An intracellular study of myenteric neurons in the mouse colon

Intracellular recordings have been made in vitro from the myenteric neurons of the distal colon of normal littermates of the piebald-lethal mouse. Out of a total of 90 neurons, 82 were classified as S/type 1 cells and 8 as AH/type 2 cells. Seventy-eight out of 82 S cells showed spontaneous fast excitatory postsynaptic potentials (EPSPs) sensitive to d-tubocurarine (dTC, 280 microM), and 22 S cells showed spontaneous action potentials (APs). Six S cells and 1 AH cell showed spontaneous nonnicotinic slow depolarizations associated with an increase in the input resistance of the cells; during the spontaneous slow depolarization in the S cells there was an increase in the frequency of nicotinic fast EPSPs and APs. Three S cells showed spontaneously occurring regular oscillations of the membrane potential (approximately mV in amplitude and approximately 4/min). Transmural nerve stimulation produced fast EPSPs with a wide range of latencies (3 ms to 20 s) in S cells; the fast EPSPs were blocked by dTC (280 microM) or solutions containing low Ca2+ (0.25 mM) and high Mg2+ (12 mM) but not by atropine (ATR, 14 microM). Single or repetitive transmural stimulation produced slow EPSPs in 24 S cells and 3 AH cells; these were not blocked by dTC (280 microM) nor ATR (14 microM). During the slow EPSPs there was an increase in the input resistance of the cells. In those S cells that showed slow EPSPs there were many long-latency fast EPSPs; long-latency fast EPSPs were also observed in 11 other S cells that did not show a slow EPSP following repetitive transmural nerve stimulation. Long-latency fast EPSPs may be related to the firing of other neurons during their slow EPSPs. The myenteric neurons in the mouse colon have similar properties to the myenteric neurons in the guinea pig small intestine. However, the colonic myenteric neurons show more ongoing synaptic activity and more prolonged activity after nerve stimulation than myenteric neurons in the guinea pig small intestine. This activity may be due to regional differences, species differences, or preparation differences (in this study the myenteric plexus was adherent to the underlying circular muscle layer).     

Excitatory synaptic transmission in cultures of rat olfactory bulb

1. Olfactory bulb neurons were dissociated from neonatal rats and plated at low density on a confluent layer of olfactory bulb astrocytes. Intracellular stimulation of presumptive mitral/tufted (M/T) cells evoked monosynaptic excitatory postsynaptic potentials (EPSPs) in adjacent neurons. Whole-cell recording techniques and a flow-pipe drug delivery system were used to compare EPSPs with voltage-clamp recordings of currents evoked by excitatory amino acids (EAA) including N-acetylaspartylglutamate (NAAG), a putative mitral cell transmitter. 2. Cultured olfactory bulb neurons were morphologically and physiologically distinct. Large pyramidal-shaped neurons were present, which were NAAG immunoreactive; stimulation of these neurons invariably evoked EPSPs, suggesting that they were M/T cells. The majority of small bipolar neurons were glutamic acid decarboxylase (GAD) immunoreactive consistent with granule or periglomerular gamma-aminobutyric acid (GABA)ergic interneurons. 3. Monosynaptic EPSPs between M/T cells could be separated into fast and slow components by the use of EAA receptor antagonists. A fast component with a time-to-peak of 7.7 +/- 1.0 (SE) ms and half-width of 31.8 +/- 7.4 ms was blocked by the non-NMDA receptor antagonist 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX, 2.5 microM). The slow component (time-to-peak = 41.4 +/- 7.2 ms; half-width = 218.9 +/- 40.4 ms) was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5, 100 microM). 4. Under voltage clamp, flow-pipe applications of NAAG (10-1,000 microM) evoked inward currents at a holding potential of -60 mV in Mg-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses of cortical neurons to stimulation of corpus callosum in vitro

1. An in vitro slice preparation of rat cingulate cortex was used to analyze the responses of layer V neurons to electrical stimulation of the corpus callosum (CC). In addition, synaptic termination of callosal afferents with layer V neurons was evaluated electron microscopically to provide a structural basis for interpreting some of the observed response sequences. 2. Layer V neurons had a resting membrane potential (RMP) of 60 +/- 0.68 (SE) mV, an input resistance of 47 +/- 4.74 M omega, a membrane time constant of 4.37 +/- 0.51 ms, an electrotonic length constant of 1.38 +/- 0.25, and produced spontaneous action potentials that were 50 +/- 0.3 mV in amplitude. Intracellular depolarizing current pulses evoked spikes that were sometimes associated with low-amplitude (2-5 mV) depolarizing (5-10 ms in duration) and hyperpolarizing (10-20 ms in duration) afterpotentials. 3. A single stimulus of increasing intensities to the CC produced one of the following response sequences: a) antidromic spike and an excitatory postsynaptic potential (EPSP), which initiated one or more spikes; b) antidromic spike, EPSP-evoked action potentials, and a hyperpolarization, which may have represented an intrinsic cell property or inhibitory synaptic activity; c) EPSP and evoked spikes only; d) high-amplitude EPSP with an all-or-none burst of action potentials. 4. Antidromically activated (AA) neurons always produced EPSPs in response to CC stimulation. When compared with nonantidromically activated neurons, AA cells had a more negative RMP, greater electrotonic length constant (LN), higher ratio of dendritic to somatic conductance (rho), and formed shorter duration, callosal-evoked EPSPs. 5. Neurons in anterior cingulate cortex produced EPSPs of longer duration than did those in posterior cortex (50 +/- 3.57 versus 26 +/- 1.56 ms, respectively). EPSPs in anterior neurons also had a higher maximum amplitude (20.5 +/- 1.0 versus 11.5 +/- 0.79 mV) and longer time to peak (11.6 +/- 2.2 versus 8.2 +/- 0.8 ms). 6. Electron microscopy of Golgi-impregnated neurons following contralateral lesions demonstrated that both pyramidal and nonpyramidal neurons received direct callosal afferents. Synaptic termination of callosal axons with the apical dendritic trees of anterior pyramidal cells was 6 times greater than it was with posterior pyramidal neurons. 7. EPSP shape differences in anterior and posterior neurons may be partially accounted for by the density and distribution of callosal afferents to these two cortices.     

GABAA-Dependent chloride influx modulates GABAB-mediated IPSPs in hippocampal pyramidal cells.

The relationship between postsynaptic inhibitory responses [the fast GABA(A)-mediated inhibitory postsynaptic potential (IPSP) and the slow GABA(B)-mediated IPSP] were investigated in hippocampal CA3 pyramidal cells. Mossy fiber-evoked GABA(B)-mediated IPSPs were, paradoxically, of greater amplitude in cells with resting membrane potential of -62 mV (13.6 +/- 0.5 mV; mean +/- SE) as compared with cells with resting membrane potential of -54 mV (7.0 +/- 0.8 mV). In addition, when a cell's membrane potential was artificially manipulated, GABA(B)-mediated IPSPs were reduced at relatively depolarized levels (-55 mV) and enhanced at relatively hyperpolarized potentials (at least -60 mV). In contrast, the preceding GABA(A)-mediated IPSPs were larger at the more positive membrane potentials and smaller as the cell was hyperpolarized. Similar voltage dependency was obtained when monosynaptic GABA(A)- and GABA(B)-mediated IPSPs were isolated in the presence of glutamatergic receptor antagonists. However, monosynaptic GABA(B)-mediated IPSPs isolated in the presence of glutamatergic and GABA(A) receptor antagonists were not reduced at the more positive membrane potentials, and were significantly larger in amplitude than GABA(B)-mediated IPSPs preceded by a monosynaptic GABA(A)-mediated IPSP. The amplitude of the isolated monosynaptic GABA(B)-mediated IPSPs recorded with potassium chloride-containing microelectrodes was significantly smaller than the comparable potential recorded with potassium acetate microelectrodes without chloride. We conclude that voltage-dependent chloride influx, via GABA(A) receptor-gated channels, modulates postsynaptic GABA(B)-mediated inhibition in hippocampal CA3 pyramidal cells.     

Voltage dependence of excitatory postsynaptic potentials of rat neocortical neurons

1. The properties of excitatory postsynaptic potentials (EPSPs) of rat neocortical neurons were investigated with a fast single-electrode current-voltage clamp in vitro. Typically, apparently pure EPSPs were obtained by selection of electric stimuli of low intensity. 2. The amplitude and time integral of the EPSP increased when the neuron was depolarized. At threshold for generation of action potentials, the amplitude of EPSPs was increased by approximately 30% [from 5.0 +/- 2.1 to 6.3 +/- 1.0 (SD) mV, n = 12]. The integral of EPSPs was maximally about fourfold (3.7 +/- 1.5, n = 16) larger than at resting membrane potential (Em). The mechanisms involved in this augmentation of EPSPs were further investigated. 3. The amplitude and the time integral of excitatory postsynaptic currents (EPSCs) decreased linearly with shifts in command potential from -100 to -60 mV. The decrease of the EPSC integral with depolarization indicates that the enhancement of the EPSP may be brought about by recruitment of a voltage-dependent inward current. 4. Evoking EPSPs at various delays after the onset of small depolarizing current pulses (0.3-0.6 nA, 600 ms) revealed that augmentation decays with time. The integral of EPSPs evoked approximately 80 ms after the onset of the current pulse was 3.7 (+/- 1.5, n = 16) times larger than at Em. The integral of EPSPs evoked at 480 ms. however, were only twofold (+/- 0.7, n = 16) larger. Hence EPSPs evoked after a delay of 80 ms were 1.7-fold (+/- 0.4, n = 24) larger than EPSPs evoked after 480 ms. EPSCs were independent of the delay of stimulation at all potentials. 5. Intracellular application of the lidocaine derivative N-(2,6-dimethyl-phenylcarbamoylmethyl) triethylammonium bromide (QX 314) at 100 mM from pipettes rapidly abolished fast action potentials and inward rectification. During comparable depolarizations the increase in EPSP integrals was much smaller in QX 314-treated neurons than in controls. On average, the integral of EPSPs evoked at 70-90 ms was 1.7 times (+/- 1.0) larger than at Em, and the integral of EPSPs evoked with larger delays was close to the value obtained at resting Em (0.9 +/- 0.3, n = 8). The ratio of EPSP integrals early versus late (1.8 +/- 0.5) is comparable to controls, suggesting that QX 314-sensitive currents are unlikely to be involved in the time-dependent enhancement. 6. Mimicking EPSPs by brief depolarizations atop long depolarizations revealed a time- and voltage-dependent enhancement comparable to that of EPSPs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monosynaptic excitatory amino acid transmission from the posterior rhombencephalic reticular nucleus to spinal neurons involved in the control of locomotion in lamprey

1. The reticulospinal neurons in the lamprey posterior rhombencephalic reticular nucleus (PRRN) and their projections to different types of spinal neurons have been investigated by the use of simultaneous paired intracellular recordings from one pre- and one postsynaptic cell. PRRN is of particular importance for the initiation of locomotion. 2. Intracellular stimulation of single PRRN neurons produced monosynaptic excitatory postsynaptic potentials (EPSPs) in simultaneously recorded motoneurons and spinal premotor interneurons of both the excitatory and inhibitory type. Individual PRRN neurons produced EPSPs in several different types of target cells, as revealed by signal averaging. Each single PRRN neuron had extensive monosynaptic connections to approximately 73% of the motoneuronal population. Conversely, several PRRN neurons converge on individual spinal neurons. The average amplitude of the EPSPs was 0.43 +/- 0.40 (SD) mV. The EPSPs varied in time course (time to peak = 7.5 +/- 2.8 ms; duration at one-half peak amplitude = 21.9 +/- 18.1 ms). 3. The EPSPs produced by reticulospinal cells were composed of either exclusively chemical, exclusively electrical, or mixed chemical and electrical components. The electrical EPSPs remained when the ordinary physiological solution was substituted for one without Ca2+ but with Mn2+. The chemical component of the EPSPs was always depressed when a broad-spectrum excitatory amino acid (EAA) antagonist, such as kynurenic acid, was applied, suggesting that the chemical component was because of EAA transmission. The chemical EPSP could have two components, one late, suppressed by N-methyl-D-aspartate (NMDA) antagonists, and one early because of activation of kainate/quisqualate receptors. 4. Three-dimensional reconstructions of Lucifer yellow-filled PRRN neurons were performed with a confocal laser scanning microscope. PRRN neurons producing monosynaptic excitatory amino acid EPSPs were found to have a fusiform cell body located near the surface of the fourth ventricle and an extensive fanlike dendritic tree extending to the ventral and lateral margin of the brain stem within the basal plate. The axons descend in the lateral funiculi of the spinal cord. 5. PRRN neurons utilizing EAA transmission are active during fictive locomotion. They presumably initiate and reinforce ongoing spinal locomotor activity by monosynaptically increasing the general excitability of the spinal premotor interneurons of the spinal locomotor networks by means of their extensive divergent and convergent monosynaptic connections.     

Synaptic and intrinsic control of membrane excitability of neostriatal neurons. II. An in vitro analysis

1. The effects of intrinsic membrane properties on the spontaneous and synaptically evoked activity of neostriatal neurons were studied in an in vitro slice preparation with the use of intracellular recordings. The recorded neurons did not show spontaneous action potentials at rest; depolarizing current pulses triggered a tonic firing pattern. 2. Subthreshold spontaneous depolarizing potentials (SDPs) were observed in 52% of the recorded neurons. The amplitude of these potentials at rest ranged between 2 and 15 mV, and their duration between 4 and 100 ms. The frequency and the amplitude of the SDPs were functions of the membrane potential: membrane depolarization by constant positive current increased the frequency of the SDPs and reduced their amplitude; hyperpolarization of the membrane decreased their frequency and increased their amplitude. Often, at membrane potentials more negative than -90 mV, SDPs were completely suppressed. 3. SDPs were blocked by low calcium-cobalt containing solutions. In the presence of tetrodotoxin (TTX, 1-3 microM), SDPs were completely abolished in 50% of the tested neurons; in the remaining neurons, small (1-4 mV) TTX-resistant SDPs were observed. In most of the neurons, bicuculline (BIC, 10-100 microM) and low concentrations of tetanus toxin (5-10 micrograms/ml) did not clearly affect the SDPs. Higher concentrations of tetanus toxin (100 micrograms/ml) blocked the SDPs as well as the synaptic potentials evoked by intrastriatal stimulation. 4. At resting membrane potential, intrastriatal stimulation produced a fast depolarizing postsynaptic potential (EPSP) that was reduced by BIC (10-100 microM). The relationship between EPSP amplitude and membrane potential was studied either by utilizing K(+)-chloride electrodes or by the use of cesium-chloride electrodes. In both these cases, the reversal potential for the EPSPs was between 0 and -14 mV. In cesium-loaded neurons, the decrease of the EPSP, usually observed at negative membrane potentials (below -85 mV), was clearly reduced. Internal cesium prolonged the duration of the SDPs and the EPSPs evoked by intrastriatal stimulation. 5. The relationship between spontaneous and evoked synaptic activity and membrane potential was studied in the presence of different external potassium blockers. 4-Aminopyridine (4AP, 0.1-1 mM) increased the EPSP amplitude and the frequency of the SDPs, but did not decrease membrane rectification and the shunt of the EPSPs present at negative membrane potentials. On the contrary, rectification of the membrane and the shunt of the EPSPs below -85 mV were clearly reduced by tetraethylammonium (TEA, 10-20 mM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Primary afferents evoke excitatory amino acid receptor-mediated EPSPs that are modulated by presynaptic GABAB receptors in lamprey.

1. The primary afferent neurons (dorsal cells) are of two types in lamprey, which are fast (touch) and slowly adapting (pressure), respectively. Intracellular stimulation of such sensory neurons evokes mono- and polysynaptic excitatory postsynaptic potentials (EPSPs) in spinobulbar neurons (giant interneurons) and in unidentified interneurons. Paired intracellular recordings between identified sensory cells and spinobulbar neurons made it possible to study the synaptic transmission in detail. It is shown that both touch and pressure primary afferents utilize excitatory amino acid (EAA) transmission and, furthermore, that these effects are subject to a presynaptic GABAB receptor modulation. 2. The monosynaptic mixed electrical and chemical EPSPs in giant interneurons had a mean peak amplitude of 3.2 +/- 1.3 (SD) mV, a time to peak of 4.7 +/- 1.2 ms, and a duration at one-half peak amplitude of 9.4 +/- 3.2 ms. Corresponding results were obtained with dorsal root or dorsal column stimulation. Seventy percent of the fast-adapting dorsal cells of the "touch" type evoked monosynaptic mixed EPSPs in giant interneurons, whereas only 3% of the slowly adapting "pressure" dorsal cells did. 3. The chemical part of the monosynaptic EPSPs evoked in giant interneurons was, in all cases tested, blocked by application of EAA antagonists, like the nonselective antagonist kynurenic acid (KYAC; 2 mM). The selective kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 microM) had a similar effect, whereas the selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-aminophosphono-5-valeric acid (AP-5; 200-400 microM) did not change the EPSP, even in the absence of magnesium ions. 4. The monosynaptic excitatory synaptic transmission was modulated by application of the selective GABAB receptor agonist L-baclofen (5-10 mM local droplet application or 100-1,000 microM bath applied) or by gamma-aminobutyric acid (GABA; 100-1,000 microM), also when GABAA receptor-evoked effects were blocked by bicuculline (10 microM). L-baclofen or GABA in combination with bicuculline did not evoke any effects in the postsynaptic neuron on membrane potential, input resistance, or spike threshold. Therefore the effects of the GABAB receptor activation most likely occurs at the presynaptic afferent level. 5. In conclusion, the monosynaptic excitation from skin mechanoreceptors evoked in spinobulbar neurons is mediated by EAA receptors of the kainate/AMPA type. GABAB receptor activation causes a depression of this EPSP, most likely because of a presynaptic action. GABA interneurons are known to form close appositions on sensory axons in the lamprey.     

Activation of N-methyl-D-aspartate receptors parallels changes in cellular and synaptic properties of dentate gyrus granule cells after kindling

1. The cellular and synaptic properties of rat dentate gyrus granule cells (GCs) were examined using intra-/extracellular and Ca2+-sensitive microelectrode recordings following epilepsy induced by kindling of the hippocampal commissures or amygdala. 2. The recordings were made in hippocampal slices prepared from sham-stimulated controls and animals that have received daily stimuli to reach stage IV-V of kindling. The average number of stimulation trials (60 Hz/1 s, 100-150 microA) required to reach full motor seizures (stage V) was 23 +/- 2 for commissural kindling and 14 +/- 1 for amygdala kindling. 3. The resting membrane potential of GCs following kindling (RMP; -72 +/- 3 mV) was not significantly different from the RMP of control GCs (-70 +/- 2 mV). Similarly, action potential height and threshold were unaffected by kindling. However, kindling altered other cellular properties of GCs regardless of the site of stimulation (hippocampal commissures or amygdala), the stage of kindling reached (IV or V), or the time elapsed between the last kindling stimulus and preparation of the hippocampal slices (24 h-6 wk). The input resistance of kindled GCs (55 +/- 4 M omega) was significantly higher than that of controls (40 +/- 3 M omega). In contrast to most control GCs, the slope conductance (GS) of kindled neurons, measured with constant-amplitude current injections at various membrane potentials, generally increased at membrane potentials more negative than rest. Furthermore, other voltage-dependent ionic conductances (see below), that were not normally encountered in control GCs, were present in kindled neurons. 4. The intracellularly recorded monosynaptic excitatory postsynaptic potentials (EPSPs) of kindled GCs, evoked through the stimulation of the lateral perforant pathway, differed significantly from the EPSPs of control GCs. The amplitudes of control EPSPs increased upon hyperpolarizations and decreased following depolarizations of the membrane, as expected for conventional EPSPs without contribution from voltage-dependent conductances. In contrast, the EPSPs of kindled GCs invariably increased in amplitude and duration at membrane potentials 5-20 mV depolarized from rest, indicating the presence of a characteristic voltage-dependent component. Frequently, following the synaptically triggered action potentials, kindled GCs displayed depolarizing afterpotentials. 5. Perfusion of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV; 30 microM) had no effect on the EPSPs of control GCs, but consistently reduced the amplitude and duration of EPSPs in kindled GCs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic organization of tectal-facial pathways in cat. II. Synaptic potentials following midbrain tegmentum stimulation.

1. The organization of the synaptic pathways underlying midbrain tegmentum influence over the facial musculature was studied with the use of an acute electrophysiological approach in the cat. Under pentobarbital sodium anesthesia, synaptic potentials were recorded intracellularly in antidromically identified facial motoneurons following electrical stimulation of the paralemniscal zone. The cells of origin and the pathways responsible for the potentials evoked from the paralemniscal zone were defined with the use of retrograde transport of horseradish peroxidase (HRP). The putative role of the paralemniscal zone with regard to the production of disynaptic, tectally evoked potentials in facial motoneurons was investigated both by inactivating this nucleus with injections of lidocaine and by making acute brain stem lesions to sever the paralemniscal-facial and other afferent pathways. 2. Following paralemniscal stimulation, monosynaptic, excitatory postsynaptic potentials (EPSPs) with latencies ranging from 0.6 to 0.9 ms, steep rising phases, and amplitudes in excess of 4.0 mV were recorded in motoneurons of the temporal and auriculoposterior subdivisions, which supply the pinna muscles. Smaller amplitude EPSPs (less than 1.0 mV) with monosynaptic latencies were observed in the zygomatic subdivision. Polysynaptic EPSPs with latencies ranging from 1.0 to 1.8 ms were also observed in all three of these subdivisions. However, only long-latency EPSPs, arriving at 2.0 ms or later, were present in ventral subdivision motoneurons. 3. Inhibitory postsynaptic potentials (IPSPs) were also frequently recorded in facial motoneurons after paralemniscal stimulation. Monosynaptic IPSPs with latencies ranging from 0.8 to 1.2 ms and amplitudes in excess of 4.0 mV were recorded in facial motoneurons of the temporozygomatic and auriculoposterior subdivisions, as were polysynaptic IPSPs with latencies ranging from 1.2 to 1.8 ms. IPSPs were sometimes observed in combination with a smaller, shorter latency EPSPs. Only long-latency IPSPs of greater than 2.0 ms were recorded in ventral subdivision motoneurons. In all cases, both the EPSPs and the IPSPs were graded in character and could be augmented by multiple stimuli. 4. The contralateral paralemniscal zone and the supraoculomotor area, bilaterally, represented the two most prominent afferent sources labeled after HRP injection of the facial nucleus. The superior colliculus and numerous reticular formation regions were also identified as facial nucleus afferents by the presence of retrogradely labeled cells. The retrogradely labeled cells in the paralemniscal zone exhibited heterogeneous soma size. HRP-labeled axons of the paralemniscal-facial pathway were observed to cross the midline by traveling ventral to the brachium conjunctivum in the caudal mesencephalon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane properties and synaptic responses of the guinea pig nucleus accumbens neurons in vitro

1. The membrane properties and synaptic responses of guinea pig nucleus accumbens neurons in vitro were studied with intracellular recording methods. 2. The population of neurons could be divided into groups of low (20-60 M omega, average 46.5 M omega) and high (60-180 M omega, average 96.5 M omega) input resistance. The resting membrane potential in both groups was approximately -70 mV. 3. Other membrane properties were quite similar in both groups. Inward rectification occurred at potentials more negative than -80 mV; this was blocked by Cs+ (2 mM). Membrane potential oscillations were observed at potentials between -65 and -55 mV; these were blocked by tetrodotoxin (TTX, 0.5 microM). Outward rectification occurred at potentials less negative than -45 mV; this was depressed by tetraethylammonium (TEA, 10 mM). 4. Action potentials elicited by small depolarizing current pulses (2-5 ms, 0.3-0.5 nA) were approximately 95 mV in amplitude and 1.0 ms in duration. The afterhyperpolarization following each action potential was less than 30 ms in duration, and no accommodation of action-potential discharge was seen at frequencies up to 40 Hz. The action potentials were reversibly blocked by TTX (0.3 microM). In addition, TTX-insensitive, Ca2+-dependent spikes were evoked by passing larger and more prolonged current pulses (greater than 40 ms, greater than 0.5 nA) across the membrane. 5. Focal electrical stimulation of the slice surface with low intensity (1 ms, less than 10 V) elicited excitatory postsynaptic potentials (EPSPs) in neurons of both high- and low-resistance groups. The reversal potential (+10.2 mV) for the EPSPs was close to the reversal potential (+7.7 mV) of the responses to glutamate applied in the superfusing solution. The N-methyl-D-aspartic acid (NMDA) receptor antagonists, D-alpha-aminoadipic acid (1 mM) and DL-2-amino-5-phosphonovaleric acid (DL-APV, 250 microM), reversibly depressed the EPSP; the glutamate uptake inhibitor, L-aspartic acid-beta-hydroxamate (50 microM), or removal of Mg2+ from the superfusate, augmented the EPSP. 6. When the intensity of the focal stimulus was increased (1 ms, greater than or equal to 10 V), a second larger depolarizing response (duration, 800 ms to 2 s) could be evoked in addition to the smoothly graded EPSP. This was seen only in cells of the high-resistance group (90-130 M omega).(ABSTRACT TRUNCATED AT 400 WORDS)     

EPSPs in rat neocortical neurons in vitro. II. Involvement of N-methyl-D-aspartate receptors in the generation of EPSPs

1. Intracellular recordings were obtained from neurons in layer II/III of rat frontal cortex. Single-electrode current- and voltage-clamp techniques were employed to compare the sensitivity of excitatory postsynaptic potentials (EPSPs) and iontophoretically evoked responses to N-methyl-D-aspartate (NMDA) to the selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-2-APV). The voltage dependence of the amplitudes of the EPSPs before and after pharmacologic changes in the neuron's current-voltage relationship was also examined. 2. NMDA depolarized the membrane potential, increased the neuron's apparent input resistance (RN), and evoked bursts of action potentials. The NMDA-induced membrane current (INMDA) gradually increased with depolarization from -80 to -40 mV. The relationship between INMDA and membrane potential displayed a region of negative slope conductance in the potential range between -70 and -40 mV which was sufficient to explain the apparent increase in RN and the burst discharges during the NMDA-induced depolarization. 3. Short-latency EPSPs (eEPSPs) were evoked by low-intensity electrical stimulation of cortical layer IV. Changes in the eEPSP waveform following membrane depolarization and hyperpolarization resembled those of NMDA-mediated responses. However, the eEPSP was insensitive to D-2-APV applied at concentrations (up to 20 microM) that blocked NMDA responses. 4. EPSPs with latencies between 10 and 40 ms [late EPSPs (lEPSPs)] were evoked by electrical stimulation using intensities just subthreshold to the activation of IPSPs. The amplitude of the lEPSP increased with hyperpolarization and decreased with depolarization. 5. The lidocaine derivative QX-314, injected intracellularly, suppressed sodium-dependent action potentials and depolarizing inward rectification. Simultaneously, the amplitude of the eEPSP significantly decreased with depolarization. Neither the amplitude of a long-latency EPSP nor the amplitude of inhibitory postsynaptic potentials (IPSPs) was significantly affected by QX-314. 6. Cesium ions (0.5-2.0 mM) added to the bathing solution reduced or blocked hyperpolarizing inward rectification. Under these conditions, the amplitude of the eEPSP increased with hyperpolarization. The amplitude of the lEPSP was unaltered or enhanced. 7. The lEPSP was reversibly blocked by D-2-APV (5-20 microM), although the voltage-dependence of its amplitude did not resemble the action of NMDA on neocortical neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Local synaptic circuits and epileptiform activity in slices of neocortex from children with intractable epilepsy.

1. Single and dual intracellular recordings were performed in neocortical slices obtained from tissue samples surgically removed from children (8 mo to 15 yr) for the treatment of intractable epilepsy. Electrical stimulation and glutamate microapplication were used to study local synaptic inputs to pyramidal cells. 2. In recordings with potassium-acetate electrodes, activation of presynaptic neocortical neurons with glutamate microdrops did not elicit a clear increase in postsynaptic potentials (PSPs) but did suppress current-evoked repetitive spike firing in recorded neurons. Bicuculline (10 microM) blocked this effect, suggesting it was caused by the activation of presynaptic gamma-aminobutyric acid (GABA) cells. In recordings with KCl electrodes, glutamate microdrops elicited an increase in the frequency and amplitude of depolarizing PSPs. Bicuculline (5-10 microM) blocked the glutamate-evoked PSPs, suggesting they were reversed GABAA-receptor-mediated inhibitory postsynaptic potentials (IPSPs). In one cell recorded with a KCl electrode (total n = 8), current-evoked spike trains elicited afterdischarges of reversed IPSPs, thus revealing a recurrent inhibitory circuit. Therefore local inhibitory synaptic circuits were robust and could be observed in tissue from patients as young as 11 mo. 3. In addition to short-latency (10-25 ms), monosynaptic excitatory postsynaptic potentials (EPSPs), electrical stimulation at low intensities sometimes elicited delayed EPSPs (20-60 ms). When GABAA-receptor-mediated synaptic inhibition was partially reduced in bicuculline (5-10 microM), electrical stimulation evoked large EPSPs at long and variable latencies (100-300 ms). Glutamate microapplication caused an increase in the frequency and amplitude of EPSPs; preliminary results suggest that glutamate microdrops were less likely to evoke EPSPs in tissue from younger patients (8-12 mo) than in slices from patients greater than 4 yr. Evidence for local excitatory synaptic circuits was thus found when synaptic inhibition was partially reduced. 4. After further reduction of inhibition in bicuculline (5-50 microM), electrical stimulation elicited epileptiform bursts. In pairs of simultaneously recorded neurons, bursts were generated synchronously from long-latency EPSPs (100-300 ms) in slices from patients as young as 8 mo. Reflected EPSPs at very long and variable latencies (500-1,100 ms) and repetitive epileptiform bursts could be evoked synchronously in pairs of cells. Glutamate activation of local presynaptic neurons elicited robust epileptiform events in recorded cells. This was seen in slices from patients as young as 16 mo. 5. These data provide physiological evidence for the presence of local inhibitory and excitatory synaptic circuits in human neocortex at least as early as 11 and 8 mo, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrophysiological responses of trigeminal root ganglion neurons in vitro

The membrane electrical properties of neurons and their responses to endogenous compounds or other neuroactive substances were investigated in vitro with intracellular recording techniques in slices of trigeminal root ganglia of guinea-pigs. The mean resting membrane potential of these neurons was -60 mV. Intracellular injections of hyperpolarizing current pulses evoked time-dependent rectification with varying degrees of dependence on membrane voltage in 107 of 110 neurons. Membrane potential oscillations were observed following the termination of the hyperpolarizing pulses and after similar injections of depolarizing current. This phenomenon appeared to be voltage-dependent at levels that were subthreshold for spike genesis; the more pronounced oscillations were evident at the more depolarized levels and were insensitive to tetrodotoxin applications. Two groups of neurons could be distinguished on the basis of certain characteristics in their action potentials. The majority exhibited short duration (0.6 ms) spikes with mean amplitude of 72 mV in response to intracellular depolarizing current. The brief (3 ms) afterhyperpolarizations that followed such spikes were blocked by intracellular injections of Cs+ or by bath applications of tetraethylammonium. Action potentials in the minority group exhibited a hump in their repolarization phase. The humped spikes had a mean peak amplitude of 78 mV and a longer duration (2 ms). Both the duration (6 ms) and the amplitude (16 mV) of the afterhyperpolarization were significantly greater in this latter group of neurons. Some fast spikes were easily blocked whereas others, including humped spikes, were resistant to tetrodotoxin (10(-6) M). Spikes which were resistant, were also not affected by perfusion with Co2+ (10(-3) M) and were reduced in amplitude during perfusion with Na+-deficient solution. Bath applications of S-glutamate (10(-4)-10(-2) M) depolarized only two of ten neurons by less than 3 mV. Similarly, 5-hydroxytryptamine produced a small depolarization in only two of thirteen neurons. Perfusion of gamma-aminobutyrate (10(-5)-10(-2) M) resulted in an increase in input conductance that waned despite continued application and was associated with a depolarization (2-14 mV) in 44/50 neurons. In some neurons, gamma-aminobutyrate application enhanced their repetitive firing ability, possibly as a result of the increased oscillatory behavior of the membrane at certain depolarized potentials. The effects of gamma-aminobutyrate were blocked by the GABAA-receptor antagonist, bicuculline (10(-4) M) but were unaffected by the GABAB-receptor agonist, baclofen (10(-4) M).(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium-dependent potassium conductance in rat supraoptic nucleus neurosecretory neurons

Intracellular recordings of rat supraoptic nucleus neurons were obtained from perfused hypothalamic explants. Individual action potentials were followed by hyperpolarizing afterpotentials (HAPs) having a mean amplitude of -7.4 +/- 0.8 mV (SD). The decay of the HAP was approximated by a single exponential function having a mean time constant of 17.5 +/- 6.1 ms. This considerably exceeded the cell time constant of the same neurons (9.5 +/- 0.8 ms), thus indicating that the ionic conductance underlying the HAP persisted briefly after each spike. The HAP had a reversal potential of -85 mV and was unaffected by intracellular Cl- ionophoresis of during exposure to elevated extracellular concentrations of Mg2+. In contrast, the peak amplitude of the HAP was proportional to the extracellular Ca2+ concentration and could be reversibly eliminated by replacing Ca2+ with Co2+, Mn2+, or EGTA in the perfusion fluid. During depolarizing current pulses, evoked action potential trains demonstrated a progressive increase in interspike intervals associated with a potentiation of successive HAPs. This spike frequency adaptation was reversibly abolished by replacing Ca2+ with Co2+, Mn2+, or EGTA. Bursts of action potentials were followed by a more prolonged afterhyperpolarization (AHP) whose magnitude was proportional to the number of impulses elicited (greater than 20 Hz) during a burst. Current injection revealed that the AHP was associated with a 20-60% decrease in input resistance and showed little voltage dependence in the range of -70 to -120 mV. The reversal potential of the AHP shifted with the extracellular concentration of K+ [( K+]o) with a mean slope of -50 mV/log[K+]o.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantal analysis of potentiating action of phorbol ester on synaptic transmission in the hippocampus

The mechanism of the potentiating action of phorbol diacetate on synaptic transmission in the hippocampus was studied by the quantal analysis technique. Thin transverse sections were prepared from guinea pig hippocampus and intracellular potentials were recorded from CA3 neurons. Unitary excitatory postsynaptic potentials (EPSPs) were induced in the impaled neurons by brief glutamate pulses administered to granule cells. The amplitude of the unitary EPSPs fluctuated according to Poisson distribution. From the mean and variance of the amplitude of the unitary EPSPs, the mean quantal content (m) and the mean quantal amplitude (q) were calculated. Before phorbol diacetate administration, the values of m and q were 9.7 +/- 1.4 and 1.1 +/- 0.28 mV (mean +/- S.D.), respectively. Potentiation of synaptic transmission by phorbol diacetate was accompanied by increases in the value of m. The value of q remained unchanged in most neurons and decreased in some. These results indicate that the phorbol ester causes an increase in release of neurotransmitter and thereby potentiates synaptic transmission.     

Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices.

1. Conventional intracellular and extracellular recording techniques were used to investigate the physiology and pharmacology of epileptiform bursts induced by 4-aminopyridine (4-AP, 50 microM) in the CA3 area of rat hippocampal slices maintained in vitro. 2. 4-AP-induced epileptiform bursts, consisting of a 25-to 80-ms depolarizing shift of the neuronal membrane associated with three to six fast action potentials, occurred at the frequency of 0.61 +/- 0.29 (SD)/s. The bursts were generated synchronously by CA3 neurons and were triggered by giant excitatory postsynaptic potentials (EPSPs). A second type of spontaneous activity consisting of a slow depolarization also occurred but at a lower rate (0.04 +/- 0.2/s). 3. The effects of 4-AP on EPSPs and inhibitory postsynaptic potentials (IPSPs) evoked by mossy fiber stimulation were studied on neurons impaled with a mixture of K acetate and 2(triethyl-amino)-N-(2,6-dimethylphenyl) acetamide (QX-314)-filled microelectrodes. After the addition of 4-AP, the EPSP became potentiated and was followed by the appearance of a giant EPSP. This giant EPSP completely obscured the early IPSP recorded under control conditions and inverted at -32 +/- 3.9 mV (n = 4), suggesting that both inhibitory and excitatory conductances were involved in its generation. IPSPs evoked by Schaffer collateral stimulation increased in amplitude and duration after 4-AP application. 4. The spontaneous field bursts and the stimulus-induced giant EPSP induced by 4-AP were not affected by N-methyl-D-aspartate (NMDA) receptor antagonists 3-3 (2-carboxy piperazine-4-yl) propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV) but were blocked by quisqualate/kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). CNQX also abolished the presence of small spontaneously occurring EPSPs, thereby disclosing the presence of bicuculline-sensitive (BMI, 20 microM) IPSPs. 5. Small, nonsynchronous EPSPs played an important role in the generation of 4-AP-induced epileptiform activity. 1) After the addition of 4-AP, small EPSPs appeared randomly on the baseline and then became clustered to produce a depolarizing envelope of irregular shape that progressively formed an epileptiform burst, 2) These small EPSPs were more numerous in the 100 ms period that preceded burst onset. 3) The frequency of occurrence of small EPSPs was positively correlated with the frequency of occurrence of synchronous bursts. 4) Small EPSPs and bursts were similarly decreased after the addition of different concentrations of CNQX (IC50 in both cases of approximately 1.2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

A slow fraction of Mg2+ unblock of NMDA receptors limits their contribution to spike generation in cortical pyramidal neurons

The timing of voltage-dependent removal of Mg(2+) block of N-methyl-d-aspartate receptors (NMDARs) is potentially critical for determining their nonlinear contribution to excitability. Here, we measure the kinetics of NMDAR unblock in nucleated patch and whole cell recordings of rat cortical pyramidal neurons during depolarizing voltage steps. At room temperature, the unblock showed a very fast component (tau < 1 ms) and a slower component (tau = 14-23 ms in nucleated patches). The slow component accounted for half of the current at +40 mV and its amplitude and time constant showed some voltage dependence. Blocking with hyperpolarization was very fast (tau < 200 micros). Voltage-clamp with action potential waveforms, at both room temperature and at 33 degrees C, showed that the rising phase of single fast action potentials unblocks far less NMDAR current than expected from the stationary voltage dependence, while a large amplitude of current is uncovered during the upstroke of slow calcium action potentials. The repolarization of fast sodium action potentials uncovers an NMDAR tail current, much bigger than the stationary level of current.     

Excitatory postsynaptic potentials evoked by ventral root stimulation in neonate rat motoneurons in vitro

1. Intracellular recordings were made from antidromically identified motoneurons in transverse (500 microns) lumbar spinal cord slices of neonatal (12-20 day) rats. 2. Electrical stimulation of ventral rootlets evoked, with or without an antidromic spike or initial segment potential, a depolarizing response (latency, 1-4.2 ms), a hyperpolarizing response (latency, 1.5-3.5 ms), or a combination of two preceding responses in 38, 6, and 8% of motoneurons investigated. 3. The hyperpolarizing response was reversibly eliminated by low Ca2+ (0.25 mM), d-tubocurarine (d-Tc; 10 microM) or strychnine (1 microM), suggesting that this response represents an inhibitory post-synaptic potential (IPSP) mediated by glycine or a related substance release from inhibitory interneurons subsequent to their activation by axon collaterals in a manner analogous to the Renshaw cell circuitry described for the cat motoneurons. 4. The depolarizing responses were excitatory postsynaptic potentials (EPSPs), because they could be graded by varying the stimulus intensity and were reversibly abolished in low Ca2+ solution. 5. Membrane hyperpolarization increased the amplitude of EPSPs, and the mean extrapolated reversal potential was -4 mV. 6. EPSPs were augmented, rather than diminished, by dihydro-beta-erythroidine (1 microM) or d-Tc, arguing against a role of recurrent motor axon collaterals in initiating the responses. 7. The conduction velocity of the fibers initiating the EPSPs ranged from 0.35 to 0.96 m/s, indicating that these fibers were unmyelinated. Furthermore, the EPSP exhibited a constant delay when the stimulus frequency was varied from 1 to 5 Hz, and the synaptic delay estimated by extrapolation was less than 1 ms, suggesting that it was a monosynaptic event. 8. After complete separation of the ventral and dorsal horns by a knife cut, stimulation of ventral rootlets could still evoke an EPSP in motoneurons. 9. Superfusion of the slices with the nonselective glutamate receptor antagonist kynurenic acid (0.2-1 mM) or the selective quisqualate/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) (0.5-1 microM) reversibly diminished the EPSPs. 10. EPSPs evoked by stimulation of dorsal and ventral rootlets exhibited different latency and waveform in the same motoneurons. 11. The results provide evidence that activation of ventral root afferents evoked an EPSP mediated by glutamate or a related substance in a population of motoneurons. Furthermore, the afferent pathway mediating the EPSP appears to be monosynaptic and confined to the ventral horn.