Dissociated Rat Cortical Neurons Research Articles

Mild membrane depolarization in neurons induces immediate early gene transcription and acutely subdues responses to a successive stimulus

Immediate early genes (IEGs) are transcribed in response to neuronal activity from sensory stimulation during multiple adaptive processes in the brain. The transcriptional profile of IEGs is indicative of the duration of neuronal activity, but its sensitivity to the strength of depolarization remains unknown. Also unknown is whether activity history of graded potential changes influence future neuronal activity. In this work with dissociated rat cortical neurons, we found that mild depolarization—mediated by elevated extracellular potassium (K+)—induces a wide array of rapid IEGs and transiently depresses transcriptional and signaling responses to a successive stimulus. This latter effect was independent of de novo transcription, translation, and signaling via calcineurin or mitogen-activated protein kinase. Furthermore, as measured by multiple electrode arrays and calcium imaging, mild depolarization acutely subdues subsequent spontaneous and bicuculline-evoked activity via calcium- and N-methyl-d-aspartate receptor–dependent mechanisms. Collectively, this work suggests that a recent history of graded potential changes acutely depress neuronal intrinsic properties and subsequent responses. Such effects may have several potential downstream implications, including reducing signal-to-noise ratio during synaptic plasticity processes.

Impairment of early neuronal maturation in anti-NMDA-receptor encephalitis.

Adequate immunotherapies for anti-NMDAR encephalitis during pregnancy produce a relatively good clinical outcome for pregnant mothers and their infants, but there are no reports about the future growth of their babies. The damage of anti-NMDAR antibodies to early neuronal development is still unknown. Serum or cerebrospinal fluid from one patient with anti-NMDAR encephalitis (the index patient) and one patient with schizophrenia (the control patient) was administered to primary cultures of dissociated rat cortical neurons, and dendritic outgrowth, centrosome elimination, and branching of dendrites were investigated. For rescue experiments, serum of the index patient was replaced with normal culture media after 3 days' administration of the index patient. Serum and cerebrospinal fluid of the index patient statistically significantly impaired dendritic outgrowth of cultured rat cortical primary neurons. Serum of the index patient also statistically significantly delayed centrosome elimination. Impaired dendritic outgrowth and delayed centrosome elimination were not perfectly rescued by changing to normal culture media. Serum of the index patient also statistically significantly reduced the branching of dendrites. This is the first demonstration of the damage by anti-NMDAR antibodies on early dendritic development in vitro. As a strategy to protect embryonic neurons, our findings may support the efficacy of early immunotherapy for anti-NMDAR encephalitis in pregnancy.

Consolidation of memory traces in cultured cortical networks requires low cholinergic tone, synchronized activity and high network excitability

In systems consolidation, encoded memories are replayed by the hippocampus during slow-wave sleep (SWS), and permanently stored in the neocortex. Declarative memory consolidation is believed to benefit from the oscillatory rhythms and low cholinergic tone observed in this sleep stage, but underlying mechanisms remain unclear. To clarify the role of cholinergic modulation and synchronized activity in memory consolidation, we applied repeated electrical stimulation in mature cultures of dissociated rat cortical neurons with high or low cholinergic tone, mimicking the cue replay observed during systems consolidation under distinct cholinergic concentrations. In the absence of cholinergic input, these cultures display activity patterns hallmarked by network bursts, synchronized events reminiscent of the low frequency oscillations observed during SWS. They display stable activity and connectivity, which mutually interact and achieve an equilibrium. Electrical stimulation reforms the equilibrium to include the stimulus response, a phenomenon interpreted as memory trace formation. Without cholinergic input, activity was burst-dominated. First application of a stimulus induced significant connectivity changes, while subsequent repetition no longer affected connectivity. Presenting a second stimulus at a different electrode had the same effect, whereas returning to the initial stimuli did not induce further connectivity alterations, indicating that the second stimulus did not erase the ‘memory trace’ of the first. Distinctively, cultures with high cholinergic tone displayed reduced network excitability and dispersed firing, and electrical stimulation did not induce significant connectivity changes. We conclude that low cholinergic tone facilitates memory formation and consolidation, possibly through enhanced network excitability. Network bursts or SWS oscillations may merely reflect high network excitability.

Divergent Synaptic Scaling of Miniature EPSCs following Activity Blockade in Dissociated Neuronal Cultures.

Neurons can respond to decreased network activity with a homeostatic increase in the amplitudes of miniature EPSCs (mEPSCs). The prevailing view is that mEPSC amplitudes are uniformly multiplied by a single factor, termed "synaptic scaling." Deviations from purely multiplicative scaling have been attributed to biological differences, or to a distortion imposed by a detection threshold limit. Here, we demonstrate in neurons dissociated from cortices of male and female mice that the shift in mEPSC amplitudes observed in the experimental data cannot be reproduced by simulation of uniform multiplicative scaling, with or without the distortion caused by applying a detection threshold. Furthermore, we demonstrate explicitly that the scaling factor is not uniform but is close to 1 for small mEPSCs, and increases with increasing mEPSC amplitude across a substantial portion of the data. This pattern was also observed for previously published data from dissociated mouse hippocampal neurons and dissociated rat cortical neurons. The finding of "divergent scaling" shifts the current view of homeostatic plasticity as a process that alters all synapses on a neuron equally to one that must accommodate the differential effect observed for small versus large mEPSCs. Divergent scaling still accomplishes the essential homeostatic task of modifying synaptic strengths in the opposite direction of the activity change, but the consequences are greatest for those synapses which individually are more likely to bring a neuron to threshold.SIGNIFICANCE STATEMENT In homeostatic plasticity, the responses to chronic increases or decreases in network activity act in the opposite direction to restore normal activity levels. Homeostatic plasticity is likely to play a role in diseases associated with long-term changes in brain function, such as epilepsy and neuropsychiatric illnesses. One homeostatic response is the increase in synaptic strength following a chronic block of activity. Research is focused on finding a globally expressed signaling pathway, because it has been proposed that the plasticity is uniformly expressed across all synapses. Here, we show that the plasticity is not uniform. Our work suggests that homeostatic signaling molecules are likely to be differentially expressed across synapses.

3D axon growth by exogenous electrical stimulus and soluble factors

Axon growth and alignment are fundamental processes during nervous system development and neural regeneration after injury. The present study investigates the effects of exogenous stimulus of electrical signals and soluble factors on axon 3D growth, using a silk protein material-based 3D brain tissue model. Electrical stimulus was delivered via embedded gold wires positioned at the interface of the scaffold region and the center matrix gel-filled region, spanning the axon growth area. This setup delivered applied electrical field directly to growing axons, and the effects were compared to micro-needle assisted local delivery of soluble factors of extracellular (ECM) components and neurotrophins. Dissociated rat cortical neurons were exposed to an alternating field of 80 mV/mm at 0.5 Hz to 2 kHz or soluble factors for up to 4 days, and evaluated by of β III-tubulin immunostaining, confocal imaging and 3D neurite tracing. 0.5–20 Hz were found to promote axon growth, with 2 Hz producing the biggest effect of ∼30% axon length increase compared to control cultures. Delivery of ECM components of laminin and fibronectin resulted significantly greater axon initial length increases compared to neurotrophic factors, such as BDNF, GDNF, NGF and NT3 (all at 1 μM). Though axon lengths under 2 Hz stimulation and LN or FN exposure were statistically similar, significant AC-induced axon alignment was found under all frequencies tested. The effects included perpendicular orientation of axons trespassing an electrode, large populations of aligned axon tracts in parallel to the field direction with a few perpendicularly aligned along the middle point of the EF. These findings are consistent with the hypothesis that an electrode in AC field could act as an alternating cathode that attracts the growing tip of the axon. These results demonstrate the use of alternating electric field stimulation to direct axon 3D length growth and orientation. Our study provides basis for further optimizing stimulation parameters, in conjunction of delivery of growth promoting soluble factors to direct axon growth in a brain mimetic 3D environment. This system provides a platform for studying the effects of exogenous signals on nervous system development and for testing neuromodulation approaches for neurological diseases.

Histone Hypervariants H2A.Z.1 and H2A.Z.2 Play Independent and Context-Specific Roles in Neuronal Activity-Induced Transcription of Arc/Arg3.1 and Other Immediate Early Genes.

The histone variant H2A.Z is an essential and conserved regulator of eukaryotic gene transcription. However, the exact role of this histone in the transcriptional process remains perplexing. In vertebrates, H2A.Z has two hypervariants, H2A.Z.1 and H2A.Z.2, that have almost identical sequences except for three amino acid residues. Due to such similarity, functional specificity of these hypervariants in neurobiological processes, if any, remain largely unknown. In this study with dissociated rat cortical neurons, we asked if H2A.Z hypervariants have distinct functions in regulating basal and activity-induced gene transcription. Hypervariant-specific RNAi and microarray analyses revealed that H2A.Z.1 and H2A.Z.2 regulate basal expression of largely nonoverlapping gene sets, including genes that code for several synaptic proteins. In response to neuronal activity, rapid transcription of our model gene Arc is impaired by depletion of H2A.Z.2, but not H2A.Z.1. This impairment is partially rescued by codepletion of the H2A.Z chaperone, ANP32E. In contrast, under a different context (after 48 h of tetrodotoxin, TTX), rapid transcription of Arc is impaired by depletion of either hypervariant. Such context-dependent roles of H2A.Z hypervariants, as revealed by our multiplexed gene expression assays, are also evident with several other immediate early genes, where regulatory roles of these hypervariants vary from gene to gene under different conditions. Together, our data suggest that H2A.Z hypervariants have context-specific roles that complement each other to mediate activity-induced neuronal gene transcription.

Feed-Forward Propagation of Temporal and Rate Information between Cortical Populations during Coherent Activation in Engineered In Vitro Networks.

Transient propagation of information across neuronal assembles is thought to underlie many cognitive processes. However, the nature of the neural code that is embedded within these transmissions remains uncertain. Much of our understanding of how information is transmitted among these assemblies has been derived from computational models. While these models have been instrumental in understanding these processes they often make simplifying assumptions about the biophysical properties of neurons that may influence the nature and properties expressed. To address this issue we created an in vitro analog of a feed-forward network composed of two small populations (also referred to as assemblies or layers) of living dissociated rat cortical neurons. The populations were separated by, and communicated through, a microelectromechanical systems (MEMS) device containing a strip of microscale tunnels. Delayed culturing of one population in the first layer followed by the second a few days later induced the unidirectional growth of axons through the microtunnels resulting in a primarily feed-forward communication between these two small neural populations. In this study we systematically manipulated the number of tunnels that connected each layer and hence, the number of axons providing communication between those populations. We then assess the effect of reducing the number of tunnels has upon the properties of between-layer communication capacity and fidelity of neural transmission among spike trains transmitted across and within layers. We show evidence based on Victor-Purpura’s and van Rossum’s spike train similarity metrics supporting the presence of both rate and temporal information embedded within these transmissions whose fidelity increased during communication both between and within layers when the number of tunnels are increased. We also provide evidence reinforcing the role of synchronized activity upon transmission fidelity during the spontaneous synchronized network burst events that propagated between layers and highlight the potential applications of these MEMs devices as a tool for further investigation of structure and functional dynamics among neural populations.

State-Dependent Propagation of Neuronal Sub-Population in Spontaneous Synchronized Bursts.

Repeating stable spatiotemporal patterns emerge in synchronized spontaneous activity in neuronal networks. The repertoire of such patterns can serve as memory, or a reservoir of information, in a neuronal network; moreover, the variety of patterns may represent the network memory capacity. However, a neuronal substrate for producing a repertoire of patterns in synchronization remains elusive. We herein hypothesize that state-dependent propagation of a neuronal sub-population is the key mechanism. By combining high-resolution measurement with a 4096-channel complementary metal-oxide semiconductor (CMOS) microelectrode array (MEA) and dimensionality reduction with non-negative matrix factorization (NMF), we investigated synchronized bursts of dissociated rat cortical neurons at approximately 3 weeks in vitro. We found that bursts had a repertoire of repeating spatiotemporal patterns, and different patterns shared a partially similar sequence of sub-population, supporting the idea of sequential structure of neuronal sub-populations during synchronized activity. We additionally found that similar spatiotemporal patterns tended to appear successively and periodically, suggesting a state-dependent fluctuation of propagation, which has been overlooked in existing literature. Thus, such a state-dependent property within the sequential sub-population structure is a plausible neural substrate for performing a repertoire of stable patterns during synchronized activity.

An in vitro method to manipulate the direction and functional strength between neural populations

We report the design and application of a Micro Electro Mechanical Systems (MEMs) device that permits investigators to create arbitrary network topologies. With this device investigators can manipulate the degree of functional connectivity among distinct neural populations by systematically altering their geometric connectivity in vitro. Each polydimethylsilxane (PDMS) device was cast from molds and consisted of two wells each containing a small neural population of dissociated rat cortical neurons. Wells were separated by a series of parallel micrometer scale tunnels that permitted passage of axonal processes but not somata; with the device placed over an 8 × 8 microelectrode array, action potentials from somata in wells and axons in microtunnels can be recorded and stimulated. In our earlier report we showed that a one week delay in plating of neurons from one well to the other led to a filling and blocking of the microtunnels by axons from the older well resulting in strong directionality (older to younger) of both axon action potentials in tunnels and longer duration and more slowly propagating bursts of action potentials between wells. Here we show that changing the number of tunnels, and hence the number of axons, connecting the two wells leads to changes in connectivity and propagation of bursting activity. More specifically, the greater the number of tunnels the stronger the connectivity, the greater the probability of bursting propagating between wells, and shorter peak-to-peak delays between bursts and time to first spike measured in the opposing well. We estimate that a minimum of 100 axons are needed to reliably initiate a burst in the opposing well. This device provides a tool for researchers interested in understanding network dynamics who will profit from having the ability to design both the degree and directionality connectivity among multiple small neural populations.

Improved PeT Molecules for Optically Sensing Voltage in Neurons

VoltageFluor (VF) dyes have the potential to measure voltage optically in excitable membranes with a combination of high spatial and temporal resolution essential to better characterize the voltage dynamics of large groups of excitable cells. VF dyes sense voltage with high speed and sensitivity using photoinduced electron transfer (PeT) through a conjugated molecular wire. We show that tuning the driving force for PeT (ΔGPeT + w) through systematic chemical substitution modulates voltage sensitivity, estimate (ΔGPeT + w) values from experimentally measured redox potentials, and validate the voltage sensitivities in patch-clamped HEK cells for 10 new VF dyes. VF2.1(OMe).H, with a 48% ΔF/F per 100 mV, shows approximately 2-fold improvement over previous dyes in HEK cells, dissociated rat cortical neurons, and medicinal leech ganglia. Additionally, VF2.1(OMe).H faithfully reports pharmacological effects and circuit activity in mouse olfactory bulb slices, thus opening a wide range of previously inaccessible applications for voltage-sensitive dyes.

Understanding the temporal evolution of neuronal connectivity in cultured networks using statistical analysis

BackgroundMicro-Electrode Array (MEA) technology allows researchers to perform long-term non-invasive neuronal recordings in-vitro while actively interacting with the cultured neurons. Despite numerous studies carried out using MEAs, many functional, chemical and structural mechanisms of how dissociated cortical neurons develop and respond to external stimuli are not yet well understood because of the lack of quantitative studies that assess how their development can be affected by chronic external stimulation.MethodsTo investigate network changes, we analyzed a large MEA data set composed of neuron spikes recorded from cultures of dissociated rat cortical neurons plated on MEA dishes with 59 recording electrodes each. Neural network activity was recorded during the first five weeks of each culture’s in-vitro development. Stimulation sessions were delivered to each of the 59 electrodes. The False Discovery Rate technique was used to quantify the temporal evolution of dissociated cortical neurons. Our analysis focused on network responses that occurred within selected time window durations, namely 50 ms, 100 ms and 150 ms after stimulus onset.ResultsOur results show an evolution in dissociated cortical neuronal network activity over time, that reflects the network synaptic evolution. Furthermore, we tested the sensitivity of our technique to different observation time windows and found that varying the time windows, allows us to capture different dynamics of the observed responses. In addition, when selecting a 150 ms observation time window, our findings indicate that cultures dissociated from the same brain tissue display trends in their temporal evolution that are more similar than those obtained from different brains.ConclusionOur results emphasize that the FDR technique can be implemented without the need to make any particular assumptions about the data a priori. The proposed technique was able to capture the well-known dissociated cortical neuron networks’ temporal evolution, that has been previously observed in in-vivo and in intact brain tissue studies. Furthermore, our findings suggest that the time window that is used to capture the stimulus-evoked network responses is a critical parameter to analyze the electrical behavioral and temporal evolution of dissociated cortical neurons.

Short-Term Memory in Networks of Dissociated Cortical Neurons

Short-term memory refers to the ability to store small amounts of stimulus-specific information for a short period of time. It is supported by both fading and hidden memory processes. Fading memory relies on recurrent activity patterns in a neuronal network, whereas hidden memory is encoded using synaptic mechanisms, such as facilitation, which persist even when neurons fall silent. We have used a novel computational and optogenetic approach to investigate whether these same memory processes hypothesized to support pattern recognition and short-term memory in vivo, exist in vitro. Electrophysiological activity was recorded from primary cultures of dissociated rat cortical neurons plated on multielectrode arrays. Cultures were transfected with ChannelRhodopsin-2 and optically stimulated using random dot stimuli. The pattern of neuronal activity resulting from this stimulation was analyzed using classification algorithms that enabled the identification of stimulus-specific memories. Fading memories for different stimuli, encoded in ongoing neural activity, persisted and could be distinguished from each other for as long as 1 s after stimulation was terminated. Hidden memories were detected by altered responses of neurons to additional stimulation, and this effect persisted longer than 1 s. Interestingly, network bursts seem to eliminate hidden memories. These results are similar to those that have been reported from similar experiments in vivo and demonstrate that mechanisms of information processing and short-term memory can be studied using cultured neuronal networks, thereby setting the stage for therapeutic applications using this platform.

A Role for Myosin VI in the Localization of Axonal Proteins

In neurons polarized trafficking of vesicle-bound membrane proteins gives rise to the distinct molecular composition and functional properties of axons and dendrites. Despite their central role in shaping neuronal form and function, surprisingly little is known about the molecular processes that mediate polarized targeting of neuronal proteins. Recently, the plus-end-directed motor Myosin Va was shown to play a critical role in targeting of transmembrane proteins to dendrites; however, the role of myosin motors in axonal targeting is unknown. Here we show that Myosin VI, a minus-end-directed motor, plays a vital role in the enrichment of proteins on the surface of axons. Engineering non-neuronal proteins to interact with Myosin VI causes them to become highly concentrated at the axonal surface in dissociated rat cortical neurons. Furthermore, disruption of either Myosin VI function or expression leads to aberrant dendritic localization of axonal proteins. Myosin VI mediates the enrichment of proteins on the axonal surface at least in part by stimulating dendrite-specific endocytosis, a mechanism that has been shown to underlie the localization of many axonal proteins. In addition, a version of Channelrhodopsin 2 that was engineered to bind to Myosin VI is concentrated at the surface of the axon of cortical neurons in mice in vivo, suggesting that it could be a useful tool for probing circuit structure and function. Together, our results indicate that myosins help shape the polarized distributions of both axonal and dendritic proteins.

Statistical Long-Term Correlations in Dissociated Cortical Neuron Recordings

The study of nonlinear long-term correlations in neuronal signals is a central topic for advanced neural signal processing. In particular, the existence of long-term correlations in neural signals recorded via multielectrode array (MEA) could provide interesting information about changes in interneuron communications. In this study we propose a new method for long-term correlation analysis of neuronal burst activity based on the periodogram alpha slope estimation of the MEA signal. We applied our method to recordings taken from cultured networks of dissociated rat cortical neurons. We show the effectiveness of the method in analyzing the activity changes as well as the temporal dynamics that take place during the development of such cultures. Results demonstrate that the alpha parameter is able to divide the network development in three well-defined stages, showing pronounced variations in the long-term correlation among bursts.

Studying the formation of large cell aggregates in patterned neuronal cultures

Patterned neuronal cultures could be produced by plating cells dissociated from rat cortices on glass coverslips, the surface of which was printed with poly- l-lysine (PLL)-positive micropatterns. Large cell aggregates, which greatly disrupted the patterned distribution of neurons, were also generated. To investigate how large cell aggregates were formed, dissociated rat cortical neurons were plated on PLL-coated coverslips in a Petri dish, the surface of which was non-adherent to cells. The cell and cell aggregate densities found later on the coverslip surface increased significantly when larger dishes were used. Most of the neurons not attaching to substratum were able to survive for at least 24 h without entering apoptosis. During this time they formed floating spherical aggregates in the medium. These aggregates subsequently were able to attach to PLL-coated coverslips and produced large aggregates resembling those found within our patterned neuronal cultures. The results suggest a causative relationship between the generation of large numbers of neurons unattached to substratum and the formation of large cell aggregates on the patterned neuronal cultures. It was further demonstrated here that patterned neuronal cultures free of large cell aggregates could be prepared by a procedure employing both stencil patterning and microcontact printing technologies.

Burst on Hurst Algorithm for Detecting Activity Patterns in Networks of Cortical Neurons

� Abstract— Electrophysiological signals were recorded from primary cultures of dissociated rat cortical neurons coupled to Micro- Electrode Arrays (MEAs). The neuronal discharge patterns may change under varying physiological and pathological conditions. For this reason, we developed a new burst detection method able to identify bursts with peculiar features in different experimental conditions (i.e. spontaneous activity and under the effect of specific drugs). The main feature of our algorithm (i.e. Burst On Hurst), based on the auto-similarity or fractal property of the recorded signal, is the independence from the chosen spike detection method since it works directly on the raw data.

Methylisothiazolinone, A Neurotoxic Biocide, Disrupts the Association of Src Family Tyrosine Kinases with Focal Adhesion Kinase in Developing Cortical Neurons

Methylisothiazolinone (MIT) is a biocide widely used in industrial and cosmetic products with potential as a neurotoxicant. We previously reported that short acute exposures to relatively high concentrations of MIT (100 microM) lead to widespread and selective neuronal death in vitro. To evaluate the biological properties of chronic exposures to MIT, freshly dissociated rat cortical neurons were continuously exposed to low concentrations (0.1-3 microM) of the biocide in serum-containing media. Although we observed minimal effects on cell viability, MIT induced a dramatic inhibition of neurite outgrowth. Immunoblotting and immunoprecipitation experiments revealed that focal adhesion kinase (FAK) phosphorylation was primarily affected by the MIT treatment. The phosphorylation level at tyrosines 576 and 861 of FAK was significantly decreased and likely contributed to the overall reduction of tyrosine phosphorylation of this protein. MIT inhibited Src family kinases (SFKs) in cell-free assays and led to the physical dissociation of FAK from the signaling complexes that it normally forms with c-Src and Fyn in developing neurons. High-density neuronal cultures were then employed to increase cell-to-cell contact. This approach resulted in an overall enhancement of SFKs and FAK phosphorylation and could overcome the deficits induced by MIT. This study suggests that a disruption of FAK-SFK complexes due to SFK inhibition leads to FAK dysfunction, with detrimental effects to immature neurons. Prolonged exposure to low levels of MIT and related compounds may have damaging consequences to the developing nervous system.

Histamine H 1 antagonists block M-currents in dissociated rat cortical neurons

We investigated the effects of histamine H 1 antagonists on acutely dissociated neurons from the rat cortex using the patch-clamp technique. First-generation antihistamines, such as pyrilamine, d-chlorpheniramine, diphenhydramine, and ketotifen, suppressed M-currents in a concentration-dependent manner with respective half-inhibition concentrations ( C 50) of 35.9, 48.5, 34.8, and 47.8 μM at a holding potential of −26.5 mV. Astemizole, a second-generation antihistamine, inhibited M-currents with a C 50 of 18.1 μM, but cetirizine did not do so, up to a concentration of 300 μM. Neither ranitidine nor cimetidine, both H 2 antagonists, suppressed M-currents. The C 50 of pyrilamine significantly decreased with membrane hyperpolarization, suggesting that it acts directly on M channel pores. The inhibition of M channels may be involved in the neurotoxic effects of histamine H 1 antagonist overdose.

Mechanism of synchronized Ca 2+ oscillations in cortical neurons

Dissociated rat cortical neurons reassociate in vitro to form synaptically connected networks. Removal of Mg 2+ from the extracellular medium then induces neurons in the network to undergo synchronized oscillations of cytoplasmic calcium. Previous studies have shown that these calcium oscillations involve the activation of NMDA receptors and that the rising phase of each calcium spike is coincident with a brief burst of action potentials (Robinson et al., Jpn. J. Physiol. 43 (Suppl. 1) (1993) S125–130; Robinson et al., J. Neurophysiol. 70 (1993) 1606–1616; Murphy et al., J. Neurosci. 12 (1992) 4834–4845). We have found that these calcium oscillations are dependent on an influx of extracellular calcium but are independent of mobilization of calcium from intracellular stores. The influx of extracellular Ca 2+ occurs primarily through L-type voltage-gated calcium channels (VGCCs), since diltiazem inhibits calcium oscillations under all conditions. On the other hand, N-, P/Q-, and T-type VGCCs are not required for calcium oscillations, although inhibitors of these channels may act as partial antagonists. In addition to removal of Mg 2+, oscillations can also be induced by the inhibition of voltage-gated K + channels with 4-aminopyridine (4-AP), a treatment known to increase neurotransmitter release. In the presence of 4-AP, synchronized calcium oscillations become independent of NMDA receptor activation, although they continue to require activation of AMPA/KA receptors. A model for the mechanism of neuronal calcium oscillations and the reason for their synchrony is presented.

Block of neuronal chloride channels by tetraethylammonium ion derivatives.

The block by the symmetric tetraethylammonium (TEA) ion derivatives tetrapropylammonium (TPrA), tetrabutylammonium (TBA), and tetrapentylammonium (TPeA) ions of fast chloride channels in acutely dissociated rat cortical neurons was studied with the excised inside-out configuration of the patch-clamp technique. When applied to the intracellular membrane surface, all three of the quaternary ammonium compounds (QAs) induced the appearance of short-lived closed states in a manner consistent with a blocking mechanism where the blocker preferentially binds to the open kinetic state and completely blocks ion current through the channel. The drug must leave the channel before the channel can return to a closed state. The mechanism of block was studied using one-dimensional dwell-time analysis. Kinetic models were fit to distributions of open and closed interval durations using the Q-matrix approach. The blocking rate constants for all three of the QAs were similar with values of approximately 12-20 x 10(6) M-1s-1. The unblocking rates were dependent on the size or hydrophobicity of the QA with the smallest derivative, TPrA, inducing a blocked state with a mean lifetime of approximately 90 microseconds, while the most hydrophobic derivative, TPeA, induced a blocked state with a mean lifetime of approximately 1 ms. Thus, it appears as though quaternary ammonium ion block of these chloride channels is nearly identical to the block of many potassium channels by these compounds. This suggests that there must be structural similarities in the conduction pathway between anion and cation permeable channels.