Type VI adenylyl cyclase negatively regulates GluN2B-mediated LTD and spatial reversal learning

The calcium-sensitive type VI adenylyl cyclase (AC6) is a membrane-bound adenylyl cyclase (AC) that converts ATP to cAMP under stimulation. It is a calcium-inhibited AC and integrates negative inputs from Ca2+ and multiple other signals to regulate the intracellular cAMP level. In the present study, we demonstrate that AC6 functions upstream of CREB and negatively controls neuronal plasticity in the hippocampus. Genetic removal of AC6 leads to cyclase-independent and N-terminus of AC6 (AC6N)-dependent elevation of CREB expression, and enhances the expression of GluN2B-containing NMDA receptors in hippocampal neurons. Consequently, GluN2B-dependent calcium signaling and excitatory postsynaptic current, long-term depression, and spatial reversal learning are enhanced in the hippocampus of AC6−/− mice without altering the gross anatomy of the brain. Together, our results suggest that AC6 negatively regulates neuronal plasticity by modulating the levels of CREB and GluN2B in the hippocampus.

The protein expression level was quantified and shown in (B). The data represent the means ± SEM of three independent experiments. Statistical analyses were conducted using Student's t-test. The data were presented as the means ± SEM for each group. *p<0.05, Student's t-test. Recording configuration (left panel): a stimulation electrode (stim) was placed in the stratum radiatum to activate the Schaffer collaterals; rec represents fEPSP recording.
Hippocampal Schaffer collateral-CA1 input/output curves (right panel) from AC6 +/+ 8 and AC6 -/mice were recorded prior to LTP induction. (B-D) Hippocampal Schaffer collateral-CA1 LTP of AC6 +/+ (n=8-25 slices from 4-13 mice) and AC6 -/mice (n=7-16 slices from 4-10 mice) was induced using (B) 2 trains of high-frequency stimulation (HFS; each train includes 100 Hz for 1 s, the train-to-train interval is 20 s), a single train of (C) HFS (100 Hz for 1 s) or (D) TBS (theta-burst stimulation, 5 bursts of 4 pulses at 100 Hz) and LTP was recorded for 1 h. The representative traces showed the fEPSP of AC6 +/+ (left) and AC6 -/-(right) mice before (black) and 1hr after (gray) the LTP induction. Scale bars: 0.2 mV, 10 ms. The summary and cumulative plot of LTP level are shown in the middle and right panels, respectively. The data were analyzed using Wilcoxon signed-rank test and presented as the means ± SEM for each group The subcellular localization of (A) AC1 and AC6, and (B) AC5 were detected using the indicated antibody. α-Tubulin, syntaxin 1, and PSD95 were used as the internal loading controls for the total, SYP/non-PSD, and PSD fractions, respectively. The protein expression level was quantified and shown in (C). The data represent the means ± SEM of three independent experiments. Statistical analyses were conducted using Student's t-test. ***p<0.001, versus control group.   Tables   Table S1. Related to Figure 4, the gene expression levels of NMDAR subunit, GluN2B and CREB were increased in the AC6 -/hippocampus without affecting AMPAR subunit, GluA1.

Gene
Gene name

Supplemental Experimental Procedures Brain tissue preparation and Nissl staining
The mice were initially anesthetized with 80 mg/kg sodium pentobarbital (i.p.) and subsequently perfused intracardially with 0.9% NaCl and 4% paraformaldehyde ( washed with distilled water, followed by a standard dehydrating procedure (85% EtOH à 95% EtOH à 100% EtOH à Xylene). The stained hippocampal sections were mounted with permanent mounting solution (Malinol; Mutoh Chemical Co., Tokyo, Japan). The images were acquired using a system microscope (BX51, Olympus Optical Co., Tokyo, Japan) equipped with a microscope digital camera system (DP70, Olympus Optical Co).

Cell culture and transfection
HEK-293T cells were cultured as previous described 1 . For primary hippocampal neurons, neurons were isolated from AC6 +/+ and AC6 -/mouse embryos at E18.5. The

Subcellular fractionation
Subcellular fractions of AC6 +/+ and AC6 -/mouse brains were obtained using biochemical fractionation techniques as previously described 3 , with slight modifications. All reagents/solutions (including H 2 O) contained protease inhibitors (cOmplete EDTA-free protease inhibitor cocktail, Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitors (PhosSTOP inhibitor cocktails, Roche Diagnostics). Briefly, the hippocampus was first homogenized in ice-cold 18 homogenization solution 1 (0.32 M sucrose, 4 mM HEPES, pH 7.4). Part of the hippocampus lysate was saved as the total lysate. The supernatant (S1) was collected after centrifuging the remaining hippocampus lysate at 800 x g at 4°C for 10 min. The pellet (P1) was resuspended in ice-cold homogenization solution 1 and centrifuged at 700 x g at 4°C for 10 min to collect the supernatant (S1'). The cytosolic fraction (S2) was obtained from centrifuging the combined supernatant (S1+S1') at 10,000 x g at

RNA extraction and RT-qPCR
RNA extraction was performed following the manufacture's protocol (Life Technologies). The hippocampus tissue (in glass Douncer grinder) or primary hippocampal neurons (in culture dish) were homogenized by using 1 ml TRIzol (Life Technologies). The homogeneous lysate was transferred to RNase-free microfuge tube and incubated at RT for 5 minutes. 0.2 ml Chloroform/Isoamyl was then added into the homogeneous lysate and mixed well by inverting the tube at RT for 3 minutes followed by centrifuged at 12,000 X g for 10 minutes. The supernatant was then transferred into a fresh RNase-free tube and added 0.5 ml isopropyl alcohol to precipitate RNA. After 10 minutes centrifugation at 12,000 X g, the supernatant was discarded the supernatant and pellet was washed twice with 0.5 ml 70% ethanol. The pellet was air-dried for 10 minutes and re-suspendeded in RNase-free distilled water.
The nucleic acid isolated was treated with DNase1 (Life Technologies) and RNA was precipitated following the manufacturer's protocol to remove genomic DNA contamination. The total RNA concentration and quality were measured by using NanoDrop spectrophotometer (ND-1000, Thermo Scientific). For complementary DNA (cDNA) synthesis, the total RNA was mixed with 10 mM dNTP and random primers followed by heating the mixture at 65 °C for 5 minutes and incubating on ice for at least 1 minute. After brief centrifugation, the 5X first-strand buffer, 0.1M DTT,  Table S3.

Motor function analyses
The motor functions of AC6 +/+ and AC6 -/mice were examined using the rotarod performance and locomotor activity tests. In rotarod performance, the motor coordination ability of each mouse was assessed using the rotarod apparatus (Ugo Basile, Comerio, Italy). Briefly, the average latency to fall from the rod (28 rpm) of each mouse was recorded for 2 min. Locomotor activity was recorded and analyzed in an activity chamber (Coulbourn Instruments, Allentown, PA, USA) equipped with 16 x 16 infrared sensors for 10 minutes. The horizontal and vertical exploration, which represent locomotion and rearing, respectively, of each mouse was recorded.

von Frey assay
One hour before the experiment, each animal (non-anesthetized) was placed in a small acrylic box (10 cm L x 6 cm W x 10.5 cm H) on wired mesh ( tungsten electrode) with increasing force a total of 5 times (each hindpaw) with a 3 min interval between tests. Data collected from left and right hindpaws were averaged together. The lowest force to evoke a withdrawal response is considered the pain threshold for the tested animal.

Anxiety-related behavior
The anxiety responses of AC6 +/+ and AC6 -/were assessed using an open field test and elevated plus maze (EPM) as previously described 4

Three-chamber sociability analyses
Sociability tests were performed as previously described 6 , with slight modifications.
For habituation, the mice were first placed into the middle compartment (12 cm x 25.4 cm) of a three-compartment chamber (52 cm L x 25.4 cm W x 23 cm H), isolating the isolated the right and left compartments (20 cm x 25.4 cm) with empty wire containment cups (12 cm diameter) in the middle using the removable walls for 5 min. For social affiliation analyses, one mouse (stranger 1) was placed inside a wire containment cup located in one compartment (left or right) of the three-compartment chamber, followed by removing the walls between the compartments. The mouse was allowed to access and explore each compartment for 10 min. For the social preference analysis, the three-compartment chamber was isolated and a second mouse (stranger 2) was placed inside the opposite compartment (right or left), and the walls between the compartments were removed. The exploring activity was recorded for 10 min. The duration spent by each mouse in each compartment was analyzed using Top Scan software (Clever Sys. Inc.).

Fear-conditioning
A fear conditioning chamber (Med Associates, Saint Albans, VT, USA) equipped with auditory cue and electric shock device was used for the fear conditioning test.
Briefly, the mice were placed and habituated in the conditioning chamber for 2 min.
The mice received a 30-second neutral conditioned stimulus (CS, white noise, 90 dB), followed by a 2-second noxious unconditioned stimulus (US, electric foot shock, 0.5 mA). After an inter-trial interval (2 min), mice received a second identical set of stimuli. To analyze the contextual memory, the mice were placed back into the conditioning chamber without a foot shock, and freezing behavior was recorded for 5 min. To analyze the cued memory, the mice were moved to a novel chamber without the steel rod for 3 min, followed by the administration of conditioning stimuli for 3 min. Separate groups of mice were used for contextual and cued memory analyses.
The freezing behavior of the mice was recorded and analyzed. Short-and long-term memories were analyzed at 1 and 24 hr, respectively, after the initial fear conditioning test session.

Morris water maze
The Morris water maze test was performed as previously described 7,8 , with modifications. Handling was performed daily (for 5 min) for five days prior to the experiment. Each mouse was subjected to a four-trial session for pre-training. Each mouse was placed on the visible platform for at least 15 sec, followed by a 60 sec swimming period. The mouse was released into the water at the water level and the releasing point of each trial was different. For spatial acquisition (training session), each mouse was trained four times per day with the hidden platform for five consecutive days. Before the first trial of the first session, each mouse was placed on the hidden platform for 15 sec. The mouse was subsequently released into the water for 60 sec and the escape latency was recorded. The recording was terminated when the mouse found the platform, where it would remain for 15 sec. To examine the reference memory, probe tests were performed on the third and fifth days of the training. The hidden platform was removed, and the mouse was allowed to swim in the pool for 60 sec. For the spatial reversal test, the hidden platform was relocated to the opposite quadrant. A new set of four trials was conducted each day for three additional days. The reversal probe trial was performed at the end of the reversal test.
The swimming path, speed, and time spent in different quadrants by the mice were analyzed using a video tracking system, TrackMot (Singa Technology, Taiwan).

Electrophysiology
The brains of AC6 +/+ and AC6 -/mice (3-6 months old) were removed and chilled in ice-cold modified oxygenated artificial cerebrospinal fluid (ACSF) (87 mM NaCl, 25 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 2.5 mM KCl, 10 mM glucose, 75 mM sucrose, 0.5 mM CaCl 2 , and 7 mM MgCl 2 ) after rapid decapitation. The mouse brains were was sectioned into transverse hippocampal slices with 250-350 µm thickness using a vibrating tissue slicer (DTK-1000; Dosaka, Kyoto, Japan) in ice-cold modified ACSF followed by incubation in the oxygenated (95% O 2 , 5% CO 2 ) solution at 34°C for 25 min and stored at room temperature until further use as previously described 9,10 . For LTD experiments, after measurement of the input-output curve, the stimulus 23 intensity was adjusted to evoke 40 -50% of the maximal fEPSP. After establishing a 15-min stable baseline (< 5% change), LTD was induced by applying low-frequency stimulation (LFS) or paired-pulse stimulation (PPS). LFS comprised 1800 pulses at 2 Hz (NMDAR-dependent LTD), whereas PPS comprised 900 pairs at 1 Hz with 50 ms (NMDAR-independent LTD) or 200 ms (NMDAR-dependent LTD) inter-pulse interval. The LTD level was determined from the averaged fEPSP slope recorded during 50-60 min after induction and normalized to the baseline. Ro25-6981 was bath applied at least 1 hr before LTD induction and throughout the entire experiment.   2058-2068 (2003)