Search

Glucagon-like peptide-1 (GLP-1) and its analogs are widely used for diabetes treatment. The paraventricular nucleus (PVN) is crucial for regulating cardiovascular activity. This study aims to determine the roles of GLP-1 and its receptors (GLP-1R) in the PVN in regulating sympathetic outflow and blood pressure. Experiments were carried out in male normotensive rats and spontaneously hypertensive rats (SHR). Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded. GLP-1 and GLP-1R expressions were present in the PVN. PVN microinjection of GLP-1R agonist recombinant human GLP-1 (rhGLP-1) or EX-4 increased RSNA and MAP, which were prevented by GLP-1R antagonist exendin 9-39 (EX9-39) or GLP-1R antagonist 1, superoxide scavenger tempol, antioxidant N-acetylcysteine, NADPH oxidase (NOX) inhibitor apocynin, adenylyl cyclase (AC) inhibitor SQ22536 or protein kinase A (PKA) inhibitor H89. PVNmicroinjection of rhGLP-1 increased superoxide production, NADPH oxidase activity, cAMP level, AC, and PKA activity, which were prevented by SQ22536 or H89. GLP-1 and GLP-1R were upregulated in the PVN of SHR. PVN microinjection of GLP-1 agonist increased RSNA and MAP in both WKY and SHR, but GLP-1 antagonists caused greater effects in reducing RSNA and MAP in SHR than in WKY. The increased superoxide production and NADPH oxidase activity in the PVN of SHR were augmented by GLP-1R agonists but attenuated by GLP-1R antagonists. These results indicate that activation of GLP-1R in the PVN increased sympathetic outflow and blood pressure via cAMP-PKA-mediated NADPH oxidase activation and subsequent superoxide production. GLP-1 and GLP-1R upregulation in the PVN partially contributes to sympathetic overactivity and hypertension. [ABSTRACT FROM AUTHOR]

Widespread release of norepinephrine (NE) throughout the forebrain fosters learning and memory via adrenergic receptor (AR) signaling, but the molecular mechanisms are largely unknown. The β² AR and its downstream effectors, the trimeric stimulatory Gs-protein, adenylyl cyclase (AC), and the cAMP-dependent protein kinase A (PKA), form a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. Phosphorylation of CaV1.2 by PKA on Ser1928 is required for the upregulation of Ca2+ influx on β2 AR stimulation and long-term potentiation induced by prolonged theta-tetanus (PTT-LTP) but not LTP induced by two 1-s-long 100-Hz tetani. However, the function of Ser1928 phosphorylation in vivo is unknown. Here, we show that S1928A knock-in (KI) mice of both sexes, which lack PTT-LTP, express deficiencies during initial consolidation of spatial memory. Especially striking is the effect of this mutation on cognitive flexibility as tested by reversal learning. Mechanistically, long-term depression (LTD) has been implicated in reversal learning. It is abrogated in male and female S1928A knock-in mice and by β2 AR antagonists and peptides that displace β2 AR from CaV1.2. This work identifies CaV1.2 as a critical molecular locus that regulates synaptic plasticity, spatial memory and its reversal, and LTD. [ABSTRACT FROM AUTHOR]

Stimulatory coupling of dopamine D1 (D1R) and adenosine A2A receptors (A2AR) to adenylyl cyclase within the striatum is mediated through a specific Gαolfβ2γ7 heterotrimer to ultimately modulate motor behaviors. To dissect the individual roles of the Gαolfβ2γ7 heterotrimer in different populations of medium spiny neurons (MSNs), we produced and characterized conditional mouse models, in which the Gng7 gene was deleted in either the D1R-or A2AR/D2R-expressing MSNs. We show that conditional loss of γ7 disrupts the cell type-specific assembly of the Gαolfβ2γ7 heterotrimer, thereby identifying its circumscribed roles acting downstream of either the D1Rs or A2ARs in coordinating motor behaviors, including in vivo responses to psychostimulants. We reveal that Gαolfβ2γ7/cAMP signal in D1R-MSNs does not impact spontaneous and amphetamine-induced locomotor behaviors in male and female mice, while its loss in A2AR/D2R-MSNs results in a hyperlocomotor phenotype and enhanced locomotor response to amphetamine. Additionally, Gαolfβ2γ7/cAMP signal in either D1R- or A2AR/D2R-expressing MSNs is not required for the activation of PKA signaling by amphetamine. Finally, we show that Gαolfβ2γ7 signaling acting downstream of D1Rs is selectively implicated in the acute locomotor-enhancing effects of morphine. Collectively, these results support the general notion that receptors use specific Gαβγ proteins to direct the fidelity of downstream signaling pathways and to elicit a diverse repertoire of cellular functions. Specifically, these findings highlight the critical role for the γ7 protein in determining the cellular level, and hence, the function of the Gαolfβ2γ7 heterotrimer in several disease states associated with dysfunctional striatal signaling. [ABSTRACT FROM AUTHOR]

Release of neuropeptides from dense core vesicles (DCVs) is essential for neuromodulation. Compared with the release of small neurotransmitters, much less is known about the mechanisms and proteins contributing to neuropeptide release. By optogenetics, behavioral analysis, electrophysiology, electron microscopy, and live imaging, we show that synapsin SNN-1 is required for cAMP-dependent neuropeptide release in Caenorhabditis elegans hermaphrodite cholinergic motor neurons. In synapsin mutants, behaviors induced by the photoactivated adenylyl cyclase bPAC, which we previously showed to depend on ACh and neuropeptides (Steuer Costa et al., 2017), are altered as in animals with reduced cAMP. Synapsin mutants have slight alterations in synaptic vesicle (SV) distribution; however, a defect in SV mobilization was apparent after channelrhodopsin-based photostimulation. DCVs were largely affected in snn-1 mutants: DCVs were; 30% reduced in synaptic terminals, and their contents not released following bPAC stimulation. Imaging axonal DCV trafficking, also in genome-engineered mutants in the serine-9 protein kinase A phosphorylation site, showed that synapsin captures DCVs at synapses, making them available for release. SNN-1 colocalized with immobile, captured DCVs. In synapsin deletion mutants, DCVs were more mobile and less likely to be caught at release sites, and in nonphosphorylatable SNN-1B(S9A) mutants, DCVs traffic less and accumulate, likely by enhanced SNN-1 dependent tethering. Our work establishes synapsin as a key mediator of neuropeptide release. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR2) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR2-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared with matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR2 in NaV1.8-positive neurons (Par2Nav1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. Par2Nav1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR2-dependent hyperexcitability of trigeminal neurons from WT female mice. Par2 deletion, LI-1, and inhibitors of adenylyl cyclase or protein kinase A (PKA) prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N terminus of PAR2 at Asn30↓Arg31, proximal to the canonical trypsin activation site. Lgmn activated PAR2 by biased mechanisms in HEK293 cells to induce Ca2+ mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not β-arrestin recruitment or PAR2 endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR2 by biased mechanisms that evoke cancer pain. [ABSTRACT FROM AUTHOR]

Chronic pain caused by spinal cord injury (SCI) is notoriously resistant to treatment, particularly by opioids. After SCI, DRG •neurons show hyperactivity and chronic depolarization of resting membrane potential (RMP) that is maintained by cAMP signaling through PKA and EPAC. Importantly, SCI also reduces the negative regulation by Gxi of adenylyl cyclase and its production of cAMP, independent of alterations in G protein-coupled receptors and/or G proteins. Opioid reduction of pain depends on coupling of opioid receptors to Gai/o family members. Combining high-content imaging and cluster analysis, we show that in male rats SCI decreases opioid responsiveness in vitro within a specific subset of small-diameter nociceptors that bind isolectin B4. This SCI effect is mimicked in nociceptors from naive animals by a modest 5 min depolarization of RMP (15 mM K+; - 4 5 mV), reducing inhibition of cAMP signaling by -opioid receptor agonists DAMGO and morphine. Disinhibition and activation of C-Raf by depolarization-dependent phosphorylation are central to these effects. Expression of an activated C-Raf reduces sensitivity of adenylyl cyclase to opioids in nonexcitable HEK293 cells, whereas inhibition of CRaf or treatment with the hyperpolarizing drug retigabine restores opioid responsiveness and blocks spontaneous activity of nociceptors after SCI. Inhibition of ERK downstream of C-Raf also blocks SCI-induced hyperexcitability and depolarization, without direct effects on opioid responsiveness. Thus, depolarization-dependent C-Raf and downstream ERK activity maintain a depolarized RMP and nociceptor hyperactivity after SCI, providing a self-reinforcing mechanism to persistently promote nociceptor hyperexcitability and limit the therapeutic effectiveness of opioids. [ABSTRACT FROM AUTHOR]

In Drosophila, the mushroom bodies (MB) constitute the central brain structure for olfactory associative memory. As in mammals, the cAMP/PKA pathway plays a key role in memory formation. In the MB, Rutabaga (Rut) adenylate cyclase acts as a coincidence detector during associative conditioning to integrate calcium influx resulting from acetylcholine stimulation and G-protein activation resulting from dopaminergic stimulation. Amnesiac encodes a secreted neuropeptide required in the MB for two phases of aversive olfactory memory. Previous sequence analysis has revealed strong homology with the mammalian pituitary adenylate cyclase-activating peptide (PACAP). Here, we examined whether amnesiac is involved in cAMP/PKA dynamics in response to dopamine and acetylcholine co-stimulation in living flies. Experiments were conducted with both sexes, or with either sex. Our data show that amnesiac is necessary for the PKA activation process that results from coincidence detection in the MB. Since PACAP peptide is cleaved by the human membrane neprilysin hNEP, we searched for an interaction between Amnesiac and Neprilysin 1 (Nepl), a fly neprilysin involved in memory. We show that when Nepl expression is acutely knocked down in adult MB, memory deficits displayed by amn hypomorphic mutants are rescued. Consistently, Nepl inhibition also restores normal PKA activation in amn mutant flies. Taken together, the results suggest that Nepl targets Amnesiac degradation to terminate its signaling function. Our work thus highlights a key role for Amnesiac in establishing within the MB the PKA dynamics that sustain middle-term memory (MTM) formation, a function modulated by Nepl. [ABSTRACT FROM AUTHOR]

Cytoskeletal proteins and post-translational modifications play a role in mood disorders. Post-translational modifications of tubulin also alter microtubule dynamics. Furthermore, tubulin interacts closely with Gas, the G-protein responsible for activation of adenylyl cyclase. Postmortem tissue derived from depressed suicide brain showed increased Gas in lipid-raft domains compared with normal subjects. Gas, when ensconced in lipid rafts, couples less effectively with adenylyl cyclase to produce cAMP, and this is reversed by antidepressant treatment. A recent in vitro study demonstrated that tubulin anchors Gas to lipid rafts and that increased tubulin acetylation (due to F1DAC6 inhibition) and antidepressant treatment decreased the proportion of Gas complexed with tubulin. This suggested that deacetylated-tubulin might be more prevalent in depression. This study examined tubulin acetylation in whole-tissue homogenate, plasma membrane, and lipid-raft membrane domains in tissue from normal control subjects, depressed suicides, and depressed nonsuicides (human males/females). While tissue homogenate showed no changes in tubulin acetylation between control, depressed suicides, and depressed nonsuicides, plasma membrane-associated tubulin showed significant decreases in acetylation from depressed suicides and depressed nonsuicides compared with controls. No change was seen in expression of the enzymes responsible for tubulin acetylation or deacetylation. These data suggest that, during depression, membrane-localized tubulin maintains a lower acetylation state, permitting increased sequestration of Gxs in lipid-raft domains, where it is less likely to couple to adenylyl cyclase for cAMP production. Thus, membrane tubulin may play a role in mood disorders, which could be exploited for diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Stress alters brain function by modifying the structure and function of neurons and astrocytes. The fine processes of astrocytes are critical for the clearance of neurotransmitters during synaptic transmission. Thus, experience-dependent remodeling of glial processes is anticipated to alter the output of neural circuits. However, the molecular mechanisms that underlie glial structural plasticity are not known. Here we show that a single exposure of male and female mice to an acute stress produced a long-lasting retraction of the lateral processes of cerebellar Bergmann glial cells. These cells express the GluA1 subunit of AMPA-type glutamate receptors, and GluA1 knockdown is known to shorten the length of glial processes. We found that stress reduced the level of GluA1 protein and AMPA receptor-mediated currents in Bergmann glial cells, and these effects were absent in mice devoid of CPEB3, a protein that binds to GluA1 mRNA and regulates GluA1 protein synthesis. Administration of a β-adrenergic receptor blocker attenuated the reduction in GluA1, and deletion of adenylate cyclase 5 prevented GluA1 suppression. Therefore, stress suppresses GluA1 protein synthesis via an adrenergic/adenylyl cyclase/CPEB3 pathway, and reduces the length of astrocyte lateral processes. Our results identify a novel mechanism for GluA1 subunit plasticity in non-neuronal cells and suggest a previously unappreciated role for AMPA receptors in stress-induced astrocytic remodeling. [ABSTRACT FROM AUTHOR]

Little is known about intracellular signaling mechanisms that persistently excite neurons in pain pathways. Persistent spontaneous activity (SA) generated in the cell bodies of primary nociceptors within dorsal root ganglia (DRG) has been found to make major contributions to chronic pain in a rat model of spinal cord injury (SCI) (Bedi et al., 2010; Yang et al., 2014). The occurrence of SCI-induced SA in a large fraction of DRG neurons and the persistence of this SA long after dissociation of the neurons provide an opportunity to define intrinsic cell signaling mechanisms that chronically drive SA in pain pathways. The present study demonstrates that SCI-induced SA requires continuing activity of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA), as well as a scaffolded complex containing AC5/6, A-kinase anchoring protein 150 (AKAP150), and PKA. SCI caused a small but significant increase in the expression of AKAP150 but not other AKAPs. DRG membranes isolated from SCI animals revealed a novel alteration in the regulation of AC. AC activity stimulated by Ca2+-calmodulin increased, while the inhibition of AC activity by Gαi showed an unexpected and dramatic decrease after SCI. Localized enhancement of the activity of AC within scaffolded complexes containing PKA is likely to contribute to chronic pathophysiological consequences of SCI, including pain, that are promoted by persistent hyperactivity in DRG neurons. [ABSTRACT FROM AUTHOR]

Transient changes in striatal dopamine (DA) concentration are considered to encode a reward prediction error (RPE) in reinforcement learning tasks. Often, a phasic DA change occurs concomitantly with a dip in striatal acetylcholine (ACh), whereas other neuromodulators, such as adenosine (Adn), change slowly. There are abundant adenylyl cyclase (AC) coupled GPCRs for these neuromodulators in striatal medium spiny neurons (MSNs), which play important roles in plasticity. However, little is known about the interaction between these neuromodulators via GPCRs. The interaction between these transient neuromodulator changes and the effect on cAMP/PKA signaling via Golf- and Gi/o-coupled GPCR are studied here using quantitative kinetic modeling. The simulations suggest that, under basal conditions, cAMP/PKA signaling could be significantly inhibited in D1R+ MSNs via ACh/M4R/Gi/o and an ACh dip is required to gate a subset of D1R/Golf-dependent PKA activation. Furthermore, the interaction between ACh dip and DA peak, via D1R and M4R, is synergistic. In a similar fashion, PKA signaling in D2+ MSNs is under basal inhibition via D2R/Gi/o and a DA dip leads to a PKA increase by disinhibiting A2aR/Golf , but D2+ MSNs could also respond to the DA peak via other intracellular pathways. This study highlights the similarity between the two types of MSNs in terms of high basal AC inhibition by Gi/o and the importance of interactions between Gi/o and Golf signaling, but at the same time predicts differences between them with regard to the sign of RPE responsible for PKA activation. [ABSTRACT FROM AUTHOR]

The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1 + mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1 + mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptor (GPCR)-mediated presynaptic inhibition is a fundamental mechanism regulating synaptic transmission in the CNS. The classical GPCR-mediated presynaptic inhibition in the CNS is produced by direct interactions between the Gβγ subunits of the G-protein and presynaptic Ca2+ channels, K+ channels, or synaptic proteins that affect transmitter release. This mode of action is shared by well known GPCRs such as the α2, GABAB, and CB1 receptors. We report that the α2 receptor-mediated inhibition of presynaptic Ca2+ channel and transmitter release in rat retinal rod bipolar cells depends on the Gα subunit via a Ga-adenylate cyclase- cAMP cascade and requires participation of the type 4 phosphodiesterase (PDE4), a new role for phosphodiesterase in neural signaling. By using the Gα instead of the Gβγ subunits, this mechanism is able to use a cyclase/PDE enzyme pair to dynamically control a cyclic nucleotide second messenger (i.e., cAMP) for the regulation of synaptic transmission, an operating strategy that shows remarkable similarity to that of dynamic control of cGMP and transmitter release from photoreceptors by the guanylate cyclase/PDE6 pair in phototransduction. Our results demonstrate a new paradigm of GPCR-mediated presynaptic inhibition in the CNS and add a new regulatory mechanism at a critical presynaptic site in the visual pathway that controls the transmission of scotopic information. They also provide a presynaptic mechanism that could contribute to neuroprotection of retinal ganglion cells by α2 agonists, such as brimonidine, in animal models of glaucoma and retinal ischemia and in glaucoma patients. [ABSTRACT FROM AUTHOR]

Neurons in the CNS do not regenerate following injury; regeneration is blocked by inhibitory proteins in myelin, such as myelin-associated glycoprotein (MAG). Elevating neuronal levels of the second messenger cAMP overcomes this blocked axonal outgrowth. One way to elevate cAMP is pretreating neurons with neurotrophins, such as brain-derived neurotrophic factor (BDNF). However, pleiotropic effects and poor bioavailability make exogenous administration of neurotrophins in vivo problematic; therefore, alternative targets must be considered. In neurons, two families of adenylyl cyclases synthesize cAMP, transmembrane adenylyl cyclases (tmACs), and soluble adenylyl cyclase (sAC). Here, we demonstrate that sAC is the essential source of cAMP for BDNF to overcome MAG-dependent inhibition of neurite outgrowth. Elevating sAC in rat and mouse neurons is sufficient to induce neurite outgrowth on myelin in vitro and promotes regeneration in vivo. These results suggest that stimulators of sAC might represent a novel therapeutic strategy to promote axonal growth and regeneration. [ABSTRACT FROM AUTHOR]

Centrins are ancient calmodulin-related Ca2+-binding proteins associated with basal bodies. In lower eukaryotes, Centrin2 (CETN2) is required for basal body replication and positioning, although its function in mammals is undefined. We generated a germline CETN2 knock-out (KO) mouse presenting with syndromic ciliopathy including dysosmia and hydrocephalus. Absence of CETN2 leads to olfactory cilia loss, impaired ciliary trafficking of olfactory signaling proteins, adenylate cyclase III (ACIII), and cyclic nucleotide-gated (CNG) channel, as well as disrupted basal body apical migration in postnatal olfactory sensory neurons (OSNs). In mutant OSNs, cilia baseanchoring of intraflagellar transport components IFT88, the kinesin-II subunit KIF3A, and cytoplasmic dynein 2 appeared compromised. Although the densities of mutant ependymal and respiratory cilia were largely normal, the planar polarity of mutant ependymal cilia was disrupted, resulting in uncoordinated flow of CSF. Transgenic expression of GFP-CETN2 rescued the Cetn2-deficiency phenotype. These results indicate that mammalian basal body replication and ciliogenesis occur independently of CETN2; however, mouse CETN2 regulates protein trafficking of olfactory cilia and participates in specifying planar polarity of ependymal cilia. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β1 (TGF-β1) protects against neuroinflammatory events underlying neuropathic pain. TGF-β signaling enhancement is a phenotypic characteristic of mice lacking the TGF-β pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which leads to an increased synaptic release of opioid peptides and to a naloxone-reversible hypoalgesic/antiallodynic phenotype. Herein, we investigated the following: (1) the effects of BAMBI deficiency on opioid receptor expression, functional efficacy, and analgesic responses to endogenous and exogenous opioids; and (2) the involvement of the opioid system in the antiallodynic effect of TGF-β1. BAMBI-KO mice were subjected to neuropathic pain by sciatic nerve crash injury (SNI). Gene (PCR) and protein (Western blot) expressions of θ- and δ-opioid receptors were determined in the spinal cord. The inhibitory effects of agonists on the adenylyl cyclase pathway were investigated. Two weeks after SNI, wild-type mice developed mechanical allodynia and the functionality of ü-opioid receptors was reduced. By this time, BAMBI-KO mice were protected against allodynia and exhibited increased expression and function of opioid receptors. Four weeks after SNI, when mice of both genotypes had developed neuropathic pain, the analgesic responses induced by morphine and RB101 (an inhibitor of enkephalin-degrading enzymes, which increases the synaptic levels of enkephalins) were enhanced in BAMBI-KO mice. Similar results were obtained in the formalin-induced chemical-inflammatory pain model. Subcutaneous TGF-β1 infusion prevented pain development after SNI. The antiallodynic effect of TGF-β1 was naloxone-sensitive. In conclusion, modulation of the endogenous opioid system by TGF-β signaling improves the analgesic effectiveness of exogenous and endogenous opioids under pathological pain conditions. [ABSTRACT FROM AUTHOR]

Animals modulate their arousal state to ensure that their sensory responsiveness and locomotor activity match environmental demands. Neuropeptides can regulate arousal, but studies of their roles in vertebrates have been constrained by the vast array of neuropeptides and their pleiotropic effects. To overcome these limitations, we systematically dissected the neuropeptidergic modulation of arousal in larval zebrafish. We quantified spontaneous locomotor activity and responsiveness to sensory stimuli after genetically induced expression of seven evolutionarily conserved neuropeptides, including adenylate cyclase activating polypeptide 1b (adcyap1b), cocaine-related and amphetamine-related transcript (cart), cholecystokinin (cck), calcitonin gene-related peptide (cgrp), galanin, hypocretin, and nociceptin. Our study reveals that arousal behaviors are dissociable: neuropeptide expression uncoupled spontaneous activity from sensory responsiveness, and uncovered modality-specific effects upon sensory responsiveness. Principal components analysis and phenotypic clustering revealed both shared and divergent features of neuropeptidergic functions: hypocretin and cgrp stimulated spontaneous locomotor activity, whereas galanin and nociceptin attenuated these behaviors. In contrast, cart and adcyap1b enhanced sensory responsiveness yet had minimal impacts on spontaneous activity, and cck expression induced the opposite effects. Furthermore, hypocretin and nociceptin induced modality-specific differences in responsiveness to changes in illumination. Our study provides the first systematic and high-throughput analysis of neuropeptidergic modulation of arousal, demonstrates that arousal can be partitioned into independent behavioral components, and reveals novel and conserved functions of neuropeptides in regulating arousal. [ABSTRACT FROM AUTHOR]

During corticogenesis, pituitary adenylate cyclase-activating polypeptide (PACAP; ADCYAP1) may contribute to proliferation control by activating PAC1 receptors of neural precursors in the embryonic ventricular zone. PAC1 receptors, specifically the hop and short iso-forms, couple differentially to and activate distinct pathways that produce pro- or anti-mitogenic actions. Previously, we found that PACAP was an anti-mitogenic signal from embryonic day 13.5 (E13.5) onward both in culture and in vivo and activated cAMP signaling through the short isoform. However, we now find that mice deficient in PACAP exhibited a decrease in the BrdU labeling index (LI) in E9.5 cortex, suggesting that PACAP normally promotes proliferation at this stage. To further define mechanisms, we established a novel culture model in which the viability of very early cortical precursors (E9.5 mouse and E10.5 rat) could be maintained. At this stage, we found that PACAP evoked intracellular calcium fluxes and increased phospho-PKC levels, as well as stimulated G1 cyclin mRNAs and proteins, S-phase entry, and proliferation without affecting cell survival. Significantly, expression of hop receptor isoform was 24-fold greater than the short isoform at E10.5, a ratio that was reversed at E14.5 when short expression was 15-fold greater and PACAP inhibited mitogenesis. Enhanced hop isoform expression, elicited by in vitro treatment of E10.5 precursors with retinoic acid, correlated with sustained pro-mitogenic action of PACAP beyond the developmental switch. Conversely, depletion of hop receptor using short-hairpin RNA abolished PACAP mitogenic stimulation at E10.5. These observations suggest that PACAP elicits temporally specific effects on cortical proliferation via developmentally regulated expression of specific receptor isoforms. [ABSTRACT FROM AUTHOR]

Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell- cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibit adenylyl cyclase (AC) and reduce PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to coregulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod-cone gap junctions but possibly including some rod-rod and cone-cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately downregulated expression of D4R and AC type 1 mRNA. We conclude that adenosine and dopamine coregulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function. [ABSTRACT FROM AUTHOR]

The formation of primary cilia is a highly choreographed process that can be disrupted in developing neurons by overexpressing neuromodulatory G-protein-coupled receptors GPCRs or by blocking intraflagellar transport. Here, we examined the effects of overexpressing the ciliary GPCRs, 5HT6 and SSTR3, on cilia structure and the differentiation of neocortical neurons. Neuronal overexpression of 5HT6 and SSTR3 was achieved by electroporating mouse embryo cortex in utero with vectors encoding these receptors. We found that overexpression of ciliary GPCRs in cortical neurons, especially 5HT6, induced the formation of long (>30 μm) and often forked cilia. These changes were associated with increased levels of intraflagellar transport proteins and accelerated ciliogenesis in neonatal neocortex, the induction of which required Kif3a, an anterograde motor critical for cilia protein trafficking and growth. GPCR overexpression also altered the complement of signaling molecules within the cilia. We found that SSTR3 and type III adenylyl cyclase (ACIII), proteins normally enriched in neuronal cilia, were rarely detected in 5HT6-elongated cilia. Intriguingly, the changes in cilia structure were accompanied by changes in neuronal morphology. Specifically, disruption of normal ciliogenesis in developing neocortical neurons, either by overexpressing cilia GPCRs or a dominant-negative form of Kif3a, significantly impaired dendrite outgrowth. Remarkably, coexpression of ACIII with 5HT6 restored ACIII to cilia, normalized cilia structure, and restored dendrite outgrowth, effects that were not observed in neurons coexpressing ACIII and dominant-negative form of Kif3a. Collectively, our data suggest the formation of neuronal dendrites in developing neocortex requires structurally normal cilia enriched with ACIII. [ABSTRACT FROM AUTHOR]

Cilia of olfactory sensory neurons are the primary sensory organelles for olfaction. The detection of odorants by the main olfactory epithelium (MOE) depends on coupling of odorant receptors to the type 3 adenylyl cyclase (AC3) in olfactory cilia. We monitored the effect of airflow on electro-olfactogram (EOG) responses and found that the MOE of mice can sense mechanical forces generated by airflow. The airflow-sensitive EOG response in the MOE was attenuated when cAMP was increased by odorants or by forskolin suggesting a common mechanism for airflow and odorant detection. In addition, the sensitivity to airflow was significantly impaired in the MOE from AC3-/- mice. We conclude that AC3 in the MOE is required for detecting the mechanical force of airflow, which in turn may regulate odorant perception during sniffing. [ABSTRACT FROM AUTHOR]

Feedback inhibition of adenylyl cyclase III (ACIII) via Ca2+-induced phosphorylation has long been hypothesized to contribute to response termination and adaptation of olfactory sensory neurons (OSNs). To directly determine the functional significance of this feedback mechanism for olfaction in vivo, we genetically mutated serine1076 of ACIII, the only residue responsible for Ca2+-induced phosphorylation and inhibition of ACIII (Wei et al., 1996,1998), to alanine in mice. Immunohistochemistry and Western blot analysis showed that the mutation affects neither the cilial localization nor the expression level of ACIII in OSNs. Electroolfactogram analysis showed no differences in the responses between wild-type and mutant mice to single-pulse odorant stimulations or in several stimulation paradigms for adaptation. These results suggest that phosphorylation of ACIII on serine1076 plays a far less important role in olfactory response attenuation than previously thought. [ABSTRACT FROM AUTHOR]

cAMP is a critical second messenger mediating activity-dependent neuronal survival and neurite growth. We investigated the expression and function of the soluble adenylyl cyclase (sAC, ADCY10) in CNS retinal ganglion cells (RGCs). We found sAC protein expressed in multiple RGC compartments including the nucleus, cytoplasm and axons. sAC activation increased cAMP above the level seen with transmembrane adenylate cyclase (tmAC) activation. Electrical activity and bicarbonate, both physiologic sAC activators, significantly increased survival and axon growth, whereas pharmacologic or siRNA-mediated sAC inhibition dramatically decreasedRGCsurvival and axon growth in vitro, and survival in vivo. Conversely, RGC survival and axon growth were unaltered in RGCs from AC1/AC8 double knock-out mice or after specifically inhibiting tmACs. These data identify a novel sAC-mediatedcAMPsignaling pathway regulating RGC survival and axon growth, and suggest new neuroprotective or regenerative strategies based on sAC modulation. [ABSTRACT FROM AUTHOR]

Consolidation of hippocampus-dependent memory is dependent on activation of the cAMP/Erk/MAPK (mitogen-activated protein kinase) signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation ofMAPKin the hippocampus during the circadian cycle.A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependentmemoryas well as adenylyl cyclase andMAPKactivities. Electrolytic lesion of the SCN2 d after training for contextual fear memory reduced contextual memory measured 2 weeks after training, indicating that maintenance of contextual memory depends on the SCN.Spatialmemorywasalsocompromisedin SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclaseandMAPKactivities inthehippocampuswasdestroyedbylesioning oftheSCN.Thesedatasuggestthathippocampus-dependentlong-termmemoryisdependenton the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus. [ABSTRACT FROM AUTHOR]

When certain memory becomes obsolete, effective suppression of the previously established memory is essential for animals to adapt to the changing environment. At the cellular level, reversal of synaptic potentiation may be important for neurons to acquire new information and to prevent synaptic saturation. Here, we investigated the function of Ca2+-stimulated cAMP signaling in the regulation of bidirectional synaptic plasticity and spatial memory formation in double knock-out mice (DKO) lacking both type 1 and 8 adenylyl cyclases (ACs). In anesthetized animals, the DKO mutants showed defective long-term potentiation (LTP) after a single high-frequency stimulation (HFS) or two spaced HFSs at 100 Hz. However, DKO mice showed normal LTP after a single HFS at 200 Hz or two compressed HFSs at 100 Hz. Interestingly, reversal of synaptic potentiation as well as de novo synaptic depression was impaired in DKO mice. In the Morris water maze, DKO mice showed defective acquisition and memory retention, although the deficits could be attenuated by overtraining or compressed trainings with a shorter intertrial interval. In the reversal platform test, DKO animals were impaired in both relearning and old memory suppression. Furthermore, the extinction of the old spatial memory was not efficient in DKO mice. These data demonstrate that Ca2+-stimulated AC activity is important not only for LTP and spatial memory formation but also for the suppression of both previously established synaptic potentiation and old spatial memory. [ABSTRACT FROM AUTHOR]

In the olfactory epithelium (OE), odorant receptor stimulation generates cAMP signals that function in both odor detection and the regulation of axon guidance molecule expression. The enzyme that synthesizes cAMP, adenylyl cyclase 3 (AC3), is coexpressed in olfactory sensory neurons (OSNs) with poly-N-acetyllactosamine (PLN) oligosaccharides determined by the glycosyltransferase 3GnT2. The loss of either enzyme results in similar defects in olfactory bulb (OB) innervation andOSNsurvival, suggesting that glycosylation may be important for AC3 function. We show here that AC3 is extensively modified with N-linked PLN, which is essential for AC3 activity and localization.OnWestern blots,AC3from the wild-typeOEmigrates diffusely as a heavily glycosylated 200 kDa band that interacts with the PLN-binding lectin LEA. AC3 from the 3GnT2-/- OE loses these PLN modifications, migrating instead as a 140 kDa glycoprotein. Furthermore, basal and forskolin-stimulated cAMP production is reduced 80-90% in the 3GnT2-/- OE. Although AC3 traffics normally to null OSN cilia, it is absent from axon projections that aberrantly target the OB. The cAMP-dependent guidance receptor neuropilin-1 is also lost from 3GnT2-/- OSNs and axons, while semaphorin-3A ligand expression is upregulated. In addition, kirrel2, a mosaically expressed adhesion molecule that functions in axon sorting, is absent from 3GnT2-/- OB projections. These results demonstrate that PLN glycans are essential in OSNs for proper AC3 localization and function. We propose that the loss of cAMPdependent guidance cues is also a critical factor in the severe axon guidance defects observed in 3GnT2-/- mice. [ABSTRACT FROM AUTHOR]

Although primary cilia are found on neurons throughout the brain, their physiological function remains elusive. Human ciliopathies are associated with cognition defects, and transgenic mice lacking proteins expressed in primary cilia exhibit defects in learning and memory. Recently, it was reported that mice lacking the G-protein-coupling receptor somatostatin receptor-3 (SSTR3), a protein expressed predominately in the primary cilia of neurons, have defective memory for novel object recognition and lower cAMP levels in the brain. Since SSTR3 is coupled to regulation of adenylyl cyclase, this suggests that adenylyl cyclase activity in primary cilia of CNS neurons may be critical for some forms of learning and memory. Because the type 3 adenylyl cyclase (AC3) is expressed in primary cilia of hippocampal neurons, we examined AC3-/- mice for several forms of learning and memory. Here, we report that AC3-/- mice show no short-term memory for novel objects and fail to exhibit extinction of contextual fear conditioning. They also show impaired learning and memory for temporally dissociative passive avoidance. Since AC3 is exclusively expressed in primary cilia, we conclude that cAMP signals generated within primary cilia contribute to some forms of learning and memory, including extinction of contextual fear conditioning. [ABSTRACT FROM AUTHOR]

The zebrafish camouflage response is an innate "hard-wired" behavior that offers an excellent opportunity to explore neural circuit assembly and function. Moreover, the camouflage response is sensitive to ethanol, making it a tractable system for understanding how ethanol influences neural circuit development and function. Here we report the identification of corticotropin-releasing factor (CRF) as a critical component of the camouflage response pathway. We further show that ethanol, having no direct effect on the visual sensory system or the melanocytes, acts downstream of retinal ganglion cells and requires the CRF-proopiomelanocortin pathway to exert its effect on camouflage. Treatment with ethanol, as well as alteration of light exposure that changes sensory input into the camouflage circuit, robustly modifies CRF expression in subsets of neurons. Activity of both adenylyl cyclase 5 and extracellular signal-regulated kinase (ERK) is required for such ethanol-induced or light-induced plasticity of crf expression. These results reveal an essential role of a peptidergic pathway in camouflage that is regulated by light and influenced by ethanol at concentrations relevant to abuse and anxiolysis, in a cAMP-dependent and ERK-dependent manner. We conclude that this ethanol-modulated camouflage response represents a novel and relevant system for molecular genetic dissection of a neural circuit that is regulated by light and sensitive to ethanol. [ABSTRACT FROM AUTHOR]

Mechanisms influencing the development of olfactory bulb glomeruli are poorly understood. While odor receptors (ORs) play an important role in olfactory sensory neuron (OSN) axon targeting/coalescence (Mombaerts et al., 1996; Wang et al., 1998; Feinstein and Mombaerts, 2004), recent work showed that G protein activation alone is sufficient to induce OSN axon coalescence (Imai et al., 2006; Chesler et al., 2007), suggesting an activity-dependent mechanism in glomerular development. Consistent with these data, OSN axon projections and convergence are perturbed in mice deficient for adenylyl cyclase III, which is downstream from the OR and catalyzes the conversion of ATP to cAMP. However, in cyclic nucleotide-gated (CNG) channel knock-out mice OSN axons are only transiently perturbed (Lin et al., 2000), suggesting that the CNG channel may not be the sole target of cAMP. This prompted us to investigate an alternative channel, the hyperpolarization-activated, cyclic nucleotide-gated cation channel (HCN), as a potential developmental target of cAMP in OSNs. Here, we demonstrate that HCN channels are developmentally precocious in OSNs and therefore are plausible candidates for affecting OSN axon development. Inhibition of HCN channels in dissociated OSNs significantly reduced neurite outgrowth. Moreover, in HCN1 knockout mice the formation of glomeruli was delayed in parallel with perturbations of axon organization in the olfactory nerve. These data support the hypothesis that the outgrowth and coalescence of OSN axons is, at least in part, subject to activity-dependent mechanisms mediated via HCN channels. [ABSTRACT FROM AUTHOR]

Synapse formation is tightly associated with neuronal excitability. We found striking synaptic overgrowth caused by Drosophila K+- channel mutations of the seizure and slowpoke genes, encoding Erg and Ca2+-activated large-conductance (BK) channels, respectively. These mutants display two distinct patterns of "satellite" budding from larval motor terminus synaptic boutons. Double-mutant analysis indicates thatBKand ErgK+ channels interact with separate sets of synaptic proteins to affect distinct growth steps. Post-synaptic L-type Ca2+ channels, Dmca1D, and PSD-95-like scaffold protein, Discs large, are required for satellite budding induced by slowpoke and seizure mutations. Pre-synaptic cacophony Ca2+ channels and the NCAM-like adhesion molecule, Fasciclin II, take part in a maturation step that is partially arrested by seizure mutations. Importantly, slowpoke and seizure satellites were both suppressed by rutabaga mutations that disrupt Ca2+/CaM-dependent adenylyl cyclase, demonstrating a convergence of K+ channels of different functional categories in regulation of excitability-dependent Ca2+ influx for triggering cAMP-mediated growth plasticity. [ABSTRACT FROM AUTHOR]

Similar to other invertebrate and vertebrate animals, cAMP-dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homolog of the vertebrate pituitary adenylate cyclaseactivating polypeptide (PACAP) and for the AC-activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this “strong” food-reward conditioning paradigm the activation of AC by PACAP was necessary forLTMto form.Wefound that in a “weak” multitrial food reward conditioning paradigm, lip touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multitrial tactile conditioning accelerated the formation of transcriptiondependent memory. Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning. [ABSTRACT FROM AUTHOR]

The chemokine SDF1 activates a cAMP-mediated signaling pathway that antagonizes retinal responses to the midline repellent slit. We show that knocking down the calmodulin-activated adenylate cyclase ADCY8 makes retinal axons insensitive to SDF1. Experiments in vivo using male and female zebrafish (Danio rerio) confirm a mutual antagonism between slit signaling and ADCY8-mediated signaling. Unexpectedly, knockdown of ADCY8 or another calmodulin-activated cyclase, ADCY1, induces ipsilateral misprojections of retinal axons that would normally cross the ventral midline. We demonstrate a cell-autonomous requirement for ADCY8 in retinal neurons for normal midline crossing. These findings are the first to show that ADCY8 is required for axonal pathfinding before axons reach their targets. They support a model in which ADCY8 is an essential component of a signaling pathway that opposes repellent signaling. Finally, they demonstrate that ADCY8 helps regulate retinal sensitivity to midline guidance cues. [ABSTRACT FROM AUTHOR]

Dopamine (DA)-dependent corticostriatal plasticity is thought to underlie incremental procedural learning.Aprimary effector of striatal DA signaling is cAMP, yet its role in corticostriatal plasticity and striatum-dependent learning remains unclear. Here, we show that genetic deletion of a striatum-enriched isoform of adenylyl cyclase, AC5 knock-out (AC5KO), impairs two forms of striatum-dependent learning and corticostriatal synaptic plasticity. AC5KO mice were severely impaired in acquisition of a response strategy in the cross maze, a striatum-dependent task requiring a correct body turn to find a goal arm. In addition, AC5KO mice were impaired in acquisition of a motor skill, as assessed by the accelerated rotarod. Slice electrophysiology revealed a deficit in corticostriatal long-term depression (LTD) after high-frequency stimulation of tissue from AC5KO mice. LTD was rescued by activation of either presynaptic cannabinoid type 1 (CB1 ) receptors or postsynaptic metabotropic glutamate receptors (mGluRs), suggesting a postsynaptic role of AC5-cAMP, upstream of endocannabinoid release. In striatopallidal-projecting medium spiny neurons, DA D2 receptors are negatively coupled to cAMP production, and activation of these receptors is required for endocannabinoid release and corticostriatal LTD. Recordings from striatopallidal neurons indicated that this is mediated by AC5, because coactivation of D2 and mGluRs could induce LTD in wild-type but not in AC5KO neurons. To further examine the role of cAMP in corticostriatal plasticity, we elevated cAMP in striatal neurons of wild-type mice via the recording electrode. Under these conditions, corticostriatal LTD was eliminated. Together, these data suggest an AC5-cAMP- endocannabinoid-CB1 signaling pathway in corticostriatal plasticity and striatum-dependent learning. [ABSTRACT FROM AUTHOR]

Hippocampus-dependent memory requires a cAMP signal that is generated by Ca2+-stimulated adenylyl cyclases (AC1, AC8). Young transgenic mice overexpressing AC1 in the forebrain (AC1+ mice) have enhanced hippocampal long-term potentiation, superior memory for novel object recognition and more persistent remote contextual memory. To determine whether increasing AC1 expression improves memory when older mice are trained, we analyzed fear, recognition, and spatial memory in mice aged to 25 months. Here we report that young adult AC1+ mice have enhanced social recognition memory, and normal fear and spatial memory. Surprisingly, aged AC1+ mice had poorer spatial memory than age-matched wild-type littermates. These data suggest that the decrease in Ca2+-stimulated adenylyl cyclase activity during aging of wild-type mice may be an adaptive mechanism required to maintain spatial memory function. [ABSTRACT FROM AUTHOR]

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca2+/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that, whereas AC1 and AC8 single knock-out mice (AC1-/- and AC8-/-) exhibit Ca2+-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knock-out (DKO) mice do not. Furthermore,DKOmice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization after chronic cocaine treatment. Because of the known role for the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated ERK (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase-positive interneurons in DKO mice relative to wild-type (WT) controls. After acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice.Wedemonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581 and cAMP response element-binding protein (pCREB) at Ser133 after acute cocaine treatment. Our results demonstrate that the Ca2+-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs. [ABSTRACT FROM AUTHOR]

Current research in the field of anxiety disorders is largely receptor-centric, leaving intracellular pathways largely unexplored. Gαs, the G-protein which stimulates adenylyl cyclase and L-type voltage-gated calcium channels, may be one intracellular molecule regulating anxiety-related behaviors as increased efficacy of Gαs signaling has been noted in patient populations that suffer from anxiety. We report here anxiety-related behaviors in two lines of transgenic mice expressing a constitutively active isoform of Gαs (or Gαs*). The first line expressed Gαs* throughout postnatal forebrain neurons, while the second line of mice conditionally expressed Gαs* selectively in the striatum (Gαs*str mice). In the open field, both lines of mice showed a significant preference for the periphery suggesting that expression of Gαs* in the striatum alone was sufficient to produce an anxiogenic phenotype. In the light/dark box, Gαs*str mice exhibited longer latencies to enter the light and spent significantly less time in the lit compartment. Similarly, Gαs*str mice showed longer latencies to enter the open quadrants and spent less time in the open quadrants of the elevated zero maze. Interestingly, these anxiety-related phenotypes were largely unrelated to developmental effects as mice expressing the Gαs*str transgene during development, but not at testing, were normal on most measures. These observations show that chronic Gαs signaling in the striatum is sufficient to trigger anxiety-related behaviors largely independent of developmental effects and suggest the cAMP pathway or L-type voltage-gated calcium channels may be viable targets for future pharmacological intervention in the treatment of anxiety disorders. [ABSTRACT FROM AUTHOR]

Although molecular mechanisms for hippocampus-dependent memory have been extensively studied, much less is known about signaling events important for remote memory. Here we report that mice lacking type 1 adenylyl cyclase (AC1) are able to establish and retrieve remote contextual memory but unable to sustain it as long as wild-type mice. Interestingly, mice overexpressing AC1 show superior remote contextual memory even though they exhibit normal hippocampus-dependent contextual memory. These data illustrate that calcium coupling to cAMP contributes to the stability of remote memory and identifies AC1 as a potential drug target site to improve long-term remote memory. [ABSTRACT FROM AUTHOR]

In associative learning, animals learn to associate external cues or their own actions with appetitive or aversive outcomes. Although the dopamine (DA) system and the striatum/nucleus accumbens have been implicated in both the pavlovian and instrumental form of associative learning, whether specific neuronal signaling mechanisms underlie one form or the other is unknown. Here, we report that the striatum-enriched isoform of adenylyl cyclase (AC), AC5, is selectively required for appetitive pavlovian learning. Mice with genetic deletion of AC5 (AC5KO) acquired instrumental responding yet were unable to use cues that predicted reward delivery. The specificity of this deficit was confirmed by an inability of AC5KO mice to learn a simple appetitive pavlovian conditioning task. Conversely, AC5KO mice showed intact aversive pavlovian learning, suggesting the deficit was specific for learning about appetitive outcomes. Our results suggest that AC5 is a critical component of DA-dependent strengthening of stimulus-reward contingencies. [ABSTRACT FROM AUTHOR]

Group I metabotropic glutamate receptors (mGluR1 and mGluR5) are important neuronal mediators of postsynaptic signaling that influence synaptic strength, plasticity, and other factors. Regulation of group I mGluR localization and function by Homer proteins appears to be a viable means for neurons to fine-tune these processes. The presence of different Homer isoforms can act as a switch to reprioritize mGluR1 and mGluR5 signaling at the point of IP3 receptor activation by promoting or reducing activation of specific downstream effectors. Furthermore, these Homer-dependent effects on mGluR signaling may mechanistically underlie many of the long-term changes in neuronal function associated with changes inHomerprotein expression described in the recent literature. However, most studies focusing on mGluR regulation by Homer proteins used relatively long-term overexpression. Thus, a definitive demonstration of mGluR1/5 signal regulation by natively expressed Homer proteins has been elusive. I examined the ability of endogenous Homer 1a to alter mGluR signaling in rat sympathetic neurons and hippocampal autapses using pituitary adenylate cyclase activating peptide (PACAP) to induce native Homer 1a expression. In sympathetic neurons, both Homer 1a overexpression and PACAP treatment reversed the decrease in mGluR1-mediated calcium current modulation associated with Homer 2b expression. In hippocampal autapses, PACAP treatment uncoupled postsynaptic mGluR5 from EPSC inhibition, similar to the effect of Homer 1a overexpression. In both cases, RNA silencing of Homer 1a but not control RNA interference treatment prevented the PACAP effect, suggesting that it resulted specifically from native Homer 1a expression. [ABSTRACT FROM AUTHOR]

cAMP regulates secretory processes through both PKA-independent and PKA-dependent signaling pathways. Their relative contributions to fast neurotransmission are unclear at present, although forskolin, which is generally believed to enhance intracellular cAMP levels by stimulation of adenylyl cyclase activity, was shown to increase vesicular release probability ( p) and the number of releasable vesicles (N) in various neuronal preparations. Using low-frequency (0.2 Hz) electrophysiological recordings in the presence of the Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP (ESCA1 ), we find that Epac activation by this analog accounts on average for 38% of the forskolin-induced increase in evoked EPSC amplitudes and for 100% of the forskolin-induced increase in miniature EPSC (mEPSC) frequency in dissociated autaptic neuronal cultures from mouse hippocampus. From paired-pulse facilitation experiments, and considering the enhancement of mEPSC frequency, we conclude that ESCA1-induced Epac activity is presynaptic in origin and increases p. In addition, preapplication of ESCA1 augmented a subsequent enhancement of evoked EPSC amplitudes by phorbol ester (PDBu). This effect was maximal when ESCA1 application preceded the PDBu application by 3 min. Because the PDBu response was abolished after downregulation of intracellular PKC activity, we conclude that ESCA1-induced Epac activation leads to presynaptic changes involving Epac-to-PKC signaling. [ABSTRACT FROM AUTHOR]

Neuropathic pain is caused by a primary lesion or dysfunction in the nervous system. Investigations have mainly focused on the spinal mechanisms of neuropathic pain, and less is known about cortical changes in neuropathic pain. Here, we report that peripheral nerve injury triggered long-term changes in excitatory synaptic transmission in layer II/III neurons within the anterior cingulate cortex (ACC). Both the presynaptic release probability of glutamate and postsynaptic glutamate AMPA receptor-mediated responses were enhanced after injury using the mouse peripheral nerve injury model. Western blot showed upregulated phosphorylation of GluR1 in the ACC after nerve injury. Finally, we found that both presynaptic and postsynaptic changes after nerve injury were absent in genetic mice lacking calcium-stimulated adenylyl cyclase 1 (AC1). Our studies therefore provide direct integrative evidence for both long-term presynaptic and postsynaptic changes in cortical synapses after nerve injury, and that AC1 is critical for such long-term changes. AC1 thus may serve as a potential therapeutic target for treating neuropathic pain. [ABSTRACT FROM AUTHOR]

Experimental evidence from mutant or genetically altered mice indicates that the formation of barrels and the proper maturation of thalamocortical (TC) synapses in the primary somatosensory (barrel) cortex depend on mechanisms mediated by neural activity. Type 1 adenylyl cyclase (AC1), which catalyzes the formation of cAMP, is stimulated by increases in intracellular Ca2+ levels in an activitydependent manner. The AC1 mutant mouse, barrelless (brl), lacks typical barrel cytoarchitecture, and displays presynaptic and postsynaptic functional defects at TC synapses. However, because AC1 is expressed throughout the trigeminal pathway, the barrel cortex phenotype of brl micemaybe a consequence of AC1 disruption in cortical or subcortical regions. To examine the role of cortical AC1 in the development of morphological barrels and TC synapses, we generated cortex-specific AC1 knock-out (CxAC1KO) mice. We found that neurons in layer IV form grossly normal barrels and TC axons fill barrel hollows in CxAC1KO mice. In addition, whisker lesion-induced critical period plasticity was not impaired in these mice. However, we found quantitative reductions in the quality of cortical barrel cytoarchitecture and dendritic asymmetry of layer IV barrel neurons in CxAC1KO mice. Electrophysiologically, CxAC1KO mice have deficits in the postsynaptic but not in the presynaptic maturation of TC synapses. These results suggest that activity-dependent postsynaptic AC1-cAMP signaling is required for functional maturation of TC synapses and the development of normal barrel cortex cytoarchitecture. They also suggest that the formation of the gross morphological features of barrels is independent of postsynaptic AC1 in the barrel cortex. [ABSTRACT FROM AUTHOR]

Glutamate generates fast postsynaptic depolarization throughout the CNS. The positive-feedback nature of glutamate signaling likely necessitates flexible adaptive mechanisms that help prevent runaway excitation. We have previously explored presynaptic adaptive silencing, a form of synaptic plasticity produced by ongoing neuronal activity and by strong depolarization. Unsilencing mechanisms that maintain active synapses and restore normal function after adaptation are also important, but mechanisms underlying such presynaptic reactivation remain unexplored. Here we investigate the involvement of the cAMP pathway in the basal balance between silenced and active synapses, as well as the recovery of baseline function after depolarization-induced presynaptic silencing. Activation of the cAMP pathway activates synapses that are silent at rest, and pharmacological inhibition of cAMP signaling silences basally active synapses. Adenylyl cyclase (AC) 1 and AC8, the major Ca2+-sensitive AC isoforms, are not crucial for the baseline balance between silent and active synapses. In cells from mice doubly deficient in AC1 and AC8, the baseline percentage of active synapses was only modestly reduced compared with wild-type synapses, and forskolin unsilencing was similar in the two genotypes. Nevertheless, after strong presynaptic silencing, recovery of normal function was strongly inhibited in AC1/AC8-deficient synapses. The entire recovery phenotype of the double null was reproduced in AC8-deficient but not AC1-deficient cells.Weconclude that, under normal conditions, redundant cyclase activity maintains the balance between presynaptically silent and active synapses, but AC8 plays a particularly important role in rapidly resetting the balance of active to silent synapses after adaptation to strong activity. [ABSTRACT FROM AUTHOR]

Ca-stimulated adenylyl cyclases (ACs) transduce neuronal stimulation-evoked increase in calcium to the production of cAMP, which impinges on the regulation of many aspects of neuronal function. Type 1 and type 8 AC (AC1 and AC8) are the only ACs that are directly stimulated by Ca. Although AC1 function was implicated in regulating reference spatial memory, the function of AC8 in memory formation is not known. Because of the different biochemical properties of AC1 and AC8, these two enzymes may have distinct functions. For example, AC1 activity is regulated by both Ca and G-proteins. In contrast, AC8 is a pure Ca sensor. It is neither stimulated by Gs nor inhibited by Gi. Recent studies also suggested that AC1 and AC8 were differentially concentrated at different subcellular domains, implicating that Ca-stimulated signaling might be compartmentalized. In this study, we used AC8 knock-out (KO) mice and found behavioral deficits in memory retention for temporal dissociative passive avoidance and object recognition memory. When examined by Morris water maze, AC8 KO mice showed normal reference memory. However, the acquisition of newer spatial information was defective in AC8 KO mice. Furthermore, AC8 KO mice were severely impaired in hippocampus-dependent episodic-like memory when examined by the delayed matching-to-place task. Because AC8 is preferentially localized at the presynaptic active zone, our results suggest a novel role of presynaptic cAMP signaling in memory acquisition and retention, as well as distinct mechanisms underlying reference and working/episodic-like memory. [ABSTRACT FROM AUTHOR]

The fragile X syndrome is caused by the lack of fragile X mental retardation protein (FMRP) attributable to silencing of the FMR1 gene. The metabotropic glutamate receptors (mGluRs) in the CNS contribute to different brain functions, including learning/memory, mental disorders, drug addiction, and persistent pain. Most of the previous studies have been focused on downstream targets of FMRP in hippocampal neurons, and fewer studies have been reported for the second-messenger signaling pathways between group I mGluRs and FMRP. Furthermore, no molecular study has been performed in the anterior cingulate cortex (ACC), a key region involved in high brain cognitive and executive functions. In this study, we demonstrate that activation of group I mGluR upregulated FMRP in ACC neurons of adult mice through the Ca2+-dependent signaling pathways. Using genetic approaches, we found that Ca22+/calmodulin-stimulated adenylyl cyclase 1 (AC1) and calcium/calmodulin-dependent kinase IV (CaMKIV) contribute to the upregulation of FMRP induced by stimulating group I mGluRs. The upregulation of FMRP occurs at the transcriptional level. The cAMP-dependent protein kinase is activated by stimulating group I mGluRs through AC1 in ACC neurons. Both AC1 and CaMKIV contribute to the regulation of FMRP by group I mGluRs probably through cAMP response element-binding protein activation. Our study has provided the first evidence for a molecular link between group I mGluRs and FMRP in ACC neurons and may help us to understand the pathogenesis of fragile X syndrome. [ABSTRACT FROM AUTHOR]

During development, axons are directed to their targets by extracellular guidance cues. The axonal response to the guidance cue netrin-1 is profoundly influenced by the concentration of cAMP within the growth cone. In some cases, cAMP affects the sensitivity of the growth cone to netrin-1, whereas in others it changes the response to netrin-1 from attraction to repulsion. The effects of cAMP on netrin-1 action are well accepted, but the critical issue of whether cAMP production is activated by a netrin-1 induced signaling cascade remains uncertain. A previous report has suggested that axon guidance in response to netrin-1 requires cAMP production mediated by soluble adenyl cyclase (sAC). We have used genetic, molecular and biochemical strategies to assess this issue. Surprisingly, we found only extremely weak expression of sAC in embryonic neurons and determined that, under conditions where netrin-1 directs axonal pathfinding, exposure to netrin-1 does not alter cAMP levels. Furthermore, although netrin-1-deficient mice exhibit major axon guidance defects, we show that pathfinding is normal in sAC-null mice. Therefore, although cAMP can alter the response of axons to netrin-1, we conclude that netrin-1 does not alter cAMP levels in axons attracted by this cue, and that sAC is not required for axon attraction to netrin-1. [ABSTRACT FROM AUTHOR]

Recent in vivo and in vitro studies have demonstrated that Gsα migrates from a Triton X-100 (TX-100)-insoluble membrane domain (lipid raft) to a TX-100-soluble nonraft membrane domain in response to chronic, but not acute, treatment with tricyclic or selective serotonin reuptake inhibitor antidepressants. This migration resulted in a more facile association with adenylyl cyclase. Our hypothesis is that Gsα maybe ensconced, to a greater extent, in lipid rafts during depression, and that one action of chronic antidepressant treatment is to reverse this. In this postmortem study, we examined Gsα membrane localization in the cerebellum and prefrontal cortex of brains from nonpsychiatric control subjects and suicide cases with confirmed unipolar depression. Sequential TX-100 and TX-114 detergent extractions were performed on the brain tissue. In the cerebellum, the ratio of TX-100/TX-114-soluble Gsα is ~2:1 for control versus depressed suicides. Results with prefrontal cortex samples from each group demonstrate a similar trend. These data suggest that depression localizes Gsα to a membrane domain (lipid rafts) where it is less likely to couple to adenylyl cyclase and that antidepressants may upregulate Gsα signaling via disruption of membrane microenvironments. Raft localization of Gsα in human peripheral tissue may thus serve as a biomarker for depression and as a harbinger of antidepressant responsiveness. [ABSTRACT FROM AUTHOR]

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca2+/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that, whereas AC1 and AC8 single knock-out mice (AC1-/- and AC8-/-) exhibit Ca2+-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knock-out (DKO) mice do not. Furthermore,DKOmice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization after chronic cocaine treatment. Because of the known role for the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated ERK (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase-positive interneurons in DKO mice relative to wild-type (WT) controls. After acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice.Wedemonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581 and cAMP response element-binding protein (pCREB) at Ser133 after acute cocaine treatment. Our results demonstrate that the Ca2+-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs. [ABSTRACT FROM AUTHOR]

Noradrenergic modulation of cortical circuits is involved in information processing, regulation of higher functions, and prevention of epileptic activity. Here, we studied the effects of noradrenaline on the functional connectivity of GABAergic networks of the hippocampus and show that electrical synapses between interneurons are a novel target of noradrenergic modulation in vitro. Application of noradrenaline or of the selective β-adrenergic agonist isoproterenol decreased gap junction-based coupling in paired recordings from stratum lacunosum-moleculare interneurons by ~40%. Similar results were obtained after pharmacological stimulation of the adenylyl cyclase with forskolin. In contrast, the adenylyl cyclase antagonistMDL12330A[cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine] or the specific protein kinase A (PKA) inhibitor H89 (N-[2-( p-bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) enhanced the basal strength of coupling by ~30%. In addition, PKA-mediated phosphorylation was critical for both isoproterenol- and forskolin-dependent regulation of coupling, because inclusion of the PKA antagonist KT5720 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i] [1,6]benzodiazocine-10-carboxylicacid hexyl ester] in the recording pipettes prevented modulation. Lastly, we studied the effects of β-adrenergic modulation on mixed polysynaptic transmission within the GABAergic network. Isoproterenol depressed propagation of GABAA receptor-mediated synaptic currents, but did not change significantly direct GABAergic input, indicating that regulation of electrical coupling adds flexibility to the information flow generated by chemical synapses. In conclusion, activation of β-adrenergic receptors in stratum lacunosum-moleculare GABAergic networks reduces electrical synaptic transmission via a cAMP/PKA signaling cascade, and affects the degree of synaptic divergence within the circuit. We propose that this dynamic modulation and interplay between electrical and chemical synaptic transmission in GABAergic networks contributes to the tuning of memory processes in vivo, and prevents hypersynchronous activity. [ABSTRACT FROM AUTHOR]

Physical traces underlying simple memories can be confined to a single group of cells in the brain. In the fly Drosophila melanogaster, the Kenyon cells of the mushroom bodies house traces for both appetitive and aversive odor memories. The adenylate cyclase protein, Rutabaga, has been shown to mediate both traces. Here, we show that, for appetitive learning, another group of cells can additionally accommodate a Rutabaga-dependent memory trace. Localized expression of rutabaga in either projection neurons, the first-order olfactory interneurons, or in Kenyon cells, the second-order interneurons, is sufficient for rescuing the mutant defect in appetitive short-term memory. Thus, appetitive learning may induce multiple memory traces in the first- and second-order olfactory interneurons using the same plasticity mechanism. In contrast, aversive odor memory of rutabaga is rescued selectively in the Kenyon cells, but not in the projection neurons. This difference in the organization of memory traces is consistent with the internal representation of reward and punishment. [ABSTRACT FROM AUTHOR]