Characterization of the presynaptic calcium channels involved in glutamate exocytosis from rat hippocampal mossy fiber synaptosomes

Calcium antagonists inhibit both the Ca2(+)-dependent and -independent release of endogenous glutamate from intact synaptosomes. In the present study, the inhibitory potency of several different classes of calcium antagonists were determined under conditions that control for an effect of these compounds on the Ca2(+)-independent component of glutamate release. The following order of inhibitory potency was derived: flunarizine and cinnarizine greater than diltiazem greater than verapamil, nifedipine and nimodipine greater than omega-conotoxin much greater than amiloride, phenytoin, gadolinium and nickel. Only the diphenylpiperazine derivatives inhibited Ca2(+)-dependent glutamate release with an IC50 value of less than 10(-5) M. This finding indicates that no one type of presynaptic calcium channel predominantly mediates Ca2(+)-dependent glutamate release from hippocampal mossy fiber terminals. It is suggested that the exocytosis of glutamate from rat hippocampal mossy fiber synaptosomes may be mediated by multiple types of calcium channels.     

Synapse-to-synapse variation of calcium channel subtype contributions in large mossy fiber terminals of mouse hippocampus

Both N- and P/Q-type voltage-dependent calcium channels are involved in fast transmitter release in the hippocampus, but are differentially regulated. Although variable contributions of voltage-dependent calcium channel subtypes to presynaptic Ca2+ influx have been suggested to give a neural network of great diversity, their presence has only been demonstrated in a culture system and has remained unclear in the brain. Here, the individual large mossy fiber presynaptic terminal was labeled with Ca2+/Sr2+-sensitive fluorescent dextrans in the hippocampal slice of the mouse. The fractional contribution of voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx was directly measured by the sensitivity of Ca2+/Sr2+-dependent fluorescent increment to subtype-selective neurotoxins, omega-conotoxin GVIA (an N-type selective blocker), omega-agatoxin IVA (a P/Q-type selective blocker) and SNX-482 (an R-type selective blocker). Synapse-to-synapse comparison of large mossy fiber terminals revealed that the contributions of N- and R-type voltage-dependent calcium channels varied more widely than that of P/Q-type. Even two large mossy fiber presynaptic terminals neighboring on the same axon differed in the fractional contributions of N- and R-type voltage-dependent calcium channels. On the other hand, these terminals were similar in the fractional contributions of P/Q-type voltage-dependent calcium channels. These results provide direct evidence that individual large mossy fiber synapses are differential in the contribution of N- and R-type voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx. We suggest that the synapse-to-synapse variation of presynaptic voltage-dependent calcium channel subtype contributions may be one of the mechanisms amplifying diversity of the hippocampal network.     

Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network

It is widely accepted that the hippocampus plays a major role in learning and memory. The mossy fiber synapse between granule cells in the dentate gyrus and pyramidal neurons in the CA3 region is a key component of the hippocampal trisynaptic circuit. Recent work, partially based on direct presynaptic patch-clamp recordings from hippocampal mossy fiber boutons, sheds light on the mechanisms of synaptic transmission and plasticity at mossy fiber synapses. A high Na⁺ channel density in mossy fiber boutons leads to a large amplitude of the presynaptic action potential. Together with the fast gating of presynaptic Ca²⁺ channels, this generates a large and brief presynaptic Ca²⁺ influx, which can trigger transmitter release with high efficiency and temporal precision. The large number of release sites, the large size of the releasable pool of vesicles, and the huge extent of presynaptic plasticity confer unique strength to this synapse, suggesting a large impact onto the CA3 pyramidal cell network under specific behavioral conditions. The characteristic properties of the hippocampal mossy fiber synapse may be important for pattern separation and information storage in the dentate gyrus-CA3 cell network.     

Loss of dynorphin-mediated inhibition of voltage-dependent Ca2+ currents in hippocampal granule cells isolated from epilepsy patients is associated with mossy fiber sprouting

The endogenous kappa receptor selective opioid peptide dynorphin has been shown to inhibit glutamate receptor-mediated neurotransmission and voltage-dependent Ca2+ channels. It is thought that dynorphin can be released from hippocampal dentate granule cells in an activity-dependent manner. Since actions of dynorphin may be important in limiting excitability in human epilepsy, we have investigated its effects on voltage-dependent Ca2+ channels in dentate granule cells isolated from hippocampi removed during epilepsy surgery. One group of patients showed classical Ammon's horn sclerosis characterized by segmental neuronal cell loss and astrogliosis. Prominent dynorphin-immunoreactive axon terminals were present in the inner molecular layer of the dentate gyrus, indicating pronounced recurrent mossy fiber sprouting. A second group displayed lesions in the temporal lobe that did not involve the hippocampus proper. All except one of these specimens showed a normal pattern of dynorphin immunoreactivity confined to dentate granule cell somata and their mossy fiber terminals in the hilus and CA3 region. In patients without mossy fiber sprouting the application of the kappa receptor selective opioid agonist dynorphin A ([D-Arg6]1-13, 1 microM) caused a reversible and dose-dependent depression of voltage-dependent Ca2+ channels in most granule cells. These effects could be antagonized by the non-selective opioid antagonist naloxone (1 microM). In contrast, significantly less dentate granule cells displayed inhibition of Ca2+ channels by dynorphin A in patients with mossy fiber sprouting (Chi-square test, P < 0.0005). The lack of dynorphin A effects in patients showing mossy fiber sprouting compares well to the loss of kappa receptors on granule cells in Ammon's horn sclerosis but not lesion-associated epilepsy. Our data suggest that a protective mechanism exerted by dynorphin release and activation of kappa receptors may be lost in hippocampi with recurrent mossy fiber sprouting.     

Pharmacological characterization of voltage-dependent calcium currents in rat hippocampal neurons.

The kinetics and pharmacology of voltage-dependent calcium (Ca) currents in primary cultures of hippocampal neurons were studied using the whole cell clamp technique. The low voltage-activated (LVA) Ca current was activated at -50 mV and completely inactivated within 100 ms. This current was insensitive to omega-conotoxin (omega-CgTx) and to the calcium agonist Bay K 8644. The high-voltage-activated (HVA) Ca current was activated at -20 mV and inactivated incompletely during pulses of 200 ms duration. The snail toxin omega-CgTx revealed two pharmacological components of the HVA Ca current, one irreversibly blocked and the other insensitive to the toxin. Bay K 8644 had a clear agonistic action mainly on the omega-CgTx insensitive component of the HVA Ca current.     

Kainate receptor-mediated inhibition of glutamate release involves protein kinase A in the mouse hippocampus

The mechanisms involved in the inhibition of glutamate release mediated by the activation of presynaptic kainate receptors (KARs) at the hippocampal mossy fiber-CA3 synapse are not well understood. We have observed a long-lasting inhibition of CA3 evoked excitatory postsynaptic currents (eEPSCs) after a brief application of kainate (KA) at concentrations ranging from 0.3 to 10 muM. The inhibition outlasted the change in holding current caused by the activation of ionotropic KARs in CA3 pyramidal cells, indicating that this action is not contingent on the opening of the receptor channels. The inhibition of the eEPSCs by KA was prevented by G protein and protein kinase A (PKA) inhibitors and was enhanced after stimulation of the adenylyl cyclase (AC) with forskolin. We conclude that KARs present at mossy fiber terminals mediate the inhibition of glutamate release through a metabotropic mechanism that involves the activation of an AC-second messenger cAMP-PKA signaling cascade.     

Kainate receptor-dependent presynaptic modulation and plasticity

Kainate-type ionotropic glutamate receptors (KARs) distribute widely and heterogenously throughout the central nervous system (CNS). There is now increasing evidence showing that, in addition to conventional action to mediate postsynaptic excitation, KAR also exerts presynaptic action modulating the amount of transmitter release at certain synapses in the CNS. The mechanism and physiological function of presynaptic KARs have been studied most extensively at the hippocampal mossy fiber (MF)-CA3 synapse, one of the CNS regions where the highest density of KAR subunits is expressed. One unique feature of presynaptic KARs is that their activation modulates transmitter release bi-directionally; weak activation enhances glutamate release, while strong activation leads to inhibition. These findings may be explained by their possible ionotropic action leading to axonal depolarization, which in turn regulates several voltage-dependent channels involved in action potential-dependent Ca2+ entry processes. Furthermore, physiological activation of presynaptic KAR involves an activity-dependent process. Large frequency facilitation, a form of short-term plasticity characteristic of the MF-CA3 synapse, is mediated, at least partly, by presynaptic KAR. Bi-directional and activity-dependent regulation of transmitter release by kainate autoreceptors might have physiological significance in information processing in the hippocampus and other CNS regions, as well as its well-known pathological action contributing to epileptogenesis.     

Presynaptic modulation of glutamate and dynorphin release by excitatory amino acids in the guinea-pig hippocampus.

Excitatory amino acid agonists and antagonists were evaluated for their ability to affect the concomitant release of endogenous L-glutamate and dynorphin A(1-8)-like immunoreactivity from guinea-pig hippocampal mossy fiber synaptosomes. Previous work in this laboratory demonstrated that L(+)2-amino-4-phosphonobutyrate inhibits the potassium-evoked release of these endogenous neurotransmitters from guinea-pig but not rat hippocampal mossy fiber synaptosomes. Therefore, the present study was conducted to evaluate excitatory amino acid agonists as indices to the functional properties of this L(+)2-amino-4-phosphonobutyrate-sensitive glutamatergic autoreceptor on mossy fiber terminals. Low micromolar concentrations of quisqualate, but not kainate, N-methyl-D-aspartate, nor RS-alpha-amino-3-hydroxy-5-methyl-4-isoazole-propionic acid, significantly inhibited the potassium-evoked release of both L-glutamate and dynorphin A(1-8)-like immunoreactivity. Quisqualate-induced inhibition of L-glutamate release from mossy fiber terminals was antagonized by the non-N-methyl-D-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. In contrast, high concentrations of kainate enhanced the potassium-evoked release of L-glutamate and dynorphin A(1-8)-like immunoreactivity, and this potentiation was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Kainate (1 mM) was the only agonist which significantly enhanced the basal release of L-glutamate, whereas the spontaneous efflux of dynorphin A(1-8)-like immunoreactivity was not affected by any of the agonists tested. The results presented in this paper suggest the existence of inhibitory and excitatory presynaptic glutamatergic autoreceptors that act to modulate the release of endogenous L-glutamate- and prodynorphin-derived peptides from guinea-pig hippocampal mossy fiber terminals. These inhibitory and excitatory autoreceptors, which are sensitive to quisqualate/L(+)2-amino-4-phosphonobutyrate or kainate, respectively, may play an important role in regulating synaptic activity at glutamatergic synapses throughout the central nervous system.     

Assessing the roles of presynaptic ryanodine receptors and adenosine receptors in caffeine-induced enhancement of hippocampal mossy fiber transmission

Caffeine robustly enhances transmitter release from the hippocampal mossy fiber terminals, although it remains uncertain whether calcium mobilization through presynaptic ryanodine receptors mediates this enhancement. In this study, we adopted a selective adenosine A1 blocker to assess relative contribution of A1 receptors and ryanodine receptors in caffeine-induced synaptic enhancement. Application of caffeine further enhanced transmission at the hippocampal mossy fiber synapse even after full blockade of adenosine A1 receptors. This result suggests that caffeine enhances mossy fiber synaptic transmission by two distinct presynaptic mechanisms, i.e., removal of A1 receptor-mediated tonic inhibition and ryanodine receptor-mediated calcium release from intracellular stores.     

Calcium signaling at single mossy fiber presynaptic terminals in the rat hippocampus

We investigated internal Ca(2+) release at mossy fiber synapses on CA3 pyramidal neurons (mossy fiber terminals, MFTs) in the hippocampus. Presynaptic Ca(2+) influx was induced by giving a brief train of 20 stimuli at 100 Hz to the mossy fiber pathway. Using Ca(2+) imaging techniques, we recorded the Ca(2+) response as DeltaF/F, which increased rapidly with stimulation, but was often accompanied by a delayed peak that occurred after the train. The rise in presynaptic [Ca(2+)] could be completely blocked by application of 400 microM Cd(2+). Furthermore, the evoked Ca(2+) signals were reduced by group II mGluR agonists. Under the same experimental conditions, we investigated the effects of several agents on MFTs that disrupt regulation of intracellular Ca(2+) stores resulting in depletion of internal Ca(2+). We found that ryanodine, cyclopiazonic acid, thapsigargin, and ruthenium red all decreased both the early and the delayed increase in the Ca(2+) signals. We applied D,L-2-amino-5-phosphonovaleric acid (D,L-APV; 50 microM) and 6,7-Dinitroquinoxaline-2,3-dione (DNQX; 20 microM) to exclude the action of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Experiments with alternative lower affinity indicators for Ca(2+) (fura-2FF and calcium green-2) and the transient K(+) channel blocker, 4-aminopyridine were performed to control for the possible saturation of fura-2. Taken together, these results strongly support the hypothesis that the recorded terminals were from the mossy fibers of the dentate gyrus and suggest that a portion of the presynaptic Ca(2+) signal in response to brief trains of stimuli is due to release of Ca(2+) from internal stores.     

Assessing the long-term role of L-type voltage dependent calcium channel blocker verapamil on short-term presynaptic plasticity at dentate gyrus of hippocampus

High-voltage-activated Ca(2+) channels on presynaptic nerve terminals are known to play an important role in neurotransmitter release at both excitatory and inhibitory synapses. Whereas there is currently debate over the contribution of L-type voltage dependent Ca(2+) channels (L-type VDCCs) on the short-term presynaptic plasticity which is a defining feature of neuronal activity, the underlying mechanisms are poorly understood. In the present study, the L-type VDCCs chronically was inhibited with different doses of verapamil (10, 20 and 50 mg/kg; orally) to evaluate hippocampal dentate gyrus (DG) inhibitory interneuron function and its involvement on short-term plasticity using paired pulse stimulation in perforant path-DG of hippocampus. Our data show that chronic oral treatment of verapamil at dose of 50 mg/kg but not at lower doses, facilitated the excitability of DG cells at inter-stimulus intervals 20, 30 and 50 ms (P<0.03, 0.01 and 0.001; respectively) in population spike amplitude ratio, which is indicative of paired pulse potentiation in perforant path-DG synapses. While there are no significant differences in field excitatory postsynaptic potential slope ratio at all doses. We suggest that DG neurons facilitation is caused by inhibition of inhibitory interneurons directly and/or indirectly via inhibition of glutamate release in hippocampal DG. Therefore, these experiments indicate that chronic use of verapamil has effect on short-term presynaptic plasticity.     

Neuropeptide Y regulates recurrent mossy fiber synaptic transmission less effectively in mice than in rats: Correlation with Y2 receptor plasticity

A unique feature of temporal lobe epilepsy is the formation of recurrent excitatory connections among granule cells of the dentate gyrus as a result of mossy fiber sprouting. This novel circuit contributes to a reduced threshold for granule cell synchronization. In the rat, activity of the recurrent mossy fiber pathway is restrained by the neoexpression and spontaneous release of neuropeptide Y (NPY). NPY inhibits glutamate release tonically through activation of presynaptic Y2 receptors. In the present study, the effects of endogenous and applied NPY were investigated in C57Bl/6 mice that had experienced pilocarpine-induced status epilepticus and subsequently developed a robust recurrent mossy fiber pathway. Whole cell patch clamp recordings made from dentate granule cells in hippocampal slices demonstrated that, as in rats, applied NPY inhibits recurrent mossy fiber synaptic transmission, the Y2 receptor antagonist (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide (BIIE0246) blocks its action and BIIE0246 enhances synaptic transmission when applied by itself. Y5 receptor agonists had no significant effect. Thus spontaneous release of NPY tonically inhibits synaptic transmission in mice and its effects are mediated by Y2 receptor activation. However, both NPY and BIIE0246 were much less effective in mice than in rats, despite apparently equivalent expression of NPY in the recurrent mossy fibers. Immunohistochemistry indicated greater expression of Y2 receptors in the mossy fiber pathway of normal mice than of normal rats. Pilocarpine-induced status epilepticus markedly reduced the immunoreactivity of mouse mossy fibers, but increased the immunoreactivity of rat mossy fibers. Mossy fiber growth into the inner portion of the dentate molecular layer was associated with increased Y2 receptor immunoreactivity in rat, but not in mouse. These contrasting receptor changes can explain the quantitatively different effects of endogenously released and applied NPY on recurrent mossy fiber transmission in mice and rats.     

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.     

The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa.

The neoglycoproteins alpha-D-mannose-bovine serum albumin (mannose-BSA) and N-acetyl-alpha-D-glucosamine-BSA (glucNAc-BSA) were shown to rapidly increase intracellular free calcium ([Ca2+]i) in human spermatozoa. The increase in [Ca2+]i induced by these neoglycoproteins accounts for the known ability of these compounds to induce the acrosome reaction in human spermatozoa. Our data support the hypothesis that mannose-BSA, but not progesterone, activates T-type Ca2+ channels in human spermatozoa for the following reasons: (i) the capacity of mannose-BSA to increase [Ca2+]i was inhibited by the specific T-type Ca2+ channel blocker mibefradil (IC50 = 10(-6) mol/l) while progesterone was not inhibited by 10(-5) M mibefradil; (ii) the effect of mannose-BSA to elevate [Ca2+]i was inhibited more potently by Ni2+ (IC50 = 0.1 mmol/l) than Cd2+ (IC50 = 0.5 mmol/l), whereas the effect of progesterone to elevate [Ca2+]i was inhibited equally by Ni2+ and Cd2+ (IC50 = 0.25 mmol/l); (iii) the effects of mannose-BSA and progesterone to increase [Ca2+]i were greater than additive. These data support the idea that mannose-BSA and progesterone were activating distinct Ca2+ channels, one of which was a T-type Ca2+ channel activated by mannose-BSA whereas the Ca2+ channel that was activated by progesterone has yet to be defined at the molecular level.     

Pancreatic acinar cells: acetylcholine-induced membrane depolarization, calcium efflux and amylase release

1. The effects of acetylcholine upon the output of amylase, Ca(2+) efflux and membrane potential of pancreatic acinar cells have been measured in segments of mouse pancreas superfused in vitro.2. Amylase output was measured continuously using an on-line automated fluorimetric method; Ca(2+) efflux was monitored by measuring the release of (45)Ca(2+) from pre-labelled tissue; and intracellular recordings of acinar transmembrane potentials were obtained with glass micro-electrodes. In some experiments membrane potentials, and in others (45)Ca(2+) efflux, were measured concomitantly with amylase release.3. Acetylcholine depolarized the acinar cells, increased tissue (45)Ca(2+) efflux and raised amylase output, each with a similar dose-dependence, i.e. a maximal response at 10(-5)M, threshold =/< 10(-8)M, and ED(50) values of 0.7 x 10(-7)M, 0.5 x 10(-7)M, and 2 x 10(-7)M for depolarization, amylase release, and (45)Ca(2+) efflux, respectively.4. In response to acetylcholine both depolarization and (45)Ca(2+) efflux preceded or coincided with the increase in amylase output.5. Acetylcholine 10(-5)M and [K](0) 47 mM were without effect on (45)Ca(2+) efflux in the presence of atropine (3 x 10(-6)M) but pancreozymin (0.3 u./ml.) still elicited a marked increase in (45)Ca(2+) release.6. These results suggest that the stimulatory action of acetylcholine on the pancreatic acinar cell involves, sequentially, a specific receptor-activated increase in membrane permeability, depolarization, Ca(2+) mobilization and amylase release. These events are discussed in relation to the integrated mechanism of stimulus-secretion coupling.     

Effect of azelastine on the release and action of leukotriene C4 and D4

The effect of azelastine on the release of leukotriene C4 and D4 (LTC4 and LTD4), and the antagonistic action of the drug against the leukotrienes were determined by using in vitro tests and compared with those of ketotifen and chlorpheniramine. Azelastine inhibited LTC4 and LTD4 release from guinea pig lung fragments passively sensitized with homologous anti-ovalbumin IgGl-b antibody. The 50% inhibitory concentration (IC50) of azelastine was 6.4 X 10(-5) M for a 15-min preincubation or 4.7 X 10(-5) M for a 30-min preincubation. Ketotifen and chlorpheniramine were inhibitory only at the highest concentration tested (3 X 10(-4) M), giving inhibitions of 35.6 and 21.3%, respectively. Azelastine also inhibited calcium ionophore A23187-induced release of leukotrienes from human polymorphonuclear leukocytes; the IC50 values were 3.6 X 10(-5) M for 15 min and 2.3 X 10(-6) M for 30 min of preincubation. Ketotifen and chlorpheniramine were inhibitory only after a 30-min preincubation, with IC50 values of 2.1 X 10(-5) and 5.9 X 10(-5) M, respectively. The potent inhibition by azelastine might be partly a result of the inhibition of 5-lipoxygenase, since 5-hydroxyeicosatetraenoic acid formation in rat basophilic leukemia cell homogenate was inhibited by azelastine. Pretreatment of guinea pig ileum with azelastine antagonized LTC4- and LTD4-induced contraction of the ileum with IC50 values of 7.0 X 10(-6) and 1.1 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hippocampal mossy fiber activity evokes Ca2+ release in CA3 pyramidal neurons via a metabotropic glutamate receptor pathway.

Mossy fiber activity can evoke Ca2+ release from internal stores in CA3 neurons, but the physiological conditions under which this occurs and the mechanisms underlying the release are not understood. Using rat hippocampal slices we report here that short trains of mossy fiber stimulation activate group I metabotropic glutamate receptors (mGluRs) on CA3 pyramidal neurons and elicit waves of Ca2+ release from inositol 1,4,5-trisphosphate (IP3) sensitive internal stores that propagate from stratum lucidum to the soma and in some cases distally out the dendrites. Activation of mGluR1,5 receptors by an agonist trans-azetidine-2,4-dicarboxylic acid (tADA) applied to stratum lucidum was also sufficient to induce waves of Ca2+ release. This release was blocked by internal heparin, but not by dantrolene, suggesting the involvement of IP3 rather than ryanodine receptors in not only the initial release but also in the maintenance of the propagating waves. Release could be facilitated by Ca2+ influx through voltage-gated Ca2+ channels, which is consistent with the known Ca2+ sensitivity of IP3 receptors.These results provide insight into the mechanisms and conditions of Ca2+ release in CA3 neurons and demonstrate the powerful influence mossy fiber input can have on these neurons.     

Assessment of the antidepressant activity of dothiepin and its metabolites by preclinical tests

The affinities of dothiepin and its principal metabolites northiaden, dothiepin sulphoxide and northiaden sulphoxide for [3H]imipramine binding sites in the rat cortical homogenates, and for [3H]spiperone and [3H]serotonin receptor sites in preparations from the rat frontal cortex and hippocampus were studied. As inhibitors of [3H]imipramine binding, the strengths of the drugs are, in terms of their IC50 (concentration corresponding to 50% inhibition): dothiepin 2.8 X 10(-6) M, northiaden 5.0 X 10(-6) M, northiaden sulphoxide 4.0 X 10(-5) M and dothiepin sulphoxide 3.2 X 10(-5) M. The potencies of the drugs in inhibiting serotonergic binding followed a similar trend. Using frontal cortical tissue suspensions and [3H]spiperone, the IC50 values were determined to be: dothiepin 4.2 X 10(-6) M, northiaden 5.0 X 10(-6) M, northiaden sulphoxide 1.6 X 10(-4) M and dothiepin sulphoxide 1.6 X 10(-4) M; whereas in hippocampal suspensions and using [3H]serotonin, the IC50 values were 2.5 X 10(-6) M, northiaden 4.0 X 10(-5) M, dothiepin sulphoxide 2.5 X 10(-4) M and northiaden sulphoxide greater than 10(-3) M. The influence of the drugs on the uptake of [14C]serotonin into human platelets was also investigated. All had an inhibitory effect upon the uptake, the order of potency being dothiepin greater than northiaden greater than northiaden sulphoxide greater than dothiepin sulphoxide. Plots of 1/v versus 1/s showed that the inhibition was competitive for all four compounds.     

Electron microscopic study of mossy fiber endings of the hippocampal formation in El mice

The effects of seizure activity on the mossy fiber endings of El mice were studied by electron microscopy. During epileptic seizures of El mice, the number of clear round vesicles (50 nm) in the mossy fiber endings of the hippocampal formation decreased, while the number of large densecore vesicles (100 nm) increased. In these endings, the large dense-core vesicles were scattered during the resting state, but after seizure activity they tended to accumulate together and attach to the presynaptic membrane. Omega-shaped profiles, which seemed to be due to exocytosis of the large dense-core vesicles, were seen in the presynaptic membrane.     

Multiple forms of LTP in hippocampal CA3 neurons use a common postsynaptic mechanism.

We investigated long-term potentiation (LTP) at mossy fiber synapses on CA3 pyramidal neurons in the hippocampus. Using Ca2+ imaging techniques, we show here that when postsynaptic Ca2+ was sufficiently buffered so that [Ca2+]i did not rise during synaptic stimulation, the induction of mossy fiber LTP was prevented. In addition, induction of mossy fiber LTP was suppressed by postsynaptic injection of a peptide inhibitor of cAMP-dependent protein kinase. Finally, when ionotropic glutamate receptors were blocked, LTP depended on the postsynaptic release of Ca2+ from internal stores triggered by activation of metabotropic glutamate receptors. These results support the conclusion that mossy fiber LTP and LTP at other hippocampal synapses share a common induction mechanism involving an initial rise in postsynaptic [Ca2+].