Regulation by equilibrative nucleoside transporter of adenosine outward currents in adult rat spinal dorsal horn neurons

A current response induced by superfusing adenosine was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 78% of the neurons examined, adenosine induced an outward current at -70 mV [18.8 +/- 1.1 pA (n = 98) at 1mM] in a dose-dependent manner (EC(50) = 177 microM). A similar current was induced by A(1) agonist N(6)-cyclopentyladenosine (1 microM), whereas A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) reversed the adenosine action. The adenosine current reversed its polarity at a potential being close to the equilibrium potential for K(+), and was attenuated by Ba(2+) (100 microM) and 4-aminopyridine (5mM) but not tetraethylammonium (5mM). The adenosine current was enhanced in duration by equilibrative nucleoside-transport (rENT1) inhibitor S-(4-nitrobenzyl)-6-thioinosine (1 microM) and adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (1 microM), and slowed in falling phase by adenosine kinase (AK) inhibitor iodotubercidine (1 microM). We conclude that a Ba(2+)- and 4-aminopyridine-sensitive K(+) channel in SG neurons is opened via the activation of A(1) receptors by adenosine whose level is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. Considering that intrathecally-administered adenosine analogues produce antinociception, the regulatory systems of adenosine may serve as targets for antinociceptive drugs.     

Tonic regulation of the activity of noradrenergic neurons in the locus coeruleus of the conscious rat studied by dual-probe microdialysis

In the present study, receptor specific compounds were infused via a microdialysis probe in the vicinity of the right locus coeruleus (LC). During the infusion of these compounds, the extracellular content of noradrenaline was recorded in the ipsilateral medial prefrontal cortex (mPFC) with a second microdialysis probe. Agonists and antagonists of various subtypes receptors that have been described to be localized on LC cells, were infused near the LC. The receptors investigated were: alpha2-adrenergic, muscarinic, nicotinic, GABAergic (GABAA and GABAB), glutamatergic (NMDA and non-NMDA). The compounds infused were: clonidine (100 microM), idazoxan (50 microM), bicuculline (50 microM), muscimol (50 microM), baclofen (50 microM), CGP52432 (100 microM), NMDA (300 microM), CPP (300 microM), kainate (100 microM), DNQX (500 microM), oxotremorine (100 microM), atropine (10 microM), nicotine (100 microM) and mecamylamine (100 microM). Evidence was provided that GABAA, NMDA, non-NMDA glutamate, and muscarinic cholinergic receptors in the LC played roles in controlling the activity of noradrenaline neurons. The LC noradrenergic neurons were not tonically excitated by glutamatergic or cholinergic afferent neurons, and were not tonically inhibited by alpha2 autoreceptors. Tonic inhibition was evident for GABAergic neurons, acting via GABAA receptors.     

Hypocretin/orexin depolarizes and decreases potassium conductance in locus coeruleus neurons

Recent studies demonstrated that noradrenergic locus coeruleus (LC) neurons are a particularly strong target of the novel neuropeptide, hypocretin (orexin). The present study sought to elucidate the action of hypocretin-B (HCRT) on LC neurons recorded intracellularly in rat brain slices. Bath (1.0 microM) or local puff application (50-100 microM in pipette) of HCRT depolarized LC neurons in rat brain slices and increased their spontaneous discharge rate. Depolarization evoked by HCRT was persistent in the presence of tetrodotoxin (TTX, 1 microM) and Co2+ (1 mM), indicating that HCRT directly activated LC neurons, and that its effect on the postsynaptic cell was not due to activation of TTX-sensitive sodium channels or Co2+-sensitive calcium channels. The apparent input resistance was significantly increased in the majority of LC neurons during the HCRT-evoked depolarization. Moreover, the HCRT-evoked depolarization was decreased in amplitude with hyperpolarization of membrane. The present results indicate that decreased potassium conductance is involved in the effect of HCRT on LC neurons.     

Functional role of alpha1-adrenoceptors in the locus coeruleus: a microdialysis study

The present study elucidates the role of alpha(1)-adrenoreceptors in the locus coeruleus (LC) using a dual-probe microdialysis in conscious rats. One probe sampled noradrenaline in the LC, whereas the second probe sampled noradrenaline in a main projection area, the prefrontal cortex (PFC). To investigate a possible tonic activation of LC neurons by alpha(1)-adrenoceptor, the alpha(1)-antagonist prazosin (10 microM) was infused into the LC. Extracellular noradrenaline in the LC decreased to about 50% of basal levels but no change of noradrenaline release was detected in the ipsilateral PFC. Next, the interaction between alpha(1)- and alpha(2)-adrenoceptors was investigated. Local administration of the alpha(2)-adrenoceptor antagonist idazoxan (100 microM) into the LC increased the noradrenaline release in the LC to about 400%, whereas noradrenaline release in the PFC rose to 150% of basal levels. A similar effect was seen when the specific alpha(2A)-adrenoceptor antagonist BRL 44408 (10 microM) was infused: extracellular noradrenaline in the LC and PFC increased to about 400 and 120% of the basal levels, respectively. When infusions of idazoxan (100 microM) or BRL 44408 (10 microM) into the LC were combined with prazosin (10 microM), the excitatory effects of the alpha(2)-adrenoceptor antagonists on the release of noradrenaline were strongly suppressed in the LC as well as in the ipsilateral PFC. It is concluded that alpha(1)-adrenoreceptors are involved in the regulation of LC activity. Apparently, alpha(1)- and alpha(2)-adrenoceptors have opposite roles in their function as autoreceptors on LC cells.     

Effects of milnacipran on neuronal excitability and synaptic transmission in neurons of the rat locus coeruleus

Effects of milnacipran (MIL), a selective serotonin and noradrenaline (NA) reuptake inhibitor, on the neuronal excitability and synaptic transmission in the rat locus coeruleus (LC) were examined by intracellular and whole-cell patch-clamp recording techniques. We compared MIL and methylphenidate (MPH), a selective NA and dopamine reuptake inhibitor, as a therapeutic agent for attention deficit/hyperactivity disorder. Application of MPH (1-100 microM) and MIL (1-100 microM) to artificial cerebrospinal fluid (ACSF) produced a hyperpolarizing response in LC neurons in a concentration-dependent manner. Spontaneous firing of LC neurons was blocked during the hyperpolarization. The MIL-induced hyperpolarization was blocked by yohimbine (1 microM), an antagonist for alpha-adrenoceptors. These results suggest that the MIL-induced hyperpolarization is mediated by NA via alpha2-adrenoceptors in LC neurons. Under the whole-cell patch-clamp condition, prolonged application of MIL produced an outward current which lasted as long as MIL existed in the ACSF. The outward current induced by NA was enhanced by MIL in LC neurons. MIL enhanced the amplitude and duration of the inhibitory postsynaptic potential, while it depressed the excitatory postsynaptic potential. The results indicated that both MIL and MPH showed almost the same effects on neuronal activity and synaptic transmission in the rat LC. These results suggest that MIL increases the concentration of NA at synaptic clefts by inhibiting the NA reuptake system in the rat LC.     

Adenosine suppresses protein kinase A- and C-induced enhancement of glutamate release in the hippocampus

Cultured hippocampal neurons from neonatal rats were used to investigate the effect of adenosine on the release of glutamate. Spontaneous tetrodotoxin-resistant miniature excitatory postsynaptic currents (mEPSCs) through AMPA receptor channels were recorded by means of the whole-cell patch-clamp technique. Adenosine (50 microM) reversibly reduced the frequency of mEPSCs by approximately 50-60%, but did not change their amplitudes. The protein kinase A inhibitor Rp-cyclic adenosine monophosphate (100-150 microM) did not block the adenosine-dependent reduction of the mEPSC frequency, showing that adenosine is not depressing synaptic transmission via a protein kinase A (PKA)-dependent mechanism. The D1 dopamine agonist SKF-38393 (250 microM), forskolin (5 microM) and 8Br-cAMP (2 mM), known to activate the cAMP/PKA-dependent signalling pathway, all enhanced the mEPSC frequency. A subsequent application of adenosine (50 microM) strongly reduced the potentiation produced by any one of these three drugs. It also reversed protein kinase C (PKC)-dependent stimulation of glutamate release induced by phorbol myristate acetate (100 nM). Taken together, adenosine not only inhibits the spontaneous release of glutamate independently of protein kinases A and C but also reverses the enhancement of exocytosis produced by protein kinases A and C activators.     

Some pharmacological differences between hippocampal excitatory and inhibitory synapses in transmitter release: an in vitro study.

The effects of adenosine, carbachol, and baclofen on synaptic transmission between neurons in cultured rat hippocampal explants were studied using the tight-seal whole cell clamp technique. In the culture, stimulations of neurites cause postsynaptic currents (PSCs) in nearby neurons under voltage-clamp condition. In the presence of 20 microM bicuculline, most PSCs were considered as glutamatergic excitatory postsynaptic currents (EPSCs), because they were blocked by glutamate antagonist, kynurenate at 1 mM. In the presence of 1 mM kynurenate, PSCs seemed to be inhibitory postsynaptic currents mediated by gamma-aminobutyric acid (GABA), because they were blocked by GABA antagonist, bicuculline at 20 microM. Adenosine at 100 microM and carbachol at 10 microM suppressed these EPSCs to about 35% of control. However, adenosine and carbachol at the same concentration did not suppress the IPSCs. Baclofen at 10 microM suppressed both EPSCs and IPSCs significantly (EPSCs: to about 40% of control, IPSCs: to about 30% of control). In contrast, membrane currents elicited by ionophoretically applied glutamate and GABA were not suppressed by 100 microM adenosine, 10 microM carbachol, and 10 microM baclofen. From these results, it is suggested that the pharmacological sensitivities of transmitter release from presynaptic terminals are different between glutamatergic excitatory synapses and GABAergic inhibitory synapses in hippocampal cultures.     

Heterogeneous population of dopaminergic neurons derived from mouse embryonic stem cells: preliminary phenotyping based on receptor expression and function

The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells; or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population; cells could be further subdivided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.     

Neuronal impulse activity in surviving sections of the guinea pig neocortex during blockade of synaptic transmission by adenosine

Background neuronal discharges were recorded extracellularly in guinea pig cerebral cortex slices maintained in vitro. Synaptic disconnection was reached by adding 100 microM of adenosine or 20-75 microM of 5-AMP. It was shown that 30-40% of neurons continued to discharge even under conditions of complete synaptic blockade, although the discharge frequency decreased. These neurons were considered as endogenously active ones. Other cells stopped their activity as soon as adenosine reached the slice, but they began to generate action potentials again after superfusion with normal solution. These cells were synaptically activated and were considered as exogenously active. In particular most cells with initially very regular discharges became silent during blockade and restored their activity after superfusion with normal saline. This fact suggests the existence of reverberating activity in neocortical slices with periods of 30-140 ms.     

Mechanisms of apoptosis induced by purine nucleosides in astrocytes

Astrocytes release adenine-based and guanine-based purines under physiological and, particularly, pathological conditions. Thus, the aim of this study was to determine if adenosine induced apoptosis in cultured rat astrocytes. Further, if guanosine, which increases the extracellular concentration of adenosine, also induced apoptosis determined using the TUNEL and Annexin V assays. Adenosine induced apoptosis in a concentration-dependent manner up to 100 microM. Inosine, hypoxanthine, guanine, and guanosine did not. Guanosine or adenosine (100 microM) added to the culture medium was metabolized, with 35% or 15%, respectively, remaining after 2-3 h. Guanosine evoked the extracellular accumulation of adenosine, and particularly of adenine-based nucleotides. Cotreatment with EHNA and guanosine increased the extracellular accumulation of adenosine and induced apoptosis. Inhibition of the nucleoside transporters using NBTI (100 microM) or propentophylline (100 microM) significantly decreased but did not abolish the apoptosis induced by guanosine + EHNA or adenosine + EHNA, respectively. Apoptosis produced by either guanosine + EHNA or adenosine + EHNA was unaffected by A(1) or A(2) adenosine receptor antagonists, but was significantly reduced by MRS 1523, a selective A(3) adenosine receptor antagonist. Adenosine + EHNA, not guanosine + EHNA, significantly increased the intracellular concentration of S-adenosyl-L-homocysteine (SAH) and greatly reduced the ratio of S-adenosyl-L-methioine to SAH, which is associated with apoptosis. These data demonstrate that adenosine mediates apoptosis of astrocytes both, via activation of A(3) adenosine receptors and by modulating SAH hydrolase activity. Guanosine induces apoptosis by accumulating extracellular adenosine, which then acts solely via A(3) adenosine receptors.     

ATP-induced synaptic potentiation in hippocampal slices

The purpose of this study was to investigate the influence of different adenosine triphosphate (ATP) concentrations (ranging from 400 nM to 250 microM) on hippocampal potentials recorded from pyramidal neurons. ATP applied at a concentration of 400 nM induced a 100% increase in the size of the population spike (potentiation). The potential started to increase 30-60 s after ATP application, reached a maximum after 20 min, and remained potentiated for longer than 1.5 h. Washing the slices with fresh Ringer solution did not reverse the effect. ATP applied at a concentration of 50-150 microM, temporarily depressed the potential. This depression, however, was transient, as the potential gradually recovered by itself and reached a value higher than that observed before ATP application. ATP applied at the concentration of 250 microM caused a long-lasting depression of the potential. The potential was not restored by washing the slices, but recovered after addition of 0.7 microM 3,4-diaminopyridine. These data show a concentration-dependent mode of ATP action on hippocampal neurons and suggest a role for ATP in regulating synaptic efficiency.     

Adenosine antagonists increase spontaneous and evoked transmitter release from neuronal cells in culture

To examine the role played by endogenous adenosine in the modulation of transmitter release in the CNS, the effect of adenosine antagonists has been studied. Two systems have been used: EPSPs recorded from pyramidal cells in organotypic hippocampal cultures; and release of newly synthesized [3H]glutamate from cerebellar granule cells in dissociated culture. Bath application of 0.1-1 microM 8-phenyltheophylline (8-PT) reversibly increased both the number and size of spontaneous EPSPs and caused bursting activity in some cells. This effect was blocked by the glutamate antagonist gamma-D-glutamylglycine (DGG) (1 mM) but not by atropine (10 microM) or bicuculline (100 microM). Another adenosine antagonist isobutylmethylxanthine (IBMX, 10 microM) had a similar effect to 8-PT. Spontaneous activity in pyramidal cells and that induced by adenosine antagonists was blocked by the adenosine agonist 2-chloroadenosine (2-CA) (0.2-20 microM). 8-PT (10 microM) markedly potentiated K+-stimulated release of newly synthesized glutamate, and also enhanced basal glutamate release. The agonist (-)-phenylisopropyladenosine ((-)-PIA, 2 microM) which is relatively selective for A1 receptors, reduced by 19 +/- 5% the 8-PT-induced enhancement, and reduced K+-stimulated glutamate release in the absence of 8-PT to a similar extent. In contrast 5'-N-ethylcarboxamido adenosine (NECA, 2 microM), which is a relatively selective A2 agonist, slightly enhanced glutamate release. From these results it is likely that 8-PT potentiates glutamate release in both systems by blocking the effect of endogenous adenosine on presynaptic A1 receptors.     

Adenosine modulates a voltage-dependent chloride conductance in cultured hippocampal neurons

Whole cell currents were recorded in cultured rat hippocampal neurons using the patch-clamp method. When the cells were held near the resting membrane potential (-60 mV) the application of adenosine (1 microM) or the adenosine analogues 2CA (100 nM) and R-PIA (40 nM) induced a steady-state inward current. This response was unchanged when extra- and intracellular media were used, in which Na+ and K+ were substituted by impermeable ions in equimolar concentrations. In contrast the current was affected by lowering the extracellular Cl- concentration and thus Cl- was considered to be the ionic carrier. Additionally an almost complete block of the current was observed after applications of DIDS (50 microM), a putative Cl- channel blocker. The modulated current was voltage-dependent and was slowly activated by hyperpolarizing voltage steps. The adenosine action was theophylline- and pertussis toxin-sensitive indicating that the modulatory effect is mediated via an A1 receptor coupled to a G protein of the Gi or Go class.     

Effect of adenosine on total and regional cerebral blood flow of the newborn piglet

The effect of adenosine on total and regional CBF, measured by radiolabeled microspheres, was assessed in 16 anesthetized and ventilated newborn (1-3 days old) piglets. They received a ventriculocisternal perfusion containing either CSF alone (controls, n = 5) or CSF mixed with two different concentrations of adenosine (15 min each) randomly assigned using the following doses: 0.1 microM, 10 microM, 100 microM, 1 mM (n = 4), or 10 mM (n = 6). Mean CSF adenosine concentration (by HPLC) before perfusion was 0.6 +/- 0.4 microM. Total and regional CBF were not altered by the perfusion of CSF alone. All adenosine concentrations, except at low doses, increased total and regional CBF, without altering the cerebral metabolic rate for oxygen. Brainstem blood flow was increased by a mean of 110, 145, 306, and 378% with 10 microM, 100 microM, 1 mM, and 10 mM concentrations, respectively. Except for the highest concentration, CBF response was dose dependent in each region of the brain with the following order of potency: brainstem greater than periventricular area greater than telencephalon, midbrain, total brain, and cerebellum. These data indicate that, in the newborn, adenosine is a potent vasodilator of cerebral vessels. If the newborn brain can synthesize appropriate concentrations of adenosine, this nucleoside may play a major role in regional CBF regulation during the neonatal period.     

Cisplatin modulates voltage gated channel currents of dorsal root ganglion neurons of rats

The anticancer drug cis-diammindichloroplatin (CDDP, cisplatin) causes severe side effects like peripheral sensitive neuropathy. The toxicity of CDDP has been linked to changes in intracellular calcium homeostasis ([Ca2+]i). Voltage activated calcium channel currents (ICa(V)) are important for the regulation of [Ca2+]i; therefore, this study was designed to examine the effect of CDDP on ICa(V) in comparison to voltage activated potassium (IK(V)) and sodium (INa(V)) channel currents using the whole cell patch clamp method on dorsal root ganglion neurons of rats. In small neurons (or=?25 microm) were less sensitive to CDDP. The peak ICa(V) was reduced by 14.1+/-2.3% and IK(V) by 12.8+/-3.4% (100 microM). The sensitivity of INa(V) in large neurons to CDDP was not different compared to small neurons. We conclude that the reduction of ICa(V) in small cells may be responsible for the neurotoxic side effects CDDP causes in sensory neurons.     

The effect of electrical stimulation of the locus coeruleus on the neuronal activity of the parietal associative cortex

In experiments performed on cats operated under ketamine anaesthesia and subsequently immobilized by myorelaxin it was demonstrated that locus coeruleus (LC) being stimulated by a train of pulses can exert influence on 79% of parietal cortex neurons. Inhibition of the background activity for 300-700 ms or a decrease in its frequency by 16-32% were observed in them after the LC stimulation. During intracellular recording neurons with background activity and "silent" neurons responded to LC stimulation by hyperpolarization (5-7 mV) lasting for 120-500 ms with latency of 30-90 ms. The duration of the inhibitory pause in background activity caused by transcallosal stimulation increased by 50-200 ms due to conditioning stimulation of LC. The duration of IPSPs evoked by transcallosal stimulation also increased by 50-100 ms under the influence of LC stimulation. It is concluded that the influence of LC stimulation on the activity of the parietal cortex neurons can be exerted either directly as inhibition of background activity and hyperpolarization or as modulation of effects of other neurotransmitters.     

Adenosine inhibits L-type Ca2+ current and catecholamine release in the rabbit carotid body chemoreceptor cells

In an in vitro preparation of the intact carotid body (CB) of the rabbit, adenosine (100 microM) inhibited hypoxia-induced catecholamine release by 25%. The specific A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 microM) prevented the inhibition and increased the response to hypoxia further. In isolated chemoreceptor cells from the same species, adenosine inhibited voltage-dependent Ca2+ currents by 29% at 1 microM (concentration producing half-maximal inhibition, IC50 = 50 nM). This inhibition was mimicked by R(-)N6-(2-phenylisopropyl)-adenosine and 2-chloroadenosine (1 microM), two purinergic agonists poorly active at the intracellular ('P') site, and persisted in the presence of dipyridamole (a blocker of adenosine uptake; 1 microM) and was fully inhibited by 8-phenyltheophylline (10 microM). The A1 antagonists DPCPX (10 microM) and 8-cyclopentyl-1,3-dimethylxantine (0.1 microM) inhibited the effect of adenosine by 93% (IC50 = 0.14 microM) and 59%, respectively. The inhibition of the Ca2+ current (I(Ca)) was reduced by nisoldipine (an L-type Ca2+ channel antagonist) by nearly 50%, and was unaltered by omega-conotoxin GVIA, a blocker of N-type Ca2+ channels. Adenosine did not affect the voltage-dependent Na+ current (I(Na)) or K+ current (I(K)). We conclude that adenosine A1 receptors are located in chemoreceptor cells and mediate the inhibition of L-type Ca2+ channels and thereby the release of catecholamines produced by hypoxia. The data also indicate that endogenous adenosine acts as a physiological negative modulator of the chemoreceptor cell function. The previously reported excitatory action of adenosine on the activity of the sensory nerve of the CB is discussed in terms of a balance between the inhibition mediated by A1 receptors and the excitation mediated by A2 receptors.     

Interactions of cyclic adenosine monophosphate,brain-derived neurotrophic factor,and glial cell line-derived neurotrophic factor treatment on the survival and growth of postnatal mesencephalic dopamine neurons in vitro

The survival of rat postnatal mesencephalic dopamine (DA) neurons in dissociated cell cultures was studied by examining the combinatorial effects of dibutyryl cyclic adenosine monophosphate (db-cAMP), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), as well as selective inhibitors of protein kinase A (PKA), and mitogen-activated protein kinase (MAPK). Postnatal DA neurons were maintained for 14 days in vitro, and were identified by immunohistochemistry using tyrosine hydroxylase antibody. The survival and growth of DA neurons was significantly increased by the inclusion of either >100 microM db-cAMP or 10 microM Forskolin plus 100 microM IBMX in the culture medium. Neither 10-50 ng/ml GDNF nor 50 ng/ml BDNF alone significantly increased DA neuron survival in vitro. However, the combined use of GDNF and BDNF did increase DA neuron survival, and the addition of either db-cAMP or IBMX/Forskolin to media containing these neurotrophins markedly increased DA neuron survival and growth. The cAMP inhibitor Rp-cAMP, the cAMP-dependent protein kinase A inhibitor H89, and the MAP kinase (MAPK) pathway inhibitor PD98059 significantly reduced the survival of DA neurons when applied alone in the absence of added growth factors. Application of GDNF plus BDNF, or db-cAMP significantly protected the DA neurons from the deleterious effects on survival of either 20 microM H89 or 20 microM PD 98059. The results suggest that BDNF, GDNF, and cAMP produce convergent signals to activate PKA and MAPK pathways which are involved in the survival of postnatal mesencephalic DA neurons in vitro.     

Diadenosine polyphosphates facilitate the evoked release of acetylcholine from rat hippocampal nerve terminals

Diadenosine polyphosphates are present in synaptic vesicles, are released upon nerve stimulation and possess membrane receptors, namely in presynaptic terminals. However, the role of diadenosine polyphosphates to control neurotransmitter release in the CNS is not known. We now show that diadenosine pentaphosphate (Ap(5)A, 3-100 microM) facilitated in a concentration dependent manner the evoked release of acetylcholine from hippocampal nerve terminals, with a maximal facilitatory effect of 116% obtained with 30 microM Ap(5)A. The selective diadenosine polyphosphate receptor antagonist, diinosine pentaphosphate (Ip(5)I, 1 microM), inhibited by 75% the facilitatory effect of Ap(5)A (30 microM), whereas the P(2) receptor antagonists, suramin (100 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 microM) only caused a 18-24% inhibition, the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (20 nM), caused a 36% inhibition and the adenosine A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3, 5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 20 nM), was devoid of effect. These results show that diadenosine polyphosphates act as neuromodulators in the CNS, facilitating the evoked release of acetylcholine mainly through activation of diadenosine polyphosphate receptors.     

Day-night variations in zinc sensitivity of GABAA receptor-channels in rat suprachiasmatic nucleus

In the suprachiasmatic nucleus (SCN), electrical activity, secretion, and other cellular functions undergo profound rhythm during day-night cycle due to oscillatory expression of clock gene constituents. Although SCN is enriched with gamma-aminobutyric acid (GABA)-ergic neurons, it is unknown whether there are circadian changes in the GABAA receptor expression and/or function. Here we investigated the possible daily variations in zinc sensitivity of GABAA channels in rat SCN neurons maintained in brain slices. Extracellular zinc inhibited GABA-induced currents in all ventrolateral (VL) and dorsomedial (DM) SCN neurons studied, as well as in neurons of non-SCN regions. In SCN neurons, the currents evoked by 30 microM GABA were inhibited by Zn2+ with an IC50 of 50.3+/-3.2 microM, whereas currents evoked by 100 microM GABA were inhibited with an IC50 of 181.6+/-32.0 microM. The antagonist action of zinc saturated at 97.4+/-0.7% for 30 microM GABA and 91.6+/-2.7% for 100 microM GABA. These observations indicate that Zn2+ inhibits SCN GABAA receptor competitively and in part non-competitively. In SCN neurons, but not in other neurons, the zinc sensitivity varied with daily time. During the day, the calculated IC50 for zinc was significantly lower than during the night (43.9+/-4.7 microM vs. 58.6+/-3.8, respectively). These results indicate that native GABAA receptors in SCN neurons display pharmacological properties of receptors having and not having gamma subunit and that the proportionality of these receptors could change during the day and night.