From the
The type I Ca
Intracellular cAMP regulates many important neuronal functions
including ion channel activity (Nairn et al., 1985),
transcription (Nigg et al., 1985; Hagiwara et al.,
1993), and synaptic plasticity (for review, see Frank and
Greenberg(1994)). For example, the late or long lasting form of long
term potentiation (LTP)
There is
increasing evidence that Ca
Synaptogenesis and the expression of LTP in rodents occur during the
first 3 weeks following birth (Pokorny and Yamamoto, 1981; Harris and
Teyler, 1984). The objective of this study was to determine if
Ca
Several previous studies have reported that basal and
forskolin-stimulated adenylyl cyclase activities in rat and mouse
brains reach a maximum during the first 2-3 weeks following birth
(Keshles and Levitzki, 1984; Broaddus and Bennet, 1990; Rius et
al., 1994). However, the developmental expression of specific
adenylyl cyclases including the Ca
Ca
The first 3 weeks following birth is an
important period for the development of the mouse and rat brain. In the
CA1 pyramidal cells, the number of basal dendrites and terminal
branches of the apical dendrites are established by P5 (Pokorny and
Yamamoto, 1981). The basal dendrites, the main shafts, and terminal
fibers branching within the stratum moleculare of the apical dendrites
develop by P10. Between P15 and P24, the lateral branches of the apical
dendrites in the stratum radiatum and its preterminal fibers branching
in the stratum lacunosum develop (Pokorny and Yamamoto, 1981). In a
study of the postnatal development of the CA1 region of the rabbit
hippocampus, cell branching and spine frequency reached near-adult
levels by 3 weeks (Schwartzkroin, et al., 1982). In addition,
it has been demonstrated that the CA1 region of the rat hippocampus
shows little LTP prior to P5, maximal potentiated response around P15,
and a subsequent decline to adult levels (Teyler et al.,
1989). Therefore, synaptogenesis, dendritic development, and the
development of LTP coincides with the developmental changes in I-AC
expression and activity in the rat and mouse brain. However, anatomical
analysis of brains from I-AC mutant and wild-type mice revealed no
significant differences. The increased expression of I-AC in the
hippocampus during the first 2 weeks following birth is, however,
consistent with other evidence, suggesting that this enzyme contributes
to signal transduction systems important for some forms of synaptic
plasticity and memory (Xia et al., 1991; Impey et
al., 1994; Wu et al., 1995).
In summary, I-AC mutant
mice are deficient in developmental expression of
Ca
We thank Dr. Jack Krupinski for providing the HEK-293
cells expressing VIII-AC. We also thank Dr. Z. Xia for providing the
mouse type I adenylyl cyclase cDNA clone and Dr. Thomas R. Hinds for
helpful discussion and advice.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-sensitive adenylyl cyclase has
been implicated in several forms of synaptic plasticity in vertebrates.
Mutant mice in which this enzyme was inactivated by targeted
mutagenesis show deficient spatial memory and altered long term
potentiation (Wu, Z. L., Thomas, S. A., Villacres, E. C., Xia, Z.,
Simmons, M. L., Chavkin, C., Palmiter, R. D., and Storm, D. R.(1995) Proc. Natl Acad Sci. U. S. A. 92, 220-224). Long term
potentiation in the CA1 region of the rat hippocampus develops during
the first 2 weeks after birth and reaches maximal expression at
postnatal day 15 with a gradual decline at later stages of development.
Here we report that Ca
-stimulated adenylyl cyclase
activity in rat hippocampus, cerebellum, and cortex increases
significantly between postnatal days 1-16. This increase appears
to be due to enhanced expression of type I adenylyl cyclase rather than
type VIII adenylyl cyclase, the other adenylyl cyclase that is directly
stimulated by Ca
and calmodulin. Type I adenylyl
cyclase mRNA in the hippocampus increased 7-fold during this
developmental period. The developmental expression of
Ca
-stimulated adenylyl cyclase activity in mouse
brain was attenuated in mutant mice lacking type I adenylyl cyclase.
Changes in expression of the type I adenylyl cyclase during the period
of long term potentiation development are consistent with the
hypothesis that this enzyme is important for neuroplasticity and
spatial memory in vertebrates.
(
)in the CA1 region of
the hippocampus is dependent upon the activity of cAMP-dependent
protein kinases (Frey et al., 1993). Dopamine receptor
antagonists block LTP in the CA1 as well as dopamine stimulation of
adenylyl cyclase (Frey et al., 1991). Furthermore, cAMP
elevating agents potentiate various forms of LTP in the hippocampus
(Stanton and Sarvey, 1985; Hopkins and Johnston, 1988; Chavez-Noriega
and Stevens, 1992, 1994; Weisskopf et al., 1994).
/calmodulin
(CaM)-stimulated adenylyl cyclases may be important for some forms of
neuroplasticity in vertebrates (Xia et al., 1991). The
physiological function of the type I adenylyl cyclase (I-AC) and its
role in neuroplasticity is of particular interest since it is
neurospecific, Ca
/CaM sensitive (Xia et al.,
1993), and expressed in those areas of the brain that exhibit synaptic
plasticity including the CA1, CA2, CA3 and dentate gyrus regions of the
hippocampal formation (Xia et al., 1991). Furthermore, mutant
mice deficient in I-AC are deficient in spatial memory and show
depression of LTP in the hippocampus (Wu et al., 1995).
However, I-AC mutant mouse brains still show residual
Ca
-stimulated adenylyl cyclase activity that is most
likely due to VIII-AC, a Ca
-sensitive adenylyl
cyclase that is also expressed in the hippocampus (Cali et
al., 1994). Approximately 50% of the
Ca
-stimulated adenylyl cyclase activity in the mouse
hippocampus is contributed by I-AC (Wu et al., 1995).
-stimulated adenylyl cyclase is expressed in the
brain during this developmental period. Our data indicate that rat
hippocampus, cerebellum, and cerebral cortex show a highly significant
increase in Ca
-stimulated adenylyl cyclase activity
during the first 2 weeks of development, which is due primarily to
increases in the expression of I-AC.
Cell Membrane Preparations
Membranes were
prepared from HEK-293 cells as described previously (Choi et
al., 1992b). Wild-type and I-AC mutant C57BL/6J mouse cerebellar
membranes (Wu et al., 1995) were prepared from mice varying in
age from postnatal day 2 (P2) to adulthood. Sprague-Dawley rat
cerebellar, hippocampal, and cerebral cortical membranes were prepared
from rats varying in age from P1 to P22. Brain tissue was resuspended
in homogenization buffer (20 mM Tris-HCl, pH 7.4, 0.5 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 5
µg/ml leupeptin, 2 mM MgCl, 0.5 mM phenylmethylsulfonyl fluoride) and homogenized at 4 °C.
Unbroken cells and nuclei were removed by centrifugation at 600
g for 2 min, and the supernatants were subjected to
centrifugation at 30,000
g for 20 min. The resulting
membrane pellet was suspended in the homogenization buffer without EGTA
and assayed for adenylyl cyclase activity.
Adenylyl Cyclase Assay
The enzyme assay was
performed at 30 °C for 20 min by adding membrane fractions
(10-75 µg of protein) to an assay solution containing 1
mM [-
P]ATP (500 cpm/pmol),
[
H]cAMP (20,000 cpm/µmol), 5 mM MgCl
, 1 mM EDTA, 2 mM cyclic AMP, 5
mM theophylline, 0.1% bovine serum albumin, 40 µg/ml CaM,
20 mM creatine phosphate, 60 units/ml creatine phosphokinase
in 20 mM Tris-HCl, pH 7.4, in a final assay volume of 250
µl. The reaction was stopped by adding 750 µl of 1.5% sodium
dodecyl sulfate. The reaction mixture was heated at 100 °C for 2
min, and the
P- and
H-labeled cyclic AMP
generated was recovered using Dowex AG 50W-X4 and neutral alumina
columns, as described by Salomon et al.(1974). The values of
adenylyl cyclase activities represent the mean of triplicate
determinations ± S.D. Protein concentrations were determined by
using the Pierce BCA protein assay kit.
Quantitation of Free Ca
Free calcium concentrations in the
adenylyl cyclase buffers were varied using a CaConcentrations
/EGTA buffer
system. Free Ca
concentrations were estimated using
the Bound and Determined computer program (Brooks and Storey, 1992).
RNA Analysis
Total RNA was isolated from frozen
tissues by the acid guanidinium thiocyanate/phenol/chloroform
extraction method (Chomczynski and Sacchi, 1987).
Poly(A)-selected RNA was isolated from total RNA using
type III oligo(dT) cellulose (Collaborative Research Inc., Bedford MA)
and analyzed on a formaldehyde, 1.2% agarose gel in MOPS buffer
(Ausubel et al., 1989). mRNA was transferred onto Nytran
membranes (Schleicher & Schuell) in 10
SSC (saline sodium
citrate) for 16-20 h, cross-linked at 80 °C for 1 h, and
prehybridized at 42 °C in a hybridization buffer containing 50%
formamide, 5
SSC, 1
Denhardt's solution, and 250
µg/ml denatured salmon sperm DNA for 4 h.
[
-
P]dCTP random primed cDNA probes
generated from the hypervariable region of a mouse I-AC clone
(corresponding to amino acids 469-663) or from the first 771
nucleotides of the coding region and 329 nucleotides of the
5`-untranslated region of the VIII-AC were used for Northern analysis.
These probes were hybridized to the immobilized RNA for 16-20 h
at 42 °C in the hybridization buffer. The membrane was washed in 2
SSC, 0.5% SDS for 10 min at room temperature and once for 5 min
at 65 °C. It was then washed 3 times for 20 min in 1
SSC,
0.5% SDS at 65 °C prior to autoradiography. The probe was stripped
by boiling in 0.1
SSC for 10 min. The membrane was then
hybridized with a
-actin probe and then rehybridized again with an
elongation factor 1
probe. PhosphorImager quantitation was
performed by exposing the radioactive blot to a phosphor screen. After
appropriate exposure, the screens were scanned using the PhosphorImager
model 400S (Molecular Dynamics, Sunnyvale, CA).
In Situ Hybridization
Rats (postnatal days 7, 14,
and adult) were decapitated, and the brains were rapidly isolated,
frozen in OCT compound (Miles, Inc., Elkhart, IN) on dry ice, and
stored at -70 °C until use. Sections (20 µm) were cut in
a cryostat, thaw mounted on a poly-L-lysine-coated microscope
slide, and stored at -70 °C. A template plasmid corresponding
to nucleotides 1557-1981 of the bovine I-AC cDNA clone was used
for the transcription of riboprobes (Xia et al., 1991). This
sequence is found in a hypervariable region of the adenylyl cyclases
and distinguishes I-AC from other adenylyl cyclases. The labeled
antisense RNA probe was transcribed from the HindIII-linearized template with T3 RNA polymerase
(Stratagene) in the presence of [S]UTP (DuPont
NEN). The corresponding sense probe was transcribed from the SmaI-linearized template with T7 RNA polymerase. The specific
activities of the probes were between 10
and 10
cpm/µg. In situ hybridizations were performed as
described previously (Yan et al., 1994). Briefly, the sections
were fixed in 4% (w/v) paraformaldehyde, pretreated with 0.2 M HCL, 2 µg/ml proteinase K, and 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8), followed by graded dehydration for 2
min each in 30, 60, 80, 95, and 100% ethanol. Before hybridization,
each section was prehybridized with 50 µl of hybridization buffer
(50% deionized formamide, 10% dextran sulfate, 0.3 M NaCl, 1
Denhardt's solution, 10 mM dithiothreitol, 1
mM EDTA, 1 mg/ml yeast tRNA, and 100 mg/ml synthetic
poly(A)
RNA) in the absence of probe under parafilm
coverslips in a humid chamber at 50 °C for 2-6 h. The
prolonged prehybridization significantly reduces the background without
affecting the specific hybridization signal intensity. The sections
were then briefly rinsed in 2
SSC, dehydrated with ethanol, and
air dried. Hybridization was performed in the hybridization buffer
containing
S-labeled antisense riboprobes (0.1
µg/ml/kilobase) at 50 °C for 16-20 h in a humid chamber
under glass coverslips sealed by rubber cement. After hybridization,
sections were washed twice with 2
SSC, 10 mM
dithiothreitol for 30 min each at 55 °C. In order to remove
nonspecifically bound single-stranded probe, the sections were treated
with a solution containing 20 µg/ml RNase A, 100 units/ml RNase T1,
0.5 M NaCl, 10 mM Tris-HCl, pH 8, and 1 mM EDTA for 30 min at 37 °C. The sections were further washed in
2
SSC, 50% formamide, 10 mM dithiothreitol for 30 min
at 55 °C and in 0.1
SSC, 10 mM dithiothreitol for
30 min at 55 °C. The sections were dried by graded dehydration with
30-100% ethanol and then coated with NTB2 emulsion (Eastman Kodak
Co.) and exposed for 1-3 weeks at 4 °C. The emulsion-coated
slides were developed, fixed, stained through the emulsion with
hematoxylin or hematoxylin/eosin, and mounted with Permount (Fisher).
Each comparative set of slides was dipped in the same batch of emulsion
and exposed for the same period of time. Nonspecific hybridization was
determined by parallel incubation of adjacent sections with
-
S-labeled sense riboprobes.
Developmental Expression of
Ca
I-AC mRNA is
expressed in the hippocampal formation, cerebellum, and cerebral cortex
of the adult rat brain, but it is absent from other areas of brain
including the brain stem (Xia et al., 1991). We measured basal
and Ca-stimulated Adenylyl Cyclase Activity in Rat
Hippocampus, Cerebellum, and Cerebral Cortex
-stimulated adenylyl cyclase activity in rat
brain membrane preparations during the first 3 weeks of postnatal
development because this is the period of development in the
hippocampus of the rat (Teyler et al., 1989).
Ca
-stimulated adenylyl cyclase activity in membranes
from the rat hippocampus increased 5.5 ± 0.3-fold between P1 and
P16 and declined somewhat after P16 (Fig. 1A). Although
basal adenylyl cyclase activity also increased during this period, the
relative increase in Ca
-stimulated adenylyl cyclase
activity was greater (5.5 ± 0.3- versus 3.5 ±
0.1-fold). Ca
/CaM stimulation relative to basal
activity was 3.6 ± 0.2-fold at P1 and 5.4 ± 0.3-fold at
P16. In the cerebellum, both Ca
-stimulated and basal
adenylyl cyclase activities increased 7-10-fold between P5 and
P18 (Fig. 1B). Basal and Ca
-stimulated
adenylyl cyclase activities in the cerebral cortex increased
3-4-fold during the same developmental period (Fig. 1C).
Figure 1:
Effect of Ca on
adenylyl cyclase activity in the developing postnatal rat hippocampus (A), cerebellum (B), and cerebral cortex (C). Membranes were prepared from Sprague-Dawley rat
hippocampus (P1-P19), cerebellum (P3-P22), and cerebral
cortex (P3-P18) and assayed for adenylyl cyclase activity in the
presence (
) or absence (
) of 300 nM free
Ca
and 2.4 µM CaM as described under
``Experimental Procedures.'' Adenylyl cyclase activities are
the mean of triplicate determinations ±
S.D.
Ca
The developmental increase in
CaSensitivity of Adenylyl Cyclase
in Rat Hippocampal Membranes
-stimulated adenylyl cyclase activity in the
hippocampus was particularly interesting because of recent data
implicating I-AC and other Ca
-stimulated adenylyl
cyclases in various forms of LTP in the hippocampus (Wu et
al., 1995; Weisskopf et al., 1994). Two
Ca
-stimulated adenylyl cyclases are expressed in the
hippocampus, I-AC (Xia etal., 1991) and VIII-AC
(Cali etal., 1994). These two enzymes are the only
known adenylyl cyclases that are directly stimulated by Ca
and CaM in vitro. Therefore, the Ca
sensitivity of adenylyl cyclase activity in the wild-type rat
hippocampal membranes was compared with I-AC and VIII-AC expressed in
HEK-293 cells to evaluate the contribution of these enzymes to
Ca
-stimulated adenylyl cyclase in this region of the
brain (Fig. 2). Half-maximal stimulation of adenylyl cyclase
activity in P10 rat hippocampal membranes was at 200 nM free
Ca
(Fig. 2A). I-AC and VIII-AC were
half-maximally stimulated at 100 and 800 nM free
Ca
, respectively (Fig. 2, B and C). Furthermore, the Ca
activation curves
for I-AC and hippocampal adenylyl cyclase activities were both bell
shaped, with a decrease in Ca
-stimulated activity
above 2.5 µM free Ca
. These data
suggest that I-AC makes a significant contribution to
Ca
-stimulated adenylyl cyclase activity in the rat
hippocampus.
Figure 2:
Comparison between the
Ca sensitivity of hippocampal adenylyl cyclase
activity and I-AC or VIII-AC. Membranes were isolated from 10-day-old
Sprague-Dawley rat hippocampi and I-AC or VIII-AC stably transfected
293 cell and assayed for adenylyl cyclase in the presence of 2.4
µM CaM as described under ``Experimental
Procedures.'' Free Ca
concentrations were varied
using EGTA/CaCl
buffers as described under
``Experimental Procedures.'' Adenylyl cyclase activities are
the mean of triplicate determinations ±
S.D.
Developmental Expression of I-AC mRNA in Rat
Brain
The developmental increase in
Ca-stimulated adenylyl cyclase activity in rat brain
could be due to changes in gene expression or modulation of adenylyl
cyclase activity by other proteins, e.g. protein kinases.
Consequently, we quantitated I-AC mRNA expression in brains from P2,
P6, and P16 rats by Northern analysis using a probe specific for I-AC.
The I-AC transcript was 11.5 kilobases, consistent with previous
reports (Xia et al., 1993). In these experiments,
poly(A)
-selected RNA was isolated from the
hippocampus, cerebellum, or whole rat brain, and the amount of I-AC
mRNA present in each sample was normalized to elongation factor 1
mRNA (Fig. 3B). In the hippocampus, cerebellum, or whole
brain, I-AC mRNA increased during the period from P2 to P16. For
example, in the hippocampus, I-AC mRNA increased 7-fold between P2 and
P16 (Fig. 3B), which is consistent with the increase in
Ca
-stimulated adenylyl cyclase activity in this
tissue (Fig. 1). In contrast, VIII-AC mRNA increased 2-fold
between P2 and P16 (data not shown). These data indicate that the
developmental increase in Ca
stimulated adenylyl
cyclase activity was due primarily to expression of I-AC.
Figure 3:
Northern
analysis of I-AC mRNA in rat hippocampi, cerebella, and whole brains
during postnatal development. A, Northern analysis for I-AC,
-actin, and elongation factor 1
(EF-1
) mRNA was
carried out as described under ``Experimental Procedures.'' B, the relative levels of I-AC mRNA were normalized to
elongation factor 1
mRNA by phosphorimaging
analysis.
In Situ Hybridization Analysis for I-AC
The
distribution of mRNA for I-AC in developing rat brains was also
examined by in situ hybridization using a bovine I-AC-specific
riproprobe corresponding to bases 1557-1981 (Fig. 4). The
strongest hybridization signals were detected in the granule cell
layers of the dentate gyrus, pyramidal cells of the CA2 region, and the
granule cell layers of the cerebellum. Moderate levels of I-AC mRNA
were observed in the pyramidal cells of the CA1 and CA3 fields. This
expression pattern was evident in P7, P14, and adult rat brains.
However, P14 rat brains showed stronger hybridization signals in the
hippocampus, cortex, and cerebellum than did brains from P7. I-AC mRNA
levels decreased somewhat from P14 to the adult stage, consistent with
the decrease in Ca-stimulated adenylyl cyclase
activity after 2 weeks of postnatal development in both rats and mice ( Fig. 1and 5A).
Figure 4:
In situ hybridization analysis
for I-AC in developing rat hippocampus and cerebellum. Sagittal
sections of rat hippocampi (i) or cerebella (ii) were
hybridized with -
S-labeled antisense riboprobes
directed to mRNA encoding I-AC and visualized using dark-field
photomicroscopy during development. A, P7; B, P14; C, adult stage; D, hybridization of the adult stage
with sense probe. CA1, CA2, and CA3, are the
pyramidal cell layers of the hippocampus. DG, dentate gyrus; GL, granular layer.
Adenylyl Cyclase Activity in the Developing Cerebellum of
the I-AC Mutant Mouse
The data discussed above indicate that
increases in Ca-stimulated adenylyl cyclase activity
during the first 2 weeks after birth may be primarily attributable to
I-AC. Consequently, adenylyl cyclase activity was measured in the
cerebellum from developing wild-type and I-AC mutant mice (Wu et
al., 1995). Like rats, wild-type mice showed a significant
increase in basal and Ca
-stimulated adenylyl cyclase
activity during the first 2 weeks of postnatal development (Fig. 5). The developmental increase in
Ca
-stimulated activity was greater than basal
activity (6.5- versus 3.0-fold). I-AC mutant mice showed only
a 2-fold increase in Ca
-stimulated adenylyl cyclase
activity between P2 and P16 and no significant increase in basal
activity. Similarly, the large increase in forskolin-stimulated
adenylyl cyclase activity between P2 and P16 in wild-type mice was due
primarily to I-AC.
Figure 5:
Developmental expression of
Ca-stimulated adenylyl cyclase activity in cerebellar
membranes from wild-type and I-AC mutant mice. Cerebellar membranes
were isolated from wild-type (
) or I-AC mutant (
) mice (P2
through adulthood) and analyzed for adenylyl cyclase activity as
described under ``Experimental Procedures.'' A, 300
nM free Ca
and 2.4 µM CaM; B, basal activity; C, 10 µM forskolin.
Adenylyl cyclase activities are the mean of triplicate determinations
± S.D.
Morphological Analysis of Brains from Wild-type and I-AC
Mutant Mice
Because of the developmental expression pattern of
I-AC, it was important to compare the structure of brains from
wild-type and I-AC mutant mice. Analysis of 7-week-old mouse brain
coronal sections stained with cresyl violet by light microscopy showed
no detectable anatomical differences in the hippocampus, neocortex, or
cerebellum (data not shown). There were also no apparent differences in
the arrangement of cell body layers of the hippocampus or cerebellum
when sections were examined at higher magnification. These data
indicate that the type I adenylyl cyclase may not be crucial for
hippocampal and cerebellar development and that the brains of mutant
mice are anatomically indistinguishable from wild-type mice.
-stimulated
adenylyl cyclase activity in brain had not been examined prior to this
study. Of the 10 adenylyl cyclases cloned to date, only I-AC and
VIII-AC are directly stimulated by Ca
in vitro and in vivo (Choi et al., 1992a; Cali et
al., 1994), but neither is stimulated by
-adrenergic agonists in vivo (Wayman et al., 1994; Cali et al.,
1994). However, I-AC is synergistically stimulated by intracellular
Ca
and receptor activation in vivo (Wayman et al., 1994), whereas VIII-AC is not. It was the objective of
this study to examine the developmental expression of
Ca
-stimulated adenylyl cyclases in rats and mice
during P2-P16, a period during which various forms of synaptic
plasticity develop in the rodent brain (Harris and Teyler, 1984).
-stimulated adenylyl cyclase activity in mouse
and rat brains increases significantly during the first 2 weeks of
development, particularly in the hippocampus. Northern analysis
suggests that this increase may be due primarily to elevations in I-AC
mRNA. These developmental increases in I-AC mRNA were also evident when
rat brain slices were analyzed by in situ hybridization.
Although we did not determine whether the increase in I-AC mRNA was due
to increased transcription or message stability, it is interesting that
the I-AC transcript has a very large 3` noncoding region (approximately
7 kilobases) that may have specific sites for interaction with mRNA
binding proteins. VIII-AC mRNA showed only minor increases during this
developmental period. The increase in Ca
-stimulated
adenylyl cyclase activity was significantly disrupted in I-AC mutant
mice confirming that I-AC is the major source for the developmental
increases in activity.
-stimulated adenylyl cyclase activity. These
observations are consistent with the proposal that I-AC may play an
important role in various forms of synaptic plasticity by coupling the
Ca
and cAMP signal transduction systems.
/CaM, the
complex of four calcium ions bound to calmodulin; I-AC, type I adenylyl
cyclase; VIII-AC, type VIII adenylyl cyclase; P, postnatal day; HEK-293
cells, human embryonic kidney-293 cells; MOPS,
4-morpholinepropanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.