The Orphan GPCR Receptor, GPR88, Interacts with Nuclear Protein Partners in the Cerebral Cortex.

GPR88 is an orphan G-protein-coupled receptor (GPCR) highly expressed in striatal medium spiny neurons (MSN), also found in cortical neurons at low level. In MSN, GPR88 has a canonical GPCR plasma membrane/cytoplasmic expression, whereas in cortical neurons, we previously reported an atypical intranuclear localization. Molecular size analysis suggests that GPR88, expressed in plasma membrane of MSN or in nuclear compartment of cortical neurons, corresponds to the full-length protein. By transfection of cortical neurons, we showed that GPR88 fluorescent chimeras exhibit a nuclear localization. This localization is contingent on the third intracytoplasmic loop and C-terminus domains, even though these domains do not contain any known nuclear localization signals (NLS). Using yeast two-hybrid screening with these domains, we identified the nuclear proteins ATRX, TOP2B, and BAZ2B, all involved in chromatin remodeling, as potential protein partners of GPR88. We also validated the interaction of GPR88 with these nuclear proteins by proximity ligation assay on cortical neurons in culture and coimmunoprecipitation experiments on cortical extracts from GPR88 wild-type (WT) and knockout (KO) mice. The identification of GPR88 subcellular partners may provide novel functional insights for nonclassical modes of GPCR action that could be relevant in the maturating process of neocortical neurons.

BMP- and neuropilin 1-mediated motor axon navigation relies on spastin alternative translation.

Functional analyses of genes responsible for neurodegenerative disorders have unveiled crucial links between neurodegenerative processes and key developmental signalling pathways. Mutations in SPG4-encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes that couple microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown. To selectively unravel their neuronal function, we blocked spastin synthesis from each initiation codon during zebrafish development and performed rescue analyses. The knockdown of each isoform led to different motor neuron and locomotion defects, which were not rescued by the selective expression of the other isoform. Notably, both morphant neuronal phenotypes were observed in a CRISPR/Cas9 spastin mutant. We next showed that M1 spastin, together with HSP proteins atlastin 1 and NIPA1, drives motor axon targeting by repressing BMP signalling, whereas M87 spastin acts downstream of neuropilin 1 to control motor neuron migration. Our data therefore suggest that defective BMP and neuropilin 1 signalling may contribute to the motor phenotype in a vertebrate model of spastin depletion.

Early reduced dopaminergic tone mediated by D3 receptor and dopamine transporter in absence epileptogenesis.

OBJECTIVE: In Genetic Absence Epilepsy Rats From Strasbourg (GAERSs), epileptogenesis takes place during brain maturation and correlates with increased mRNA expression of D3 dopamine receptors (D3R). Whether these alterations are the consequence of seizure repetition or contribute to the development of epilepsy remains to be clarified. Here, we addressed the involvement of the dopaminergic system in epilepsy onset in GAERSs. METHODS: Experiments were performed using rats at different stages of brain maturation from three strains according to their increasing propensity to develop absence seizures: nonepileptic control rats (NECs), Wistar Hannover rats, and GAERSs. Changes in dopaminergic neurotransmission were investigated using different behavioral and neurochemical approaches: autoradiography of D3R and dopamine transporter, single photon emission computed tomographic imaging, acute and chronic drug effects on seizure recordings (dopaminergic agonists and antagonists), quinpirole-induced yawns and dopamine synaptosomal uptake, microdialysis, brain tissue monoamines, and brain-derived neurotrophic factor quantification. RESULTS: Autoradiography revealed an increased expression of D3R in 14-day-old GAERSs, before absence seizure onset, that persists in adulthood, as compared to age-matched NECs. This was confirmed by increased yawns, a marker of D3R activity, and increased seizures when animals were injected with quinpirole at low doses to activate D3R. We also observed a concomitant increase in the expression and activity of the dopamine transporter in GAERSs before seizure onset, consistent with both lowered dopamine basal level and increased phasic responses. SIGNIFICANCE: Our data show that the dopaminergic system is persistently altered in GAERSs, which may contribute not only to behavioral comorbidities but also as an etiopathogenic factor in the development of epilepsy. The data suggest that an imbalanced dopaminergic tone may contribute to absence epilepsy development and seizure onset, as its reversion by a chronic treatment with a dopamine stabilizer significantly suppressed epileptogenesis. Our data suggest a potential new target for antiepileptic therapies and/or improvement of quality of life of epileptic patients.

Characterization of a Human Point Mutation of VGLUT3 (p.A211V) in the Rodent Brain Suggests a Nonuniform Distribution of the Transporter in Synaptic Vesicles.

The atypical vesicular glutamate transporter type 3 (VGLUT3) is expressed by subpopulations of neurons using acetylcholine, GABA, or serotonin as neurotransmitters. In addition, VGLUT3 is expressed in the inner hair cells of the auditory system. A mutation (p.A211V) in the gene that encodes VGLUT3 is responsible for progressive deafness in two unrelated families. In this study, we investigated the consequences of the p.A211V mutation in cell cultures and in the CNS of a mutant mouse. The mutation substantially decreased VGLUT3 expression (-70%). We measured VGLUT3-p.A211V activity by vesicular uptake in BON cells, electrophysiological recording of isolated neurons, and its ability to stimulate serotonergic accumulation in cortical synaptic vesicles. Despite a marked loss of expression, the activity of the mutated isoform was only minimally altered. Furthermore, mutant mice displayed none of the behavioral alterations that have previously been reported in VGLUT3 knock-out mice. Finally, we used stimulated emission depletion microscopy to analyze how the mutation altered VGLUT3 distribution within the terminals of mice expressing the mutated isoform. The mutation appeared to reduce the expression of the VGLUT3 transporter by simultaneously decreasing the number of VGLUT3-positive synaptic vesicles and the amount of VGLUT3 per synapses. These observations suggested that VGLUT3 global activity is not linearly correlated with VGLUT3 expression. Furthermore, our data unraveled a nonuniform distribution of VGLUT3 in synaptic vesicles. Identifying the mechanisms responsible for this complex vesicular sorting will be critical to understand VGLUT's involvement in normal and pathological conditions.SIGNIFICANCE STATEMENT VGLUT3 is an atypical member of the vesicular glutamate transporter family. A point mutation of VGLUT3 (VGLUT3-p.A211V) responsible for a progressive loss of hearing has been identified in humans. We observed that this mutation dramatically reduces VGLUT3 expression in terminals (∼70%) without altering its function. Furthermore, using stimulated emission depletion microscopy, we found that reducing the expression levels of VGLUT3 diminished the number of VGLUT3-positive vesicles at synapses. These unexpected findings challenge the vision of a uniform distribution of synaptic vesicles at synapses. Therefore, the overall activity of VGLUT3 is not proportional to the level of VGLUT3 expression. These data will be key in interpreting the role of VGLUTs in human pathologies.

Cooperative Interactions between 480 kDa Ankyrin-G and EB Proteins Assemble the Axon Initial Segment.

The axon initial segment (AIS) is required for generating action potentials and maintaining neuronal polarity. Significant progress has been made in deciphering the basic building blocks composing the AIS, but the underlying mechanisms required for AIS formation remains unclear. The scaffolding protein ankyrin-G is the master-organizer of the AIS. Microtubules and their interactors, particularly end-binding proteins (EBs), have emerged as potential key players in AIS formation. Here, we show that the longest isoform of ankyrin-G (480AnkG) selectively associates with EBs via its specific tail domain and that this interaction is crucial for AIS formation and neuronal polarity in cultured rodent hippocampal neurons. EBs are essential for 480AnkG localization and stabilization at the AIS, whereas 480AnkG is required for the specific accumulation of EBs in the proximal axon. Our findings thus provide a conceptual framework for understanding how the cooperative relationship between 480AnkG and EBs induces the assembly of microtubule-AIS structures in the proximal axon. SIGNIFICANCE STATEMENT: Neuronal polarity is crucial for the proper function of neurons. The assembly of the axon initial segment (AIS), which is the hallmark of early neuronal polarization, relies on the longest 480 kDa ankyrin-G isoform. The microtubule cytoskeleton and its interacting proteins were suggested to be early key players in the process of AIS formation. In this study, we show that the crosstalk between 480 kDa ankyrin-G and the microtubule plus-end tracking proteins, EBs, at the proximal axon is decisive for AIS assembly and neuronal polarity. Our work thus provides insight into the functional mechanisms used by 480 kDa ankyrin-G to drive the AIS formation and thereby to establish neuronal polarity.

Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane.

Dopamine (DA) is a major regulator of sensorimotor and cognitive functions. The DA transporter (DAT) is the key protein that regulates the spatial and temporal activity of DA release into the synaptic cleft via the rapid reuptake of DA into presynaptic termini. Several lines of evidence have suggested that transporter-interacting proteins may play a role in DAT function and regulation. Here, we identified the tetratricopeptide repeat domain-containing protein Ctr9 as a novel DAT binding partner using a yeast two-hybrid system. We showed that Ctr9 is expressed in dopaminergic neurons and forms a stable complex with DAT in vivo via GST pulldown and co-immunoprecipitation assays. In mammalian cells co-expressing both proteins, Ctr9 partially colocalizes with DAT at the plasma membrane. This interaction between DAT and Ctr9 results in a dramatic enhancement of DAT-mediated DA uptake due to an increased number of DAT transporters at the plasma membrane. We determined that the binding of Ctr9 to DAT requires residues YKF in the first half of the DAT C terminus. In addition, we characterized Ctr9, providing new insight into this protein. Using three-dimensional modeling, we identified three novel tetratricopeptide repeat domains in the Ctr9 sequence, and based on deletion mutation experiments, we demonstrated the role of the SH2 domain of Ctr9 in nuclear localization. Our results demonstrate that Ctr9 localization is not restricted to the nucleus, as previously described for the transcription complex Paf1. Taken together, our data provide evidence that Ctr9 modulates DAT function by regulating its trafficking.

Excitation-transcription coupling via calcium/calmodulin-dependent protein kinase/ERK1/2 signaling mediates the coordinate induction of VGLUT2 and Narp triggered by a prolonged increase in glutamatergic synaptic activity.

Homeostatic scaling of glutamatergic and GABAergic transmission is triggered by prolonged alterations in synaptic neuronal activity. We have previously described a presynaptic mechanism for synaptic homeostasis and plasticity that involves scaling the level of vesicular glutamate (VGLUT1) and gamma-aminobutyric acid (GABA) (VGAT) transporter biosynthesis. These molecular determinants of vesicle filling and quantal size are regulated by neuronal activity in an opposite manner and bi-directionally. Here, we report that a striking induction of VGLUT2 mRNA and synaptic protein is triggered by a prolonged increase in glutamatergic synaptic activity in mature neocortical neuronal networks in vitro together with two determinants of inhibitory synaptic strength, the neuronal activity-regulated pentraxin (Narp), and glutamate decarboxylase (GAD65). Activity-dependent induction of VGLUT2 and Narp exhibits a similar intermediate-early gene response that is blocked by actinomycin D and tetrodotoxin, by inhibitors of ionotropic glutamate receptors and L-type voltage-gated calcium channels, and is dependent on downstream signaling via calmodulin, calcium/calmodulin-dependent protein kinase (CaMK) and extracellular signal-regulated kinase 1/2 (ERK1/2). The co-induction of VGLUT2 and Narp triggered by prolonged gamma-aminobutyric acid type A receptor blockade is independent of brain-derived nerve growth factor and TrkB receptor signaling. VGLUT2 protein induction occurs on a subset of cortically derived synaptic vesicles in excitatory synapses on somata and dendritic processes of multipolar GABAergic interneurons, recognized sites for the clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptors by Narp. We propose that VGLUT2 and Narp induction by excitation-transcription coupling leads to increased glutamatergic transmission at synapses on GABAergic inhibitory feedback neurons as part of a coordinated program of Ca(2+)-signal transcription involved in mechanisms of homeostatic plasticity after prolonged hyperactivity.

Structural and Functional Characterization of the Interaction of Snapin with the Dopamine Transporter: Differential Modulation of Psychostimulant Actions.

The importance of dopamine (DA) neurotransmission is emphasized by its direct implication in several neurological and psychiatric disorders. The DA transporter (DAT), target of psychostimulant drugs, is the key protein that regulates spatial and temporal activity of DA in the synaptic cleft via the rapid reuptake of DA into the presynaptic terminal. There is strong evidence suggesting that DAT-interacting proteins may have a role in its function and regulation. Performing a two-hybrid screening, we identified snapin, a SNARE-associated protein implicated in synaptic transmission, as a new binding partner of the carboxyl terminal of DAT. Our data show that snapin is a direct partner and regulator of DAT. First, we determined the domains required for this interaction in both proteins and characterized the DAT-snapin interface by generating a 3D model. Using different approaches, we demonstrated that (i) snapin is expressed in vivo in dopaminergic neurons along with DAT; (ii) both proteins colocalize in cultured cells and brain and, (iii) DAT and snapin are present in the same protein complex. Moreover, by functional studies we showed that snapin produces a significant decrease in DAT uptake activity. Finally, snapin downregulation in mice produces an increase in DAT levels and transport activity, hence increasing DA concentration and locomotor response to amphetamine. In conclusion, snapin/DAT interaction represents a direct link between exocytotic and reuptake mechanisms and is a potential target for DA transmission modulation.

Motor axon navigation relies on Fidgetin-like 1-driven microtubule plus end dynamics.

During neural circuit assembly, extrinsic signals are integrated into changes in growth cone (GC) cytoskeleton underlying axon guidance decisions. Microtubules (MTs) were shown to play an instructive role in GC steering. However, the numerous actors required for MT remodeling during axon navigation and their precise mode of action are far from being deciphered. Using loss- and gain-of-function analyses during zebrafish development, we identify in this study the meiotic clade adenosine triphosphatase Fidgetin-like 1 (Fignl1) as a key GC-enriched MT-interacting protein in motor circuit wiring and larval locomotion. We show that Fignl1 controls GC morphology and behavior at intermediate targets by regulating MT plus end dynamics and growth directionality. We further reveal that alternative translation of Fignl1 transcript is a sophisticated mechanism modulating MT dynamics: a full-length isoform regulates MT plus end-tracking protein binding at plus ends, whereas shorter isoforms promote their depolymerization beneath the cell cortex. Our study thus pinpoints Fignl1 as a multifaceted key player in MT remodeling underlying motor circuit connectivity.

Homeostatic scaling of vesicular glutamate and GABA transporter expression in rat neocortical circuits.

Homeostatic control of pyramidal neuron firing rate involves a functional balance of feedforward excitation and feedback inhibition in neocortical circuits. Here, we reveal a dynamic scaling in vesicular excitatory (vesicular glutamate transporters VGLUT1 and VGLUT2) and inhibitory (vesicular inhibitory amino acid transporter VIAAT) transporter mRNA and synaptic protein expression in rat neocortical neuronal cultures, using a well established in vitro protocol to induce homeostatic plasticity. During the second and third week of synaptic differentiation, the predominant vesicular transporters expressed in neocortical neurons, VGLUT1 and VIAAT, are both dramatically upregulated. In mature cultures, VGLUT1 and VIAAT exhibit bidirectional and opposite regulation by prolonged activity changes. Endogenous coregulation during development and homeostatic scaling of the expression of the transporters in functionally differentiated cultures may serve to control vesicular glutamate and GABA filling and adjust functional presynaptic excitatory/inhibitory balance. Unexpectedly, hyperexcitation in differentiated cultures triggers a striking increase in VGLUT2 mRNA and synaptic protein, whereas decreased excitation reduces levels. VGLUT2 mRNA and protein are expressed in subsets of VGLUT1-encoded neocortical neurons that we identify in primary cultures and in neocortex in situ and in vivo. After prolonged hyperexcitation, downregulation of VGLUT1/synaptophysin intensity ratios at most synapses is observed, whereas a subset of VGLUT1-containing boutons selectively increase the expression of VGLUT2. Bidirectional and opposite regulation of VGLUT1 and VGLUT2 by activity may serve as positive or negative feedback regulators for cortical synaptic transmission. Intracortical VGLUT1/VGLUT2 coexpressing neurons have the capacity to independently modulate the level of expression of either transporter at discrete synapses and therefore may serve as a plastic interface between subcortical thalamic input (VGLUT2) and cortical output (VGLUT1) neurons.

Activity-dependent regulation of vesicular glutamate and GABA transporters: a means to scale quantal size.

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.

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.

SNAT2 amino acid transporter is regulated by amino acids of the SLC6 gamma-aminobutyric acid transporter subfamily in neocortical neurons and may play no role in delivering glutamine for glutamatergic transmission.

System A transporters SNAT1 and SNAT2 mediate uptake of neutral alpha-amino acids (e.g. glutamine, alanine, and proline) and are expressed in central neurons. We tested the hypothesis that SNAT2 is required to support neurotransmitter glutamate synthesis by examining spontaneous excitatory activity after inducing or repressing SNAT2 expression for prolonged periods. We stimulated de novo synthesis of SNAT2 mRNA and increased SNAT2 mRNA stability and total SNAT2 protein and functional activity, whereas SNAT1 expression was unaffected. Increased endogenous SNAT2 expression did not affect spontaneous excitatory action-potential frequency over control. Long term glutamine exposure strongly repressed SNAT2 expression but increased excitatory action-potential frequency. Quantal size was not altered following SNAT2 induction or repression. These results suggest that spontaneous glutamatergic transmission in pyramidal neurons does not rely on SNAT2. To our surprise, repression of SNAT2 activity was not limited to System A substrates. Taurine, gamma-aminobutyric acid, and beta-alanine (substrates of the SLC6 gamma-aminobutyric acid transporter family) repressed SNAT2 expression more potently (10x) than did System A substrates; however, the responses to System A substrates were more rapid. Since ATF4 (activating transcription factor 4) and CCAAT/enhancer-binding protein are known to bind to an amino acid response element within the SNAT2 promoter and mediate induction of SNAT2 in peripheral cell lines, we tested whether either factor was similarly induced by amino acid deprivation in neurons. We found that glutamine and taurine repressed the induction of both transcription factors. Our data revealed that SNAT2 expression is constitutively low in neurons under physiological conditions but potently induced, together with the taurine transporter TauT, in response to depletion of neutral amino acids.

Identification of endophilins 1 and 3 as selective binding partners for VGLUT1 and their co-localization in neocortical glutamatergic synapses: implications for vesicular glutamate transporter trafficking and excitatory vesicle formation.

1. Selective protein-protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation. 2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1-endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3. 3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.

Differential expression and regulation of the high-affinity choline transporter CHT1 and choline acetyltransferase in neurons of superior cervical ganglia.

Previous studies revealed that leukemia inhibitory factor (LIF) and retinoic acid (RA) induce a noradrenergic to cholinergic switch in cultured sympathetic neurons of superior cervical ganglia (SCG) by up-regulating the coordinate expression of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. Here, we examined the effect of both factors on high-affinity choline uptake (HACU) and on expression of the high-affinity choline transporter CHT1. We found that HACU and CHT1-mRNA levels are up-regulated by LIF and down-regulated by RA in these neurons. Thus, in contrast to LIF, RA differentially regulates the expression of the presynaptic cholinergic proteins. Moreover, we showed that untreated SCG neurons express HACU and CHT1-mRNAs at much higher levels than ChAT activity and transcripts. In intact SCG, CHT1-mRNAs are abundant and synthesized by the noradrenergic neurons themselves. This study provides the first example of CHT1 expression in neurons which do not use acetylcholine as neurotransmitter.

Quantification of transcriptional activities of reporter gene constructs in primary cultures of sympathetic neurons.

Primary cultures of sympathetic neurons provide an attractive cellular model for investigating the mechanisms of neurotransmitter phenotypic plasticity. However, it has not been possible to transfect these neurons by conventional techniques, and this has been a major impediment to molecular investigations of neuronal gene expression in this system. Here, reporter plasmids were transferred into the nuclei of cultured sympathetic neurons by microinjection. We developed and improved this procedure and were able to measure the transcriptional activities of two coinjected promoters in small groups of neurons, and even from a single neuron. Promoter activities can thus be quantified and normalized relative to that of a constitutively expressed promoter, allowing correction for variability in the injection and assay procedures. High and low promoter activities can be reliably quantified. Importantly, this method can be used not only for reporter plasmids but also for DNA fragments containing only a promoter and reporter gene without any vector sequence that might interfere with promoter. Using this approach, we measured neuronal promoter activities and found that one promoter region of the gene encoding choline acetyltransferase was up-regulated by more than sevenfold by leukemia inhibitory factor. This method thus provides the means to investigate the function of neuronal genes and the mechanisms that regulate their transcription in cultured sympathetic neurons.