Kv1.1/1.2 channels are downstream effectors of nitric oxide on synaptic GABA release to preautonomic neurons in the paraventricular nucleus

The paraventricular nucleus (PVN) of the hypothalamus is important for the neural regulation of cardiovascular function. Nitric oxide (NO) increases synaptic GABA release to presympathetic PVN neurons through the cyclic guanosine monophosphate (cGMP)/protein kinase G signaling pathway. However, the downstream signaling mechanisms underlying the effect of NO on synaptic GABA release remain unclear. In this study, whole-cell voltage-clamp recordings were performed on retrograde-labeled spinally projecting PVN neurons in rat brain slices. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly increased the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons. A specific antagonist of cyclic ADP ribose, 8-bromo-cyclic ADP ribose (8-Br-cADPR), had no significant effect on l-arginine-induced potentiation of mIPSCs. Surprisingly, blocking of voltage-gated potassium channels (Kv) with 4-aminopyridine or alpha-dendrotoxin eliminated the effect of l-arginine on mIPSCs in all labeled PVN neurons tested. The membrane permeable cGMP analog mimicked the effect of l-arginine on mIPSCs, and this effect was blocked by alpha-dendrotoxin. Furthermore, the specific Kv channel blocker for Kv1.1 (dendrotoxin-K) or Kv1.2 (tityustoxin-Kalpha) abolished the effect of l-arginine on mIPSCs in all neurons tested. SNAP failed to inhibit the firing activity of labeled PVN neurons in the presence of dendrotoxin-K, Kalpha. Additionally, the immunoreactivity of Kv1.1 and Kv1.2 subunits was colocalized extensively with synaptophysin in the PVN. These findings suggest that NO increases GABAergic input to PVN presympathetic neurons through a downstream mechanism involving the Kv1.1 and Kv1.2 channels at the nerve terminals.     

Increased asynchronous release and aberrant calcium channel activation in amyloid precursor protein deficient neuromuscular synapses

Despite the critical roles of the amyloid precursor protein (APP) in Alzheimer's disease pathogenesis, its physiological function remains poorly established. Our previous studies implicated a structural and functional activity of the APP family of proteins in the developing neuromuscular junction (NMJ). Here we performed comprehensive analyses of neurotransmission in mature neuromuscular synapse of APP deficient mice. We found that APP deletion led to reduced paired-pulse facilitation and increased depression of synaptic transmission with repetitive stimulation. Readily releasable pool size and total releasable vesicles were not affected, but probability of release was significantly increased. Strikingly, the amount of asynchronous release, a measure sensitive to presynaptic calcium concentration, was dramatically increased, and pharmacological studies revealed that it was attributed to aberrant activation of N- and L-type Ca(2+) channels. We propose that APP modulates synaptic transmission at the NMJ by ensuring proper Ca(2+) channel function.     

Vesicle recycling at ribbon synapses in the finely branched axon terminals of mouse retinal bipolar neurons

In retinal bipolar neurons, synaptic ribbons mark the presence of exocytotic active zones in the synaptic terminal. It is unknown, however, where compensatory vesicle retrieval is localized in this cell type and by what mechanism(s) excess membrane is recaptured. To determine whether endocytosis is localized or diffuse in mouse bipolar neurons, we imaged FM4-64 to track vesicles in cells whose synaptic ribbons were tagged with a fluorescent peptide. In synaptic terminals, vesicle retrieval occurred at discrete sites that were spatially consistent over multiple stimuli, indicative of endocytotic “hot spots.” Retrieval sites were spatially correlated with fluorescently labeled synaptic ribbons. Electron microscopy (EM) analysis of bipolar cell terminals after photoconversion of internalized FM dye revealed that almost all of the dye was contained within vesicles ∼30 nm in diameter. Clathrin-coated vesicles were observed budding from the plasma membrane and within the cytosol, and application of dynasore, a dynamin inhibitor, arrested membrane retrieval just after the budding stage. We conclude that synaptic vesicles in the fine branches of mouse bipolar axon terminals are retrieved locally near active zones, at least in part via a clathrin-mediated pathway.     

Differential control of two forms of glutamate release by group III metabotropic glutamate receptors at rat entorhinal synapses

Neurotransmitter release at CNS synapses occurs via both action potential-dependent and independent mechanisms, and it has generally been accepted that these two forms of release are regulated in parallel. We examined the effects of activation of group III metabotropic glutamate receptors (mGluRs) on stimulus-evoked and spontaneous glutamate release onto entorhinal cortical neurones in rats, and found a differential regulation of action potential-dependent and independent forms of release. Activation of presynaptic mGluRs depressed the amplitude of stimulus-evoked excitatory postsynaptic currents, but concurrently enhanced the frequency of spontaneous excitatory currents. Moreover, these differential effects on glutamate release were mediated by pharmacologically separable mechanisms. Application of the specific activator of adenylyl cyclase, forskolin, mimicked the effect of mGluR activation on spontaneous, but not evoked release, and inhibition of adenylyl cyclase with 9-tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) blocked mGluR-mediated enhancement of spontaneous release, but not depression of evoked release. Occlusion studies with calcium channel blockers suggested that the group III mGluRs might depress evoked release through inhibition of both N and P/Q, but not R-type calcium channels. We suggest that the concurrent depression of action potential-evoked, and enhancement of action potential-independent glutamate release operate through discrete second messenger/effector systems at excitatory entorhinal terminals in rat brain.     

GABAA and strychnine-sensitive glycine receptors modulate N-methyl-D-aspartate-evoked acetylcholine release from rat spinal motoneurons: a possible role in neuroprotection

Increasing experimental and clinical evidence suggests that abnormal glutamate transmission might play a major role in a vast number of neurological disorders. As a measure of glutamatergic excitation, we have studied the acetylcholine (ACh) release induced by N-methyl-d-aspartate (NMDA) receptor stimulation in primary cultured rat ventral horn spinal neurons and we have evaluated the possibility to limit the consequences of the hyperactivation of glutamatergic receptors, by recruiting the inhibitory transmission mediated by GABA and glycine. For this purpose, we have exposed cell cultures, previously loaded with [(3)H]choline, to NMDA, which increased the spontaneous tritium efflux in a concentration-dependent manner. Tritium release is dependent upon external Ca(2+), tetrodotoxin, Cd(2+) ions and omega-conotoxin GVIA, but not on omega-conotoxin MVIIC nor nifedipine, suggesting the involvement of N-type voltage-sensitive calcium channels. NMDA-mediated [(3)H]ACh release was completely prevented by MK-801, 5,7-diclorokynurenic acid and ifenprodil, while it was strongly inhibited by a lower external pH, suggesting that the involved NMDA receptors contain NR1 and NR2B subunits. Muscimol inhibited NMDA-evoked [(3)H]ACh release and its effect was antagonized by SR95531 and potentiated by diazepam, indicating the involvement of benzodiazepine-sensitive GABA(A) receptors. Also glycine, via strychnine-sensitive receptors, inhibited the effect of NMDA. It is concluded that glutamate acts on the NMDA receptors situated on spinal motoneurons to evoke ACh release, which can be inhibited through the activation of GABA(A) and glycine receptors present on the same neurons. These data suggest that glutamatergic overload of receptors located onto spinal cord motoneurons might be decreased by activating GABA(A) and glycine receptors.     

Calcium influx through presynaptic 5-HT3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies

Serotonin 5-hydroxytryptamine type 3 receptors (5HT3R) are Ca2+-permeant, non-selective cation channels that have been localized to presynaptic terminals and demonstrated to modulate neurotransmitter release. In the present study the effect of 5-HT on GABA release in the hippocampus was characterized using both electrophysiological and biochemical techniques. 5-HT elicited a burst-like, 6- to 10-fold increase in the frequency of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) measured with whole-cell voltage-clamp recordings of CA1 neurons in hippocampal slices. When tetrodotoxin was used to block action potential propagation, the 5-HT-induced burst of IPSCs was still observed. Stimulation of hippocampal synaptosomes with 5-HT resulted in a significant increase in the amount of [3H]GABA released by hyperosmotic saline. In both preparations, the 5-HT effect was shown to be mediated by 5HT3Rs, as it was mimicked by the selective 5HT3R agonist m-chlorophenyl biguanide and blocked by the selective 5HT3R antagonist 3-tropanylindole-3-carboxylate hydrochloride. The 5HT3R-mediated increase in GABA release was blocked by 100 microM cadmium or by omitting Ca2+ in external solutions, indicating the Ca2+-dependence of the effect. The high voltage-activated Ca2+ channel blockers omega-conotoxin GVIA and omega-conotoxin MVIIC and 10 microM cadmium had no significant effect on the 5-HT3R-mediated enhancement of GABA release, indicating that Ca2+ influx through the 5-HT3R facilitates GABA release. Taken together, these data provide direct evidence that Ca2+ entry via presynaptic 5HT3Rs facilitates the release of GABA from hippocampal interneurons.     

N-methyl-D-aspartate triggers neonatal rat hypoglossal motoneurons in vitro to express rhythmic bursting with unusual Mg2+ sensitivity

The brainstem nucleus hypoglossus innervates the tongue which must contract rhythmically during respiration, chewing and swallowing. Such rhythmic discharges are due to network bursting mediated by AMPA receptor-dependent glutamatergic transmission. The contribution by hypoglossal motoneurons themselves to rhythmicity remains, however, unclear as they might simply express cyclic patterns produced by premotoneurons or, in analogy to spinal motoneurons, might participate to bursting due to activation of their N-methyl-D-aspartate (NMDA) receptors. Using patch clamp recording from hypoglossal motoneurons in slice preparations of neonatal rat brainstem, we observed that NMDA directly depolarized motoneurons to generate various discharge patterns. Most motoneurons produced transient bursts which were consistently restored by repolarizing membrane potential to rest. Fewer motoneurons generated either sustained bursting or random firing. Rhythmic bursts were recorded from XII nerve rootlets even when single motoneuron bursting required hyperpolarization. NMDA evoked bursts were blocked by the Ca2+ antagonist Cd2+, the gap junction blocker carbenoxolone, or Mg2+ free solution, and partially inhibited by tetrodotoxin or nifedipine. Under voltage clamp, NMDA-induced bursting persisted at negative or positive potentials and was resistant to high extracellular Mg2+ in accordance with the observation of widespread motoneuron expression of NMDA 2D receptor subunits that confer poor Mg2+ sensitivity. It is proposed that NMDA depolarized motoneurons with the contribution of Mg2+ insensitive channels, and triggered bursting via cyclic activation/deactivation of voltage-dependent Na+, Ca2+ and K+ currents spread through gap junctions. The NMDA-evoked bursting pattern was similar to the rhythmic discharges previously recorded from the XII nerve during milk sucking by neonatal rats.     

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors modulate release of inhibitory amino acids in rat spinal cord dorsal horn

Local inhibition within the spinal cord dorsal horn is mediated by the neurotransmitters GABA and glycine and strongly influences nociceptive and temperature signaling. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are expressed by inhibitory interneurons and have been shown to modulate GABA release in other regions of the CNS. In the spinal cord, there is morphological evidence for presynaptic AMPA receptor subunits in GABAergic dorsal horn neurons, but functional data are lacking. To determine if AMPA receptors are indeed functional at presynaptic terminals of inhibitory neurons, we recorded evoked and miniature inhibitory postsynaptic currents (mIPSPs) in the superficial dorsal horn of the rat spinal cord. We show that AMPA receptor activation enhances spontaneous release of inhibitory amino acids in the presence of tetrodotoxin onto both lamina II neurons and NK1 receptor-expressing (NK1R+) lamina I neurons. This effect is sensitive to the concentration of extracellular Ca2+, yet is not fully blocked in most neurons in the presence of Cd2+, suggesting possible Ca2+ entry through AMPA receptors. Postsynaptic Ca2+ elevation is not required for these changes. AMPA-induced increases in mIPSP frequency are also seen in more mature dorsal horn neurons, indicating that these receptors may play a role in nociceptive processing in the adult. In addition, we have observed AMPA-induced depression of evoked release of GABA and glycine onto lamina I NK1R+ neurons. Taken together these data support a role for presynaptic AMPA receptors in modulating release of GABA and glycine in the superficial dorsal horn. Because inhibition in the dorsal horn is important for controlling pain signaling, presynaptic AMPA receptors acting to modulate the inhibitory inputs onto dorsal horn neurons would be expected to impact upon pain signaling in the spinal cord dorsal horn.     

Angiotensin II regulates endothelial cell migration through calcium influx via T-type calcium channel in human umbilical vein endothelial cells

Aim: The T-type calcium channel is expressed in vascular endothelial cells, but its role in endothelial cell function is yet to be elucidated. We analysed the endothelial functional role of T-type calcium channel-dependent calcium under angiotensin II (Ang II) stimulation. Methods: Human umbilical vein endothelial cells were co-incubated with hormone at 10⁻⁷ m and either Efonidipine 10⁻⁵ m or Verapamil 10⁻⁵ m or Mibefradil 10⁻⁵ m or Wortmannin 10⁻⁶ m . The contribution of Ang II receptors was evaluated using PD123319 10⁻⁷ m and ZD 7155 10⁻⁷ m . The calcium ion concentration was observed using Fluo-3 acetossimetil ester. The cells were observed after 3, 6, 9 and 12 h. Results: The microfluorescence method points out that Ang II induces intracellular calcium modulation in time by distinct mechanisms. AT2 receptor blockade is necessary to observe significant increase in [Ca²⁺]_i levels. Pre-treatment with Mibefradil abolishes Ang II -induced cell migration. Conclusions: Our data show that Ang II, via AT1 receptor, modulates calcium concentration involving T-type calcium channel and L-type calcium channel but only the calcium influx via T-type calcium channels regulates endothelial cell migration which is essential for angiogenesis.     

Multiple mechanisms of manganese-induced quenching of fura-2 fluorescence in rat mast cells

Whole-cell patch-clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca²⁺]_i) were used to study Mn²⁺ influx in rat peritoneal mast cells. The calcium-selective current, activated by depletion of intracellular calcium stores (I _CRAC for calcium release-activated calcium current), supports a small but measurable Mn²⁺ current. In the presence of intracellular BAPTA, a Mn²⁺ current through I _CRAC was recorded in isotonic MnCl₂ (100 mM) without a significant quenching of fura-2 fluorescence. Its amplitude was 10% of that measured in physiological solution containing 10 mM Ca²⁺. However, following store depletion, a significant quenching of fura-2 fluorescence could be measured only when intracellular BAPTA was omitted, so that all the incoming Mn²⁺ could be captured by the fluorescent dye. Two other ionic currents activated by receptor stimulation also induced Mn²⁺ quenching of fura-2 fluorescence: a small current through non-specific cation channels of 50-pS unitary conductance and a distinct cationic current of large amplitude. In addition to these influx mechanisms, Mn²⁺ was taken up into calcium stores and was subsequently co-released with Ca²⁺ by Ca²⁺-mobilizing agonists.     

Vesicular GABA release delays the onset of the Purkinje cell terminal depolarization without affecting tissue swelling in cerebellar slices during simulated ischemia

Neurosteroids that can enhance GABA_A receptor sensitivity protect cerebellar Purkinje cells against transient episodes of global brain ischemia, but little is known about how ischemia affects GABAergic transmission onto Purkinje cells. Here we use patch-clamp recording from Purkinje cells in acutely prepared slices of rat cerebellum to determine how ischemia affects GABAergic signaling to Purkinje cells. In voltage-clamped Purkinje cells, exposing slices to solutions designed to simulate brain ischemia caused an early, partial suppression of the frequency of spontaneous inhibitory post synaptic currents (sIPSCs), but after 5–8 min GABA accumulated in the extracellular space around Purkinje cells, generating a large (∼17 nS), sustained GABA_A receptor-mediated conductance. The sustained GABA_A conductance occurred in parallel with an even larger (∼117 nS) glutamate receptor-mediated conductance, but blocking GABA_A receptors did not affect the timing or magnitude of the glutamate conductance, and blocking glutamate receptors did not affect the timing or magnitude of the GABA_A conductance. Despite the lack of interaction between GABA and glutamate, blocking GABA_A receptors significantly accelerated the onset of the Purkinje cell “ischemic” depolarization (ID), as assessed with current-clamp recordings from Purkinje cells or field potential recordings in the dendritic field of the Purkinje cells. The Purkinje cell ID occurred ∼2 min prior to the sustained glutamate release under control conditions and a further 1–2 min earlier when GABA_A receptors were blocked. Tissue swelling, as assessed by monitoring light transmittance through the slice, peaked just after the ID, prior to the sustained glutamate release, but was not affected by blocking GABA_A receptors. These data indicate that ischemia induces the Purkinje cell ID and tissue swelling prior to the sustained glutamate release, and that blocking GABA_A receptors accelerates the onset of the ID without affecting tissue swelling. Taken together these data may explain why Purkinje cells are one of the most ischemia sensitive neurons in the brain despite lacking NMDA receptors, and why neurosteroids that enhance GABA_A receptor function protect Purkinje cells against transient episodes of global brain ischemia.     

gamma-Aminobutyric acidA (GABAA) receptors modulate [3H]GABA release from isolated neuronal growth cones in the rat

Potassium-induced release of gamma-[3H]aminobutyric acid [( 3H]GABA) from a growth cone-enriched fraction isolated from neonatal rat forebrain was inhibited by the GABA mimetic muscimol in a dose-dependent manner (IC50 15 nM). The GABA antagonist bicuculline completely reversed the effect of muscimol. Bicuculline alone slightly potentiated the K+-induced release of [3H]GABA. Baclofen, a proposed selective agonist for a bicuculline-insensitive GABAB receptor, was found to cause only a slight reduction in the K+-induced release of [3H]GABA. These results are compatible with the presence of a negative feedback mechanism mediated by GABAA receptors for controlling [3H]GABA release from growth cones of the developing rat forebrain.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Calcium dysregulation in Alzheimer's disease: From mechanisms to therapeutic opportunities

Calcium is involved in many facets of neuronal physiology, including activity, growth and differentiation, synaptic plasticity, and learning and memory, as well as pathophysiology, including necrosis, apoptosis, and degeneration. Though disturbances in calcium homeostasis in cells from Alzheimer's disease (AD) patients have been observed for many years, much more attention was focused on amyloid-β (Aβ) and tau as key causative factors for the disease. Nevertheless, increasing lines of evidence have recently reported that calcium dysregulation plays a central role in AD pathogenesis. Systemic calcium changes accompany almost the whole brain pathology process that is observed in AD, including synaptic dysfunction, mitochondrial dysfunction, presenilins mutation, Aβ production and Tau phosphorylation. Given the early and ubiquitous involvement of calcium dysregulation in AD pathogenesis, it logically presents a variety of potential therapeutic targets for AD prevention and treatment, such as calcium channels in the plasma membrane, calcium channels in the endoplasmic reticulum membrane, Aβ-formed calcium channels, calcium-related proteins. The review aims to provide an overview of the current understanding of the molecular mechanisms involved in calcium dysregulation in AD, and an insight on how to exploit calcium regulation as therapeutic opportunities in AD.