Dendritic spikes as a mechanism for cooperative long-term potentiation

Strengthening of synaptic connections following coincident pre- and postsynaptic activity was proposed by Hebb as a cellular mechanism for learning. Contemporary models assume that multiple synapses must act cooperatively to induce the postsynaptic activity required for hebbian synaptic plasticity. One mechanism for the implementation of this cooperation is action potential firing, which begins in the axon, but which can influence synaptic potentiation following active backpropagation into dendrites. Backpropagation is limited, however, and action potentials often fail to invade the most distal dendrites. Here we show that long-term potentiation of synapses on the distal dendrites of hippocampal CA1 pyramidal neurons does require cooperative synaptic inputs, but does not require axonal action potential firing and backpropagation. Rather, locally generated and spatially restricted regenerative potentials (dendritic spikes) contribute to the postsynaptic depolarization and calcium entry necessary to trigger potentiation of distal synapses. We find that this mechanism can also function at proximal synapses, suggesting that dendritic spikes participate generally in a form of synaptic potentiation that does not require postsynaptic action potential firing in the axon.     

The spread of Na+ spikes determines the pattern of dendritic Ca2+ entry into hippocampal neurons.

The dendrites of many types of neurons contain voltage-dependent Na+ and Ca2+ conductances that generate action potentials (see ref. 1 for review). The function of these spikes is not well understood, but the Ca2+ entry stimulated by spikes probably affects Ca(2+)-dependent processes in dendrites. These include synaptic plasticity, cytotoxicity and exocytosis. Several lines of evidence suggest that dendritic spikes occur within subregions of the dendrites. To study the mechanism that govern the spread of spikes in the dendrites of hippocampal pyramidal cells, we imaged Ca2+ entry with Fura-2 (ref. 9) and Na+ entry with a newly developed Na(+)-sensitive dye. Our results indicate that Ca2+ entry into dendrites is triggered by Na+ spikes that actively invade the dendrites. The restricted spatial distribution of Ca2+ entry seems to depend on the spread of Na+ spikes in the dendrites, rather than on a limited distribution of Ca2+ channels. In addition, we have observed an activity-dependent process that modulates the invasion of spikes into the dendrites and progressively restricts Ca2+ entry to more proximal dendritic regions.     

Rapid BDNF-induced retrograde synaptic modification in a developing retinotectal system

In cultures of hippocampal neurons, induction of long-term synaptic potentiation or depression by repetitive synaptic activity is accompanied by a retrograde spread of potentiation or depression, respectively, from the site of induction at the axonal outputs to the input synapses on the dendrites of the presynaptic neuron. We report here that rapid retrograde synaptic modification also exists in an intact developing retinotectal system. Local application of brain-derived neurotrophic factor (BDNF) to the Xenopus laevis optic tectum, which induced persistent potentiation of retinotectal synapses, led to a rapid modification of synaptic inputs at the dendrites of retinal ganglion cells (RGCs), as shown by a persistent enhancement of light-evoked excitatory synaptic currents and spiking activity of RGCs. This retrograde effect required TrkB receptor activation, phospholipase Cgamma activity and Ca2+ elevation in RGCs, and was accounted for by a selective increase in the number of postsynaptic AMPA-subtype glutamate receptors at RGC dendrites. Such retrograde information flow in the neuron allows rapid regulation of synaptic inputs at the dendrite in accordance to signals received at axon terminals, a process reminiscent of back-propagation algorithm for learning in neural networks.     

Analysis of calcium channels in single spines using optical fluctuation analysis

Most synapses form on small, specialized postsynaptic structures known as dendritic spines. The influx of Ca2+ ions into such spines--through synaptic receptors and voltage-sensitive Ca2+ channels (VSCCs)--triggers diverse processes that underlie synaptic plasticity. Using two-photon laser scanning microscopy, we imaged action-potential-induced transient changes in Ca2+ concentration in spines and dendrites of CA1 pyramidal neurons in rat hippocampal slices. Through analysis of the large trial-to-trial fluctuations in these transients, we have determined the number and properties of VSCCs in single spines. Here we report that each spine contains 1-20 VSCCs, and that this number increases with spine volume. We are able to detect the opening of a single VSCC on a spine. In spines located on the proximal dendritic tree, VSCCs normally open with high probability (approximately 0.5) following dendritic action potentials. Activation of GABA(B) receptors reduced this probability in apical spines to approximately 0.3 but had no effect on VSCCs in dendrites or basal spines. Our studies show that the spatial distribution of VSCC subtypes and their modulatory potential is regulated with submicrometre precision.     

Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons.

Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.     

NMDA receptor agonists selectively block N-type calcium channels in hippocampal neurons.

The modulation of voltage-dependent calcium channels by various neurotransmitters has been demonstrated in many neurons. Because of the critical role of Ca2+ in transmitter release and, more generally, in transmembrane signalling, this modulation has important functional implications. Hippocampal neurons possess low-threshold (T-type) Ca2+ channels and both L- and N-type high voltage-activated Ca2+ channels. N-type Ca2+ channels are blocked selectively by omega-conotoxin and adenosine. These substances both block excitatory synaptic transmission in the hippocampus, whereas dihydropyridines, which selectively block L-type channels, are ineffective. Excitatory synaptic transmission in the hippocampus displays a number of plasticity phenomena that are initiated by Ca2+ entry through ionic channels operated by N-methyl-D-aspartate (NMDA) receptors. Here we report that NMDA receptor agonists selectively and effectively depress N-type Ca2+ channels which are involved in neurotransmitter release from presynaptic sites. The inhibitory effect is eliminated by the competitive NMDA antagonist D-2-amino-5-phosphonovalerate, does not require Ca2+ entry into the cell, and is probably receptor-mediated. This phenomenon may provide a negative feedback between the liberation of excitatory transmitter and entry of Ca2+ into the cell, and could be important in presynaptic inhibition and in the regulation of synaptic plasticity.     

Spike-timing-dependent synaptic plasticity depends on dendritic location

In the neocortex, each neuron receives thousands of synaptic inputs distributed across an extensive dendritic tree. Although postsynaptic processing of each input is known to depend on its dendritic location, it is unclear whether activity-dependent synaptic modification is also location-dependent. Here we report that both the magnitude and the temporal specificity of spike-timing-dependent synaptic modification vary along the apical dendrite of rat cortical layer 2/3 pyramidal neurons. At the distal dendrite, the magnitude of long-term potentiation is smaller, and the window of pre-/postsynaptic spike interval for long-term depression (LTD) is broader. The spike-timing window for LTD correlates with the window of action potential-induced suppression of NMDA (N-methyl-D-aspartate) receptors; this correlation applies to both their dendritic location-dependence and pharmacological properties. Presynaptic stimulation with partial blockade of NMDA receptors induced LTD and occluded further induction of spike-timing-dependent LTD, suggesting that NMDA receptor suppression underlies LTD induction. Computer simulation studies showed that the dendritic inhomogeneity of spike-timing-dependent synaptic modification leads to differential input selection at distal and proximal dendrites according to the temporal characteristics of presynaptic spike trains. Such location-dependent tuning of inputs, together with the dendritic heterogeneity of postsynaptic processing, could enhance the computational capacity of cortical pyramidal neurons.     

Conservation of total synaptic weight through balanced synaptic depression and potentiation

Memory is believed to depend on activity-dependent changes in the strength of synapses. In part, this view is based on evidence that the efficacy of synapses can be enhanced or depressed depending on the timing of pre- and postsynaptic activity. However, when such plastic synapses are incorporated into neural network models, stability problems may develop because the potentiation or depression of synapses increases the likelihood that they will be further strengthened or weakened. Here we report biological evidence for a homeostatic mechanism that reconciles the apparently opposite requirements of plasticity and stability. We show that, in intercalated neurons of the amygdala, activity-dependent potentiation or depression of particular glutamatergic inputs leads to opposite changes in the strength of inputs ending at other dendritic sites. As a result, little change in total synaptic weight occurs, even though the relative strength of inputs is modified. Furthermore, hetero- but not homosynaptic alterations are blocked by intracellular dialysis of drugs that prevent Ca2+ release from intracellular stores. Thus, in intercalated neurons at least, inverse heterosynaptic plasticity tends to compensate for homosynaptic long-term potentiation and depression, thus stabilizing total synaptic weight.     

Direct cortical input modulates plasticity and spiking in CA1 pyramidal neurons

The hippocampus is necessary for the acquisition and retrieval of declarative memories. The best-characterized sensory input to the hippocampus is the perforant path projection from layer II of entorhinal cortex (EC) to the dentate gyrus. Signals are then processed sequentially in the hippocampal CA fields before returning to the cortex via CA1 pyramidal neuron spikes. There is another EC input-the temporoammonic (TA) pathway-consisting of axons from layer III EC neurons that make synaptic contacts on the distal dendrites of CA1 neurons. Here we show that this pathway modulates both the plasticity and the output of the rat hippocampal formation. Bursts of TA activity can, depending on their timing, either increase or decrease the probability of Schaffer-collateral (SC)-evoked CA1 spikes. TA bursts can also significantly reduce the magnitude of synaptic potentiation at SC-CA1 synapses. The TA-CA1 synapse itself exhibits both long-term depression (LTD) and long-term potentiation (LTP). This capacity for bi-directional plasticity can, in turn, regulate the TA modulation of CA1 activity: LTP or LTD of the TA pathway either enhances or diminishes the gating of CA1 spikes and plasticity inhibition, respectively.     

Optical imaging of calcium accumulation in hippocampal pyramidal cells during synaptic activation

The dynamic response of nerve cells to synaptic activation and the spatial distribution of biochemical processes regulated by ion concentration are critically dependent on the cell-surface distribution of ion channels. In the hippocampus, intracellular calcium-ion concentration is thought to influence the biochemical events associated with kindling, excitotoxicity, and long-term potentiation. Computer models of hippocampal pyramidal cells also indicate that calcium-channel location influences dynamic characteristics such as bursting. Here, we have used in situ microfluorometric imaging in brain slices to directly measure the spatial distribution of calcium accumulation in guinea-pig CA1 pyramidal cells during trains of orthodromic synaptic stimulation. Calcium accumulation is substantial throughout the entire proximal section of the apical and basal dendrites. Most of this accumulation results from influx through non-NMDA (N-methyl-D-aspartate) voltage-gated calcium channels, and in the apical dendrite it drops steeply as the dendrite enters stratum moleculare, the termination zone of perforant path afferents. These results demonstrate a marked segregation of calcium-channel activity and directly show a spatial distribution of calcium accumulation during orthodromic synaptic activation.     

Dendritic spines as individual neuronal compartments for synaptic Ca2+ responses.

The possibility that postsynaptic spines on neuronal dendrites are discrete biochemical compartments for Ca(2+)-activated processes involved in synaptic plasticity is a widely proposed concept that has eluded experimental demonstration. Using microfluorometry on CA3 neurons in hippocampal slices, we show here that with weak presynaptic stimulation of associative/commissural fibres, Ca2+ accumulates in single postsynaptic spines but not in the parent dendrite. Stronger stimulation also promotes changes in dendrites. The NMDA-receptor antagonist AP-5 blocks changes in Ca2+ in spines. Sustained steep Ca2+ gradients between single spines and the parent dendrite, often lasting several minutes, develop with repeated stimulation. The observed compartmentalization allows for the specificity, cooperativity and associativity displayed by memory models such as long-term potentiation.     

Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses

Marijuana affects brain function primarily by activating the G-protein-coupled cannabinoid receptor-1 (CB1), which is expressed throughout the brain at high levels. Two endogenous lipids, anandamide and 2-arachidonylglycerol (2-AG), have been identified as CB1 ligands. Depolarized hippocampal neurons rapidly release both anandamide and 2-AG in a Ca2+-dependent manner. In the hippocampus, CB1 is expressed mainly by GABA (gamma-aminobutyric acid)-mediated inhibitory interneurons, where CB1 clusters on the axon terminal. A synthetic CB1 agonist depresses GABA release from hippocampal slices. These findings indicate that the function of endogenous cannabinoids released by depolarized hippocampal neurons might be to downregulate GABA release. Here we show that the transient suppression of GABA-mediated transmission that follows depolarization of hippocampal pyramidal neurons is mediated by retrograde signalling through release of endogenous cannabinoids. Signalling by the endocannabinoid system thus represents a mechanism by which neurons can communicate backwards across synapses to modulate their inputs.     

Reversible uncoupling of inactivation in N-type calcium channels.

N-type calcium channels are thought to be expressed specifically in neuronal cells and to have a dominant role in the control of neurotransmitter release from sympathetic neurons. But their unitary properties are poorly understood and the separation of neuronal Ca2+ current into components carried by N-type or L-type Ca2+ channels is controversial. Here we show that individual N-type Ca2+ channels in sympathetic neurons can carry two kinetically distinct components of current, one that is rapidly transient and one that is long lasting. The mechanism that gives rise to these two components is unexpected for Ca2+ channels: a test depolarization elicits either a rapidly inactivating, single short burst with an average duration of 40 ms, or sustained, noninactivating channel activity lasting for over 1 s. The switching between inactivating and noninactivating activity is a slow process, the occurrence of each type of unitary kinetic behaviour remaining statistically correlated over several seconds. Variable coupling of inactivation in N-type Ca2+ channels could be an effective mechanism for the modulation of neuronal excitability and synaptic plasticity.     

Calcium stores regulate the polarity and input specificity of synaptic modification

Activity-induced synaptic modification is essential for the development and plasticity of the nervous system. Repetitive correlated activation of pre- and postsynaptic neurons can induce persistent enhancement or decrement of synaptic efficacy, commonly referred to as long-term potentiation or depression (LTP or LTD). An important unresolved issue is whether and to what extent LTP and LTD are restricted to the activated synapses. Here we show that, in the CA1 region of the hippocampus, reduction of postsynaptic calcium influx by partial blockade of NMDA (N-methyl-D-aspartate) receptors results in a conversion of LTP to LTD and a loss of input specificity normally associated with LTP, with LTD appearing at heterosynaptic inputs. The induction of LTD at homo- and heterosynaptic sites requires functional ryanodine receptors and inositol triphosphate (InsP3) receptors, respectively. Functional blockade or genetic deletion of type 1 InsP3 receptors led to a conversion of LTD to LTP and elimination of heterosynaptic LTD, whereas blocking ryanodine receptors eliminated only homosynaptic LTD. Thus, postsynaptic Ca2+, deriving from Ca2+ influx and differential release of Ca2+ from internal stores through ryanodine and InsP3 receptors, regulates both the polarity and input specificity of activity-induced synaptic modification.     

Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated.

At excitatory synapses in the central nervous system, the number of glutamate molecules released from a vesicle is much larger than the number of postsynaptic receptors. But does release of a single vesicle normally saturate these receptors? Answering this question is critical to understanding how the amplitude and variability of synaptic transmission are set and regulated. Here we describe the use of two-photon microscopy to image transient increases in Ca2+ concentration mediated by NMDA (N-methyl-D-aspartate) receptors in single dendritic spines of CA1 pyramidal neurons in hippocampal slices. To test for NMDA-receptor saturation, we compared responses to stimulation with single and double pulses. We find that a single release event does not saturate spine NMDA receptors; a second release occurring 10 ms later produces approximately 80% more NMDA-receptor activation. The amplitude of spine NMDA-receptor-mediated [Ca2+] transients (and the synaptic plasticity which depends on this) may thus be sensitive to the number of quanta released by a burst of action potentials and to changes in the concentration profile of glutamate in the synaptic cleft.     

Two components of long-term potentiation induced by different patterns of afferent activation

Long-term potentiation (LTP) of excitatory synaptic transmission could be a mechanism underlying memory. Induction of LTP requires Ca2+ influx into postsynaptic neurons through ion channels gated by NMDA (N-methyl-D-aspartate) receptors in hippocampus (area CA1 and dentate gyrus) and neocortex. Here we report that a component of LTP not requiring the activation of NMDA receptors can be induced in area CA1. The component is dependent on tetanus frequency, requires increases in postsynaptic intracellular Ca2+ concentrations, and is suppressed by an antagonist of voltage-dependent Ca2+ channels.     

Locally dynamic synaptic learning rules in pyramidal neuron dendrites

Long-term potentiation (LTP) of synaptic transmission underlies aspects of learning and memory. LTP is input-specific at the level of individual synapses, but neural network models predict interactions between plasticity at nearby synapses. Here we show in mouse hippocampal pyramidal cells that LTP at individual synapses reduces the threshold for potentiation at neighbouring synapses. After input-specific LTP induction by two-photon glutamate uncaging or by synaptic stimulation, subthreshold stimuli, which by themselves were too weak to trigger LTP, caused robust LTP and spine enlargement at neighbouring spines. Furthermore, LTP induction broadened the presynaptic-postsynaptic spike interval for spike-timing-dependent LTP within a dendritic neighbourhood. The reduction in the threshold for LTP induction lasted approximately 10 min and spread over approximately 10 microm of dendrite. These local interactions between neighbouring synapses support clustered plasticity models of memory storage and could allow for the binding of behaviourally linked information on the same dendritic branch.     

Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. Alpha-neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that alpha-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.