Suppression of presynaptic calcium influx by metabotropic glutamate receptor agonists in neonatal rat hippocampus

Mechanisms of presynaptic inhibition by metabotropic glutamate receptor (mGluR) agonists were investigated in neonatal rat hippocampal CA1 region using the optical recording technique recently developed. Following selective loading of presynaptic terminals with a fluorescent Ca²⁺ indicator dye rhod-2 AM, changes in Ca²⁺ signals and the corresponding field excitatory postsynaptic potentials (EPSPs) induced by single electrical stimuli to the Schaffer collateral-commissural (SCC) pathway were recorded simultaneously. Application of a mGluR agonist, 1 S,3 R-1-aminocyclopentane-1,3-dicar?ylic acid (1 S,3 R-ACPD; 100 μM) or (±)-1-aminocyclopentane-trans-1,3-dicar?ylic acid (trans-ACPD; 100 μM), reversibly reduced both the field EPSP and the presynaptic Ca²⁺ transient, and the quantitative relationship between them was quite similar to that observed during application of Cd²⁺, a non-selective Ca²⁺ channel blocker, or in a Ca²⁺-free solution. Application of 4-aminopyridine (4-AP; 1 mM), a blocker of certain subtypes of voltage-dependent K⁺ channels, significantly inhibited the 1 S,3 R-ACPD effect. Application of DCG-IV, a novel mGluR2/mGluR3-selective agonist, suppressed field EPSPs only slightly even at a high dose (3 μM). These results suggest that activation of presynaptic mGluR different from mGluR2/mGluR3 suppresses the action potential-triggered Ca²⁺ influx, probably via 4-AP-sensitive mechanisms, and thereby reduces glutamate release in neonatal rat hippocampal CA1 region.     

AMPA receptor subunit mRNAs and intracellular [Ca] in cultured mouse and rat cerebellar granule cells

Cultured mouse cerebellar granule cells differ from their rat counterparts in that they survive well when grown in non-depolarising medium (5 mM K⁺). However, when chronically stimulated by added glutamate agonists, including (RS)α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), rat cerebellar granule cells also survive well in non-depolarising medium. We hypothesised that the relatively good survival of mouse cerebellar granule cells in the absence of added glutamate agonists might reflect AMPA receptors resistant to desensitisation. These receptors might be stimulated by endogenous glutamate. We tested this hypothesis by comparing cultured mouse and rat cerebellar granule cells grown in depolarising (25 mM K⁺) and non-depolarising (5 mM K⁺) medium. We studied the AMPA-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), using the fluorescent Ca²⁺ chelator, Fluo-3, and the relative concentrations of mRNAs for the four AMPA receptor subunits, GluR1–4. GluR1–4 mRNAs were measured by restriction enzyme analysis of a PCR product containing cDNA with a composition proportional to the four subunit mRNAs. We found that the [Ca²⁺]_i-response to AMPA receptor activation in cultured cerebellar granule cells is determined mainly by the desensitisation properties of the AMPA receptors rather than by their ion permeability. We also found that mouse cerebellar granule cells express AMPA receptors which are more resistant to desensitisation than the corresponding rat AMPA receptors. Thus, relatively slow AMPA receptor desensitisation kinetics may contribute to the survival of mouse cerebellar granule cells in non-depolarising medium.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

On the role of calcium ions in the presynaptic alpha-receptor mediated inhibition of [H]noradrenaline release from rat brain cortex synaptosomes

Rat brain cortex synaptosomes, previously labeled by incubation with [³H]noradrenaline ([³H]NA) were continuously superfused with Krebs-Ringer media. Release of [³H]NA was induced by superfusion with medium containing either 15 mM K⁺, 20 μM veratrine or 1 μM of the calcium-ionophore A 23187 and was strongly dependent on the concentration of Ca²⁺ in the medium. Noradrenaline (1μM, in the presence of the uptake inhibitor desipramine) inhibited K⁺-induced [³H]NA release by activation of presynaptic alpha-receptors. When the Ca²⁺-concentration in the medium was reduced, or the Mg²⁺-concentration increased, [³H]NA release appeared to be more susceptible to alpha-receptor mediated inhibition.Noradrenaline (1 μM) inhibited [³H]NA release induced by 15 mM K⁺, in the presence of 0.075 Ca²⁺ and 10 mM Mg²⁺, by 86%. Veratrine-induced release was also inhibited by alpha-receptor activation. However, [³H]NA release induced by the calcium-ionophore was not affected by alpha-receptor agonists. These results strongly support the view that alpha-receptor activation results in a decrease of the availability of Ca²⁺ for stimulus-secretion coupling processes. Presumably this is effected by an inhibition of voltage-sensitive calcium channels in the neuronal membrane associated with neurotransmitter release.     

Arachidonic acid inhibits both K and Ca currents in isolated type I cells of the rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K⁺ and Ca²⁺ channels in isolated rat type I carotid body cells. AA (2–20 μM) produced a concentration-dependent inhibition of both K⁺ currents and Ca²⁺ channel currents. The effects of AA on K⁺ currents were unaffected by indomethacin (5 μM), phenidone (5 μM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.     

Metabotropic glutamate receptor 1 activity generates persistent, N-methyl-d-aspartate receptor-dependent depression of hippocampal pyramidal cell excitability

Metabotropic glutamate receptors (mGluRs) are involved in many forms of neuronal plasticity. In the hippocampus, they have well‐defined roles in long‐lasting forms of both synaptic and intrinsic plasticity. Here, we describe a novel form of long‐lasting intrinsic plasticity that we call (S)‐3,5‐dihydroxyphenylglycine (DHPG)‐mediated long‐term depression of excitability (DHPG‐LDE), and which is generated following transient pharmacological activation of group I mGluRs. In extracellular recordings from hippocampal slices, DHPG‐LDE was expressed as a long‐lasting depression of antidromic compound action potentials (cAPs) in CA1 or CA3 cells following a 4‐min exposure to the group I mGluR agonist (S)‐DHPG. A similar phenomenon was also seen for orthodromic fibre volleys evoked in CA3 axons. In single‐cell recordings from CA1 pyramids, DHPG‐LDE was manifest as persistent failures in antidromic action potential generation. DHPG‐LDE was blocked by (S)‐(+)‐a‐amino‐4‐carboxy‐2‐methylbenzeneacetic acid (LY367385), an antagonist of mGluR1, but not 2‐methyl‐6‐(phenylethynyl)pyridine hydrochloride (MPEP), an mGluR5 inhibitor. Although insensitive to antagonists of α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate/kainate and γ‐aminobutyric acid_A receptors, DHPG‐LDE was blocked by antagonists of N‐methyl‐d ‐aspartate (NMDA) receptors. Similarly, in single‐cell recordings, DHPG‐mediated antidromic spike failures were eliminated by NMDA receptor antagonism. Long after (S)‐DHPG washout, DHPG‐LDE was reversed by mGluR1 antagonism. A 4‐min application of (S)‐DHPG also produced an NMDA receptor‐dependent persistent depolarization of CA1 pyramidal cells. This depolarization was not solely responsible for DHPG‐LDE, because a similar level of depolarization elicited by raising extracellular K⁺ increased the amplitude of the cAP. DHPG‐LDE did not involve HCN channels or protein synthesis, but was eliminated by blockers of protein kinase C or tyrosine phosphatases.     

Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea

The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 ± 2.3 mV from the resting membrane potential of −58.5 ± 5.9 mV (n = 48). This hyperpolarization was associated with an outward current ( 97.7 ± 22.2, pA, n = 15). The hyperpolarization was inhibited by 300 μM quinine, 5 mN Ba²⁺ and increasing the extracellular K⁺ to 30 mM from 5 mM. In the absence of extracellular Ca²⁺ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50 μM GdCl₃, which is known to block strecch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50 μM GdCl₃ also inhibited [Ca²⁺]_i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca²⁺]_i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca²⁺ influx. In turn, this [Ca²⁺]_i increase leads to an activation of the Ca²⁺-activated K⁺ channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K⁺ efflux.     

Muscarinic receptor-mediated calcium signaling in spiral ganglion neurons of the mammalian cochlea

Using indo-1 microspectrofluorometry, we examined the effects of cholinergic agonists on the concentration of intracellular Ca²⁺ ions ([Ca²⁺]_i) in spiral ganglion neurons, isolated from rat cochleae at different stages of post-natal development (from P3 to P30). Extracellular application of acetylcholine (ACh) or carbamylcholine generated a rapid and transient increase in [Ca²⁺]_i. The ACh concentration-response curve indicated an apparent dissociation constant (K_d) of 8 μM and a Hill coefficient of 1.0. Removing extracellular free Ca²⁺ did not suppress the ACh-induced Ca²⁺ responses suggesting an intracellular Ca²⁺-release mechanism. When we compared the cholinergic response at different stages of postnatal development, there were no significant differences on the aspect of the Ca²⁺ response and the percentage of responsive neurons, which ranged between 50 and 65% per cochlear preparation. The application of muscarine triggered reversible Ca²⁺ responses similar to those observed with ACh, with an apparent K_d of 10 μM and a Hill coefficient of 1.0. The cholinergic-induced Ca²⁺ response was reversibly blocked by muscarinic antagonists with the following order of potency, atropine>4-DAMP>methoctramine>pirenzepine. Nicotine (10 to 100 μM) did not evoke Ca²⁺ responses and the nicotinic antagonist curare (10 μM) did not block the ACh-evoked responses. The present study is the first direct demonstration of functional muscarinic receptors (mAChRs) in spiral ganglion neurons. These mAChRs activated by the cholinergic lateral efferent system may participate in the regulation of the electrical activity of the afferent auditory fibers contacting the inner hair cells.     

Effects of Pb and Cd on acetylcholine release and Ca movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

In this work we examined the effects of Pb²⁺ and Cd²⁺ on (a) [³H]ACh release and voltage-sensitive Ca²⁺ channels in rat brain synaptosomes, and (b)⁴⁵Ca²⁺ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb²⁺ (K_i ≈ 1.1 μM) and Cd²⁺ (K_i ≈ 2.2) competitively block the K⁺-evoked influx of⁴⁵Ca²⁺ through the ‘fast’ calcium channels in synaptosomes. The K_is obtained with synaptosomes are in good agreement with the K_i values obtained from electrophysiological experiments at the frog neuromuscular junction (K_Pb:0.99 μM, K_Cd: 1.7 μM)⁷. The K_i for the inhibition of ACh release from synaptosomes by Cd²⁺ is 4.5 μM. Pb²⁺ is a less effective inhibitor of transmitter release (K_i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd²⁺ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb²⁺ on spontaneous release is (a) not abolished by omission of Ca²⁺ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb²⁺ exposure, (c) is reversed upon the removal of Pb²⁺. In the presence of physiological concentrations of ATP (1 mM), Mg²⁺ (1 mM) and P_i (2 mM), 1–10 μM Pb²⁺ inhibits calcium uptake but Pb²⁺ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb²⁺-induced enhancement of P_i uptake. Cd²⁺ inhibits Ca²⁺ binding at all concentrations tested (1–50 μM) and reduces the Pb²⁺-induced Ca²⁺-binding to organelles. Neither Pb²⁺ nor Cd²⁺ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with⁴⁵Ca. In summary, these data are consistent with the notion that Pb²⁺ and Cd²⁺ are potent blockers of presynaptic voltage-sensitive Ca²⁺ channels and the evoked release of transmitter which is contingent on Ca²⁺ influx through these channels. Our results are not consistent with the hypothesis that Pb²⁺ augments spontaneous release by interfering with intraterminal Ca²⁺-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.     

Activation of central muscarinic receptors inhibit Ca/Mg ATPase and ATP-dependent Ca transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ accumulation activity, with aK_m forCa²⁺ 0.5 μM, close to the cytosolic concentration of Ca²⁺. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1–20 μM), both Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5–1.0 μM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca²⁺ channels or Na⁺-Ca²⁺ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca²⁺ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca²⁺ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

Subunit selective decrease of AMPA and metabotropic glutamate receptor mRNA expression in rat brain by systemic administration of the NMDA receptor blocker MK-801

Glutamate mediates its effects in mammals through both ionotropic and metabotropic receptors. Antagonists of ionotropic N-methyl-d-aspartate (NMDA) glutamate receptors elicit neuroprotective and neurotropic effects that have been attributed to Ca²⁺ block through the membrane ion channel. Nonetheless, molecular and biochemical effects of NMDA receptor antagonism on other glutamate receptor subunits remain poorly understood. We investigated the effects of acute administration of the noncompetitive NMDA receptor antagonist MK-801 on the mRNA expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and metabotropic glutamate receptor (mGluR) subunits to determine the contribution of different glutamate receptors in response to blockade of NMDA receptor channels. In situ hybridization to rat brain sections revealed that AMPA receptor subunits GluR3 and GluR4, and mGluR3 were modestly but significantly decreased ∼10–20%, 8 h following 5 mg/kg MK-801 administration. A time course and dose response study revealed that the effect on mGluR3 was reversed by 24 h and occurred significantly at a dose range from 1 to 5 mg/kg. These results indicate that selected AMPA and mGluR subunit mRNAs respond at the RNA level to the blockade of NMDA receptors.     

Stimulation of GABA release from retinal horizontal cells by potassium and acidic amino acid agonists

The release of [³H]GABA from horizontal cells of goldfish retina was studied by biochemical analysis of perfused isolated retina. Retinas were incubated for 15 min in 0.72 μM[³H]GABA, rinsed for 30 min and then perfused with 1 min pulses of increasing concentrations of K⁺ and acidic amino acid agonists under a variety of conditions. Radioactivity in the perfusate was determined by liquid scintillation spectroscopy. The main findings are: (1) virtually all of the [³H]GABA released byl-glutamate (l-Glu) andl-aspartate (l-Asp) and 50% of the K⁺-evoked release, is calcium independent; (2) K⁺-evoked [³H]GABA release is only 10% of that released byl-Glu; (3) threshold [³H]GABA release occurs with 320 μMl-Glu, 1175 μMl-Asp, 4μM quisqualic acid (QA), 4μM kainic acid (KA) and 53 μM N-methyl-dl-aspartate (NMDLA); (4) the quisqualate antagonist glutamic acid diethyl ester (GDEE), has no specific inhibitory action on any of the agonists, whereasd-α-aminoadipic acid (DαAA), an NMDA antagonist, potently inhibits the action of NMDLA andl-Asp; (5) the presence of Mg²⁺, even at 1 mM, totally inhibits NMDLA and also inhibits the action ofl-Glu andl-Asp below 1 mM; (6)d-Asp potentiates the action ofl-Glu by 0.6–0.8 log units and completely inhibits the action ofl-Asp; (7)l-Asp at a ratio of 3:1 potentiates the effect ofl-Glu. From these and other results one concludes that: (a) [³H]GABA release from H1 cells is calcium independent and depends on factors other than passive depolarization, probably sodium; (2) the likely transmitter of red cones isl-Glu acting on quisqualate or kainate receptors, and (3)l-Asp acts predominantly on NMDA receptors and may provide a modulatory role in the outer retina by potentiating the action ofl-Glu.     

Different pathways by which extracellular Ca promotes calcitonin gene-related peptide release from central terminals of capsaicin-sensitive afferents of guinea pigs: effect of capsaicin, high K and low pH media

Different modes by which Ca²⁺, entering the nerve terminal, promotes transmitter secretion as well as the ability of protons to release neuropeptides, have been shown in peripheral endings of capsaicin-sensitive afferents. We have studied these two aspects in the central endings of these neurons by measuring the release of calcitonin-gene related peptide-like immunoreactivity (CGRP-LI) from slices of the dorsal half of the guinea pig spinal cord. Altough capsaicin (1 μM) released both CGRP-LI and substance P-like immunoreactivity (SP-LI), CGRP-LI was chosen as the sole suitable marker of peptides released from central terminals of capsaicin-sensitive afferents, since after in vitro desensitization to capsaicin (1μM capsaicin for 20 min), high K⁺ (80 mM) failed to evoke CGRP-LI release, whereas SP-LI release was still observed. The capsaicin (1 μM)-evoked CGRP-LI release was entirely dependent on extracellular Ca²⁺. It was unaffected by 0.3 μM tetrodotoxin (TTX), slightly reduced by 0.1 μM ω-conotoxin (CTX) and blocked by 10 μM Ruthenium red (RR). The Ca²⁺-dependent K⁺ (80 mM)-evoked CGRP-LI release was unaffected by TTX, markedly reduced by CTX and only moderatedly inhibited by RR. Low pH (pH 5) produced a remarkable increase in CGRP-LI outflow that was abolished after exposure to capsaicin, reduced by about 50% in Ca²⁺-free medium and unaffected by TTX (0.3 μM). The Ca²⁺-dependent component of the proton-evoked CGRP-LI release was abolished in the presence of RR (10 μM) and slightly inhibited by CTX (0.1 μM). The mode by which capsaicin or high K⁺ promote Ca²⁺ entry into the central endings of capsaicin-sensitive afferents, and hence promote neuropeptide release may be distinguished on a pharmacological basis. Protons release CGRP in the spinal cord by a mechanism that shares a common pathway with that activated by capsaicin.     

Tetraethylammonium ions block the nicotinic cholinergic receptors of cochlear outer hair cells

Cochlear outer hair cells (OHCs) express nicotinic acetylcholine receptors (nAChRs) whose calcium permeability allow the activation of co-localized Ca²⁺-sensitive K⁺ channels (SK-type). The large organic cation tetraethylammonium (TEA) is known to block at millimolar concentration voltage-gated and Ca²⁺-activated K⁺ currents in OHCs. In the present study, we show that extracellular TEA blocked much more efficiently, at micromolar concentrations, ACh-evoked K⁺ currents in isolated guinea pig OHCs. The dose–inhibition curve indicated an IC₅₀ of 60 μM, a value two orders of magnitude lower than the one reported on SK or BK channels. The site of the blocking action was on the extracellular side of the plasma membrane since 10 mM intracellular TEA did not prevent or change the characteristics of the ACh-evoked K⁺ current. The block of this K⁺ current in OHCs was mainly explained by a direct action of TEA at the nAChRs. Indeed, we demonstrated that extracellular TEA inhibited directly the ionotropic cation current flowing through the nAChRs (IC₅₀=30 μM). This study demonstrated for the first time that extracellular TEA is an effective blocker of the OHCs’ nAChRs.     

Pharmacological characterization of adenosine A1 receptors and its functional role in brown trout (Salmo trutta) brain

The adenosine receptor agonist N⁶-cyclohexyl[³H]adenosine ([³H]CHA) was used to identify and pharmacologically characterize adenosine A1 receptors in brown trout (Salmo trutta) brain. In membranes prepared from trout whole brain, the A1 receptor agonist [³H]CHA bound saturably, reversibly and with high affinity (K_d=0.69±0.04 nM; B_max=0.624±0.012 pmol/mg protein) to a single class of binding sites. In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [³H]CHA from high-affinity binding sites with the rank order of potency characteristic for an adenosine A1 receptors. A1 receptor density appeared not age-related (from 3 months until 4 years), and was similar in different brain areas. The specific binding was inhibited by guanosine 5′-triphosphate (IC₅₀=0.778±0.067 μM). GTP (5 μM) induced a low affinity state of A1 receptors. In superfused trout cerebral synaptosomes, 30 mM K⁺ stimulated the release of glutamate in a calcium dependent manner. Glutamate-evoked release was dose-dependently reduced by CHA, and the inhibition was reversed by the A1 antagonist 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, 30 mM K⁺ as well as 1 mM glutamate stimulated the release of adenosine in a Ca²⁺-independent manner and tetrodotoxin insensitive. These findings show that in trout brain adenosine A1 receptors are present which are involved in the modulation of glutamate transmitter release. Moreover, the stimulation of adenosine release by K⁺ depolarisation or glutamate support the hypothesis that, as in mammalian brain, a cross-talk between adenosine and glutamate systems exists also in trout brain.     

Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization

Ethanol and nerve growth factor (NGF) affect the survival of cholinergic neurons in the rat medial septum. To investigate whether calcium (Ca²⁺) homeostasis in these neurons is affected by ethanol or NGF treatment, changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) were studied in embryonic (E21) cultured medial septal neurons before stimulation (basal) and during stimulation with high potassium (K⁺). Changes in [Ca²⁺]; across time were measured in cultures of neurons treated without ethanol or with 100, 2110, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (-NGF). Changes in [Ca²⁺]_i were analyzed from fluorescence images, using indo-1. The effect of ethanol or NGF treatment was to reduce the rise in basal [Ca²⁺]_i. The combination of ethanol and NGF treatment in +NGF neurons led to increases in basal [Ca²⁺]_i with the greatest increase in basal [Ca²⁺]_i occurring with 200 mg% ethanol. The effect of ethanol or NGF was to increase [Ca²⁺]_i; during stimulation with high K⁺. The greatest increases in [Ca⁺]_i occurred with 100 and 800 mg% ethanol. Together, ethanol and NGF treatment in +NGF-treated neurons led to significantly greater increases or decreases in K⁺ stimulated changes in [Ca²⁺]_i compared to similarly treated -NGF neurons. We conclude that in medial septal neurons (before and during depolarization) changes in Ca²⁺ homeostasis occur in the presence of ethanol or NGF. The changes in [Ca²⁺]_i, following ethanol treatment are greater when NGF is present.     

Presynaptic effect of 7-OH-DPAT on evoked [H]-acetylcholine release in rat striatal synaptosomes

The objective of the present experiments was to study the presynaptic effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT, a D₂-like dopamine receptor agonist) on [³H]-acetylcholine ([³H]-ACh) release induced by potassium (15 mM, 25 mM and 60 mM), potassium channel-blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA and quinine) and veratridine to gain insight into the mechanisms involved in the activation of the D₂ dopamine-receptor subtype located at striatal cholinergic nerve terminals. 7-OH-DPAT (1 μM) inhibited the evoked [³H]-ACh release induced by K⁺ 15 mM in a similar percentage than that obtained during basal conditions (30% and 27%, respectively). Nevertheless, in the presence of 25 mM and 60 mM of K⁺ the inhibitory effect of 7-OH-DPAT was completely abolished. 4-AP (1–100 μM) and TEA (1 and 5 mM) significantly enhanced [³H]-ACh release, showing 69.32%±7.60% (P<0.001) and 52.27%±5.64% (P<0.001), respectively, at the highest concentrations tested. In these conditions, 7-OH-DPAT (1 μM) inhibited the release induced by potassium channel-blockers 25–27%. Quinine (0.1–1 μM) did not alter [³H]-ACh release either in the presence or absence of 7-OH-DPAT. Veratridine 10 μM evoked [³H]-ACh release in the presence of a low-calcium medium, but in such conditions 7-OH-DPAT (1 μM) did not modify the neurotransmitter release in the absence or presence of veratridine. Present data indicate that activation of the presynaptic D₂ dopamine receptor inhibits the [³H]-ACh release by increasing K⁺ conductance, as high K⁺ concentrations abolished the inhibitory control of 7-OH-DPAT on [³H]-ACh release. This effect could be mediated by potassium channels different from those sensitive to 4-AP, TEA and quinine. In addition, the presynaptic D₂ dopamine-receptor activation seems to not involve changes in intracellular Ca²⁺.