Cloning and characterization of an olfactory cyclic nucleotide-gated channel expressed in mouse heart

Regulation of ionic currents in the heart is partly achieved by signaling cascades which alter intracellular levels of cyclic nucleotides. Changes in cyclic nucleotide levels can regulate channels either directly, like the direct binding of cAMP to the i(f) channel in pacemaker tissues, or indirectly through phosphorylation of channels by cAMP-dependent, or cGMP-dependent protein kinases. These types of regulation generally alter the voltage sensitivities of channels. A class of voltage-insensitive channels, first discovered in retinal rods and olfactory neurons, were recently identified in the heart. These channels are opened by the direct binding of cyclic nucleotides, providing a means of regulating ionic currents outside the influence of membrane voltage. Since different isoforms have different affinities for cAMP and cGMP, it is important to determine which isoforms are expressed in heart in order to predict their roles in heart function. We have cloned the olfactory channel from mouse heart, and find that although the message is very rare, Western blot analysis indicates the olfactory channel protein is stable in heart sarcolemma. Our data also suggest the olfactory channel protein forms homomeric channels in the heart since other isoforms or splice variants were not detected either by PCR amplification or by RNase protection. In addition, we have isolated and sequenced the mouse olfactory cyclic nucleotide-gated channel gene, and show the genomic organization is remarkably similar to that found in the human retinal channel gene. Part of this work was presented in abstract form.     

Synaptic tagging: implications for late maintenance of hippocampal long-term potentiation

A novel property of hippocampal LTP, 'variable persistence', has recently been described that is, we argue, relevant to the role of LTP in information storage. Specifically, new results indicate that a particular pattern of synaptic activation can give rise, either to a relatively short-lasting LTP, or to a longer-lasting LTP as a function of the history of activation of the neuron. This has led to the idea that the induction of LTP is associated with the setting of a'synaptic tag' at activated synapses, whose role is to sequester plasticity-related proteins that then serve to stabilize temporary synaptic changes and so extend their persistence. In this article, we outline the synaptic tag hypothesis, compare predictions it makes with those of other theories about the persistence of LTP, and speculate about the cellular identity of the tag. In addition, we outline the requirement for aminergic activation to induce late LTP and consider the functional implications of the synaptic tag hypothesis with respect to long-term memory.     

An isoform of the rod photoreceptor cyclic nucleotide-gated channel beta subunit expressed in olfactory neurons

Sensory transduction in olfactory neurons involves the activation of a cyclic nucleotide-gated (CNG) channel by cAMP. Previous studies identified a CNG channel alpha subunit (CNG2) and a beta subunit (CNG5), which when heterologously expressed form a channel with properties similar but not identical to those of native olfactory neurons. We have cloned a new type of CNG channel beta subunit (CNG4. 3) from rat olfactory epithelium. CNG4.3 derives from the same gene as the rod photoreceptor beta subunit (CNG4.1) but lacks the long, glutamic acid-rich domain found in the N terminus of CNG4.1. Northern blot and in situ hybridization revealed that CNG4.3 is expressed specifically in olfactory neurons. Expression of CNG4.3 in human embryonic kidney 293 cells did not lead to detectable currents. Coexpression of CNG4.3 with CNG2 induced a current with significantly increased sensitivity for cAMP whereas cGMP affinity was not altered. Additionally, CNG4.3 weakened the outward rectification of the current in the presence of extracellular Ca2+, decreased the relative permeability for Ca2+, and enhanced the sensitivity for L-cis diltiazem. Upon coexpression of CNG2, CNG4.3, and CNG5, a conductance with a cAMP sensitivity greater than that of either the CNG2/CNG4.3 or the CNG2/CNG5 channel and near that of native olfactory channel was observed. Our data suggest that CNG4.3 forms a subunit of the native olfactory CNG channel. The expression of various CNG4 isoforms in retina and olfactory epithelium indicates that the CNG4 subunit may be necessary for normal function of both photoreceptor and olfactory CNG channels.     

Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels

Native cylic nucleotide-gated (CNG) channels are composed of alpha and beta subunits. Olfactory CNG channels were expressed from rat cDNA clones in Xenopus oocytes and studied in inside-out patches. Using tandem dimers composed of linked subunits, we investigated the stoichiometry and arrangement of the alpha and beta subunits. Dimers contained three subunit types: alphawt, betawt, and alpham. The alpham subunit lacks an amino-terminal domain that greatly influences gating, decreasing the apparent affinity of the channel for ligand by 9-fold, making it a reporter for inclusion in the tetramer. Homomeric channels from injection of alphawtalphawt dimers and from alphawt monomers were indistinguishable. Channels from injection of alphawtalpham dimers had apparent affinities 3-fold lower than alphawt homomultimers, suggesting a channel with two alphawt and two alpham subunits. Channels from coinjection of alphawtalphawt and betabeta dimers were indistinguishable from those composed of alpha and beta monomers and shared all of the characteristics of the alpha+beta phenotype of heteromeric channels. Coinjection of alphawtalpham and beta beta dimers yielded channels also of the alpha+beta phenotype but with an apparent affinity 3-fold lower, indicating the presence of alpham in the tetramer and that alpha+beta channels have adjacent alpha-subunits. To distinguish between an alpha-alpha-alpha-beta and an alpha-alpha-beta-beta arrangement, we compared apparent affinities for channels from coinjection of alphawtalphawt and betaalphawt or alphawtalphawt and betaalpham dimers. These channels were indistinguishable. To further argue against an alpha-alpha-alpha-beta arrangement, we quantitatively compared dose-response data for channels from coinjection of alphawtalpham and beta beta dimers to those from alpha and beta monomers. Taken together, our results are most consistent with an alpha-alpha-beta-beta arrangement for the heteromeric olfactory CNG channel.     

Cognitive enhancers and hippocampal long-term potentiation in vitro

We review our works on the pharmacological modulation of long-term potentiation (LTP) at guinea pig hippocampal mossy fiber-CA3 synapses in vitro. The magnitude of tetanus-induced LTP at the mossy fiber synapse was augmented by perfusion of slices with several cognitive enhancers, such as bifemelane (1 microM). The mossy fiber LTP was enhanced by somatostatin (0.32 microM) and inhibited in somatostatin-depleted slices from cysteamine-treated guinea pigs. An involvement of the 5-HT3 receptor also showed that granisetron (0.1 microM) enhanced the mossy fiber LTP. The above-mentioned enhancements by perfused agents were commonly reversed, at least in part, by muscarinic antagonists. However, the magnitude of mossy fiber LTP was bidirectionally modulated by muscarinic stimulations of slices with physostigmine or carbachol at different concentrations. The enhancing effects of high-concentration carbachol was antagonized by pirenzepine, and in contrast, the inhibition by low-concentration carbachol was antagonized in the presence of AF-DX116. When guinea pigs were preinjected with the cholinotoxin AF64A, the magnitude of LTP was decreased in the slices prepared from AF64A-treated animals. These results suggest that endogenous acetylcholine dominantly plays facilitatory roles through muscarinic M1 receptors in the induction of mossy fiber LTP. The pharmacological characterization of mossy fiber LTP may be of help to the evaluation of cognitive enhancers at a neuronal circuit level.     

Acute alcohol blocks neurosteroid modulation of synaptic transmission and long-term potentiation in the rat hippocampal slice

Effects of ethanol (22 mM) on the modulation of synaptic transmission and long-term potentiation (LTP) by the neurosteroid dehydroepiandrosterone sulfate (DHEAS; 10 microM) was examined in the in vitro rat hippocampal slice preparation. The synaptic responses were elicited by Schaffer collateral stimulation and recorded extracellularly in the somatic and dendritic regions of CA1 pyramidal neurons. LTP induction produced an increase (approximately 55% to 75%) in the amplitude of synaptic responses in ethanol and ethanol plus DHEAS (ethanol/DHEAS) treated slices. These increases were significantly smaller than the approximately 130% increase observed previously in slices treated with DHEAS, but were not significantly different from the approximately 82% increase observed in control slices. These results indicate that an ethanol/DHEAS interaction prevents the enhancement of LTP normally observed with DHEAS treatment of hippocampal slices. An ethanol/DHEAS interaction also altered DHEAS's effects on individual synaptic components of the synaptic response to Schaffer collateral stimulation. Ethanol applied before but not after DHEAS prevented DHEAS's enhancement of the NMDA receptor-mediated synaptic component. DHEAS's depression of the GABAA receptor-mediated synaptic component was also blocked by ethanol. Ethanol or DHEAS individually had no effect on the AMPA receptor-mediated synaptic component, but application of ethanol after DHEAS resulted in a small enhancement of this synaptic component, an effect that was not observed if ethanol was applied before DHEAS. These results show that ethanol and DHEAS interact, altering DHEAS's effects on synaptic transmission and LTP in the hippocampus. Such an interaction may be involved in ethanol's actions on the CNS and raises the possibility that ethanol and DHEAS may act via a common site or pathway.     

Modified hippocampal long-term potentiation in PKC gamma-mutant mice

Calcium-phospholipid-dependent protein kinase (PKC) has long been suggested to play an important role in modulating synaptic efficacy. We have created a strain of mice that lacks the gamma subtype of PKC to evaluate the significance of this brain-specific PKC isozyme in synaptic plasticity. Mutant mice are viable, develop normally, and have synaptic transmission that is indistinguishable from wild-type mice. Long-term potentiation (LTP), however, is greatly diminished in mutant animals, while two other forms of synaptic plasticity, long-term depression and paired-pulse facilitation, are normal. Surprisingly, when tetanus to evoke LTP was preceded by a low frequency stimulation, mutant animals displayed apparently normal LTP. We propose that PKC gamma is not part of the molecular machinery that produces LTP but is a key regulatory component.     

Src activation in the induction of long-term potentiation in CA1 hippocampal neurons

Long-term potentiation (LTP) is an activity-dependent strengthening of synaptic efficacy that is considered to be a model of learning and memory. Protein tyrosine phosphorylation is necessary to induce LTP. Here, induction of LTP in CA1 pyramidal cells of rats was prevented by blocking the tyrosine kinase Src, and Src activity was increased by stimulation producing LTP. Directly activating Src in the postsynaptic neuron enhanced excitatory synaptic responses, occluding LTP. Src-induced enhancement of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor-mediated synaptic responses required raised intracellular Ca2+ and N-methyl-D-aspartate (NMDA) receptors. Thus, Src activation is necessary and sufficient for inducing LTP and may function by up-regulating NMDA receptors.     

Calcium-dependent modulation of the agonist affinity of the mammalian olfactory cyclic nucleotide-gated channel by calmodulin and a novel endogenous factor

Capillary electrophoresis has been successfully employed to determine the level of drugs in a variety of pharmaceutical preparations. A large number of reports have shown agreement between CE results and HPLC data or with label claim. Currently the use of CE for main component assays constitutes 26% of the routine usage of CE within drug companies and is the most frequent application. The choice between adopting CE or HPLC for a particular application is very dependent upon the relative merits of each technique to the individual assay. Often, CE can have advantages in terms of reduced sample pretreatment, consumable costs, and analysis time. The ability to separate a wide range of solutes using a single set of operating conditions is a strong advantage of CE. This paper extensively reviews the literature reports of the use of CE for main peak assay and indicates the validation performance data achieved. The specific requirements relating to optimized accuracy and precision in CE assay are discussed in some detail. The use of an appropriate internal standard to improve performance for precision, accuracy, and linearity, and to reduce the impact of sample matrix effects, is experimentally shown by results from the analysis of levothyroxine samples. The applications are subdivided into those samples analyzed by free solution capillary electrophoresis (FSCE) at low or high pH or those separated by micellar electrokinetic capillary electrophoresis (MECC).     

Aging differentially alters forms of long-term potentiation in rat hippocampal area CA1

Long-term potentiation (LTP) of the Schaffer collateral/commissural inputs to CA1 in the hippocampus was shown to consist of N-methyl-D-aspartate receptor (NMDAR) and voltage-dependent calcium channel (VDCC) dependent forms. In this study, the relative contributions of these two forms of LTP in in vitro hippocampal slices from young (2 mo) and old (24 mo) Fischer 344 rats were examined. Excitatory postsynaptic potentials (EPSP) were recorded extracellularly from stratum radiatum before and after a tetanic stimulus consisting of four 200-Hz, 0.5-s trains given 5 s apart. Under control conditions, a compound LTP consisting of both forms was induced and was similar, in both time course and magnitude, in young and old animals. NMDAR-dependent LTP (nmdaLTP), isolated by the application of 10 microM nifedipine (a voltage-dependent calcium channel blocker), was significantly reduced in magnitude in aged animals. The VDCC dependent form (vdccLTP), isolated by the application of 50 microM D,L-2-amino-5-phosphonvalerate (APV), was significantly larger in aged animals. Although both LTP forms reached stable values 40-60 min posttetanus in young animals, in aged animals vdccLTP increased and nmdaLTP decreased during this time. In both young and old animals, the sum of the two isolated LTP forms approximated the magnitude of the compound LTP, and application of APV and nifedipine or genestein (a tyrosine kinase inhibitor) together blocked potentiation. These results suggest that aging causes a shift in synaptic plasticity from NMDAR-dependent mechanisms to VDCC-dependent mechanisms. The data are consistent with previous findings of increased L-type calcium current and decreased NMDAR number in aged CA1 cells and may help explain age-related deficits in learning and memory.     

Galanin inhibits long-term potentiation at Schaffer collateral-CA1 synapses in guinea-pig hippocampal slices

CNSs AND OTHER Master's-prepared nurses are usually expected to lead research utilization (RU) efforts in organizations, but they often feel unprepared to do so. In this article, the need for clinical RU is discussed and ideas for implementation, using an adaptation of the Conduct and Utilization of Research in Nursing model, are described. The RU process is applied to research about once-a-day temperatures in afebrile patients. To assist nurses who are implementing research-based practice, we list studies that were used for the practice change, summarize the research base on the topic, and offer an example of clinical use of practice guidelines.     

Amygdala beta-noradrenergic influence on hippocampal long-term potentiation in vivo

We have previously shown that hippocampal long-term potentiation (LTP), one form of synaptic plasticity that may underlie learning and memory, is attenuated by blocking neuron activity of the basolateral amygdala (BLA). In the present study we investigated the amygdala noradrenergic or cholinergic contribution to hippocampal LTP formation. When propranolol, a beta-adrenoceptor antagonist, was injected into the BLA 10 min before tetanus, the formation of LTP in the perforant path-dentate granule cell synapses was significantly impaired. Scopolamine, a muscarinic cholinergic receptor antagonist, did not affect the formation of LTP. These results suggest that amygdala beta-noradrenergic activity plays a critical role in modulation of hippocampal LTP.     

Spatial working memory is independent of hippocampal CA1 long-term potentiation in rats.

This study investigated the relationship between spatial working memory and hippocampal long-term potentiation (LTP) using the allocentric place discrimination task (APDT) in rats, in which the selection accuracy is a good index for spatial working memory. Either the selective M1 muscarinic receptor antagonist pirenzepine (50 μg) or the choline uptake inhibitor hemicholinium-3 (5 μg) impaired APDT selection accuracy, but neither affected the induction of LTP in the hippocampal CA1 region in anesthetized rats. In contrast, the selective N-methyl-{d}-aspartate receptor antagonist D-amino-5-phosphonopentanoate (200 nmol) did not impair APDT selection accuracy but completely blocked hippocampal CA1 LTP. These results suggest that spatial working memory is independent of hippocampal CA1 LTP and that the central cholinergic system is involved in spatial working memory, but not through the modulation of hippocampal CA1 LTP. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Protected-site phosphorylation of protein kinase C in hippocampal long-term potentiation

One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the alpha and betaII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a "protected site" model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.     

The projection from hippocampal area CA1 to the subiculum sustains long-term potentiation

Long-term potentiation (LTP) is a popular model of the synaptic plasticity which may be engaged by the biological processes underlying learning and memory. Most available studies of LTP have concentrated on the analysis of LTP occurring in 'early' components of the hippocampal circuit (for example, dentate gyrus and area CA1). We examine here, for the first time, LTP as it occurs in the massive, unidirectional projection from CA1 to the subiculum in vivo. We show that this projection sustains high-frequency stimulus-induced LTP (10 trains of 20 stimuli at 200 Hz; intertrain interval 2 s; LTP 181 +/- 9% at 30 min post-LTP induction). In addition, input-output (I/O) curves show a leftward shift for all stimulation values.     

Early undernutrition impairs hippocampal long-term potentiation in adult rats.

Investigated the effects of early malnutrition on the ability to establish and maintain hippocampal long-term potentiation (LTP), a relatively permanent enhancement of synaptic and cellular responses induced by high-frequency stimulation. Six control male rats and 8 previously undernourished Ss were used in the acute preparation, and 4 controls and 6 previously undernourished Ss in the chronic preparation. Following high-frequency stimulation of hippocampal dentate granule cells, potentiation was difficult to achieve in undernourished Ss. LTP showed a significant decline within 3–6 hrs and was completely absent at 24 hrs. Further trains of stimulation resulted in only small benefits in undernourished Ss. Coupled with previously reported morphological and behavioral deficits, these findings indicate a marked hippocampal dysfunction resulting from early undernutrition and provide a potentially valuable approach for relating nutritionally induced behavioral impairments to brain function. (26 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Involvement of dendritic adhesion molecule telencephalin in hippocampal long-term potentiation

Telencephalin (TLCN) is a cell adhesion molecule belonging to the immunoglobulin superfamily whose expression is restricted to neurons within the most highly developed brain segment, telencephalon. Immunoelectronmicroscopic study revealed that in the hippocampal CA1 region, TLCN was localized at the surface membrane of postsynaptic spines of pyramidal cell dendrites but not at that of axonal terminals. Blocking of TLCN function using anti-TLCN antibody or recombinant soluble TLCN protein caused a striking suppression of the long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses. The suppression was observed even when the blocking was initiated immediately after the tetanic stimuli. These observations suggest a role for TLCN-mediated cell-cell interactions as a key step in the development of LTP.     

Molecular and pharmacological analysis of cyclic nucleotide-gated channel function in the central nervous system

Most functional studies of cyclic nucleotide-gated (CNG) channels have been confined to photoreceptors and olfactory epithelium, in which CNG channels are abundant and easy to study. The widespread distribution of CNG channels in tissues throughout the body has only recently been recognized and the functions of this channel family in many of these tissues remain largely unknown. The molecular biological and pharmacological properties of the CNG channel family are summarized in order to put in context studies aimed at probing CNG channel functions in these tissues using pharmacological and genetic methods. Compounds have now been identified that are useful in distinguishing CNG channel activated pathways from cAMP/cGMP dependent-protein kinases or other pathways. The ways in which these interact with CNG channels are understood and this knowledge is leading to the identification of more potent and more specific CNG channel subtype-specific agonists or antagonists. Recent molecular and genetic analyses have identified novel roles of CNG channels in neuronal development and plasticity in both invertebrates and vertebrates. Targeting CNG channels via specific drugs and genetic manipulation (such as knockout mice) will permit better understanding of the role of CNG channels in both basic and higher orders of brain function.     

Endogenous serine protease inhibitor modulates epileptic activity and hippocampal long-term potentiation

Protease nexin-1 (PN-1), a member of the serpin superfamily, controls the activity of extracellular serine proteases and is expressed in the brain. Mutant mice overexpressing PN-1 in brain under the control of the Thy-1 promoter (Thy 1/PN-1) or lacking PN-1 (PN-1-/-) were found to develop epileptic activity in vivo and in vitro. Theta burst-induced long-term potentiation (LTP) and NMDA receptor-mediated synaptic transmission in the CA1 field of hippocampal slices were augmented in Thy 1/PN-1 mice and reduced in PN-1-/- mice. Compensatory changes in GABA-mediated inhibition in Thy 1/PN-1 mice suggest that altered brain PN-1 levels lead to an imbalance between excitatory and inhibitory synaptic transmission.