Heightened sympathetic neuron activity and altered cardiomyocyte properties in spontaneously hypertensive rats during the postnatal period.

The Spontaneously Hypertensive Rat (SHR) has increased sympathetic drive to the periphery that precedes and contributes to the development of high blood pressure, making it a useful model for the study of neurogenic hypertension. Comparisons to the normotensive Wistar Kyoto (WKY) rat have demonstrated altered active and intrinsic properties of SHR sympathetic neurons shortly before the onset of hypertension. Here we examine the structural and functional plasticity of postnatal SHR and WKY sympathetic neurons cultured alone or co-cultured with cardiomyocytes under conditions of limited extrinsic signaling. SHR neurons have an increased number of structural synaptic sites compared to age-matched WKY neurons, measured by the co-localization of presynaptic vesicular acetylcholine transporter and postsynaptic shank proteins. Whole cell recordings show that SHR neurons have a higher synaptic charge than WKY neurons, demonstrating that the increase in synaptic sites is associated with increased synaptic transmission. Differences in synaptic properties are not associated with altered firing rates between postnatal WKY and SHR neurons and are not influenced by interactions with target cardiomyocytes from either strain. Both SHR and WKY neurons show tonic firing patterns in our cultures, which are depleted of non-neuronal ganglionic cells and provide limited neurotrophic signaling. This suggests that the normal mature, phasic firing of sympathetic neurons requires extrinsic signaling, with potentially differential responses in the prehypertensive SHR, which have been reported to maintain tonic firing at later developmental stages. While cardiomyocytes do not drive neuronal differences in our cultures, SHR cardiomyocytes display decreased hypertrophy compared to WKY cells and altered responses to co-cultured sympathetic neurons. These experiments suggest that altered signaling in SHR neurons and cardiomyocytes contributes to changes in the cardiac-sympathetic circuit in prehypertensive rats as early as the postnatal period.

Satellite glial cells modulate cholinergic transmission between sympathetic neurons.

Postganglionic sympathetic neurons and satellite glial cells are the two major cell types of the peripheral sympathetic ganglia. Sympathetic neurons project to and provide neural control of peripheral organs and have been implicated in human disorders ranging from cardiovascular disease to peripheral neuropathies. Here we show that satellite glia regulate synaptic activity of cultured postnatal sympathetic neurons, providing evidence for local ganglionic control of sympathetic drive. In addition to modulating neuron-to-neuron cholinergic neurotransmission, satellite glia promote synapse formation and contribute to neuronal survival. Examination of the cellular architecture of the rat sympathetic ganglia in vivo shows this regulation of neuronal properties takes place during a developmental period in which neuronal morphology and density are actively changing and satellite glia enwrap sympathetic neuronal somata. Cultured satellite glia make and release factors that promote neuronal activity and that can partially rescue the neurons from cell death following nerve growth factor deprivation. Thus, satellite glia play an early and ongoing role within the postnatal sympathetic ganglia, expanding our understanding of the contributions of local and target-derived factors in the regulation of sympathetic neuron function.

Targeted disruption of glycogen synthase kinase-3ß in cardiomyocytes attenuates cardiac parasympathetic dysfunction in type 1 diabetic Akita mice.

Type 1 diabetic Akita mice develop severe cardiac parasympathetic dysfunction that we have previously demonstrated is due at least in part to an abnormality in the response of the end organ to parasympathetic stimulation. Specifically, we had shown that hypoinsulinemia in the diabetic heart results in attenuation of the G-protein coupled inward rectifying K channel (GIRK) which mediates the negative chronotropic response to parasympathetic stimulation due at least in part to decreased expression of the GIRK1 and GIRK4 subunits of the channel. We further demonstrated that the expression of GIRK1 and GIRK4 is under the control of the Sterol Regulatory element Binding Protein (SREBP-1), which is also decreased in response to hypoinsulinemia. Finally, given that hyperactivity of Glycogen Synthase Kinase (GSK)3ß, had been demonstrated in the diabetic heart, we demonstrated that treatment of Akita mice with Li+, an inhibitor of GSK3ß, increased parasympathetic responsiveness and SREBP-1 levels consistent with the conclusion that GSK3ß might regulate IKACh via an effect on SREBP-1. However, inhibitor studies were complicated by lack of specificity for GSK3ß. Here we generated an Akita mouse with cardiac specific inducible knockout of GSK3ß. Using this mouse, we demonstrate that attenuation of GSK3ß expression is associated with an increase in parasympathetic responsiveness measured as an increase in the heart rate response to atropine from 17.3 ± 3.5% (n = 8) prior to 41.2 ± 5.4% (n = 8, P = 0.017), an increase in the duration of carbamylcholine mediated bradycardia from 8.43 ± 1.60 min (n = 7) to 12.71 ± 2.26 min (n = 7, P = 0.028) and an increase in HRV as measured by an increase in the high frequency fraction from 40.78 ± 3.86% to 65.04 ± 5.64 (n = 10, P = 0.005). Furthermore, patch clamp measurements demonstrated a 3-fold increase in acetylcholine stimulated peak IKACh in atrial myocytes from GSK3ß deficiency mice compared with control. Finally, western blot analysis of atrial extracts from knockout mice demonstrated increased levels of SREBP-1, GIRK1 and GIRK4 compared with control. Taken together with our prior observations, these data establish a role of increased GSK3ß activity in the pathogenesis of parasympathetic dysfunction in type 1 diabetes via the regulation of IKACh and GIRK1/4 expression.

Alphaxalone Binds in Inner Transmembrane ß+-α- Interfaces of α1ß3γ2 γ-Aminobutyric Acid Type A Receptors.

BACKGROUND: Neurosteroids like alphaxalone are potent anxiolytics, anticonvulsants, amnestics, and sedative-hypnotics, with effects linked to enhancement of γ-aminobutyric acid type A (GABAA) receptor gating in the central nervous system. Data locating neurosteroid binding sites on synaptic αßγ GABAA receptors are sparse and inconsistent. Some evidence points to outer transmembrane ß-α interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggests that steroids bind more intracellularly in ß-α interfaces. METHODS: The authors created 12 single-residue ß3 cysteine mutations: ß3T262C and ß3T266C in ß3-M2; and ß3M283C, ß3Y284C, ß3M286C, ß3G287C, ß3F289C, ß3V290C, ß3F293C, ß3L297C, ß3E298C, and ß3F301C in ß3-M3 helices. The authors coexpressed α1 and γ2L with each mutant ß3 subunit in Xenopus oocytes and electrophysiologically tested each mutant for covalent sulfhydryl modification by the water-soluble reagent para-chloromercuribenzenesulfonate. Then, the authors assessed whether receptor-bound alphaxalone, etomidate, or propofol blocked cysteine modification, implying steric hindrance. RESULTS: Eleven mutant ß3 subunits, when coexpressed with α1 and γ2L, formed functional channels that displayed varied sensitivities to the three anesthetics. Exposure to para-chloromercuribenzenesulfonate produced irreversible functional changes in ten mutant receptors. Protection by alphaxalone was observed in receptors with ß3V290C, ß3F293C, ß3L297C, or ß3F301C mutations. Both etomidate and propofol protected receptors with ß3M286C or ß3V290C mutations. Etomidate also protected ß3F289C. In α1ß3γ2L structural homology models, all these protected residues are located in transmembrane ß-α interfaces. CONCLUSIONS: Alphaxalone binds in transmembrane ß-α pockets of synaptic GABAA receptors that are adjacent and intracellular to sites for the potent anesthetics etomidate and propofol.

Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

The acetylcholine-activated inward rectifier potassium current ( IKACh) is constitutively active in persistent atrial fibrillation (AF). We tested the hypothesis that the blocking of IKACh with the small molecule chloroquine terminates persistent AF. We used a sheep model of tachypacing-induced, persistent AF, molecular modeling, electrophysiology, and structural biology approaches. The 50% inhibition/inhibitory concentration of IKACh block with chloroquine, measured by patch clamp, was 1 µM. In optical mapping of sheep hearts with persistent AF, 1 µM chloroquine restored sinus rhythm. Molecular modeling suggested that chloroquine blocked the passage of a hydrated potassium ion through the intracellular domain of Kir3.1 (a molecular correlate of IKACh) by interacting with residues D260 and F255, in proximity to I228, Q227, and L299. 1H 15N heteronuclear single-quantum correlation of purified Kir3.1 intracellular domain confirmed the modeling results. F255, I228, Q227, and L299 underwent significant chemical-shift perturbations upon drug binding. We then crystallized and solved a 2.5 Å X-ray structure of Kir3.1 with F255A mutation. Modeling of chloroquine binding to the mutant channel suggested that the drug's binding to the pore becomes off centered, reducing its ability to block a hydrated potassium ion. Patch clamp validated the structural and modeling data, where the F255A and D260A mutations significantly reduced IKACh block by chloroquine. With the use of numerical and structural biology approaches, we elucidated the details of how a small molecule could block an ion channel and exert antiarrhythmic effects. Chloroquine binds the IKACh channel at a site formed by specific amino acids in the ion-permeation pathway, leading to decreased IKACh and the subsequent termination of AF.-Takemoto, Y., Slough, D. P., Meinke, G., Katnik, C., Graziano, Z. A., Chidipi, B., Reiser, M., Alhadidy, M. M., Ramirez, R., Salvador-Montañés, O., Ennis, S., Guerrero-Serna, G., Haburcak, M., Diehl, C., Cuevas, J., Jalife, J., Bohm, A., Lin,Y.-S., Noujaim, S. F. Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

Monoaminergic orchestration of motor programs in a complex C. elegans behavior.

Monoamines provide chemical codes of behavioral states. However, the neural mechanisms of monoaminergic orchestration of behavior are poorly understood. Touch elicits an escape response in Caenorhabditis elegans where the animal moves backward and turns to change its direction of locomotion. We show that the tyramine receptor SER-2 acts through a Gαo pathway to inhibit neurotransmitter release from GABAergic motor neurons that synapse onto ventral body wall muscles. Extrasynaptic activation of SER-2 facilitates ventral body wall muscle contraction, contributing to the tight ventral turn that allows the animal to navigate away from a threatening stimulus. Tyramine temporally coordinates the different phases of the escape response through the synaptic activation of the fast-acting ionotropic receptor, LGC-55, and extrasynaptic activation of the slow-acting metabotropic receptor, SER-2. Our studies show, at the level of single cells, how a sensory input recruits the action of a monoamine to change neural circuit properties and orchestrate a compound motor sequence.

ACR-12 ionotropic acetylcholine receptor complexes regulate inhibitory motor neuron activity in Caenorhabditis elegans.

Heterogeneity in the composition of neurotransmitter receptors is thought to provide functional diversity that may be important in patterning neural activity and shaping behavior (Dani and Bertrand, 2007; Sassoè-Pognetto, 2011). However, this idea has remained difficult to evaluate directly because of the complexity of neuronal connectivity patterns and uncertainty about the molecular composition of specific receptor types in vivo. Here we dissect how molecular diversity across receptor types contributes to the coordinated activity of excitatory and inhibitory motor neurons in the nematode Caenorhabditis elegans. We show that excitatory and inhibitory motor neurons express distinct populations of ionotropic acetylcholine receptors (iAChRs) requiring the ACR-12 subunit. The activity level of excitatory motor neurons is influenced through activation of nonsynaptic iAChRs (Jospin et al., 2009; Barbagallo et al., 2010). In contrast, synaptic coupling of excitatory and inhibitory motor neurons is achieved through a second population of iAChRs specifically localized at postsynaptic sites on inhibitory motor neurons. Loss of ACR-12 iAChRs from inhibitory motor neurons leads to reduced synaptic drive, decreased inhibitory neuromuscular signaling, and variability in the sinusoidal motor pattern. Our results provide new insights into mechanisms that establish appropriately balanced excitation and inhibition in the generation of a rhythmic motor behavior and reveal functionally diverse roles for iAChR-mediated signaling in this process.

Pharmacological studies of methoxycarbonyl etomidate's carboxylic acid metabolite.

BACKGROUND: Methoxycarbonyl etomidate (MOC-etomidate) is a rapidly metabolized and ultrashort-acting etomidate analog that does not produce prolonged adrenocortical suppression after bolus administration. Its metabolite (MOC-ECA) is a carboxylic acid whose pharmacology is undefined. We hypothesized that MOC-ECA possesses significantly lower pharmacological activity than MOC-etomidate, accounting for the latter's very brief duration of hypnotic action and inability to produce prolonged adrenocortical suppression after bolus administration. To test this hypothesis, we compared the potencies of MOC-ECA and MOC-etomidate in 3 biological assays. METHODS: The hypnotic potency of MOC-ECA was assessed in tadpoles using a loss-of-righting reflexes assay. The γ-aminobutyric acid type A (GABA(A)) receptor modulatory potencies of MOC-ECA and MOC-etomidate were compared by defining the concentrations of each required to directly activate α(1)(L264T)ß(2)γ(2L) GABA(A) receptors. The adrenocortical inhibitory potencies of MOC-ECA and MOC-etomidate were compared by defining the concentrations of each required to inhibit in vitro cortisol production by adrenocortical cells. RESULTS: MOC-ECA's 50% effective concentration for loss-of-righting reflexes in tadpoles was 2.8 ± 0.64 mM as compared with a previously reported value of 8 ± 2 µM for MOC-etomidate. The 50% effective concentrations for direct activation of GABA(A) receptors were 3.5 ± 0.63 mM for MOC-ECA versus 10 ± 2.5 µM for MOC-etomidate. The half-maximal inhibitory concentration for inhibiting in vitro cortisol production by adrenocortical cells was 30 ± 7 µM for MOC-ECA versus 0.10 ± 0.02 µM for MOC-etomidate. CONCLUSIONS: In all 3 biological assays, MOC-ECA's potency was approximately 300-fold lower than that of MOC-etomidate.

Impaired dopamine release and synaptic plasticity in the striatum of parkin-/- mice.

Parkin is the most common causative gene of juvenile and early-onset familial Parkinson's diseases and is thought to function as an E3 ubiquitin ligase in the ubiquitin-proteasome system. However, it remains unclear how loss of Parkin protein causes dopaminergic dysfunction and nigral neurodegeneration. To investigate the pathogenic mechanism underlying these mutations, we used parkin-/- mice to study its physiological function in the nigrostriatal circuit. Amperometric recordings showed decreases in evoked dopamine release in acute striatal slices of parkin-/- mice and reductions in the total catecholamine release and quantal size in dissociated chromaffin cells derived from parkin-/- mice. Intracellular recordings of striatal medium spiny neurons revealed impairments of long-term depression and long-term potentiation in parkin-/- mice, whereas long-term potentiation was normal in the Schaeffer collateral pathway of the hippocampus. Levels of dopamine receptors and dopamine transporters were normal in the parkin-/- striatum. These results indicate that Parkin is involved in the regulation of evoked dopamine release and striatal synaptic plasticity in the nigrostriatal pathway, and suggest that impairment in evoked dopamine release may represent a common pathophysiological change in recessive parkinsonism.

Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial Parkinsonism-linked gene DJ-1.

The manifestations of Parkinson's disease are caused by reduced dopaminergic innervation of the striatum. Loss-of-function mutations in the DJ-1 gene cause early-onset familial parkinsonism. To investigate a possible role for DJ-1 in the dopaminergic system, we generated a mouse model bearing a germline disruption of DJ-1. Although DJ-1(-/-) mice had normal numbers of dopaminergic neurons in the substantia nigra, evoked dopamine overflow in the striatum was markedly reduced, primarily as a result of increased reuptake. Nigral neurons lacking DJ-1 were less sensitive to the inhibitory effects of D2 autoreceptor stimulation. Corticostriatal long-term potentiation was normal in medium spiny neurons of DJ-1(-/-) mice, but long-term depression (LTD) was absent. The LTD deficit was reversed by treatment with D2 but not D1 receptor agonists. Furthermore, DJ-1(-/-) mice displayed hypoactivity in the open field. Collectively, our findings suggest an essential role for DJ-1 in dopaminergic physiology and D2 receptor-mediated functions.

Stimulation-dependent regulation of the pH, volume and quantal size of bovine and rodent secretory vesicles.

Trapping of weak bases was utilized to evaluate stimulus-induced changes in the internal pH of the secretory vesicles of chromaffin cells and enteric neurons. The internal acidity of chromaffin vesicles was increased by the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP; in vivo and in vitro) and by high K+ (in vitro); and in enteric nerve terminals by exposure to veratridine or a plasmalemmal [Ca2+]o receptor agonist (Gd3+). Stimulation-induced acidification of chromaffin vesicles was [Ca2+]o-dependent and blocked by agents that inhibit the vacuolar proton pump (vH+-ATPase) or flux through Cl- channels. Stimulation also increased the average volume of chromaffin vesicles and the proportion that displayed a clear halo around their dense cores (called active vesicles). Stimulation-induced increases in internal acidity and size were greatest in active vesicles. Stimulation of chromaffin cells in the presence of a plasma membrane marker revealed that membrane was internalized in endosomes but not in chromaffin vesicles. The stable expression of botulinum toxin E to prevent exocytosis did not affect the stimulation-induced acidification of the secretory vesicles of mouse neuroblastoma Neuro2A cells. Stimulation-induced acidification thus occurs independently of exocytosis. The quantal size of secreted catecholamines, measured by amperometry in cultured chromaffin cells, was found to be increased either by prior exposure to L-DOPA or stimulation by high K+, and decreased by inhibition of vH+-ATPase or flux through Cl- channels. These observations are consistent with the hypothesis that the content of releasable small molecules in secretory vesicles is increased when the driving force for their uptake is enhanced, either by increasing the transmembrane concentration or pH gradients.