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Fluorescent Ca2+ indicators are widely used to measure the intracellular Ca2+ concentration ([Ca2+]i) in living cells, including neurons. By calibrating an indicator in solutions that mimic the main ionic constituents of the actual cytoplasm, [Ca2+]i can be determined from the measured fluorescence intensity. However, different studies have reported considerably different Ca2+-binding affinities (Kd) for the same indicator, even though they used calibrating solutions with similar compositions. In this paper, we present a method to accurately determine the Kd values of non-ratiometric Ca2+ indicators in solutions that mimicked a standard patch-clamp internal solution. The free Ca2+ concentration ([Ca2+]) in these solutions, which was set by either EGTA or HEDTA, was measured with a Ca2+-selective macroelectrode. We found that such a measurement was critical for an accurate calibration of the Ca2+ indicators. The Kd values of OGB-1, OGB-6F, fluo-5F, and fluo-4FF were 0.26 ± 0.01, 8.7 ± 0.4, 1.00 ± 0.05, and 23.0 ± 0.7 μM, respectively. Calculating [Ca2+] with Maxchelator, a widely used computer program, led to a significant underestimation of the Kd values of OGB-6F, fluo-5F, and fluo-4FF. This is because the purity of EGTA was considerably less than that advertised by the manufacturer. In addition, the Kd value of HEDTA was overestimated by Maxchelator. Therefore, besides batch-to-batch variations, the fact that [Ca2+] in the calibrating solutions of many studies was estimated with Maxchelator is very likely a reason for the different published values of Kd of Ca2+ indicators. Using a reaction-diffusion model to reproduce Ca2+ rises in a nerve terminal, we further showed that incorrect calibration of fluorescent Ca2+ indicators can underlie the large variation of the endogenous Ca2+ binding ratio between different types of excitatory synapses.

Although the endoplasmic reticulum (ER) is present throughout axons, and IP3 and ryanodine receptors are widely expressed in nerve terminals, whether Ca2+ release from presynaptic stores contributes to action potential (AP)-evoked Ca2+ transients remains controversial. We investigated the release of Ca2+ from ER stores in boutons en passant of neocortical layer 5 pyramidal neurons. A hallmark of these stores is that they spontaneously release Ca2+ at a low frequency. Using a high-affinity Ca2+ indicator, we documented and characterised such spontaneous Ca2+ transients (sCaTs), which occurred at a rate of ~0.2 per min and raised the intracellular Ca2+ concentration ([Ca2+]i) by ~2 µM in the absence of exogenous buffers. Caffeine increased the average frequency of sCaTs by 90%, without affecting their amplitude and decay kinetics. Therefore, presynaptic ryanodine receptors were likely involved. To determine if presynaptic ER stores contribute to intracellular Ca2+ accumulation during repetitive stimulation, we measured [Ca2+]i during 2 s long trains of APs evoked at 10-50 Hz. We found that for frequencies

1. Coordinated oscillations in diameter occur spontaneously in cerebral vessels and depend on the opening of voltage dependent calcium channels. However, the mechanism that induces the initial depolarisation has remained elusive. We investigated the involvement of canonical transient receptor potential (TRPC) channels, which encode nonselective cation channels passing Na(+) and Ca(2+) currents, by measuring changes in diameter, intracellular Ca(2+) and membrane potential in branches of juvenile rat basilar arteries. 2. Removal of extracellular Ca(2+) abolished vasomotion and relaxed arteries, but paradoxically produced depolarisation. 3. Decrease in temperature to 24 degrees C or inhibition of phospholipase C (PLC) abolished vasomotion, hyperpolarised and relaxed arteries and decreased intracellular Ca(2+). 4. Reduction in the driving force for Na(+) through decrease in extracellular Na(+) produced similar effects and prevented the depolarisation elicited by removal of extracellular Ca(2+). 5. Nonselective TRP channel blockers, SKF96365 and gadolinium, mimicked the effects of inhibition of the PLC pathway. 6. Depolarisation of vessels in which TRP channels were blocked with SKF96365 reinstated vascular tone and vasomotion. 7. Quantitative polymerase chain reaction revealed TRPC1 as the predominantly expressed TRPC subtype. 8. Incubation with a function blocking TRPC1 antibody delayed the onset of vasomotion. 9. We conclude that nonselective cation channels contribute to vasoconstriction and vasomotion of cerebral vessels by providing an Na(+)-induced depolarisation that activates voltage dependent calcium channels. Our antibody data suggest the involvement of TRPC1 channels that might provide a target for treatment of therapy-refractory vasospasm.

There are two types of excitatory neurons within layer IV of rat somatosensory cortex: star pyramidal (SP) and spiny stellate cells (SS). We examined the intrinsic properties and connectivity between these neurons to determine differences in function. Eighty-four whole cell recordings of pairs of neurons were examined in slices of rat barrel cortex at 36 ± 1°C. Only minimal differences in intrinsic properties were found; however, differences in synaptic strength could clearly be shown. Connections between homonymous pairs (SS–SS or SP–SP) had a higher efficacy than heteronymous connections. This difference was mainly a result of quantal content. In 42 pairs, synaptic dynamics were examined. Sequences of action potentials (3–20 Hz) in the presynaptic neuron consistently caused synaptic depression ( Ē2/ Ē1 = 0.53 ± 0.18). The dominant component of depression was release-independent; this depression occurred even when preceding action potentials had failed to cause a response. The release-dependence of depression was target specific; in addition, release-independence was greater for postsynaptic SPs. In a subset of connections formed only between SP and any other cell type (43%), synaptic efficacy was dependent on the presynaptic membrane potential ( Vm); at −55 mV, the connections were almost silent, whereas at −85 mV, transmission was very reliable. We suggest that, within layer IV, there is stronger efficacy between homonymous than between heteronymous excitatory connections. Under dynamic conditions, the functional connectivity is shaped by synaptic efficacy at individual connections, by Vm, and by the specificity in the types of synaptic depression.

Synaptic transmission between pairs of excitatory neurones in layers V (N= 38) or IV (N= 6) of somatosensory cortex was examined in a parasagittal slice preparation obtained from young Wistar rats (14–18 days old). A combined experimental and theoretical approach reveals two characteristics of short-term synaptic depression. Firstly, as well as a release-dependent depression, there is a release-independent component that is evident in smaller postsynaptic responses even following failure to release transmitter. Secondly, recovery from depression is activity dependent and is faster at higher input frequencies. Frequency-dependent recovery is a Ca2+-dependent process and does not reflect an underlying augmentation. Frequency-dependent recovery and release-independent depression are correlated, such that at those connections with a large amount of release-independent depression, recovery from depression is faster. In addition, both are more pronounced in experiments performed at physiological temperatures. Simulations demonstrate that these homeostatic properties allow the transfer of rate information at all frequencies, essentially linearizing synaptic responses at high input frequencies.

Voltage-clamp recordings from layer II neurones in somatosensory cortex of rats aged between 12 and 17 days showed a high frequency of spontaneous postsynaptic currents (sPSCs), which on average was 33 +/- 13 Hz (s.d.). sPSCs were mediated largely by glutamatergic AMPA receptors. Their rates and amplitudes were independent of blocking sodium channels with 1 microM tetrodotoxin (TTX). Most of them, therefore, represent genuine miniature excitatory postsynaptic currents (mEPSCs). The rise time of the fastest (10 %) mEPSCs was 288 +/- 86 micros (10-90 %) and the half-width was 1073 +/- 532 micros. The amplitude was -5.9 +/- 1.1 pA with a coefficient of variation (CV) of 0.44 +/- 0.14. The rate of mEPSCs was very temperature sensitive with a Q(10) (33-37 degrees C) of 8.9 +/- 0.9. Due to this temperature sensitivity, we estimated that the microscope lamp contributed an increase in temperature of about 4 degrees C to the tissue in the focal volume of the condenser. Cell-type differences in the rate of mEPSCs were found between pyramidal/multipolar and bipolar cells. The latter had a frequency of about a third of that seen in the other cell groups. Recordings in layer II are ideally suited to investigate mechanisms of spontaneous transmitter release.

The role of glycinergic and GABAergic systems in mediating spontaneous synaptic transmission in newly formed neural networks was examined in motoneurons in the developing rat spinal cord. Properties of action potential–independent miniature inhibitory postsynaptic currents (mIPSCs) mediated by glycine and GABAA receptors (GlyR and GABAAR) were studied in spinal cord slices of 17- to 18-day-old embryos ( E17–18) and 1- to 3-day-old postnatal rats ( P1–3). mIPSC frequency and amplitude significantly increased after birth, while their decay time decreased. To determine the contribution of glycinergic and GABAergic synapses to those changes, GlyR- and GABAAR-mediated mIPSCs were isolated based on their pharmacological properties. Two populations of pharmacologically distinct mIPSCs were recorded in the presence of glycine or GABAA receptors antagonists: bicuculline-resistant, fast-decaying GlyR-mediated mIPSCs, and strychnine-resistant, slow-decaying GABAAR-mediated mIPSCs. The frequency of GABAAR-mediated mIPSCs was fourfold higher than that of GlyR-mediated mIPSCs at E17–18, indicating that GABAergic synaptic sites were functionally dominant at early stages of neural network formation. Properties of GABAAR-mediated mIPSC amplitude fluctuations changed from primarily unimodal skewed distribution at E17–18 to Gaussian mixtures with two to three discrete components at P1–3. A developmental shift from primarily long-duration GABAergic mIPSCs to short-duration glycinergic mIPSCs was evident after birth, when the frequency of GlyR-mediated mIPSCs increased 10-fold. This finding suggested that either the number of glycinergic synapses or the probability of vesicular glycine release increased during the period studied. The increased frequency of GlyR-mediated mIPSCs was associated with more than a twofold increase in their mean amplitude, and in the number of motoneurons in which mIPSC amplitude fluctuations were best fitted by multi-component Gaussian curves. A third subpopulation of mIPSCs was apparent in the absence of glycine and GABAA receptor antagonists: mIPSCs with both fast and slow decaying components. Based on their dual-component decay time and their suppression by either strychnine or bicuculline, we assumed that these were generated by the activation of co-localized postsynaptic glycine and GABAA receptors. The contribution of mixed glycine-GABA synaptic sites to the generation of mIPSCs did not change after birth. The developmental switch from predominantly long-duration GABAergic inhibitory synaptic currents to short-duration glycinergic currents might serve as a mechanism regulating neuronal excitation in the developing spinal networks.

Most excitatory synapses on neocortical pyramidal cells are located on dendrites, which are endowed with a variety of active conductances. The main origin for action potentials is thought to be at the initial segment of the axon, although local regenerative activity can be initiated in the dendrites. The transfer characteristics of synaptic voltage and charge along the dendrite to the soma remains largely unknown, although this is an essential determinant of neural input-output transformations. Here we perform dual whole-cell recordings from layer V pyramidal cells in slices from somatosensory cortex of juvenile rats. Steady-state and sinusoidal current injections are applied to characterize the voltage transfer characteristics of the apical dendrite under resting conditions. Furthermore, dendrosomatic charge and voltage transfer are determined by mimicking synapses via dynamic current-clamping. We find that around rest, the dendrite behaves like a linear cable. The cutoff frequency for somatopetal current transfer is around 4 Hz, i.e., synaptic inputs are heavily low-pass filtered. In agreement with linearity, transfer resistances are reciprocal in opposite directions, and the centroids of the synaptic time course are on the order of the membrane time constant. Transfer of excitatory postsynaptic potential (EPSP) charge, but not peak amplitude, is positively correlated with membrane potential. We conclude that the integrative properties of dendrites in infragranular neocortical pyramidal cells appear to be linear near resting membrane potential. However, at polarized potentials charge transferred is voltage-dependent with a loss of charge at hyperpolarized and a gain of charge at depolarized potentials.

Cowan, A. I., C. Stricker, L. J. Reece, and S. J. Redman. Long-term plasticity at excitatory synapses on aspinous interneurons in area CA1 lacks synaptic specificity. J. Neurophysiol. 79: 13–20, 1998. The synaptic specificity of long-term potentiation (LTP) was examined at synapses formed on aspinous dendrites of interneurons whose somata were located in the pyramidal cell layer of hippocampal area CA1. Intracellular recordings from slices prepared from rats were used to monitor excitatory postsynaptic potentials (EPSPs) elicited by extracellular stimulation in stratum radiatum. Two synaptic inputs were evoked at 0.5 Hz by stimulating axons adjacent to stratum pyramidale and s. lacunosum-moleculare. After obtaining baseline recordings (≥10 min), one of the EPSPs was conditioned. The protocol involved tetanic stimulation, sometimes combined with somatic depolarization. Low-frequency stimulation of the two pathways was then resumed and EPSPs were recorded for

1. Dual intracellular recordings were made from synaptically coupled pyramidal cell-to-interneurone pairs (n = 5) of the cat visual cortex in vitro. Pre- and postsynaptic neurones were labelled with biocytin, followed by correlated light and electron microscopic analysis to determine all sites of synaptic interaction. 2. Pyramidal neurones in layers II-III elicited monosynaptic EPSPs in three distinct classes of smooth dendritic local-circuit neurones, namely basket cells (n = 3), a dendrite-targeting cell (n = 1) and a double bouquet cell (n = 1). Unitary EPSPs in basket cells were mediated by one, two, and two synaptic junctions, whereas the pyramid-to-dendrite-targeting cell and pyramid-to-double bouquet cell interaction were mediated by five and seven synaptic junctions, respectively. Recurrent synaptic junctions were found on all somato-dendritic compartments, with a tendency to be clustered close to the soma on the double bouquet and dendrite-targeting cells. The latter interneurones were reciprocally connected with pyramidal cells. 3. Unitary EPSPs had an average peak amplitude of 1005 +/- 518 microV, fast rise times (10-90%; 0.67 +/- 0.25 ms) and were of short duration (at half-amplitude, 4.7 +/- 1.0 ms). Their decay was monoexponential (tau = 7.8 +/- 4.3 ms) at hyperpolarized membrane potentials and appeared to be shaped by passive membrane properties (tau = 9.2 +/- 8.5 ms). All parameters of concomitantly recorded spontaneous EPSPs were remarkably similar (mean amplitude, 981 +/- 433 microV; mean rise time, 0.68 +/- 0.18 ms; mean duration, 4.7 +/- 1.7 ms). 4. In all three pyramidal-to-basket cell pairs, closely timed (10-50 ms) pairs of presynaptic action potentials resulted in statistically significant paired-pulse depression, the mean of the averaged second EPSPs being 80 +/- 11% of the averaged conditioning event. The overall degree of paired-pulse modulation was relatively little affected by either the amplitude of the preceding event or the inter-event interval. 5. The probability density function of the peak amplitudes of the unitary EPSPs could be adequately fitted with a quantal model. Without quantal variance, however, the minimum number of components in the model, excluding the failures, exceeded the number of electron microscopically determined synaptic junctions for all five connections. In contrast, incorporating quantal variance gave a minimum number of components which was compatible with the number of synaptic junctions, and which fitted the data equally well as models incorporating additional components but no quantal variance. For this model with quantal variance with the minimum number of components the estimate of the quantal coefficient of variation ranged between 0.33 and 0.46, and the corresponding quantal sizes ranged between 260 and 657 microV. The peak EPSP amplitudes in two of the four connections with more than one synaptic junction could be adequately described by a uniform binomial model for transmitter release. 6. In conclusion, at least three distinct interneurone classes receive local excitatory pyramidal cell input which they relay to different compartments on their postsynaptic target neurones. The reliability of transmission is high, but the fast time course of the EPSPs constrains their temporal summation. Due to the relatively small amplitude of unitary EPSPs several convergent inputs will therefore be required to elicit suprathreshold responses.

1. EPSCs were evoked in CA1 pyramidal neurones of young rats in vitro by extracellular stimulation of axons in a restricted stratum radiatum field, and were recorded using the whole-cell technique. 2. Quantal fluctuations in EPSC amplitude could be demonstrated for nineteen of fifty EPSCs analysed. Quantal currents (at the soma) ranged from 2.6 to 9.5 pA (after correction for the access resistance) with a mean of 4.0 +/- 2.0 pA. 3. Quantal variance was negligible for the majority (13/19) of the EPSCs. However, a large quantal variance (with a coefficient of variation > 0.4) is one possible reason why a large number of the EPSCs (29/50) could not be shown to have quantal fluctuations. 4. The statistical pattern of fluctuations in the amplitude of the majority of the quantal EPSCs (18/19) could not be described by conventional models of transmitter release. 5. The time course of the EPSC and a compartmental model of CA1 pyramidal neurones were used to calculate synaptic location. The quantal current (at the soma) was independent of the electrotonic location of the synapse at which it was evoked. The peak quantal conductance generating each quantal current ranged from 0.5 to 6.8 nS (mean 1.3 +/- 1.4 nS), its magnitude increasing with distance from the soma. The mean peak conductance is likely to be generated by the opening of at least 60-160 AMPA channels.

Loading slices of rat barrel cortex with 50 microM BAPTA-AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca(2+) stores with 20 microM cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca(2+) stores, results in a similar reduction of mEPSCs to that observed with BAPTA-AM, indicating that the source for intracellular Ca(2+) is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 microM ryanodine or 10 mM caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca(2+) release from ryanodine stores. Blocking IP(3) receptors with 14 microM 2-aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP(3) stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 microM (RS)-3,5-dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP(3) receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca(2+)-induced Ca(2+) release (CICR) from ryanodine stores after the spontaneous opening of IP(3) stores.

1. Long-term potentiation (LTP) was induced in EPSCs evoked in CA1 pyramidal neurones of young rats in vitro by extracellular stimulation of stratum radiatum. Low frequency stimulation was paired with postsynaptic depolarization to induce LTP, using whole-cell recording techniques. 2. Sufficient control and potentiated records were obtained under stable recording conditions to allow a quantal analysis of eleven EPSCs. The fluctuations in amplitude of all eleven EPSCs were quantized before conditioning stimulation, and they remained quantized after LTP induction, usually with an increased quantal variance. 3. Quantal current was increased by conditioning for nine out of eleven EPSCs. The increase in quantal current was correlated with the percentage increase in the EPSC. For only two EPSCs could the entire potentiation be attributed to an increase in quantal current. 4. The amplitude fluctuations of five control EPSCs could be described by binomial statistics, but after conditioning the binomial description held for only one of these EPSCs. For this EPSC, conditioning caused the release probability to increase from 0.39 +/- 0.05 to 0.47 +/- 0.02. 5. Quantal content was increased by conditioning stimulation for ten out of eleven EPSCs. The increase in quantal content was correlated with the percentage increase in the EPSC. However, for only four EPSCs could the entire potentiation be attributed to an increase in quantal content. 6. Most EPSCs were evoked with a high proportion of response failures. The probability of response failures decreased in eight out of eleven EPSCs following the induction of LTP. There was a negative correlation between the change in the probability of response failures and the amount of LTP. 7. The minimal number of sites at which transmission occurred increased for ten out of eleven EPSCs following LTP induction. Increases in the minimal number of active sites following conditioning were associated with decreases in the probability of response failures for seven out of eleven EPSCs. 8. The induction of LTP usually resulted in changes in the time course of the EPSCs. Cable analysis using a passive compartmental model of a CA1 pyramidal cell suggested that these time course changes were associated with shifts in the average electrotonic location of the active sites following LTP induction, rather than being caused by an increased duration of synaptic current. 9. LTP expression involves postsynaptic modifications to enhance the synaptic current at active sites. New sites are recruited, and our data cannot be used to determine if this is a result of a pre- or a postsynaptic change. Evidence for an increase in release probability was found for one EPSC.