The vesicular acetylcholine transporter is required for neuromuscular development and function

Abstract

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.

FIG. 1.

Generation of VAChT^del/del mice. (A) Generation of VAChT del mice using the Cre-LoxP system. Boxes represent the different exons of ChAT or VAChT. Open boxes represent the ORF of VAChT and ChAT. Note that the VAChT gene is within the first intron of ChAT. (B) Schematic representation of the VAChT gene locus, the floxed allele, and the del allele. P1, P2, P3, and P4 indicate the positions of PCR primers used for genotyping. (C) Sequence analysis of the 329-bp fragment amplified with primers P2 and P4. Restriction sites and LoxP sequences are indicated. (D) VAChT mutant mice (VAChT^del/del) died rapidly after birth in cyanosis (not shown). Embryos from E18.5 exhibited flaccid limbs and kyphosis (hunchback). (E) Southern blot analysis confirmed the presence of the del allele in VAChT mutant mice. (F) Genotype of VAChT mutant mice by PCR.

FIG. 2.

VAChT expression and ACh release in VAChT^del/del E18.5 mice. (A) Quantitative analysis of VAChT mRNA levels by qPCR. PCR products were run in a polyacrylamide gel. *, statistically different from wt; **, statistically different from wt/del {one-way ANOVA with Bonferroni post hoc [F(2,6 = 920)]; P < 0.0001; n = 4}. Lanes labeled with a − show the respective negative control without the sample. (B) Western blot analysis of VAChT, synaptophysin, and tubulin in spinal cord extracts. (C) Average values for the amount of VAChT from densitometric analyses of several Western blots. Tubulin immunoreactivity was used to normalize protein loading. Data are presented as percentages of VAChT^wt/wt levels. *, statistically different from wt; **, statistically different from wt/del {one-way ANOVA with Bonferroni post hoc [F(2,9 = 927.9)]; P < 0.0001; n = 4}. (D) Effects of KCl-induced depolarization on [³H]ACh release from synaptosomes. *, statistically different from wt/wt (P < 0.05 by t test). (E) MEPPs from the NMJ. MEPPs recorded in VAChT^wt/del muscle show no significant change in amplitude compared to VAChT^wt/wt mice. VAChT-null mice showed the existence of scarce MEPPs with decreased amplitude compared to VAChT^wt/wt and VAChT^wt/del mice. (F) d-Tubocurarine abrogated MEPPs in both VAChT^wt/wt and VAChT^del/del mice.

FIG. 3.

Neuromuscular function in VAChT^wt/del mice. (A) Grip force measured for VAChT^wt/wt and VAChT^wt/del mice. There is no significant difference between the two genotypes. (B) Time spent hanging upside down from a wire netting for VAChT^wt/wt and VAChT^wt/del mice. No significant difference was observed. (C) Spontaneous locomotor activity is not changed between the genotypes.

FIG. 4.

(A) Choline uptake in HEK293 cells transiently transfected with empty vector (pcDNA3.1) or L531A CHT1 cDNAs. The data represent the means ± SEM of data from five independent experiments (in duplicates) and are normalized to data for cells expressing empty vector (pcDNA3.1). *, significant difference (P < 0.05 by t test). (B) ACh competition assay using HEK293 cells transiently transfected with empty vector (pcDNA3.1) or L531A CHT1 cDNAs. The data represent the means ± SEM of data from four independent experiments and are normalized to data for cells expressing the empty vector (pcDNA3.1). *, significantly different from control uptake. (C) ACh uptake in HEK293 cells transiently transfected with empty vector (pcDNA3.1) or L531A CHT1 cDNAs. The data represent the means ± SEM of data from three independent experiments and are normalized to data for cells expressing the empty vector (pcDNA3.1).

FIG. 5.

Neurochemical alterations in VAChT^del/del mice. (A) Intracellular ACh contents in brains of VAChT mutant mouse embryos. Data are means ± SEM (three to five mice). An asterisk indicates a statistically significant difference by one-way ANOVA with Bonferroni post hoc test [F(2,10 = 12.72)]. (B) Intracellular ACh content in brains from adult VAChT^wt/wt and VAChT^wt/del mice (n = 4 to 6 brains) (***, P < 0.001). (C) ChAT mRNA levels detected by qPCR from E18.5 mouse brains [F(2,9 = 18.28)] (n = 4). (D) CHT1 mRNA levels detected by qPCR from E18.5 mouse brains. *, statistically different from VAChT^wt/wt mice; **, statistically different from VAChT^wt/del mice [F(2,11 = 12.52)] (n = 5). (E) ChAT and CHT1 protein expression in E18.5 spinal cords. (F) Quantification of protein expression (three to four animals) (P < 0.05) [CHT1 F(2,6) = 35.21; ChAT F(2,15) = 4.599]. *, statistically different from wt/wt; **, statistically different from wt/del.

FIG. 6.

Alterations in MN number and in NMJ morphology in VAChT^del/del E18.5 mice. (A) VAChT immunoreactivity was easily detected in presynaptic termini of VAChT^wt/wt and VAChT^wt/del diaphragms but was absent in VAChT^del/del mice. (B) CHT1 immunoreactivity was detected in all genotypes, although VAChT^del/del mice showed an altered distribution of nerve endings (see below and Fig. 5). (C) Abnormal distribution of nAChR in VAChT^del/del NMJs. Images show that nAChRs from VAChT^del/del mice present stronger labeling and are distributed over a broader region than those from wt and VAChT^wt/del mice. (D) Quantification of nAChR fluorescence. Four independent experiments were performed, and the results are expressed as means ± SEM. An asterisk indicates a statistically significant difference (one-way ANOVA with Bonferroni post hoc) [F(2,20 = 5.632)]. A.U., arbitrary units. (E) The number of lumbar MNs was significantly increased in VAChT and ChAT-null mice. Clear bars, wt mice; dark bars, homozygous mutant mice.

FIG. 7.

Synaptic alteration in VAChT^del/del E18.5 mice. (A) Quantification of CHT1 fluorescence (arbitrary units [A.U.]) in nerve terminals. An asterisk indicates a statistically significant difference (one-way ANOVA with Bonferroni post hoc test) [F(2,20 = 5.632)]. (B) Density of nerve terminals immunolabeled for CHT1 in hemidiaphragms from VAChT^wt/wt, VAChT^wt/del, and VAChT^del/del mice. *, statistically different by ANOVA with Bonferroni post hoc test [F(2,17) = 18.43]. (C) Density of nerve terminals stained with FM1-43fx in hemidiaphragms of VAChT^wt/wt and VAChT^del/del mice (*, P = 0.0218 for VAChT^wt/wt versus VAChT^del/del mice by unpaired t test; n = 6). (D) Number of nerve terminals stained with FM1-43fx per hemidiaphragm (*, P = 0.0260 for VAChT^wt/wt versus VAChT^del/del mice by unpaired t test; n = 6). (E) Representative images of NMJs stained with FM1-43fx in hemidiaphragms of VAChT^wt/wt and VAChT^del/del mice (scale bar, 10 μm). (F) Average area of single nerve terminals in mouse hemidiaphragms stained with FM1-43fx (*, P = 0.0018 for VAChT^wt/wt versus VAChT^del/del mice by unpaired t test). At least 30 end plates were analyzed for each genotype.

FIG. 8.

Altered morphology at the NMJ of VAChT^del/del E18.5 mice. Whole diaphragms were stained with anti-neurofilament antibody (red), and nAChRs were labeled with α-bungarotoxin (green). Confocal stacks were obtained, and maximal projections are shown in the images. The image is representative of three experiments. Note the large increase in axonal sprouting in VAChT-null mice.

FIG. 9.

Muscle morphology of E18.5 embryos from VAChT^wt/wt, VAChT^wt/del, and VAChT^del/del genotypes. Skeletal muscles (gastrocnemius) were stained with hematoxylin and eosin. Black arrows indicate a loss of normal myofibrillar architecture. Bar, 250 μm.