Featured Review Cellscience Reviews Vol 2 No.1 ISSN 1742-8130

Ca²⁺-stimulated Adenylyl Cyclases and Hippocampal Neuroplasticity

Hongbing Wang^{1, 2} and Daniel R. Storm³

Introduction

Studies with invertebrates and vertebrates have established that the cAMP/PKA (cAMP-dependent protein kinase) signaling pathway regulates different forms of synaptic plasticity. Ca²⁺-stimulated adenylyl cyclases (AC) play a key role in regulating long-lasting synaptic changes; they integrate the two major second messengers, Ca²⁺ and cAMP. Among the identified ACs, type 1 and type 8 (AC1 and AC8) are the only Ca²⁺-stimulated ACs identified in the brain. In this review, we will describe some of the major findings for the role of AC1 and AC8 in hippocampal plasticity, including long-term potentiation (LTP) and long-term memory (LTM) formation.

Cyclic AMP signal transduction pathway and neuroplasticity

Extensive evidence indicates the essential role of cAMP signaling in regulating different forms of neuroplasticity, including learning and memory. In Aplysia, facilitation at the connection between the sensory neurons and their target cells (interneurons and motorneurons) provided a classical model for learning and memory. Ghiradi and colleagues demonstrated that inhibition of PKA by Rp-cAMPS blocked the presynaptic facilitation [1]. In contrast, an active form of PKA (catalytic subunit) was sufficient to support long-term facilitation [2]. The long-term facilitation depends on de novo gene transcription and new protein synthesis, and may be regulated by the cAMP responsive element binding protein (CREB) [3].

In Drosophila, learning and memory mutants were identified by the associative learning tests, in which flies learn to associate a particular odor with an electric shock. One mutant, Rutabaga, is deficient in Ca²⁺/CaM-sensitive AC activity. Another, the dunce mutation, is deficient in cAMP phosphodiesterase activity [4-7]. Both mutants did not learn to avoid the odor associated with the electric shock, even though they were normal at odor detection. This indicates that the level of cAMP may be critical for the signaling in learning and memory. Similar to Aplysia, long-term memory (LTM) in Drosophila requires new protein synthesis, and short-term memory (STM) does not. The protein synthesis and gene transcription may be mediated through cAMP responsive element (CRE), because the induction of a dominant negative CREB in Drosophila blocks long-term memory [8]. Conversely, the induction of an active isoform of dCREB2 enhances LTM [9].

In mammals, LTP was used as an electrophysiological and cellular model to study neuroplasticity [10]. Potentiated post-synaptic responses are observed after the application of multiple electric stimulations at high frequency [11]. Short-term potentiation (STP) or decremental LTP (D-LTP) does not require new protein synthesis, and normally lasts less than 2 hours. Long-lasting LTP or late-phase LTP (L-LTP) requires both translation and gene transcription, and lasts longer than 3 hours in vitro. Application of a membrane-permeable analog of cAMP (Sp-cAMPS) onto hippocampal slices induced L-LTP, and inhibition of PKA blocked L-LTP [12]. Moreover, stimuli that induce L-LTP lead to increase of cAMP level and PKA activation, as well as phosphorylation of several PKA substrates [13-15]. By over-expressing an inhibitory form of PKA regulatory subunit, R(AB), using transgenic techniques, Abel and colleagues generated mice with reduced PKA activity. The mutant mice are functionally deficient in L-LTP, spatial memory, and long-term contextual memory [16]. Consistently, CREB mutant mice are deficient in long-term memory [17].

AC1 and AC8 are the only Ca²⁺-stimulated adenylyl cyclases present in brain

Adenylyl cyclases (AC), the enzymes that convert intracellular ATP to cAMP, provide the principal regulation of cAMP signaling. They are membrane-bound proteins, and regulated by heterotrimeric G proteins, G-coupled receptors, Ca²⁺/calmodulin (CaM), and protein kinases. Using CaM-sepharose affinity chromatography, Westcott and colleagues separated and demonstrated two forms of ACs: one being sensitive to Ca²⁺/CaM, and the other being insensitive [18]. The development of forskolin affinity matrix greatly facilitated the purification of the enzymes [19]. This finally led to the molecular cloning of type 1 adenylyl cyclase (AC1), which is sensitive to Ca²⁺/CaM [20]. Based on the nucleotide sequence of AC1, other ACs were subsequently cloned by cDNA library screening and polymerase chain reaction techniques [21-28].

Because Ca²⁺ and cAMP are the two major second messengers regulating neural activity, it is hypothesized that the integration of the two signals by Ca²⁺-stimulated adenylyl cyclases plays fundamental role in neuroplasticity. Among all cloned ACs, type 1, type 3, and type 8, are stimulated by Ca²⁺/CaM. The Ca²⁺-stimulated AC1 is the only member of the AC family found exclusively in the nervous system. Its mRNA was found in specific brain areas, including hippocampus, neocortex, entorhinal cortex, cerebellum, olfactory cortex and pineal gland [29-32], strongly suggesting its function in learning and memory, circadian regulation, and motor coordination. Remarkable increases in AC1 activity can be stimulated by Ca²⁺ and CaM, both in vitro and in vivo, at nanomolar concentrations [33-35]. Further, a synergistic activation of AC1 by Ca²⁺ and Gs-coupled receptors was observed.

Stimulation of Ca²⁺ through CaM was also observed for AC8, though to a lesser extent. AC8 mRNA was detected in distinct regions of the brain, as well as in other tissues including lung and parotid gland [22, 36]. In contrast to AC1, AC8 activity is neither inhibited by Gi-coupled receptors, nor stimulated by Gs, suggesting that AC8 is likely a pure Ca²⁺ sensor [37, 38]. Another Ca²⁺-sensitive AC, AC3, is mainly expressed in the olfactory epithelium, suggesting its function in olfaction [21].

Studies using knockout technology provided direct evidence for the function of AC1 and AC8 in brain. AC1 knockout (AC1KO) and AC8 knockout (AC8KO) mice were generated by homologous recombination [39-41]. Ca²⁺-stimulated AC activity dropped roughly 60% in the hippocampus and 40% in the cerebellum of AC1KO mice. In AC8 KO mice, the Ca²⁺-stimulated activity decreased 30% and 50% in hippocampus and cerebellum, respectively. There was no Ca²⁺-stimulated AC activity in AC1/AC8 double knockout (DKO) mice [40], indicating that AC1 and AC8 are the only Ca²⁺-stimulated ACs present in the brain.

The function of AC1 and AC8 in hippocampal LTP

Since the functional enhancement of synaptic efficacy by neuronal activity was discovered in the early 1970s [42], this phenomenon (now called LTP) has been investigated extensively, and is considered to be the cellular mechanism underlying information storage [10]. After high frequency stimulation (HFS), the synaptic response (in some cases measured by the size of field potentials) is potentiated. Such potentiation can last several hours in vitro, and days, or even months in vivo. Hence, it is called long-term potentiation or LTP. Within the hippocampus, a brain region associated with learning and memory, Ca²⁺/cAMP-stimulation of mitogen-activated protein kinase (MAPK) and PKA signaling pathways is found to regulate either the induction or the maintenance of LTP [43-45].

To study the role of AC1 and AC8 in LTP, AC1KO and AC8KO mice were generated by specific gene targeting. LTP in the CA1 region of the hippocampus was measured by stimulating the Schaffer collateral pathway. Both AC1KO and AC8KO showed normal basal neural transmission. They also displayed normal short-term plasticity, as indicated by comparable paired-pulse facilitation (PPF) to wild type (WT) mice. Although L-LTP (induced by 4 trains of HFS) was normal in either AC1KO or AC8KO mice, the double knockout mice (DKO, mutants for both AC1 and AC8) showed significant deficits. This indicates an essential function of Ca²⁺-stimulated AC in the transcription-dependent form of LTP, and that either AC1 or AC8 alone is sufficient to support L-LTP [40]. Interestingly, D-LTP (induced by a single HFS) was impaired in AC1KO mice [46]. We also found that the rising phase of LTP was slower in the AC1KO mice [39]. These data suggest that AC1 may also be involved in the expression of LTP. The function of AC8 in D-LTP remains to be elucidated. Although cAMP has been implicated in regulating neuroplasticity in the dentate gyrus (DG) region of the hippocampus [47], DG LTP was normal in AC1KO [46] and AC8KO mice [48]. DG LTP properties in DKO mice has not as yet been determined.

Compared to LTP in CA1 and DG regions of the hippocampus, which depends upon the activation of NMDA receptors [10], mossy fiber/CA3 LTP (MF-LTP) is mechanistically different [49, 50]. Because MF-LTP can be induced without the postsynaptic activation of NMDA receptors [51, 52], it is proposed that presynaptic Ca²⁺ increases activate cAMP production and cause a persistent enhancement of glutamate release [53, 54]. This hypothesis is supported by the observation that the synaptic protein Rab3A, which is regulated by PKA through rabphilin and RIM, is required for mossy fiber LTP [55-58]. It has also been suggested that cAMP-dependent modification of hyperpolarization-dependent cation channels (Ih) causes long-term depolarization of Mossy Fiber terminals [59] (but see also [60]). Furthermore, mice mutants in Cβ1 and RIβ isoforms of PKA exhibited a loss in MF-LTP [61]. In addition, application of a PKA inhibitor impairs both early and late phases of MF-LTP, both of which are stimulated by PKA activators forskolin and Sp-cAMPS. However, post-synaptic mechanisms for mossy fiber LTP have also been suggested. For example, Yeckel and colleagues showed that mossy fiber LTP is prevented by postsynaptic injection of a peptide inhibitor of PKA [62].

We have measured MF-LTP in AC1KO, AC8KO and DKO mice. In contrast to CA1 LTP, MF-LTP requires Ca²⁺-stimulated AC activity from both AC1 and AC8. Significant deficits in MF-LTP were observed in both AC1KO [46] and AC8KO [48]. Interestingly, AC1KO, AC8KO and DKO showed comparable reduction in MF-LTP [48]. Although the basal neural transmission at the Mossy Fiber-CA3 synapses was normal in both AC1KO and AC8KO, the PPF was defective in AC8KO but not in AC1KO [48]. This is possibly due to the different sub-cellular localization of these two enzymes. AC8 is functioned expressed within excitatory synapses, whereas AC1 is not [48]. We are currently investigating the possibility that AC1 and AC8 differentially regulate MF plasticity at post-and pre-synaptic sites, respectively.

Table 1. Summary of the phenotypes of ACKO mice in hippocampal LTP.

The function of AC1 and AC8 in hippocampus-dependent LTM

As described earlier, cAMP and PKA activity regulate learning and memory formation in both invertebrates and vertebrates. We have applied a battery of behavioral tests to address the role of AC1 and AC8 in hippocampus-dependent memory formation. Spatial memory was tested using the Morris water maze. Mice were trained to navigate in a circular swimming pool, their memory task being to find the hidden platform to allow them to escape from the water. At the end of the training session, mice were subject to a probe test, in which the hidden platform was removed. If the mice learned to use the environmental cues to find the position of the platform and remembered, they would spend more time in the “target” area, which was the location of the hidden platform during training. AC1KO displayed normal learning with reduced escape latency during training. However, they spent significantly less time around the target area in the probe test [39], compared to the wild type mice.

AC1KO, AC8KO and DKO were also tested by other hippocampus-dependent learning paradigms, including contextual fear conditioning and passive avoidance. When trained for contextual fear conditioning, mice learn to associate the contextual cues with an aversive stimulus such as a mild foot shock. During training, mice were introduced to a plastic chamber. A mild electric foot shock (0.7mA for 2 seconds) was delivered 2 min later (Fig. 1A). When tested, mice were re-introduced to the same chamber and tested for their contextual memory. If mice learned to associate the contextual cues with the aversive stimulus (foot shock), they would exhibit fear response (Fig. 1B). The fear response was scored by the percentage of freezing, defined as immobility (Fig. 1B). When tested 1 day after training, AC1KO, AC8KO and DKO mice showed significant freezing. However, when tested 8 days after training, only AC1KO and AC8KO, but not DKO, showed retention for contextual memory[40]. Therefore, mice lacking both AC1 and AC8 are able to learn and exhibit short-term memory, but cannot form long-term memory in the contextual fear conditioning task. Similarly, LTM for passive avoidance was normal for both AC1KO and AC8KO mice. DKO mice learned the passive avoidance task, but showed no memory retention when tested 30min after training [40]. It is important to note that DKO mice showed normal LTM for cued fear conditioning, which depends upon the amygdala [40]. These data strongly support specific function of Ca²⁺-stimulated AC in hippocampus-dependent memory formation.

How do AC1 and AC8 regulate the long-lasting synaptic changes? The current hypothesis for the molecular mechanism underlying LTP and LTM suggests requirements for new translation and transcription. Ca²⁺-stimulated cAMP signaling may be required to activate CREB/CRE-mediated gene expression. The influx of Ca²⁺ upon neuronal activation stimulates both mitogen-activated kinase (MAPK) and Ca²⁺-stimulated ACs (AC1 and AC8). Increased levels of cAMP activate both MAPK and PKA [63]. Recently, it has been discovered that the MAPK pathway is obligatory for CRE-mediated transcription in both PC12 cells and in cultured hippocampal neurons [64, 65]. Sequential activation of MAPK and Rsk2 in the cascade results in phosphorylation and activation of CREB. The activation and nuclear translocation of MAPK requires PKA activity [65]. The formation of a complex of phosphorylated CREB with CREB-binding protein (CBP) interacts with the basal transcription machinery and leads to increased gene expression [66].

In addition to the extensive documented evidence for the activation of both cAMP and MAPK signaling pathways during long-term modifications of the nervous system, direct measurement of CRE-mediated gene expression has been conducted. Transgenic mice were constructed within our group using β-galactosidase (LacZ) as the reporter molecule. CRE sequences were incorporated 5’ to a minimum RSV promoter to control the expression of LacZ. The expression of LacZ was then used to evaluate levels of CRE-mediated transcription during LTP and LTM. HFS was applied to induce L-LTP in the CA1 region of the CRE-LacZ transgenic mice. The LacZ expression in the CA1 region was significantly increased in hippocampal slices stimulated by HFS, but not by low frequency stimulation (LFS) [67]. Consistently, the active phosphorylated form of CREB was also induced by HFS. Furthermore, the late phase of LTP and the induction of LacZ were both abolished by PKA inhibitors and by the MAPK inhibitor PD98059. CRE-mediated transcription was further addressed by subjecting CRE-LacZ mice to memory training. Specific regional induction of LacZ was observed for different training protocols. Hippocampus-dependent contextual fear conditioning and passive avoidance both stimulated LacZ expression in the hippocampus, whereas no LacZ expression was induced by the auditory cued fear conditioning, an amygdala-dependent learning paradigm [68]. We have also demonstrated that CRE-mediated transcription is required for contextual memory. CRE decoy oligonucleotides were injected into the dorsal hippocampus to compete for CREB with the endogenous CRE-containing promoters. Mice which received CRE decoy before contextual training showed no memory formation [69]. Further investigation is needed to determine whether CRE-mediated transcription is ablated in DKO mice.

Over-expression of AC1 enhances LTP and recognition memory

The strength of cAMP signaling could, in theory, be regulated by molecules with opposing enzymatic activities. For example, Gs stimulates AC activity, and Gi inhibits it. The activity of AC is also counter-balanced by phosphodiesterases (PDEs) which break down cAMP. Furthermore, the activation of protein phosphatase (such as protein phosphatase-1, PP1) may dephosphorylate PKA substrates. Therefore, in principal, it might be possible to enhance memory by shifting the balance between positive and negative regulation through pharmacological or genetic manipulations. We have focused on ACs, and tested the effects of enhanced AC activity upon memory formation.

To remove the negative constraints on AC activity, we injected pertussis toxin (ptx) into the dorsal hippocampus to suppress Gi activity, and tested hippocampus-dependent memory formation. Surprisingly, ptx-treated mice were impaired in both passive avoidance and contextual memory formation [70]. We further examined the specific function of Giα1 by using knockout mice. Giα1 protein expression is reduced and depleted in Giα1 heterozygous and homozygous mutant mice respectively. Consequently, AC activity was significantly higher in the mutant hippocampus. No compensatory effects were observed for Giα2, Giα3, Gsα, and Golf in the mutant hippocampus. Although CA1 LTP was greatly enhanced in Giα1 KO mice, impairments in passive avoidance and contextual memory were observed [70]. These data indicated that the general removal of tonic cAMP constraint might be detrimental to memory formation. It could be due to several reasons. First of all, Giα1 is broadly expressed in numerous regions of the brain, and other tissues outside the central nervous system. Secondly, Giα1 not only inhibits Ca²⁺-stimulated AC (AC1 but not AC8), but also inhibits other ACs. Thus the consequence of activating different isoforms of AC upon neuronal activity, which itself increases intracellular Ca²⁺ levels, may be lower in Giα1 KO mice. Consistently, the expression of a constitutively active isoform of Gsα and a mutation of PDE in flies disrupted associative learning [71, 72]. Interestingly, PDE inhibitors attenuated memory defects in aged animals [73].

We took another approach to increase cAMP signaling by over-expressing AC1, which is neuro-specific and stimulated by Ca²⁺. Transgenic AC1 was under the control of the αCaMKII promoter to achieve postnatal and forebrain specific expression. A FLAG tag was attached to the N-terminus of AC1 to trace gene integration and transgene expression. In AC1 transgenic mice (AC1TG), FLAG-AC1 mRNA was detected only in cortex and hippocampus, but not in cerebellum. AC1TG also exhibited higher cAMP and PKA activity in the hippocampus [74].

Because Ca²⁺-stimulated AC activity is crucial for different forms of LTP, we measured field potentials in the CA1 region by stimulating Schaffer collateral pathway. WT and AC1TG mice showed a similar input/output curve and PPF, indicating that the basal neural transmission and short-term plasticity were normal in mice over-expressing AC1. After 2 trains of HFS, both WT and AC1TG showed significant and sustained LTP, however, the degree of both post-tetanic potentiation (PTP) and LTP were higher in AC1TG. In WT mice, a single train of HFS usually induces D-LTP, which decays within one hour. When stimulated by the same protocol, AC1TG mice showed sustained LTP [74].

We found that the activity of both MAPK and CREB was elevated in the hippocampus of AC1TG mice, and examined several forms of hippocampus-dependent memory within these animals. Although we observed normal contextual memory, AC1TG showed enhanced memory retention for the object recognition test. During training, mice were presented with two objects of different shapes. When tested, one of the old objects was replaced by a novel object. If mice had memory retention for the old objects, they would show preference for the new object and spend more time with it. Both WT and AC1TG showed strong recognition memory one hour after training. However, after one day, only AC1TG, but not WT mice, showed memory retention for the old object [74].

Another important property of memory is the phenomenon of memory extinction. When mice were repeatedly subjected to the conditioned stimulus without pairing with the unconditioned stimulus, the originally formed memory will become extinct. In contrast to forgetting, memory extinction involves new learning and the active suppression of pre-formed memory. To examine contextual memory extinction, mice were trained on day 1, when a foot shock was delivered in the contextual chamber. Then the trained mice were repeatedly exposed to the contextual chamber without any associated shock. Memory extinction is reflected by the decrease in freezing. Compare to WT mice, AC1TG showed slower rate of extinction [74]. Although enhanced levels of cAMP and MAPK in AC1TG may cause defects in memory extinction, the slower rate of extinction may be due to a stronger initial memory formation.

Table 2. Summary of the phenotypes of ACKO and AC1TG in hippocampus-dependent LTM.

Summary

The combination of molecular, physiological and behavioral studies indicates a pivotal role of Ca²⁺ and cAMP within neuroplasticity, including learning and memory. Long-term changes in the nervous system require new protein synthesis and gene transcription. Ca²⁺-stimulated ACs function as integrators for Ca²⁺ and cAMP signaling, through which CRE-mediated gene expression is induced with increased neural activity. Furthermore, the MAPK and PKA pathways may interact with each other to finely tune neuroplasticity. By using AC1 and AC8 mutant mice as animal models, we have demonstrated the functional involvement of Ca²⁺-stimulated ACs in different aspects of synaptic plasticity. Thorough understanding of cAMP signaling pathways may lead to potential treatments to enhance learning and memory, and to ameliorate neuronal deficits associated with normal aging [73] and neurodegenerative diseases [75, 76].

Acknowledgements

We thank the members of the lab for their suggestions and comments on this review.

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