Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials

We compared the transient increase of Ca²⁺ in single spines on basal dendrites of rat neocortical layer 5 pyramidal neurons evoked by subthreshold excitatory postsynaptic potentials (EPSPs) and back-propagating action potentials (APs) by using calcium fluorescence imaging. AP-evoked Ca²⁺ transients were detected in both the spines and in the adjacent dendritic shaft, whereas Ca²⁺ transients evoked by single EPSPs were largely restricted to a single active spine head. Calcium transients elicited in the active spines by a single AP or EPSP, in spines up to 80 μm for the soma, were of comparable amplitude. The Ca²⁺ transient in an active spine evoked by pairing an EPSP and a back-propagating AP separated by a time interval of 50 ms was larger if the AP followed the EPSP than if it preceded it. This difference reflected supra- and sublinear summation of Ca²⁺ transients, respectively. A comparable dependence of spinous Ca²⁺ transients on relative timing was observed also when short bursts of APs and EPSPs were paired. These results indicate that the amplitude of the spinous Ca²⁺ transients during coincident pre- and postsynaptic activity depended critically on the relative order of subthreshold EPSPs and back-propagating APs. Thus, in neocortical neurons the amplitude of spinous Ca²⁺ transients could encode small time differences between pre- and postsynaptic activity.     

Dynamics of dendritic calcium transients evoked by quantal release at excitatory hippocampal synapses

Synaptic N-methyl-D-aspartate (NMDA) receptors detect coincident pre- and postsynaptic activity and play a critical role in triggering changes in synaptic strength at central synapses. Despite intensive study of synaptic plasticity, relatively little is known about the magnitude and duration of calcium accumulation caused by unitary events at individual synapses. We used fluorescence imaging to detect NMDA receptor-mediated miniature synaptic calcium transients (MSCTs) caused by spontaneous release of synaptic vesicles in dendrites of cultured hippocampal neurons. MSCTs originated focally in dendritic regions <1 microm in length and decayed with a time constant of 0.35 +/- 0.03 s. Multiple occurrences of MSCTs recorded at single sites had fluctuating amplitudes, with a coefficient of variation of 0.34. From the reduction in the spatial spread of MSCTs with decreasing concentration of indicator dye, we estimated that the dominant endogenous calcium buffer in dendrites is relatively immobile (diffusion coefficient between 10 and 50 microm(2)/s). We conclude that calcium rise caused by spontaneous quantal synaptic NMDA receptor activation (i) is variable, (ii) lasts for a time period briefer than previous measurements indicate, and (iii) is confined by endogenous calcium buffers to local dendritic regions even when synapses are not on spines.     

Active summation of excitatory postsynaptic potentials in hippocampal CA3 pyramidal neurons

The manner in which the thousands of synaptic inputs received by a pyramidal neuron are summed is critical both to our understanding of the computations that may be performed by single neurons and of the codes used by neurons to transmit information. Recent work on pyramidal cell dendrites has shown that subthreshold synaptic inputs are modulated by voltage-dependent channels, raising the possibility that summation of synaptic responses is influenced by the active properties of dendrites. Here, we use somatic and dendritic whole-cell recordings to show that pyramidal cells in hippocampal area CA3 sum distal and proximal excitatory postsynaptic potentials sublinearly and actively, that the degree of nonlinearity depends on the magnitude and timing of the excitatory postsynaptic potentials, and that blockade of transient potassium channels linearizes summation. Nonlinear summation of synaptic inputs could have important implications for the computations performed by single neurons and also for the role of the mossy fiber and perforant path inputs to hippocampal area CA3.     

N-methyl-D-aspartate receptors contribute to excitatory postsynaptic potentials of cat lateral geniculate neurons recorded in thalamic slices.

Neurons of the cat's dorsal lateral geniculate nucleus were recorded intracellularly to study the contribution of N-methyl-D-aspartate (NMDA) receptors to excitatory postsynaptic potentials (EPSPs) and low-threshold calcium spikes. EPSPs were evoked by stimulation of retinogeniculate axons in the optic tract and/or corticogeniculate axons in the optic radiations; EPSPs from both sources were similar. These EPSPs had one or two components, and the second component had several characteristics of NMDA receptor-mediated events. For example, EPSP amplitude decreased when neurons were hyperpolarized and increased when stimulus frequency was increased; these EPSPs could also be blocked reversibly by application of the selective NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV). We also studied the influence of NMDA receptors on low-threshold calcium spikes, which are large, voltage- and calcium-dependent depolarizations that are often accompanied by high-frequency action potential discharge. APV blocked synaptically activated low-threshold calcium spikes, but APV had no effect on low-threshold calcium spikes that were elicited by current injection. Therefore, APV does not appear to have a direct effect on the T-type calcium channel that is involved in generation of low-threshold calcium spikes. The voltage and frequency dependence of the NMDA receptor-mediated component of the EPSPs, as well as its ability to trigger low-threshold calcium spikes, provide for complex signal processing in the lateral geniculate nucleus.     

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.     

Sodium channels in dendrites of rat cortical pyramidal neurons.

The voltage-dependent properties that have been directly demonstrated in Purkinje cell and hippocampal pyramidal cell dendrites play an important role in the integrative capacities of these neurons. By contrast, the properties of neocortical pyramidal cell dendritic membranes have been more difficult to assess. Active dendritic conductances near sites of synaptic input would have an important effect on the input-output characteristics of these neurons. In the experiments reported here, we obtained direct evidence for the existence of voltage-dependent Na+ channels on the dendrites of neocortical neurons by using cell-attached patch and whole cell recordings from acutely isolated rat neocortical pyramidal cells. The qualitative and quantitative properties of dendritic and somatic currents were indistinguishable. Insofar as Na+ currents are concerned, the soma and primary apical dendrite can be considered as one relatively uniform compartment. Similar dendritic Na+ currents on dendrites in mature neurons would play an important role in determining the integrative properties of these cortical units.     

Spatial distribution of calcium channels and cytosolic calcium transients in growth cones and cell bodies of sympathetic neurons.

Ca2+ imaging and single-channel recording were used to study the regulation of cytosolic free Ca2+ ([Ca2+]i) in local regions of frog sympathetic neurons. Digital imaging with the fluorescent Ca2+ indicator fura-2 demonstrated: (i) resting [Ca2+]i of 70-100 nM; (ii) significant increases in [Ca2+]i in growth cones and cell bodies following depolarization induced by extracellular electrical stimulation or increased external K+; (iii) in cell bodies, large transient increases in [Ca2+]i following exposure to caffeine and sustained oscillations in [Ca2+]i in the presence of elevated K+ and caffeine; and (iv) in growth cones, smaller and briefer changes in [Ca2+]i in response to caffeine. The nature of the depolarization-induced Ca2+ entry was studied with cell-attached patch recordings (110 mM Ba2+ in recording pipette). Ca2+ channel activity was observed in 18 of 20 patches on cell bodies, 3 of 5 patches along neurites, and 36 of 41 patch recordings from growth cones. We observed two types of Ca2+ channels: L-type channels, characterized by a 28-pS slope conductance, sensitivity to dihydropyridine Ca2+ channel agonist, and availability even with depolarizing holding potentials; and N-type channels, characterized by a 15-pS slope conductance, resistance to dihydropyridines, and inactivation with depolarized holding potentials. Both types of channels were found on growth cones and along neurites as well as on cell bodies; channels often appeared concentrated in local hot spots, sometimes dominated by one channel type.     

pH transients evoked by excitatory synaptic transmission are increased by inhibition of extracellular carbonic anhydrase.

Excitatory synaptic transmission has been associated with a rapid alkalinization of the brain extracellular space. These pH shifts are markedly increased by acetazolamide, an inhibitor of carbonic anhydrase. Although this effect can be readily explained by inhibition of extracellular carbonic anhydrase, this enzyme has been considered strictly intracellular in the central nervous system. To determine whether these alkaline shifts are regulated by extracellular carbonic anhydrase, we studied the effects of a membrane impermeant, dextran-bound inhibitor of this enzyme. Extracellular alkaline transients, measured with pH-sensitive microelectrodes, were generated in the CA1 region of rat hippocampal slices by repetitive electrical stimulation of Schaeffer collateral fibers or by local ejection of glutamate. More direct alkalinizations were elicited by focal ejection of NaOH in the vicinity of a pH microelectrode. These pH transients were reversibly enhanced by addition of the dextran-bound inhibitor. We conclude that there is significant carbonic anhydrase activity in the extracellular space of the brain. We postulate that this enzyme functions in the regulation and modulation of extracellular pH transients associated with neuronal activity.     

Long-term depression of climbing fiber-evoked calcium transients in Purkinje cell dendrites

In recent years much has been learned about the molecular requirements for inducing long-term synaptic depression (LTD) in various brain regions. However, very little is known about the consequences of LTD induction for subsequent signaling events in postsynaptic neurons. We have addressed this issue by examining homosynaptic LTD at the cerebellar climbing fiber (CF)-Purkinje cell (PC) synapse. This synapse is built for reliable and massive excitation: Activation of a single axon produces an unusually large alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-mediated synaptic current, the depolarization of which drives a regenerative complex spike producing a large, widespread Ca(2+) transient in PC dendrites. Here we test whether CF LTD has an impact on dendritic, complex spike-evoked Ca(2+) signals by simultaneously performing long-term recordings of complex spikes and microfluorimetric Ca(2+) measurements in PC dendrites in rat cerebellar slices. Our data show that LTD of the CF excitatory postsynaptic current produces a reduction in both slow components of the complex spike waveform and complex spike-evoked dendritic Ca(2+) transients. This LTD of dendritic Ca(2+) signals may provide a neuroprotective mechanism and/or constitute "heterosynaptic metaplasticity" by reducing the probability for subsequent induction of those forms of use-dependent plasticity, which require CF-evoked Ca(2+) signals such as parallel fiber-PC LTD and interneuron-PC LTP.     

Substance P-mediated slow excitatory postsynaptic potential elicited in dorsal horn neurons in vivo by noxious stimulation.

The original proposal that substance P is involved in the regulation of nociceptive information at the first sensory synapse in the spinal cord has been substantiated by a wide range of evidence, but definitive support has been lacking, due primarily to the lack of evidence that a specific nociceptive response in the dorsal horn can be blocked by a substance P antagonist. Here, we present evidence that CP-96,345, a specific substance P (NK-1) receptor antagonist, selectively blocks a slow, prolonged excitatory postsynaptic potential following noxious cutaneous stimulation or a train of intense electrical stimuli to sensory nerves but does not affect the response to innocuous input or the brief response to single electrical stimuli to C fibers. These results indicate the specific involvement of substance P in the mediation of a prolonged after-excitation to noxious stimulation. This may have important implications for the etiology and treatment of chronic pain and for plastic changes in nociceptive pathways.     

Neurally evoked calcium transients in terminal Schwann cells at the neuromuscular junction.

We examined the effects of motor-nerve stimulation on the intracellular Ca2+ levels of Schwann cells, the glial cells at the frog neuromuscular junction. Schwann cells, which were loaded with the fluorescent Ca2+ indicator fluo-3 and examined by confocal microscopy, showed a transient increase in free Ca2+ within a few seconds of the onset of tetanic stimulation of the motor nerve. The Ca2+ response was specific to the synapse in that it was found in the terminal Schwann cells at the junction but not in the myelinating Schwann cells along the axon. The Ca2+ transients occurred in the presence of d-tubocurare, indicating that they were not mediated by nicotinic acetylcholine receptors and recurred when the stimulus was repeated. The Ca2+ response persisted after degeneration of the postsynaptic muscle fiber, demonstrating that the terminal Schwann cell was stimulated directly by presynaptic activity. The finding that terminal Schwann cells at the neuromuscular junction respond to presynaptic activity suggests that glial-cell function is modulated by synaptic transmission.     

Calcium transients in isolated cardiac myocytes are altered by 1,1,1-trichloroethane.

1,1,1-Trichloroethane is a widely used solvent that is annually linked to several cases of sudden death following accidental exposure or abuse. Sudden death is believed to be due to ventricular fibrillation or myocardial depression. The purpose of this study was to investigate the mechanism of myocardial depression by assessing the influence of 1,1,1-trichloroethane on intracellular Ca transients in single neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2-Ca binding. Cells were exposed to 1,1,1-trichloroethane in Hanks' balanced salt solution aliquoted as a 0.2% DMSO solution by a single pass suffusion in an environmentally controlled chamber. 1,1,1-Trichloroethane (0.25 mM-8 mM) reduced the height of electrically (1 Hz, 60 V, 10 ms) induced Ca transients concentration dependently and reversibly to a maximum of about 50% with no effect on diastolic Ca concentration. Video motion analysis revealed an inhibition of contractility in the same concentration range. 1,1,1-Trichloroethane inhibited cytosolic Ca increase in response to KCl-induced (90 mM) depolarizations and further decreased the limited Ca transients in ryanodine (1 microM) pretreated myocytes. Increased external Ca (5 mM) antagonized the effect of 0.5 mM 1,1,1-trichloroethane on the Ca transients. 1,1,1-Trichloroethane reduced the caffeine (10 mM) releasable Ca pool in myocytes. These results show that 1,1,1-trichloroethane inhibits Ca mobilization during excitation-contraction coupling in ventricular myocytes. An inhibitory action on the influx of extracellular Ca as well as on sarcoplasmic reticulum Ca release and sequestration is likely to be responsible for this action.     

Layer-specific programs of development in neocortical projection neurons.

How are long-range axonal projections from the cerebral cortex orchestrated during development? By using both passively and actively transported axonal tracers in fetal and postnatal ferrets, we have analyzed the development of projections from the cortex to a number of thalamic nuclei. We report that the projections of a cortical area to its corresponding thalamic nuclei follow highly cell-specific programs of development. Axons from cells in the deepest layers of the cerebral cortex (layer 6 and superficial subplate neurons) appear to grow very slowly and be delayed for several weeks in the cerebral white matter, reaching the thalamus over a protracted period. Neurons of layer 5, on the other hand, develop their projections much faster; despite being born after the neurons of deeper layers, layer 5 neurons are the first to extend their axons out of the cortical hemisphere and innervate the thalamus. Layer 5 projections are massive in the first postnatal weeks but may become partly eliminated later in development, being overtaken in number by layer 6 cells that constitute the major corticothalamic projection by adulthood. Layer 5 projections are area-specific from the outset and arise as collateral branches of axons directed to the brainstem and spinal cord. Our findings show that the early development of corticofugal connections is determined not by the sequence of cortical neurogenesis but by developmental programs specific for each type of projection neuron. In addition, they demonstrate that in most thalamic nuclei, layer 5 neurons (and not subplate or layer 6 neurons) establish the first descending projections from the cerebral cortex.     

Modeling back propagating action potential in weakly excitable dendrites of neocortical pyramidal cells.

Simultaneous recordings from the soma and apical dendrite of layer V neocortical pyramidal cells of young rats show that, for any location of current input, an evoked action potential (AP) always starts at the axon and then propagates actively, but decrementally, backward into the dendrites. This back-propagating AP is supported by a low density (-gNa = approximately 4 mS/cm2) of rapidly inactivating voltage-dependent Na+ channels in the soma and the apical dendrite. Investigation of detailed, biophysically constrained, models of reconstructed pyramidal cells shows the following. (i) The initiation of the AP first in the axon cannot be explained solely by morphological considerations; the axon must be more excitable than the soma and dendrites. (ii) The minimal Na+ channel density in the axon that fully accounts for the experimental results is about 20-times that of the soma. If -gNa in the axon hillock and initial segment is the same as in the soma [as recently suggested by Colbert and Johnston [Colbert, C. M. & Johnston, D. (1995) Soc. Neurosci. Abstr. 21, 684.2]], then -gNa in the more distal axonal regions is required to be about 40-times that of the soma. (iii) A backward propagating AP in weakly excitable dendrites can be modulated in a graded manner by background synaptic activity. The functional role of weakly excitable dendrites and a more excitable axon for forward synaptic integration and for backward, global, communication between the axon and the dendrites is discussed.     

Bidirectional plasticity of excitatory postsynaptic potential (EPSP)-spike coupling in CA1 hippocampal pyramidal neurons

Integration of synaptic excitation to generate an action potential (excitatory postsynaptic potential-spike coupling or E-S coupling) determines the neuronal output. Bidirectional synaptic plasticity is well established in the hippocampus, but whether active synaptic integration can display potentiation and depression remains unclear. We show here that synaptic depression is associated with an N-methyl-d-aspartate receptor-dependent and long-lasting depression of E-S coupling. E-S depression is input-specific and is expressed in the presence of gamma-aminobutyric acid type A and B receptor antagonists. In single neurons, E-S depression is observed without modification of postsynaptic passive properties. We conclude that a decrease in intrinsic excitability underlies E-S depression and is synergic with glutamatergic long-term depression.     

Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors.

Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism--increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.     

Sodium action potentials in the dendrites of cerebellar Purkinje cells.

We report here that in cerebellar Purkinje cells from which the axon has been removed, positive voltage steps applied to the voltage-clamped soma produce spikes of active current. The spikes are inward, are all-or-none, have a duration of approximately 1 ms, and are reversibly eliminated by tetrodotoxin, a Na channel poison. From cell to cell, the amplitude of the spikes ranges from 4 to 20 nA. Spike latency decreases as the depolarizing step is made larger. These spikes clearly arise at a site where the voltage is not controlled, remote from the soma. From these facts we conclude that Purkinje cell dendrites contain a sufficient density of Na channels to generate action potentials. Activation by either parallel fiber or climbing fiber synapses produces similar spikes, suggesting that normal input elicits Na action potentials in the dendrites. These findings greatly alter current views of how dendrites in these cells respond to synaptic input.     

Maintenance of presynaptic function by AMPA receptor-mediated excitatory postsynaptic activity in adult brain

Activity-dependent synaptic modification occurs in both developing and mature animals. For reliable information transfer and storage, however, once established, synapses must be maintained stably. We investigated how chronic blockade of neuronal activity or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors affects excitatory climbing fiber (CF) to Purkinje cell (PC) synapses in adult mouse cerebellum. Both treatments caused reduced glutamate concentration transient at the synaptic cleft, decreased frequency of quantal excitatory postsynaptic current, and diminished CF innervation of PC shaft dendrites but no change in CF's release probability. These results indicate that, in the mature cerebellum, AMPA receptor-mediated excitatory postsynaptic activity maintains CF's functional glutamate-release sites and its innervation of PC shaft dendrites.     

Subtype-selective reconstitution of synaptic transmission in sympathetic ganglion neurons by expression of exogenous calcium channels

Fast cholinergic neurotransmission between superior cervical ganglion neurons (SCGNs) in cell culture is initiated by N-type Ca(2+) currents through Ca(v)2.2 channels. To test the ability of different Ca(2+)-channel subtypes to initiate synaptic transmission in these cells, SCGNs were injected with cDNAs encoding Ca(v)1.2 channels, which conduct L-type currents, Ca(v)2.1 channels, which conduct P/Q-type Ca(2+) currents, and Ca(v)2.3 channels, which conduct R-type Ca(2+) currents. Exogenously expressed Ca(v)2.1 channels were localized in nerve terminals, as assessed by immunocytochemistry with subtype-specific antibodies, and these channels effectively initiated synaptic transmission. Injection with cDNA encoding Ca(v)2.3 channels yielded a lower level of presynaptic labeling and synaptic transmission, whereas injection with cDNA encoding Ca(v)1.2 channels resulted in no presynaptic labeling and no synaptic transmission. Our results show that exogenously expressed Ca(2+) channels can mediate synaptic transmission in SCGNs and that the specificity of reconstitution of neurotransmission (Ca(v)2.1 > Ca(v)2.3 > Ca(v)1.2) follows the same order as in neurons in vivo. The specificity of reconstitution of neurotransmission parallels the specificity of trafficking of these Ca(v) channels to nerve terminals.