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The Journal of Neuroscience, November 1, 1999, 19(21):9298-9305
Multiorgan Autonomic Dysfunction in Mice Lacking the 
2 and the
4 Subunits of Neuronal Nicotinic Acetylcholine Receptors
Wei
Xu1,
Avi
Orr-Urtreger6,
Filippo
Nigro3,
Shari
Gelber4,
Cara Ballard
Sutcliffe1,
Dawna
Armstrong2,
James W.
Patrick3,
Lorna W.
Role4, 5,
Arthur L.
Beaudet1, and
Mariella
De
Biasi3
Departments of 1 Molecular and Human Genetics and
2 Pathology and 3 Division of Neuroscience,
Baylor College of Medicine, Houston, Texas, 77030, 4 Center
for Neurobiology and Behavior and 5 Department of Anatomy
and Cell Biology, Columbia University, New York, New York, 10032, and
6 The Genetics Institute, Tel-Aviv Sourasky Medical
Center, Tel-Aviv, Israel
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ABSTRACT |
Transcripts for the 
2 and the 4 nicotinic acetylcholine
receptor (nAChR) subunits are found throughout the CNS and the
peripheral nervous system. These two subunits can form
heteromultimeric channels with any of the 
2, 3, 4, or 5
subunits in heterologous expression systems. Nonetheless, the subunit
composition of native nAChRs and the role of different nAChR subtypes
in vivo remain unclear. We prepared null mutations for
the 2 and the 4 genes and bred 2
/4/ mice by mating
mice of identical 2/4+/ or 2+/4/ genotype.
The 2/ and the 4/ single-mutant mice grow to adulthood
with no visible phenotypic abnormalities. The 2/4/ double
mutants survive to birth but have impaired growth and increased
perinatal mortality. They also present enlarged bladders with dribbling
urination and develop urinary infection and bladder stones. The ocular
pupils are widely dilated and do not constrict in response to light.
Histological studies revealed no significant abnormalities of brain or
peripheral tissues except for hyperplasia in the bladder mucosa of
4/ and 2/4/ mutants. Bladder strips from
2/4/ mice did not respond to nicotine but contracted when
stimulated with a muscarinic agonist or electric field stimulation.
Bladder strips from 4 mutants did not respond to nicotine despite
the absence of major bladder dysfunction in vivo.
Acetylcholine-activated whole-cell currents were absent in superior
cervical ganglion neurons from 2/4/ mice and reduced
in neurons from 4/ mice. Although there is apparent redundancy
and a superficially normal phenotype in 2/ and 4/ mice,
physiological studies indicate major deficits in the 4/ mice.
Our previous description of a similar phenotype in 3/ mice and
the current data suggest that the 3 and the 4 subunits are major
components in autonomic nAChRs. The phenotype of the
2/4/ and 3/ mice resembles the autosomal
recessive megacystis-microcolon-hypoperistalsis syndrome in humans.
Key words:
neuronal nicotinic subunits; autonomic ganglia; autonomic dysfunction; knock-out mice; bladder; eye
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INTRODUCTION |
Neuronal nicotinic acetylcholine
receptors (nAChRs) are expressed throughout the CNS and the peripheral
nervous system (PNS) and can be formed by the assembly of both and
subunits (Anand et al., 1991
; Cooper et al., 1991; Sargent, 1993;
McGehee and Role, 1995). Eleven distinct genes are currently known to
encode nAChR subunits, but the composition of native neuronal nAChRs remains undetermined. In heterologous expression systems, the injection
of cDNA encoding 7, 8, or 9 subunits is sufficient to form
functional nAChRs alone, whereas the 2, 3, or 4 nAChR subunits
require the presence of either 2 or 4 cDNA for the assembly of
functional receptors. The 5, 6, and 3 nAChR subunits form
functional channels only in the presence of other and subunits
(Ramirez-Latorre et al., 1996; Wang et al., 1996; Colquhoun and
Patrick, 1997; Gerzanich et al., 1997; Fucile et al., 1998).
Each of the 11 genes encoding neuronal nAChRs (2-9 and
2-4) is expressed in the CNS and PNS, but their relative
abundance varies (Listerud et al., 1991; Mandelzys et al., 1994; Rust
et al., 1994; Poth et al., 1997). The 4 and the 2 genes, although present in the PNS, are highly expressed in several areas of the CNS
(Goldman et al., 1987; Morris et al., 1990) and account for 90% of the
high-affinity [3H]nicotine-binding sites
found in the mammalian brain (Flores et al., 1992; Picciotto et al.,
1995). Mice lacking the 2 subunit have a superficially normal
phenotype but show abnormal passive avoidance and increased
neurodegenation on aging (Picciotto et al., 1995; Zoli et al., 1999).
Furthermore, the anti-nociceptive effects of nicotine are reduced in
both 4- and 2-deficient mice (Marubio et al., 1999).
The 3 subunit is found in the CNS (Wada et al., 1989) but is highly
expressed in the PNS (Listerud et al., 1991; Mandelzys et al., 1994;
Rust et al., 1994). Recently we demonstrated that mice lacking 3
have several physiological abnormalities including poor growth,
decreased survival, absence of bladder contractility, and widely
dilated ocular pupils (Xu et al., 1999).
Transcripts for the 4 gene are found only in restricted brain
regions (Duvoisin et al., 1989; Boulter et al., 1990; Dineley-Miller and Patrick, 1992; Zoli et al., 1998) but are abundantly expressed in
peripheral neurons (Mandelzys et al., 1994; McGehee and Role, 1995;
Poth et al., 1997; Zhang et al., 1998). The concurrent expression of
2 and 4 in the PNS suggests that 2 and 4 might be
functionally related in the formation of autonomic nAChRs in mammals.
However, because the subunit composition of native nAChRs mediating
synaptic transmission in the PNS is unknown, the relative contribution of 2- and/or 4-containing nAChRs remains elusive. Here we show that mice lacking the 4 subunit are superficially normal and grow to
adulthood. In contrast, mice lacking both 2 and 4 have severe
autonomic dysfunction, growth retardation, and premature death.
Similarly to the 3/ mice (Xu et al., 1999), both the 2/4/ and the 4/ mutants lack bladder contractile
responses to nicotine. In addition, nicotine gated whole-cell currents
are absent in the superior cervical ganglion neurons from
2/4/ mice and are significantly reduced in 4/
mice. These and previous findings (Xu et al., 1999) suggest that 3
and the 4 are requisite participants in the majority of functional
ganglionic nAChRs.
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MATERIALS AND METHODS |
Targeted deletions of 2 and 4 genes. The mouse
gene for the nAChR 2 subunit was isolated by screening a mouse
129/SvEv genomic library (a gift from Richard Baehringer; M. D. Anderson Cancer Center, Houston, TX) with a rat cDNA probe (Wada et
al., 1989). The isolated genomic clone contained the region from exons 1 to 6, and the detailed restriction map and intron and exon boundaries were obtained. A construct with the 8 kb region from exons 1 to 5, which contains three out of the four transmembrane domains, replaced
with a neomycin resistance cassette (Neo) was electroporated into the
AB2.1 embryonic stem (ES) cells and transmitted into the germline as
described previously (Bradley, 1987; Xu et al., 1999). Chimeric mice
were bred with C57BL/6J mice. The mice used in the studies reported
here were maintained on the mixed background of 129/SvEv and C57BL/6J.
The mutant allele can be identified as a 6 kb fragment instead of a 12 kb fragment on Southern blot analysis using a flanking genomic probe.
Three-way PCR with the following primers was also designed to determine
the genotype for the mutation: 2 wild-type forward,
5'-CTCTGACTGTAAAGGCAGTGGTTGCTATAG-3'; 2 wild-type reverse,
5'TAGCTATTGACGACGTCTTTAAGATCC-3'; and 2 mutant reverse,
5'-GAGACTAGTGAGACGTGCTACTTCCATTTG. The wild-type product is 250 bp,
and the mutant product is 400 bp.
Similarly, screening a mouse 129/SvJ genomic library (catalog #946305;
Stratagene, La Jolla, CA) with a 4 cDNA fragment identified a clone
containing the 4-coding region (A. Orr-Urtregers, unpublished data). A partial physical map and intron and exon boundaries
were determined via subcloning and sequencing. A construct with a 4.1 kb region containing exon 5, which encodes for the three transmembrane domains, replaced with a puromycin resistance gene, loxP
site, and exons 3-9 of the hypoxanthine phosphorybosyl
transferase (hprt) gene (Ramirez-Solis et al., 1995),
was electroporated into AB2.2 ES cells. The targeted ES cells
were injected into blastocysts and transmitted into the germline
(Bradley, 1987; Xu et al., 1999). The 4 mutant mice used for the
present studies were also maintained in a mixed 129/SvEv and C57BL/6J
background. The mutant allele was identified as a 5.8 kb fragment
instead of a 7.8 kb one in wild type by the flanking genomic probe when
using a SacI digest in Southern blot analysis. The 4 null
mutation was also identified by three-way PCR with the primer sequences
as follows: 4 forward, 5'-TGTAGAGCGAGCATCCGAACA-3'; 4 wild-type
reverse, 5'-TCTCTACTTAGGCTGCCTGTCT; and 4 mutant reverse,
5'-AGTACCTTCTGAGGCGGAAAGA-3'). The wild-type product is 300 bp, and
the mutant product is 150 bp.
The mice with null mutations of both 2 and 4 subunits were
generated via breeding of mice with a single-gene mutation. The expression of 2 was assessed by Northern blot analysis of the brain
total RNA probed with rat 2 cDNA (Wada et al., 1989). The expression
of 4 was not detectable by Northern blot, but reverse transcription-PCR (RT-PCR) was used to test the expression of 4. The total RNA from mouse brains was reverse transcribed with random hexamers as primers, and PCR was performed with hprt and 4 primers. The sequences of hprt primers were
5'-ATGACCTAGATTTGTTTGTATACC-3' (hprt-1) and
5'-GTAGCTCTTCAGTCTGATAAAATCTAC-3' (hprt-2), and the sequences of 4 primers were 5'-GCATCTGGAGAGCGATGACCGA GATCAAAG-3' (4 RT-1 forward) and 5'-TAGCCTAGGAGTCCTTGGAGGGTGCGTGGA-3'
(4 RT-2 reverse).
Cell culture and whole-cell measurements. Primary cultures
of sympathetic neurons were prepared from the superior cervical ganglia
of neonatal mice (within 24 hr of birth). Ganglia were removed,
desheathed, and cut into pieces in Ca+2-
and Mg+2-free PBS. These pieces
were incubated in trypsin (150 µg/ml type IIIS; Sigma, St. Louis, MO)
for 30 min and resuspended in DMEM (Life Technologies,
Gaithersburg, MD) supplemented with horse serum (10%), penicillin (50 U/ml), streptomycin (50 µg/ml), glutamine (2 mM), 2.5S
nerve growth factor (100 ng/ml; Harlan Bioproducts for Science,
Madison, WI), and glucose (10 mM). After mechanical dispersion by repeated passage through a fire-polished Pasteur pipette,
the ganglion cells were plated on a 0.1% poly-L-ornithine substrate. To prevent the growth of non-neuronal cells, we supplemented the cultures with 5'-fluorodeoxyuridine (10 µM; Sigma)
after 2 d. A separate culture was prepared from each pup from
litters born to heterozygote parents. Experiments were performed after 5-7 d in culture.
Whole-cell currents were elicited with 300 µM nicotine
using standard whole-cell techniques. In each experiment, the plating medium was removed, and the neurons were rinsed gently with
extracellular recording solution consisting of 150 mM NaCl,
3 mM KCl, 2.5 mM CaCl2,
10 mM glucose, 10 mM HEPES, and 1 µM tetrodotoxin, titrated to pH 7.2 with 1 M
NaOH. The culture dish was placed on the stage of a Zeiss inverted
microscope equipped with phase-contrast optics. Cells were viewed at
400× with a 40× objective. All experiments were done at room temperature.
Patch pipettes were pulled from Kimax capillary tubing (Kimble Glass,
Vineland, NJ) on a List vertical puller (List, Darmstadt, Germany).
Pipette resistances were 2-3 M
. The intracellular recording solution consisted of (in mM): 3 NaCl, 150 KCl, 1 MgCl2, 1 EGTA, 5 MgATP, 0.3 NaGTP, and 10 HEPES,
titrated to pH 7.2 with 1 M KOH. Cells were held at 60
mV, and the series resistance and capacitance compensation were
adjusted to eliminate transients. Macroscopic currents were recorded
with an Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster
City, CA) and a VR-10B analog-to-digital converter (Instrutech
Corporation, Great Neck, NY). Currents were acquired and digitally
filtered (1 kHz), and the peaks of the currents were detected with
Clampex (pClamp6 software; Axon Instruments). Nicotine was
dissolved in extracellular recording solution and applied locally by
pressure ejection from an ~2 µm tip diameter pipette for 5 sec (12 psi; Picospritzer; General Valve, Fairfield, NJ).
Histological, morphological, and physiological analysis. The
mice were examined for gross and microscopic pathology. The animals were killed and dissected after fixation in 10% formalin. Organs were
inspected and prepared in blocks. The head and brain were cut in the
coronal plane into six blocks. The tissues were dehydrated and
impregnated with paraffin, sectioned at 5 µm, and stained with
hematoxylin and eosin (H&E). The blocks containing the bladder were reacted with anti-neurofilament antibody (anti-neurofilament monoclonal; 1:2000; Dako, Carpinteria, CA) after antigen retrieval in
0.1 M citric acid, pH 6.0, in the microwave oven three
times for 5 min each. The blocks of head and brain were sectioned at seven levels. One level was prepared with anti-glial fibrillary acidic
protein (anti-GFAP; Dako; at 1:1000 dilution). Appropriate controls
were used for each study.
To observe the growth and lethality of the mice, we determined the
genotype of some newborn mice at birth by PCR. The number and weight of
surviving mice were monitored daily to construct growth and survival
curves. Blood was drawn from anesthetized mice by cardiac puncture
followed by death. Serum was tested for glucose, creatinine, urea
nitrogen, K+,
Na+, Ca+2,
Cl
, and PO4 3.
Urine was drawn directly from the bladder of anesthetized mice with a 29 gauge needle syringe and tested for creatinine, glucose, Na+, K+,
Ca+2, PO43,
osmolarity, and bacterial growth. The composition of urinary stones was
determined at the Mayo Medical Laboratories (Rochester, MN).
In vitro contractility of mouse urinary bladders.
Bladder strips from neonatal mice (2-5 d after birth) were studied as
described previously (Xu et al., 1999). Bladder strips were exposed to
electrical field stimulation or drugs after a 1 hr equilibration into a
Krebs-Henseleit solution aerated with a mixture of 5%
CO2/95% O2. The
Krebs-Henseleit solution had the following composition (in
mM): 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 25 NaHCO3, 1.5 MgSO4,
11.0 D-glucose, and 2.5 CaCl2.
Contractions were measured with an isometric Transducer (Grass
Instruments, Quincy, MA) and recorded on a chart recorder (Gould
Instruments, Cleveland, OH). Frequency-response curves (1-40 Hz) were
elicited by stimulating the bladder strips for 5 sec with pulses of 5 msec duration at supramaximal voltage (50 V) every 60 sec. Nicotine was
added as a single dose (0.1 mM) directly to the organ bath
and was washed out after a maximum contraction was recorded. To
determine the relative response for each strip, we normalized the
nicotine data to the contraction induced by the muscarinic agonist
carbamylcholine (CCH). A single dose of 0.1 mM CCH was
added after a 1 hr interval with several changes of solution.
Dose-response curves for CCH were constructed by adding cumulatively
the drug from 0.1 µM to 0.3 mM. During the
CCH experiments the bladder strips were preexposed to 20 µM hexamethonium for 20 min. Dose-response curves were
fitted with a logistic equation to determine EC50 values.
Noninvasive measurement of intestinal peristalsis in newborn
mice. Three- to five-day-old mice were separated from the mother and kept warm on an electrical blanket. After a 4 hr fasting period the
mice were administered by mouth 20 µl of a blue-1 color food dye (Adams Extract, Austin, TX) in 5% glucose solution. The intraoral administration was performed with sterile Eppendorf microloaders (Eppendorf, Hamburg, Germany). Thirty minutes after the administration, mice were killed, and the stomach and intestines were carefully dissected. Intestinal propulsion was calculated as the percentage of
the distance traveled by the colored solution per total length of the
small intestine (from pylorus to ileocecal junction).
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RESULTS |
Preparation of mice with null mutations for both the 2 and
4 subunits
Mice deficient in the 2 subunit were generated by replacing the
8 kb region containing exons 1 to 5 with Neo in ES cells followed by transmission to the germline (Fig.
1A). Southern blot analysis with HindIII and the 2-flanking probe revealed a
6 kb fragment from the mutant allele in homozygous (/) mice, a
12 kb fragment in the wild-type (+/+) mice, and both fragments in heterozygous (+/) mice. Similarly, mice lacking the 4 nAChR subunit were prepared by introducing a 4.1 kb deletion including most
of the exon 5-coding sequence in ES cells and further transmitted into
the germline (Fig. 1B). Southern blot analysis using
the 4-flanking probe revealed a length change of the SacI
fragment from 7.8 kb for the wild-type allele to 5.8 kb for the 4
mutant allele. Three breeding schemes (Fig. 1C) were used to
compare 2/4/ with 2+/+4/ littermates (Fig.
1C, left), 2/4/ with
2/4+/+ littermates (Fig. 1C, center),
and 2+/+4/ with 2+/+4+/+ (wild-type) littermates (Fig.
1C, right). Examples of mice of various genotypes
are shown by Southern blot in Figure 1D. The
wild-type allele and mutant allele can also be distinguished by PCR
using three oligonucleotides as described in Materials and Methods.
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Figure 1.
Gene targeting of the neuronal nAChR 2 and 4
subunits. A, Partial genomic structure of the murine
2 gene including the region from exons 1 to 6 (solid
boxes) and the structure of the targeting vector are
shown. The targeting vector contains Neo as a
positive selectable marker. A homologous recombination event generated
the deletion from exons 1 to 5. The diagnostic probe at the 5'-flanking
region is shown as an open box.
B, Partial genomic structure of the murine 4 gene and
4-targeting vector are depicted with restriction enzyme sites and
exons 5 and 6 (solid boxes). The
targeting vector contains a puromycin resistance cassette
(Puro) as a positive selectable marker and the Herpes
simplex thymidine kinase gene (TK) as a negative
selectable marker to obtain a replacement mutation. The diagnostic
flanking probe is indicated as an open
box. In both A and B the
restriction enzyme sites are as follows: E,
EcoRI; H, HindIII;
P, PstI; S,
SacI; Sa, SalI; and
X, XhoI. C, The three breeding schemes
used to generate the mice studied are shown. Left,
2+/+4/ were compared with 2/4/ littermates.
Center, Similarly, this breeding allowed comparison of
2/4+/+ mice with 2/4/ littermates.
Right, Finally, in this breeding, 2+/+4/ mice
were compared with 2+/+4+/+ (wild-type) littermates.
D, Southern blot analysis identified the mice with the
indicated genotypes using 2- and 4-flanking probes as indicated
in A and B. E, Northern
blot analysis of the expression of 2 mRNA in the brains of
2+/+4+/+, 2/4+/+, 2+/+4/, and
2/4/ mice is shown. The probes are the rat cDNA of 2
and a cDNA for the control gene glyceraldehyde-3-phosphate
dehydrogenase (gapdh). F, Reverse
transcription-PCR analysis of 4 expression in the brains of
2+/+4+/+, 2/4+/+, 2+/+4/, and
2/4/ mice is shown. Reverse transcription was performed
with the addition of reverse transcriptase (RT)
or absence of the enzyme () as the negative control, followed by PCR
with primers of either the 4 gene or the hprt gene.
The size of the PCR product is 176 bp for the 4 gene and 266 bp for
the hprt gene.
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Expression of 2 mRNA was not detectable by Northern blot in
2/ mice, indicating that the 2 mutation is a null allele (Fig. 1E). Reverse transcription-PCR analysis
indicated that the expression of 4 mRNA was not detectable in tissue
from 4/ mice; therefore, the 4 mutation also is a null allele
(Fig. 1F). The expression of either 2 or 4 was
not noticeably affected in the mice with the null mutation of the other
gene, and the expression of both genes was not detectable in mice with
the double mutation (2/4/; Fig.
1E,F).
Retarded growth and postnatal lethality in
2/4/ mice
The null mice for either the 2 or 4 nAChR subunits
(2/4+/+ and 2+/+4/) were viable and present in the
expected ratio when generated from the mating of heterozygous mice
(data not shown). The mice grew to normal size without showing any
obvious physical or neurological deficits. The mice homozygous for both 2 and 4 mutations, generated by mating 2+/4/ mice
(Fig. 1C, left) or 2/4+/ mice (Fig.
1C, center), were present in the expected ratio
at birth. However, the 2/4/ mice died over the first 3 weeks of life, and <10% of the mice survived beyond 10 d after
birth (Fig. 2A). Their
growth was significantly impaired compared with that of the littermates
containing two copies of the wild-type allele of either 2 or 4
(Fig. 2B). The double mutants also displayed retarded
development, such as delayed hair growth and delayed opening and
flattening of the ears after birth, and their eyelids remained closed
after postnatal day 15.
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Figure 2.
Phenotypic findings in mice lacking both 2 and
4 nAChR subunits. A, Survival curve of 30 2/4/ mice ( ) and 34 2/4+/+ mice ( ) in
the first 11 d after birth. The survival curve of the
2/4+/+ mice did not differ from that of age-matched wild-type
animals (data not shown). B, Body weight growth of
2/4/ mice (; n = 30 at day 1 and
n = 6 at day 9) compared with that of
2/4+/+ littermates (; n = 34). The
body weight of the 2/4+/+ mice did not differ from that
of age-matched 2+/+4/ or wild-type animals (data not shown).
Error bars represent the SD from the mean body weight for each
group of animals. C, D, Comparison between the eye of a
16-d-old 2/4+/+ mouse (C) and that of a
2/4/ littermate (D). The
2/4/ mouse had widely dilated pupils that did not
constrict in response to light. The eyes of 2+/+4/ and
2/4+/+ mice did not differ from those of wild-type
animals.
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Dilated ocular pupils in 2/4/ mice
In older 2/4/ mice (>18 d), the palpebral fissures
were noticeably smaller than those in controls or mice with a
single-gene mutation, and the eyelids remained closed. Careful
examination revealed that the ocular pupils were widely dilated
compared with those in the other genotypes (Fig. 2C,D) and
did not constrict in response to light when the lids were pried open.
The wild-type mice as well as mice carrying at least one normal allele
for 2 or 4 (2/4+/+ or 2/4+/ and
2+/4/ or 2+/+4/) opened their eyes around
postnatal day 15 and showed normal pupillary reflex to light.
Absence of ACh-activated nAChR currents in the superior cervical
ganglion neurons of 2/4/ mice
To test whether the genetic deletion of 2 and/or 4 subunits
altered the nAChR-mediated responses of sympathetic ganglion neurons,
we examined the macroscopic currents elicited by applied nicotine.
Neurons were dispersed from the superior cervical ganglia (SCG) of
individual neonatal pups and maintained in vitro as
described in Materials and Methods. Application of maximal
concentrations of nicotine (300 µM) elicited
robust inward currents in all neurons from wild-type mice
(Iavg = 3098 ± 188 pA;
n = 10), whereas there was no detectable response to
applied nicotine in the 2/4/ mice at any concentration
tested (ranging from 10 µM to 3 mM; n = 36, 5 animals;
Fig. 3A). ACh (300 µM) and cytisine (300 µM) were equally ineffective in eliciting
currents from these mice (n = 6; data not shown).
Analysis of the nicotine-evoked macroscopic currents recorded in
2+/+4/ or in 2/4+/+ mice indicated that both
subunits can participate in functional nAChRs in mouse SCG (Fig.
3B). However, comparison of the magnitude of the nicotinic responses in 2+/+4/ versus 2/4+/+ mice suggests
that 4-containing nAChRs underlie a significant portion of the
wild-type, nicotine-evoked currents. Thus the average peak of
nicotine-gated macroscopic currents in 4-deficient neurons was 2%
that of wild-type or 2-deficient neurons [2+/+4/
(Iavg = 73 ± 7 pA) vs
2/4+/+ (Iavg = 3123 ± 212 pA); n = 13 and 24, respectively).
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Figure 3.
Comparison of nicotine-evoked currents from
wild-type, 2/4+/+, 2+/+4/, and
2/4/ superior cervical ganglia neurons. A,
Vertical bars represent average peak
currents in response to a 5 sec pulse of nicotine (300 µM). Nicotine induced a large-amplitude desensitizing
current in 100% of 2+/+4+/+ (I = 3098 ± 188 pA; n = 10) and 2/4+/+
(I = 3123 ± 212 pA; n = 24) neurons and a small-amplitude desensitizing current in 100% of
2+/+4/ neurons (I = 73 ± 7 pA;
n = 13). In contrast, there was no response to
applied nicotine in any of the 2/4/ neurons
(n = 36). B, Traces
are representative whole-cell currents from superior cervical ganglia
neurons of each genotype tested and shown in A.
Calibration: 2+/+4+/+, 2/4+/+, 1000 nA, 5 sec;
2+/+4/, 2/4/, 250 pA, 5 sec.
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Hypoperistalsis in 2/4/ mice
We examined intestinal propulsion in 2/4/ mice and
their littermate controls by administration of blue-1 color food. Thirty minutes after oral administration, the blue-colored solution had
traveled 45.1 ± 2.1% (± SEM) and 36.9 ± 4.2% along the
intestinal tract of 2/4+/+ (n = 23) and
2/4/ (n = 13) mice, respectively (p < 0.05). The length of the small intestine,
from the pylorus to the ileocecal valve, was 12.3 ± 0.4 cm (± SEM) for the 2/4+/+ (n = 23) mice and
10.2 ± 0.4 cm for the 2/4/ (n = 13)
mice. Although significantly different, the shorter intestinal length in the 2/4/ mice was in accordance with their smaller
size. Intestinal motility in wild-type animals did not differ from that of the 2/4+/+ mice.
Histochemical analysis of the double-mutant mice
Gross and microscopic examination of heart, lungs,
gastrointestinal tract, liver, spleen, gonads, kidney, adrenal glands, pancreas, and skeletal muscle from each of the four groups of animals
studied showed no consistent abnormality. The cerebral cortex,
hippocampus, basal ganglia, brain stem, and cerebellum also revealed no
abnormalities using H&E and anti-GFAP staining. The bladders of seven
out of seven 2/4/ mice (ages, 4-24 d) were abnormal
(Fig. 4B). Three
bladders were dilated, and all had abnormalities of the mucosa
consisting of increased thickness of the mucosal layer (focal or
diffuse), evidence of mitotic activity in four, and disorganization or
dysplastic change in three. One bladder showed mucosal calcification.
Intramural nerves were identified in all of the bladders using
anti-neurofilament antibodies. These alterations were less severe but
resembled those seen in bladders of the 3/ mice (Xu et al.,
1999), as illustrated in Figure 4C. Bladders from
2+/+4/ mice revealed focal hyperplasia or dysplasia in three
out of four animals and mitotic activity in two out of four animals. In
five wild-type mice (ages, 4-33 d), bladder mucosa was normal in
three, one showed focal thickening, and one showed a single mitotic
figure (Fig. 4A). In 2/4+/+ mice (ages,
3-41 d), the mucosa showed focal thickening without mitosis in five
out of six animals. Nerves were present in all of the bladder walls as
indicated by positive staining with anti-neurofilament mAb.
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Figure 4.
Altered bladder epithelium in 2/4/
mice. A, Microscopy of a 2+/+4+/+ bladder wall
with epithelium and underlying muscle. The epithelium is composed of
several layers of regular cells. H&E staining, 400×. B,
Bladder epithelium of a 2/4/ mouse that exhibits
dysplasia. The number of epithelial cells is increased, and the cells
are irregularly arranged. Some of the cells have decreased amounts of
cytoplasm with an altered nuclear-to-cytoplasmic ratio. H&E staining,
400×. C, Bladder epithelium and underlying smooth
muscle from an 3/ mouse. The epithelium is dysplastic with an
increased number of cells, which are irregularly arranged. Some cells
show hyperchromatic nuclei and have decreased amounts of cytoplasm. Two
cells exhibit mitotic figures. H&E staining, 400×.
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Lack of bladder contractility in 4/ and
2/4/ mice
Similar to the mice deficient in the 3 nAChR subunit (Xu et
al., 1999), the 2/4/ mice displayed distended urinary
bladders and dribbling urination. Their urine became infected with
Gram-negative and Gram-positive bacteria and often contained stones of
calcium phosphate and magnesium phosphate as described for the
3-deficient mice (Xu et al., 1999). Blood chemistry measurements
were performed to determine glucose, electrolytes, phosphate, calcium,
urea nitrogen, and creatinine, and no consistent differences were found
between control and single- or double-mutant mice. Urine chemistries
included glucose, electrolytes, phosphate, calcium, and creatinine and did not reveal significant differences among the groups studied.
To study functional responses mediated by the intramural postganglionic
parasympathetic innervation, we measured the contraction to 0.1 mM nicotine in bladder strips from wild-type,
2+/+4/, 2/4+/+, and 2/4/ mice (Fig.
5A). The responses of
2/4+/+ mice were superimposable with those of their wild-type
littermate controls. In the 2/4/ mice, similar to the
mice lacking the 3 subunit, the contractility to nicotine was
impaired. Surprisingly, bladder responses were also significantly
reduced in the 2+/+4/ mice, despite the absence of bladder
distension and urine retention. Nicotine responses in wild-type mice
were 44% of the contraction elicited by direct muscarinic stimulation
of the bladder smooth muscle with CCH. Nicotine responses in the
2+/+4/ mice were only 3% of the contraction in response to
0.1 mM CCH.
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Figure 5.
Reduced bladder contraction in response to
nicotine in 2+/+4/ and 2/4/ mice.
A, Responses to nicotine in 2+/+4+/+
(n = 4), 2/4+/+ (n = 18), 2+/+4/ (n = 17), and
2/4/ (n = 12) mice are shown. Data
represent the contractile response to nicotine as a percentage of that
to CCH (± SEM; **p < 0.01). B,
Bladder contractions in response to CCH were superimposable in
2+/+4+/+ () and 2/4+/+ ( ) mice and were similar
in 2+/+4/ ( ) and 2/4/ ( ) mice. The
contractility responses of both 2+/+4/ and 2/4/
mice were significantly different from those of 2+/+4+/+ and
2/4+/+ mice (**p < 0.01;
*p < 0.05). C. Bladder contractions in response to
field stimulation were similar for 2+/+4+/+ () and
2/4+/+ () mice and for 2+/+4/ () and
2/4/ () mice. The contractility responses of
2+/+4/ and 2/4/ mice were significantly
different from those of 2+/+4+/+ and 2/4+/+ mice
(*p < 0.05).
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To decipher the mechanisms of bladder dysfunction further, we analyzed
bladder strips for contractile responses to CCH, a stable ACh analog.
Figure 5B shows the contractile responses of bladder smooth
muscle strips to CCH. In the 2/4+/+ mice, when neuronal
nAChRs were blocked by preincubation with 20 µM
hexamethonium, the concentration-response curves for CCH were
superimposable with those of the wild-type mice. In the same
experimental conditions, we observed a leftward shift of the CCH
concentration-response curves for both the 2+/+4/ and the
2/4/ mutants. The latter results suggest that the ability
of bladder smooth muscles to respond to muscarinic receptor stimulation
is preserved in the mutant mice. Muscarinic receptor supersensitivity,
suggested by the leftward shift of the concentration-response curves,
might be a consequence of reduced ACh release onto the bladder smooth muscle in the 4-deficient mice.
We also tested whether neurotransmitter release could be elicited in
the mutant mice by exposing bladder strips to electric field
stimulation. Figure 5C shows that frequency-response curves were similar for 2/4+/+ and wild-type mice, whereas those for
2+/+4/ and 2/4/ mice were shifted to the left.
These results, together with the histochemistry results showing normal intramural innervation of the bladder, suggest that bladder innervation in 2+/+4/ and 2/4/ mutants is normal and that
neurotransmitters can be released via mechanisms that bypass nAChR stimulation.
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DISCUSSION |
The simultaneous absence of the 2 and the 4 nAChR subunits
produces severe autonomic dysfunction with megacystis, hypoperistalsis, mydriasis, and palpebral ptosis being among the most obvious phenotypic characteristics. In addition, the rate of perinatal mortality is highly
increased, and in the surviving mice, the postnatal growth is retarded.
The ptosis of the upper eyelid in 2/4/ mice might reflect
developmental delay and poor growth or might be explained by the
absence of sympathetic inputs to the eyelid muscle innervated by
postganglionic sympathetic fibers from the superior cervical ganglion
(Yamashita and Honjin, 1982). The absence of ACh-elicited currents in
2/
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ABSTRACT
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