Nonlinear-microscopy optical-pulse sources based on mode-locked semiconductor lasers.

We developed picosecond optical-pulse sources suitable for multiphoton microscopy based on mode-locked semiconductor lasers. Using external-cavity geometry, stable hybrid mode locking was achieved at a repetition rate of 500 MHz. Semiconductor optical amplifiers driven by synchronized electric pulses reached subharmonic optical-pulse repetition rates of 1-100 MHz. Two-stage Yb-doped fiber amplifiers produced optical pulses of 2 ps duration, with a peak power of a few kilowatts at a repetition rate of 10 MHz. These were employed successfully for nonlinear-optic bio-imaging using two-photon fluorescence, second-harmonic generation, and sum-frequency generation of synchronized two-color pulses.

Calcium-dependent persistent facilitation of spike backpropagation in the CA1 pyramidal neurons.

Sodium-dependent action potentials initiated near the soma are known to backpropagate over the dendrites of CA1 pyramidal neurons in an activity-dependent manner. Consequently, later spikes in a train have smaller amplitude when recorded in the apical dendrites. We found that depolarization and resultant Ca(2+) influx into dendrites caused a persistent facilitation of spike backpropagation. Dendritic patch recordings were made from CA1 pyramidal neurons in mouse hippocampal slices under blockade of fast excitatory and inhibitory synaptic inputs. Trains of 10 backpropagating action potentials induced by antidromic stimulation showed a clear decrement in the amplitude of later spikes when recorded in the middle apical dendrites. After several depolarizing current pulses, the amplitude of later spikes increased persistently, and all spikes in a train became almost equal in size. BAPTA (10 mm) contained in the pipette or low-Ca(2+) perfusing solution abolished this depolarization-induced facilitation, indicating that Ca(2+) influx is required. This facilitation was present in Galpha(q) knock-out mice that lack the previously reported muscarinic receptor-mediated enhancement of spike backpropagation. Therefore, these two forms of facilitation are clearly distinct in their intracellular mechanisms. Intracellular injection of either calmodulin binding domain (100 micrometer) or Ca(2+)/calmodulin-kinase II (CaMKII) inhibitor 281-301 (10 micrometer) blocked the depolarization-induced facilitation. Bath application of a membrane-permeable CaMKII inhibitor KN-93 (10 micrometer) also blocked the facilitation, but KN-92 (10 micrometer), an inactive isomer of KN-93, had no effect. These results suggest that increases in [Ca(2+)](i) cause persistent facilitation of spike backpropagation in the apical dendrite of CA1 pyramidal neuron by CaMKII-dependent mechanisms.

Hippocalcin-deficient mice display a defect in cAMP response element-binding protein activation associated with impaired spatial and associative memory.

Hippocalcin is a member of the neuronal calcium sensor (NCS) protein family that is highly expressed in hippocampal pyramidal cells and moderately expressed in the neurons of cerebral cortex, cerebellum and striatum. Here we examined the physiological roles of hippocalcin using targeted gene disruption. Hippocalcin-deficient (-/-) mice displayed no obvious structural abnormalities in the brain including hippocampal formation at the light microscopic level. Deletion of hippocalcin did not result in up-regulation of the hippocalcin-related proteins; neural visinin-like Ca(2+)-binding proteins (NVP) 1, 2, and 3. The synaptic excitability of hippocampal CA1 neurons appeared to be normal, as estimated by the shape of field excitatory postsynaptic potentials elicited by single- and paired-pulse stimuli, and by tetanic stimulation. However, N-methyl-d-aspartate stimulation- and depolarization-induced phosphorylation of cAMP-response element-binding protein (CREB) was significantly attenuated in -/- hippocampal neurons, suggesting an impairment in an activity-dependent gene expression cascade. In the Morris water maze test, the performance of -/- mice was comparable to that of wild-type littermates except in the probe test, where -/- mice crossed the previous location of the platform significantly less often than +/+ mice. Hippocalcin-deficient mice were also impaired on a discrimination learning task in which they needed to respond to a lamp illuminated on the left or right side to obtain food reinforcement. No abnormalities were observed in motor activity, anxiety behavior, or fear learning. These results suggest that hippocalcin plays a crucial role in the Ca(2+)-signaling pathway that underlies long-lasting neural plasticity and that leads to spatial and associative memory.

Control of Na+ spike backpropagation by intracellular signaling in the pyramidal neuron dendrites.

The integrative function of neurons depends on the somato-dendritic distribution and properties of voltage-gated ion channels. Sodium, potassium, calcium, and hyperpolarization-activated cyclic nucleotide-gated K+ (HCN) channels expressed in the dendrites can be modulated by a number of neurotransmitters and second-messenger systems. For example, activation of protein kinases leads to an increase in dendritic excitability by removing a slow inactivation of Na+ channels and decreasing the activity of transient K+ channels in the apical dendrites of hippocampal pyramidal neurons. Consequently, action potentials propagating along the dendrites can be modified significantly by a variety of neuromodulatory synaptic inputs.

Block of long-term potentiation by intracellular application of anti-phosphatidylinositol 4,5-bisphosphate antibody in hippocampal pyramidal neurons.

We examined the effects of black widow spider toxin and anti-phosphatidylinositol 4,5-bisphosphate antibody on the changes in excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents accompanying long-term potentiation using whole-cell recording from hippocampal CA1 pyramidal neurons of rodents. In the presence of black widow spider toxin, tetanic stimulation of input fibers produced a short-lived potentiation followed by a gradual decline of the excitatory postsynaptic current amplitude. With an anti-phosphatidylinositol 4,5-bisphosphate antibody containing pipette, tetanus elicited only decremental potentiation of excitatory postsynaptic currents with a reduced frequency of spontaneous excitatory postsynaptic currents, suggesting inhibition of retrograde reinforcement from the antibody-injected neuron. With both black widow spider toxin and anti-phosphatidylinositol 4,5-bisphosphate antibody, neurons showed a rapid depression of excitatory postsynaptic currents after tetanus. The results indicate that time-dependent interactions between presynaptic terminals and the postsynaptic spikes take place during long-term potentiation.

Abnormal Ca2+ homeostasis before cell death revealed by whole cell recording of ischemic CA1 hippocampal neurons.

Slices were made from the hippocampus of gerbils following transient ischemia achieved by clamping the carotid arteries for 5 min, and changes in the electrophysiology of CA1 pyramidal neurons were studied by whole cell patch-clamp recording as well as conventional intracellular recording. The great majority of CA1 neurons in slices made 2.5-3 days after ischemia showed reduced resting potentials and were easily depolarized by prolonged low-frequency stimulation or by tetanic stimulation of the Schaffer collateral/commissural input. This stimulus-induced depolarization was accelerated by intracellular injection of D-myo-inositol 1,4,5-triphosphate, which depolarized membrane potentials towards 0 mV without synaptic input stimulation. Intracellular application of BAPTA, a Ca2+ chelator, effectively blocked the stimulus-induced depolarization. When recording from ischemic neurons with patch pipettes containing both D-myo-inositol 1,4,5-triphosphate and BAPTA, excitatory postsynaptic currents were transiently potentiated by stimulation, but the membrane potential did not show stimulus-induced depolarization and remained steady for long periods. These results lend support to the view that the intracellular Ca2+ regulation system is severely disturbed following ischemia, and that input fiber stimulation leads to abnormal Ca2+ accumulation in ischemic neurons resulted in neuronal death. The reduction of free Ca2+ inside the ischemic neuron by BAPTA apparently saves neurons which are otherwise destined to delayed neuronal death.

Inositol 1,3,4,5-tetrakisphosphate as a mediator of neuronal death in ischemic hippocampus.

Selective death of CA1 pyramidal neurons after transient forebrain ischemia has attracted interest for its possible relation to the pathogenesis of memory deficits and dementia. Using whole cell patch-clamp recording from CA1 pyramidal neurons in hippocampal slices of gerbils after ischemia we studied the intracellular signaling mechanisms related to the phosphoinositide cycle. Intracellular application of an antibody against phosphatidylinositol 4,5-bisphosphate rescued ischemic neurons from stimulus-induced irreversible depolarization. Furthermore, application of inositol 1,3,4,5-tetrakisphosphate in normal cells caused an irreversible depolarization in response to synaptic input, which mimicked the deterioration of ischemic neurons. Depolarization of both ischemic and normal neurons in the presence of inositol 1,3,4,5-tetrakisphosphate was prevented by the addition of the Ca2+ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetate. Application of antibody against inositol 1,4,5-triphosphate 3-kinase, which blocks formation of inositol 1,3,4,5-tetrakisphosphate, also protected against cell deterioration. Our results suggest that the vulnerability of hippocampal pyramidal neurons following ischemia is caused by a disturbed phosphoinositide cascade, with one metabolite, inositol 1,3,4,5-tetrakisphosphate, playing a key role in the induction of Ca2+ accumulation, which leads to neuronal death.

Single glutamate channels in CA1 pyramidal neurones after transient ischaemia.

Patch clamp recordings were made from CA1 pyramidal neurones to study changes in the glutamate receptor subtypes in the gerbil hippocampus after transient ischaemia. In whole-cell recordings, the maximum chord conductances of AMPA currents in ischaemic neurones were increased over those of control neurones but NMDA-induced currents in the ischaemic neurones were smaller than the control. In AMPA-activated single channel currents, an open time histogram of the control neurones was well fitted by a single exponential function whereas in the ischaemic patches it was fitted by a double exponential function, indicating that currents consisted of at least two kinetically different types. These functional changes of the glutamate receptor channels may contribute to the abnormalities of the excitatory synaptic currents recorded in post-ischaemic CA1 neurones.

Regional differences in the cat caudate nucleus as to the effectiveness in inducing contraversive head-turning by electrical stimulation.

An attempt was made to re-examine regional differences in the cat caudate nucleus as to the effectiveness in inducing contraversive head-turning by electrical stimulation and to analyze the time course of head-turning quantitatively. In 5 of the total 9 cats, the right sensorimotor cortex and its surrounding areas had been ablated chronically. While the awake, unrestrained cat maintained a stable standing posture facing forward, stimulation was applied systematically to various points in and around the caudate nucleus with a movable stimulating electrode. Trains of stimulating current pulses of less than 300 microA were given, mostly at a rate of 100 Hz for 5 s. In most experiments in which stimulation was given to the side of the intact cerebral cortex, stimulation of caudal portions of the head of the caudate nucleus was effective in inducing contraversive head-turning, but that of its rostral portions was ineffective. In experiments on the side of chronic cortical ablation, similar results were obtained. These results suggested that head-turning induced by stimulation of the caudate nucleus was brought about not by the activation of the corticofugal fibers from these cortical areas by a current spreading to the internal capsule, but by the activation of caudate neurons. Hence, it was demonstrated that there were regional differences in the cat caudate nucleus as to the effectiveness in inducing head-turning. The mean of the shortest latencies of the onset of head-turning for individual stimulation points was 396 ms (S.D., 210 ms) for the side of the intact cerebral cortex, and 454 ms (S.D., 289 ms) for the side of the cortical ablation. Statistically, there was no significant difference between them. Therefore, it was further revealed that the elimination of the sensorimotor cortex did not affect the caudate-induced head-turning in terms of the latency of its onset.

EMG activities of neck muscles underlying lateral flexion of the neck during head-turning induced by electrical stimulation of the caudate nucleus in cats.

Patterns of EMG activities of neck muscles underlying the initiation of head-turning, induced by stimulation of the caudate nucleus, were analyzed with special reference to temporal relations between the onset of head-turning and that of changes in EMG activities. These patterns were compared with those associated with the initiation of lateral flexion of the neck which occurred without electrical stimulation of the caudate nucleus in order to examine whether the caudate-induced head-turning was initiated via the same muscular system as that used in non-caudate-induced head movements. Experiments were carried out using 5 awake, unrestrained cats which were trained to stand still with one limb on each of 4 footplates. Trains of stimulating current pulses were applied to several stimulation points in the caudate nucleus while the animal maintained a stable standing posture with its neck extended. Head movements in the horizontal plane and EMGs of 6 neck muscles (splenius, longissimus cervicis, obliquus capitis caudalis, biventer cervicis, complexus and cervical multifidus) were recorded. Patterns of EMG activities of neck muscles around the onset of the caudate-induced head-turning were characterized by an increase in activity of the splenius, the longissimus cervicis and the obliquus capitis caudalis muscles, and by a decrease in activity of the complexus, the biventer cervicis and the cervical multifidus on the side of flexion. It is suggested that an increase in activity of the splenius, the longissimus cervicis and the obliques capitis caudalis muscles was responsible for the initiation of this evoked response. In non-caudate-induced lateral flexion of the neck, patterns of activities of neck muscles were similar to those in caudate-induced head-turning. It is therefore concluded that the caudate-induced head-turning as an evoked behavioral response was initiated through a muscular system similar to that utilized for similar head movements occurring without electrical stimulation of the caudate nucleus, although the pathways involved are thought to be different.

Stimulation of the caudate nucleus induces contraversive saccadic eye movements as well as head turning in the cat.

The effects of stimulation of the caudate nucleus were investigated in alert cats, with special reference to the induction of eye and head movements. Stimulation of caudal portions of the caudate nucleus on one side with trains of current pulses induced gaze shifts towards the contralateral side. When the head of the animal was restrained, the majority of evoked eye movements were single conjugate saccades. The amplitude and direction of the evoked saccade varied depending on the initial eye position. The amplitude of the horizontal component tended to be larger for saccades initiated from more ipsilateral positions, and became gradually smaller as the initial eye position shifted to the contralateral side. If the eye was far into the contralateral positions, no saccades were induced. Furthermore, the saccades tended to have a downward component when the eye was initially focused upward, and an upward component when the eye was focused downward. When the head was made free to move, the same stimulation induced a sequence of contraversive staircase gaze shifts composed of coordinated eye and head movements. The eye movements in the orbit resembled nystagmus, consisting of contraversive saccades followed by reverse compensatory movements. The head turning, though smooth and continuous, was also suggested to consist of a series of movements coupled with saccadic eye movements. This study indicates a potential role of the caudate nucleus in the control of orienting reflexes.

Cyclic changes in NMDA receptor activation in hippocampal CA1 neurons after ischemia.

We studied N-methyl-D-aspartate (NMDA) receptor-mediated synaptic potentials in CA1 pyramidal neurons using hippocampal slices of gerbils after transient forebrain ischemia. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and bicuculline, stimulation of Schaffer collateral/commissural fibers induced field excitatory postsynaptic potentials (fEPSP) activated by NMDA receptors. We found that in many slices after ischemia, prolonged low-frequency stimulation (0.1-10 Hz) caused repeated depression and potentiation of the NMDA-mediated fEPSP. Changes in fEPSP amplitude were dependent on stimulus frequency and the cycle frequency ranged from 0.08 to 2.5 cycles/min. These cyclic changes were blocked by application of BAPTA-AM, a membrane-permeable Ca2+ chelator, but were little affected by application of verapamil or by lowering the Ca2+ in bathing solution. Intracellular recordings from CA1 neurons revealed that low-frequency stimulation caused periodic depolarizations of membrane potential accompanied by depression of the excitatory postsynaptic potentials. The cyclic changes of fEPSPs were blocked by inhibitors of protein kinase C (PKC) but were unaffected by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) or myosin light-chain kinase (MLCK). These results suggest that stimulus-dependent NMDA-receptor activation, mediated by PKC, takes place in the postischemic CA1 neurons and that the cyclic change may reflect abnormal intracellular Ca2+ signaling processes leading to neuronal degeneration.

Modulation of synaptic transmission in hippocampal CA1 neurons by a novel neurotoxin (beta-pompilidotoxin) derived from wasp venom.

We examined the effects of beta-pompilidotoxin (beta-PMTX), a neurotoxin derived from wasp venom, on synaptic transmission in the mammalian central nervous system (CNS). Using hippocampal slice preparations of rodents, we made both extracellular and intracellular recordings from the CA1 pyramidal neurons in response to stimulation of the Schaffer collateral/commissural fibers. Application of 5-10 microM beta-PMTX enhanced excitatory postsynaptic potentials (EPSPs) but suppressed the fast component of the inhibitory postsynaptic potentials (IPSPs). In the presence of 10 microM bicuculline, beta-PMTX potentiated EPSPs that were composed of both non-NMDA and NMDA receptor-mediated potentials. Potentiation of EPSPs was originated by repetitive firings of the presynaptic axons, causing summation of EPSPs. In the presence of 10 microM CNQX and 50 microM APV, beta-PMTX suppressed GABA(A) receptor-mediated fast IPSPs but retained GABA(B) receptor-mediated slow IPSPs. Our results suggest that beta-PMTX facilitates excitatory synaptic transmission by a presynaptic mechanism and that it causes overexcitation followed by block of the activity of some population of interneurons which regulate the activity of GABA(A) receptors.

Spontaneous excitatory postsynaptic currents in hippocampal CA1 pyramidal neurons of the gerbil after transient ischemia.

The changes in the spontaneous excitatory postsynaptic currents (sEPSCs) after transient cerebral ischemia were studied using whole-cell recording from CA1 pyramidal neurons in the gerbil. In neurons recorded 1-2 days after ischemia, sEPSCs had a slowed time course with the decay time constant fitted by a single exponential and it progressively increased after ischemia. Frequency and amplitude distribution of sEPSCs in ischemic neurons were not significantly different from those in the control neurons. The results support the view that abnormal non-N-methyl-D-aspartic acid currents originate at the degenerated postsynaptic site, unrelated to the presynaptic releasing mechanisms.

Frequency-dependent signal processing in apical dendrites of hippocampal CA1 pyramidal cells.

Depending on an animal's behavioral state, hippocampal CA1 pyramidal cells receive distinct patterns of excitatory and inhibitory synaptic inputs. The time-dependent changes in the frequencies of these inputs and the nonuniform distribution of voltage-gated channels lead to dynamic fluctuations in membrane conductance. In this study, using a whole-cell patch-clamp method, we attempted to record and analyze the frequency dependencies of membrane responsiveness in Wistar rat hippocampal CA1 pyramidal cells following noise current injection directly into dendrites and somata under pharmacological blockade of all synaptic inputs. To estimate the frequency-dependent properties of membrane potential, membrane impedance was determined from the voltage response divided by the input current in the frequency domain. The cell membrane of most neurons showed low-pass filtering properties in all regions. In particular, the properties were strongly expressed in the somata or proximal dendrites. Moreover, the data revealed nonuniform distribution of dendritic impedance, which was high in the intermediate segment of the apical dendritic shaft (∼220-260µm from the soma). The low-pass filtering properties in the apical dendrites were more enhanced by membrane depolarization than those in the somata. Coherence spectral analysis revealed high coherence between the input signal and the output voltage response in the theta-gamma frequency range, and large lags emerged in the distal dendrites in the gamma frequency range. Our results suggest that apical dendrites of hippocampal CA1 pyramidal cells integrate synaptic inputs according to the frequency components of the input signal along the dendritic segments receiving the inputs.

Muscarinic modulation of spike backpropagation in the apical dendrites of hippocampal CA1 pyramidal neurons.

In pyramidal neurons from the CA1 region of the rat hippocampus, Na+-dependent action potentials backpropagate over the dendrites in an activity-dependent manner. Consequently, later spikes in a train have smaller amplitudes when recorded in the apical arbors. We studied the effect of the cholinergic agonist carbachol (CCh) on this pattern of activity when spikes were evoked synaptically or antidromically in the transverse slice preparation. Concentrations as low as 1 microM were effective in reversing the modulation, making the amplitude of all spikes in a train equal and independent of the frequency of spike firing. CCh did not change the propagation of the first spike in a train. These effects of CCh were blocked by 1 microM atropine, showing that only muscarinic receptors were involved. The effects of CCh on the pattern of spike propagation were observed in the proximal and middle dendrites, but recordings in the distal dendrites (>300 micron from the soma) showed that CCh did not boost the amplitude in this region. Intracellular BAPTA (10 mM) or EGTA (10 mM) had no effect on activity-dependent backpropagation but blocked the effect of CCh. Backpropagating spikes caused increases in [Ca2+]i at all dendritic locations. In the middle and distal dendrites these increases normally peaked at the time of the first few large action potentials. In association with the enhancement of spike backpropagation, CCh increased the amplitude and duration of the train-evoked [Ca2+]i changes. These effects of CCh on dendritic spike potentials and associated [Ca2+]i changes may be important in modulating synaptic integration and plasticity in these neurons.

IPSPs modulate spike backpropagation and associated [Ca2+]i changes in the dendrites of hippocampal CA1 pyramidal neurons.

1. We studied the effects of synaptic inhibition on backpropagating Na+ spikes in the apical dendrites of CA1 pyramidal neurons in transverse slices from the rat hippocampus. Action potentials were evoked synaptically by stimulation in the stratum radiatum or antidromically by stimulation in the alveus. 2. Inhibitory postsynaptic potentials, evoked by stimulation in the stratum lacunosum moleculare, reduced the amplitude of single spikes in the distal dendrites but did not change the amplitudes in the somatic or proximal regions. Inhibition also reduced the spike-associated [Ca2+]i changes in the distal dendrites but had little effect on the changes in the proximal part of the cell. Both of these results are consistent with inhibition converting actively backpropagating spikes into passively spreading potentials at some point in the arbor. 3. In most cells, the spike amplitude reduction in the distal dendrites was blocked by bicuculline methiodide (10 microM) and inhibition was most effective when evoked in a time window < 10 ms preceding the action potential. This suggests that the amplitude reduction was due to a conductance shunt activated by gamma-aminobuturic acid-A (GABAA) receptors. Synaptically evoked GABAB responses were detected but usually did not block spike propagation. 4. Direct hyperpolarization in the distal dendrites was also effective in blocking antidromically evoked spike backpropagation but probably does not contribute when the action potentials are evoked synaptically. 5. This effect of inhibition is different from its usual function in synaptic integration because spike generation and propagation down the axon are not significantly affected. This kind of inhibition might be important in regulating transient [Ca2+]i changes in the dendrites including individual dendritic branches.

Elevation of intracellular Na+ induced by hyperpolarization at the dendrites of pyramidal neurones of mouse hippocampus.

1. Whole-cell recordings were made from CA1 pyramidal cells in mouse hippocampal slices with patch pipettes containing the sodium indicator dye SBFI (sodium binding benzofuran isophthalate). Using a high-speed imaging system, we investigated changes in intracellular sodium concentration, [Na+]i, in response to hyperpolarizing pulses applied to the soma. 2. In current-clamp recordings, we detected increases in [Na+]i during negative current injection. Hyperpolarization-induced [Na+]i elevation was more prominent in the middle apical dendrites than in the soma. 3. In the voltage-clamp mode, hyperpolarization induced rapid increases in [Na+]i at the apical dendrites that were significantly faster than those at the soma. The signals were not affected by bath application of 1 microM TTX, but were reduced by 5 mM CsCl. 4. Changes in membrane potential recorded from the apical dendrites in response to negative currents were significantly smaller than those recorded from the soma. In the presence of 5 mM CsCl, the I-V relationships measured at the soma and the dendrites became almost identical, indicating that CsCl-sensitive components are predominantly in the apical dendrites. 5. These results suggest that hyperpolarization-induced [Na+]i elevations reflect Na+ influx through the non-selective cation channel (Ih channel), and that this channel is distributed predominantly in the apical dendrites. The non-uniform Na+ influx may contribute to integrative functions of the dendrites.

Ca(2+)-dependent non-NMDA receptor-mediated synaptic currents in ischemic CA1 hippocampal neurons.

1. The changes in excitatory postsynaptic currents (EPSCs) after transient cerebral ischemia were studied using whole-cell recording from CA1 pyramidal neurons in gerbils. In 64% (18 of 28) neurons recorded 1.5-3 days after ischemia, EPSCs showed a markedly slowed time course that was never seen in normal control neurons. 2. The slow EPSCs were not affected by an N-methyl-D-aspartate (NMDA) receptor antagonist [DL-2-aminophosphonovalerate (APV); 100 microM] but were abolished by a non-NMDA receptor antagonist [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); 10 microM], indicating that the slow EPSCs were mostly composed of non-NMDA current. 3. The slow non-NMDA EPSCs had rise times ranging from 1.2 to 7.3 ms and decay time constants between 11.5 and 56.3 ms. In normal neurons the rise time of the non-NMDA component of EPSCs ranged from 1.6 to 7.5 ms and the decay time constants ranged from 4.9 to 27.3 ms. 4. The reversal potential of the slow EPSCs in ischemic neurons was not changed by replacing 50% of the NaCl in the external solution with sodium isethionate. Bath application of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 100 microM) had no effect on the slow EPSCs. Therefore Cl- current is not responsible for the slow EPSCs. 5. When external Ca2+ concentration was reduced to half of control, the decay time constant of the slow EPSCs decreased to 50 +/- 25%, mean +/- SD. In addition, bath application of a cell-permeable Ca2+ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,-N',N'-tetraacetyl,tetr aacetoxymethyl ester(BAPTA-AM), reduced the decay time constant.(ABSTRACT TRUNCATED AT 250 WORDS)