Temporal regularity of neurotransmitter release at single terminal in cultured hippocampal neurons

The whole-cell GABA-mediated inhibitory postsynaptic currents were studied using the patch-clamp technique on synaptically connected cultured hippocampal neurons. The stimulus-evoked inhibitory postsynaptic currents were recorded in the tetrodotoxin-containing solution in response to the low-amplitude long (10-20ms) extracellular depolarization of a single presynaptic terminal. During each depolarization the postsynaptic response in a form of several superimposed independent events was recorded. The amplitudes of these responses fluctuated randomly, irrespective of the number of the event. In all the investigated neurons the distributions of delays revealed regularly spaced multiple peaks. The number of peaks increased with the duration of stimulus. The distance between the peaks was on average 2.97+/-0.86ms (n=58). The mean intervals between successive releases were distributed exponentially indicating the independence of the release sites. Thus neurotransmitter release might occur with maximal probability at the most probable times irrespective of the presence or failure of the previous event. The increase in stimulating pulse amplitude led to a decrease in the number of clearly detectable peaks in distributions. The decrease in the number of peaks in the distribution of delays was not accompanied with a decrease in the distance between peaks within the range of reliable resolution of the peaks. The amplitude distribution also revealed regularly spaced multiple peaks. The absence of significant correlation between the amplitude of the first and the second event demonstrated the independence of the succeeding release on the preceding release during long stimulation. Results of statistical analysis of our experimental data supports the hypothesis of multiquantal neurotransmitter release in a single inhibitory hippocampal synapse. Neurotransmitter release during long stimulation may occur at certain times with maximal probability, keeping the mean inter-release interval constant. Thus the interval is not determined directly by the depletion of vesicles and the number of vesicles which may be released at the most probable time is random.     

Reduction of neurotransmitter release by the exogenous H3 domain peptide of HPC-1/syntaxin 1A in cultured rat hippocampal neurons

A recent study has shown that a genetic variation in the Cathepsin D (catD) gene is a major risk factor for the development of Alzheimer's disease (AD). CatD is an intracellular aspartyl protease involved in neurodegeneration. A C-->T (Ala-->Val) transition at position 224 has been associated with altered intracellular maturation. Recently, a significant overrepresentation of the T allele of the catD gene in AD patients compared with controls was reported. However, this finding has not yet been confirmed. We analyzed the distribution of catD and apolipoprotein E polymorphisms in Italian patients with sporadic and familial AD (FAD). Our studies revealed that the distribution of catD polymorphism did not differ in AD and FAD patients and controls. Thus, our data do not support a role for the catD gene as a genetic risk factor in the development of AD.     

Corticosterone increases serotonin type-3 receptor mRNA in rat pheochromocytoma-12 cells

We investigated nocturnal secretion pattern of melatonin in patients with cardiac syndrome X and healthy subjects. The present study performed in five patients with cardiac syndrome X and in nine healthy controls. Blood samples from all subjects were collected every 2 h intervals between 22:00 and 08:00 h. Melatonin levels were measured with a radioimmunoassay kit. Patients with cardiac syndrome X secreted less nocturnal melatonin at 02:00 h than control subjects (P=0.04). Peak and Delta melatonin (peak-lowest melatonin) were found lower in patients with cardiac syndrome X (P=0.039 and P=0.028, respectively). In conclusion patients with cardiac syndrome X show a markedly decreased nocturnal melatonin synthesis. Our study provides useful information about melatonin synthesis and release in patients with cardiac syndrome X might help physicians in managing these patients.     

Enhancement of leukocyte response to lipopolysaccharide by secretory group IIA phospholipase A2.

Secretory nonpancreatic group IIA phospholipase A2 (sPLA2), a lipolytic enzyme found in plasma, is thought to play an important role in inflammation. In patients with sepsis, a strong positive correlation is observed between the plasma level of sPLA2 and poor clinical outcome in sepsis. We have thus asked whether sPLA2 could play a role in enabling responses of cells to bacterial lipopolysaccharide (LPS), a key contributor to sepsis. In the presence of sPLA2, cellular responses to LPS were significantly increased. This was demonstrated in assays of LPS-stimulated interleukin-6 (IL-6) production in whole blood and binding of freshly isolated human polymorphonuclear neutrophils (PMN) to fibrinogen-coated surfaces. We further found that sPLA2 enhanced binding of labeled LPS to PMN, and that the sPLA2-mediated cell responses to LPS were all blocked by monoclonal antibodies directed against membrane CD14. Two properties ofsPLA2 may contribute to its activity to mediate responses to LPS. sPLA2 appears to bind LPS because pre-exposure of sPLA2 to LPS led to a dose-dependent increase in its ability to hydrolyze phospholid substrate, and incubation of sPLA2 with BODIPY-LPS micelles resulted in enhanced fluorescence, presumably from the disaggregation of the LPS aggregates. Additional studies demonstrated that the esterolytic function of sPLA2 is also needed both for the disaggregation of LPS and CD14-dependent cell stimulation. The precise mechanisms by which LPS-binding and esterolytic activity contribute to sPLA2 activity are not clear but our data strongly suggest that these activities result in interaction of LPS with CD14 and subsequent cell activation.     

Expression of group IIA secretory phospholipase A2 is elevated in prostatic intraepithelial neoplasia and adenocarcinoma

Phospholipase A2 (PLA2) enzymes release arachidonic acid from cellular phospholipids in a variety of mammalian tissues, including prostate. Group IIa secretory PLA2 (sPLA2) can generate arachidonate from cellular phospholipids. We examined the group IIa sPLA2 expression in benign prostatic tissues, prostatic intraepithelial neoplasia (PIN), and adenocarcinoma to determine whether sPLA2 expression is altered in the carcinogenesis of human prostatic cancer. Thirty-three of 74 total cases (45%) of benign prostatic tissue showed positive immunohistochemical staining for group IIA sPLA2, whereas 63 of 69 total cases (91%) of high-grade PINs and 70 of 78 total cases (90%) of adenocarcinomas gave positive results. Four of 10 cases of low-grade PIN showed positive immunoreactivity for sPLA2. The number of cells staining for sPLA2 was significantly less in benign epithelium (4%) and low-grade PIN (4%) compared to high-grade PIN (40%) or adenocarcinoma (38%) (P < 0.001). There was no significant difference between high-grade PIN and adenocarcinoma in the number of cells staining positively for sPLA2. The intensity of sPLA2 immunoreactivity was also different among benign prostatic tissue, low-grade PIN, high-grade PIN, and prostatic adenocarcinoma specimens. The malignant cells demonstrated more intense immunohistochemical staining (moderate to strong staining in 81% and 69% cases for high-grade PIN and adenocarcinoma, respectively) than benign glands (moderate staining in 11% of cases). No strong staining was observed in benign glands or low-grade PIN. Our data are consistent with the contention that group IIA sPLA2 expression is elevated in neoplastic prostatic tissue and support the hypothesis that dysregulation of sPLA2 may play a role in prostatic carcinogenesis.     

A chloride channel in excised patches from cultured rat hippocampal neurons

A non-calcium-dependent chloride channel of conductance 62 pS, found in isolated inside-out patches of cultured fetal rat hippocampal neurons, is described. The channel does not require the presence of any neurotransmitter to be active and probably plays a part in maintaining the normal resting membrane potential.     

Overexpression of APP stimulates basal and constitutive exocytosis in PC12 cells

The mechanisms that underlie the altered neurotransmitter system in Alzheimer's disease (AD) are not well understood. Amyloid precursor protein (APP) is a precursor protein for beta-amyloid, an important trigger protein in the pathogenesis of AD. Duplication of the APP gene as well as APP genes that contain certain mutations has been reported to be associated with familial AD (FAD), and a role of APP in neurotransmission has been suggested recently. This study examines the role of APP in exocytosis in PC12 cells using transfected human growth hormone (hGH) as a reporter for secretion. It was found that overexpression of APP or expression of the Swedish FAD mutation (APPsw) in PC12 cells significantly increased the basal secretion and constitutive secretion of hGH. Expression of an APP phosphorylation-deficient mutant decreased both basal and constitutive secretion relative to the APP wild-type, suggesting a role for APP-Thr668 phosphorylation in secretion in PC12 cells. Overexpression of X11alpha, a protein that stabilizes cellular APP, also increased the basal secretion of hGH but, contrary to APP, decreased the constitutive secretion of hGH, suggesting that basal and constitutive secretion is likely to proceed via distinct pathways and that the increase in the basal secretion of hGH may result from APP-X11alpha interaction. These results demonstrate an unknown role for APP in secretion, and suggest that elevated levels of APP or APP mutation in FAD brains contribute to the altered neurotransmitter pathology of AD through stimulation of basal and constitutive secretion.     

Extracellular phospholipase A2 and histamine release from rat peritoneal mast cells.

Phospholipase A2 (PLA2) from cobra (Naja naja) venom and PLA2 from porcine pancreas accelerated IgE antibody-mediated histamine release from rat peritoneal mast cells. These enhancements were clearly abrogated by heating the enzymes and pretreatment with parabromophenacyl bromide, mepacrine and antiflammin. Indomethacin (cyclooxygenase inhibitor) and AA-861 (lipoxygenase inhibitor) did not affect the enhancement by PLA2. These results indicate that extracellular PLA2 enhances the IgE antibody-mediated histamine release from rat peritoneal mast cells without the participation of arachidonate.     

Increased type IIA secretory phospholipase A2 expression contributes to oxidative stress in end-stage renal disease

End-stage renal disease (ESRD) patients exhibit increased in vivo oxidative stress conceivably contributing to cardiovascular mortality. The type IIA secretory phospholipase A₂ (sPLA₂) has proatherogenic activity. We explored the hypothesis that sPLA₂ contributes to oxidative stress generation and endothelial dysfunction in ESRD patients and transgenic (tg) mice. Patients with ESRD had increased in vivo oxidative stress as assessed by plasma isoprostane levels (p < 0.001). Active sPLA₂ in plasma was substantially increased compared with healthy controls (1,156 ± 65 versus 184 ± 5 ng/dL, p < 0.001) and correlated with plasma isoprostanes (r = 0.61, p < 0.001). Correspondingly, human sPLA₂ tg mice display increased generation of reactive oxygen species within aortic vascular smooth muscle cells, leading to severe endothelial dysfunction (maximal vasodilation in response to 10 μmol/L acetylcholine, sPLA₂ 36 ± 8%, controls 80 ± 2% of phenylephrine-induced vasoconstriction). Increased vascular oxidative stress in sPLA₂ tg mice is dependent on the induction of vascular cyclooxygenase (COX)2 expression. Conversely, ESRD patients show increased formation of COX2-derived prostaglandins (p < 0.05) correlated with plasma sPLA₂ (r = 0.71, p < 0.05). Our data indicate that increased expression of sPLA₂ might represent a novel causative risk factor contributing to the increased cardiovascular disease morbidity and mortality in ESRD.     

Single voltage-dependent potassium channels in cultured rat hippocampal neurons

Single-channel recordings using the gigohm seal patch-clamp technique were carried out on the somatic membranes of dissociated embryonic rat hippocampal neurons grown in cell culture. The recording medium contained tetrodotoxin to block the voltage-dependent Na+ conductance and Cd2+ to block Ca2+ and Ca2+-activated conductances. In the cell-attached configuration, depolarizing voltage steps activated outward directed single-channel currents with conductance 15-20 pS. The channel openings exhibited a moderate degree of flickering. The mean burst lifetimes ranged from 5 to 13 ms with a tendency to increase slightly at more depolarized potentials (T = 21-25 degrees C). Reversal potential measurements using excised membrane patches indicated that the channels behaved as expected of a K+-selective membrane pore. Channel opening occurred in Ca2+-free EGTA-containing solutions but was never observed in the presence of tetraethylammonium (TEA; 20 mM). The frequency of channel opening increased as the membrane was depolarized by up to 50 mV from resting potential; the fraction of time spent in the open state during the first 300 ms following a step depolarization increased e-fold for a 8-25 mV change in potential. First-latency histograms and simulations of the macroscopic current based on channel data obtained during repeated depolarizing voltage steps indicated that the probability of the channel being in the open state increases gradually with time after a step depolarization. During repeated depolarizing steps the channels appeared to randomly enter and exit a long-lived inactive state. It is concluded that these channels may underly the slowly activating, very slowly inactivating, TEA-sensitive voltage-dependent K+ current (IK) in cultured hippocampal neurons.     

Excision-activated chloride channels in cultured postnatal rat hippocampal neurons

Chloride permeable intermediate conductance single channel events activated on patch excision were found in outside-out patches from cultured postnatal hippocampal neurons. A majority of the channels had a conductance of 83 ± 2.1 pS when recorded in a symmetrical TEAC1 solution. Two other populations of channels with conductance values of 62 ± 2.1 pS and 145 ± 1.9 pS were also observed. The reversal potentials for these intermediate conductance Cl⁻ channels coincided with that of the GABA activated channels. The channels characteristically appeared 5–15 min after patch excision, suggesting that these channels may be blocked by some diffusible factors under physiological conditions. Based on the measurements of channel burst durations while the channel was partially blocked, and the channel open times after complete relief from the block, the mechanism of blockade does not appear to be a simple open channel blockade. The high prevalence and its potential regulation by cytosolic factors suggest an important physiological role for these Cl⁻ channels coupling neuronal excitability with cellular metabolism.     

CRMP-2 induces axons in cultured hippocampal neurons

In cultured hippocampal neurons, one axon and several dendrites differentiate from a common immature process. Here we found that CRMP-2/TOAD-64/Ulip2/DRP-2 (refs. 2-4) level was higher in growing axons of cultured hippocampal neurons, that overexpression of CRMP-2 in the cells led to the formation of supernumerary axons and that expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant-negative manner. Thus, CRMP-2 seems to be critical in axon induction in hippocampal neurons, thereby establishing and maintaining neuronal polarity.     

Non-synaptic exocytosis enhanced in rat cerebellar granule neurons cultured under survival-promoting conditions

Cultured rat cerebellar granule neurons (CGNs) are often used to analyze activity-dependent neuronal selection occurring during brain development. The CGNs survive long only when the culture medium contains a depolarizing agent. However, it is argued whether the depolarization critical for survival is of presynaptic or postsynaptic compartment. Since CGNs form no synapses among them, it is generally assumed that the latter would be the case. But it is possible that the depolarization would induce exocytosis of survival-promoting substances whether or not CGNs form synapses. Here we directly examined the exocytotic activities of CGNs under survival-promoting and survival-limiting conditions by electron microscopy to support this possibility. CGNs possessed clusters of synaptic vesicle-like vesicles (SVVs) in neuritic varicosities. CGNs cultured in high-KCl medium had significantly smaller SVV clusters than those cultured in low-KCl medium. The number of SVVs increased when the high KCl-cultured CGNs were transferred to low-KCl medium, indicating a sustained high rate of exocytosis in high-KCl medium. The majority of the varicosities containing SVVs were not apposed to definite postsynaptic structures, indicating that exocytosis occurs from a non-synaptic surface. Fluorescence Ca(2+) imaging revealed that the high KCl-cultured CGNs had spots of high Ca(2+) along their neurites, corresponding to the varicosities.     

Desensitization of GABA-induced currents in cultured rat hippocampal neurons.

The basic characteristics of desensitization of the GABAA receptor were investigated in cultured rat hippocampal neurons (three days to four weeks in vitro) using whole cell patch clamp techniques. GABA at 10-500 microM was perfused on to neurons for 30 or 60 s, with 60 s intervals of wash with control bath solution between perfusions. Desensitization, evaluated by peak-to-plateau ratio and time constants of current decay (tau), was dose-dependent and culture age-dependent. Desensitization was observed as early as three days in culture, the earliest time tested. At all ages, higher concentrations of GABA induced both larger and faster desensitization. Desensitization was markedly voltage-dependent and decreased with depolarization; peak-to-plateau ratio went from 6.3 to 1.4 and tau went from 4.6 to 26.8 s when holding potentials were changed from -80 mV to +30 mV. Low concentrations of GABA (1-2 microM) perfused for 2-60 s, which did not induce any current, had no effect on the maximal response nor desensitization produced by a subsequent application of 100 microM GABA. This finding suggests that GABA receptors were not desensitized without first being activated.     

Molecular correlates of the M-current in cultured rat hippocampal neurons

M-type K⁺ currents ( I _K(M)) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K⁺ channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I _K(M) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I _K(M) with an IC₅₀ of 3.6 ± 1.5 μM. In 70 % of these cells, TEA fully suppressed I _K(M) with an IC₅₀ of 0.7 ± 0.1 m m . In the remaining cells, TEA maximally reduced I _K(M) by only 59.7 ± 5.2 % with an IC₅₀ of 1.4 ± 0.3 m m ; residual I _K(M) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I _K(M) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.     

Effect of serum from cardiovascular patients on catalytic activity of secretory phospholipase A2 (IIA)

Incubation of patients’ serum catalytically active by type IIA secretory phospholipase A₂ (SP-IIA) with serum containing the enzyme in a high concentration but exhibiting no catalytic activity in 1:1 volume ratio led to a significant inhibition of SP-IIA catalytic activity. Donor and patient sera with low levels of SP-IIA had no effect on the serum with SP-IIA activity under these conditions. However, the increase in the content of patients’ serum with a low level of SP-IIA in the incubation mixture to 1:2 (v/v) and of donor serum to 1:3 (v/v) also led to blockade of SP-IIA catalytic activity. These results indicate that human serum contains an SP-IIA inhibitor and its concentration decreases significantly in sera with SP-IIA activity. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 11, pp. 525–527, November, 2006     

Effects of secretory phospholipase A2 enzymes on mast cells of rat,guinea pig and human

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