Reliability Of Synaptic Transmission Research Articles

Оценка эффективности β2-адреноагониста формотерола в компенсации электрофизиологических проявлений стероидной миопатии в модельных экспериментах на животных

Цель исследования - изучение с помощью стимуляционной электромиографии в экспериментах на крысах эффективности β2-адреноагониста формотерола в компенсации электрофизиологических нарушений передней большеберцовой мышцы (m. tibialis anterior), вызванных длительным введением дексаметазона. Методика. Исследования выполнены на 40 половозрелых крысах-самках в возрасте 4-5 мес массой 180-200 г. Животные были разделены на 4 группы: контрольную (К - группа, без каких-либо воздействий, n=10) и 3 экспериментальных по 10 животных в каждой. Крысы 1-й группы на протяжении 30 сут получали дексаметазон, 2-й группы - дексаметазон в комплексе с формотеролом, 3-я - получала только формотерол. Дексаметазон вводили внутрибрюшинно (0,25 мг/кг) 1 раз в 2 сут. Формотерол (1,5 мкг/кг) вводили ежедневно под кожу. Через 30 сут на наркотизированных животных (тиопентал натрия 100мг/кг внутрибрюшинно) проводили острый опыт. Изучали электрофизиологические параметры передней большеберцовой мышцы с помощью экспериментальной двухканальной установки, оба канала были связаны с регистрирующим устройством - запоминающим цифровым осциллографом Tektronix (TDS2004C). Результаты. Установлено, что формотерол в комплексе с дексаметазоном, предотвращал уменьшение количества активируемых двигательных единиц мышцы и удлинение латентного периода М-ответов. А также обусловливал существенное повышение амплитуды М-ответов (на 115% в сравнении с контролем). Вместе с тем, формотерол в комплексе с дексаметазоном не предотвращал появление полифазных М-ответов, но компенсировал снижение их амплитуды. Введение дексаметазона с формотеролом уменьшало частоту случаев сниженной надежности синаптической передачи (до 30% против 70% в группе крыс, получавших дексаметазон изолированно). Вместе с тем, в случае комплексного введения дексаметазона с формотеролом наблюдалось удлинение латентного периода М-ответов после выполнения утомляющей работы. Заключение. Полученные данные указывают на высокую эффективность β2-адреноагониста формотерола в предотвращении электрофизиологических нарушений в мышце, вызванных длительным введением дексаметазона. Вместе с тем формотерол в комплексе с дексаметазоном, хотя и снижал частоту случаев низкой надежности синаптической передачи в группе (до 30% против 70% в группе, получавших дексаметазон изолированно), в полной мере не предотвращал данного эффекта стероидной миопатии. Aim. To study efficacy of β2-adrenergic agonist formoterol (F) in compensation of electrophysiological disorders in mixed-type skeletal muscles (m. tibial anterior) induced by long-term dexamethasone (D) treatment. Methods. Experiments were performed on sexually mature female rats (180-210 g) divided into four groups: 1) control, group C (intact rats, n=10); 2) first experimental group, 30D (30-day dexamethasone treatment, n=10); 3) second experimental group, 30D+F (30-day dexamethasone plus formoterol treatment, n=10); and third experimental group, 30F (30-day formoterol treatment, n=10). Dexamethasone (KRKA, Slovenia) was administered every two days, i.p., at a dose of 0.25 mg/kg, which was equivalent to the clinical therapeutic dose. Formoterol (Foradil, Novartis, Switzerland) was administered daily at a dose of 1.5 µ/kg, s.c. On day 30, rats were anesthetized with sodium thiopental (100 mg/kg), and stimulation electromyography was performed in an acute experiment. The anterior tibial muscle was stimulated with suprathreshold electrical current via the fibular nerve, and electrophysiological parameters of the muscle were recorded. Results. Formoterol in combination with dexamethasone prevented the decrease in the number of activated muscle motor units and the prolongation of M-response latency, which were typical for the 30D group. Also, formoterol not only corrected the decreased M-response amplitude but even significantly increased it in the 30D+F group by 115% compared to the control (p<0.05). At the same time, formoterol in combination with dexamethasone did not prevent the emergence of polyphasic M-responses (as observed in 50% of animals in the 30D+F group and 40% of animals in the 30D group) but compensated for the decrease in their amplitude. Administration of F+D reduced the incidence of less reliable synaptic transmission (up to 30% in the 30D+F group vs. 70% in the 30D group) but did not completely prevent it. Formoterol administered together with dexamethasone did not prevent marked facilitation of synaptic transmission in 50% of rats at the optimal neuromuscular stimulation rate (30 imp/s). However, this combination increased the amplitude of the first M-response in a series, which indicated the absence of blocked synapses in the 30D+F group. Formoterol in combination with dexamethasone prevented the typical for D-group disorder of M-response, which was more pronounced than in control, and the decrease in the number of activated muscle motor units after performing a fatigable work. At the same time, the D+F treatment increased the M-response latency after performing the fatigable work, which was typical for the 30D group but was not characteristic of the control. This fact confirmed a lower reliability of synaptic transmission both in 30D and 30D+F groups. Conclusion. The study demonstrated a high efficacy of the β2-adrenergic agonist formoterol for preventing electrophysiological disorders in the muscle induced by long-term administration of dexamethasone, which indicated myopathic alterations. At the same time, formoterol in combination with dexamethasone, although reduced the incidence of low-reliability synaptic transmission (up to 30% vs. 70% in the group receiving dexamethasone alone), did not completely prevent this manifestation of steroid myopathy.

Модулирующее влияние адреналина на развитие стероидной миопатии у белых крыс, индуцированной длительным введением гидрокортизона

Цель исследования - изучение эффективности адреналина в компенсации негативных эффектов длительно вводимого гидрокортизона на скелетную мышцу смешанного типа. Методика. Эксперименты проводились на половозрелых крысах-самках (190-220 г), разделенных на 3 группы: контрольную (n = 10), I опытную (n = 10, ежедневно на протяжении 30 сут. получали гидрокортизона ацетат, Г-группа) и II опытную (n = 10, ежедневно на протяжении 30 сут. получали гидрокортизона ацетат в комплексе с адреналина гидрохлоридом, (Г+А)-группа). Гидрокортизона ацетат («Фармак», Украина) вводили внутрибрюшинно в дозе, адекватной терапевтической для человека, - 3 мг/кг. Адреналина гидрохлорид («Здоровье», Украина) вводили подкожно в дозе 0,2 мг/кг. На наркотизированных животных (тиопентал натрия, 100 мг/кг) с помощью методов электромиографии и миографии изучали параметры функционального состояния передней большеберцовой мышцы в условиях ее сокращения, индуцированного раздражением сверхпороговым электрическим током малоберцового нерва. Результаты. Длительное (в течение 30 сут.) введение гидрокортизона вызывало развитие симптомов стероидной миопатии, о чем свидетельствует появление низкоамплитудных М-ответов нормальной длительности, возрастание частоты полифазных потенциалов (до 30%), уменьшение количества активируемых двигательных единиц (на 33%) и мышечной массы (на 14%). Гидрокортизоновый гиперкортицизм сопровождался удлинением латентного периода одиночного сокращения передней большеберцовой мышцы (на 37%) и фазы его укорочения (на 20%), уменьшением амплитуды одиночных сокращений (на 32%), объема выполненной мышцей внешней работы (на 40%) и развиваемой мощности (на 41%) при тетаническом сокращении. Отмечены нарушения в целостном процессе нервно-мышечной передачи, что проявлялось в уменьшении степени ее надежности (у 30% особей), выраженном облегчении (более 85% у 50% особей) или депрессии (до -42% у 40% особей) при стимуляции нервно-мышечного аппарата с оптимальной частотой (30 имп/с). Адреналин, вводимый в комплексе с гидрокортизоном, предотвращал появление ряда симптомов миопатии, таких, как снижение амплитуды М-ответов, уменьшение количества активируемых двигательных единиц мышцы, ее массы, внешней работы и мощности, а также обусловливал уменьшение частоты генерации полифазных М-ответов (с 30% до 10%). Кроме того, адреналин ослаблял негативный эффект гидрокортизона на синаптический аппарат, что проявлялось в уменьшении частоты встречаемости сниженной надежности синаптической передачи (с 30% до 10%), выраженного ее облегчения (с 50% до 20%) и депрессии (с 40% до 10%) у животных (Г+А)-группы в сравнении с Г-группой. Заключение. Полученные в модельных экспериментах на животных в условиях in situ данные свидетельствуют о способности адреналина эффективно компенсировать ряд негативных эффектов, развивающихся в скелетной мышце при длительном введении гидрокортизона. Based on the beneficial effect of chronic adrenergic stimulation on the neuromuscular apparatus function, the aim of this study was to evaluate efficacy of adrenaline at therapeutic doses in compensation for adverse effects of chronically administered hydrocortisone on mixed-fiber type skeletal muscles ( m. tibialis anterior ). Method. Experiments were performed on sexually mature female rats (190-220 g) divided into 3 groups: control (n = 10), experimental group 1 (H group, n = 10, hydrocortisone acetate, for 30 days daily), and experimental group 2 (H+A group, n = 10, hydrocortisone acetate in combination with adrenaline hydrochloride, for 30 days daily). Hydrocortisone acetate (Farmak, Ukraine) was injected i.p. at a dose of 0.2 mg/kg, which was equivalent to the human therapeutic dose of 3 mg/kg. Adrenaline hydrochloride (Zdorovje, Ukraine) was injected s.c. at a dose of 0.2 mg/kg. Parameters of the tibialis anterior muscle function were studied on anesthetized animals (sodium thiopental, 100 mg/kg) using electromyography and myography methods. Muscle contractions were induced by suprathreshold electrical stimulation of the fibular nerve. Results. Chronic administration (30 days) of hydrocortisone caused symptoms of steroid myopathy evident as low-amplitude M-waves of normal duration, increased frequency of polyphase potentials (by 30%), decreased quantity of activated motor units (by 33%), and decreased muscle mass (by 14%). Hydrocortisone-induced hypercorticoidism was associated with prolonged latent period (by 37%) and shortening phase (by 20%) and decreased amplitude (by 32%), amount of muscular external work (by 40%) in a single tibialis anterior muscle contraction, and decreased muscular power (by 41%) developed in a tetanic contraction. Following repeated hydrocortisone injections, the overall process of neuromuscular transmission was disturbed, which was evident as its impaired reliability (30% of rats) and pronounced facilitation (>85% in 50% of rats) or depression (<-42% in 40% of rats) at an optimum stimulation frequency (30 imp/s). Adrenaline administered in combination with hydrocortisone prevented formation of myopathy symptoms, such as decreases in the M-wave amplitude, number of activated motor units, muscle weight, external work and power, and it also decreased the frequency of polyphase M-waves (from 30% to 10%). In addition, adrenaline attenuated the adverse effect of hydrocortisone on the synaptic apparatus by decreasing the incidence of reduced reliability of synaptic transmission (from 30% to 10%), facilitation (from 50% to 20%), and depression (from 40% to 10%) in the H+A-group compared to the H group. Conclusion. The experimental data obtained in model experiments on animals in situ support the adrenaline ability to effectively compensate for a number of adverse effects of chronic hydrocortisone treatment on the skeletal muscle.

The extracellular matrix molecule brevican is an integral component of the machinery mediating fast synaptic transmission at the calyx of Held.

The proteoglycan brevican is a major component of the extracellular matrix of perineuronal nets and is highly enriched in the perisynaptic space suggesting a role for synaptic transmission. We have introduced the calyx of Held in the auditory brainstem as a model system to study the impact of brevican on dynamics and reliability of synaptic transmission. In vivo extracellular single-unit recordings at the calyx of Held in brevican-deficient mice yielded a significant increase in the action potential (AP) transmission delay and a prolongation of pre- and postsynaptic APs. The changes in dynamics of signal transmission were accompanied by the reduction of presynaptic vGlut1 and ultrastructural changes in the perisynaptic space. These data show that brevican is an important mediator of fast synaptic transmission at the calyx of Held. The extracellular matrix is an integral part of the neural tissue. Its most conspicuous manifestation in the brain are the perineuronal nets (PNs) which surround somata and proximal dendrites of distinct neuron types. The chondroitin sulfate proteoglycan brevican is a major component of PNs. In contrast to other PN-comprising proteoglycans (e.g. aggrecan and neurocan), brevican is mainly expressed in the perisynaptic space closely associated with both the pre- and postsynaptic membrane. This specific localization prompted the hypothesis that brevican might play a role in synaptic transmission. In the present study we specifically investigated the role of brevican in synaptic transmission at a central synapse, the calyx of Held in the medial nucleus of the trapezoid body, by the use of in vivo electrophysiology, immunohistochemistry, biochemistry and electron microscopy. In vivo extracellular single-unit recordings were acquired in brevican-deficient mice and the dynamics and reliability of synaptic transmission were compared to wild-type littermates. In knockout mice, the speed of pre-to-postsynaptic action potential (AP) transmission was reduced and the duration of the respective pre- and postsynaptic APs increased. The reliability of signal transmission, however, was not affected by the lack of brevican. The changes in dynamics of signal transmission were accompanied by the reduction of (i) presynaptic vGlut1 and (ii) the size of subsynaptic cavities. The present results suggest an essential role of brevican for the functionality of high-speed synaptic transmission at the calyx of Held.

Characteristics of the Transmission of Excitation in Rat Neuromuscular Synapses at Different Periods of Postnatal Development

Studies on diaphragm muscle neuromuscular preparations from rats at different states of postnatal development compared the morphological characteristics and functions of the synaptic apparatus, including the time parameters of evoked secretion. Along with nerve ending areas smaller than those in adults, neonates also showed a reduced rate of conduction along motor nerves, reduced intensities of evoked and spontaneous quantum secretion, and very marked fluctuations in true synaptic delays in single-quantum endplate currents. The high level of asynchronicity in the phasic secretion of acetylcholine quanta, along with the longer lifetime of the open state of ion channels in the synapses of neonates, partially compensated for the decrease in the reliability of synaptic transmission due to the reduction in the quantum composition of the postsynaptic response.

Motile Axonal Mitochondria Contribute to the Variability of Presynaptic Strength

One of the most notable characteristics of synaptic transmission is the wide variation in synaptic strength in response to identical stimulation. In hippocampal neurons, approximately one-third of axonal mitochondria are highly motile, and some dynamically pass through presynaptic boutons. This raises a fundamental question: can motile mitochondria contribute to the pulse-to-pulse variability of presynaptic strength? Recently, we identified syntaphilin as an axonal mitochondrial-docking protein. Using hippocampal neurons and slices of syntaphilin knockout mice, we demonstrate that the motility of axonal mitochondria correlates with presynaptic variability. Enhancing mitochondrial motility increases the pulse-to-pulse variability, whereas immobilizing mitochondria reduces the variability. By dual-color live imaging at single-bouton levels, we further show that motile mitochondria passing through boutons dynamically influence synaptic vesicle release, mainly by altering ATP homeostasis in axons. Thus, our study provides insight into the fundamental properties of the CNS to ensure the plasticity and reliability of synaptic transmission.

Nanodomain Coupling at an Excitatory Cortical Synapse

The coupling distance between presynaptic Ca(2+) influx and the sensor for vesicular transmitter release determines speed and reliability of synaptic transmission. Nanodomain coupling (<100 nm) favors fidelity and is employed by synapses specialized for escape reflexes and by inhibitory synapses involved in synchronizing fast network oscillations. Cortical glutamatergic synapses seem to forgo the benefits of tight coupling, yet quantitative detail is lacking. The reduced transmission fidelity of loose coupling, however, raises the question whether it is indeed a general characteristic of cortical synapses. Here we analyzed excitatory parallel fiber to Purkinje cell synapses, major processing sites for sensory information and well suited for analysis because they typically harbor only a single active zone. We quantified the coupling distance by combining multiprobability fluctuation analyses, presynaptic Ca(2+) imaging, and reaction-diffusion simulations in wild-type and calretinin-deficient mice. We found a coupling distance of <30 nm at these synapses, much shorter than at any other glutamatergic cortical synapse investigated to date. Our results suggest that nanodomain coupling is a general characteristic of conventional cortical synapses involved in high-frequency transmission, allowing for dense gray matter packing and cost-effective neurotransmission.

Gephyrin Regulates GABAergic and Glutamatergic Synaptic Transmission in Hippocampal Cell Cultures

Gephyrin is a scaffold protein essential for stabilizing glycine and GABA(A) receptors at inhibitory synapses. Here, recombinant intrabodies against gephyrin (scFv-gephyrin) were used to assess whether this protein exerts a transynaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrin-transfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NLG2), a protein that, interacting with neurexins, ensures the cross-talk between the post- and presynaptic sites. Interestingly, hampering gephyrin function also produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Overexpressing NLG2 in gephyrin-deprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. Pulldown experiments demonstrated that gephyrin interacts not only with NLG2 but also with NLG1, the isoform enriched at excitatory synapses. These results suggest a key role of gephyrin in regulating transynaptic signaling at both inhibitory and excitatory synapses.

The Calyx of Held Develops Adult-Like Dynamics and Reliability by Hearing Onset in the MouseIn Vivo

The development of the auditory system has received increasing attention since the mechanisms of patterned, spontaneous activity in prehearing mammals were discovered. This early activity originates in the cochlea and is assumed to be of importance for the establishment and refinement of synaptic connections in the auditory system. In the present study we investigate synaptic transmission and its interplay with spontaneous discharges in the developing auditory system. We used the calyx of Held as a model system, where this question can be investigated in vivo over a broad range of ages [postnatal day 8 (P8)-P28]. To precisely quantify the timing and reliability of synaptic transmission, we developed a novel fitting approach which decomposes the extracellularly recorded signal into its presynaptic and postsynaptic components. In prehearing mice, we found signal transmission to be unreliable, with high variability in the transmission delay and in the amplitude of postsynaptic components. These timing and amplitude changes were strongly correlated with the preceding activity. Around hearing onset (P12-P14), the properties of signal transmission converged to the adult-like state which was characterized by high transmission reliability as well as high consistency in timing and amplitude. Although activity-dependent depression was still found in action potentials, EPSP depression no longer played a prominent role. In conclusion, the maturation of synaptic transmission at the calyx of Held seems to be precisely timed to achieve its adult potential by the time acoustically evoked signal processing commences.

Rapid structural alterations of the active zone lead to sustained changes in neurotransmitter release

The likelihood with which an action potential elicits neurotransmitter release, the release probability (p(r)), is an important component of synaptic strength. Regulatory mechanisms controlling several steps of synaptic vesicle (SV) exocytosis may affect p(r), yet their relative importance in determining p(r) and eliciting temporal changes in neurotransmitter release at individual synapses is largely unknown. We have investigated whether the size of the active zone cytomatrix is a major determinant of p(r) and whether changes in its size lead to corresponding alterations in neurotransmitter release. We have used a fluorescent sensor of SV exocytosis, synaptophysin-pHluorin, to measure p(r) at individual synapses with high accuracy and employed a fluorescently labeled cytomatrix protein, Bassoon, to quantify the amount of active zone cytomatrix present at these synapses. We find that, for synapses made by a visually identified presynaptic neuron, p(r) is indeed strongly correlated with the amount of active zone cytomatrix present at the presynaptic specialization. Intriguingly, active zone cytomatrices are frequently subject to synapse-specific changes in size on a time scale of minutes. These spontaneous alterations in active zone size are associated with corresponding changes in neurotransmitter release. Our results suggest that the size of the active zone cytomatrix has a large influence on the reliability of synaptic transmission. Furthermore, they implicate mechanisms leading to rapid structural alterations at active zones in synapse-specific forms of plasticity.

Stabilization of visual responses through cholinergic activation

Neuronal processing of sensory information requires that rapidly changing synaptic inputs are continuously transformed into action potentials. Variability of spike firing is generally considered as noise and might therefore interfere with the reliability of synaptic transmission in sensory systems. In a system in which the number of spikes is a variable that determines the quality of neuronal transmission, variability of spike counts is a paradoxical attribute. In contrast, in a system in which precisely correlated spike firing can influence synaptic integration, response variability might be used as an additional mechanism for coding information. As acetylcholine has been shown to reduce spike-frequency adaptation and enhance gamma frequency (21–70 Hz) oscillations, we set out to study the influence of cholinergic modulation on the variability of spike counts and gamma oscillations. Iontophoretic application of carbachol, a cholinergic agonist, in cat primary visual cortex or electrical stimulation of the mesencephalic reticular formation reduced the spike count variability and stabilized gamma frequency oscillations of visually induced responses. Response stabilization was correlated with enhancement of gamma-frequency oscillations but not with averaged firing rates. Lowering variability of sensory responses might be a mechanism to stabilize and improve reliability of neuronal transmission. Cholinergic activation may therefore influence the efficacy of neuronal transmission by modulating the precise timing of neuronal responses.

Reliability of Synaptic Transmission at the Synapses of Held In Vivo under Acoustic Stimulation

BackgroundThe giant synapses of Held play an important role in high-fidelity auditory processing and provide a model system for synaptic transmission at central synapses. Whether transmission of action potentials can fail at these synapses has been investigated in recent studies. At the endbulbs of Held in the anteroventral cochlear nucleus (AVCN) a consistent picture emerged, whereas at the calyx of Held in the medial nucleus of the trapezoid body (MNTB) results on the reliability of transmission remain inconsistent. In vivo this discrepancy could be due to the difficulty in identifying failures of transmission.Methods/FindingsWe introduce a novel method for detecting unreliable transmission in vivo. Based on the temporal relationship between a cells' waveform and other potentials in the recordings, a statistical test is developed that provides a balanced decision between the presence and the absence of failures. Its performance is quantified using simulated voltage recordings and found to exhibit a high level of accuracy. The method was applied to extracellular recordings from the synapses of Held in vivo. At the calyces of Held failures of transmission were found only rarely. By contrast, at the endbulbs of Held in the AVCN failures were found under spontaneous, excited, and suppressed conditions. In accordance with previous studies, failures occurred most abundantly in the suppressed condition, suggesting a role for inhibition.Conclusions/SignificanceUnder the investigated activity conditions/anesthesia, transmission seems to remain largely unimpeded in the MNTB, whereas in the AVCN the occurrence of failures is related to inhibition and could be the basis/result of computational mechanisms for temporal processing. More generally, our approach provides a formal tool for studying the reliability of transmission with high statistical accuracy under typical in vivo recording conditions.

Peculiarities of synaptic vesicle recycling in frog and mouse motor nerve terminals

Using electrophysiology and fluorescence microscopy (dye FM1-43), comparative study of neurotransmitter secretion, synaptic vesicle exo-endocytosis, and recycling has been carried out in frog and mouse motor nerve terminals during a long strong stimulation (3 min; 20 imp/s). The obtained data have revealed three synaptic vesicle pools and two recycling ways existing on motor nerve terminals. The strong stimulation induced consecutive depletion of readily releasable, mobilized, and reserve vesicle pools of frog nerve terminals. The exocytosis rate exceeded the endocytosis rate; predominant was the slow synaptic vesicle recycling that replenished the reserve pool. In mouse nerve endings, vesicles of the readily releasable and mobilized pools were only exocytosed, the pools being replenished by fast recycling. At the same time, exo- and endocytosis occurred nearly in parallel and vesicles of the reserve pool did not participate in the neurotransmitter secretion. In is suggested that evolution of motor nerve terminals was directed to a decrease of the vesicle pool size, economic spending, and effective reuse of synaptic vesicles. This is achieved by an increase of endocytosis and recycling rates. These features can provide a long maintenance of a quite level of neurotransmitter secretion in nerve terminals of homoiothermal animals to preserve reliability of synaptic transmission during the high-frequency activity.

Stochastic Simulations on the Reliability of Action Potential Propagation in Thin Axons

It is generally assumed that axons use action potentials (APs) to transmit information fast and reliably to synapses. Yet, the reliability of transmission along fibers below 0.5 μm diameter, such as cortical and cerebellar axons, is unknown. Using detailed models of rodent cortical and squid axons and stochastic simulations, we show how conduction along such thin axons is affected by the probabilistic nature of voltage-gated ion channels (channel noise). We identify four distinct effects that corrupt propagating spike trains in thin axons: spikes were added, deleted, jittered, or split into groups depending upon the temporal pattern of spikes. Additional APs may appear spontaneously; however, APs in general seldom fail (<1%). Spike timing is jittered on the order of milliseconds over distances of millimeters, as conduction velocity fluctuates in two ways. First, variability in the number of Na channels opening in the early rising phase of the AP cause propagation speed to fluctuate gradually. Second, a novel mode of AP propagation (stochastic microsaltatory conduction), where the AP leaps ahead toward spontaneously formed clusters of open Na channels, produces random discrete jumps in spike time reliability. The combined effect of these two mechanisms depends on the pattern of spikes. Our results show that axonal variability is a general problem and should be taken into account when considering both neural coding and the reliability of synaptic transmission in densely connected cortical networks, where small synapses are typically innervated by thin axons. In contrast we find that thicker axons above 0.5 μm diameter are reliable.

Ceramidase Regulates Synaptic Vesicle Exocytosis and Trafficking

A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C(5)-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50-70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50-70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission.

Declusterization of GABAA Receptors Affects the Kinetic Properties of GABAergic Currents in Cultured Hippocampal Neurons

Speed and reliability of synaptic transmission are essential for information coding in neuronal networks and require the presence of clustered neurotransmitter receptors at the plasma membrane in precise apposition to presynaptic terminals. Receptor clusterization is the result of highly regulated processes involving functional and structural proteins. Among the structural elements, microtubules are known to play a crucial role in anchoring of gamma-aminobutyric acid, type A (GABA(A)) receptors. Here we show that microtubule depolymerization with nocodazole induces the declusterization of GABA(A) receptors and modifies the kinetic properties of GABAergic currents in cultured hippocampal neurons. In particular, this drug, applied either in the bath or via the patch pipette, induced the acceleration of the onset kinetics of miniature inhibitory postsynaptic currents (mIPSCs) without significantly affecting their frequency, thus suggesting a main postsynaptic site of action. After nocodazole treatment, current responses to ultrafast applications of GABA exhibited a faster rise time and an accelerated onset of desensitization. A quantitative analysis of GABA-evoked currents and model simulations suggest that declusterization affects the gating properties of GABA(A) receptors. In particular, a faster entry into the desensitized state of declustered GABA(A) receptors may account for the changes in the kinetic properties of mIPSCs after nocodazole treatment. Hence it appears that the clustered condition of GABA(A) receptors contributes in shaping GABAergic currents.

Transmitter release modulation by intracellular Ca2+ buffers in facilitating and depressing nerve terminals of pyramidal cells in layer 2/3 of the rat neocortex indicates a target cell-specific difference in presynaptic calcium dynamics.

1. In connections formed by nerve terminals of layer 2/3 pyramidal cells onto bitufted interneurones in young (postnatal day (P)14-15) rat somatosensory cortex, the efficacy and reliability of synaptic transmission were low. At these connections release was facilitated by paired-pulse stimulation (at 10 Hz). In connections formed by terminals of layer 2/3 pyramids with multipolar interneurones efficacy and reliability were high and release was depressed by paired-pulse stimulation. In both types of terminal, however, the voltage-dependent Ca2+ channels that controlled transmitter release were predominantly of the P/Q- and N-subtypes. 2. The relationship between unitary EPSP amplitude and extracellular calcium concentration ([Ca2+]o) was steeper for facilitating than for depressing terminals. Fits to a Hill equation with nH = 4 indicated that the apparent KD of the Ca2+ sensor for vesicle release was two- to threefold lower in depressing terminals than in facilitating ones. 3. Intracellular loading of pyramidal neurones with the fast and slowly acting Ca2+ buffers BAPTA and EGTA differentially reduced transmitter release in these two types of terminal. Unitary EPSPs evoked by pyramidal cell stimulation in bitufted cells were reduced by presynaptic BAPTA and EGTA with half-effective concentrations of approximately 0.1 and approximately 1 mM, respectively. Unitary EPSPs evoked in multipolar cells were reduced to one-half of control at higher concentrations of presynaptic BAPTA and EGTA (approximately 0.5 and approximately 7 mM, respectively). 4. Frequency-dependent facilitation of EPSPs in bitufted cells was abolished by EGTA at concentrations of > or = 0.2 mM, suggesting that accumulation of free Ca2+ is essential for facilitation in the terminals contacting bitufted cells. In contrast, facilitation was unaffected or even slightly increased in the terminals loaded with BAPTA in the concentration range 0.02-0.5 mM. This is attributed to partial saturation of exogenously added BAPTA. However, BAPTA at concentrations > or = 1 mM also abolished facilitation. 5. Frequency-dependent depression of EPSPs in multipolar cells was not significantly reduced by EGTA. With BAPTA, the depression decreased at concentrations > 0.5 mM, concomitant with a reduction in amplitude of the first EPSP in a train. 6. An analysis is presented that interprets the effects of EGTA and BAPTA on synaptic efficacy and its short-term modification during paired-pulse stimulation in terms of changes in [Ca2+] at the release site ([Ca2+]RS) and that infers the affinity of the Ca2+ sensor from the dependence of unitary EPSPs on [Ca2+]o. 7. The results suggest that the target cell-specific difference in release from the terminals on bitufted or multipolar cells can be explained by a longer diffusional distance between Ca2+ channels and release sites and/or lower Ca2+ channels density in the terminals that contact bitufted cells. This would lead to a lower [Ca2+] at release sites and would also explain the higher apparent K(D) of the Ca2+ sensor in facilitating terminals.