A frequency-dependent switch from inhibition to excitation in a hippocampal unitary circuit

The hippocampus, a brain structure essential for memory and cognition, is classically represented as a trisynaptic excitatory circuit. Recent findings challenge this view, particularly with regard to the mossy fibre input to CA3, the second synapse in the trisynaptic pathway. Thus, the powerful mossy fibre input to CA3 pyramidal cells might mediate both synaptic excitation and inhibition. Here we show, by recording from connected cell pairs in rat entorhinal-hippocampal slice cultures, that single action potentials in a dentate granule cell evoke a net inhibitory signal in a pyramidal cell. The hyperpolarization is due to disynaptic feedforward inhibition, which overwhelms monosynaptic excitation. Interestingly, this net inhibitory synaptic response changes to an excitatory signal when the frequency of presynaptic action potentials increases. The process responsible for this switch involves the facilitation of monosynaptic excitatory transmission coupled with rapid depression of inhibitory circuits. This ability to immediately switch the polarity of synaptic responses constitutes a novel synaptic mechanism, which might be crucial to the state-dependent processing of information in associative hippocampal networks.     

Latent synaptic pathways revealed after tetanic stimulation in the hippocampus

Synaptic plasticity may result from changes at existing synapses or from alterations in the number of functional synaptic connections. In the hippocampus excitatory synaptic strength is persistently enhanced after tetanic stimulation. Here we report that latent synaptic pathways may also become functional. Simultaneous intracellular recordings were made from pairs of CA3 pyramidal cells in slices from guinea pig hippocampus. After stimulating afferent fibres repetitively, polysynaptic excitatory pathways between previously unconnected cells became apparent. The efficacy of recurrent inhibitory circuits was also reduced. The loss of inhibitory control is of interest because latent excitatory pathways are revealed after pharmacological suppression of inhibition. This plasticity in local synaptic circuits leads to the emergence of synchronous firing in groups of CA3 cells. The formation of groups of associated cells and the ability of some cells to initiate synchronous firing in a larger cell group through recurrent pathways is reminiscent of several models of information storage and recall in the cortex.     

Semaphorin 3A is a chemoattractant for cortical apical dendrites

The apical dendrites of pyramidal neurons integrate inputs from various cortical layers and are central to information processing. Here we show that the growth of apical dendrites towards the pial surface is regulated by a diffusible chemoattractant present at high levels near the marginal zone. A major component of this signal is semaphorin 3A (Sema3A), which was previously characterized as a chemorepellant for cortical axons. Soluble guanylate cyclase is asymmetrically localized to the developing apical dendrite, and is required for the chemoattractive effect of Sema3A. Thus the asymmetric localization of soluble guanylate cyclase confers distinct Sema3A responses to axons and dendrites. These observations reveal a mechanism by which a single chemotropic signal can pattern both axons and dendrites during development.     

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.     

A new cellular mechanism for coupling inputs arriving at different cortical layers

Pyramidal neurons in layer 5 of the neocortex of the brain extend their axons and dendrites into all layers. They are also unusual in having both an axonal and a dendritic zone for the initiation of action potentials. Distal dendritic inputs, which normally appear greatly attenuated at the axon, must cross a high threshold at the dendritic initiation zone to evoke calcium action potentials but can then generate bursts of axonal action potentials. Here we show that a single back-propagating sodium action potential generated in the axon facilitates the initiation of these calcium action potentials when it coincides with distal dendritic input within a time window of several milliseconds. Inhibitory dendritic input can selectively block the initiation of dendritic calcium action potentials, preventing bursts of axonal action potentials. Thus, excitatory and inhibitory postsynaptic potentials arising in the distal dendrites can exert significantly greater control over action potential initiation in the axon than would be expected from their electrotonically isolated locations. The coincidence of a single back-propagating action potential with a subthreshold distal excitatory postsynaptic potential to evoke a burst of axonal action potentials represents a new mechanism by which the main cortical output neurons can associate inputs arriving at different cortical layers.     

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.     

Fast neurotransmitter release triggered by Ca influx through AMPA-type glutamate receptors

Feedback inhibition at reciprocal synapses between A17 amacrine cells and rod bipolar cells (RBCs) shapes light-evoked responses in the retina. Glutamate-mediated excitation of A17 cells elicits GABA (gamma-aminobutyric acid)-mediated inhibitory feedback onto RBCs, but the mechanisms that underlie GABA release from the dendrites of A17 cells are unknown. If, as observed at all other synapses studied, voltage-gated calcium channels (VGCCs) couple membrane depolarization to neurotransmitter release, feedforward excitatory postsynaptic potentials could spread through A17 dendrites to elicit 'surround' feedback inhibitory transmission at neighbouring synapses. Here we show, however, that GABA release from A17 cells in the rat retina does not depend on VGCCs or membrane depolarization. Instead, calcium-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs), activated by glutamate released from RBCs, provide the calcium influx necessary to trigger GABA release from A17 cells. The AMPAR-mediated calcium signal is amplified by calcium-induced calcium release (CICR) from intracellular calcium stores. These results describe a fast synapse that operates independently of VGCCs and membrane depolarization and reveal a previously unknown form of feedback inhibition within a neural circuit.     

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.     

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.     

Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons

Integration and processing of electrical signals in individual neurons depend critically on the spatial distribution of ion channels on the cell surface. In hippocampal pyramidal neurons, voltage-sensitive calcium channels have important roles in the control of Ca2(+)-dependent cellular processes such as action potential generation, neurotransmitter release, and epileptogenesis. Long-term potentiation of synaptic transmission in the hippocampal pyramidal cell, a form of neuronal plasticity that is thought to represent a cellular correlate of learning and memory, is dependent on Ca2+ entry mediated by synaptic activation of glutamate receptors that have a high affinity for NMDA (N-methyl(-D-aspartate) and are located in distal dendrites. Stimuli causing long-term potentiation at these distal synapses also cause a large local increase in cytosolic Ca2+ in the proximal regions of dendrites. This increase has been proposed to result from activation of voltage-gated Ca2+ channels. At least four types of voltage-gated Ca2+ channels, designated N, L. T and P, may be involved in these processes. Here we show that L-type Ca2+ channels, visualized using a monoclonal antibody, are located in the cell bodies and proximal dendrites of hippocampal pyramidal cells and are clustered in high density at the base of major dendrites. We suggest that these high densities of L-type Ca2+ channels may serve to mediate Ca2+ entry into the pyramidal cell body and proximal dendrites in response to summed excitatory inputs to the distal dendrites and to initiate intracellular regulatory events in the cell body in response to the same synaptic inputs that cause long-term potentiation at distal dendritic synapses.     

Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo

Many lines of evidence suggest that memory in the mammalian brain is stored with distinct spatiotemporal patterns. Despite recent progresses in identifying neuronal populations involved in memory coding, the synapse-level mechanism is still poorly understood. Computational models and electrophysiological data have shown that functional clustering of synapses along dendritic branches leads to nonlinear summation of synaptic inputs and greatly expands the computing power of a neural network. However, whether neighbouring synapses are involved in encoding similar memory and how task-specific cortical networks develop during learning remain elusive. Using transcranial two-photon microscopy, we followed apical dendrites of layer 5 pyramidal neurons in the motor cortex while mice practised novel forelimb skills. Here we show that a third of new dendritic spines (postsynaptic structures of most excitatory synapses) formed during the acquisition phase of learning emerge in clusters, and that most such clusters are neighbouring spine pairs. These clustered new spines are more likely to persist throughout prolonged learning sessions, and even long after training stops, than non-clustered counterparts. Moreover, formation of new spine clusters requires repetition of the same motor task, and the emergence of succedent new spine(s) accompanies the strengthening of the first new spine in the cluster. We also show that under control conditions new spines appear to avoid existing stable spines, rather than being uniformly added along dendrites. However, succedent new spines in clusters overcome such a spatial constraint and form in close vicinity to neighbouring stable spines. Our findings suggest that clustering of new synapses along dendrites is induced by repetitive activation of the cortical circuitry during learning, providing a structural basis for spatial coding of motor memory in the mammalian brain.     

Long-lasting self-inhibition of neocortical interneurons mediated by endocannabinoids

Neocortical GABA-containing interneurons form complex functional networks responsible for feedforward and feedback inhibition and for the generation of cortical oscillations associated with several behavioural functions. We previously reported that fast-spiking (FS), but not low-threshold-spiking (LTS), neocortical interneurons from rats generate a fast and precise self-inhibition mediated by inhibitory autaptic transmission. Here we show that LTS cells possess a different form of self-inhibition. LTS, but not FS, interneurons undergo a prominent hyperpolarization mediated by an increased K+-channel conductance. This self-induced inhibition lasts for many minutes, is dependent on an increase in intracellular [Ca2+] and is blocked by the cannabinoid receptor antagonist AM251, indicating that it is mediated by the autocrine release of endogenous cannabinoids. Endocannabinoid-mediated slow self-inhibition represents a powerful and long-lasting mechanism that alters the intrinsic excitability of LTS neurons, which selectively target the major site of excitatory connections onto pyramidal neurons; that is, their dendrites. Thus, modulation of LTS networks after their sustained firing will lead to long-lasting changes of glutamate-mediated synaptic strength in pyramidal neurons, with consequences during normal and pathophysiological cortical network activities.     

Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential

Traditionally, neuronal operations in the cerebral cortex have been viewed as occurring through the interaction of synaptic potentials in the dendrite and soma, followed by the initiation of an action potential, typically in the axon. Propagation of this action potential to the synaptic terminals is widely believed to be the only form of rapid communication of information between the soma and axonal synapses, and hence to postsynaptic neurons. Here we show that the voltage fluctuations associated with dendrosomatic synaptic activity propagate significant distances along the axon, and that modest changes in the somatic membrane potential of the presynaptic neuron modulate the amplitude and duration of axonal action potentials and, through a Ca2+-dependent mechanism, the average amplitude of the postsynaptic potential evoked by these spikes. These results indicate that synaptic activity in the dendrite and soma controls not only the pattern of action potentials generated, but also the amplitude of the synaptic potentials that these action potentials initiate in local cortical circuits, resulting in synaptic transmission that is a mixture of triggered and graded (analogue) signals.     

A physiological role for GABAB receptors in the central nervous system

The role of GABA in synaptic transmission in the mammalian central nervous system is more firmly established than for any other neurotransmitter. With virtually every neuron studied, the synaptic action of GABA is mediated by bicuculline-sensitive GABAA receptors which selectively increase chloride conductance. However, it has been shown that GABA has a presynaptic inhibitory action on transmitter release that is insensiive to bicuculline and is selectively mimicked by baclofen. The receptors involved in this action are referred to as GABAB receptors, to distinguish them from the classic bicuculline-sensitive GABAA receptors. In hippocampal pyramidal cells an additional postsynaptic action of GABA and baclofen has been reported that is also insensitive to GABAA antagonists, and may be mediated by GABAB receptors on the postsynaptic neuron. This action of GABA and baclofen involves an increase in potassium conductance. Synaptic activation of pathways converging on hippocampal pyramidal cells results in a slow inhibitory postsynaptic potential which involves an increase in potassium conductance, and it has been suggested that GABAB receptors might be responsible for this synaptic potential. However, to establish convincingly that GABAB receptors are physiologically important in the central nervous system, a selective GABAB antagonist is required. Here we provide this missing evidence. Using the hippocampal slice preparation, we now report that the phosphonic acid derivative of baclofen, phaclofen, is a remarkably selective antagonist of both the postsynaptic action of baclofen and the bicuculline-resistant action of GABA, and that it selectively abolishes the slow inhibitory postsynaptic potential in pyramidal cells.     

Organization of cell assemblies in the hippocampus

Neurons can produce action potentials with high temporal precision. A fundamental issue is whether, and how, this capability is used in information processing. According to the 'cell assembly' hypothesis, transient synchrony of anatomically distributed groups of neurons underlies processing of both external sensory input and internal cognitive mechanisms. Accordingly, neuron populations should be arranged into groups whose synchrony exceeds that predicted by common modulation by sensory input. Here we find that the spike times of hippocampal pyramidal cells can be predicted more accurately by using the spike times of simultaneously recorded neurons in addition to the animals location in space. This improvement remained when the spatial prediction was refined with a spatially dependent theta phase modulation. The time window in which spike times are best predicted from simultaneous peer activity is 10-30 ms, suggesting that cell assemblies are synchronized at this timescale. Because this temporal window matches the membrane time constant of pyramidal neurons, the period of the hippocampal gamma oscillation and the time window for synaptic plasticity, we propose that cooperative activity at this timescale is optimal for information transmission and storage in cortical circuits.     

Turning on and off recurrent balanced cortical activity

The vast majority of synaptic connections onto neurons in the cerebral cortex arise from other cortical neurons, both excitatory and inhibitory, forming local and distant 'recurrent' networks. Although this is a basic theme of cortical organization, its study has been limited largely to theoretical investigations, which predict that local recurrent networks show a proportionality or balance between recurrent excitation and inhibition, allowing the generation of stable periods of activity. This recurrent activity might underlie such diverse operations as short-term memory, the modulation of neuronal excitability with attention, and the generation of spontaneous activity during sleep. Here we show that local cortical circuits do indeed operate through a proportional balance of excitation and inhibition generated through local recurrent connections, and that the operation of such circuits can generate self-sustaining activity that can be turned on and off by synaptic inputs. These results confirm the long-hypothesized role of recurrent activity as a basic operation of the cerebral cortex.     

Brain-state- and cell-type-specific firing of hippocampal interneurons in vivo

Neural-network oscillations at distinct frequencies have been implicated in the encoding, consolidation and retrieval of information in the hippocampus. Some GABA (gamma-aminobutyric acid)-containing interneurons fire phase-locked to theta oscillations (4-8 Hz) or to sharp-wave-associated ripple oscillations (120-200 Hz), which represent different behavioural states. Interneurons also entrain pyramidal cells in vitro. The large diversity of interneurons poses the question of whether they have specific roles in shaping distinct network activities in vivo. Here we report that three distinct interneuron types--basket, axo-axonic and oriens-lacunosum-moleculare cells--visualized and defined by synaptic connectivity as well as by neurochemical markers, contribute differentially to theta and ripple oscillations in anaesthetized rats. The firing patterns of individual cells of the same class are remarkably stereotyped and provide unique signatures for each class. We conclude that the diversity of interneurons, innervating distinct domains of pyramidal cells, emerged to coordinate the activity of pyramidal cells in a temporally distinct and brain-state-dependent manner.     

Recombinant human lipocortin 1 inhibits thromboxane release from guinea-pig isolated perfused lung

The guinea-pig perfused isolated lung, used in conjunction with the cascade superfusion system to measure the release of thromboxane A2(TXA2), is a simple and convenient model for assessing the inhibition by glucocorticoids of eicosanoid formation. Dexamethasone inhibits the release of TXA2 from the lung when it is stimulated by agents such as RCS-RF2 of leukotrienes, but not when bradykinin or arachidonic acid are used. Using this model we have shown that the glucocorticoids suppress eicosanoid generation by cells through the induction of a family of phospholipase A2-inhibitory proteins now termed the 'lipocortins'. Recently the primary structure of one form of lipocortin has been elucidated and the human gene cloned. Lipocortin 1 is a polar monomeric protein with anti-phospholipase properties in vitro and we now report that when infused into guinea-pig lung preparations this protein has the same inhibitory profile as the glucocorticoids but with a more rapid onset of action. This is the first demonstration that eicosanoid formation can be inhibited by a recombinant phospholipase inhibitory protein applied extracellularly.     

Experience-dependent and cell-type-specific spine growth in the neocortex

Functional circuits in the adult neocortex adjust to novel sensory experience, but the underlying synaptic mechanisms remain unknown. Growth and retraction of dendritic spines with synapse formation and elimination could change brain circuits. In the apical tufts of layer 5B (L5B) pyramidal neurons in the mouse barrel cortex, a subset of dendritic spines appear and disappear over days, whereas most spines are persistent for months. Under baseline conditions, new spines are mostly transient and rarely survive for more than a week. Transient spines tend to be small, whereas persistent spines are usually large. Because most excitatory synapses in the cortex occur on spines, and because synapse size and the number of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are proportional to spine volume, the excitation of pyramidal neurons is probably driven through synapses on persistent spines. Here we test whether the generation and loss of persistent spines are enhanced by novel sensory experience. We repeatedly imaged dendritic spines for one month after trimming alternate whiskers, a paradigm that induces adaptive functional changes in neocortical circuits. Whisker trimming stabilized new spines and destabilized previously persistent spines. New-persistent spines always formed synapses. They were preferentially added on L5B neurons with complex apical tufts rather than simple tufts. Our data indicate that novel sensory experience drives the stabilization of new spines on subclasses of cortical neurons. These synaptic changes probably underlie experience-dependent remodelling of specific neocortical circuits.     

Spike train dynamics predicts theta-related phase precession in hippocampal pyramidal cells

According to the temporal coding hypothesis, neurons encode information by the exact timing of spikes. An example of temporal coding is the hippocampal phase precession phenomenon, in which the timing of pyramidal cell spikes relative to the theta rhythm shows a unidirectional forward precession during spatial behaviour. Here we show that phase precession occurs in both spatial and non-spatial behaviours. We found that spike phase correlated with instantaneous discharge rate, and processed unidirectionally at high rates, regardless of behaviour. The spatial phase precession phenomenon is therefore a manifestation of a more fundamental principle governing the timing of pyramidal cell discharge. We suggest that intrinsic properties of pyramidal cells have a key role in determining spike times, and that the interplay between the magnitude of dendritic excitation and rhythmic inhibition of the somatic region is responsible for the phase assignment of spikes.