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Keywords:

  • Choline acetyltransferase promoter;
  • Choline acetyltransferase;
  • Vesicular acetylcholine transporter;
  • Cholinergic neurons

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. DNA cloning
  5. RESULTS
  6. A 3,402-bp fragment from the 5′-noncoding region of the mouse ChAT gene restricts the expression of the LacZ reporter gene to selected neurons of the CNS
  7. DISCUSSION
  8. Acknowledgements

Abstract : Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5′-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system ; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5′-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.

One of the main neurotransmitters in the mammalian nervous system is acetylcholine (ACh). Neurons displaying the cholinergic phenotype require the expression of proteins involved in the synthesis, storage, and release of ACh. Choline acetyltransferase (ChAT ; acetyl-CoA : choline O-acetyltransferase, EC 2.3.1.6) is the rate-limiting enzyme in the synthesis of ACh and is a specific cholinergic marker in the CNS. In all species so far examined, ChAT is encoded by a single gene (for review, see Wu and Hersh, 1994). In rodents, the ChAT gene can give rise to seven differentially spliced mRNA isoforms, M, N1, N2, R1, R2, R3, and R4, all of which encode the same ChAT protein and differ only in their noncoding 5′-ends (Misawa et al., 1992 ; Kengaku et al., 1993).

The protein responsible for translocating cytoplasmic ACh into synaptic vesicles is the vesicular ACh transporter (VAChT) (Song et al., 1997). The entire coding region of the VAChT gene is located within the first intron of the ChAT gene and in the same transcriptional orientation as ChAT. The nested arrangement of the VAChT/ChAT locus is conserved phylogenetically from nematodes to mammals (Alfonso et al., 1993 ; Béjanin et al., 1994 ; Erickson et al., 1994 ; Roghani et al., 1994 ; Naciff et al., 1997 ; for review, see Eiden, 1998). The unique gene organization of the VAChT/ChAT locus offers the possibility of coordinated regulation of two proteins required to express the cholinergic phenotype. Misawa et al. (1995) have addressed this issue using primary cultures of sympathetic superior cervical neurons from newborn rats treated with the cytokine differentiation factor/leukemia inhibitor factor (CDF/LIF) or ciliary neurotrophic factor (CNTF). They found that both agents induce the expression of VAChT and ChAT mRNA in those cells in a parallel fashion. Berrard et al. (1995) found essentially the same results using CDF/LIF or retinoic acid in the same type of neurons. Interestingly, Misawa et al. (1995) found that the cytokines induced only the VAChT mRNA that does not contain the R-type exon and not the other two mRNA species identified by Béjanin et al. (1994) that contain the R-type exon. In a murine septal cell line (SN56), Berse and Blusztajn (1995) found that the treatment of these cells with retinoids, cyclic AMP, or LIF/CNTF also increased the mRNA levels for both VAChT and ChAT in a coordinated fashion. These results suggest that common signaling pathways control the coordinated up-regulation of ChAT and VAChT mRNA levels, perhaps through the same promoter at the VAChT/ChAT locus.

The complete set of regulatory elements required to restrict the cholinergic-specific expression of the VAChT/ChAT locus is still unknown. Recently, Cervini et al. (1995) analyzed the 5′-flanking region of the rat VAChT gene and found that within the first 1,370 bp flanking the translation initiation codon of VAChT, there are two overlapping promoters responsible for the specific transcription of the V1 and V2 types of VAChT mRNA. Both promoter regions were highly active in noncholinergic cell lines (PC-G2 and 293), indicating that they do not contain regulatory elements responsible for the cholinergic specificity of VAChT gene expression. Lönnerberg et al. (1995) studied the expression pattern of a 2.3-kb DNA fragment from the 5′-flanking region of the rat ChAT gene located upstream of the R-type exon, fused to a noncholinergic promoter in an orientation-independent manner. These authors measured relative chloramphenicol acetyltransferase (CAT) activity in tissue homogenates and did not determine the cellular expression of their transgene. Although they found CAT activity in regions of the CNS containing cholinergic neurons, the possibility of noncholinergic cells located in the same areas expressing the transgene remains in question.

To identify a promoter region that would be useful in targeting transgene expression to cholinergic neurons, we evaluated two different DNA fragments from the 5′-flanking region of the mouse ChAT gene in transgenic mice. Our results indicate that a 6,417-bp DNA fragment, flanking the mouse ChAT gene (from -6,371 to +46, +1 is defined as the translational start site) (Misawa et al., 1992), contains a set of regulatory elements that restrict the expression of the reporter gene LacZ to cholinergic neurons of transgenic mice. The identification of this cholinergic-specific promoter will be useful to evaluate the mechanism of diseases characterized by dysfunction of cholinergic neurons, such as Alzheimer's disease and amyotrophic lateral sclerosis, and will be valuable in designing and exploring strategies directed to treat such neurological disorders.

DNA cloning

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. DNA cloning
  5. RESULTS
  6. A 3,402-bp fragment from the 5′-noncoding region of the mouse ChAT gene restricts the expression of the LacZ reporter gene to selected neurons of the CNS
  7. DISCUSSION
  8. Acknowledgements

In the initial characterization of the 5′-flanking region of the mouse ChAT gene (Misawa et al., 1992), an EcoRI-HindIII DNA fragment (4,060 bp) of the genomic clone MG35 was used. For the current study, this segment was digested with HindIII and BamHI to release a 3,402-bp fragment, which encompassed the entire N- and M-type exons as well as intervening sequences of the mouse ChAT gene. Recently, we have analyzed a larger fragment of the 5′-flanking region of the mouse ChAT gene (Naciff et al., 1997), which encompasses the initial 4,060-bp fragment, and additional sequences located further upstream of the EcoRI site, up to a BamHI site, in the same genomic clone MG35. This genomic DNA clone (6,417 bp in length) contains the entire coding region of the VAChT, 633 bp of the 5′-flanking region of this gene, additional sequences downstream of the VAChT gene, located at the 5′ end of the N-type exons (see Fig. 1), and the entire sequence of the other construct. The HindIII site is located within the open reading frame of the ChAT gene (position +46 bp, from initial ATG) ; therefore, the transgenes tested included this sequence.

image

Figure 1. Schematic representation of the 5′-flanking region of the mouse ChAT gene. To identify the promoter region of the mouse ChAT gene that would target cholinergic-specific expression, two different DNA fragments from the 5′-flanking region of this gene were evaluated for their capacity to drive the expression of a reporter gene (LacZ) in transgenic mice. The first construct included 3,356 bp upstream from the Kozak initiation sequence from the ChAT gene and 46 bp from the coding sequence of this gene (ChAT-3402). The second construct included this region and additional sequences further upstream, up to -6,371 (ChAT-6417). Sequence analysis indicated the presence of an intronless open reading frame (1,590 bp) encoding the entire mouse VAChT, and it has been described (Naciff et al., 1997). Open and filled boxes represent noncoding and coding sequences, respectively. R, N, and M are the three ChAT noncoding exons initially described by Misawa et al. (1992). The R-type exon is not contained in our genomic clone (MG35), but it is positioned based on analogy to the rat VAChT/ChAT gene locus. Selected restriction sites used for subcloning are indicated.

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Targeted expression of β-galactosidase to cholinergic neurons in transgenic mice

The β-galactosidase (LacZ) gene from Escherichia coli was used as a reporter gene to determine the expression pattern of two domains of the 5′-flanking region of the mouse ChAT gene. One domain was obtained from digesting the genomic clone with BamHI and HindIII (see Fig. 1), which released a DNA fragment of 3,402 bp that was subcloned into pBluescript SK (Stratagene). This DNA was used to assemble the transgene by linking it to the LacZ gene, derived from pCH110 (Pharmacia), at the HindIII site. The LacZ gene contained a Kozak consensus sequence for eukaryotic translation initiation (Kozak, 1991) and an SV40 polyadenylation sequence. The second construct included a 6,417-bp NotI-HindIII fragment from the 5′-flanking region of the mouse ChAT gene. The transgene was assembled by subcloning the LacZ gene, excised from pCH110 (Pharmacia) by BamHI and HindIII digestion, into pBK-CMV (Stratagene) containing the NotI-HindIII fragment from the 5′-flanking region of the mouse ChAT gene, previously subcloned into pBluescript SK. Proper orientation and position of the different components of both constructs were confirmed by sequencing the double-stranded DNA, in both directions, by the dideoxy method of Sanger et al. (1977) using Sequenase 2.0 (U.S. Biochemical, Cleveland, OH, U.S.A.). The first type of construct was excised from pBluescript SK by BamHI digestion, releasing a transgene of 7.2 kb. The second construct, 10.2-kb transgene, was excised from pBK-CMV by NotI and SalI digestion. The transgenes were separated in a 1% agarose gel and purified by Geneclean (Bio 101). This DNA was dissolved in 10 mM Tris (pH 8.3) with 0.1 mM EDTA and microinjected into the male pronucleus of one-cell mouse embryos of strain FVB/N by the University of Cincinnati Transgenic Animal Core Facility. Founder mice were identified by PCR amplification of a 755-bp fragment from genomic DNA isolated from tail clippings, using the LacZ gene-specific primers 5′-AACAGCACCTCGAACTGAGC-3′ and 5′-CTTCAGCCTCCAGTACAGCG-3′. As an internal control, to confirm the integrity of the genomic DNA, a 386-bp fragment from a single copy gene present in all the animals, β-thyroid-stimulating hormone, was amplified using the primers 5′-TCCTCAAAGATGCTCATTAG-3′ and 5′-GCAAGCAAGAGTTCTATTCA-3′. The PCR was performed at 95°C for 30 s, 53°C for 30 s, and 72°C for 1 min for 30 cycles in a thermocycler (Amplitron II : Thermolyne). PCR products were separated on a 2% agarose (GibcoBRL) gel and visualized by ethidium bromide staining. Founder mice and their offspring were mated to FVB/N mice to maintain hemizygous lines.

From the pronuclear injection of the transgene composed of the 3,402 bp of the 5′-noncoding region of the mouse ChAT gene, upstream of the LacZ gene, we identified 12 transgenic animals by PCR and Southern blot analysis of genomic DNA, isolated from tail clips of 58 pups at weaning stage. Eleven of the 12 transgenic mouse lines generated transmitted stably the transgene to the offspring, and 10 of them gave rise to hemizygotes that expressed β-galactosidase during adulthood with identical regional distribution. Three of these mouse lines were analyzed thoroughly. When the 6,417-bp fragment from the ChAT gene was used to build the transgene, 38 pups were obtained, and two transgenic mouse lines were generated. When analyzed thoroughly, both mouse lines transmitted stably the transgene to their offspring and both mouse lines gave rise to hemizygotes that expressed the LacZ gene during adulthood with identical regional distribution.

LacZ expression in transgenic mice

To determine the spatiotemporal expression pattern of the LacZ gene, the activity of β-galactosidase was evaluated in whole-mount tissues as well as in tissue sections from brain (including the cerebellum), brainstem, spinal cord, liver, lung, and heart of transgenic mice, using X-Gal (4-chloro-5-bromo-3-indolyl-β-d-galactoside) as a substrate. The tissues were processed as described by Bonnerot and Nicolas (1993). Briefly, adult mice (25-30 g) were deeply anesthetized with pentobarbital sodium and perfused transcardially with 10% formalin in phosphate-buffered saline (PBS ; pH 7.4). The tissues were dissected and postfixed by immersion in the same fixative for 20 min at 4°C, then washed with seven changes of PBS and transferred into a histochemical reaction mixture : 1 mg/ml X-Gal, 4 mM K4Fe(CN)6· 3H2O, 4 mM K3Fe(CN)6, 2 mM MgCl2 in PBS. The samples were incubated at 30°C in a humidified incubator for 24 h. The tissues were transferred to PBS for 2 h and then to 30% sucrose in PBS at 4°C. Frozen sections (30-60 μm in thickness) were obtained, dehydrated, and mounted for analysis. In all cases, the entire brain and spinal cord were sectioned. In tissue sections, β-galactosidase activity was visualized as follows : Sections were washed in PBS (5 × 5 min) and then incubated in the histochemical reaction mixture for 6-8 h at 30°C in a humidified incubator. After this, sections were washed in PBS (5 × 5 min), dehydrated in graded ethanols, washed in xylene, and coverslipped with Permount (Fisher Scientific, Houston, TX, U.S.A.). Records of the expression pattern of LacZ activity found in tissue sections were photomicrographed on Kodak Ektachrome Elite 200 film, using a Nikon Optiphot microscope, and then digitized using a Nikon LS-20E (Coolscan II) film scanner.

Indirect immunofluorescence of tissue sections

To determine the distribution pattern of the VAChT, adult (25-30 g) wild-type and transgenic littermates were used, processed as above. Frozen adjacent sections (30-60 μm in thickness) from the cervical region of the spinal cord and from different regions of the brain were obtained and processed as free-floating sections to reveal β-galactosidase activity (as indicated above) or for immunodetection of VAChT using a polyclonal goat anti-VAChT antibody (Chemicon International, Temecula, CA, U.S.A.) diluted 1:1,500. After incubation for 20 h at 4°C, sections were washed in PBS and further incubated with fluorescein-conjugated rabbit anti-goat IgG (30 min at 37°C). Records of the expression pattern of LacZ activity and the VAChT expression pattern found in tissue sections were photomicrographed on Kodak Ektachrome Elite 200 film, using a Nikon Optiphot microscope, and then digitized using a Nikon LS-20E (Coolscan II) film scanner.

Retrograde labeling of spinal cord neurons with Fluoro-Gold

Retrograde labeling of motor neurons of the spinal cord was performed using the fluorescent dye Fluoro-Gold (Fluorochrome, Englewood, CO, U.S.A.) as described by Nacidff et al. (1996). Tissue sections from the spinal cord (lumbar region) were processed to reveal β-galactosidase activity and for detection of retrogradely transported Fluoro-Gold. The β-galactosidase activity was visualized under light microscopy, and the Fluoro-Gold staining of the same field was visualized under epifluorescence. The same field was photographed on Kodak Ektachrome Elite 200 film, using a Nikon Optiphot epifluorescence microscope, and then digitized using a Nikon LS-20-E (Coolscan II) film scanner.

A 3,402-bp fragment from the 5′-noncoding region of the mouse ChAT gene restricts the expression of the LacZ reporter gene to selected neurons of the CNS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. DNA cloning
  5. RESULTS
  6. A 3,402-bp fragment from the 5′-noncoding region of the mouse ChAT gene restricts the expression of the LacZ reporter gene to selected neurons of the CNS
  7. DISCUSSION
  8. Acknowledgements

Previous work identified a region of the 5′-untranslated region of the mouse ChAT gene, from -2,752 to +46, that contains regulatory elements capable of directing neuron-specific expression of the CAT reporter gene in vitro (Misawa et al., 1992, 1993 ; Pu et al., 1993). Here, we tested the ability of this region to confer cholinergic specificity to the expression of the LacZ reporter gene in vivo. A 3,402-bp fragment from the 5′-flanking region of the mouse ChAT gene, encompassing the sequence from -3,356 to +46, was used to direct the expression of the bacterial β-galactosidase reporter gene in transgenic mice (Fig. 1, ChAT-3402). Cell specificity of LacZ transgene expression was examined by β-galactosidase staining of tissue sections and whole-mount staining of brain, spinal cord, liver, lung, and heart. These tissues were chosen because they display characteristic patterns of ChAT protein expression and represent CNS (brain and spinal cord), autonomic nervous system (thoracic spinal cord), and control nonneuronal tissues (liver, lung, and heart).

The 3.4-kb fragment of the 5′-flanking region of the mouse ChAT gene contains regulatory elements that restricted the expression of the reporter gene to select regions of the nervous system (Fig. 2). Nonneuronal cells in tissues like liver, lung, and heart (Fig. 2L) did not express the transgene. In the CNS, the main sites of transgene expression correspond to areas containing ChAT-expressing neurons or areas that receive cholinergic innervation. These areas include the olfactory bulb (Fig. 2A) and anterior olfactory nucleus. In the basal forebrain, β-galactosidase was expressed in the nuclei of the horizontal and vertical diagonal band, medial septum, and caudate putamen (Fig. 2B). In the brain cortex, LacZ gene expression was strongest in layer II, with a decreasing gradient of expression into layers III-IV (Fig. 2C). In the hippocampal formation, the transgene was expressed in CA1-3 fields and dentate gyrus (Fig. 2D) and also in the medial habenular nucleus (Fig. 2E). The transgene was additionally expressed in neurons and terminals located in the median eminence, particularly in the arcuate nucleus, epithalamus, thalamus, hypothalamus, amygdala, and globus pallidus (Fig. 2F). In the brainstem, some of the motor nuclei expressed the transgene (Fig. 2G and H) as well as motor neurons of the spinal cord (Fig. 2J and K). Neurons of the autonomic nervous system (thoracic spinal cord) expressed the transgene (Fig. 2K). In the cerebellum, the LacZ gene was expressed in cells and terminals located primarily in the molecular layer (Fig. 2I), although β-galactosidase activity can also be detected in the granular layer and the white matter.

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Figure 2. Reporter gene expression pattern of the ChAT-3402 promoter. A 3,402-bp DNA fragment (from -3,356 to +46) from the 5′-flanking region of the mouse ChAT gene contains adequate regulatory elements that restrict the expression of the reporter LacZ gene to selected neurons of the CNS in transgenic mice ; it does not confer absolute cholinergic specificity. β-Galactosidase staining (blue-green) was performed on tissue sections obtained from different regions of the CNS of transgenic mice. The staining pattern indicates the expression pattern of the LacZ gene driven by a 3,402-bp fragment, encompassing the sequence from -3,356 to +46 (+1 is defined as the transcriptional start site) from the 5′-flanking region of the mouse ChAT gene. Shown are sections of olfactory bulb (A), medial septal nucleus and horizontal and vertical nuclei of the diagonal band (B) ; prefrontal cortex (C) ; hippocampal formation (D) ; medial habenular nucleus (E) ; epithalamus, thalamus, hypothalamus, amygdala, and globus pallidus (F) ; facial motor nucleus (G) ; dorsal motor nucleus of the vagus and the hypoglossal nucleus (H) ; cerebellum (I) ; and spinal cord at the cervical (J) and thoracic (K) levels. Nonneuronal tissue, ventricular and auricular walls (L), are also shown. 7, facial nucleus ; 10, dorsal motor nucleus of the vagus ; 12, hypoglossal nucleus ; a, auricle ; Amy, amygdala ; CA1-3, fields CA1-3 of Ammon's horn ; cc, central canal ; CC, corpus callosum ; CPu, caudate putamen ; DG, dentate gyrus ; DH, dorsal horn ; E/OV, ependyma or subependymal layer/olfactory ventricle ; EPI, external plexiform layer ; Fc, frontal cortex ; I-VI, layers of cerebral cortex, as defined by Nissl staining ; GI, glomerular layer ; Gr, granular layer ; HDB/VDB, horizontal and vertical diagonal band nuclei ; Hi, hippocampus ; LV, lateral ventricle ; MGP, medial globus pallidus ; MHb, medial habenular nucleus ; Mi, mitral cell layer ; Mol, molecular layer ; MS, medial septal nucleus ; Pir, piriform cortex ; PVP, paraventricular hypothalamic nucleus ; v, ventricle ; VH, ventral horn ; WM, white matter.

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Expression of the ChAT-3402-directed transgene was not detected in some areas of the CNS known to contain cholinergic neurons. This property was particularly evident in the cranial nerves. The facial nucleus (Fig. 2G), dorsal motor nucleus of the vagus and the hypoglossal nucleus (Fig. 2H), and laterodorsal tegmental nucleus consist of defined cholinergic neurons but did not express the transgene. Furthermore, this region of the 5′-flanking region of the mouse ChAT gene does not contain all the regulatory elements required to restrict the expression of the reporter gene to all cholinergic neurons. Some noncholinergic cells located in different regions of the CNS expressed the transgene, particularly in the brainstem (Fig. 2H) and the dorsal horns of the spinal cord (Fig. 2J and K, inset in K). It seems that the ChAT-3402 fragment (Fig. 1) from the 5′-flanking region of the mouse ChAT gene lacks regulatory elements necessary to confer cholinergic-specific expression of our transgene.

A 6.4-kb fragment from the 5′-flanking region of the mouse ChAT gene encompasses the regulatory elements required to confer cholinergic-specific expression of a reporter gene in transgenic mice

To include and identify regulatory elements required to confer cholinergic-specific expression absent from the region -3,356 to +46, included in the first transgene, a larger fragment from the 5′-noncoding region of the mouse ChAT gene, encompassing the sequence from -6,371 to +46 (Fig. 1, ChAT-6417), was used to drive the expression of the LacZ reporter gene. The final fragment used as a promoter (6,417 bp) contains the entire coding region of the VAChT gene, 633 bp of the 5′-flanking region of this gene, additional sequences down-stream of the VAChT gene located at the 5′-end of the N-type exon (Naciff et al., 1997), and the entire sequence of the previous construct from -3,356 to +46 (Fig. 1). The expression pattern of the transgene was examined by β-galactosidase staining of tissue sections and wholemount staining of brain, spinal cord, liver, lung, and heart.

The β-galactosidase expression pattern driven by the 6,417-bp DNA fragment is summarized in Table 1. This fragment of the 5′-flanking region of the mouse ChAT gene encompasses enough regulatory elements to confer cholinergic-specific expression to the LacZ reporter gene (Fig. 3). Nonneuronal tissues (liver, lung, and heart) did not express the transgene. The transgene was expressed in ChAT- and VAChT-expressing neurons of the olfactory bulb, local circuit neurons and puncta fibers innervating the glomerular layer (Fig. 3A), and in the olfactory tubercle. In the basal forebrain, cholinergic neurons from the nuclei of the diagonal band of Broca, vertical and horizontal limbs of the diagonal band, medial septum, and caudate putamen expressed the transgene (Fig. 3B). In the brain cortex (Fig. 3C), the expression pattern of β-galactosidase activity followed the laminar expression pattern of ChAT and VAChT proteins, being more abundant in nonpyramidal cells located in layers II, V, and VI. In the entorhinal cortex, the LacZ gene was expressed strongly in fibers and cells located in superficial layers II and III (Fig. 3F). In the hippocampal formation (Fig. 3D and F), the LacZ gene was expressed in local circuit neurons as well as in fibers with a distinctive regional and laminar distribution. β-Galactosidase-expressing cells were evident in all layers of the dentate gyrus, in strata oriens and lacunosum moleculare of the hippocampus, and in the subiculum. The highest densities of β-galactosidase-expressing fibers were observed in the plexuses situated just above and below the granule cell layer (Fig. 3D and F), with a progressive decrease in the expression from CA3-2 into CA1. The transgene was specifically expressed in the medial habenular nucleus (Fig. 3E) and in cholinergic cells and terminals from the median eminence, particularly in the arcuate nucleus, the parafascicular, anterior, medial, and ventromedial thalamic nuclei, and the basolateral amygdaloid nucleus (Fig. 3F). In contrast to the transgene containing the ChAT-3402 DNA fragment, the LacZ gene under the control of the ChAT-6417 DNA fragment was specifically expressed in cholinergic neurons of the brainstem. β-Galactosidase activity was detected in neurons of the pedunculopontine tegmental and laterodorsal tegmental nuclei (Fig. 3G), facial motor nucleus (Fig. 3H), parabigeminal, oculomotor, trochlear, dorsal motor nucleus of the vagus, and hypoglossal nucleus (Fig. 3I). The transgene was also expressed in areas known to contain cholinergic neurons and/or to receive cholinergic input, such as the entopeduncular nucleus, zona incerta, and superior colliculus. In the cerebellum, β-galactosidase activity was expressed in some cells located mostly within the molecular layer, in a subpopulation of mossy fibers and puncta in the cerebellar nuclei, in fiber plexuses around Purkinje cells, granule cells, and parallel fibers in the cerebellar cortex (Fig. 3J). Barmack et al. (1992) reported similar distribution patterns for ChAT activity and ChAT immunoreactivity in the cerebellum of rat, rabbit, and cat. The transgene was also highly expressed in multiple cholinergic fibers innervating the choroid plexus (Fig. 3F) found in the lateral, third, and fourth ventricles attached to the ventricular walls.

Table 1. Tissue expression pattern of LacZ reporter gene directed by 6,417-bp fragment from 5′-flanking region of mouse ChAT gene in transgenic mice Tissues from adult transgenic mice were processed to reveal the β-galactosidase activity, indicating LacZ expression, as described in Materials and Methods. The relative density of LacZ-positive somata or puncta/fibers was subjectively rated as follows : (-) absent, (+) low, (++) moderate, (+++) high, and (++++) very high.
TissueRelative density of neurons LacZ-positive Relative density of puncta/fibers LacZ-positive
Spinal cord  
Dorsal horn-/+++
Intermediate zone+++
Ventral horn++++++++
Cranial nerves  
Oculomotor nucleus++++
Trochlear nucleus++++
Motor nucleus of trigeminal++++++
Spinal nucleus of trigeminal-+++
Abducens nucleus+++
Facial motor nucleus+++++++
Nucleus ambiguus++++
Dorsal motor nucleus of vagus+++++
Nucleus of solitary tract-+++
Hypoglossal++++++
Cochlear nucleus+++
Dorsal raphe-+
Pontine nuclei+++++
Parabigeminal/lemniscal nucleus++
Pedunculopontine and laterodorsal tegemental nucleus++++
Interpeduncular nucleus++++++
Superior colliculus-++
Amygdala++++
Hippocampal formation  
CA1+++
CA2-3++++
Dentate gyrus-++
Medial habenula++++
Striatum++++
Basal forebrain  
Medial septum++++++
Vertical/horizontal diagonal band++++++
Nucleus basalis of Meynert++++++
Islands of Calleja+++++
Olfactory tubercle++++++
Olfactory cortex++++
Olfactory bulb+++++
Cerebral cortex  
Layer I-++
Layer II-III++
Layer IV-++
Layer V+++
Layer VI-+
image

Figure 3. Reporter gene expression pattern of the ChAT-6417 promoter. A 6,417-bp DNA fragment (from -6,371 to +46) from the 5′-flanking region of the mouse ChAT gene contains the required transcriptional information to target the expression of the reporter LacZ gene to cholinergic neurons of transgenic mice. Adult transgenic mouse tissue sections from different regions of the CNS, processed to reveal β-galactosidase activity, show that the LacZ gene is expressed only in cholinergic neurons when it is driven by a 6,417-bp fragment, encompassing the sequence from -6,371 to +46 (+1 is defined as the translational start site) from the 5′-flanking region of the mouse ChAT gene. Shown are sections of olfactory bulb (A), medial septal nucleus and horizontal and vertical nuclei of the diagonal band (B) ; prefrontal cortex (C) ; hippocampal formation (D) ; medial habenular nucleus (E) ; epithalamus, thalamus, hypothalamus, amygdala, and globus pallidus (F) ; laterodorsal tegmental nucleus (G) ; facial motor nucleus (H) ; dorsal motor nucleus of the vagus and the hypoglossal nucleus (I) ; cerebellum (J) ; and spinal cord at the cervical (K) and thoracic (L) levels. LTDg, laterodorsal tegmental nucleus ; other abbreviations as in Fig. 2.

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In the spinal cord, motor neurons located within the medial, central, and lateral motor columns of the ventral horn (Fig. 3K and inset, and L) expressed the transgene. Cholinergic partition neurons as well as cholinergic fibers present throughout the spinal cord expressed β-galactosidase. In the dorsal horn and the central gray matter (Fig. 3K and L), small cholinergic cell bodies expressed the transgene. Small neurons clustered around the central canal and scattered in laminae III-VI of the dorsal horn, ChAT-positive (Phelps et al., 1984), also express the transgene. These neurons have processes branched to the ependyma, surrounding the central canal, that strongly expressed β-galactosidase activity (Fig. 3K and L). At autonomic spinal levels (thoracic is shown in Fig. 3L), preganglionic sympathetic or parasympathetic cholinergic neurons also expressed β-galactosidase activity under the control of the 6.4-kb fragment of the ChAT promoter region.

To confirm the cholinergic specificity conferred by 6.4-kb DNA fragment from the 5′-flanking region of the mouse ChAT gene, the regional expression pattern of the LacZ gene driven by this promoter region was compared with the VAChT immunolocalization pattern in different regions of the CNS of both wild-type and transgenic littermates. The distribution and appearance of VAChT immunoreactivity (Fig. 4A, C, and E) and β-galactosidase activity (Fig. 4B and D) in the cervical region of the spinal cord and the hippocampal formation (Fig. 4H-K) closely match each other.

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Figure 4. VAChT and LacZ expression in the spinal cord and hippocampus. The VAChT immunolocalization pattern in the cervical region of the spinal cord from wild-type (A) and transgenic (C and E) littermates is mirrored by the LacZ gene expression pattern under the control of the 6.4-kb DNA fragment from the 5′-flanking region of the mouse ChAT gene (B, D, and F, arrows). B-C and D-E are adjacent coronal cryosections. By retrograde transport, motor neurons that project through the sciatic nerve of adult transgenic mice (F and G) were labeled with Fluoro-Gold, a fluorescent dye. Co-localization of β-galactosidase activity (F) and Fluoro-Gold (G) in the same neurons of the ventral horn (arrows) confirmed these cells to be cholinergic neurons. In the hippocampal formation (H-K), the VAChT expression pattern (J and K) is similar in the wild-type (H and J) and the transgenic animals (I and K), and it matches closely the expression patterns of the LacZ gene (I). Abbreviations as in Fig. 2.

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In the spinal cord, motor neurons located within the medial, central, and lateral motor columns of the ventral horn (Fig. 4A-F) expressed VAChT as well as cholinergic partition neurons. Multiple large varicosities (bouton-like structures) containing high levels of VAChT are in contact with perikarya and dendrites of motor neurons (Fig. 4E), clearly positive for β-galactosidase activity (Fig. 4D and E, arrows). The identity of the cells expressing LacZ gene located in the ventral horns of the spinal cord was confirmed by retrograde labeling with the fluorescent dye Fluoro-Gold. The dye was injected into the perineurium of the right sciatic nerve, near the trochanter, of mature transgenic mice and by retrograde transport marked motor neurons that project through the sciatic nerve. Co-localization of Fluoro-Gold and β-galactosidase activity (Fig. 4F and G) in the same neurons (arrows) of the right ventral horn confirmed these cells to be cholinergic.

In the CA1-3 region of the hippocampal formation, the immunodetection of VAChT-positive fibers in wildtype (Fig. 4J) and ChAT-6417 transgenic (Fig. 4K) animals showed the highest density of cholinergic fibers in the pyramidal cell layer. This VAChT immunodetection pattern mirrors the expression pattern of the β-galactosidase (Fig. 4I) when directed by the 6.4-kb DNA fragment from the 5′-flanking region of the mouse ChAT gene. In wild-type and transgenic animals, VAChT-positive neurons were observed in the olfactory tubercle, cerebral cortex, basal forebrain, including the nuclei of the diagonal band of Broca, vertical and horizontal limbs of the diagonal band, medial septum, and caudate putamen ; the medial habenular nucleus ; the median eminence, particularly in the arcuate nucleus ; the parafascicular, anterior, medial, and ventromedial thalamic nuclei ; and the basolateral amygdaloid, pedunculopontine tegmental, and laterodorsal tegmental nuclei, facial motor nucleus, parabigeminal, oculomotor, trochlear, dorsal motor nucleus of the vagus, and hypoglossal nucleus. VAChT immunoreactivity was also observed in axonal fibers in areas such as the olfactory bulb, cerebral cortex, striatum, basal forebrain, amygdala, mesopontine complex, thalamic nuclei, superior colliculus, and cranial and spinal motor nuclei. The VAChT immunolocalization pattern in different regions of the CNS of both wild-type and transgenic littermates matches closely the expression pattern of the β-galactosidase activity (Figs. 3 and 4). These results confirm that within the first 6.4-kb fragment from the 5′-flanking region of the mouse ChAT gene resides a complete set of regulatory elements that confer cholinergic-specific expression to our reporter gene.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. DNA cloning
  5. RESULTS
  6. A 3,402-bp fragment from the 5′-noncoding region of the mouse ChAT gene restricts the expression of the LacZ reporter gene to selected neurons of the CNS
  7. DISCUSSION
  8. Acknowledgements

We have identified a promoter that provides specific cholinergic expression of the β-galactosidase (LacZ) reporter gene in transgenic mice. This promoter is located within a 6,417-bp DNA fragment flanking in the 5′-end of the mouse ChAT gene. This region encompasses 633 bp of the 5′-flanking region of the mouse VAChT gene, the entire open reading frame of the VAChT gene (Naciff et al., 1997), contained within the first intron of the ChAT gene, sequences upstream of the initiation codon of the ChAT gene, and the first 46 bp of the coding region for ChAT. The transgenic expression pattern of the LacZ reporter gene under the control of this 6,417-bp fragment was very similar to the distribution pattern of the cholinergic neurons previously defined by immuno-histochemistry for ChAT and VAChT in the rat and other species (Houser et al., 1983 ; Eckenstein et al., 1988 ; Levey et al., 1984 ; Martínez-Murillo and Rodrigo, 1995 ; Mufson et al., 1986 ; Butcher et al., 1992 ; Gilmor et al., 1996 ; Schäfer et al., 1994, 1995 ; Usdin et al., 1995 ; Erickson et al., 1996 ; Weihe et al., 1996 ; Arvidsson et al., 1997 ; Ichikawa et al., 1997).

The expression pattern of VAChT immunoreactivity in wild-type and transgenic littermates closely follows the expression pattern of the LacZ gene in transgenic animals when the reporter gene is under the control of the 6.4-kb DNA fragment from the 5′-flanking region of the mouse ChAT gene (Figs. 3 and 4). Both VAChT and β-galactosidase activity are co-expressed in the same neurons and their fibers. These results indicate that the promoter region contained within the sequence from -6,371 to +46 of the VAChT/ChAT gene locus encompasses a repertoire of regulatory elements required to confer cholinergic cell-specific expression.

There are, however, some areas in which the reporter gene is expressed under the control of the ChAT-6417 promoter region, but the existence of cholinergic neurons in such areas is controversial. These areas include the cerebral cortex, olfactory bulb, anterior olfactory nucleus, hippocampus, and cerebellum. For example, in rodents, ChAT-positive cortical neurons have been identified by immunohistochemistry with several different ChAT-specific antibodies (Eckenstein et al., 1988 ; Butcher et al., 1992 ; Butcher, 1995 ; Gilmor et al., 1996 ; Ichikawa et al., 1997). Although the identity of the local circuit cholinergic neurons has not been confirmed by in situ hybridization histochemistry for ChAT by different authors (Butcher et al., 1992 ; Oh et al., 1992 ; Ichikawa et al., 1997), Lauterborn et al. (1993) detected ChAT mRNA in neurons located in layers II and III of the rat brain cortex. We have determined the expression of VAChT immunoreactivity in those areas. Furthermore, the presence of cortical neurons expressing VAChT was described recently by Gilmor et al. (1996) and Weihe et al. (1996). As the well known cholinergic nuclei are the major groups of cells expressing the reporter gene, we suggest that the expression of the LacZ gene in the cerebral cortex, olfactory bulb, anterior olfactory nucleus, hippocampus, and cerebellum represents the use of the ChAT promoter in authentic local circuit cholinergic neurons. The expression of the LacZ gene in those local circuit neurons offers a new way of labeling them, by using a fluorescein-based galactosidase substrate to reveal the LacZ gene-expressing cells in vivo, to study their properties in acutely dissociated tissue cultures or tissue sections.

At the DNA sequence level, there is 95% identity between the 6,417-bp fragment from the 5′-flanking region of the mouse ChAT gene and the rat counterpart (Béjanin et al., 1994 ; Erickson et al., 1994 ; Roghani et al., 1994 ; Naciff et al., 1997 ; this study), although in the rat's case, the promoter activity of this fragment has not been characterized in vivo. Lönnerberg et al. (1995) reported that a 2,343-bp segment from the 5′-flanking region of the R-type exon (Fig. 1), a noncoding region of the rat ChAT gene, combined with the herpes simplex virus thymidine kinase heterologous basal promoter, directed CAT activity expression in several regions of the CNS containing cholinergic cells. The biochemical assay in tissue homogenates performed by Lönnerberg et al. did not distinguish individual cell types, only particular regions sectioned from the CNS. This region of the rat ChAT gene promoter contains the sequence from -3,865 to -1,523 bp (+1 being the transcriptional start site for the ChAT mRNA containing the R-type exon) of the 5′-untranslated region of the rat ChAT gene (Ibáñez and Persson, 1993 ; Lönnerberg et al., 1995). We cannot compare their results directly with our study because the genomic regions analyzed are different. None of our transgenes included sequences from the 5′-flanking region of the R-type exon or this exon. Our results clearly demonstrate that the cis-acting elements located down-stream of the R-type exon present in the ChAT-6417 transgene, containing the sequence from -6,371 to +46 of the VAChT/ChAT gene locus, confer cholinergic specificity. However, the results of Lönnerberg et al. (1995) and the fact that in rodents the R-type exon is present in both VAChT and ChAT transcripts (Misawa et al., 1992, 1995 ; Kengaku et al., 1993Béjanin et al., 1994) clearly indicate the activity of a promoter located upstream of the R-type exon.

Our results indicate that the proximal region of the 5′-flanking region of the mouse ChAT gene (-3,356 to +46) contains a neuronal-restrictive silencer-like element (NRSLE) that is active in selecte neurons of the CNS and also a cholinergic-specific enhancer (CSE) that is used by some, but not all, cholinergic neurons. The presence of regulatory elements similar to the ones we are postulating to be located in the proximal part of the 5′-flanking region of the ChAT gene has been reported for ChAT and other neuron-specific genes (Ibáñez and Persson, 1991 ; Misawa et al., 1993, 1995 ; Pu et al., 1993 ; Erickson et al., 1994, 1996 ; Berrard et al., 1995 ; Berse and Blusztajn, 1995 ; Cervini et al., 1995 ; Li et al., 1995 ; Lönnerberg et al., 1995, 1996 ; Usdin et al., 1995 ; Hersh et al., 1996 ; Bessis et al., 1997). This DNA segment (Fig. 1, ChAT-3402) is apparently missing an additional NRSLE that is required to restrict LacZ expression to cholinergic cells and also lacks at least one other CSE used by all cholinergic neurons. These two putative regulatory elements are located further upstream of ChAT-3402, on the 5′-flanking region of the mouse ChAT gene, and were included in our second transgene (Fig. 1, ChAT-6417). The identity of those regulatory elements is unknown. Sequence analysis of the DNA fragment from -6,371 to +46 of the 5′-end of the mouse ChAT gene indicates the presence of multiple potential NRSLE sequences that do not have 100% homology with the NRSLE described by Lönnerberg et al. (1996) for the rat ChAT gene promoter and which functionality in the mouse has yet to be addressed. Within the first 6,417 bp of the 5′-flanking region of the mouse ChAT gene, there are multiple regulatory elements conserved among different species, such as AP-1, AP-4, CRE, CNTF-RE, GRE, IRE, SP-1, and SRE (see the nucleotide sequences deposited in the GeneBank/EMBL Data Bank). These and other regulatory elements, acting individually or cooperatively, are required to direct specific gene expression to cholinergic neurons (Hersh et al., 1993, 1996 ; Pu et al., 1993 ; Baskin et al., 1997 ; Berse and Blusztajn, 1997 ; Hahm et al., 1997 ; Tanaka et al., 1998).

In Drosophila melanogaster, the cholinergic gene locus is arrayed similarly to that found in mammals (Kitamato et al., 1998). It has been suggested that in Drosophila, correct cholinergic expression from the VAChT/ChAT gene locus is achieved by usage of control elements located upstream of the R-type exon (Sugihara et al., 1991Kitamato and Salvaterra, 1995 ; Kitamato et al., 1995, 1998). The mammalian VAChT/ChAT gene locus seems to contain regulatory elements that direct the initiation of the transcription of VAChT and ChAT genes in different ways. A transcriptional start site located upstream of the R-type exon results in production of different mRNA transcripts by alternative splicing with a common 5′-exon (the R-type) for both VAChT and ChAT. From separate initiation sites, within or downstream of the R-type exon, different transcripts of VAChT and ChAT are produced with unique 5′-flanking sequences. Our results indicate that a cholinergic-specific promoter resides downstream of the R-type exon. We previously reported that the 6.4-kb DNA fragment of the 5′-flanking region of the mouse ChAT gene results in overexpression of the VAChT mRNA in transgenic mice (Naciff et al., 1997), indicating that regulatory elements that allow initiation of transcription of the VAChT mRNA reside within the first 633 bp of the 5′-flanking region of the mouse VAChT gene. Whether this 633-bp region encompasses sufficient regulatory elements to confer cholinergic-specific expression remains to be tested. The data in the present study clearly demonstrate that the first 6,417 bp of the 5′-flanking region of the mouse ChAT gene encompasses sufficient regulatory elements to direct the cholinergic-specific expression of LacZ. However, subtle differences between the expression pattern of β-galactosidase activity and those of endogenous ChAT and VAChT proteins may exist, as a reflection of a role of the flanking sequences located at the 5′-end of the R-type exon. The expression pattern of the reporter gene under the control of the two fragments from the 5′-region of the mouse ChAT gene clearly demonstrates the presence of regulatory elements that influence transcription of the VAChT/ChAT gene locus completely independently of the elements located upstream of the R exon.

The 6,417-bp region of the 5′-flanking region of the mouse ChAT gene analyzed in this study can be used to selectively target gene products to cholinergic neurons to develop animal models for human diseases where injury and degeneration of cholinergic neurons are involved, as in Alzheimer's disease and amyotrophic lateral sclerosis. The creation of conditional mutant depends on the cell target specificity ; therefore, the identification of a cholinergic-specific promoter is of great value. Cholinergic targeted expression will allow production of conditional site-specific recombination controlled by ligand-regulated transgenes to introduce somatic mutations for different gene products at a chosen time (Feil et al., 1996 ; Akagi et al., 1997) to design and explore strategies directed at understanding and treating cholinergic neurological disorders.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. DNA cloning
  5. RESULTS
  6. A 3,402-bp fragment from the 5′-noncoding region of the mouse ChAT gene restricts the expression of the LacZ reporter gene to selected neurons of the CNS
  7. DISCUSSION
  8. Acknowledgements

This work was supported by National Institutes of Health grants DK 46433 (to J. R. Dedman) and DK-41740-S1 (to J. M. Naciff). We thank Glenn Doerman for his artistic expertise in preparing the graphics.

  • 1
    Akagi K., Sandig V., Vooijs M., Van der Valk M., Giovannini M., Strauss M., Berns A. (1997) Cre-mediated somatic site-specific recombination in mice.Nucleic Acids Res. 25,17661773.
  • 2
    Alfonso A., Grundahl K., Duerr J.S., Han H.P., Rand J.B. (1993) The Caenorhabditis elegans unc-17 gene : a putative vesicular acetylcholine transporter.Science 261,617619.
  • 3
    Arvidsson U., Riedl M., Elde R., Meister B. (1997) Vesicular acetylcholine transporter (VAChT) protein : a novel and unique marker for cholinergic neurons in the central and peripheral nervous systems.J. Comp. Neurol. 378,454467.DOI: 10.1002/(SICI)1096-9861(19970224)378:4<454::AID-CNE2>3.3.CO;2-5
  • 4
    Barmack N.H., Baughman R.W., Eckenstein F.P. (1992) Cholinergic innervation of the cerebellum of the rat, rabbit, cat, and monkey as revealed by choline acetyltransferase activity and immunohistochemistry,J. Comp. Neurol. 317,233249.
  • 5
    Baskin F., Li Y., Hersh L.B., Davis R.M., Rosenberg R.N. (1997) An AP-2 binding sequence within exon 1 of human and porcine choline acetyltransferase genes enhances transcription in neural cells.Neuroscience 76,821827.
  • 6
    Béjanin S., Cervini R., Mallet J., Berrard S. (1994) A unique gene organization for two cholinergic markers, choline acetyltrans-ferase and a putative vesicular transporter for acetylcholine.J. Biol. Chem. 269,2194421947.
  • 7
    Berrard S., Varoqui H., Cervini R., Israël M., Mallet J., Diebler M. -F. (1995) Coregulation of two embedded gene products, choline acetyltransferase and the vesicular acetylcholine transporter.J. Neurochem.65,939942.
  • 8
    Berse B. & Blusztajn J.K. (1995) Coordinated up-regulation of choline acetyltransferase and vesicular acetylcholine transporter gene expression by retinoic acid receptor α, cAMP, and leukemia inhibitory factor/ciliary neurotrophic factor signaling pathways in a murine septal cell line. J. Biol. Chem. 270,2210122104.
  • 9
    Berse B. & Blusztajn J.K. (1997) Modulation of cholinergic locus expression by glucocorticoids and retinoic acid is cell-type specific.FEBS Lett. 410,175179.
  • 10
    Bessis A., Champtiaux N., Chatelin L., Changeux J.P. (1997) The neuron-restrictive silencer element : a dual enhancer/silencer crucial for patterned expression of a nicotinic receptor gene in the brain.Proc. Natl. Acad. Sci. USA 94,59065911.
  • 11
    Bonnerot C. & Nicolas J.F. (1993) Application of LacZ gene fusions to postimplantation development.Methods Enzymol.225,451469.
  • 12
    Butcher L.L. (1995) Cholinergic neurons and networks, inThe Rat Nervous System, 2nd ed. (Paxinos G., ed), pp. 10031015. Academic Press, San Diego.
  • 13
    Butcher L.L., Oh J.D., Woolf N.J., Edwards R.H., Roghani A. (1992) Organization of central cholinergic neurons revealed by combined in situ hybridization histochemistry and choline-O-acetyltransferase immunocytochemistry. Neurochem. Int. 21,429445.
  • 14
    Cervini R., Houhou L., Pradat P.F., Béjanin S., Mallet J., Berrard S. (1995) Specific vesicular acetylcholine transporter promoters lie within the first intron of the rat choline acetyltransferase gene.J. Biol. Chem. 270,2465424657.
  • 15
    Eckenstein F.P., Baughman R.W., Quinn J. (1988) An anatomical study of cholinergic innervation in rat cerebral cortex.Neuroscience 25,457474.
  • 16
    Eiden L.E. (1998) The cholinergic gene locus.J. Neurochem.70,22272240.
  • 17
    Erickson J.D., Varoqui H., Schäfer M.K., Modi W., Diebler M., Weihe E., Rand J., Eiden L.E., Bonner T.I., Usdin T.B. (1994) Functional identification of a vesicular acetylcholine transporter and its expression from a “cholinergic” gene locus.J. Biol. Chem. 269,2192921932.
  • 18
    Erickson J.D., Weihe E., Schafer M.K., Neale E., Williamson L., Bonner T.I., Tao-Cheng J., Eiden L.E. (1996) The VAChT/ChAT “cholinergic gene locus” : new aspects of genetic and vesicular regulation of cholinergic function.Prog. Brain Res. 109,6982.
  • 19
    Feil R., Brocard J., Mascrez B., LeMuer M., Metzger D., Chambon P. (1996) Ligand-activated site-specific recombination in mice.Proc. Natl. Acad. Sci. USA 93,1088710890.
  • 20
    Gilmor M.L., Nash N.R., Roghani A., Edwards R.H., Yi H., Hersch S.M., Levey A.I. (1996) Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles.J. Neurosci.16,21792190.
  • 21
    Hahm M., Chen L., Patel C., Erickson J., Bonner T.I., Weihe E., Schafer M.K., Edien L.E. (1997) The human cholinergic gene locus : upstream sequencing, in vivo transcription patterns and role of the NRSE/RE-1 and other control elements in VAChT expression.J. Mol. Neurosci.9,223236.
  • 22
    Hersh L.B., Kong C.F., Sampson C., Mues G., Li Y., Fisher A., Hilt D., Baetge E.E. (1993) Comparison of the promoter region of the human and porcine choline acetyltransferase genes : localization of an important enhancer region.J. Neurochem.61,306314.
  • 23
    Hersh L.B., Inoue H., Li Y. -P. (1996) Transcriptional regulation of the human choline acetyltransferase gene.Prog. Brain Res. 109,4754.
  • 24
    Houser C.R., Crawford G.D., Barber R.P., Salvaterra P.M., Vaughn J.E. (1983) Organization and morphological characterization of cholinergic neurons : an immunocytochemical study with a monoclonal antibody to choline acetyltransferase.Brain Res. 266,97119.
  • 25
    Ibáñez C.F. & Persson H. (1991) Localization of sequences determining cell type specificity and NGF responsiveness in the promoter region of the rat choline acetyltransferase gene.Eur. J. Neurosci.3,13091315.
  • 26
    Ichikawa T., Ajiki K., Matsuura J., Misawa H. (1997) Localization of two cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter in the central nervous system of the rat : in situ hybridization histochemistry and immunohistochemistry.J. Chem. Neuroanat.13,2339.
  • 27
    Kengaku M., Misawa H., Deguchi T. (1993) Multiple mRNA species of choline acetyltransferase from rat spinal cord.Mol. Brain Res. 18,7176.
  • 28
    Kitamato T. & Salvaterra P.M. (1995) APOU homeo domain protein related to dPOU-19/pdm-1 binds to the regulatory DNA necessary for vital expression of the Drosophila choline acetyltransferase gene.J. Neurosci.15,35093518.
  • 29
    Kitamato T., Ikeda K., Salvaterra P.M. (1995) Regulation of choline acetyltransferase/LacZ fusion gene expression in putative cholinergic neurons of Drosophila melanogaster.J. Neurobiol.28,7081.
  • 30
    Kitamato T., Wang W., Salvaterra P.M. (1998) Structure and organization of the Drosophila cholinergic locus.J. Biol. Chem. 273,27072713.
  • 31
    Kozak M. (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation.J. Biol. Chem. 266,1986719870.
  • 32
    Lauterborn J.C., Isackson P.J., Montalvo R., Gall C.M. (1993) In situ hybridization localization of choline acetyltransferase mRNA in adult rat brain and spinal cord.Mol. Brain Res. 17,5969.
  • 33
    Levey A.I., Wainer B.H., Rye D.B., Mufson E.J., Mesulam M. -M. (1984) Choline acetyltransferase-immunoreactive neurons intrinsic to rodent cortex and distinction from acetylcholinesterase-positive neurons.Neuroscience 13,341353.
  • 34
    Li Y.P., Baskin F., Davis R., Wu D., Hersh L.B. (1995) A cell type-specific silencer in the human choline acetyltransferase gene requiring two distinct and interactive E boxes.Mol. Brain Res. 30,106114.
  • 35
    Lönnerberg P., Lendahl U., Funakoshi H., Auml;rhlund-Richter L., Persson H., Ibáñez C.F. (1995) Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.Proc. Natl. Acad. Sci. USA 92,40464050.
  • 36
    Lönnerberg P., Schoenherr C.J., Anderson D.J., Ibáñez C.F. (1996) Cell type-specific regulation of choline acetyltransferase gene expression. Role of the neuron-restrictive silencer element and cholinergic-specific enhancer sequences.J. Biol. Chem. 271,3335833365.
  • 37
    Martínez-Murillo R. & Rodrigo J. (1995) The localization of cholinergic neurons and markers in the CNS, inCNS Neurotransmitters and Neuromodulators. Acetylcholine (Stone T. W., ed), pp. 137. CRC Press Inc., Boca Raton, Florida.
  • 38
    Misawa H., Ishi K., Deguchi T. (1992) Gene expression of mouse choline acetyltransferase. Alternative splicing and identification of a highly active promoter region.J. Biol. Chem. 267,2039220399.
  • 39
    Misawa H., Takahashi R., Deguchi T. (1993) Transcriptional regulation of choline acetyltransferase gene by cyclic AMP.J. Neurochem.60,13831387.
  • 40
    Misawa H., Takahashi R., Deguchi T. (1995) Coordinate expression of vesicular acetylcholine transporter and choline acetyltransferase in sympathetic cervical neurons.Neuroreport 6,965968.
  • 41
    Mufson E.J., Martin T.L., Mash D.C., Wainer B.H., Mesulam M. -M. (1986) Cholinergic projections from the parabigeminal nucleus (Ch 8) to the superior colliculus in the mouse : a combined analysis of horseradish peroxidase transport and choline acetyltransferase immunohistochemistry.Brain Res. 370,144148.
  • 42
    Naciff J.M., Behbehani M.M., Kaetzel M.A., Dedman J.R. (1996) Annexin VI modulates Ca2+ and K+ conductances of spinal cord and dorsal root ganglion neurons.Am. J. Physiol.271, ( Cell Physiol. 40) C2004C2015.
  • 43
    Naciff J.M., Misawa H., Dedman J.R. (1997) Molecular characterization of the mouse vesicular acetylcholine transporter gene.Neuroreport 8,34673473.
  • 44
    Oh J.D., Woolf N.J., Roghani A., Edwards R.H., Butcher L.L. (1992) Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of choline acetyltransferase mRNA.Neuroscience 47,807822.
  • 45
    Phelps P.E., Barber R.P., Houser C.R., Crawford G.D., Salvaterra P.M., Vaughn J.E. (1984) Postnatal development of neurons containing choline acetyltransferase in rat spinal cord : an immunocytochemical study.J. Comp. Neurol. 229,347361.
  • 46
    Pu H., Zhai P., Gurney M. (1993) Enhancer, silencer, and growth factor responsive regulatory sequences in the promoter for the mouse choline acetyltransferase gene.Mol. Cell. Neurosci.4,131142.DOI: 10.1006/mcne.1993.1017
  • 47
    Roghani A., Feldman J., Kohan S.A., Shirzadi A., Gundersen C.B., Brecha N., Edwards R.H. (1994) Molecular cloning of a putative vesicular transporter for acetylcholine.Proc. Natl. Acad. Sci. USA 91,1062010624.
  • 48
    Sanger F., Niklen S., Coulson A.R. (1977) DNA sequencing with chain-terminating inhibitors.Proc. Natl. Acad. Sci. USA 74,54635467.
  • 49
    Schäfer M.K., Weihe E., Varoqui H., Eiden L.E., Erickson J.D. (1994) Distribution of the vesicular acetylcholine transporter (VAChT) in the central and peripheral nervous system of the rat.J. Mol. Neurosci.5,118.
  • 50
    Schäfer M.K., Weihe E., Erickson J.D., Eiden L.E. (1995) Human and monkey cholinergic neurons visualized in paraffinembedded tissues by immunoreactivity for VAChT, the vesicular acetylcholine transporter.J. Mol. Neurosci.6,225236.
  • 51
    Song H., Ming G.L., Fon E., Bellochio E., Edwards R.H., Poo M.M. (1997) Expression of a putative vesicular acetylcholine transporter facilitates quantal transmitter packaging.Neuron 18,815826.
  • 52
    Sugihara H., Andrisani V., Salvaterra P.M. (1991) Genomic organization of Drosophila choline acetyltransferase.J. Neurochem.57,16361642.
  • 53
    Tanaka H., Zhao Y., Wu D., Hersh L.B. (1998) The use of DNase I hypersensitivity site mapping to identify regulatory regions of the human cholinergic gene locus.J. Neurochem.70,17991808.
  • 54
    Usdin T.B., Eiden L.E., Bonner T.I., Erickson J.D. (1995) Molecular biology of the vesicular ACh transporter.Trends Neurosci.18,218224.
  • 55
    Weihe E., Tao-Cheng J., Schäfer M.K., Erickson J.D., Eiden L.E. (1996) Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.Proc. Natl. Acad. Sci. USA 93,35473552.
  • 56
    Wu D. & Hersh L.B. (1994) Choline acetyltransferase : celebrating its fiftieth year.J. Neurochem.62,16531663.