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Previous Article | Next Article
The Journal of Neuroscience, January 1, 1999, 19(1):180-192
Regulation and Immunohistochemical Localization of
-Stimulated Adenylyl Cyclases in Mouse Hippocampus
Lauren P. Baker, Mark D. Nielsen, Soren Impey, Beth M. Hacker, Steven W. Poser, Mandy Y. M. Chan, and Daniel R. Storm Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280
ABSTRACT
Top
Abstract
Introduction
Specific forms of synaptic plasticity such as long-term potentiation (LTP) are modulated by or require increases in cAMP. The various adenylyl cyclase isoforms possess unique regulatory properties, and thus cAMP increases in a given cell type or tissue in response to converging signals are subject to the properties of the adenylyl cyclase isoforms expressed. In most tissues, adenylyl cyclase activity is stimulated by neurotransmitters or hormones via stimulatory G-protein (Gs)-coupled receptors and is inhibited via inhibitory G-protein (Gi)-linked receptors. However, in the hippocampus, stimulation of Gi-coupled receptors potentiates Gs-stimulated cAMP levels. This effect may be associated with the regulatory properties of adenylyl cyclase types 2 and 4 (AC2 and AC4), isoforms that are potentiated by the
subunit of Gi in vitro. Although AC2 has been shown to be stimulated by
Key words: adenylyl cyclase; cAMP; G-protein;
INTRODUCTION
The regulation of adenylyl cyclases by G-protein-coupled receptors is a classically described mechanism in which a stimulatory G-protein (Gs) couples neurotransmitters to activation of adenylyl cyclase, whereas an inhibitory G-protein (Gi) couples to inhibition of adenylyl cyclase. In certain tissues, Gi signaling can potentiate Gs input (Andrade, 1993
; Gereau and Conn, 1994
Although the Ca2+-stimulated adenylyl cyclases (AC1 and AC8) are believed to play a role in synaptic plasticity in the hippocampus (Choi et al., 1993
To date, nine mammalian adenylyl cyclase isotypes (AC1-AC9) have been identified (Krupinski et al., 1989
The in vitro regulatory properties of AC2 and AC4 are consistent with the possibility of a role in Gi-mediated potentiation of Gs-stimulated cAMP. The activity of both isoforms is potentiated in vitro by Gi in the presence of Gs (Feinstein et al., 1991
MATERIALS AND METHODS
Materials. Isoproterenol, 3-methyl-1-isobutylxanthine (IBMX), serotonin, and somatostatin-14 were purchased from Sigma (St. Louis, MO). Pertussis toxin was obtained from List Biologic (Campbell, CA). Restriction endonucleases and DNA ligase were purchased from New England Biolabs (Beverly, MA). Polyclonal antibodies generated against adenylyl cyclases 2 or 4 were generously provided by Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies generated against MAP-2 or synaptophysin were purchased from Sigma.
Cell culture. HEK 293 cells were grown at 37°C in HEPES-buffered (H)-DMEM supplemented with 10% bovine calf serum (BCS) and 1% penicillin and streptomycin in a humidified 95% O2/5% CO2 incubator. Cell culture materials were obtained from Life Technologies (Gaithersburg, MD) unless otherwise indicated.
Expression of the 5-HT7A receptor and AC2 or AC4 in HEK 293 cells. The AC4 cDNA clone was generously provided by Drs. Wei-Jen Tang and Alfred G. Gilman (University of Texas Southwestern Medical Center, Dallas, TX). The AC4 insert was released from pBluescript (Stratagene, La Jolla, CA) by digestion with KpnI and BamHI and was ligated into the pCEP4 expression vector (Invitrogen, San Diego, CA). The AC2 cDNA was generously provided by Dr. Randall R. Reed (Johns Hopkins School of Medicine, Baltimore, MD) and was ligated into pCEP4. The 5-HT7A receptor cDNA clone was a gift from Dr. Mark Hamblin (Veterans Administration Medical Center, Seattle, WA) and was ligated into pcDNAIII (Invitrogen). Polyclonal populations of G418- (500 µg/ml; Calbiochem, La Jolla, CA) and/or hygromycin- (400 µg/ml; Calbiochem) resistant 293 cells were obtained by stable transfection of the pcDNAIII or pCEP4 expression vector and/or pcDNAIII-5-HT7A or pCEP4-AC4 (or pCEP4-AC2 alone for analysis of AC2 expression by immunocytochemistry) using the calcium phosphate method (Chen and Okayama, 1987
cAMP accumulation assay. Changes in intracellular cAMP were measured by determining the ratio of [3H]cAMP to the total ATP, ADP, and AMP pool in [3H]adenine-loaded cells as described previously (Wong et al., 1991
Transient coexpression of AC4 with the C terminal of
-adrenergic receptor kinase 1 or transducin
in HEK 293 cells. The peptide minigene construct encoding the C terminal of
Description of AC2 and AC4 antibodies. The anti-AC2 and -AC4 antibodies generated by Santa Cruz Biotechnology are rabbit polyclonal antibodies generated against 20 amino acid peptides corresponding to unique intracellular C-terminal amino acid sequences in AC2 and AC4, respectively. The sequence used to generate the AC2 antibody corresponds to amino acids 1071-1090 (KTYFVNTEMSRSLSQSNLAS) of rat AC2, which is 95% identical to the corresponding human sequence. The sequence used to generate the AC4 antibody corresponds to amino acids 1045-1064 (QLCTYFLNTDLTRTGSPSAS) of rat AC4. Database searches using GCG Blast revealed high-score matches only to rat and human AC2 for the AC2 antigen peptide and to rat AC4 for the AC4 antigen peptide.
Western blot analysis of AC2 and AC4 expression in HEK 293 cells. HEK 293 cells expressing the pCEP4 vector alone or cells stably transfected with AC2 or AC4 were grown to confluence in 100 mm culture dishes and harvested in 20 mM Tris HCl, pH 7.4, 1 mM EDTA, 2 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, 3.2 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.5 µg/ml pepstatin A. Homogenates were centrifuged at 600 × g for 5 min, and the supernatant was retained and centrifuged at 90,000 × g for 30 min. Membrane pellets were resuspended in buffer C (1% NP-40, 50 mM sodium phosphate, pH 7.4, 10 mM EDTA, 1 mM DTT, 2 mM MgCl2, 0.5 mM PMSF, 5 µg/ml leupeptin, and 2 µg/ml aprotinin) and were used immediately or frozen at
80°C for later use. Protein concentrations were determined according to the method of Bradford, using BSA as a standard (Bradford, 1976
HEK 293 cell immunocytochemistry. HEK 293 cells alone or stably transfected with AC2 or AC4 were plated onto poly-D-lysine-coated coverslips (60 µg/ml poly-D-lysine) and grown in appropriate growth medium for 2-4 d. Cells were fixed with 4% paraformaldehyde in PBS, pH 7.4, for 20 min, rinsed with PBS, and permeabilized and blocked for 1 hr with PBS plus 0.5% Triton X-100, 4% cold fish gelatin, and 1% normal goat serum. Cells were incubated with 100 ng/ml anti-AC2 or -AC4 in PBS plus 0.5% cold fish gelatin overnight at 4°C on a rocker, rinsed, and then incubated with 1 µg/ml lissamine-rhodamine-conjugated goat anti-rabbit IgG for 1 hr. After a final rinse, the coverslips were mounted onto slides with Gelmount (Biomedia, Foster City, CA). Where indicated, peptide antigen specific for AC2 or AC4 (Santa Cruz Biotechnology) was included at a 20-fold molar excess to antibody as a control.
Immunohistochemistry on mouse hippocampal sections. C57Bl/6J mice were killed by decapitation, and the brains were quickly removed. Coronal brain slices (500 µm) were cut using a vibratome (Campden Instruments, Leicestershire, UK). Slices were fixed for 4-6 hr with 4% paraformaldehyde in PBS, pH 7.4. After fixation, slices were cryoprotected in PBS plus 30% sucrose and 0.02% NaN3 and were maintained at 4°C until further use. For immunolabeling, 40 µm sections were cut from the fixed 500 µm slices on a sliding microtome and placed free-floating into a blocking solution of PBS, pH 7.4, plus 0.025% Triton X-100 (PBST) supplemented with 5% BSA. Sections were blocked for 1 hr at room temperature and then incubated in PBST plus 0.5% BSA and 250 or 125 ng/ml rabbit polyclonal antibody against AC2 or AC4, respectively, for 18-24 hr. Where indicated, peptide antigens were added at a 20-fold molar excess to antibody immediately before incubation. After incubation with the primary antibody, sections were washed four times for 10 min in PBST at room temperature, after which 1 µg/ml lissamine-rhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) was added to the sections for 1 hr at room temperature. Sections were then washed and mounted onto slides using Gelmount (Biomedia) or Vectashield (Vector Laboratories, Burlingame, CA). For double labeling, a 1 hr incubation in a 1:400 dilution of mouse monoclonal anti-synaptophysin (clone SVP-38, ascites fluid; Sigma) or a 1:500 dilution of mouse monoclonal anti-MAP2 (clone HM-2, ascites fluid; Sigma) was followed by incubation in 1 µg/ml FITC-conjugated goat anti-mouse antibody (Jackson ImmunoResearch). Immunolabeling was analyzed using a Bio-Rad MRC 600 confocal microscope (William M. Keck Imaging Center, University of Washington, Seattle, WA). Labeling of discrete brain structures was determined according to the mouse brain atlas of Franklin and Paxinos (1997)
RESULTS
G-protein-coupled receptors stimulate AC4 in intact cells
HEK 293 cells were transfected with AC4 cDNA, and cells from the stably selected polyclonal cell line were treated with the
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Figure 1. Gs-coupled receptors stimulate AC4 in HEK 293 cells. A, HEK 293 cells stably expressing AC4 were treated with increasing concentrations of isoproterenol. B, HEK 293 cells stably coexpressing AC4 with the Gs-coupled 5-HT7 receptor were treated with increasing concentrations of serotonin. Relative cAMP accumulation was determined as described in Materials and Methods, and endogenous HEK 293 cell cAMP accumulation was subtracted from the cAMP measured in AC4-expressing cells. The data are the means ± SD of triplicate assays.
Gi-mediated potentiation is inhibited by pertussis toxin and by
G-protein
stimulation of AC2 (Tang and Gilman, 1991
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Figure 2. Pertussis toxin,
To determine whether somatostatin increases AC4 activity by stimulating the release of
Characterization of AC2 and AC4 antibodies in HEK 293 cells
Before determining the expression of AC2 and AC4 in the hippocampus, we characterized the specificity of the AC2 and AC4 polyclonal antibodies by Western blot and immunolabeling studies. By Western blot, the AC2 antibody recognized AC2 overexpressed in HEK 293 cells under native conditions and very little under denaturing conditions (Fig. 3A). HEK 293 cells express low levels of AC2 endogenously (Hellevuo et al., 1993
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Figure 3. Western blot detection of AC2 and AC4 in HEK 293 cell membranes. A, Whole-cell lysate from HEK 293 cells expressing the pCEP4 vector alone (293) or AC2-pCEP4 (293-AC2) was subjected to native PAGE conditions and Western blotted as described in Materials and Methods. Blots were developed using enhanced chemiluminescence and scanned on a Hewlett-Packard Scan Jet II CX scanner. B, Membranes from HEK 293 cells were prepared and treated with or without N-glycosidase F as described in Materials and Methods. Twenty micrograms of membranes from 293-pCEP4 cells (293; lanes 1 and 2) and 293-AC4 cells (293-AC4; lanes 3 and 4) were separated by SDS-PAGE on 7.5% acrylamide gels. Protein was transferred to nitrocellulose and incubated with anti-AC4 antibody. Molecular weight markers equal 206, 117, 89, and 47 kDa. Note the conversion of the glycosylated form(s) to the lower molecular weight nonglycosylated form after treatment with N-glycosidase F. A minor band at ~60 kDa is only seen in AC4-expressing cells and is therefore probably a proteolytic fragment of AC4 that is recognized by the AC4 antibody.
Immunolabeling studies using untransfected HEK 293 cells or cells expressing AC2 or AC4 were also performed (Fig. 4). The AC2 antibody produced a low level of labeling in HEK 293 cells transfected with the empty expression vector (Fig. 4A). In contrast, pronounced membrane labeling was observed in cells stably expressing AC2 (Fig. 4B), which was completely abrogated after AC2 peptide adsorption (Fig. 4C). No AC2 labeling over endogenous levels was observed in AC4-expressing cells (Fig. 4D). Similarly, the AC4 antibody labeled cells expressing AC4 (Fig. 4F) but not vector-transfected cells (Fig. 4E) or cells expressing AC2 (Fig. 4H). Adsorption of the AC4 antibody with the AC4 peptide blocked all immunolabeling with the AC4 antibody (Fig. 4G). The AC2 and AC4 antibodies also did not recognize other adenylyl cyclases (AC1, AC3, AC5, AC6, AC8, and AC9) expressed in HEK 293 cells (data not shown).
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Figure 4. Immunocytochemical detection of AC2 and AC4 stably expressed in HEK 293 cells. A, E, HEK 293 cells transfected with the pCEP4 vector (designated HEK 293) were processed for immunocytochemistry with the AC2 (A) or AC4 (E) antibody as described in Materials and Methods. B, F, HEK 293 cells expressing AC2 or AC4 (designated 293-AC2 or 293-AC4) were labeled with the AC2 (B) or AC4 (F) antibody. C, Adsorption of the AC2 antibody with AC2 peptide antigen (PEP) blocked AC2 immunolabeling in HEK 293 cells expressing AC2. D, Cells expressing AC4 did not label with the AC2 antibody. G, Adsorption of the AC4 antibody with AC4 peptide antigen blocked AC4 immunolabeling in HEK 293 cells expressing AC4. H, Cells expressing AC2 did not label with the AC4 antibody. Scale bar, 25 µm.
Analysis of AC2 and AC4 expression in mouse hippocampal formation
To define the expression and localization of AC2 and AC4 proteins in the mouse hippocampal formation, we performed immunhistochemical studies. The AC4 antibody was also used to immunoprecipitate AC4 from mouse hippocampal membranes (data not shown). The AC2 antibody was not useful for immunoprecipitation. For immunohistochemistry, 40 µm hippocampal slices were labeled using either the AC2 or AC4 antibody and were examined by confocal microscopy. Specific labeling for both adenylyl cyclase isoforms was observed throughout the hippocampal formation, and the labeling pattern was more or less indistinguishable for both AC2 and AC4. In the dentate gyrus, immunolabeling for AC2 and AC4 was concentrated in the proximal dendrites of the granule cells and extended into the dendritic field in the molecular layer (Fig. 5A,B). A lower level of labeling was also observed in the cell bodies. Double labeling was performed for AC2 or AC4 and either a dendritic marker (MAP2; Figs. 6, 7) or a synaptic terminal marker (synaptophysin; Figs. 8, 9). AC signal was coincident with the MAP2 labeling in the dendrites (Fig. 6C,F,C',F'; yellow indicates coincident signal) and did not colocalize at all with the synaptophysin signal (Fig. 8), indicating a postsynaptic localization of these adenylyl cyclase isoforms. Scattered cells in the hilus were also labeled using the AC2 and AC4 antibodies.
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Figure 5. Immunohistochemical detection of AC2 and AC4 in the mouse hippocampal formation. Mouse brain sections (40 µm) were processed for immunohistochemistry as described in Materials and Methods. A, AC2 labeling in the dentate gyrus (DG). B, AC4 labeling in dentate gyrus. Note the concentration of AC2 and AC4 label in the proximal dendrites (arrows) and into the dendritic field in the molecular layer (arrowheads). C, AC2 labeling in area CA3. D, AC4 labeling in area CA3. Note the concentration of labeling in the CA3 neuron dendrites in the stratum lucidum (the dendrites are viewed in the coronal plane in these tissue sections). The boxed areas in C and D show specific examples of the described labeling pattern. E, AC2 labeling in area CA1. F, AC4 labeling in area CA1. Note the accumulation of label in the CA1 cell bodies and along the membrane of the CA1 dendrites (arrows) in the stratum radiatum. HIL, Hilus; LUC, stratum lucidum; STR RAD, stratum radiatum. Scale bar, 10 µm.
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Figure 6. Top. Immunohistochemical detection of AC2 and AC4 in the mouse dentate gyrus. Mouse brain sections (40 µm) were processed for immunohistochemistry as described in Materials and Methods. A, AC2 labeling (red). B, MAP2 labeling of the same image shown in A (green). C, Merged image of A and B (yellow indicates coincident localization). D, AC4 labeling (red). E, MAP2 labeling of the same image shown in D (green). F, Merged image of D and E (yellow as described in C). C', F', Higher magnification images of the boxed areas in C and F, respectively. Scale bar, 50 µm. Boxed areas in A and B correspond to that in C. Boxed areas in D and E correspond to that in F.
Figure 7. Bottom. Immunohistochemical detection of AC2 and AC4 in the CA1 region of mouse hippocampus. Mouse brain sections (40 µm) were processed for immunohistochemistry as described in Materials and Methods. A, AC2 labeling (red). B, MAP2 labeling of the same image shown in A (green). C, Merged image of A and B (yellow indicates coincident localization). D, AC4 labeling (red). E, MAP2 labeling of the same image shown in D (green). F, Merged image of D and E (yellow as described in C). C', F', Higher magnification images of the boxed areas in C and F, respectively. Scale bar, 50 µm. Boxed areas in A and B correspond to that in C. Boxed areas in D and E correspond to that in F.
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Figure 8. Top. Immunohistochemical localization of AC2 and AC4 relative to synaptophysin in the mouse dentate gyrus. Mouse brain sections (40 µm) were processed for immunohistochemistry as described in Materials and Methods. A, AC2 labeling. B, Synaptophysin (Syn) labeling of the same image shown in A. Note that the labeling pattern for AC2 is not coincident with that for synaptophysin. C, Peptide adsorption of AC2 eliminating AC2 labeling in the dentate gyrus. D, AC4 labeling. E, Synaptophysin labeling of the same image shown in D. Note that the labeling pattern for AC4 is not coincident with that for synaptophysin. F, Peptide adsorption of AC4 eliminating AC4 labeling in the dentate gyrus. Scale bar, 50 µm.
Figure 9. Bottom. Immunohistochemical localization of AC2 and AC4 relative to synaptophysin in the CA1 region of the mouse hippocampus. Mouse brain sections (40 µm) were processed for immunohistochemistry as described in Materials and Methods. A, AC2 labeling. B, Synaptophysin labeling of the same image shown in A. Note that the labeling pattern for AC2 is not coincident with that for synaptophysin. C, Peptide adsorption of AC2 eliminating AC2 labeling in CA1. D, AC4 labeling. E, Synaptophysin labeling of the same image shown in D. Note that the labeling pattern for AC4 is not coincident with that for synaptophysin. F, Peptide adsorption of AC4 eliminating AC4 labeling in CA1. Scale bar, 50 µm.
In area CA3, labeling for both AC2 and AC4 was concentrated in short, coronally sectioned CA3 pyramidal cell dendrites throughout the stratum lucidum (Fig. 5C,D). Upon closer examination, the labeling was coincident with MAP2 signal (Fig. 10C,F,C',F'). Similar to the pattern observed in the dentate gyrus, the labeling pattern was not colocalized with synaptophysin signal (Fig. 11C,F,C',F'). In fact, the adenylyl cyclase label appeared in many instances to be surrounded by synaptophysin labeling, similar to what has been described (Lim et al., 1997
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Figure 10. Top. Immunohistochemical localization of AC2 and AC4 with MAP2 in the CA3 region of the mouse hippocampus. Mouse brain sections (40 µm) were processed for immunohistochemistry as described in Materials and Methods. A, AC2 labeling (red). B, MAP2 labeling of the same image shown in A (green). C, Merged image of A and B (yellow indicates coincident localization of AC2 and MAP2). D, AC4 labeling (red). E, MAP2 labeling of the same image shown in D (green). F, Merged image of D and E (yellow as described in C). C', F', Higher magnification images of the boxed areas in C and F, respectively. Scale bar, 50 µm. Boxed areas in A and B correspond to that in C. Boxed areas in D and E correspond to that in F.
Figure 11. Bottom. Immunohistochemical localization of AC2 and AC4 with synaptophysin in the CA3 region of mouse hippocampus. Mouse brain sections (40 µm) were processed for immunohistochemistry as described in Materials and Methods. A, AC2 labeling (red). B, Synaptophysin labeling of the same image shown in A (green). C, Merged image of A and B (note lack of yellow labeling, indicating a lack of coincident localization of AC2 and synaptophysin). D, AC4 labeling (red). E, Synaptophysin labeling of the same image shown in D (green). F, Merged image of D and E. C', F', Higher magnification images of the boxed areas in C and F, respectively. Scale bar, 50 µm. Boxed areas in A and B correspond to that in C. Boxed areas in D and E correspond to that in F.
In area CA1, immunofluorescence for both AC2 and AC4 was concentrated along the apical dendrites throughout the stratum radiatum, with some labeling in the CA1 pyramidal cell bodies as well (Fig. 5E,F). As with the labeling in the dentate gyrus and CA3, the signal was coincident with the signal for the postsynaptic marker MAP2 (Fig. 7C,F,C',F') and was not colocalized with labeling for synaptophysin (Fig. 9). Very little labeling was seen in the processes in the stratum oriens, although occasional small cell bodies did label for AC2 or AC4. The labeling for AC2 and AC4 throughout the hippocampal formation was completely blocked by adsorption with the appropriate peptide antigen (Figs. 8C,F, 9C,F). Double labeling for AC2 and AC4 in the same tissue sections using a combination of fluorophore-labeled IgG and Fab fragments demonstrated that AC2 and AC4 are expressed in a very similar manner in the mouse hippocampal formation (data not shown). The only consistent difference in labeling was a slightly higher level of AC2 over AC4 signal in the dentate gyrus granule somas (Fig. 5A,B).
Immunohistochemical labeling for AC4 was also observed in several forebrain regions. In the neocortex and piriform cortex, a moderate percentage of neurons were labeled and included pyramidal cell bodies and dendrites (data not shown). Discrete labeling of neurons in the septum, medial habenular nucleus, induseum griseum, and the paraventricular thalamic nucleus was also seen (data not shown).
DISCUSSION
Each of the adenylyl cyclase isoforms possesses distinct regulatory properties, and all of the cloned mammalian adenylyl cyclases are expressed in the brain. Therefore, cAMP levels in neurons can be modulated by a variety of signals, including receptor activation, changes in intracellular Ca2+, and activation of protein kinases (for review, see Sunahara et al., 1996
Whereas most mammalian tissues express adenylyl cyclase activity that is inhibited by activation of Gi-coupled receptors, in the brain and lung, Gi-coupled receptors actually potentiate Gs-stimulated cAMP production (Andrade, 1993
In this study, we demonstrated that AC4 is stimulated by Gs-coupled receptors in intact HEK 293 cells. This is consistent with previous in vitro data (Gao and Gilman, 1991
A second aspect of this study was directed at elucidating immunohistochemically the localization of AC2 and AC4 in the mouse hippocampal formation. To this end, we demonstrated that both AC2 and AC4 proteins are expressed in the hippocampus. The immunohistochemical labeling pattern for both AC2 and AC4 was for the most part indistinguishable and colocalized with that for MAP2, a dendritic and/or postsynaptic marker, in all regions of the hippocampal formation. In the dentate gyrus, labeling was found in the proximal dendrites and extended into the dendritic field in the molecular layer. In CA3, AC2 and AC4 immunolabeling was concentrated in the CA3 pyramidal cell dendrites throughout the stratum lucidum. In the CA1 region, AC2 and AC4 labeling was concentrated along the apical dendrites of the pyramidal cells and extended throughout the stratum radiatum. This postsynaptic pattern is similar to that described by Mons et al. (1995)
Possible roles for
A possible role for AC4 in synaptic plasticity in the hippocampus may be to integrate Gs and Gi inputs, perhaps as a complement to the signal generated by AC2. The presence of AC2 and AC4 in the hippocampus thus allows for flexibility in the generation of robust cAMP increases necessary for synaptic plasticity. In addition to relying on the Ca2+-stimulated adenylyl cyclases as a means of eliciting cAMP-mediated long-term adaptive changes, hippocampal neurons may have evolved with several distinct mechanisms for expression of synaptic plasticity. It is possible that there exist two (or more) parallel pathways involving cAMP that encode different forms of synaptic plasticity. With respect to the AC4 immunolabeling observed in the forebrain, it is intriguing that several of these areas are sites in which synaptic plasticity has been demonstrated (e.g., neocortex and piriform cortex) (Hasselmo and Barkai, 1995
In summary, these results demonstrate that AC4 can act as a coincidence detector of paired Gs and Gi inputs. The finding that both AC2 and AC4 proteins are expressed postsynaptically in hippocampus suggests that these adenylyl cyclase isoforms may play a role in certain forms of hippocampal synaptic plasticity.
FOOTNOTES Received July 22, 1998; revised Sept. 28, 1998; accepted Oct. 15, 1998.
This work was supported by National Institutes of Health Grant 20498 to D.R.S. We thank Drs. Karl Obrietan for providing thick tissue sections, Jean-Christophe Deloulme for aid with Western blot analysis, and Ruth Westenbroek for valuable discussions regarding the immunohistochemistry. We thank Lisa Prichard, Scott Wong, Kien Trinh, Jaime Athos, Bill Watt, and Drs. Guy Chan, Ulrika Lernmark, and Karl Obrietan for critical reading of this manuscript. Confocal imaging and analysis were performed at the William M. Keck Imaging Center (University of Washington, Seattle, WA).
Correspondence should be addressed to Dr. Daniel R. Storm, Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280.
Drs. Baker and Nielsen are coprimary authors.
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