GluN3-Containing NMDA Receptors in the Rat Nucleus Accumbens Core Contribute to Incubation of Cocaine Craving.

Cue-induced cocaine craving progressively intensifies (incubates) after withdrawal from cocaine self-administration in rats and humans. In rats, the expression of incubation ultimately depends on Ca2+-permeable AMPARs that accumulate in synapses onto medium spiny neurons (MSNs) in the NAc core. However, the delay in their accumulation (∼1 month after drug self-administration ceases) suggests earlier waves of plasticity. This prompted us to conduct the first study of NMDAR transmission in NAc core during incubation, focusing on the GluN3 subunit, which confers atypical properties when incorporated into NMDARs, including insensitivity to Mg2+ block and Ca2+ impermeability. Whole-cell patch-clamp recordings were conducted in MSNs of adult male rats 1-68 d after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 d of cocaine withdrawal, and this persisted for at least 68 d of withdrawal. The earliest functional alterations were mediated through increased contributions of GluN2B-containing NMDARs, followed by increased contributions of GluN3-containing NMDARs. As predicted by GluN3-NMDAR incorporation, fewer MSN spines exhibited NMDAR-mediated Ca2+ entry. GluN3A knockdown in NAc core was sufficient to prevent incubation of craving, consistent with biotinylation studies showing increased GluN3A surface expression, although array tomography studies suggested that adaptations involving GluN3B also occur. Collectively, our data show that a complex cascade of NMDAR and AMPAR plasticity occurs in NAc core, potentially through a homeostatic mechanism, leading to persistent increases in cocaine cue reactivity and relapse vulnerability. This is a remarkable example of experience-dependent glutamatergic plasticity evolving over a protracted window in the adult brain.SIGNIFICANCE STATEMENT "Incubation of craving" is an animal model for the persistence of vulnerability to cue-induced relapse after prolonged drug abstinence. Incubation also occurs in human drug users. AMPAR plasticity in medium spiny neurons (MSNs) of the NAc core is critical for incubation of cocaine craving but occurs only after a delay. Here we found that AMPAR plasticity is preceded by NMDAR plasticity that is essential for incubation and involves GluN3, an atypical NMDAR subunit that markedly alters NMDAR transmission. Together with AMPAR plasticity, this represents profound remodeling of excitatory synaptic transmission onto MSNs. Given the importance of MSNs for translating motivation into action, this plasticity may explain, at least in part, the profound shifts in motivated behavior that characterize addiction.

Human-Induced Neurons from Presenilin 1 Mutant Patients Model Aspects of Alzheimer's Disease Pathology.

Traditional approaches to studying Alzheimer's disease (AD) using mouse models and cell lines have advanced our understanding of AD pathogenesis. However, with the growing divide between model systems and clinical therapeutic outcomes, the limitations of these approaches are increasingly apparent. Thus, to generate more clinically relevant systems that capture pathological cascades within human neurons, we generated human-induced neurons (HiNs) from AD and non-AD individuals to model cell autonomous disease properties. We selected an AD patient population expressing mutations in presenilin 1 (mPS1), which is linked to increased amyloid production, tau pathology, and calcium signaling abnormalities, among other features. While these AD components are detailed in model systems, they have yet to be collectively identified in human neurons. Thus, we conducted molecular, immune-based, electrophysiological, and calcium imaging studies to establish patterns of cellular pathology in this patient population. We found that mPS1 HiNs generate increased Aß42 and hyperphosphorylated tau species relative to non-AD controls, and exaggerated ER calcium responses that are normalized with ryanodine receptor (RyR) negative allosteric modulators. The inflammasome product, interleukin-18 (IL-18), also increased PS1 expression. This work highlights the potential for HiNs to model AD pathology and validates their role in defining cellular pathogenesis and their utility for therapeutic screening.

Emerging pathways driving early synaptic pathology in Alzheimer's disease.

The current state of the AD research field is highly dynamic is some respects, while seemingly stagnant in others. Regarding the former, our current lack of understanding of initiating disease mechanisms, the absence of effective treatment options, and the looming escalation of AD patients is energizing new research directions including a much-needed re-focusing on early pathogenic mechanisms, validating novel targets, and investigating relevant biomarkers, among other exciting new efforts to curb disease progression and foremost, preserve memory function. With regard to the latter, the recent disappointing series of failed Phase III clinical trials targeting Aß and APP processing, in concert with poor association between brain Aß levels and cognitive function, have led many to call for a re-evaluation of the primacy of the amyloid cascade hypothesis. In this review, we integrate new insights into one of the earliest described signaling abnormalities in AD pathogenesis, namely intracellular Ca2+ signaling disruptions, and focus on its role in driving synaptic deficits - which is the feature that does correlate with AD-associated memory loss. Excess Ca2+release from intracellular stores such as the endoplasmic reticulum (ER) has been well-described in cellular and animal models of AD, as well as human patients, and here we expand upon recent developments in ER-localized release channels such as the IP3R and RyR, and the recent emphasis on RyR2. Consistent with ER Ca2+ mishandling in AD are recent findings implicating aspects of SOCE, such as STIM2 function, and TRPC3 and TRPC6 levels. Other Ca2+-regulated organelles important in signaling and protein handling are brought into the discussion, with new perspectives on lysosomal regulation. These early signaling abnormalities are discussed in the context of synaptic pathophysiology and disruptions in synaptic plasticity with a particular emphasis on short-term plasticity deficits. Overall, we aim to update and expand the list of early neuronal signaling abnormalities implicated in AD pathogenesis, identify specific channels and organelles involved, and link these to proximal synaptic impairments driving the memory loss in AD. This is all within the broader goal of identifying novel therapeutic targets to preserve cognitive function in AD.

Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/- /PS1 +/-). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aß load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

Design and Fabrication of a Three-Dimensional Multi-Electrode Array for Neuron Electrophysiology.

Neural recording and stimulation with high spatial and temporal resolution are highly desirable in the study of neurocommunication and diseases. Planar multiple microelectrode arrays (MEA) or quasi-three-dimensional (3D) MEA with fixed height have been proposed by many researchers and become commercially available. In this paper, we present the design, fabrication, and test of a novel true 3D multiple electrode array for brain slice stimulation and recording. This MEA is composed of 105 microelectrodes with 50 µm diameter and 125 µm center-to-center spacing integrated in a 1.2 × 1.2 mm2 area. This "true" 3D MEA allows us to precisely position the individual electrodes by piezoelectric-based actuators to penetrate the inactive tissue layer and to approach the active neurons so as to optimize the recording and stimulation of electrical field potential. The capability to stimulate nerve fibers and record postsynaptic field potentials was demonstrated in an experiment using mouse brain hippocampus slice.

Lithium Provides Broad Therapeutic Benefits in an Alzheimer's Disease Mouse Model.

BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative disorder with a progressive loss of cognitive function. Currently, no effective treatment regimen is available. Lithium, a mood stabilizer for bipolar disorder, exerts broad neuroprotective and neurotrophic actions and improves cognitive function. OBJECTIVE: The study investigated if lithium stabilizes Ca2+ signaling abnormalities in hippocampal neurons and subsequently normalize downstream effects on AD neuropathology and synaptic plasticity in young AD mice. METHODS: Four-month-old 3xTg-AD mice were treated with a LiCl diet chow for 30 days. At the end of the lithium treatment, a combination of two-photon Ca2+ imaging, electrophysiology, and immunohistochemistry assays were used to assess the effects of the LiCl treatment on inositol trisphosphate receptor (IP3R)-dependent endoplasmic reticulum (ER) Ca2+ and voltage-gated Ca2+ channel (VGCC)-mediated Ca2+ signaling in CA1 neurons, neuronal nitric oxide synthase (nNOS) and hyperphosphorylated tau (p-tau) levels and synaptic plasticity in the hippocampus and overlying cortex from 3xTg-ADmice. RESULTS: Thirty-day LiCl treatment reduced aberrant IP3R-dependent ER Ca2+ and VGCC-mediated Ca2+ signaling in CA1 pyramidal neurons from 3xTg-AD mice and restored neuronal nitric oxide synthase (nNOS) and hyperphosphorylated tau (p-tau) levels to control levels in the hippocampal subfields and overlying cortex. The LiCl treatment enhanced post-tetanic potentiation (PTP), a form of short-term plasticity in the hippocampus. CONCLUSION: The study found that lithium exerts therapeutic effects across several AD-associated early neuronal signaling abnormalities including aberrant Ca2+ signaling, nNOS, and p-tau formation and enhances short-term synaptic plasticity. Lithium could serve as an effective treatment or co-therapeutic for AD.

Engineered neurogenesis in naïve adult rat cortex by Ngn2-mediated neuronal reprogramming of resident oligodendrocyte progenitor cells.

Adult tissue stem cells contribute to tissue homeostasis and repair but the long-lived neurons in the human adult cerebral cortex are not replaced, despite evidence for a limited regenerative response. However, the adult cortex contains a population of proliferating oligodendrocyte progenitor cells (OPCs). We examined the capacity of rat cortical OPCs to be re-specified to a neuronal lineage both in vitro and in vivo. Expressing the developmental transcription factor Neurogenin2 (Ngn2) in OPCs isolated from adult rat cortex resulted in their expression of early neuronal lineage markers and genes while downregulating expression of OPC markers and genes. Ngn2 induced progression through a neuronal lineage to express mature neuronal markers and functional activity as glutamatergic neurons. In vivo retroviral gene delivery of Ngn2 to naive adult rat cortex ensured restricted targeting to proliferating OPCs. Ngn2 expression in OPCs resulted in their lineage re-specification and transition through an immature neuronal morphology into mature pyramidal cortical neurons with spiny dendrites, axons, synaptic contacts, and subtype specification matching local cytoarchitecture. Lineage re-specification of rat cortical OPCs occurred without prior injury, demonstrating these glial progenitor cells need not be put into a reactive state to achieve lineage reprogramming. These results show it may be feasible to precisely engineer additional neurons directly in adult cerebral cortex for experimental study or potentially for therapeutic use to modify dysfunctional or damaged circuitry.

SAR of α7 nicotinic receptor agonists derived from tilorone: exploration of a novel nicotinic pharmacophore.

The well-known interferon-inducer tilorone was found to possess potent affinity for the agonist site of the α7 neuronal nicotinic receptor (K(i)=56 nM). SAR investigations determined that both basic sidechains are essential for potent activity, however active monosubstituted derivatives can also be prepared if the flexible sidechains are replaced with conformationally rigidified cyclic amines. Analogs in which the fluorenone core is replaced with either dibenzothiophene-5,5-dioxide or xanthenone also retain potent activity.

Sustained Hippocampal Synaptic Pathophysiology Following Single and Repeated Closed-Head Concussive Impacts.

Traumatic brain injury (TBI), and related diseases such as chronic traumatic encephalopathy (CTE) and Alzheimer's (AD), are of increasing concern in part due to enhanced awareness of their long-term neurological effects on memory and behavior. Repeated concussions, vs. single concussions, have been shown to result in worsened and sustained symptoms including impaired cognition and histopathology. To assess and compare the persistent effects of single or repeated concussive impacts on mediators of memory encoding such as synaptic transmission, plasticity, and cellular Ca2+ signaling, a closed-head controlled cortical impact (CCI) approach was used which closely replicates the mode of injury in clinical cases. Adult male rats received a sham procedure, a single impact, or three successive impacts at 48-hour intervals. After 30 days, hippocampal slices were prepared for electrophysiological recordings and 2-photon Ca2+ imaging, or fixed and immunostained for pathogenic phospho-tau species. In both concussion groups, hippocampal circuits showed hyper-excitable synaptic responsivity upon Schaffer collateral stimulation compared to sham animals, indicating sustained defects in hippocampal circuitry. This was not accompanied by sustained LTP deficits, but resting Ca2+ levels and voltage-gated Ca2+ signals were elevated in both concussion groups, while ryanodine receptor-evoked Ca2+ responses decreased with repeat concussions. Furthermore, pathogenic phospho-tau staining was progressively elevated in both concussion groups, with spreading beyond the hemisphere of injury, consistent with CTE. Thus, single and repeated concussions lead to a persistent upregulation of excitatory hippocampal synapses, possibly through changes in postsynaptic Ca2+ signaling/regulation, which may contribute to histopathology and detrimental long-term cognitive symptoms.

In vitro pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107.

Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

In vitro pharmacological characterization of a novel allosteric modulator of alpha 7 neuronal acetylcholine receptor, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), exhibiting unique pharmacological profile.

Targeting alpha7 neuronal acetylcholine receptors (nAChRs) with selective agonists and positive allosteric modulators (PAMs) is considered a therapeutic approach for managing cognitive deficits in schizophrenia and Alzheimer's disease. In this study, we describe a novel type II alpha7 PAM, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), that exhibits a unique pharmacological profile. In oocytes expressing alpha7 nAChRs, A-867744 potentiated acetylcholine (ACh)-evoked currents, with an EC(50) value of approximately 1 microM. At highest concentrations of A-867744 tested, ACh-evoked currents were essentially nondecaying. At lower concentrations, no evidence of a distinct secondary component was evident in contrast to 4-naphthalen-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), another type II alpha7 PAM. In the presence of A-867744, ACh concentration responses were potentiated by increases in potency, Hill slope, and maximal efficacy. When examined in rat hippocampus CA1 stratum radiatum interneurons or dentate gyrus granule cells, A-867744 (10 microM) increased choline-evoked alpha7 currents and recovery from inhibition/desensitization, and enhanced spontaneous inhibitory postsynaptic current activity. A-867744, like other alpha7 PAMs tested [1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)urea (NS1738), TQS, and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596)], did not displace the binding of [(3)H]methyllycaconitine to rat cortex alpha7(*) nAChRs. However, unlike these PAMs, A-867744 displaced the binding of the agonist [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539) in rat cortex, with a K(i) value of 23 nM. A-867744 neither increased agonist-evoked responses nor displaced the binding of [(3)H]A-585539 in an alpha7/5-hydroxytryptamine(3) (alpha7/5-HT(3)) chimera, suggesting an interaction distinct from the alpha7 N terminus or M2-3 loop. In addition, A-867744 failed to potentiate responses mediated by 5-HT(3A) or alpha3beta4 and alpha4beta2 nAChRs. In summary, this study identifies a novel and selective alpha7 PAM showing activity at recombinant and native alpha7 nAChRs exhibiting a unique pharmacological interaction with the receptor.

Broad-spectrum efficacy across cognitive domains by alpha7 nicotinic acetylcholine receptor agonism correlates with activation of ERK1/2 and CREB phosphorylation pathways.

The alpha7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel alpha7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (Ki = 10.8 nM) and human (Ki = 16.7 nM) alpha7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the alpha7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that alpha7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of alpha7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked alpha7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that alpha7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia.

High- and low-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChRs.

Previous studies in other laboratories have shown that alpha4beta2 nicotinic acetylcholine receptor (nAChR) exhibits a biphasic concentration-response relationship for ACh with low and high EC50 components, and that the low EC50 component can be augmented by decreasing the alpha4:beta2 message ratio or incubating overnight in nicotine or at low temperature (Zwart and Vijverberg, 1998; Covernton and Connolly, 2000; Buisson and Bertrand, 2001; Nelson et al., 2003; Zhou et al., 2003). In the process of cloning ferret nAChR subunits, we found alpha4 and beta2 messages with long untranslated regions (UTRs), as well as those with no UTRs. Combinations of these messages revealed that the presence of UTRs influenced the ability to exclusively express high-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChRs. Injection of oocytes with alpha4 and beta2 RNAs lacking UTRs (1:1 ratio) led to expression of a biphasic concentration-response relationship for ACh with EC50 values of 0.5 (high sensitivity) and 114 microM(low sensitivity). Decreasing the alpha4:beta2 message ratio to as much as 1:120 increased the high-sensitivity component slightly, but the ACh concentration response remained biphasic. In contrast, injection of messages with UTRs (1:1 ratio) led to expression of a monophasic concentration response to ACh and a high-sensitivity EC50 value of 2.3 microM, as shown in Fig. 1.

Therapeutic correction of ApoER2 splicing in Alzheimer's disease mice using antisense oligonucleotides.

Apolipoprotein E receptor 2 (ApoER2) is an apolipoprotein E receptor involved in long-term potentiation, learning, and memory. Given its role in cognition and its association with the Alzheimer's disease (AD) risk gene, apoE, ApoER2 has been proposed to be involved in AD, though a role for the receptor in the disease is not clear. ApoER2 signaling requires amino acids encoded by alternatively spliced exon 19. Here, we report that the balance of ApoER2 exon 19 splicing is deregulated in postmortem brain tissue from AD patients and in a transgenic mouse model of AD To test the role of deregulated ApoER2 splicing in AD, we designed an antisense oligonucleotide (ASO) that increases exon 19 splicing. Treatment of AD mice with a single dose of ASO corrected ApoER2 splicing for up to 6 months and improved synaptic function and learning and memory. These results reveal an association between ApoER2 isoform expression and AD, and provide preclinical evidence for the utility of ASOs as a therapeutic approach to mitigate Alzheimer's disease symptoms by improving ApoER2 exon 19 splicing.

Loss of functional neuronal nicotinic receptors in dorsal root ganglion neurons in a rat model of neuropathic pain.

Recent evidence has suggested that the anti-allodynic effect of neuronal acetylcholine receptor (nAChR) agonists may have a peripheral component [L.E. Rueter, K.L. Kohlhaas, P. Curzon, C.S. Surowy, M.D. Meyer, Peripheral and central sites of action for A-85380 in the spinal nerve ligation model of neuropathic pain, Pain 103 (2003) 269-276]. In further studies of the peripheral anti-allodynic mechanisms of nAChR agonists, we investigated the function of nAChRs in acutely isolated dorsal root ganglion (DRG) neurons from allodynic [L5-L6 spinal nerve ligation (SNL)] and naive adult rats. Following determination of cell diameter and membrane capacitance, responses to rapid applications of nAChR agonists were recorded under whole cell patch clamp. nAChR inward currents were observed in approximately 60% of naive neurons, across small, medium, and large diameter cells. Evoked nAChR currents could be clustered into three broad classes: fast transient, biphasic, and slow desensitizing currents, consistent with multiple subtypes of nAChR expressed in DRG [J.R. Genzen, W. Van Cleve, D.S. McGehee, Dorsal root ganglion neurons express multiple nicotinic acetylcholine receptor subtypes, J. Neurophysiol. 86 (2001) 1773-1782]. In contrast, in neurons from allodynic animals, the occurrence and amplitude of responses to nAChR agonists were significantly reduced. Reduced responsiveness to nAChR agonists covered the range of DRG neuron sizes. The decrease in the responsiveness to nAChR agonists was not seen in neighboring uninjured L4 neurons. The significant decrease in the number of cells with nAChR agonist responses, compounded with the significant decrease in response amplitude, indicates that there is a marked down regulation of functional nAChRs in DRG somata associated with SNL.

Potent desensitization of human P2X3 receptors by diadenosine polyphosphates.

In this study, the receptor desensitizing effects of diadenosine polyphosphates at recombinant human P2X3 (hP2X3) receptors were examined. Administration of Ap3A, Ap4A, Ap5A or Ap6A inhibited the hP2X3 receptor-mediated response to a subsequent application of 3 muM alphabeta-methyleneATP (alphabeta-meATP), in a concentration-dependent manner, with IC50 values 2707, 42, 59 and 46 nM, respectively. These agonists did not desensitize alphabeta-meATP responses mediated by the slowly desensitizing heteromeric human P2X2/3 receptor. hP2X3 receptor desensitization was reversible and was not observed following the increase in intracellular Ca2+ levels produced by carbachol. A similar pattern of desensitization evoked by Ap5A was also observed using electrophysiological recordings of Xenopus oocytes expressing hP2X3 receptors. These data demonstrate that diadenosine polyphosphates, found endogenously in the central nervous system, can readily desensitize hP2X3 receptors at nanomolar concentrations that are 10-fold lower than are required to produce agonist-induced receptor activation. Thus, P2X3 receptor desensitization by diadenosine polyphosphates may provide an important modulatory mechanism of P2X3 receptor activation in vivo.

ß amyloid peptide plaques fail to alter evoked neuronal calcium signals in APP/PS1 Alzheimer's disease mice.

Alzheimer's disease (AD) is a multifactorial disorder of unknown etiology. Mechanistically, beta amyloid peptides (Aß) and elevated Ca(2+) have been implicated as proximal and likely interactive features of the disease process. We tested the hypothesis that proximity to Aß plaque might exacerbate activity-dependent neuronal Ca(2+) signaling in hippocampal pyramidal neurons from APPSWE/PS1M146V mice. Using combined approaches of whole cell patch clamp recording and 2-photon imaging of neuronal Ca(2+) signals with thioflavin-S plaque labeling in hippocampal slices, we found no correlation between thioflavin-S labeled Aß plaque proximity and Ca(2+) responses triggered by ryanodine receptor (RyR) activation or action potentials in either dendrites or somata of AD mice, regardless of age. Baseline and RyR-stimulated spontaneous excitatory postsynaptic potentials also showed little difference in relation to Aß plaque proximity. Consistent with previous studies, RyR-evoked Ca(2+) release in APPSWE/PS1M146V mice was greater than in nontransgenic controls. Within the soma, RyR-evoked Ca(2+) release was elevated in older APPSWE/PS1M146V mice compared with younger APPSWE/PS1M146V mice, but was still independent of plaque proximity. The results indicate that early Ca(2+) signaling disruptions can become yet more severe with age through mechanisms independent of Aß plaques, suggesting that alternative pathogenic mechanisms might contribute to AD-associated dysfunction.

Importance of M2-M3 loop in governing properties of genistein at the α7 nicotinic acetylcholine receptor inferred from α7/5-HT3A chimera.

Genistein and 5-hydroxyindole (5-HI) potentiate the α7 nicotinic acetylcholine receptor current by primarily increasing peak amplitude, a property of type I α7 positive allosteric modulation. In this study, the effects of these two compounds were investigated at two different α7/5-HT(3) chimeras (chimera 1, comprising of extracellular α7 N-terminus fused to the remainder of 5-HT(3A), and chimera 2 containing an additional α7 encoded M2-M3 loop), and wild-type α7 and 5-HT(3A) receptors. Agonist-evoked responses, examined by expression of the chimeras in Xenopus laevis oocytes or HEK-293 cells, revealed that currents decayed slower and compounds {rank order: N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)~2-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5-phenyl-1,3,4-oxadiazole (NS6784)>acetylcholine>choline} were more potent in chimera 2 than chimera 1 or α7 receptors. In chimera 2, genistein and 5-HI potentiated agonist-evoked responses (EC(50): 4-5 µM for genistein and 300-500 µM for 5-HI) and at higher concentrations evoked current directly consistent with ago-allosteric modulation. At chimera 1 and 5-HT(3A) receptors, neither compound directly evoked any current and 5-HI, only at chimera 1, was able to potentiate agonist-evoked responses. Genistein and 5-HI did not inhibit the binding of the α7 agonist [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1] heptane ([(3)H]A-585539) to rat brain or chimera 2. In summary, this study supports the role of the M2-M3 loop being critical for the positive allosteric effect of genistein, but not 5-HI, and in agonist-evoked response fine-tuning. The identification of distinct α7 receptor modulatory sites offers unique opportunities for developing CNS therapeutics and understanding its pharmacology.

Selectivity of ABT-089 for alpha4beta2* and alpha6beta2* nicotinic acetylcholine receptors in brain.

Numerous pharmaceutical efforts have targeted neuronal nicotinic receptors (nAChRs) for amelioration of cognitive deficits. While alpha4beta2 and alpha7 are the more prominent nAChR in brain, other heteromeric nAChR can have important impact on agonist pharmacology. ABT-089 is a pioneer nAChR agonist found to enhance cognitive function with an exceptionally low incidence of adverse effects. To further investigate the mechanism of action of ABT-089, we evaluated its function in mouse brain preparations in which we have characterized the subunit composition of native nAChR. Among alpha4beta2*-nAChR, ABT-089 had partial agonist activity (7-23% of nicotine) and high selectivity for alpha4alpha5beta2 nAChR as evidenced by loss of activity in thalamus of alpha5(-/-) mice. ABT-089 stimulated [(3)H]-dopamine release (57%) exceeded the activity at alpha4beta2* nAChR, that could be explained by the activity at alpha6beta2* nAChR. The concentration-response relationship for ABT-089 stimulation of alpha6beta2* nAChR was biphasic. EC(50) and efficacy values for ABT-089, respectively, were 28 microM and 98% at the less sensitive alpha6beta2* nAChR and 0.11 microM and 36% at the more sensitive subtype (the most sensitive target for ABT-089 identified to date). ABT-089 had essentially no agonist or antagonist activity at concentrations