骨髓基质细胞分化为神经元样细胞后与皮质神经元间突触的建立

目的 观察与大脑皮质神经元共培养的骨髓基质细胞(BMSCs)经诱导分化成神经元样细胞后,与脑皮质神经元之间形成功能性突触的情况.方法 无菌条件下取绿色荧光蛋白(GFP)转基因小鼠骨髓,用贴壁筛选法体外培养获得GFP转基因小鼠BMSCs(GFP-GM-BMSCs),在体外培养、扩增、纯化.取第3代GFP-GM-BMSCs,种植到源于小鼠大脑的原代皮质神经元和胶质细胞中,培养介质为加有20 ng/mL表皮生长因子(EGF)、20 ng/mL碱性成纤维细胞生长因子(bFGF)的无血清培养基(Neurobasal-A+2%B27),体外模拟建立细胞移植的共培养体系.共培养第10天,利用FM1-43荧光染料染色活动突触小泡的特性,通过荧光显微镜观察共培养的两种细胞之间形成的突触.结果 与神经元共培养的GFP-GM-BMSCs在含有EGF、bFGF的无血清培养基中7 d后分化为神经元样细胞.共培养10 d后,FM1-43染色阳性的突触囊泡明显增加,主要位于神经元样细胞胞体、突起及其末端结构上.结论 在体外模拟细胞移植共培养体系中,分化自GFP-GM-BMSCs的神经元样细胞能与神经元之间形成突触样连接.     

Fibroblast growth factor-2 increases functional excitatory synapses on hippocampal neurons

The effect of fibroblast growth factor-2 (FGF-2) on synapse formation was investigated using rat cultured hippocampal neurons. Treatment with FGF-2 (0.4-10 ng/mL) for 6 days enhanced synaptogenesis on these neurons by approximately 50%, as determined by counting puncta immunostained for presynaptic- or postsynaptic-specific proteins. This enhancement was statistically significant, and was abolished by a specific inhibitor of mitogen-activated protein kinase (MAPK). The majority of neurons expressed FGF receptors (types 1-3) abundantly on the membrane of somata, dendrites, and growth cones, and in these regions phosphorylation of MAPK was enhanced after FGF-2 application. Furthermore, our experiments showed that the majority of synapses formed in cultures containing FGF-2 were positive both for presynaptic proteins and postsynaptic excitatory synapse-specific proteins, and that these synapses had a similar capacity to recycle the fluorescent styryl dye FM4-64 as those in the control culture. These results indicate that: (i) FGF-2 increases excitatory synapses on hippocampal neurons by activating MAPK activity through FGF receptors; and (ii) synapses formed in FGF-2-treated culture are capable of cycling vesicles.     

GABAA receptors can initiate the formation of functional inhibitory GABAergic synapses

The mechanisms that underlie the selection of an inhibitory GABAergic axon's postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABA_A receptors (GABA_ARs) themselves – the essential functional postsynaptic components of GABAergic synapses – can be sufficient to initiate formation of synaptic contacts, a novel co‐culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e. α1/β2/γ2‐GABA_ARs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse‐like contacts in these co‐cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle‐luminal domain‐specific antibody; and they supported spontaneous and action potential‐driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N‐methyl‐d ‐aspartate receptor‐expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin‐2 was co‐expressed with GABA_ARs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABA_ARs can promote the adhesion of inhibitory axons and the development of functional synapses.     

GABA and GABAA receptors at hippocampal mossy fibre synapses

Anatomical and electrophysiological evidence has raised the possibility that corelease of GABA and glutamate occurs at hippocampal mossy fibre synapses which, however, lack the vesicular GABA transporter VGAT. Here, we apply immunogold cytochemistry to show that GABA, like glutamate, has a close spatial relation to synaptic vesicles in rat mossy fibre terminals, implying that a mechanism exists to package GABA in synaptic vesicles. We also show that GABAA and AMPA receptors are colocalized at mossy fibre synapses. The expression of GABA and GABAA receptors is, however, weaker than in inhibitory synapses. Electrical stimuli that recruit mossy fibres evoke monosynaptic GABAA receptor-mediated signals in post-synaptic targets that show marked frequency-dependent facilitation and sensitivity to group II metabotropic receptors, two features that are characteristic of mossy fibre transmission. These results provide further evidence for GABA and glutamate cotransmission at mossy fibre synapses, although paired pre- and post-synaptic recordings will be required to determine the role of GABA at this unusual synapse.     

Specific inhibitory synapses shift the balance from feedforward to feedback inhibition of hippocampal CA1 pyramidal cells

Feedforward and feedback inhibition are two fundamental modes of operation widespread in the nervous system. We have functionally identified synaptic connections between rat CA1 hippocampal interneurons of the stratum oriens (SO) and interneurons of the stratum lacunosum moleculare (SLM), which can act as feedback and feedforward interneurons, respectively. The unitary inhibitory postsynaptic currents (uIPSCs) detected with K-gluconate-based patch solution at −50 mV had an amplitude of 20.0 ± 2.0 pA, rise time 2.2 ± 0.2 ms, decay time 25 ± 2.2 ms, jitter 1.4 ± 0.2 ms (average ± SEM, n  = 39), and were abolished by the γ-aminobutyric acid (GABA)_A receptor antagonist 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR 95531). Post hoc anatomical characterization revealed that all but one of the identified presynaptic neurons were oriens-lacunosum moleculare (O-LM) cells, whereas the postsynaptic neurons were highly heterogeneous, including neurogliaform ( n  = 4), basket ( n  = 4), Schaffer collateral-associated ( n  = 10) and perforant path-associated ( n  = 9) cells. We investigated the short-term plasticity expressed at these synapses, and found that stimulation at 10–40 Hz resulted in short-term depression of uIPSCs. This short-term plasticity was determined by presynaptic factors and was not target-cell specific. We found that the feedforward inhibition elicited by the direct cortical input including the perforant path onto CA1 pyramidal cells was modulated through the inhibitory synapses we have characterized. Our data show that the inhibitory synapses between interneurons of the SO and SLM shift the balance between feedback and feedforward inhibition onto CA1 pyramidal neurons.     

FM1-43造影观察PC12神经元与原代皮层神经元间的突触形成

目的 观察种植到原代皮层神经元中的PCI2细胞，在神经生长因子(nerve growth factor NGF)作用下分化成的神经元样细胞与原代皮层神经元之间功能性突触的形成，研究细胞移植环境中宿主细胞和移植细胞形成神经连接的可能及过程。方法 将绿色荧光蛋白(enhenced green flourescent protein EGFP)标记的PC12细胞种植到原代神经元中，建立共培养系统。NGF诱导其中的PC12细胞分化为神经元样细胞，可能与共培养的原代神经元形成突触连接。高钾离子的去极化刺激使突触末端囊泡被FM143染色，激光共聚焦扫描显微镜下观察，电子图像显示出活性的突触末端。结果 和对照组相比共培养系统中PC12神经元更成熟，FM1—43造影显示这些新的神经元与原代神经元之间产生了功能性突触囊泡活动。结论 与原代的神经细胞共培养有利于PCI2细胞在NGF作用下分化为神经元样细胞及形成神经突起，新形成的神经元可以和宿主神经元形成突触连接。     

Reduced number of functional glutamatergic synapses in hippocampal neurons overexpressing full-length TrkB receptors.

Brain-derived neurotrophic factor (BDNF) acutely modulates the efficacy of central glutamatergic synapses via activation of the receptor tyrosine kinase TrkB. On a longer time scale, recent evidence suggests an additional role of TrkB signaling in the formation of excitatory synaptic connections. Here, we have overexpressed full-length TrkB receptors (fl-TrkB) in hippocampal neurons, to investigate the contribution of BDNF signaling to the maturation of glutamatergic synapses. Using patch clamp recordings, we show a three-fold reduction in glutamatergic excitatory autaptic and synaptic current amplitudes in neurons overexpressing fl-TrkB, and application of saturating concentrations of BDNF and NT-4/5 completely reverses this effect. Compatible with these overexpression data, in untransfected neurons, scavenging of endogenous BDNF and NT-4/5 by TrkB-IgGs reduces excitatory autaptic current (EAC) amplitudes. By overexpression of truncated TrkB receptors (TrkB.T1, TrkB.T2) and a chimeric receptor containing only the intracellular domain of fl-TrkB, we show that intra- and extracellular domains of fl-TrkB are necessary to observe the EAC reduction. Labeling of presynaptic terminals with FM 4-64 revealed, that the reduced EAC amplitudes in fl-TrkB overexpressing neurons are accompanied by a two-fold reduction in synapse number. These results suggest, that ligand-independent signaling through fl-TrkB receptors can decrease glutamatergic synaptic strength, if sufficient amounts of BDNF or NT-4/5 are not available.     

Tuning afferent synapses of hippocampal interneurons by neuropeptide Y

Cholecystokinin (CCK)‐expressing basket cells encompass a subclass of inhibitory GABAergic interneurons that regulate memory‐forming oscillatory network activity of the hippocampal formation in accordance to the emotional and motivational state of the animal, conveyed onto these cells by respective extrahippocampal afferents. Various excitatory and inhibitory afferent and efferent synapses of the hippocampal CCK basket cells express serotoninergic, cholinergic, cannabinoid, and benzodiazepine sensitive receptors, all contributing to their functional plasticity. We explored whether CCK basket cells are modulated by neuropeptide Y (NPY), one of the major local neuropeptides that strongly inhibits hippocampal excitability and has significant effect on its memory function. Here, using GAD65‐GFP transgenic mice for prospective identification of CCK basket cells and whole‐cell patch‐clamp recordings, we show for the first time that excitatory and inhibitory inputs onto CCK basket cells in the dentate gyrus of the hippocampus are modulated by NPY through activation of NPY Y2 receptors. The frequency of spontaneous and miniature EPSCs, as well as the amplitudes of stimulation‐evoked EPSCs were decreased. Similarly, the frequency of both spontaneous and miniature IPSCs, and the amplitudes of stimulation‐evoked IPSCs were decreased after NPY application. Most of the effects of NPY could be attributed to a presynaptic site of action. Our data provide the first evidence that the excitatory and inhibitory inputs onto the CCK basket cells could be modulated by local levels of NPY, and may change the way these cells process extrahippocampal afferent information, influencing hippocampal function and its network excitability during normal and pathological oscillatory activities. © 2009 Wiley‐Liss, Inc.     

Phorbol ester uncouples adenosine inhibition of presynaptic Ca2+ transients at hippocampal synapses

Synaptic transmission involves Ca2+ influx at presynaptic terminals. Adenosine receptors inhibit transmission, and this effect can be abolished by activation of PKC with phorbol esters. Whether protein kinase C (PKC) acts via alterations in Ca2+ entry at the presynaptic terminal is unknown. In the present study, we recorded the presynaptic Ca2+ transients (preCa(delta)) in hippocampal stratum radiatum, using fluorescence photometry. The calcium dye Fura-2 AM was used to load the Schaffer collateral/commissural tract and its terminals. Tetrodotoxin (TTX)-sensitive Na+ channels and Cd2+-sensitive, high-voltage activated Ca2+ channels (HVACCs) were required to elicit the preCa(delta). Application of the phorbol ester phorbol-12,13-dibutyrate (PDBu) abolished the adenosine inhibition of both preCa(delta) and the field excitatory postsynaptic potentials (fEPSPs). PDBu consistently potentiated fEPSPs, and also increased preCa(delta) in a large majority of the slices examined. Regardless of whether potentiation was observed, PDBu always prevented adenosine inhibition of preCa(delta). In contrast, the inactive phorbol ester, 4alpha-phorbol, did not alter adenosine inhibition of preCa(delta), indicating that PKC activation is necessary for the occurrence of the observed effects. Our findings suggest that PKC activation abolishes adenosine's inhibitory effect on synaptic activity involving presynaptic Ca2+ entry.     

GABAA and GABAC receptors on mammalian rod bipolar cells

Rod bipolar (RB) cells of mammalian retinae receive synapses from different γ-aminobutyric acid (GABAergic) amacrine cells in the inner plexiform layer (IPL). We addressed the question whether RB cells of the rabbit and of the rat retina express different types of GABA receptors at these synapses. RB cells were immunolabeled in vertical sections of rat retinae with an antibody against protein kinase C (PKC). The sections were double-labled for the α1, α2, α3, or γ2 subunits of the GABA_A receptor. Punctate immunofluorescence, which represents synaptic localization, was found for all four subunits. Many of the α1-, α3-, or γ2-immunoreactive puncta coincided with the axon terminals of the PKC-immunolabeled RB cells. Sections and wholemounts of rabbit retinae were also double labeled for PKC and the ρ subunits of the GABA_C receptor. Rabbit RB cells were decorated by many ρ-immunoreactive puncta, which were shown by electron microscopy to represent synaptic localization. Previous work from our laboratory has shown that the α1, α2, α3, and ρ subunits are not found within the same synapse but are expressed at different synaptic sites. Taken together, these results suggest that RB cells of mammalian retinae express at least three different types of GABA receptors at synaptic sites in the IPL: GABA_C receptors, GABA_Areceptors containing the α1 subunit, and GABA_A receptors containing the α3 subunit. J. Comp. Neurol. 396:351–365, 1998. © 1998 Wiley-Liss, Inc.     

Two distinct classes of muscarinic action on hippocampal inhibitory synapses: M2-mediated direct suppression and M1/M3-mediated indirect suppression through endocannabinoid signalling

The cholinergic system in the CNS plays important roles in higher brain functions, primarily through muscarinic acetylcholine receptors. At cellular levels, muscarinic activation produces various effects including modulation of synaptic transmission. Here we report that muscarinic activation suppresses hippocampal inhibitory transmission through two distinct mechanisms, namely a cannabinoid-dependent and cannabinoid-independent mechanism. We made paired whole-cell recordings from cultured hippocampal neurons of rats and mice, and monitored inhibitory postsynaptic currents (IPSCs). When cannabinoid receptor type 1 (CB1) was blocked, oxotremorine M (oxo-M), a muscarinic agonist, suppressed IPSCs in a subset of neuron pairs. This suppression was associated with an increase in paired-pulse ratio, blocked by the M(2)-preferring antagonist gallamine, and was totally absent in neuron pairs from M(2)-knockout mice. When CB1 receptors were not blocked, oxo-M suppressed IPSCs in a gallamine-resistant manner in cannabinoid-sensitive pairs. This suppression was associated with an increase in paired-pulse ratio, blocked by the CB1 antagonist AM281, and was completely eliminated in neuron pairs from M(1)/M(3)-compound-knockout mice. Our immunohistochemical examination showed that M(2) and CB1 receptors were present at inhibitory presynaptic terminals of mostly different origins. These results indicate that two distinct mechanisms mediate the muscarinic suppression. In a subset of synapses, activation of M(2) receptors at presynaptic terminals suppresses GABA release directly. In contrast, in a different subset of synapses, activation of M(1)/M(3) receptors causes endocannabinoid production and subsequent suppression of GABA release by activating presynaptic CB1 receptors. Thus, the muscarinic system can influence hippocampal functions by controlling different subsets of inhibitory synapses through the two distinct mechanisms.     

Time and exposure to serotonin affect releasability of recycled vesicles at crayfish claw opener muscle synapses

The crayfish claw opener neuromuscular junction is a biological model for studying presynaptic neuromodulation by serotonin (5HT) and synaptic vesicle recycling. It has been hypothesized that 5HT enhances release by recruiting a population of either previously nonrecycling or “reluctant” vesicles to increase the readily releasable pool. To determine if 5HT activates a distinct population of synaptic vesicles, recycling membranes were labeled with the membrane dye, FM1-43. Unloading (destaining) protocols could not resolve a population of vesicles that were only releasable in the presence of 5HT. Instead, we conclude synaptic vesicles change behavior in axon terminals independent of 5HT, becoming less likely to exocytose and unload dye over periods of >1 hr after recycling. We hypothesized this to be due to the slow conversion of a portion of recycled vesicles to a difficult to release state. The possibility that vesicles in these pools were spatially separated within the terminal was tested using photoconversion of FM1-43 and transmission electron microscopy. The location of FM1-43-labeled vesicles fixed 2 min following 3 min of 20-Hz stimulation did not reveal preferential localization of recycling vesicles specifically near release sites and the distribution of labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 s following stimulation contained a significant proportion of vesicular structures equivalent in diameter to 2–5 regular vesicles, with multivesicular bodies and calveoli rarely seen, suggesting that endocytosis during sustained release at crayfish terminals occurs via multiple routes, most commonly through large “vesicle” intermediates.     

The α5GABAA receptor modulates the induction of long‐term potentiation at ventral but not dorsal CA1 hippocampal synapses

The hippocampal synapses display conspicuous ability for long‐term plasticity which is thought to underlie learning and memory. Growing evidence shows that this ability differs along the long axis of the hippocampus, with the ventral CA1 hippocampal synapses displaying remarkably lower ability for long‐term potentiation (LTP) compared with their dorsal counterpart when activated with high‐frequency stimulation. Here, we show that low frequency, 10 Hz stimulation induced LTP more reliably in dorsal than in ventral CA1 field. Blockade of alpha5 subunit‐containing GABA_A receptors eliminated the difference between dorsal and ventral hippocampus. We propose that α5GABA_A receptor‐mediated activity plays a crucial role in regulating the threshold for induction of LTP especially at the ventral CA1 hippocampal synapses. This might have important implications for the functional specialization along the hippocampus. Synapse, 2014. © 2014 Wiley Periodicals, Inc.     

Differential expression of GABAA and glycine receptors in ALS-resistant vs. ALS-vulnerable motoneurons: possible implications for selective vulnerability of motoneurons

Summary Amyotrophic lateral sclerosis (ALS) is a devastating motoneuronal degenerative disease, which is inevitably fatal in adults. ALS is characterized by an extensive loss of motoneurons in the cerebrospinal axis, except for those motoneurons that control eye movements and bladder contraction. The reason for this selectivity is not known. Systematic differences have been found in the organization of excitatory synaptic transmission in ALS-resistant vs. ALS-susceptible motor nuclei. However, although motoneurons express high levels of glycine receptors (GlyR) and GABA(A) receptors (GABA(A)R), no such studies have been carried out yet for inhibitory synaptic transmission. In this study, we compared the subunit composition, patterns of expression, density and synaptic localization of inhibitory synaptic receptors in ALS-resistant (oculomotor, trochlear and abducens) and ALS-vulnerable motoneurons (trigeminal, facial and hypoglossi). Triple immunofluorescent stainings of the major GABA(A)R subunits (alpha1, alpha2, alpha3, and alpha5), the GlyR alpha1 subunit and gephyrin, were visualized by confocal microscopy and analysed quantitatively. A strong correlation was observed between the vulnerability of motoneurons and the subunit composition of GABA(A)R, the GlyR/GABA(A)R density ratios and the incidence of synaptic vs. extrasynaptic GABA(A)R. These differences contrast strikingly with the uniform gephyrin cluster density and synaptic GlyR levels recorded in all motor nuclei examined. These results suggest that the specific patterns of inhibitory receptor organization observed might reflect functional differences that are relevant to the physiopathology of ALS.     

Molecular reconstitution of functional GABAergic synapses with expression of neuroligin-2 and GABAA receptors

Trans-synaptic cell adhesion molecules neuroligins and neurexins play an important role in promoting central synapse formation. We report here a molecular reconstruction of functional GABAergic synapses in non-neuronal cells with the coexpression of postsynaptic cell adhesion molecule neuroligin-2 (NL-2) and GABA(A) receptors. HEK 293T cells were co-transfected with GABA(A) receptors and NL-2 or its homologue neuroligin-1 (NL-1), and then cocultured with hypothalamic cultures which are enriched with GABAergic neurons. Both spontaneous and action potential-evoked GABAergic events were readily detected in HEK 293T cells coexpressing GABA(A) receptors with NL-2, but not with NL-1. Aggregating NL-2 with specific antibodies in live cells resulted in coaggregation of GABA(A) receptors. Expression of NL-2 in HEK 293T cells also induced stronger GABAergic presynaptic differentiation than that of NL-1 in neuronal cocultures. These results suggest that NL-2 may potentially serve as a central organizer for GABAergic synapse assembly by interacting with both presynaptic neurexins and postsynaptic GABA(A) receptors.     

Glutamate‐dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons

The idea that astrocytes merely provide structural and trophic support for neurons has been challenged by the demonstration that astrocytes can regulate neuronal calcium levels. However, the physiological consequences of astrocyte–neuron signalling are unknown. Using mixed cultures of rat hippocampal astrocytes and neurons we have determined functional consequences of elevating astrocyte calcium levels on co-cultured neurons. Electrical or mechanical stimulation of astrocytes to increase their calcium level caused a glutamate-dependent slow inward current (SIC) in associated neurons. Microinjection of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) into astrocytes to prevent the stimulus-dependent increase in astrocyte calcium level, blocks the appearance of the neuronal SIC. Pharmacological manipulations indicate that this astrocyte-dependent SIC is mediated by extracellular glutamate acting on N-methyl-d -aspartate (NMDA) and non-NMDA glutamate receptors. Additionally, stimulation of astrocytes reduced the magnitude of action potential-evoked excitatory and inhibitory postsynaptic currents through the activation of metabotropic glutamate receptors. The demonstration that astrocytes modulate neuronal currents and synaptic transmission raises the possibility that astrocytes play a neuromodulatory role by controlling the extracellular level of glutamate.     

Membrane traffic in outer hair cells of the adult mammalian cochlea

Outer hair cells (OHCs), the sensory-motor cells responsible for the extraordinary frequency selectivity and dynamic range of the cochlea, rapidly endocytose membrane and protein at their apical surface. Endocytosis and transcytosis in isolated OHCs from the mature guinea-pig cochlea were investigated using the amphipathic membrane probe FM1-43. We observed membrane transport from the apical surface to both the basolateral wall and the subnuclear pole. By double-labelling with DiOC6, a stain for endoplasmic reticulum, and aspiration of the plasma membrane, we showed that the basolateral target was the subsurface cisternae. The fluorescent signal was about three times weaker at the basal than at the apical pole. The speed of vesicle transport to the subnuclear pole was approximately 0.4 microm/s. Changing extracellular Ca2+ concentration from 25 microM to 2 mM accelerated rapid endocytosis. Extracellular application of BAPTA-AM (25 microM), an intracellular Ca2+ chelator, and TFP (20 microM), a specific inhibitor of calmodulin, reduced endocytic activity, as did depolarization of the whole cell. The presence of extracellular Cd2+ (200 microM), a Ca2+-channel blocker, had no effect on the voltage dependence of endocytosis at the apical pole, and inhibited the voltage dependence at the subnuclear pole. These results suggest that rapid endocytosis is a Ca2+/calmodulin-dependent process, with extracellular Ca2+ entering through voltage-gated Ca2+ channels at the basal pole. The two distinct destinations of endocytosed membrane are consistent with the functional polarization of the OHC, with the basolateral wall being dedicated to electromechanical transduction and the subnuclear pole being dedicated to electrochemical transduction processes.     

Stratum oriens stimulation-evoked modulation of hippocampal long-term potentiation involves the activation of muscarinic acetylcholine receptors and the inhibition of Kv7/M potassium ion channels

Acetylcholine is considered to be an endogenous modulator of hippocampal neurotransmission and synaptic plasticity. The activation of muscarinic acetylcholine receptors (mAChRs) reportedly enhances hippocampal synaptic plasticity, which plays an important role in memory function; however, the mechanism by which it enhances synaptic plasticity remains unclear. Here, we examined the involvement of the inhibition of Kv7/M K(+) channels, which are targets of mAChR modulation, during mAChR activation-induced enhancement of long-term potentiation (LTP) at rat hippocampal Schaffer collateral (SC)-CA1 synapses. When an electrical stimulus was applied to the stratum oriens before tetanic stimulation of the SCs, the magnitude of the induced SC-CA1 synapse LTP was enhanced as compared with that induced without stratum oriens stimulation. In the presence of the mAChR antagonist atropine, tetanic stimulation induced stable LTP, but the stratum oriens stimulation-evoked enhancement of LTP was abolished. The additional application of XE991, a selective blocker of Kv7/M K(+) channels, rescued the atropine-induced inhibition of LTP enhancement. The phospholipase C (PLC) inhibitor U-73122 inhibited the stratum oriens stimulation-evoked enhancement of LTP. Application of the T/R-type voltage-dependent Ca(2+) channel (VDCC) blocker Ni(2+) abolished the stratum oriens stimulation-evoked enhancement of LTP. In addition, tetanic stimulation with preceding stratum oriens stimulation was able to induce LTP during N-methyl-d-aspartate receptor blockade. We therefore propose that stratum oriens stimulation inhibits Kv7/M K(+) channels through mAChR activation-induced PLC activation, which leads to VDCC activation, and hence causes sufficient Ca(2+) influx to enhance LTP.     

Activation of CB1 cannabinoid receptors inhibits neurotransmitter release from identified synaptic sites in rat hippocampal cultures

The effects of cannabinoids on synaptic transmission were measured optically in rat hippocampal cultures. Synaptic release sites were labeled with the fluorescent dye FM1-43 in a stimulus-dependent manner. Action potential-induced release of FM1-43 required extracellular Ca2+ and was inhibited 65 +/- 3% by blockade of high-threshold voltage-gated Ca2+ channels with omega-grammotoxin SIA (300 nM). The cannabimimetic drug, Win 55212-2 (300 nM), inhibited FM1-43 release by 51 +/- 3%. The inhibition produced by Win55212-2 was blocked by the CB1 cannabinoid receptor antagonist, SR141716 (1 microM). The intensity of FM1-43 labeled puncta ranged 4-fold, although the inhibition produced by Win55212-2 was distributed normally across synaptic sites of various labeling intensities. The FM1-43-based optical method appears promising for the study of the effects of cannabinoids and other drugs on synaptic networks. These results indicate that cannabimimetics act presynaptically to inhibit the release of neurotransmitter and that this inhibition is observed uniformly at boutons of varied activity levels.     

Distribution of high-voltage-activated calcium channels in cultured gamma-aminobutyric acidergic neurons from mouse cerebral cortex.

The localization of voltage-gated calcium channel (VGCC) alpha(1) subunits in cultured GABAergic mouse cortical neurons was examined by immunocytochemical methods. Ca(v)1.2 and Ca(v)1.3 subunits of L-type VGCCs were found in cell bodies and dendrites of GABA-immunopositive neurons. Likewise, the Ca(v)2.3 subunit of R-type VGCCs was expressed in a somatodendritic pattern. Ca(v)2.2 subunits of N-type channels were found exclusively in small varicosities that were identified as presynaptic nerve terminals based on their expression of synaptic marker proteins. Two splice variants of the Ca(v)2.1 subunit of P/Q-type VGCCs showed widely differing expression patterns. The rbA isoform displayed a purely somatodendritic staining pattern, whereas the BI isoform was confined to axon-like fibers and nerve terminals. The nerve terminals of these cultured GABAergic neurons express Ca(v)2.2 either alone or in combination with Ca(v)2.1 (BI isoform) but never express Ca(v)2.1 alone. The functional association between VGCCs and the neurotransmitter release machinery was probed using the FM1-43 dye-labeling technique. N-type VGCCs were found to be tightly coupled to exocytosis in these cultured cortical neurons, and P-type VGCCs were also important in a fraction of the cells. The predominant role of N-type VGCCs in neurotransmitter release and the specific localization of the BI isoform of Ca(v)2.1 in the nerve terminals of these neurons distinguish them from previously studied central neurons. The complementary localization patterns observed for two different isoforms of the Ca(v)2.1 subunits provide direct evidence for alternative splicing as a means of generating functional diversity among neuronal calcium channels.