Synaptic vesicle dynamics in the mossy fiber-CA3 presynaptic terminals of mouse hippocampus

The mossy fiber (MF)-CA3 synapse in the hippocampus is unique in the CNS because of its wide dynamic range of transmitter release during short- and long-term plasticity. The presynaptic mechanisms underlying the fidelity of transmission were investigated for the MF-CA3 synapses. The relative size of readily releasable pool (RRP) of vesicles was estimated by counting the number of docked vesicles at an active zone (AZ) on the transmission electron microscopy (TEM) image. The size of the releasable pool and the exo-endocytosis kinetics were directly measured from individual large MF boutons in hippocampal slices of transgenic mice that selectively express synaptopHluorin (SpH), a pH-sensitive GFP fused to the lumenal aspect of one of the vesicular membrane proteins, VAMP-2, in these boutons. Here we found (1) there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool, (2) the initially docked vesicles are easily depleted and the RRP is maintained by refilling from the reserve subpopulation of releasable pool (“reserve” releasable pool), and (3) the contribution of rapid reuse of recycled vesicles is relatively small. Therefore, the fidelity of transmission is suggested to be ensured by the rapid refilling rate of RRP.     

Synaptic vesicle dynamics in the mossy fiber-CA3 presynaptic terminals of mouse hippocampus

The mossy fiber (MF)-CA3 synapse in the hippocampus is unique in the CNS because of its wide dynamic range of transmitter release during short- and long-term plasticity. The presynaptic mechanisms underlying the fidelity of transmission were investigated for the MF-CA3 synapses. The relative size of readily releasable pool (RRP) of vesicles was estimated by counting the number of docked vesicles at an active zone (AZ) on the transmission electron microscopy (TEM) image. The size of the releasable pool and the exo-endocytosis kinetics were directly measured from individual large MF boutons in hippocampal slices of transgenic mice that selectively express synaptopHluorin (SpH), a pH-sensitive GFP fused to the lumenal aspect of one of the vesicular membrane proteins, VAMP-2, in these boutons. Here we found (1) there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool, (2) the initially docked vesicles are easily depleted and the RRP is maintained by refilling from the reserve subpopulation of releasable pool ("reserve" releasable pool), and (3) the contribution of rapid reuse of recycled vesicles is relatively small. Therefore, the fidelity of transmission is suggested to be ensured by the rapid refilling rate of RRP.     

Sprouting of mossy fibers and presynaptic inhibition by group II metabotropic glutamate receptors in pilocarpine-treated rat hippocampal slice cultures

Mossy fibre sprouting (MFS) is a phenomenon observed in the epileptic hippocampus. We have studied MFS, in 7, 14 and 21 day in vitro (DIV) organotypic slice cultures, or in slice cultures treated with pilocarpine (0.5 mM) or pilocarpine and atropine (0.1 mM or 0.5 mM) for 48-72 h at 5 DIV and tested at 21 DIV. Acute application of pilocarpine directly activated hilar neurons and elicited epileptic-like discharges in CA3 pyramids and mossy cells of 5-8 DIV cultures, without causing substantial cell death, as assessed by lactate dehydrogenase measurements. Timm staining revealed increases in MFS in chronic pilocarpine-treated cultures, which was prevented by prior application of atropine. Extracellular synaptic responses were recorded in the granule cell layer and elicited by antidromic mossy fibre stimulation. The GABA(A) antagonist 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid (1 microM) induced a greater increase in the coastline bursting index in pilocarpine-treated cultures than in 21 DIV controls. However, there was no significant increase in the frequency of spontaneous or miniature synaptic events recorded in granule cells from pilocarpine-treated cultures. Granule cells were filled with biocytin and morphometric analysis revealed that the length of axon collaterals in the granule and molecular layer was longer in pilocarpine-treated cultures than in 21 DIV controls. Dual recordings between granule cells and between granule and hilar neurons showed that pilocarpine-treated cultures had a larger proportion of monosynaptic and polysynaptic connections. The group II metabotropic glutamate receptor (mGluR) agonist LY354740 (0.5 microM) suppressed excitatory but not inhibitory monosynaptic currents. LY354740 also inhibited antidromically evoked action currents in granule cells from pilocarpine- and to a lesser extent in pilocarpine and atropine-treated cultures, suggesting that group II mGluRs can reside along the axon and suppress action potential invasion. We provide direct evidence for the development of functional MFS and suggest a novel, axonal mechanism by which presynaptic group II mGluRs can inhibit selected synapses.     

Cooling blocks rat hippocampal neurotransmission by a presynaptic mechanism: observations using 2-photon microscopy

Over the past decade there has been great interest in the therapeutic potential of brain cooling for epilepsy, stroke, asphyxia and other neurological diseases. However, there is still no consensus regarding the neurophysiological effect(s) of brain cooling. We employed standard physiological techniques and 2-photon microscopy to directly examine the effect of temperature on evoked neurotransmitter release in rat hippocampal slices. We observed a monotonic decline in extracellular synaptic potentials and their initial slope over the temperature range 33–20°C, when the slices were cooled to a new set point in less than 5 s. Imaging the fluorescent synaptic marker FM1-43 with 2-photon microscopy showed that the same cooling protocol dramatically reduced transmitter release between 33 and 20°C. Cooling also reduced the terminal FM1-43 destaining that was induced by direct depolarization with elevated K⁺, indicating that axonal conduction block cannot account for our observations. The temperature dependence of FM1-43 destaining correlated well with the effect of temperature on field potential slope, compatible with a presynaptic explanation for our electrophysiological observations. Optical measurement of FM1-43 dissociation from cell membranes was not affected by temperature, and rapid cooling of slices loaded with FM1-43 did not increase their fluorescence. Our experiments provide visible evidence that a major neurophysiological effect of cooling in the mammalian brain is a reduction in the efficacy of neurotransmitter release. This presynaptic effect may account for some of the therapeutic benefits of cooling in epilepsy and possibly stroke.     

Inhibition of glutamatergic synaptic input to spinal lamina II(o) neurons by presynaptic alpha(2)-adrenergic receptors

Activation of spinal alpha(2)-adrenergic receptors by the descending noradrenergic system and alpha(2)-adrenergic agonists produces analgesia. However, the sites and mechanisms of the analgesic action of spinally administered alpha(2)-adrenergic receptor agonists such as clonidine are not fully known. The dorsal horn neurons in the outer zone of lamina II (lamina II(o)) are important for processing nociceptive information from C-fiber primary afferents. In the present study, we tested a hypothesis that activation of presynaptic alpha(2)-adrenergic receptors by clonidine inhibits the excitatory synaptic input to lamina II(o) neurons. Whole cell voltage-clamp recordings were performed on visualized lamina II(o) neurons in the spinal cord slice of rats. The miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presence of tetrodotoxin, bicuculline, and strychnine. The evoked EPSCs were obtained by electrical stimulation of the dorsal root entry zone or the attached dorsal root. Both mEPSCs and evoked EPSCs were abolished by application of 6-cyano-7-nitroquinoxaline-2,3-dione. Clonidine (10 microM) significantly decreased the frequency of mEPSCs from 5.8 +/- 0.9 to 2.7 +/- 0.6 Hz (means +/- SE) without altering the amplitude and the decay time constant of mEPSCs in 25 of 27 lamina II(o) neurons. Yohimbine (2 microM, an alpha(2)-adrenergic receptor antagonist), but not prazosin (2 microM, an alpha(1)-adrenergic receptor antagonist), blocked the inhibitory effect of clonidine on the mEPSCs. Clonidine (1-20 microM, n = 8) also significantly attenuated the peak amplitude of evoked EPSCs in a concentration-dependent manner. The effect of clonidine on evoked EPSCs was abolished in the presence of yohimbine (n = 5). These data suggest that clonidine inhibits the excitatory synaptic input to lamina II(o) neurons through activation of alpha(2)-adrenergic receptors located on the glutamatergic afferent terminals. Presynaptic inhibition of glutamate release from primary afferents onto lamina II(o) neurons likely plays an important role in the analgesic action produced by activation of the descending noradrenergic system and alpha(2)-adrenergic agonists.     

Novel mechanism for presynaptic inhibition: GABA(A) receptors affect the release machinery

Presynaptic inhibition is produced by increasing Cl(-) conductance, resulting in an action potential of a smaller amplitude at the excitatory axon terminals. This, in turn, reduces Ca(2+) entry to produce a smaller release. For this mechanism to operate, the "inhibitory" effect of shunting should last during the arrival of the "excitatory" action potential to its terminals, and to achieve that, the inhibitory action potential should precede the excitatory action potential. Using the crayfish neuromuscular preparation which is innervated by one excitatory axon and one inhibitory axon, we found, at 12 degrees C, prominent presynaptic inhibition when the inhibitory action potential followed the excitatory action potential by 1, and even 2, ms. The presynaptic excitatory action potential and the excitatory nerve terminal current (ENTC) were not altered, and Ca(2+) imaging at single release boutons showed that this "late" presynaptic inhibition did not result from a reduction in Ca(2+) entry. Since 50 microM picrotoxin blocked this late component of presynaptic inhibition, we suggest that gamma-aminobutyric acid-A (GABA(A)) receptors reduce transmitter release also by a mechanism other than affecting Ca(2+) entry.     

Elastolysis by proteinase 3 and its inhibition by alpha(1)-proteinase inhibitor: a mechanism for the incomplete inhibition of ongoing elastolysis

An excess of proteinase 3 (Pr3) is an assumed risk factor for elastin loss in chronic obstructive pulmonary disease. This study compared the degradation of [(14)C]elastin by Pr3 and its inhibition by alpha(1)-proteinase inhibitor (alpha(1)-PI) with the analogous reactions involving two other neutrophil serine proteases, human leukocyte elastase (HLE) and cathepsin G (CatG). The elastolytic rate catalyzed by Pr3 was estimated to be half of that of CatG and one-eighth of that of HLE. Evidence was obtained that indicated that absorption of Pr3 by the substrate was much less than that of HLE or CatG, and that the majority of absorbed Pr3 was highly mobile. These properties are consistent with the observation that elastolysis by Pr3 was almost completely and stoichiometrically inhibited by alpha(1)-PI even under conditions in which the protease had been preincubated with the substrate. In contrast, alpha(1)-PI in large molar excess was unable to inhibit completely ongoing elastolysis of the same substrate by HLE or CatG. An interfacial nonisotropic reaction mechanism has been proposed to address the incomplete inhibition of ongoing elastolysis. Pr3 was identified as being the most abundant neutrophil serine protease. However, two findings reported here, namely the low rate of elastolysis by Pr3 and the high efficacy of alpha(1)-PI against ongoing elastolysis by Pr3, imply that Pr3 might not necessarily be a major contributor to neutrophil-mediated elastin loss.     

Acute clozapine suppresses synchronized pyramidal synaptic network activity by increasing inhibition in the ferret prefrontal cortex

Recent studies have indicated that impaired neural circuitry in the prefrontal cortex is a prominent feature of the neuropathology of schizophrenia. Clozapine is one of the most effective antipsychotic drugs used for this debilitating disease. Despite its effectiveness, the mechanism by which clozapine acts on prefrontal cortical circuitry remains poorly understood. In this study, in vitro multiple whole cell recordings were performed in slices of the ferret prefrontal cortex. Clozapine, which effectively inhibited the spontaneous synchronized network activities in the prefrontal neurons, achieved the suppressive effect by decreasing the recurrent excitation among pyramidal neurons and by enhancing the inhibitory inputs onto pyramidal cells through a likely network mechanism. Indeed, under the condition of disinhibition, the depressing effects were reversed and clozapine enhanced the recurrent excitation. These results suggest that the therapeutic actions of clozapine in alleviating the positive symptoms of schizophrenia are achieved, at least partially, through the readjustment of synaptic balance between the excitation and inhibition in the prefrontal cortical circuitry.     

Presynaptic inhibition produced by an identified presynaptic inhibitory neuron. II. Presynaptic conductance changes caused by histamine

We have examined the morphology and pharmacology of the L32 neurons, identified cells that mediate presynaptic inhibition in the Aplysia abdominal ganglion, to gain insight into the putative transmitter released by the L32 cells. We analyzed the fine structure of the synaptic release sites of L32 cells stained with horseradish peroxidase. Each varicosity of L32 was found to contain two general classes of vesicles. One class of vesicles is large (mean long diameter of 98 nm) and contains an electron-dense core that typically filled or nearly filled each vesicle profile. The second class of vesicles is smaller (mean long diameter of 67 nm) and relatively electron lucent. The size, distribution, and morphology of the vesicle population in L32's terminals was similar to that described at the synapses of the identified histaminergic neuron C2 in Aplysia (2). These morphological observations suggested that L32 cells might be histaminergic. Among the various putative transmitters tested, histamine was most effective in mimicking the postsynaptic effects of L32 cells onto L10, and onto other follower cells of L32 in the abdominal ganglion. Histamine also caused inhibition of transmitter output from L10. Both the IPSP produced by L32 in L10 and the response of L10 to histamine could be reversibly blocked by cimetidine, a histamine antagonist in Aplysia (14). These results support, but do not establish the identification of histamine as the putative transmitter of L32 cells. Histamine mimics the action of L32 in mediating presynaptic inhibition allowing us to examine in more detail the conductance changes in L10 underlying presynaptic inhibition. Voltage-clamp analysis revealed that histamine blocked the voltage-dependent Ca2+ current and increased a voltage-dependent K+ current in L10, much as did L32. Both of these changes are likely to act synergistically to inhibit transmitter release. Reduction of Ca2+ current in L10 would directly inhibit transmitter release from L10 directly by decreasing the amount of Ca2+ entering during spike depolarization. The increase in K+ current would act indirectly to reduce transmitter release from L10, by hyperpolarizing L10 and decreasing the amplitude and duration of spikes in L10, as well as reducing the steady-state Ca2+ influx. These results support the idea that in Aplysia presynaptic inhibition is caused primarily by a direct transmitter-mediated reduction in presynaptic Ca2+ current and secondarily by a hyperpolarization of the presynaptic neuron due to a transmitter-mediated increase in a K+ current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maturation of evoked mossy fiber input to rat cerebellar Purkinje cells (II)

Summary The aim of this work was to establish a time sequence for the functional maturation of the mossy fiber afferent system to the cerebellum, in order to place it in the context of overall cerebellar cortical development. Rat pups at various ages were anesthetized with 0.5% halothane, and Purkinje cell activity was monitored extracellularly as limbs were electrically stimulated. The results showed that Purkinje cells can receive input from the periphery via the mossy fiber system at least by the seventh postnatal day, which is relatively early in overall cerebellar development. It is clear that synaptic transmission begins soon after the mossy fiber terminals and granule cell dendrites are in physical proximity. Initially, such input has a long latency (66 msec), is easily fatigued (at 10/sec), and has a prolonged duration of excitatory effect on Purkinje cells (27 msec). These and other functional parameters become mature by the third postnatal week (6–9 msec latency, following frequency above 20/sec), which is well before overall cerebellar cortical cytological development is completed. The maturation of many functional parameters of the cerebellar afferent systems and the acquisition of new motor behaviors are shown to emerge in parallel. Motor function appears to correlate with the establishment of a mature time scale of operation of the cerebellar circuitry.Supported by N. I. H. Grant 5-R01-GM00133 and N.S.F. Grant GB 43301     

Postsynaptic and presynaptic GABA(B) receptor-mediated inhibition in rat superficial superior colliculus neurons

It has been reported that GABA(B) receptors are abundantly expressed in the superficial superior colliculus (sSC). To elucidate the action of GABA(B) receptors in the sSC, we tested the effect of a specific GABA(B) receptor agonist baclofen on sSC neurons in slices obtained from rats (16-22 days postnatal) using the whole-cell patch clamp technique. Bath application of baclofen (10-30 microM) elicited hyperpolarization associated with an increase in potassium conductance in morphologically different types of neurons including putative excitatory and inhibitory neurons. In addition, baclofen presynaptically suppressed electrically induced excitatory postsynaptic currents and inhibitory postsynaptic currents. These effects of baclofen were antagonized by a specific GABA(B) receptor antagonist CGP55845A (1 microM). The results suggest that the GABA(B) receptors have an inhibitory action at both post- and pre-synaptic sites in the sSC.     

John Carew Eccles在获诺贝尔奖时的讲演(续)

(上接20卷第3期325页) 通过向细胞内加入5 pA的氯离子电流致使胞内的氯离子浓度增加三倍,图7A中所示的IPSP即转为去极化方向的电位,如B中所示.而当向另一个细胞里注入两倍的硫酸根离子电流时,抑制性突触电流没有改变(图7E,F).这个简单的实验证实:在抑制性递质的作用下,突触膜区对氯离子的通透性增加,而对硫酸根离子不通透.从图7I和J的结果中可以看出在两种类型的抑制性突触作用下,抑制性突触的膜区对亚硝酸根离子通透性增加,注入该离子后产生的效应在2分钟之后可以完全恢复.     

The ionic mechanism of postsynaptic inhibition（续）

通过向细胞内加入5 pA的氯离子电流致使胞内的氯离子浓度增加三倍，图7A中所示的IPSP即转为去极化方向的电位，如B中所示。而当向另一个细胞里注入两倍的硫酸根离子电流时，抑制性突触电流没有改变(图7E，F)。这个简单的实验证实：在抑制性递质的作用下，突触膜区对氯离子的通透性增加，而对硫酸根离子不通透。从图7I和J的结果     

The inhibition mechanism of histamine release by N-(3,4-dimethoxycinnamoyl) anthranilic acid

N-(3,4-Dimethoxycinnamoyl) anthranilic acid (N-5') is an inhibitor of IgE-mediated histamine release from mast cells. To elucidate inhibition mechanism, effects of N-5' were examined under various conditions using peritoneal exudate cells and isolated mast cells of rats. N-5' inhibited histamine release induced by antigen, ionophore A23187, ATP, dextran and phospholipase A2. But the release induced by compound 48/80 or ionophore X537A was not inhibited. Influx of Ca++ into mast cells and ATP consumption were inhibited. Based on these results, it is presumed that N-5' interferes with the energy-requiring system and/or Ca++ influx resulting in the inhibition of histamine release.     

On the permeability of the presynaptic terminal of the crayfish neuromuscular junction during synaptic inhibition and the action of γ-aminobutyric acid

1. The contribution of chloride to presynaptic inhibition of the crayfish neuromuscular junction during the action of γ-aminobutyric acid (GABA) and the inhibitory transmitter was investigated. Chloride in van Harreveld's solution was replaced with propionate, acetate, methylsulphate or glycerophosphate and electrical changes were recorded intracellularly and extracellularly with micro-electrodes.

2. When the preparation was soaked in Cl-deficient solutions, the quantum content of the excitatory junctional potentials (e.j.p.s), calculated from the number of failures, was increased.

3. The presynaptic inhibitory action of GABA was reduced or almost absent when the preparation was soaked in Cl-deficient solution. In Cl-deficient solution the stimulation of the inhibitory axon showed little or no inhibitory action on e.j.p.s.

4. If GABA was applied shortly after the outside solution was changed to the Cl-deficient one, the frequency of the spontaneous e.j.p.s was increased transiently.

5. It is suggested that GABA or the inhibitory transmitter act on the presynaptic excitatory terminal and predominantly increase its permeability to chloride.