Modulation of Excitatory Synaptic Transmission During Cannabinoid Receptor Activation.

The present research has reported that cannabinoid receptor 1 (CB1) agonist, delta-(9)-tetrahydrocannabinol (THC) modulates synaptogenesis during overexcitation. Microtubule and synaptic distribution, poly(ADP)-ribose (PAR) accumulation were estimated during overexcitation and in the presence of THC. Low concentration of THC (10 nM) increased synaptophysin expression and neurite length, while high concentration of THC (1 µM) induced neurotoxicity. Glutamate caused the loss of neurons, reducing the number and the length of neurites. The high concentration of THC in the presence of glutamate caused the PAR accumulation in the condensed nuclei. Glutamate upregulated genes that are involved in synaptogenesis and excitatory signal cascade. Glutamate downregulated transcription of beta3 tubulin and microtubule-associated protein 2. THC partially regulated gene expression that is implicated in the neurogenesis and excitatory pathways. This suggests that CB1 receptors play a role in neurite growth and the low concentration of THC protects neurons during overexcitation, whereas the high concentration of THC enhances the neurotoxicity.

Ca2+ dependent surface trafficking of norepinephrine transporters depends on threonine 30 and Ca2+ calmodulin kinases.

The antidepressant-sensitive norepinephrine (NE) transporter (NET) inactivates NE released during central and peripheral neuronal activity by transport into presynaptic cells. Altered NE clearance due to dysfunction of NET has been associated with the development of mental illness and cardiovascular diseases. NET activity in vivo is influenced by stress, neuronal activity, hormones and drugs. We investigated the mechanisms of Ca2+ regulation of NET and found that Ca2+ influenced both Vmax and Km for NE transport into cortical synaptosomes. Changes in extracellular Ca2+ triggered rapid and bidirectional surface trafficking of NET expressed in cultured cells. Deletion of residues 28-47 in the NET NH2-terminus abolished the Ca2+ effect on surface trafficking. Mutagenesis studies identified Thr30 in this region as the essential residue for both Ca2+- dependent phosphorylation and trafficking of NET. Depolarization of excitable cells increased surface NET in a Thr30 dependent manner. A proteomic analysis, RNA interference, and pharmacological inhibition supported roles of CaMKI and CaMKII in Ca2+-modulated NE transport and NET trafficking. Depolarization of primary noradrenergic neurons in culture with elevated K+ increased NET surface expression in a process that required external Ca2+ and depended on CaMK activity. Hippocampal NE clearance in vivo was also stimulated by depolarization, and inhibitors of CaMK signaling prevented this stimulation. In summary, Ca2+ signaling influenced surface trafficking of NET through a CaMK-dependent mechanism requiring Thr30.

Regulation of NADPH oxidase gene expression with PKA and cytokine IL-4 in neurons and microglia.

Neuronal excitation is mediated by the activation of NMDA receptor and associated with the formation of reactive oxygen species due to the activation of NADPH oxidase complex proteins. The activation of Gs protein coupled receptors (GPCRs) induces neuronal activation in the cAMP-dependent protein kinase A (PKA)-mediated signal cascade and regulates NADPH oxidase activity. However, it is unknown whether PKA regulates NADPH oxidase gene expression in neurons and microglia. In the present research, the NADPH oxidase gene expression was studied in rat cortical neurons and microglia in vitro. Purified microglial cells were identified with OX-42 antibody and they also expressed apolipoprotein E (ApoE). The time-dependent effect of cytokine interleukin-4 (IL-4) (20 ng/ml) in NADPH oxidase gene expression was studied in microglial cells. The levels of mRNA were determined by quantitative RT-PCR. The expression of NOX1, NOX2, and NCF2 was upregulated after IL-4 treatment for 4 h, but it was downregulated after 8-24 h. The expression of NCF1 was suppressed during any time of cytokine effect. IL-4 upregulated arginase1 (Arg1) and serine racemase1 (SRR1) gene expressions in microglia. Amyloid beta (Ab) suppressed NOX2, NCF1, and NCF2 gene expressions and upregulated glutamate cystine transporter (xCT), although IL-4 attenuated the effect of Ab (500 µM) in the upregulation of xCT gene expression. The activation of PKA with agonist dibutyryl cAMP (dbcAMP) (100 µM) induced the upregulation of Arg1 gene expression in microglia involving in the process of microglial activation. The transcription of NOX1, NOX2, and NCF1 was suppressed in microglial cells after dbcAMP treatment within 24 h. Neurons were identified with the microtubule-associated protein tau. The uniform distribution of tau along axons was established in normal neurons. Tau protein was redistributed after PKA agonist dbcAMP treatment for 24 h. L-glutamate (50 µM) caused the apoptotic processes and the accumulation of tau in the soma of neurons and along axons. The activation of PKA for 24 h induced the transcriptional upregulation of NOX1 and NCF1 in cortical neurons. However, L-glutamate suppressed NOX1 gene expression in neurons. These data demonstrate that the effects of IL-4 and dbcAMP are similar in the regulation of SRR1, Arg1, and NADPH oxidase complex gene expressions in neurons and microglia. IL-4 prevents glutamate release from microglia suppressing xCT expression induced by Ab. These findings suggest that the activation of GPCR in PKA-mediated pathway leads to transcriptional regulation of NADPH oxidase complex. The modulation of GPCR activation may inhibit the oxidative stress in neurons.

Regulation of neuronal activation by Alpha2A adrenergic receptor agonist.

Stress factors induce neuronal activation in brain areas that are related to anxiety and fear. High doses of caffeine induce neuronal activation with Ca2+ influx followed by expression of the immediate early gene c-fos. In the present study, we investigated c-Fos protein expression in stress-responsive brain areas induced by caffeine, as well as the role of alpha2A receptor in the regulation of neuronal activation. Immunohistochemical analysis showed that an acute effect of caffeine induced c-Fos protein expression in the hippocampus, the bed nucleus of stria terminalis (BNST), the lateral septum, the basolateral and central amygdala, the paraventricular hypothalamic nucleus (PVN), the locus coeruleus, and the lateral parabrachial nucleus (LPBN). However, c-Fos expression was attenuated after repeated treatment of caffeine, spaced 24 h apart, compared to a single acute effect. Alpha2A receptor activation with the agonist guanfacine attenuated the acute effect of caffeine in terms of c-Fos expression in neurons in the CA1-CA3 areas of hippocampus, the locus coeruleus and the LPBN as compared with effect of caffeine alone, whereas the number of c-Fos expressing neurons increased in the lateral septum, the dorsal BNST, the central amygdala, and the PVN, areas that are densely innervated by noradrenergic neurons. Guanfacine alone induced c-Fos protein expression in neurons in the central amygdala, the dorsal BNST, the PVN, the LPBN, and the caudal nucleus of the solitary tract. Guanfacine alone also induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in neurons expressing c-Fos in the dorsal BNST, the central amygdala, and the LPBN. These results suggest that alpha2A receptor activation modulates synaptic transmission in neuronal circuits that are correlated with stress in vivo.

Manganese exposure is cytotoxic and alters dopaminergic and GABAergic neurons within the basal ganglia.

Manganese is an essential nutrient, integral to proper metabolism of amino acids, proteins and lipids. Excessive environmental exposure to manganese can produce extrapyramidal symptoms similar to those observed in Parkinson's disease (PD). We used in vivo and in vitro models to examine cellular and circuitry alterations induced by manganese exposure. Primary mesencephalic cultures were treated with 10-800 microM manganese chloride which resulted in dramatic changes in the neuronal cytoskeleton even at subtoxic concentrations. Using cultures from mice with red fluorescent protein driven by the tyrosine hydroxylase (TH) promoter, we found that dopaminergic neurons were more susceptible to manganese toxicity. To understand the vulnerability of dopaminergic cells to chronic manganese exposure, mice were given i.p. injections of MnCl(2) for 30 days. We observed a 20% reduction in TH-positive neurons in the substantia nigra pars compacta (SNpc) following manganese treatment. Quantification of Nissl bodies revealed a widespread reduction in SNpc cell numbers. Other areas of the basal ganglia were also altered by manganese as evidenced by the loss of glutamic acid decarboxylase 67 in the striatum. These studies suggest that acute manganese exposure induces cytoskeletal dysfunction prior to degeneration and that chronic manganese exposure results in neurochemical dysfunction with overlapping features to PD.

Norepinephrine modulates glutamatergic transmission in the bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BNST) and its adrenergic input are key components in stress-induced reinstatement and maintenance of drug use. Intra-BNST injections of either beta-adrenergic receptor (beta-AR) antagonists or alpha2-adrenergic receptor (alpha2-AR) agonists can inhibit footshock-induced reinstatement and maintenance of cocaine- and morphine-seeking. Using electrophysiological recording methods in an in vitro slice preparation from C57/Bl6j adult male mouse BNST, we have examined the effects of adrenergic receptor activation on excitatory synaptic transmission in the lateral dorsal supracommissural BNST (dBNST) and subcommissural BNST (vBNST). Alpha2-AR activation via UK-14,304 (10 microM) results in a decrease in excitatory transmission in both dBNST and vBNST, an effect predominantly dependent upon the alpha2A-AR subtype. Beta-AR activation via isoproterenol (1 microM) results in an increase in excitatory transmission in dBNST, but not in vBNST. Consistent with the work with receptor subtype specific agonists, application of the endogenous ligand norepinephrine (NE, 100 microM) elicits two distinct effects on glutamatergic transmission. In dBNST, NE elicits an increase in transmission (62% of dBNST NE experiments) or a decrease in transmission (38% of dBNST NE experiments). In vBNST, NE elicits a decrease in transmission in 100% of the experiments. In dBNST, the NE-induced increase in synaptic transmission is blocked by beta1/beta2- and beta2-, but not beta1-specific antagonists. In addition, this increase is also reduced by the alpha2-AR antagonist yohimbine and is absent in the alpha2A-AR knockout mouse. In vBNST, the NE-induced decrease in synaptic transmission is markedly reduced in the alpha2A-AR knockout mouse. Further experiments demonstrate that the actions of NE on glutamatergic transmission can be correlated with beta-AR function.

Lethal impairment of cholinergic neurotransmission in hemicholinium-3-sensitive choline transporter knockout mice.

Presynaptic acetylcholine (ACh) synthesis and release is thought to be sustained by a hemicholinium-3-sensitive choline transporter (CHT). We disrupted the murine CHT gene and examined CHT-/- and +/- animals for evidence of impaired cholinergic neurotransmission. Although morphologically normal at birth, CHT-/- mice become immobile, breathe irregularly, appear cyanotic, and die within an hour. Hemicholinium-3-sensitive choline uptake and subsequent ACh synthesis are specifically lost in CHT-/- mouse brains. Moreover, we observe a time-dependent loss of spontaneous and evoked responses at CHT-/- neuromuscular junctions. Consistent with deficits in synaptic ACh availability, we also observe developmental alterations in neuromuscular junction morphology reminiscent of changes in mutants lacking ACh synthesis. Adult CHT+/- mice overcome reductions in CHT protein levels and sustain choline uptake activity at wild-type levels through posttranslational mechanisms. Our results demonstrate that CHT is an essential and regulated presynaptic component of cholinergic signaling and indicate that CHT warrants consideration as a candidate gene for disorders characterized by cholinergic hypofunction.

Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.

The role of acetylcholine (ACh) as a key neurotransmitter in the central and peripheral nervous system is well established. However, the role of ACh may be broader because ACh may also function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues. To begin to establish ACh of nonneuronal origin as a paracrine hormone in lung, we have examined neonatal and adult monkey bronchial epithelium for the components involved in nicotinic cholinergic signaling. Using immunohistochemistry and RT-PCR, we have demonstrated in lung bronchial epithelial cells (BECs) expression of choline acetyltransferase, the vesicular ACh transporter, the choline high-affinity transporter, alpha7, alpha4, and beta2 nicotinic ACh receptor (nAChR) subunits, and the nAChR accessory protein lynx1. Confocal microscopy demonstrates that these factors are expressed in epithelial cells and are clearly distinct from neighboring nerve fibers. Confirmation of RNA identity has been confirmed by partial sequence analysis of PCR products and by cDNA cloning. Primary culture of BECs confirms the synthesis and secretion of ACh and the activity of cholinesterases. Thus, ACh meets all the criteria for an autocrine/paracrine hormone in lung bronchial epithelium. The nonneuronal cholinergic signaling pathway in lung provides a potentially important target for cholinergic drugs. This pathway may also explain some of the effects of nicotine on fetal development and also provides additional mechanisms by which smoking affects lung cancer growth and development.

Cell surface trafficking of the antidepressant-sensitive norepinephrine transporter revealed with an ectodomain antibody.

The antidepressant-sensitive L-norepinephrine (NE) transporter (NET;SLC6A2) is a critical determinant of neurotransmitter inactivation following NE release at synapses. Although regulated trafficking of NET has been documented in transfected cells, a lack of reagents suitable for reporting native NET surface exposition has limited validation of this concept in neurons. In the current report, we document the utility of a novel antibody (43408) directed at conserved sequences in the NET second extracellular loop. Using human NET (hNET) stably transfected cells, we document loss of NET surface expression following acute (30 min) phorbol ester treatments. In superior cervical ganglion (SCG) cultures, NET surface expression is prominent on varicosities defined by FM1-43 labeling of living neurons or synaptophysin labeling of fixed preparations. Moreover, NET surface density can be rapidly augmented by brief depolarization (5 min, 40 mM K(+)). Similarly, in brainstem cultures, we demonstrate an increase in NET surface labeling following either depolarization or angiotensin II stimulation. These findings provide the first evidence for regulated trafficking of NET in neurons and support the suggestion that activity-dependent NET trafficking may provide additional modulatory capacity for noradrenergic signaling.

Vesicular localization and activity-dependent trafficking of presynaptic choline transporters.

Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.

A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity.

Norepinephrine (NE) transporters (NETs) terminate noradrenergic synaptic transmission and represent a major therapeutic target for antidepressant medications. NETs and related transporters are under intrinsic regulation by receptor and kinase-linked pathways, and clarification of these pathways may suggest candidates for the development of novel therapeutic approaches. Syntaxin 1A, a presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, interacts with NET and modulates NET intrinsic activity. NETs colocalize with and bind to syntaxin 1A in both native preparations and heterologous systems. Protein kinase C activation disrupts surface NET/syntaxin 1A interactions and downregulates NET activity in a syntaxin-dependent manner. Syntaxin 1A binds the NH(2) terminal domain of NET, and a deletion of this domain both eliminates NET/syntaxin 1A associations and prevents phorbol ester-triggered NET downregulation. Whereas syntaxin 1A supports the surface trafficking of NET proteins, its direct interaction with NET limits transporter catalytic function. These two contradictory roles of syntaxin 1A on NET appear to be linked and reveal a dynamic cycle of interactions that allow for the coordinated control between NE release and reuptake.