Rat Neocortical Neurons Research Articles

Effects of riluzole on rat cortical neurones: an in vitro electrophysiological study.

1. The electrophysiological effects of riluzole on rat prefrontal and frontal cortical neurones were investigated by using both extracellular (field) and intracellular recording techniques in brain slices. 2. Bath applied riluzole (3-200 microM) depressed the cortico-cortical stimulus-evoked field potential in a concentration-related manner (EC50 = 29.5 microM). 3. Riluzole (3-100 microM) reduced the tonic firing of the neocortical neurones which was caused by intracellular current injection, while it did not have any effect on the resting membrane potential and apparent input resistance of these cells. 4. In the presence of tetrodotoxin (1 microM) and tetraethylammonium (30 mM), the injection of a depolarizing current step generated a calcium spike in the neocortical neurones. Riluzole (30 microM) abolished this calcium-dependent action potential. However, when the amount of the depolarizing current was increased the calcium-dependent regenerative potential was evoked again. 5. The depolarization of the membrane (10-20 mV) caused by brief (8-15 s) bath applications of glutamate (300 microM-1 mM) were not changed in the presence of riluzole (30 microM). 6. It is concluded that riluzole has direct actions on rat neocortical neurones: (a) it blocks the repetitive discharge of sodium action potentials and (b) it increases the threshold for the generation of the calcium spike. These two cellular mechanisms might at least in part account for the depression of the cortico-cortical field potential caused by this drug.

122 Different distribution of L- and N-type Ca 2+ channel in rat neocortical neurons

Melanin-concentrating hormone (MCH) and neuropeptide glutamic acid-isoleucine (NEI) are expressed in neurons that are located mainly in the hypothalamus and project widely throughout the rat central nervous system. One of the main targets of melanin-concentrating hormone is the hippocampal formation, although the exact origin of the projections is unknown. By using injections of the retrograde tracer True Blue into the hippocampus, together with immunohistochemical analysis, we observed retrogradely labeled melanin-concentrating hormone-containing neurons in the lateral hypothalamic area, incerto-hypothalamic area, perifornical area, the periventricular nucleus of the hypothalamus, and in the internuclear area (between the dorsomedial and ventromedial nuclei of the hypothalamus), as well as a few retrogradely labeled and melanin-concentrating hormone-immunoreactive cells in the supramammillary nucleus. The afferents from the lateral hypothalamic area were confirmed using injection of the anterograde tracer biotinylated dextran amine, which enabled us to use histochemical analysis in order to visualize fibers and terminals in the hippocampal formation. In the medial septal nucleus, we found cholinergic neurons that are also putatively innervated by melanin-concentrating hormone immunoreactive fibers and project to the hippocampal formation. Finally, using two different protocols for immunoperoxidase, we were able to show GABAergic basket cells presumably innervated by melanin-concentrating hormone-immunoreactive fibers in the hippocampal formation. On the basis of the data collected herein, we hypothesize that the MCH/NEI projections from hypothalamic nuclei participate in spatial memory and learning through direct and indirect pathways. These pathways would enable the animal to organize its exploratory behavior during foraging.

Secreted form of -amyloid precursor protein activates protein kinase C in cultured rat neocortical neurons

Secreted form of -amyloid precursor protein activates protein kinase C in cultured rat neocortical neurons

816 Secreted form of β-amyloid precursor protein activates protein kinase C in cultured rat neocortical neurons

Hybrid biomedical structures have been used widely in various tissue-regenerating materials because they effectively induce exceptional physical and cellular responses. In this study, a new hybrid process was used to design a three-dimensional (3D) biomedical hybrid scaffold with a controlled pore-structure and high mechanical strength. A melt-dispensing method was used to obtain mechanical properties and electrohydrodynamic direct-jet (EHD-DJ) printing was used to provide microsized fibrous structures for the scaffold. Furthermore, the poly(ε-caprolactone) (PCL) hybrid scaffolds were coated biomimetically with type-I collagen to increase bioactive interactions between cells and scaffolds. The fabricated scaffolds showed similar mechanical properties to the two control scaffolds; however, the results of culturing osteoblast-like (MG63) cells showed significant increases in in vitro cellular activities (cell viability > twofold and calcium deposition > sevenfold). Based on these results, we propose a newly designed hybrid scaffold that can support significant in vitro cellular activities at the interface between cells and the 3D micro-pore structure for soft and hard tissue regeneration.

Two types of BK channels in immature rat neocortical pyramidal neurons.

1. The properties of large conductance Ca(2+)-activated K+ channels (BK channels) were investigaed in neocortical infragranular pyramidal neurons by the use of inside-out patch recordings. Neurons were acutely isolated from slices of newborn to 28-day-old rats (P0-P28) by using minimal protease exposure followed by trituration with a vibrating glass probe. Two types of BK channels, slow-gating and fast-gating, were observed in immature neurons (P0-P5), whereas only slow-gating BK were found in more mature neurons. Fast-gating BK channels differed in conductance, voltage dependence, and kinetics from the slow-gating ones. 2. The properties of fast-gating channels included a conductance of 145 +/- 12.9 (SE) pS; frequent openings with short mean open times that were relatively voltage-independent, mean closed times that showed a voltage-dependent increase, a voltage-dependent decrease in open probability (Po). The properties of slow-gating channels contrasted with those of the fast-gating ones, in that the former had a conductance of 181 +/- 3.9 pS, longer mean open times that showed a voltage-dependent increase, mean closed times that showed a marked voltage-dependent decrease, a voltage-dependent increase in Po, and slight inward rectification. The significance of these developmental variations in channel properties is discussed.

Cultured astrocytes express biglycan, a chondroitin/dermatan sulfate proteoglycan supporting the survival of neocortical neurons

Astrocyte-conditioned medium (ACM) supports the survival of rat E15 neocortical neurons. Using a microtiter assay for neuronal survival, we demonstrated that part of the survival activity is associated with a proteoglycan fraction obtained after two chromatographic steps: (1) preparative Q-Sepharose anion-exchange chromatography under non-denaturating conditions and (2) MonoQ chromatography in the presence of 8 M urea. Analytical SDS-polyacrylamide gradient gel electrophoresis of pooled active MonoQ-fractions (MQ-pool) revealed a broad proteoglycan band migrating with an apparent M r in the range of 150–400 kDa. Digestion of the MQ-pool with chondroitin-ABC-lyase yielded a major core protein of 50 kDa. In Western blots the high molecular weight (150–400 kDa) material as well as the 50 kDa core protein band were immunoreactive to chicken polyclonal antibodies raised against purified biglycan from rat meningeal fibroblasts. Northern blot analysis of total RNA prepared from highly enriched astrocyte cultures revealed a single 2.9 kb biglycan transcript. By using in situ hybridization we demonstrated that essentially all cells in these cultures expressed biglycan mRNA. Furthermore, highly purified biglycan from bovine cartilage was shown to markedly enhance survival of rat neocortical neurons. In conclusion, we have shown that astrocytes synthesize and release the small chondroitin/dermatan sulfate proteoglycan (CS/DSPG) biglycan, a molecule that was found to support survival of neocortical neurons in vitro.

Molecular Cloning and Characterization of Annexin V‐Binding Proteins with Highly Hydrophilic Peptide Structure

We previously reported that annexin V promoted the survival of cultured rat neocortical neurons. In an effort to elucidate the mechanism underlying this neurotrophic activity of annexin V, we have attempted to identify the target or binding proteins of annexin V in neuronal cells. Herein, we screened an embryonic day 17 rat brain cDNA library by western blot using glutathione S-transferase-annexin V fusion protein as a probe and then isolated four clones showing binding to annexin V in a Ca2+ - and phospholipid-dependent manner. Although these cDNAs encoded different polypeptides, all four polypeptides shared the unique feature of containing highly hydrophilic stretches with high Lys, Glu, and Ser contents. Deduced amino acid sequences of two clones showed high homology with human X-linked Helicase2 (XH2) and DNA (cytosine-5) methyltransferase (DMTase) sequences, whereas the other two were not related to any known peptide sequence. These results suggest that XH2 and DMTase are candidates for annexin V-binding proteins and thus may mediate the biological activity of annexin V.

Development of low magnesium-induced spontaneous synchronized bursting and GABAergic modulation in cultured rat neocortical neurons

Development of spontaneous synchronized bursting in the early stages of rat neocortical neuronal cultures was studied by whole-cell and extracellular recordings. Neocortical neurons from rat embryos were cultured on planar electrode arrays, and low Mg2+-induced spontaneous activity was recorded from 5 to 16 days in vitro (DIV). At 5–6 DIV the current synchronized to the bursting had only a slow component lasting 3–5 s, whereas in older cultures a fast transient component was dominant. A γ-aminobutyric acid (GABA)A receptor antagonist, bicuculline methiodide, had little effect on the spontaneous activity at 5–6 DIV, whereas in older cultures it had a marked effect on the slow current component. These results suggest a role of GABAergic transmission in the development of synchronized activities.

1132 Expression of immunoglobulin superfamily genes in rat neocortical neurons as revealed by in situ hybridization and retrograde double labeling

1132 Expression of immunoglobulin superfamily genes in rat neocortical neurons as revealed by in situ hybridization and retrograde double labeling

L-AP4 inhibits high voltage-activated Ca2+ currents in pyramidal cortical neurones.

We tested the ability of L-AP4 to modulate high voltage-activated (HVA) calcium (Ca2+) currents in pyramidal neurones acutely isolated from the adult rat (4-8 weeks). Whole cell recordings, with barium (Ba2+) ions as the charge carrier, were performed. L-AP4 reduced HVA Ca2+ conductances in 86% of the recorded cells. Saturating concentrations of L-AP4 inhibited about 21% of the current (+/- 8.3%, n = 8), although great variability was observed. Interestingly, low micromolar concentrations of (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD) and (2S,3S,4R)-alpha-(carboxy-cyclopropyl)-glycine (1-CCGI) had weaker effects than L-AP4. MAP4 fully antagonized the L-AP4-mediated reduction of HVA Ca2+ currents. These findings suggest the involvement of the AP4-sensitive receptor in the control of both cellular excitability and transmitter release in rat neocortical neurones.

Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurones.

1. Developmental changes in electrophysiological properties of pyramidal neurones correlated with the developmental decline in gap junction-dependent dye coupling were investigated in coronal slices of rat prefrontal and sensorimotor cortex. Effects of intracellular acidification induced by application of weak organic acids on neuronal dye coupling, electrotonic parameters as well as synaptic potentials were examined using the patch clamp technique. Optical monitoring of intracellular pH revealed an acidic shift of 0.4-0.5 pH units following sodium propionate application. 2. Dye coupling between layer II-III neurones was prominent during the first two postnatal weeks. During this period, pre-incubation of slices with 30 mM of the sodium salts of weak organic acids reduced the number of cells coupled to the injected neurones by 64%. 3. Between postnatal days 1 and 18, the mean neuronal input resistance decreased significantly (by 81.0%). Both the membrane time constant (tau 0) and the first equalizing time constant (tau 1) also showed a significant developmental decline of 25.8 and 65.8%, respectively. Electrotonic length decreased by 34.9%. The electrophysiological properties of neurones displayed a pronounced intercellular variability which decreased with on-going development. 4. During the first two postnatal weeks, intracellular acidification led to a mean increase in neuronal input resistance of 55.9% and a mean decreae in electrotonic length of 22.2%. The membrane time constant was reduced by approximately 25% in the majority of neurones tested. Significant electrophysiological effects induced by intracellular acidification were not detected in uncoupled neurones from 18-day-old rats. 5. EPSP width at half-maximal amplitude showed a substantial reduction of approximately 50%, while rise times of the non-NMDA receptor-mediated EPSP components displayed no significant change during development. Both weak organic acids, as well as the gap junction blocker 1-octanol, reduced excitatory synaptic transmission independent of developmental age. 6. We conclude that gap junction permeability is regulated by intracellular pH in developing layer II-III pyramidal cells in the rat neocortex. The prominent correlation between pH-induced reduction in dye coupling and changes in electrophysiological cell properties suggests a significant influence of gap junctions on synaptic integration and information transfer in the immature neocortex.

Purification of a meningeal cell-derived chondroitin sulphate proteoglycan with neurotrophic activity for brain neurons and its identification as biglycan.

Serum-free cultures of meningeal fibroblasts synthesize and release a chondroitin sulphate proteoglycan (CSPG) that markedly enhances survival but not adhesion of embryonic rat (embryonic day 15) neocortical neurons in vitro. The active molecule was purified from conditioned medium (meningeal cell-conditioned medium, MCM) in three steps by means of fast-performance liquid chromatography fractionation combined with a quantitative microphotometric bioassay: (i) preparative Q-Sepharose anion exchange chromatography under native conditions; (ii) rechromatography of biologically active Q-Sepharose fractions on a MonoQ column in the presence of 8 M urea; and (iii) final gel filtration of active MonoQ fractions on Superose 6 in the presence of 4 M guanidinium hydrochloride. Analytical sodium dodecyl sulphate-polyacrylamide gradient gel electrophoresis of active Superose 6 fractions revealed a single broad glycoprotein band with a molecular mass in the range of 220-340 kDa. Further characterization of the purified molecule with glycosaminoglycan:lyases revealed a core protein of 50 kDa and the nearly complete loss of neurotrophic activity after chondroitinase digestion, whereas heparitinase treatment changed neither electrophoretic mobility nor biological activity. Amino-terminal sequencing of the purified CSPG core protein revealed identity with the amino acid sequence of rat biglycan. Biglycan purified from bovine cartilage supported neuron survival with virtually the same activity as the CSPG purified from MCM (half-maximal activity approximate to 10(-8) M). In conclusion, we isolated a neurotrophic CSPG from meningeal cells with strong survival-enhancing activity for brain neurons that was identified as biglycan, a molecule not previously related to neural functions.

Dye coupling between pyramidal neurons in developing rat prefrontal and frontal cortex is reduced by protein kinase A activation and dopamine

During early postnatal development, lamina II/III pyramidal cells in rat neocortex are extensively coupled via gap junctions. The factors regulating gap junction permeability, as well as the mechanisms underlying the developmental uncoupling process are not understood. To investigate the influence of protein kinase A-mediated phosphorylation on dye coupling in the developing neocortex, pyramidal cells in slices of rat frontal and prefrontal cortex were injected intracellularly with the tracer neurobiotin. Control injections revealed clusters of about 30 dye-coupled neurons. Preincubation with forskolin or direct activation of protein kinase A with Sp-cAMPS reduced the number of coupled cells by about 70%. A significant reduction in dye coupling was also observed following incubation with dopamine. Application of receptor selective agonists and antagonists revealed that the uncoupling was mediated by both dopamine D1 and D2 receptors. The protein kinase A inhibitor Rp-cAMPS reduced the effect of dopamine, suggesting that the neurotransmitter regulates gap junction permeability via protein kinase A activation. In the presence of either forskolin, Sp-cAMPS, or dopamine, neurons displayed a significantly higher input resistance compared to control conditions. During the second postnatal week, transient application of forskolin to single neurons reversibly increased input resistance. At later developmental stages when coupling incidence had declined, this action of forskolin was no longer observed. Our data demonstrate a dependence of gap junction permeability on protein kinase A activity and on dopamine receptor activation in developing rat neocortical neurons. These mechanisms may modulate junctional permeability during the period of circuit formation.

Spatial profile of dendritic calcium transients evoked by action potentials in rat neocortical pyramidal neurones.

1. Simultaneous measurements of intracellular free calcium concentration ([Ca2+]i) and intrasomatic and intradendritic membrane potential (Vm) were performed using fura-2 fluorimetry and whole-cell recording in neocortical layer V pyramidal neurones in rat brain slices. 2. Back-propagating action potentials (APs) evoked [Ca2+]i transients in the entire neurone including the soma, the axon initial segment, the apical dendrite up to the distal tuft branches, and the oblique and basal dendrites, indicating that following suprathreshold activation the entire dendritic tree is depolarized sufficiently to open voltage-dependent calcium channels (VDCCs). 3. The [Ca2+]i transient peak evoked by APs showed large differences between various compartments of the neurone. Following a single AP, up to 6-fold differences were measured, ranging from 43 +/- 14 nM in the soma to 267 +/- 109 nM in the basal dendrites. 4. Along the main apical dendrite, the [Ca2+]i transients evoked by single APs or trains of APs had the largest amplitude and the fastest decay in the proximal region; the [Ca2+]i transient peak and decay time constant following a single AP were 128 +/- 25 nM and 420 +/- 150 ms, respectively, and following a train of five APs (at 10-12 Hz), 710 +/- 214 nM and 390 +/- 150 ms, respectively. The [Ca2+]i transients gradually decreased in amplitude and broadened in more distal portions of the apical dendrite up to the main bifurcation. 5. In the apical tuft branches, the profile of the [Ca2+]i transients was dependent on AP frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenoreceptor activation reduces dye-coupling between immature rat neocortical neurones.

The present study examined the effect of activation of adrenergic receptors by noradrenaline and the beta-receptor selective agonist isoproterenol on dye-coupling between developing lamina II/III pyramidal neurones in rat prefrontal and sensorimotor cortex. To assess dye-coupling neurones were intracellularly injected with neurobiotin. Under control conditions injections performed in slices obtained from neonatal rats (7-10 postnatal days) resulted in clusters of more than 30 tracer-coupled neurones. Preincubation with either isoproterenol or noradrenaline reduced the number of coupled cells by 60-80%. The effect of isoproterenol was suppressed by the beta 1-adrenoreceptor antagonist atenolol. Our results indicate that modulation of gap junction coupling between differentiating neocortical neurones might be one important function of noradrenergic afferents during early postnatal development of the neocortex.

An adenosine triphosphate (ATP)-sensitive K + channel of rat neocortical neurons is bi-gated by intracellular ATP and voltage: a novel channel gating mechanism?

To determine whether the adenosine triphosphate (ATP)-sensitive K + (K-ATP) channels, which are suggested to be mainly regulated by intracellular ATP or other kinds of triphosphate nucleotides, are gated by membrane potentials ( Vm), single K-ATP channel currents were studied on inside-out membrane patches of neurons acutely dissociated from Sprague-Dawley rat neocortex. The K-ATP channels recorded have a unitary conductance from 96.97 ± 5.32 pS ( n = 11) at potentials of ∼ 10–60 mV to 98.31 ± 3.26 pS ( n = 11) at ∼ −10 to −60 mV. Besides being inhibited by cytoplasmic ATP, channel kinetics was also affected by Vm. Open- and closed-time histograms were well fitted by 2 exponentials, suggesting that the channels have 2 open and closed states. Mean open time ( τ om), open probability increased while mean closed time ( τ cm) decreased with depolarization. The fitted equations of the relationships between Vm and those kinetic parameters may be described as: τ om = -159.26ln Vm + 403.64, P o = −0.01 Vm 2 + 0.08 Vm + 0.87 and τ cm = 0.17 Vm 3 −2.5 Vm 2 + 10.35 Vm − 7.68, respectively. We suggest that the K-ATP channels be bi-gated by both intracellular ATP and membrane potentials. This property of the neuronal K-ATP channels may be related to their pathophysiological functions.

Dendritic calcium transients evoked by single back-propagating action potentials in rat neocortical pyramidal neurons.

1. Dendrites of rat neocortical layer V pyramidal neurons were loaded with the Ca2+ indicator dye Calcium Green-1 (CG-1) or fluo-3, and the mechanisms which govern action potential (AP)-evoked transient changes in dendritic cytosolic Ca2+ concentration ([Ca2+]i) were examined. APs were initiated either by synaptic stimulation or by depolarizing the soma or dendrite by current injection, and changes in fluorescence of the indicator dye were measured in the proximal 170 microns of the apical dendrite. 2. Simultaneous two-pipette recordings of APs from the soma and apical dendrite, and dendritic fluorescence imaging indicated that a single AP propagating from the soma into the apical dendrite evokes a rapid transient increase in fluorescence indicating a transient increase in [Ca2+]i. At 35-37 degrees C the decay time constant of the fluorescence transient following an AP was around 80 ms. 3. Voltage-activated Ca2+ channels (VACCs) of several subtypes mediated the AP-evoked fluorescence transient in the proximal (100-170 microns) apical dendrite. The AP-evoked fluorescence transient resulted from Ca2+ entry through L-type (nifedipine sensitive; 25%), N-type (omega-conotoxin GVIA sensitive; 28%) and P-type (omega-agatoxin IVA sensitive; 10%) Ca2+ channels and through Ca2+ channels (R-type) not sensitive to L-, N- and P-type Ca2+ channel blockers (cadmium ion sensitive; 37%). 4. The decay time course of the dendritic fluorescence transient was prolonged by the blockers of endoplasmic reticulum (ER) Ca(2+)-ATPase, cyclopiazonic acid and thapsigargin, suggesting that uptake of Ca2+ into the ER in dendrites governs clearance of dendritic Ca2+. 5. The decay time course of the fluorescence transient was slightly prolonged by benzamil, a blocker of plasma membrane Na(+)-Ca2+ exchange and by calmidazolium, a blocker of the calmodulin-dependent plasma membrane Ca(2+)-ATPase, suggesting that these pathways are less important for dendrite Ca2+ clearance following a single AP. Neither the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) nor the blocker of Ca2+ uptake into mitochondria, Ruthenium Red, had any measurable effect on the decay time course of the fluorescence transient. 6. Dendritic fluorescence transients measured during trains of dendritic APs began to summate at impulse frequencies of 5 APs s-1. At higher frequencies APs caused a concerted and maintained elevation of dendritic fluorescence during the train.(ABSTRACT TRUNCATED AT 400 WORDS)

A potassium current controls burst termination in rat neocortical neurons after GABA withdrawal

Bursting activities were investigated under conditions of reduced outward K + currents in neocortical slices obtained from rats presenting the γ-aminobutyric acid (GABA)-withdrawal syndrome (GWS), a focal epilepsy consecutive to the interruption of a chronic intracortical GABA infusion into the somatomotor cortex. These bursts were induced by intracellular depolarizing current injection and/or by white matter stimulation. Tetraethylammonium (TEA) at doses which did not change input resistance, spike duration or first interspike time interval abolished the burst terminating process and induced plateau-like potentials (up to 500 ms) which were tetrodotoxin-resistant and blocked by Ca 2+ antagonists Cd 2+ and Co 2+. Therefore, it appears that bursts during GWS are generated by Ca 2+-dependent plateau potentials which are terminated by a K + current highly sensitive to TEA.

Characterization of pharmacologically identified voltage-gated calcium channel currents in acutely isolated rat neocortical neurons. II. Postnatal development

1. Whole cell recordings were obtained from pyramidal neurons acutely dissociated from the sensorimotor cortex of adult (from Lorenzon and Foehring, companion paper) and immature rats postnatal day 1 (P1) to adult. 2. Whole cell calcium channel currents were similar in appearance at all ages. Current amplitudes and estimated densities were initially low (approximately 16 pA/pF at ages < P6) and increased gradually, attaining adult values at approximately 4-5 wk postnatally (approximately 100 pA/pF). 3. L-type current was operationally defined as that blocked by 5 microM nifedipine, N-type current as that blocked by 1 microM omega-conotoxin GVIA, and P-type current as that blocked by 100 nM omega-agatoxin IVA. A resistant current remained in the presence of the combination of these three blockers. The proportions of these four current types did not change during ontogeny. 4. Few biophysical differences were found between the pharmacologically defined current components in adult or 1-wk-old cells. At both ages the resistant current had a more rapid time-to-peak and inactivated more completely and rapidly than the other three types. Resistant currents also activated at more negative potentials. N-, L-, and P-type currents activated at more positive potentials in 1-wk-old cells than in adult cells. For the resistant current, the voltage dependence of activation was not significantly different between the two ages.

Characterization of pharmacologically identified voltage-gated calcium channel currents in acutely isolated rat neocortical neurons. I. Adult neurons

1. Whole cell recordings were obtained from pyramidal neurons acutely dissociated from the sensorimotor cortex of adult rats. 2. Whole cell calcium channel currents were similar in appearance when elicited from holding potentials of -90 or -40 mV. With 5 mM Ba2+ as the charge carrier, currents began to activate at approximately -45 mV, peaked at approximately -10 mV, and had an apparent reversal potential of approximately +45 mV. Current amplitude and voltage dependence varied with the concentration and identity of the charge carrier (Ca2+ vs. Ba2+). Calcium channel currents were blocked completely by > 200 microM Cd2+ (IC50 approximately 3.5 microM). 3. We determined saturating doses for blockade of currents by nifedipine (Nif), omega-conotoxin GVIA (CgTx), and omega-agatoxin IVA (AgTx) in adult cells. We also tested the selectivity of these compounds by applying them in combination and in different orders. We found the three compounds to be highly, but not perfectly, specific. 4. L-type current was operationally defined as that blocked by 5 microM Nif, N-type current as that blocked by 1 microM CgTx, and P-type current as that blocked by 100 nM AgTx. In adult cells, each of these compounds blocked 30-35% of the current. When all three blockers were applied concurrently, approximately 80% of the current was blocked (20% of current was resistant to the 3 blockers). 5. Few biophysical differences were found between the pharmacologically defined current components in adult cells. The resistant current had a more rapid time-to-peak, inactivated more rapidly and completely, and activated at more negative potentials than the other three types.