Congenital myasthenic syndrome with episodic apnea in patients homozygous for a CHAT missense mutation.

The syndrome of congenital myasthenia with episodic apnea (CMS-EA) was previously found to be due to mutations in the choline acetyltransferase gene (CHAT). To identify the mutations underlying CMS-EA in a Turkish multiplex family. Direct sequencing of the CHAT gene. A consanguineous Turkish family with 2 siblings affected by muscular weakness and episodic respiratory distress. The sequencing of CHAT coding exons identified a previously unknown missense mutation that affected a highly conserved amino acid residue (I336T). The mutation was absent in 164 control chromosomes. The high degree of conservation in different species strongly suggests that I336T is a functionally important amino acid residue. The absence of I336T from a large control sample further supports the pathogenic role of I336T in CMS-EA. This is the second report of CHAT mutations causing presynaptic CMS.

Regulation of cholinergic gene expression by the neuron restrictive silencer factor/repressor element-1 silencing transcription factor

The role of protein kinase A in regulating transcription of the cholinergic gene locus, which contains both the vesicular acetylcholine transporter gene and the choline acetyltransferase gene, was investigated in PC12 cells and a protein kinase A deficient PC12 mutant, A126.1B2 in which transcription of the locus is reduced. The site of action of protein kinase A was localized to a neuron restrictive silencer element/repressor element-1 (NRSE/RE-1) within the upstream region of the cholinergic gene locus. The neuron restrictive silencer factor/repressor element-1 silencing transcription factor (NRSF/REST), the transcription factor which binds to NRSE/RE-1, was expressed at similar levels in both PC12 and A126.1B2. Although nuclear extracts containing NRSF/REST from A126.1B2 exhibited binding to NRSE/RE-1, nuclear extracts from PC12 cells did not. The NRSF/REST isoform repressor element-1 silencing transcription factor-4 (REST4) was found to be expressed in PC12 cells, but not in the protein kinase A deficient PC12 cell line. REST4 inhibited the binding of NRSF/REST to NRSE/RE-1 as determined by gel mobility shift assays. Co-immunoprecipitation was used to demonstrate interaction between NRSF/REST and REST4. Expression of recombinant REST4 in the protein kinase A deficient PC12 cell line was sufficient to transcriptionally activate the cholinergic gene locus. Thus in PC12 cells protein kinase A promotes the production of REST4, which in turn de-represses of the cholinergic gene locus by inactivating the transcription repressor NRSF/REST.

Exploring the regulation of the expression of ChAT and VAChT genes in NG108-15 cells: implication of PKA and PI3K signaling pathways.

Involvement of different protein kinases regulated by cAMP and implication of muscarinic receptors in the regulation of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA levels and ChAT activity has been studied in NG108-15 cells. Dibutyryl cAMP enhanced both ChAT and VAChT mRNA levels and stimulated ChAT activity. Muscarinic stimulation or inhibition did not change ChAT activity or the receptor subtype mRNA pattern. MEK1/2 did not affect the regulation of ChAT and VAChT mRNA levels. However, PKA plays a major role in regulating ChAT and VAChT mRNA levels, because H89 decreased both. Strikingly, inhibition of PI3K by LY294002 had two opposite effects: ChAT mRNA level was decreased and VAChT mRNA level was increased. Such a result consolidates the observation that ChAT and VAChT genes, despite their unusual organization in a single "cholinergic locus," can be differentially or synergistically regulated, depending on the activated signaling pathways.

Identification of a single nucleotide polymorphism in the choline acetyltransferase gene associated with Alzheimer's disease

The dysfunction of the cholinergic system in Alzheimer's disease (AD) supports the hypothesis that a decline in choline acetyltransferase (ChAT) activity in memory as well as in cognitive functions in AD might be functionally linked. To assess the physiological relevance of an allelic variation in the ChAT gene we investigated the presence of a possible polymorphism in AD patients and in elderly non-demented subjects as controls. By using polymerase chain reaction, single stranded conformation polymorphism or the LightCycler analysis we detected a single nucleotide polymorphism in the first common coding exon of the ChAT gene. We found a G→A transition which occurred at position +4 of the coding sequence. The association between AD and the AA genotype or A alleles were found to be significant (odds ratio 3.7 and 2.4, respectively). The frequency of the AA genotype was three times higher in AD patients than in age – matched controls. This G→A change raises the possibility that it may influence ATG usage resulting in attenuation of translation efficacy of ChAT messenger RNA. We suggest that such a polymorphism might be one of the events conferring an increased risk for deterioration of memory and cognition functions in AD.

Synergistic activation of the human choline acetyltransferase gene by c-Myb and C/EBPβ

To elucidate regulatory mechanisms at the transcriptional level of the human choline acetyltransferase gene (hChAT) we performed cotransfections assays in NG108-15 and SN56 cells using ChAT-CAT reporter plasmids with c-Myb and C/EBPβ expression plasmids. The hChAT gene has several promoters, one of which (promoter P2 or M-type) is both c-Myb and C/EBPβ inducible as 3–4-fold trans-activation was obtained in both cell lines when using either c-Myb or C/EBPβ expression vectors alone. The simultaneous expression of c-Myb and C/EBPβ in the absence or presence of NGFI-C (egr4) leads respectively to a 15-fold and 32-fold synergistic transcriptional activation of promoter P2. In the region upstream of exon M (P2) we identified a functional composite element including a c-Myb next to a C/EBP binding site. An oligonucleotide containing the composite element confers c-Myb and C/EBPβ responsiveness to a heterologous promoter which is reduced after mutation of the c-Myb binding site. We also show that the coactivators CBP/p300 are required for c-Myb and C/EBPβ trans-activation function and that RARα, RXRα and T3R have an inhibitory action on the synergistic transcriptional activity of c-Myb and C/EBPβ and propose a model to explain the phenomena. Taken together, the results suggest that the synergistic effect of c-Myb and C/EBPβ, previously observed in the hematopoietic system, functions equally in the neuronal system.

Developmental expression of ascidian neurotransmitter synthesis genes

To identify cholinergic neurons, we isolated a choline acetyltransferase (Ci-ChAT) gene from Ciona intestinalis by PCR methods. In the cloning process, we also obtained the gene encoding the vesicular acetylcholine transporter (Ci-vAChTP). These two genes shared the same 5'-UTR sequence as well as similar expression patterns. In both cases, the gene expression was first detected by whole-mount in situ hybridization in the anterior-dorsal region of the caudal nerve cord at the early tailbud stage. In the larva, the expression was seen in several cells of the visceral ganglion. These results suggest that ascidian larval motor neurons exist in the visceral ganglion.

Mitogen-activated protein kinase kinase negatively modulates ciliary neurotrophic factor-activated choline acetyltransferase gene expression.

The expression of the choline acetyltransferase (ChAT) enzyme that synthesizes the neurotransmitter acetylcholine (ACh) is upregulated by ciliary neurotrophic factor (CNTF). We studied the involvement of the mitogen-activated protein kinase (MAPK) pathway in regulating ChAT expression in a murine septal cell line. Surprisingly, we found that PD98059 and U0126, two structurally distinct inhibitors of MAPK kinase (MEK1), increased both basal and CNTF-induced ACh production. Transient transfections with ChAT promoter-luciferase reporter construct demonstrated synergy between PD98059 and CNTF at the transcriptional level. Moreover, in cotransfection studies, overexpression of constitutively activated MEK1 completely abrogated the CNTF-mediated induction of the reporter. Blocking MEK1 did not significantly alter CNTF-induced Tyr705 phosphorylation of the principal mediator of the CNTF pathway, the transcription factor Stat3. However, PD98059 inhibited Ser727 phosphorylation of Stat3, demonstrating that the latter is MEK1-dependent. Taken together, these results indicate that activation of the MEK1/MAPK pathway inhibits the CNTF-mediated stimulation of ChAT expression, possibly as a part of a feedback mechanism.

More than one way to toy with ChAT and VAChT

Expression of choline acetyltransferase (ChAT) and of the vesicular acetylcholine transporter (VAChT) is required for the acquisition and the maintenance of the cholinergic phenotype. The ChAT and VAChT genes have been demonstrated to share a common gene locus and this suggests a coordinate regulation of their expression. In the present work, we examined the effects of several differentiating treatments on the modulation of ChAT and VAChT expression at the mRNA and protein levels in growing and differentiating NG108-15 cells. In cells grown in the presence of serum, all the agents tested—retinoic acid, dexamethasone and dibutyrylcyclicAMP (dbcAMP)—induced an increase of ChAT and VAChT mRNA levels but with different efficacy. Treatment with dbcAMP plus dexamethasone resulted in the largest increase of VAChT mRNA amount while retinoic acid mostly enhanced ChAT mRNA level. However, while ChAT activity was increased by all agents, no change in the VAChT protein level was detected. In cells differentiated by serum deprivation, only retinoic acid was effective in inducing an increase of VAChT and ChAT mRNA and ChAT activity, while we observed a downregulation by dbcAMP and dexamethasone. Treatment with the antimitotic agent cytosine arabinoside led to an increase of ChAT activity which was further largely enhanced by the addition of dbcAMP plus dexamethasone, but to only a slight change in VAChT activity. We further showed that complex glycosylation processes which might play a role in targeting and/or stability of the membrane protein VAChT are deficient in these cells. Indeed, in transient transfection assays with the reporter soluble enzyme luciferase placed under regulatory and promoter regions of the VAChT gene, we observed a modulation of luciferase expression by differentiating agents. These data illustrate the complexity of the processes which participate to the expression of the ChAT and VAChT genes, both at the transcriptional and posttranslational levels.

A novel untranslated 'exon H' of the human choline acetyltransferase gene in placenta.

To investigate the existence of 5'-region(s) of human choline acetyltransferase (hChAT) mRNA in placenta we analyzed the presence or absence of ChAT 5'-untranslated regions (UTR) in human neuronal and non-neuronal cells. Total RNA from human spinal cord, placenta, cultured choriocarcinoma JEG-3 and neuroblastoma CHP126 and MC-IXC cells was reverse transcribed and used for polymerase chain reaction amplification (RT-PCR). We used a sense primer located in the 5'-flanking region, in the previously defined intronic sequence and an anti-sense primer located in the common coding exon 2 of the hChAT gene. An amplified product of 567 bp in size was obtained only in human placenta and in JEG-3 cells whereas it was absent in spinal cord, CHP126 and MC-IXC cells. It was designated 'H-type' of ChAT mRNA. Whereas CHP126 produced the R- and N-type of ChAT mRNAs, no transcript of the N-and R-type was detected in JEG-3 and human placenta. In addition, CHP126 and JEG-3 cells and placenta showed the expression of the M-type of ChAT mRNA. The identity of the amplified 567 bp product (H-type) was confirmed by Southern hybridization and sequencing. The nucleotide sequence of the amplified fragment in placenta revealed the existence of a previously unknown type of ChAT mRNA produced by alternative splicing. Using primer extension we further determined the transcription initiation site of the H-type hChAT mRNA in placenta. These results demonstrate the expression of a novel ChAT mRNA isoform in human placenta in addition to the M-type. These data may be possibly explained by the presence of a placenta specific promoter in the ChAT gene, which might be the proximal promoter P1.

Independent patterns of transcription for the products of the rat cholinergic gene locus.

The cholinergic phenotype requires the expression of the vesicular acetylcholine transporter and choline acetyltransferase proteins. Both genes are encoded at one chromosomal location called the cholinergic gene locus. We have identified by in situ hybridization histochemistry distinct patterns of transcription from the cholinergic gene locus in the subdivisions of the rat cholinergic nervous system. The vesicular acetylcholine transporter and choline acetyltransferase are co-expressed in cholinergic neurons at all developmental stages in all major types of cholinergic neurons. The relative levels of vesicular acetylcholine transporter and choline acetyltransferase transcripts, however, change substantially during development in the CNS. They also differ dramatically in distinct subdivisions of the mature cholinergic nervous system, with vesicular acetylcholine transporter mRNA expressed at high levels relative to choline acetyltransferase mRNA in the peripheral nervous system, but at equivalent levels in the CNS. Expression of the R-exon, the presumptive first non-coding exon common to both the vesicular acetylcholine transporter and choline acetyltransferase, was not detectable at any developmental stage in any of the cholinergic neuronal subtypes in the rat nervous system. Thus, in contrast to less complex metazoan organisms, production of the vesicular acetylcholine transporter and choline acetyltransferase via a common differentially spliced transcript does not seem to occur to a significant extent in the rat. We suggest that separate transcriptional start sites within the cholinergic gene locus control vesicular acetylcholine transporter and choline acetyltransferase transcription, while additional elements are responsible for the specific transcriptional control of the entire locus in cholinergic versus non-cholinergic neurons. Independent transcription of the vesicular acetylcholine transporter and choline acetyltransferase genes provides a mechanism for regulating the relative expression of these two proteins to fine-tune acetylcholine quantal size in different types of cholinergic neurons, both centrally and peripherally.

Is RE1/NRSE a Common cis-Regulatory Sequence for ChAT and VAChT Genes?

Choline acetyltransferase (ChAT), the biosynthetic enzyme of acetylcholine, and the vesicular acetylcholine transporter (VAChT) are both required for cholinergic neurotransmission. These proteins are encoded by two embedded genes, the VAChT gene lying within the first intron of the ChAT gene. In the nervous system, both ChAT and VAChT are synthesized only in cholinergic neurons, and it is therefore likely that the cell type-specific expression of their genes is coordinately regulated. It has been suggested that a 2336-base pair genomic region upstream from the ChAT and VAChT coding sequences drives ChAT gene expression in cholinergic structures. We investigated whether this region also regulates VAChT gene transcription. Transfection assays showed that this region strongly represses the activity of the native VAChT promoters in non-neuronal cells, but has no major effect in neuronal cells whether or not they express the endogenous ChAT and VAChT genes. The silencer activity of this region is mediated solely by a repressor element 1 or neuron-restrictive silencer element (RE1/NRSE). Moreover, several proteins, including RE1-silencing transcription factor or neuron-restrictive silencer factor, are recruited by this regulatory sequence. These data suggest that this upstream region and RE1/NRSE co-regulate the expression of the ChAT and VAChT genes.

The distribution of cholinergic neurons in the human central nervous system.

Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine, is presently the most specific marker for identifying cholinergic neurons in the central and peripheral nervous systems. The present article reviews immunohistochemical and in situ hybridization studies on the distribution of neurons expressing ChAT in the human central nervous system. Neurons with both immunoreactivity and in situ hybridization signals of ChAT are observed in the basal forebrain (diagonal band of Broca and nucleus basalis of Meynert), striatum (caudate nucleus, putamen and nucleus accumbens), cerebral cortex, mesopontine tegmental nuclei (pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus and parabigeminal nucleus), cranial motor nuclei and spinal motor neurons. The cerebral cortex displays regional and laminal differences in the distribution of neurons with ChAT. The medial septal nucleus and medial habenular nucleus contain immunoreactive neurons for ChAT, which are devoid of ChAT mRNA signals. This is probably because there is a small number of cholinergic neurons with a low level of ChAT gene expression in these nuclei of human. Possible connections and speculated functions of these neurons are briefly summarized.

A retrograde signal is involved in activity-dependent remodeling at a C. elegans neuromuscular junction.

We have characterized how perturbations of normal synaptic activity influence the morphology of cholinergic SAB motor neurons that innervate head muscle in C. elegans. Mutations disrupting components of the presynaptic release apparatus, acetylcholine (ACh) synthesis or ACh loading into synaptic vesicles each induced sprouting of SAB axonal processes. These sprouts usually arose in the middle of the normal innervation zone and terminated with a single presynaptic varicosity. Sprouting SAB neurons with a similar morphology were also observed upon reducing activity in muscle, either by using mutants lacking a functional nicotinic ACh receptor subunit or through muscle-specific expression of a gain-of-function potassium channel. Analysis of temperature-sensitive mutants in the choline acetyltransferase gene revealed that the sprouting response to inactivity was developmentally regulated; reduction of synaptic activity in early larval stages, but not in late larval stages, induced both sprouting and addition of varicosities. Our results indicate that activity levels regulate the structure of certain synaptic connections between nerve and muscle in C. elegans. One component of this regulatory machinery is a retrograde signal from the postsynaptic cell that mediates the formation of synaptic connections.