- Email: [email protected]
From the cholinergic gene locus to the cholinergic neuron
JPhysiolog~ 0 Elsevier,
(Au-is) Paris
(1998)92,
385-388
From the cholinergic gene locus to the cholinergic neuron Eberhard Weihea, Martin K.-H. Schafera, Burkhard Schiitza, Martin Anlaufa, Candan Depboylua, Christian Bretta, Liangbiao Chenb, Lee E. Eidenb “Department of Anutomy and Cell Biology, Philipps Uniwrsity, Robert-Koch Strclsse 6, Mrrrburg 35033, Germane hSection on Molecular Neuroscience, Laborutory of Cellular nnd Molecular Regulation, National Institute qf Mental Health, NIH, 36 Convent Drive, MSC 4090. Bethesda, MD 20892-4090, USA
Abstract - The cholinergic gene locus (CGL) was first identified in 1994 as the site (human chromosome IOql 1.2) at which choline acetyltransferase and a functional vesicular acetylcholine transporter are co-localized. Here, we present recent neuroanatomical. devrlopmental, and evolutionary insights into the chemical coding of cholinergic neurotransmission that have been gleaned from the study of the CGL, and its protein products VAChT and ChAT, which comprise a synthesis-sequestration pathway that functionally defines the cholinergic phenotype. (OElsevier, Paris)
R&urn& - Du locus CGL) a CtC identifie mosome humain IOql sion cholincrgique synthese-sequestration synaptic
g6nktique cholinergique au en 1994 et regroupe les genes 1.2). Nous presentons ici des obtenus Zr partir de I’ttude definit fonctionnellement le
vesicle I acetylcholine
I VAChT
/ ChAT
neurone cholinergique. Le locus genetique cholinergique (cholinergic gene locus. de la choline acetyltransferase et d’un recepteur vesiculaire d’acttylcholine (chro don&es sur la neuroanatomie, le dtveloppement et l’evolution de la neurotransmi\du CGL et de ses produits proteiques. VAChT et ChAT: ce systbme de phenotype cholinergique. (OElsevier, Paris)
/ peripheral
and central
The cholinergic gene locus (CGL) comprises an approximately 80 kb region of the mammalian genome that encodes choline acetyltransferase (ChAT). the vesicular acetylcholine transporter (VAChT) and the regulatory sequences responsible for expression of VAChT and ChAT in cholinergic neurons. The CGL is so named because it encodes the two proteins that functionally define the cholinergic neuronal phenotype, within a single regulatory domain [ 161. Our current view based on extensive analysis of the rat and primate nervous systems is that VAChT and ChAT mRNA and protein are invariably co-expressed, and thus are both adequate markers for the cholinergic phenotype (see [3] for review). VAChT immunohistochemistry offers certain advantages in surveying fixed tissue, and has allowed the unambiguous identification of peripheral muscular vasculature and the hypothalamus in the central nervous system, as targets of cholinergic innervation ([ 10, 1l] and references therein). Most notably, VAChT immunohistochemistry has revealed that noradrenergic and cholinergic phenotypes are co-expressed in developing autonomic neurons of both the parasympathetic and sympathetic lineages. The former become exclusively cholinergic and the latter mainly noradrenergic through a process of phenotypic restriction, rather than noradrenergic/cholinergic ‘switching’, during development [ 12, 131. Improved VAChT immunohistochemistry is perfectly suited to delineate peripheral cholinergic neurons and termi-
nervous
system
/ ontogeny
I cholinergic
differentiation
nals and to characterize peptidergic cotransmitters. Our recent immunohistochemical and in situ hybridization studies indicate that virtually all enteric neurons of the upper gastrointestinal tract co-express VIP and acetylcholine, suggesting that the ‘non-adrenergic/non-cholinergic (NANC)’ component of the enteric nervous system is much less extensive than previously believed (Anlauf et al., unpublished ohservations). VAChT immunoreactivity is present in interneurons of both rodent and primate cerebral cortex ([ 101 and references therein). In addition, the patterns of cholinergic innervation of the primate cortex. now examined in detail in paraftin-embedded tissues, suggest a decidedly lamina- and area-specific cholinergic innervation. A detailed picture of the cholinergic innervation of the telencephalon provides the foundation for a clearer understanding of cholrnergic function in cognition, arousal, and neurodegenerative diseases such as Alzheimer’s disease. With respect to the suggested neuroinflammatory background of these diseases it is of interest that the prostaglandin synthesizing enzyme cyclooxygcnase 2 (COX-2) is preferentially co-expressed with VAChT in primate cholinergic projection neurons of the basal forebrain (Depboylu et al., unpublished observations). While expression of both VAChT and ChAT from the CGL is required for the cholinergic phenotype, non-concordance in VAChT and ChAT expression in vivo could function as a mechanism to fine-tune cho-
386
E. Weihe et al.
linergic function. For example, the ratio of VAChT to ChAT mRNA in at least some parts of the peripheral nervous system of the adult rat is higher than in the central nervous system cfigure I). This may be related to an early pattern of VAChT overexpression relative to ChAT both centrally and peripherally during development [6] (Schiitz et al., unpublished observations). How might the CGL be regulated at the transcriptional level to allow differential expression of these two gene products, and to what purpose? In the nematode, VAChT and ChAT are transcribed from separatemessengerRNAs that arise from a common primary transcript, with presumably a single transcriptional start site, via differential splicing [2]. This is also the case in Drosophila [7]. However, the ratio of VAChT to ChAT mRNA is quite different in the head and body of Drosophila [7], similar to the situation in peripheral versus central cholinergic neurons in mammals. Various laboratories have inventoried VAChT and ChAT
transcripts of the central and peripheral nervous systems, or cell lines derived therefrom, and the segments of the CGL that are responsible for VAChT and/or ChAT expression in transgenic animals and cell lines (see [3, 18, 201 for reviews). At present, it appears that separate promoter/enhancers may drive VAChT and ChAT expression in a cell-specific manner, while a common exon for VAChT and ChAT (the ‘R’ exon) suggeststhat transcription from a single start site generating alternatively spliced VAChT and ChAT transcripts also occurs. Which is the dominant transcriptional mode in mammals? The answer may depend in part on physiological and anatomical context. In the brain, ‘R’ exon expression is much less than that for VAChT or ChAT orf-containing mRNAs lfigure 2) This implies that ‘R’ may be a minor transcriptional pathway, and that separate promoters drive expression of VAChT and ChAT from the CGL, at least under normal physiological conditions in the adult mammalian central nervous
Figure 1. Comparative expressionof VAChT andChAT mRNA in brain and parasympathetic ganglion of adult rat. VAChT (A) and ChAT (B) mRNA levelsare similarin adjacentbrain sections.Note higherlevelsof VAChT mRNA (C) as compared to ChAT mRNA (D) in adjacent sections of the otic ganglion. Rat (r) VAChT and rChAT riboprobes of uniform sizewere synthesized and calibrated for hybridization efficiency as described by Hahm et al. [.5].
Xth International Symposium on Cholinergic Mechanisms
r(=@4T270 _, I ., ,: .:, :. ,&.” i : ,.
:,
system. Consistent with this possibility, Naciff et al. report that a genomic fragment that excludes the R exon and includes only about 600 bases of the CGL upstream of the ATG that initiates the VAChT orf is sufficient for correct cholinergic expression in vivo
ISI.
The catalytic properties of the VAChT and ChAT proteins themselves suggest that precise regulation of the CGL is required to maintain cholinergic function both centrally and peripherally. The affinity of acetylcholine for the transporter is reckoned to be approximately 1 mM [ 17, 181,close to the estimated concentration of acetylcholine synthesized in the cytoplasm of motor neurons by ChAT. Thus changes in the ratio of VAChT to ChAT mRNAs, if reflected in differential protein abundance, could directly affect the size of ACh quanta in cholinergic neurons. Increased cholinergic quanta1 size in Xerzc>pu.rneurons expressing human VAChT support this assertion [ 141. VAChT is targeted to small synaptic vesicles (SSVs) both in vivo and in cell lines [4. 191, yet appears to be transported to the nerve terminal via large dense-core vesicles [ 151. The vesicular monoamine transporter type 2 (VMAT2) localizes preferentially to large dense-core vesicles (LDCVs) in PC 12 cells, and appears to be transported to the nerve terminal in LDCVs as well. Since VMAT2 is localized to both small and large synaptic vesicles in brain [9], PC12 cells apparently produce ‘cholinergic’ but not ‘noradrenergic’ SSVs. This finding implies that there are special properties of each type of vesicle besides the mere presence of the appro-
387
Figure 2. Relativeexpressionof tht: R, VAChTorf, andChAT first coding exon in the rat central nervousxystern. Sequentialsectionsthroughrat brain stemhybridized with specific probesfor RVAChTorf (A), R-exon (B) andrCHAT orf (C, D). Note the faint hybridization signalfor the Rexon. Datafrom Hahmet al. IS].
priate transporter. Why so many different kind of ve-sicles?In cholinergic neurons, differential releaseof ACh and neuropeptides from two different vesicular compartmentsprovides variation in the mtiOS of ACh and VIP, for example, released at different rates of stimulation (e.g., [I I). Differential targeting to subpopulations of small synaptic vesicles may also be physiologically relevant, for example at developmental stages in which autonomic neurons express both cholinergic and noradrenergic traits [ 131. Expression of VAChT and ChAT from the CGL. which makesa neuron primordially cholinergic, also makes the cholinergic neuron amenable to genetic, ontogenic, pathophysiological. and neuroanatomical analysis. Understanding the transcriptional, translational and biochemical regulation of these two proteins ultimately will aid in unlocking the mechanisms by which chemically coded transmis-sion arises during development, serves the needs of the organism. and perhaps goes awry in motor and cognitive neurodegenerative diseases. Acknowledgments We wish to acknowledge thecollaboration of our laboram ries with those of Jim Rand. at the Oklahoma Medical Re-search Foundation,
and Marie-Francoise
Diebler,
at the
Departement de Neurochimie,Laboratoirede Neurobiologic Cellulaire,Gif-stir-Yvette, in the originalcharacterizationof the mammalian cholinergic gene locus. Supported in part b\ the German Research Foundation swagenstiftung.
(SFB 397) and Volk-
E. Weihe et al.
VAChT. the vesicular acetylcholine transporter I. Central nervous system, Neuroscience 84 (1998) 331-359. Schafer M.K.-H.. Eiden L.E., Weihe E., Cholinergic neurons and terminal fields revealed by immunohistochemistry for VAChT, the vesicular acetylcholine transporter II. Peripheral nervous system, Neuroscience 84 (1998) 361-376.
References [II
Agoston D.V.. Conlon J.M., Whittaker V.P., Selective depletion of the acetylcholine and vasoactive intestinal polypeptide of the guinea-pig myenteric plexus by differential mobilization of distinct transmitter pools, Exp. Brain Res. 72 (1988) 535-542.
VI
Alfonso A., Grundahl K., McManus J.R., Asbury J.M.. Rand J.B., Alternative splicing leads to two cholinergic proteins in C. elegans, J. Mol. Biol. 241 (1994) 627-630.
[31
Eiden L.E., The cholinergic (1998) 2227-2240.
141
Gilmor M.L., Nash N.R., Roghani A., Edwards R.H., Yi H., Hersch S.M., Levey AI., Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles, J. Neurosci. 16 (1996) 2179-2 190.
[51
gene locus,
J. Neurochem.
70
Hahm S.H., Chen L., Pate1 C., Erickson .I., Banner T.I., Weihe E., Schafer MK-H., Eiden L.E.. Upstream sequencing and functional characterization of the human cholinergic gene locus, J. Mol. Neurosci. 9 (1997) 223-236.
[61
Holler T., Berse B., Cermak J.M., Diebler M.-F., Blusztajn J.K.. Differences in the developmental expression of the vesicular acetylcholine transporter and choline acetyltransferase in the rat brain, Neurosci. Lett. 212 (1996) 107-I 10.
171
Kitamoto T., Wang W., Salvaterra P.M., Structure and organization of the Drosophi/a cholinergic locus, J. Biol. Chem. 273 (1998) 27062713.
181
Naciff J.M., Misawa H., Dedman J.R., Molecular characterization of the mouse vesicular acetylcholine transporter gene, NeuroReport 8 (1997) 3467-3473.
191
Nirenberg M.J., Chan J., Liu Y., Edwards R.H., Pickel V.M., Ultrastructural localization of the vesicular monoamine transporter-2 in midbrain dopaminergic neurons: Potential sties for somatodendritic storage and release of dopamine, J. Neurosci. 16 (1996) 41354145.
[lOI
Schafer M.K.-H., Eiden L.E., Weihe E., Cholinergic neurons and terminal fields revealed by immunohistochemistry for
[I21
Schafer M.K.-H.. Schiltz B., Weihe E., Eiden L.E.. Targetindependent cholinergic differentiation in the rat sympathitic nervous system, Proc. Nat]. Acad. Sci. USA 94 (1997) 4149-4154.
Eiden LE., Weihe E.. Vesicular 1131 Schiitz B., Schafer M.K.-H., amine transporter expression and isoform selection in developing brain, peripheral nervous system and gut, Dev. Brain Res. 106 (1998) 181-204. E., Edwards R.H., 1141 Song H., Ming Cl., Fon E., Bellocchio Poo M., Expression of a putative vesicular acetylcholine transporter facilitates quanta1 transmitter packaging, Neuron 18 (1997) 815-826. S., Eiden L.E., The vesicular monoamine trans1151 Tao-Cheng porter VMATZ is targeted to large dense-core vesicles, and the vesicular acetylcholine transporter VAChT to small synaptic vesicles, in PC12 cells, Adv. Pharmacol. 11998), in press. U61 Usdin T., Eiden L.E.. Bonner T.I., Erickson J.D., ,Molecular biology of vesicular acetylcholine transporters (VAChTs), TINS 18 (1995) 218-224. [171
Varoqui H., Erickson J.D., Active transport of acetylcholine by the human vesicular acetylcholine transporter, J. Biol. Chem. 27 1 (I 996) 27229-27232.
1181 Varoqui
H., Erickson J.D., Vesicular neurotransmitter transporters, Mol. Neurobiol. I5 (1997) 165-191. Erickson J.D.. 1191 Weihe E.. Tao-Cheng J.-H., Schafer M.K.-H., Eiden LE., Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles. Proc. Nat]. Acad. Sci. USA 93 (1996) 3547-3552.
1201 Wu
D., Hersh L.B., Choline its fiftieth year, J. Neurochem.
acetyltransferase: celebrating 62 (1994) 1653-1663.
(Au-is) Paris
(1998)92,
385-388
From the cholinergic gene locus to the cholinergic neuron Eberhard Weihea, Martin K.-H. Schafera, Burkhard Schiitza, Martin Anlaufa, Candan Depboylua, Christian Bretta, Liangbiao Chenb, Lee E. Eidenb “Department of Anutomy and Cell Biology, Philipps Uniwrsity, Robert-Koch Strclsse 6, Mrrrburg 35033, Germane hSection on Molecular Neuroscience, Laborutory of Cellular nnd Molecular Regulation, National Institute qf Mental Health, NIH, 36 Convent Drive, MSC 4090. Bethesda, MD 20892-4090, USA
Abstract - The cholinergic gene locus (CGL) was first identified in 1994 as the site (human chromosome IOql 1.2) at which choline acetyltransferase and a functional vesicular acetylcholine transporter are co-localized. Here, we present recent neuroanatomical. devrlopmental, and evolutionary insights into the chemical coding of cholinergic neurotransmission that have been gleaned from the study of the CGL, and its protein products VAChT and ChAT, which comprise a synthesis-sequestration pathway that functionally defines the cholinergic phenotype. (OElsevier, Paris)
R&urn& - Du locus CGL) a CtC identifie mosome humain IOql sion cholincrgique synthese-sequestration synaptic
g6nktique cholinergique au en 1994 et regroupe les genes 1.2). Nous presentons ici des obtenus Zr partir de I’ttude definit fonctionnellement le
vesicle I acetylcholine
I VAChT
/ ChAT
neurone cholinergique. Le locus genetique cholinergique (cholinergic gene locus. de la choline acetyltransferase et d’un recepteur vesiculaire d’acttylcholine (chro don&es sur la neuroanatomie, le dtveloppement et l’evolution de la neurotransmi\du CGL et de ses produits proteiques. VAChT et ChAT: ce systbme de phenotype cholinergique. (OElsevier, Paris)
/ peripheral
and central
The cholinergic gene locus (CGL) comprises an approximately 80 kb region of the mammalian genome that encodes choline acetyltransferase (ChAT). the vesicular acetylcholine transporter (VAChT) and the regulatory sequences responsible for expression of VAChT and ChAT in cholinergic neurons. The CGL is so named because it encodes the two proteins that functionally define the cholinergic neuronal phenotype, within a single regulatory domain [ 161. Our current view based on extensive analysis of the rat and primate nervous systems is that VAChT and ChAT mRNA and protein are invariably co-expressed, and thus are both adequate markers for the cholinergic phenotype (see [3] for review). VAChT immunohistochemistry offers certain advantages in surveying fixed tissue, and has allowed the unambiguous identification of peripheral muscular vasculature and the hypothalamus in the central nervous system, as targets of cholinergic innervation ([ 10, 1l] and references therein). Most notably, VAChT immunohistochemistry has revealed that noradrenergic and cholinergic phenotypes are co-expressed in developing autonomic neurons of both the parasympathetic and sympathetic lineages. The former become exclusively cholinergic and the latter mainly noradrenergic through a process of phenotypic restriction, rather than noradrenergic/cholinergic ‘switching’, during development [ 12, 131. Improved VAChT immunohistochemistry is perfectly suited to delineate peripheral cholinergic neurons and termi-
nervous
system
/ ontogeny
I cholinergic
differentiation
nals and to characterize peptidergic cotransmitters. Our recent immunohistochemical and in situ hybridization studies indicate that virtually all enteric neurons of the upper gastrointestinal tract co-express VIP and acetylcholine, suggesting that the ‘non-adrenergic/non-cholinergic (NANC)’ component of the enteric nervous system is much less extensive than previously believed (Anlauf et al., unpublished ohservations). VAChT immunoreactivity is present in interneurons of both rodent and primate cerebral cortex ([ 101 and references therein). In addition, the patterns of cholinergic innervation of the primate cortex. now examined in detail in paraftin-embedded tissues, suggest a decidedly lamina- and area-specific cholinergic innervation. A detailed picture of the cholinergic innervation of the telencephalon provides the foundation for a clearer understanding of cholrnergic function in cognition, arousal, and neurodegenerative diseases such as Alzheimer’s disease. With respect to the suggested neuroinflammatory background of these diseases it is of interest that the prostaglandin synthesizing enzyme cyclooxygcnase 2 (COX-2) is preferentially co-expressed with VAChT in primate cholinergic projection neurons of the basal forebrain (Depboylu et al., unpublished observations). While expression of both VAChT and ChAT from the CGL is required for the cholinergic phenotype, non-concordance in VAChT and ChAT expression in vivo could function as a mechanism to fine-tune cho-
386
E. Weihe et al.
linergic function. For example, the ratio of VAChT to ChAT mRNA in at least some parts of the peripheral nervous system of the adult rat is higher than in the central nervous system cfigure I). This may be related to an early pattern of VAChT overexpression relative to ChAT both centrally and peripherally during development [6] (Schiitz et al., unpublished observations). How might the CGL be regulated at the transcriptional level to allow differential expression of these two gene products, and to what purpose? In the nematode, VAChT and ChAT are transcribed from separatemessengerRNAs that arise from a common primary transcript, with presumably a single transcriptional start site, via differential splicing [2]. This is also the case in Drosophila [7]. However, the ratio of VAChT to ChAT mRNA is quite different in the head and body of Drosophila [7], similar to the situation in peripheral versus central cholinergic neurons in mammals. Various laboratories have inventoried VAChT and ChAT
transcripts of the central and peripheral nervous systems, or cell lines derived therefrom, and the segments of the CGL that are responsible for VAChT and/or ChAT expression in transgenic animals and cell lines (see [3, 18, 201 for reviews). At present, it appears that separate promoter/enhancers may drive VAChT and ChAT expression in a cell-specific manner, while a common exon for VAChT and ChAT (the ‘R’ exon) suggeststhat transcription from a single start site generating alternatively spliced VAChT and ChAT transcripts also occurs. Which is the dominant transcriptional mode in mammals? The answer may depend in part on physiological and anatomical context. In the brain, ‘R’ exon expression is much less than that for VAChT or ChAT orf-containing mRNAs lfigure 2) This implies that ‘R’ may be a minor transcriptional pathway, and that separate promoters drive expression of VAChT and ChAT from the CGL, at least under normal physiological conditions in the adult mammalian central nervous
Figure 1. Comparative expressionof VAChT andChAT mRNA in brain and parasympathetic ganglion of adult rat. VAChT (A) and ChAT (B) mRNA levelsare similarin adjacentbrain sections.Note higherlevelsof VAChT mRNA (C) as compared to ChAT mRNA (D) in adjacent sections of the otic ganglion. Rat (r) VAChT and rChAT riboprobes of uniform sizewere synthesized and calibrated for hybridization efficiency as described by Hahm et al. [.5].
Xth International Symposium on Cholinergic Mechanisms
r(=@4T270 _, I ., ,: .:, :. ,&.” i : ,.
:,
system. Consistent with this possibility, Naciff et al. report that a genomic fragment that excludes the R exon and includes only about 600 bases of the CGL upstream of the ATG that initiates the VAChT orf is sufficient for correct cholinergic expression in vivo
ISI.
The catalytic properties of the VAChT and ChAT proteins themselves suggest that precise regulation of the CGL is required to maintain cholinergic function both centrally and peripherally. The affinity of acetylcholine for the transporter is reckoned to be approximately 1 mM [ 17, 181,close to the estimated concentration of acetylcholine synthesized in the cytoplasm of motor neurons by ChAT. Thus changes in the ratio of VAChT to ChAT mRNAs, if reflected in differential protein abundance, could directly affect the size of ACh quanta in cholinergic neurons. Increased cholinergic quanta1 size in Xerzc>pu.rneurons expressing human VAChT support this assertion [ 141. VAChT is targeted to small synaptic vesicles (SSVs) both in vivo and in cell lines [4. 191, yet appears to be transported to the nerve terminal via large dense-core vesicles [ 151. The vesicular monoamine transporter type 2 (VMAT2) localizes preferentially to large dense-core vesicles (LDCVs) in PC 12 cells, and appears to be transported to the nerve terminal in LDCVs as well. Since VMAT2 is localized to both small and large synaptic vesicles in brain [9], PC12 cells apparently produce ‘cholinergic’ but not ‘noradrenergic’ SSVs. This finding implies that there are special properties of each type of vesicle besides the mere presence of the appro-
387
Figure 2. Relativeexpressionof tht: R, VAChTorf, andChAT first coding exon in the rat central nervousxystern. Sequentialsectionsthroughrat brain stemhybridized with specific probesfor RVAChTorf (A), R-exon (B) andrCHAT orf (C, D). Note the faint hybridization signalfor the Rexon. Datafrom Hahmet al. IS].
priate transporter. Why so many different kind of ve-sicles?In cholinergic neurons, differential releaseof ACh and neuropeptides from two different vesicular compartmentsprovides variation in the mtiOS of ACh and VIP, for example, released at different rates of stimulation (e.g., [I I). Differential targeting to subpopulations of small synaptic vesicles may also be physiologically relevant, for example at developmental stages in which autonomic neurons express both cholinergic and noradrenergic traits [ 131. Expression of VAChT and ChAT from the CGL. which makesa neuron primordially cholinergic, also makes the cholinergic neuron amenable to genetic, ontogenic, pathophysiological. and neuroanatomical analysis. Understanding the transcriptional, translational and biochemical regulation of these two proteins ultimately will aid in unlocking the mechanisms by which chemically coded transmis-sion arises during development, serves the needs of the organism. and perhaps goes awry in motor and cognitive neurodegenerative diseases. Acknowledgments We wish to acknowledge thecollaboration of our laboram ries with those of Jim Rand. at the Oklahoma Medical Re-search Foundation,
and Marie-Francoise
Diebler,
at the
Departement de Neurochimie,Laboratoirede Neurobiologic Cellulaire,Gif-stir-Yvette, in the originalcharacterizationof the mammalian cholinergic gene locus. Supported in part b\ the German Research Foundation swagenstiftung.
(SFB 397) and Volk-
E. Weihe et al.
VAChT. the vesicular acetylcholine transporter I. Central nervous system, Neuroscience 84 (1998) 331-359. Schafer M.K.-H.. Eiden L.E., Weihe E., Cholinergic neurons and terminal fields revealed by immunohistochemistry for VAChT, the vesicular acetylcholine transporter II. Peripheral nervous system, Neuroscience 84 (1998) 361-376.
References [II
Agoston D.V.. Conlon J.M., Whittaker V.P., Selective depletion of the acetylcholine and vasoactive intestinal polypeptide of the guinea-pig myenteric plexus by differential mobilization of distinct transmitter pools, Exp. Brain Res. 72 (1988) 535-542.
VI
Alfonso A., Grundahl K., McManus J.R., Asbury J.M.. Rand J.B., Alternative splicing leads to two cholinergic proteins in C. elegans, J. Mol. Biol. 241 (1994) 627-630.
[31
Eiden L.E., The cholinergic (1998) 2227-2240.
141
Gilmor M.L., Nash N.R., Roghani A., Edwards R.H., Yi H., Hersch S.M., Levey AI., Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles, J. Neurosci. 16 (1996) 2179-2 190.
[51
gene locus,
J. Neurochem.
70
Hahm S.H., Chen L., Pate1 C., Erickson .I., Banner T.I., Weihe E., Schafer MK-H., Eiden L.E.. Upstream sequencing and functional characterization of the human cholinergic gene locus, J. Mol. Neurosci. 9 (1997) 223-236.
[61
Holler T., Berse B., Cermak J.M., Diebler M.-F., Blusztajn J.K.. Differences in the developmental expression of the vesicular acetylcholine transporter and choline acetyltransferase in the rat brain, Neurosci. Lett. 212 (1996) 107-I 10.
171
Kitamoto T., Wang W., Salvaterra P.M., Structure and organization of the Drosophi/a cholinergic locus, J. Biol. Chem. 273 (1998) 27062713.
181
Naciff J.M., Misawa H., Dedman J.R., Molecular characterization of the mouse vesicular acetylcholine transporter gene, NeuroReport 8 (1997) 3467-3473.
191
Nirenberg M.J., Chan J., Liu Y., Edwards R.H., Pickel V.M., Ultrastructural localization of the vesicular monoamine transporter-2 in midbrain dopaminergic neurons: Potential sties for somatodendritic storage and release of dopamine, J. Neurosci. 16 (1996) 41354145.
[lOI
Schafer M.K.-H., Eiden L.E., Weihe E., Cholinergic neurons and terminal fields revealed by immunohistochemistry for
[I21
Schafer M.K.-H.. Schiltz B., Weihe E., Eiden L.E.. Targetindependent cholinergic differentiation in the rat sympathitic nervous system, Proc. Nat]. Acad. Sci. USA 94 (1997) 4149-4154.
Eiden LE., Weihe E.. Vesicular 1131 Schiitz B., Schafer M.K.-H., amine transporter expression and isoform selection in developing brain, peripheral nervous system and gut, Dev. Brain Res. 106 (1998) 181-204. E., Edwards R.H., 1141 Song H., Ming Cl., Fon E., Bellocchio Poo M., Expression of a putative vesicular acetylcholine transporter facilitates quanta1 transmitter packaging, Neuron 18 (1997) 815-826. S., Eiden L.E., The vesicular monoamine trans1151 Tao-Cheng porter VMATZ is targeted to large dense-core vesicles, and the vesicular acetylcholine transporter VAChT to small synaptic vesicles, in PC12 cells, Adv. Pharmacol. 11998), in press. U61 Usdin T., Eiden L.E.. Bonner T.I., Erickson J.D., ,Molecular biology of vesicular acetylcholine transporters (VAChTs), TINS 18 (1995) 218-224. [171
Varoqui H., Erickson J.D., Active transport of acetylcholine by the human vesicular acetylcholine transporter, J. Biol. Chem. 27 1 (I 996) 27229-27232.
1181 Varoqui
H., Erickson J.D., Vesicular neurotransmitter transporters, Mol. Neurobiol. I5 (1997) 165-191. Erickson J.D.. 1191 Weihe E.. Tao-Cheng J.-H., Schafer M.K.-H., Eiden LE., Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles. Proc. Nat]. Acad. Sci. USA 93 (1996) 3547-3552.
1201 Wu
D., Hersh L.B., Choline its fiftieth year, J. Neurochem.
acetyltransferase: celebrating 62 (1994) 1653-1663.