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.     

A correlative physiological and morphological analysis of monosynaptically connected propriospinal axon-motoneuron pairs in the lumbar spinal cord of frogs

Intracellular stimulation of single propriospinal axons evoked excitatory postsynaptic potentials (EPSPs) in lumbar motoneurons. Mean EPSP amplitudes differed by two orders of magnitude when measured in different connections. After analyzing the distribution of mean amplitudes of 47 single-fiber EPSPs, two populations of responses could be defined: (1) those with mean amplitudes between 0.1 and 1.2 mV (mean+/-S.D.: 0.48+/-0.30 mV, 34 pairs), which is in the range of values typical for single-fiber EPSPs evoked by stimulation of supraspinal fibers and primary muscle afferents, (2) those with mean amplitudes between 1.6 and 8 mV (4.2+/-2.0 mV, 13 pairs). Both populations of responses had similarly short latencies and rise times and responded similarly to paired-pulse stimulation, consistent with monosynaptic transmission. However, the high-efficacy connections had significantly smaller coefficients of variation of EPSPs, as well as increased quantal content and quantal size. Tetanic stimulation gradually depressed the amplitude of large EPSPs by 81-86%, but did not affect small EPSPs. Recovery of large EPSPs was exponential with a time constant of 3-5.6 min. During post-tetanic depression the amplitude ratio between the test and conditioned EPSPs evoked by paired-pulse stimulation was not changed but the coefficient of variation was increased, suggesting that the depression was due to depletion of synaptic vesicles available for release.Intracellular labeling of seven electrophysiologically studied propriospinal axon-motoneuron pairs revealed that the number of axon varicosities establishing close appositions with dendrites of the labeled motoneuron was higher for connections where large-amplitude EPSPs were recorded. These varicosities were more often located on proximal dendrites of motoneurons than those of low-efficacy connections. In addition, the number of boutons in highly effective connections was several times lower than the maximal number of available quanta estimated from physiological data, implying that the large EPSPs may be generated by multivesicular release from presynaptic boutons.We conclude that the efficacy and related mode of use-dependent modulation of propriospinal connections is determined by a number of factors, including the number and position of synaptic contacts and the number of active zones or vesicles available for release.     

Role of EPSPs in initiation of spontaneous synchronized burst firing in rat hippocampal neurons bathed in high potassium

1. Spontaneous discharges that resemble interictal spikes arise in area CA3 b/c of rat hippocampal slices bathed in 8.5 mM [K+]o. Excitatory postsynaptic potentials (EPSPs) also appear at irregular intervals in these cells. The role of local synaptic excitation in burst initiation was examined with intracellular and extracellular recordings from CA3 pyramidal neurons. 2. Most (70%) EPSPs were small (less than 2 mV in amplitude), suggesting that they were the product of quantal release or were evoked by a single presynaptic action potential in another cell. It is unlikely that most EPSPs were evoked by a presynaptic burst of action potentials. Indeed, intrinsic burst firing was not prominent in CA3 b/c pyramidal cells perfused in 8.5 mM [K+]o. 3. The likelihood of occurrence and the amplitude of EPSPs were higher in the 50-ms interval just before the onset of each burst than during a similar interval 250 ms before the burst. This likely reflects increased firing probability of CA3 neurons as they emerge from the afterhyperpolarization (AHP) and conductance shunt associated with the previous burst. 4. Perfusion with 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a potent quisqualate receptor antagonist, decreased the frequency of EPSPs in CA3 b/c neurons from 3.6 +/- 0.9 to 0.9 +/- 0.3 (SE) Hz. Likewise, CNQX reversibly reduced the amplitude of evoked EPSPs in CA3 b/c cells. 5. Spontaneous burst firing in 8.5 mM [K+]o was abolished in 11 of 31 slices perfused with 2 microM CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in perforant path quantal size in aged F-344 rats.

The data presented here confirm and extend the evidence for fewer, but stronger, perforant path synaptic connections onto the granule cells of the hippocampus in old F-344 rats. The old animals used in the present report were drawn from a population that showed deficits in the retention of a spatial problem in the Morris water task. Using the method of minimal-stimulation of perforant path afferents, unitary granule cell EPSPs were found to be larger in the 25-month than in the 6- and 9-month age groups. Furthermore, applying statistical methods for quantal analysis, data are presented that suggest that the larger synaptic responses of the old rats come about through an increase in quantal size. These experiments therefore suggest that individual synapses become more powerful in the perforant pathway of old rats, and that this strengthening occurs through an increase in quantal size. The implications of these findings for hippocampal information processing are discussed.     

Postsynaptic glutamate receptors and integrative properties of fast-spiking interneurons in the rat neocortex.

The glutamate-mediated synaptic responses of neocortical pyramidal cell to fast-spiking interneuron (pyramidal-FS) connections were studied by performing paired recordings at 30-33 degrees C in acute slices of 14- to 35-day-old rats (n = 39). Postsynaptic fast-spiking (FS) cells were recorded in whole cell configuration with a patch pipette, and presynaptic pyramidal cells were impaled with sharp intracellular electrodes. At a holding potential of -72 mV (near the resting membrane potential), unitary excitatory postsynaptic potentials (EPSPs) had a mean amplitude of 2.1 +/- 1.3 mV and a mean width at half-amplitude of 10.5 +/- 3.7 ms (n = 18). Bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist D(-)2-amino-5-phosphonovaleric acid (D-AP5) had minor effects on both the amplitude and the duration of unitary EPSPs, whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) almost completely blocked the synaptic responses. In voltage-clamp mode, the selective antagonist of AMPA receptors 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3, 4-dihydro-5H-2,3-benzodiazepine (GYKI 53655; 40-66 microM) blocked 96 +/- 1.9% of D-AP5-insensitive unitary excitatory postsynaptic currents (EPSCs), confirming the predominance of AMPA receptors, as opposed to kainate receptors, at pyramidal-FS connections (n = 3). Unitary EPSCs mediated by AMPA receptors had fast rise times (0.29 +/- 0.04 ms) and amplitude-weighted decay time constants (2 +/- 0.8 ms; n = 16). In the presence of intracellular spermine, these currents showed the characteristic rectifying current-voltage (I-V) curve of calcium-permeable AMPA receptors. A slower component mediated by NMDA receptors was observed when unitary synaptic currents were recorded at a membrane potential more positive than -50 mV. In response to short trains of moderately high-frequency (67 Hz) presynaptic action potentials, we observed only a limited temporal summation of unitary EPSPs, probably because of the rapid kinetics of AMPA receptors and the absence of NMDA component in these subthreshold synaptic responses. By combining paired recordings with extracellular stimulations (n = 11), we demonstrated that EPSPs elicited by two different inputs were summed linearly by FS interneurons at membrane potentials below the action potential threshold. We estimated that, in our in vitro recording conditions, 8 +/- 5 pyramidal cells (n = 18) should be activated simultaneously to make FS interneurons fire an action potential from -72 mV. The low level of temporal summation and the linear summation of excitatory inputs in FS cells favor the role of coincidence detectors of these interneurons in neocortical circuits.     

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)     

Rat hippocampal neurons in culture: responses to electrical and chemical stimuli

Intracellular activity was recorded from dissociated rat hippocampal neurons maintained in tissue culture conditions for 4-6 wk. The cells developed dense interconnections and had typical morphological characteristics similar to hippocampal neurons in situ. The recorded neurons possessed similar electrophysiological properties to those observed in situ or in a slice preparation. Their input resistance (42 M omega), resting membrane potential (-60 mV), membrane time constant (16.2 ms), total electrotonic length (0.92), and spike size (68.3 mV) were similar to values obtained in hippocampal cells in a slice. The connections among adjacent neurons were largely inhibitory. The inhibitory postsynaptic potentials (IPSPs) had longer durations than excitatory postsynaptic potentials (EPSPs) when these were detected. Synaptic delay varied between 0.3 and 3.0 ms. There were no electrotonic connections among neurons. Reciprocal connections were common. Most neurons reacted to acetylcholine (ACh) by an increase in frequency of spontaneous EPSPs, action-potential discharges, and IPSPs. Concurrently, there was a marked reduction in the magnitude of the evoked PSPs tested in pairs of cells. This effect is probably presynaptic to the recorded neurons. A statistical analysis of quantal properties of the synaptic interactions among neurons revealed that ACh causes a reduction of magnitude of PSPs by reducing the number of releasing elements (m). This effect is different from the reduction of evoked PSPs caused by postsynaptic depolarization.     

Voltage-clamp analysis of taurine-induced suppression of excitatory postsynaptic potentials in frog spinal motoneurons

1. The depressant actions of taurine applications on lumbar motoneurons in the isolated frog spinal cord were studied using conventional intracellular recordings and the two-electrode voltage-clamp technique. 2. With microelectrodes containing K+-acetate, 0.75-2 mM taurine mostly induced a hyperpolarization that often faded or turned into depolarization during the continuous application. A higher concentration (5-7.5 mM) depolarized a majority of cells. The effects on the membrane potential were associated with an increase in input conductance (approximately 285%). 3. The reversal potential of the taurine-induced currents was approximately -70 mV, with microelectrodes containing K+-acetate. In recordings using KCl-filled electrodes, taurine (less than or equal to 2 mM) produced a large depolarization (greater than or equal to 20 mV) at resting potentials near -50 mV, thereby indicating that the reversal potential was positively shifted by loading the cell with Cl-. These results suggest that the taurine potentials were mediated predominantly by an increased Cl- permeability. 4. Voltage-dependent relaxations of taurine currents were observed in 10 of 14 neurons. 5. A linear relation was found between the input conductance and the amount of current required to generate a 1-mV increment in EPSP at resting potential. 6. Polysynaptic excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were more susceptible to taurine than the monosynaptic responses. Taurine (less than 1 mM) seemed to suppress the interneurons mediating polysynaptic pathways. 7. Monosynaptic EPSPs and EPSCs were decreased with higher concentrations of taurine (greater than 1 mM). The percent reduction of EPSPs and that of the corresponding EPSCs had a positive correlation (r = 0.95), whereas, there was no significant correlation between changes in EPSPs and in input conductance, and between changes in EPSCs and in input conductance. The amount of current required to produce a 1-mV increment of EPSP was increased in the presence of taurine, in association with the increased input conductance. 8. Taurine suppressed synaptic potentiation of EPSPs evoked by paired stimuli, at an interval of 60-180 ms. Gamma-D-glutamylglycine, an antagonist of receptors for excitatory amino acids, greatly reduced the amplitude of EPSPs, but had little effect on synaptic potentiation. 9. Taurine suppressed glutamate currents evoked at membrane potentials, clamped near rest in low Ca2+, high Mg2+ solution. 10. These findings suggest that the taurine-induced reduction of EPSPs is due mainly to suppression of EPSCs, through both presynaptic and postsynaptic mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuromuscular system of the flexible arm of the octopus: physiological characterization

The octopus arm is an outstanding example of an efficient boneless and highly flexible appendage. We have begun characterizing the neuromuscular system of the octopus arm in both innervated muscle preparations and dissociated muscle cells. Functionally antagonistic longitudinal and transverse muscle fibers showed no differences in membrane properties and mode of innervation. The muscle cells are excitable but have a broad range of linear membrane properties. They are electrotonically very compact so that localized synaptic inputs can control the membrane potential of the entire muscle cell. Three distinct excitatory neuronal inputs to each arm muscle cell were identified; their reversal potentials were extrapolated to be about -10 mV. These appear to be cholinergic as they are blocked by hexamethonium, D-tubocurarine, and atropine. Two inputs have low quantal amplitude (1-7 mV) and slow rise times (4-15 ms), whereas the third has a large size (5-25 mV) and fast rise time (2-4 ms). This large synaptic input is most likely due to exceptionally large quantal events. The probability of release is rather low, suggesting a stochastic activation of muscle cells. All inputs demonstrated a modest activity-dependent plasticity typical of fast neuromuscular systems. The pre- and postsynaptic properties suggest a rather direct relation between neuronal activity and muscle action. The lack of significant electrical coupling between muscle fibers and the indications for the small size of the motor units suggest that the neuromuscular system of the octopus arm has evolved to ensure a high level of precise localization in the neural control of arm function.     

Vestibuloocular reflex of the adult flatfish. III. A species-specific reciprocal pattern of excitation and inhibition

In juvenile flatfish the vestibuloocular reflex (VOR) circuitry that underlies compensatory eye movements adapts to a 90 degrees relative displacement of vestibular and oculomotor reference frames during metamorphosis. VOR pathways are rearranged to allow horizontal canal-activated second-order vestibular neurons in adult flatfish to control extraocular motoneurons innervating vertical eye muscles. This study describes the anatomy and physiology of identified flatfish-specific excitatory and inhibitory vestibular pathways. In antidromically identified oculomotor and trochlear motoneurons, excitatory postsynaptic potentials (EPSPs) were elicited after electrical stimulation of the horizontal canal nerve expected to provide excitatory input. Electrotonic depolarizations (0.8-0.9 ms) preceded small amplitude (<0.5 mV) chemical EPSPs at 1.2-1.6 ms with much larger EPSPs (>1 mV) recorded around 2.5 ms. Stimulation of the opposite horizontal canal nerve produced inhibitory postsynaptic potentials (IPSPs) at a disynaptic latency of 1.6-1.8 ms that were depolarizing at membrane resting potentials around -60 mV. Injection of chloride ions increased IPSP amplitude, and current-clamp analysis showed the IPSP equilibrium potential to be near the membrane resting potential. Repeated electrical stimulation of either the excitatory or inhibitory horizontal canal vestibular nerve greatly increased the amplitude of the respective synaptic responses. These observations suggest that the large terminal arborizations of each VOR neuron imposes an electrotonic load requiring multiple action potentials to maximize synaptic efficacy. GABA antibodies labeled axons in the medial longitudinal fasciculus (MLF) some of which were hypothesized to originate from horizontal canal-activated inhibitory vestibular neurons. GABAergic terminal arborizations were distributed largely on the somata and proximal dendrites of oculomotor and trochlear motoneurons. These findings suggest that the species-specific horizontal canal inhibitory pathway exhibits similar electrophysiological and synaptic transmitter profiles as the anterior and posterior canal inhibitory projections to oculomotor and trochlear motoneurons. Electron microscopy showed axosomatic and axodendritic synaptic endings containing spheroidal synaptic vesicles to establish chemical excitatory synaptic contacts characterized by asymmetrical pre/postsynaptic membrane specializations as well as gap junctional contacts consistent with electrotonic coupling. Another type of axosomatic synaptic ending contained pleiomorphic synaptic vesicles forming chemical, presumed inhibitory, synaptic contacts on motoneurons that never included gap junctions. Altogether these data provide electrophysiological, immunohistochemical, and ultrastructural evidence for reciprocal excitatory/inhibitory organization of the novel vestibulooculomotor projections in adult flatfish. The appearance of unique second-order vestibular neurons linking the horizontal canal to vertical oculomotor neurons suggests that reciprocal excitation and inhibition are a fundamental, developmentally linked trait of compensatory eye movement circuits in vertebrates.     

Quantal synaptic transmission in phrenic motor nucleus.

1. The quantal nature of excitatory synaptic transmission was studied in respiratory interneurons and phrenic motoneurons of intact neonatal rat brain stem-spinal cord preparations in vitro. Synaptic currents were recorded with whole-cell patch-clamp recording techniques. 2. Because the most important factor for quantal detection is the ratio of quantal size to quantal standard deviation, factors that influence this ratio were evaluated so that experimental techniques that enhance this ratio could be defined. 3. Under favorable conditions, we directly observed quantal amplitude fluctuations in spontaneous excitatory postsynaptic currents (EPSCs) in spinal cord respiratory neurons. The quantal conductance size was 55-100 pS. With fast decay of these EPSCs, the charge reaching the soma for a single quantum is only approximately 15 fC (Vh = -80 mV). 4. We also studied miniature EPSC amplitude distributions. These were skewed, as previously reported; however, distinct quantal intervals were observed. Furthermore, in three cells tested, the quantal size in the miniature EPSC amplitude distribution was similar to the quantal size in the spontaneous EPSC amplitude distribution. 5. We conclude that excitatory synaptic transmission in the mammalian spinal cord is quantal and that the apparent skewness of miniature EPSC distributions results from summation of events with multiple quantal peak amplitudes.     

Quantal analysis of excitatory postsynaptic potentials induced in hippocampal neurons by activation of granule cells

Summary The values of quantal content (m) and quantal amplitude (q) of excitatory postsynaptic potentials (EPSPs) elicited in CA3 neurons by activation of granule cells were estimated in thin hippocampal sections maintained in vitro. For this purpose, DL-homocysteate was administered to granule cells, and trains of EPSPs that were typical for single granule cell activation were recorded from individual CA3 neurons. The amplitudes of the first and second EPSPs in each train were measured. From the mean and variance of the amplitudes of the EPSPs, the values of q and m were calculated. The values of m and q for the first EPSPs were estimated at 8.3 and 0.28 mV, respectively, on the average. Potentiation of the second EPSPs was accompanied by a two-fold increase in the values of m without changes in the values of q. Therefore, frequency potentiation in synapses between mossy fibers and CA3 neurons may be explained by an increase in number of released quanta. Amplitudes of EPSPs were found to fluctuate in a manner described by Poisson's law.Supported by a grant from the Ministry of Education of Japan     

Primary afferent-evoked synaptic responses and slow potential generation in rat substantia gelatinosa neurons in vitro

1. Primary afferent fiber-evoked synaptic responses and the mechanisms of spike and slow potential generation have been examined in adult rat substantia gelatinosa (SG) neurons in an in vitro transverse spinal cord slice preparation in which an attached dorsal root is retained. Intracellular recordings were made from SG neurons identified by morphological and electrophysiological criteria. Afferent fiber-evoked fast excitatory postsynaptic potentials (fast EPSPs) and slow EPSPs have been analyzed. 2. SG neurons had mean resting membrane potentials of -67.1 +/- 0.5 mV (mean +/- SE), mean input resistance of 257 +/- 17.7 (SE) M omega, and a mean time constant of 21.3 +/- 1.9 ms and exhibited spontaneous EPSPs. 3. Single low-intensity stimuli applied to the dorsal root using a suction electrode produced, in 70% of SG neurons, short-latency, presumed monosynaptic fast EPSPs which had a half decay time of 10-30 ms and an amplitude of 8-28 mV. The conduction velocity of afferent fibers evoking fast EPSPs was 2-7 m/s, corresponding to that of thinly myelinated A-delta-fibers. Dorsal root stimulation at higher intensities evoked, in 10% of SG neurons, long-latency and apparently monosynaptic EPSPs which had a time course and amplitude similar to that evoked by low-intensity stimulation. The conduction velocity of fibers evoking long-latency EPSPs was 0.4-2 m/s, suggesting that they constitute predominantly C-fibers. A-delta- and C-fiber-mediated fast EPSPs were detected in 20% of SG neurons examined. 4. Low-intensity stimuli produced slow EPSPs in 20% of SG neurons. Slow EPSPs were 3-15 mV in amplitude and of up to 2 min in duration. A-delta-fibers appeared to be responsible for the generation of slow EPSPs. Slow EPSPs were associated with an increase in membrane resistance and were decreased in amplitude with membrane hyperpolarization. 5. Action potentials in SG neurons had a mean amplitude of 76.3 +/- 1.1 mV and a mean duration of 1.0 +/- 0.07 ms. Na+ ions represent the main charge carrier during the rising phase of the action potential and Ca2+ ions contribute to the shoulder on the falling phase. 6. In 20% of SG neurons, subthreshold depolarizing pulses were followed by long-lasting slow-inactivating depolarizing potentials which were able to initiate spikes. The slow depolarizing potentials were blocked by TTX and enhanced by application of TEA and Ba2+, suggesting that Na+ and K+ are involved in this slow-inactivating potential.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alterations of synaptic action in chromatolysed motoneurones of the cat

1. Monosynaptic EPSPs in lumbosacral motoneurones undergoing chromatolysis were studied by intracellular recording from 7 to 20 days after section of the appropriate ventral roots of the cat.2. The maximum monosynaptic EPSPs evoked in chromatolysed motoneurones by afferent volleys from the biceps-semitendinosus or the triceps surae muscles ranged from 1.0 to 9.5 mV in amplitude. The time-to-peak of these EPSPs was 1.7 msec on the average. These values were significantly smaller and longer, respectively, than the amplitude and the time-to-peak of monosynaptic EPSPs observed in normal motoneurones. The long time-to-peak of EPSPs in chromatolysed motoneurones could not be accounted for by asynchronous transmitter release.3. The mean number of unit EPSPs responding to a single afferent impulse (m) in chromatolysed motoneurones was comparable to that found in normal motoneurones.4. The amplitude of unit EPSPs estimated from the mean EPSP amplitude and the m value following stimulation of a single afferent fibre was significantly smaller in chromatolysed motoneurones than in normal motoneurones. This difference was attributed to a difference in synaptic location.5. The shape of monosynaptic EPSPs evoked in chromatolysed motoneurones by stimulation of single afferent fibres was analysed on the basis of Rall's compartment model. The analysis suggested that there is a lack of the excitatory synaptic input to the cell body in chromatolysed motoneurones.6. Similar alterations were also found in IPSPs. The degree of change in synaptic responses evoked by stimulation of various pathways appears to depend on the synaptic location.7. Following the study of the interaction of several inputs on the motoneurone and of their dependence on membrane potential, a tentative model of the synaptic distribution of different pathways is proposed.     

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.     

APV-sensitive dorsal root afferent transmission to neonate rat sympathetic preganglionic neurons in vitro

1. Intracellular recordings were made from antidromically identified sympathetic preganglionic neurons (SPNs) in transverse thoracolumbar spinal cord slices from neonate (12- to 22-day-old) rats. 2. Electrical stimulation of dorsal roots or dorsal root entry zone elicited in SPNs an excitatory postsynaptic potential (EPSP) or multiple EPSPs of varying latencies. The EPSP could be graded by varying the stimulus intensity and, on reaching the threshold, discharged an action potential. 3. The dorsal root-evoked EPSPs had a mean synaptic latency of 2.6 ms (range: 1.2-11 ms), suggesting a polysynaptic pathway. The EPSPs were characteristically slow in onset with a mean rise time and half-decay time of 8.3 and 23 ms, respectively. 4. At the resting membrane potential of -50 to -60 mV, the amplitude of EPSPs recorded in normal (1.3 mM Mg2+) Krebs solution was reduced by membrane hyperpolarization or depolarization. In Mg2(+)-free solution, EPSPs were potentiated and reached threshold for spike discharge. 5. The EPSPs were suppressed by the nonselective glutamate receptor antagonist kynurenic acid (0.1-0.5 mM) and by the N-methyl-D-aspartate (NMDA) receptor antagonists D-2-amino-5-phosphonovaleric acid (APV; 1-10 microM) and ketamine (5-10 microM), but not by the quisqualate (QA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 1-10 microM). The latter depressed the EPSPs elicited by stimulation of lateral funiculus in the same SPNs. 6. NMDA applied by pressure elicited a depolarization in the SPNs. In normal Krebs solution the response was voltage dependent with the peak amplitude occurring around -60 mV; conditioning depolarization or hyperpolarization diminished the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unitary, quantal and miniature GABA-activated synaptic chloride currents in cultured neurons from the rat superior colliculus.

The aim of this study was to identify the conductance change induced by one quantum of gamma-aminobutyric acid from axonal release sites on cultured superior colliculus neurons. Unitary (single cell-activated) inhibitory postsynaptic currents and spontaneous synaptic activity were recorded with patch clamp techniques in the whole cell configuration while superfusing the entire neuron with normal saline. Miniature inhibitory postsynaptic currents were recorded in the presence of tetrodotoxin and in reduced [Ca2+]o/[Mg2+]o. In addition, the membrane area contributing to synaptic activity was limited to a narrow window of 50 microns. Smaller neurons were chosen for recording to render a standard deviation of the "instrumental" noise of less than 1.5 pA at a holding voltage of -80 mV. After two weeks in vitro, the percentage of synaptically connected tectal neurons exceeded 50%. At holding voltages of -80 mV (Cl- equilibrium potential -12 mV) minimal amplitudes of unitary inhibitory postsynaptic currents were as low as 7-10 pA, while maximal amplitudes exceeded 500 pA. The mean time to peak and time constant of decay were 3.0 and 34.4 ms, respectively (n = 31). Fluctuating unitary inhibitory postsynaptic currents were deemed to be compound postsynaptic responses. Multiple Gaussian equations could be fitted to the amplitude histograms of unitary postsynaptic currents. This procedure rendered a quantal size between 5.0 and 10.9 pA (mean 7.1 pA; S.D. 1.78 pA) in five neurons from mature cultures. The amplitudes of statistically determined quantal inhibitory postsynaptic currents were slightly smaller than the independent estimate from somatic miniature inhibitory postsynaptic currents. The latter had a mean amplitude of 9.1 pA (S.D. 3.3 pA, n = 23), a mean time to peak of 1.65 ms (n = 9), and a mean time constant of decay of 16.2 ms (n = 9). Single channel recording from outside-out patches showed three to four main conductance states ranging from 9 to 22 pS. Single channel closures at the 21-24 pS level were occasionally observed during relaxation of miniature currents. The small size of whole cell quantal inhibitory postsynaptic currents and somatic miniature currents indicates that one GABA quantum opened only 5-15 single Cl- channels.     

Mechanism for increased hippocampal synaptic strength following differential experience

Exposure to novel environments or behavioral training is associated with increased strength at hippocampal synapses. The present study employed quantal analysis techniques to examine the mechanism supporting changes in synaptic transmission that occur following differential behavioral experience. Measures of CA1 synaptic strength were obtained from hippocampal slices of rats exposed to novel environments or maintained in individual cages. The input/output (I/O) curve of extracellularly recorded population excitatory postsynaptic potentials (EPSPs) increased for animals exposed to enrichment. The amplitude of the synaptic response of the field potential was related to the fiber potential amplitude and the paired-pulse ratio, however, these measures were not altered by differential experience. Estimates of biophysical parameters of transmission were determined for intracellularly recorded unitary responses of CA1 pyramidal cells. Enrichment was associated with an increase in the mean unitary synaptic response, an increase in quantal size, and a trend for decreased input resistance and reduction in the stimulation threshold to elicit a unitary response. Paired-pulse facilitation, the percent of response failures, coefficient of variance, and estimates of quantal content were not altered by experience but correlated well with the mean unitary response amplitude. The results suggest that baseline synaptic strength is determined, to a large extent, by presynaptic release mechanisms. However, increased synaptic transmission following environmental enrichment is likely due to an increase in the number or efficacy of receptors at some synapses and the emergence of functional synaptic contacts between previously unconnected CA3 and CA1 cells.     

Excitatory postsynaptic potentials trigger a plateau potential in rat subthalamic neurons at hyperpolarized states

The subthalamic nucleus (STN) directly innervates the output structures of the basal ganglia, playing a key role in basal ganglia function. It is therefore important to understand the regulatory mechanisms for the activity of STN neurons. In the present study, we aimed to investigate how the intrinsic membrane properties of STN neurons interact with their synaptic inputs, focusing on their generation and the properties of the long-lasting, plateau potential. Whole cell recordings were obtained from STN neurons in slices prepared from postnatal day 14 (P14) to P20 rats. We found that activation of glutamate receptor-mediated excitatory synaptic potentials (EPSPs) evoked a plateau potential in a subpopulation of STN neurons (n = 13/22), in a voltage-dependent manner. Plateau potentials could be induced only when the cell was hyperpolarized to more negative than about -75 mV. Plateau potentials, evoked with a depolarizing current pulse, again only from a hyperpolarized state, were observed in about half of STN neurons tested (n = 162/327). Only in neurons in which a plateau potential could be evoked by current injection did EPSPs evoke plateau potentials. L-type Ca(2+) channels, Ca(2+)-dependent K(+) channels, and TEA-sensitive K(+) channels were found to be involved in the generation of the potential. The stability of the plateau potential, tested by the injection of a negative pulse current during the plateau phase, was found to be robust at the early phase of the potential, but decreased toward the end. As a result the early part of the plateau potential was resistant to membrane potential perturbations and would be able to support a train of action potentials. We conclude that excitatory postsynaptic potentials, evoked in a subpopulation of STN neurons at a hyperpolarized state, activate L-type Ca(2+) and other channels, leading to the generation of a plateau potential. Thus about half of STN neurons can transform short-lasting synaptic excitation into a long train of output spikes by voltage-dependent generation of a plateau potential.     

Interaction of dopamine D1 and NMDA receptors mediates acute clozapine potentiation of glutamate EPSPs in rat prefrontal cortex

The atypical antipsychotic drug clozapine effectively alleviates both negative and positive symptoms of schizophrenia via unclear cellular mechanisms. Clozapine may modulate both glutamatergic and dopaminergic transmission in the prefrontal cortex (PFC) to achieve part of its therapeutic actions. Using whole cell patch-clamp techniques, current-clamp recordings in layers V-VI pyramidal neurons from rat PFC slices showed that stimulation of local afferents (in 2 microM bicuculline) evoked mixed [AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors] glutamate receptor-mediated excitatory postsynaptic potentials (EPSPs). Clozapine (1 microM) potentiated polysynaptically mediated evoked EPSPs (V(Hold) = -65 mV), or reversed EPSPs (rEPSP, V(Hold) = +20 mV) for >30 min. The potentiated EPSPs or rEPSPs were attenuated by elevating [Ca(2+)](O) (7 mM), by application of NMDA receptor antagonist 2-amino5-phosphonovaleric acid (50 microM), or by pretreatment with dopamine D1/D5 receptor antagonist SCH23390 (1 microM) but could be further enhanced by a dopamine reuptake inhibitor bupropion (1 microM). Clozapine had no significant effect on pharmacologically isolated evoked NMDA-rEPSP or AMPA-rEPSPs but increased spontaneous EPSPs without changing the steady-state resting membrane potential. Under voltage clamp, clozapine (1 microM) enhanced the frequency, and the number of low-amplitude (5-10 pA) AMPA receptor-mediated spontaneous EPSCs, while there was no such changes with the mini-EPSCs (in 1 microM TTX). Taken together these data suggest that acute clozapine can increase spike-dependent presynaptic release of glutamate and dopamine. The glutamate stimulates distal dendritic AMPA receptors to increase spontaneous EPSCs and enabled a voltage-dependent activation of neuronal NMDA receptors. The dopamine released stimulates postsynaptic D1 receptor to modulate a lasting potentiation of the NMDA receptor component of the glutamatergic synaptic responses in the PFC neuronal network. This sequence of early synaptic events induced by acute clozapine may comprise part of the activity that leads to later cognitive improvement in schizophrenia.