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Dr. Bert Sakmann (b. 1942) studied at the Universities of Tuebingen, Freiburg, Berlin, Paris, and Munich, graduating in 1967. Much of his professional life has been spent in various institutes of the Max Planck Society. In 1971, a British Council Fellowship took him to the Department of Biophysics of University College London to work with Bernard Katz (1911-2003). In 1974, he obtained his Ph.D. from the University of Goettingen and, with Erwin Neher (b. 1944) at the Max Planck Institute for Biophysical Chemistry, began work that would transform cellular biology and neuroscience, resulting in the 1991 Nobel Prize for Physiology or Medicine. In 2008, Dr. Sakmann returned to Munich, where he headed the research group "Cortical Columns in Silico" at the Max Planck Institute of Neurobiology in Martinsried. Here, their group discovered the cell-type specific sensory activation patterns in different layers of a column in the vibrissal area of rodents' somatosensory cortices.

Understanding the communication between nerve cells in the brain is key to understanding how the brain works. Communication between nerve cells involves chemical messages sent from one cell that get translated into electrical activity in the receiving cell. This electrical activity is the core language of nerve cells and of the entire brain. How does a chemical message released in one cell results in electrical activity in another nerve cell, and how did we discovery this? Let us dive together into the electrifying world of nerve cell communication. I will tell you about our experiments, which enabled us to find the most basic component of electrical activity in the brain—ion channels. The discovery of ion channels paved the way to understanding the origin of electrical activity in the brain and other organs like the heart. This discovery provided new insights into the development of drugs for treating various electrical-related diseases, such as epilepsy and heart-rate disorders.

It has become increasingly clear that the neurons in the cerebral cortex are not randomly interconnected. This wiring specificity can result from synapse formation mechanisms that interconnect neurons depending on their activity or genetically defined identity. Here we report that in addition to these synapse formation mechanisms, the structural composition of the neuropil provides a third prominent source by which wiring specificity emerges in cortical networks. This structurally determined wiring specificity reflects the packing density, morphological diversity and similarity of the dendritic and axonal processes. The higher these three factors are, the more recurrent the topology of the networks. Conversely, low density, diversity and similarity yield feedforward networks. These principles predict connectivity patterns from subcellular to network scales that are remarkably consistent with empirical observations from a rich body of literature. Thus, cortical network architectures reflect the specific morphological properties of their constituents to much larger degrees than previously thought.

Dissecting hyperactivation in AD Progressive accumulation of amyloid β (Aβ) in the brain is a defining feature of Alzheimer's disease (AD). At late stages of AD, pathological Aβ accumulations cause neurodegeneration and cell death. However, neuronal dysfunction, consisting of an excessively increased activity in a fraction of brain neurons, already occurs in early stages of the disease. Zott et al. explored the cellular basis of this hyperactivity in mouse models of AD (see the Perspective by Selkoe). Aβ-mediated hyperactivation was linked to a defect in synaptic transmission exclusively in active neurons, with the most-active neurons having the highest risk of hyperactivation. Aβ-containing brain extracts from human AD patients sustained this vicious cycle, underscoring the potential relevance of this pathological mechanism in humans. Science , this issue p. 559 ; see also p. 540

Pyramidal tract neurons (PTs) represent the major output cell type of the neocortex. To investigate principles of how the results of cortical processing are broadcasted to different downstream targets thus requires experimental approaches, which provide access to the in vivo electrophysiology of PTs, whose subcortical target regions are identified. On the example of rat barrel cortex (vS1), we illustrate that retrograde tracer injections into multiple subcortical structures allow identifying the long-range axonal targets of individual in vivo recorded PTs. Here we report that soma depth and dendritic path lengths within each cortical layer of vS1, as well as spiking patterns during both periods of ongoing activity and during sensory stimulation, reflect the respective subcortical target regions of PTs. We show that these cellular properties result in a structure–function parameter space that allows predicting a PT’s subcortical target region, without the need to inject multiple retrograde tracers., The major output cell type of the neocortex – pyramidal tract neurons (PTs) – send axonal projections to various subcortical areas. Here the authors combined in vivo recordings, retrograde tracings, and reconstructions of PTs in rat somatosensory cortex to show that PT structure and activity can predict specific subcortical targets.

Amyloid-β (Aβ) is thought to play an essential pathogenic role in Alzheimer´s disease (AD). A key enzyme involved in the generation of Aβ is the β-secretase BACE, for which powerful inhibitors have been developed and are currently in use in human clinical trials. However, although BACE inhibition can reduce cerebral Aβ levels, whether it also can ameliorate neural circuit and memory impairments remains unclear. Using histochemistry, in vivo Ca2+ imaging, and behavioral analyses in a mouse model of AD, we demonstrate that along with reducing prefibrillary Aβ surrounding plaques, the inhibition of BACE activity can rescue neuronal hyperactivity, impaired long-range circuit function, and memory defects. The functional neuronal impairments reappeared after infusion of soluble Aβ, mechanistically linking Aβ pathology to neuronal and cognitive dysfunction. These data highlight the potential benefits of BACE inhibition for the effective treatment of a wide range of AD-like pathophysiological and cognitive impairments.

Cortical layer 5B (L5B) thick-tufted pyramidal neurons have reliable responses to whisker stimulation in anesthetized rodents. These cells drive a corticothalamic pathway that evokes spikes in thalamic posterior medial nucleus (POm). While a subset of POm has been shown to integrate both cortical L5B and paralemniscal signals, the majority of POm neurons are suggested to receive driving input from L5B only. Here, we test this possibility by investigating the origin of whisker-evoked responses in POm and specifically the contribution of the L5B-POm pathway. We compare L5B spiking with POm spiking and subthreshold responses to whisker deflections in urethane anesthetized mice. We find that a subset of recorded POm neurons shows early (< 50 ms) spike responses and early large EPSPs. In these neurons, the early large EPSPs matched L5B input criteria, were blocked by cortical inhibition, and also interacted with spontaneous Up state coupled large EPSPs. This result supports the view of POm subdivisions, one of which receives whisker signals predominantly via L5B neurons.

Sensory information reaches the cortex through synchronously active thalamic axons, which provide a strong drive to layer 4 (L4) cortical neurons. Because of technical limitations, the dendritic signaling processes underlying the rapid and efficient activation of L4 neurons in vivo remained unknown. Here we introduce an approach that allows the direct monitoring of single dendritic spine Ca(2+) signals in L4 spiny stellate cells of the vibrissal mouse cortex in vivo. Our results demonstrate that activation of N-methyl-D-aspartate (NMDA) receptors is required for sensory-evoked action potential (AP) generation in these neurons. By analyzing NMDA receptor-mediated Ca(2+) signaling, we identify whisker stimulation-evoked large responses in a subset of dendritic spines. These sensory-stimulation-activated spines, representing predominantly thalamo-cortical input sites, were denser at proximal dendritic regions. The amplitude of sensory-evoked spine Ca(2+) signals was independent of the activity of neighboring spines, without evidence for cooperativity. Furthermore, we found that spine Ca(2+) signals evoked by back-propagating APs sum linearly with sensory-evoked synaptic Ca(2+) signals. Thus, our results identify in sensory information-receiving L4 cortical neurons a linear mode of dendritic integration that underlies the rapid and reliable transfer of peripheral signals to the cortical network.

The cortex connects to the thalamus via extensive corticothalamic (CT) pathways, but their function in vivo is not well understood. We investigated "top-down" signaling from cortex to thalamus via the cortical layer 5B (L5B) to posterior medial nucleus (POm) pathway in the whisker system of the anesthetized mouse. While L5B CT inputs to POm are extremely strong in vitro, ongoing activity of L5 neurons in vivo might tonically depress these inputs and thereby block CT spike transfer. We find robust transfer of spikes from the cortex to the thalamus, mediated by few L5B-POm synapses. However, the gain of this pathway is not constant but instead is controlled by global cortical Up and Down states. We characterized in vivo CT spike transfer by analyzing unitary PSPs and found that a minority of PSPs drove POm spikes when CT gain peaked at the beginning of Up states. CT gain declined sharply during Up states due to frequency-dependent adaptation, resulting in periodic high gain-low gain oscillations. We estimate that POm neurons receive few (2-3) active L5B inputs. Thus, the L5B-POm pathway strongly amplifies the output of a few L5B neurons and locks thalamic POm sub-and suprathreshold activity to cortical L5B spiking.

Although patch pipettes were initially designed to record extracellularly the elementary current events from muscle and neuron membranes, the whole-cell and loose cell-attached recording configurations proved to be useful tools for examination of signalling within and between nerve cells. In this Paton Prize Lecture, I will initially summarize work on electrical signalling within single neurons, describing communication between the dendritic compartments, soma and nerve terminals via forward- and backward-propagating action potentials. The newly discovered dendritic excitability endows neurons with the capacity for coincidence detection of spatially separated subthreshold inputs. When these are occurring during a time window of tens of milliseconds, this information is broadcast to other cells by the initiation of bursts of action potentials (AP bursts). The occurrence of AP bursts critically impacts signalling between neurons that are controlled by target-cell-specific transmitter release mechanisms at downstream synapses even in different terminals of the same neuron. This can, in turn, induce mechanisms that underly synaptic plasticity when AP bursts occur within a short time window, both presynaptically in terminals and postsynaptically in dendrites. A fundamental question that arises from these findings is: 'what are the possible functions of active dendritic excitability with respect to network dynamics in the intact cortex of behaving animals?' To answer this question, I highlight in this review the functional and anatomical architectures of an average cortical column in the vibrissal (whisker) field of the somatosensory cortex (vS1), with an emphasis on the functions of layer 5 thick-tufted cells (L5tt) embedded in this structure. Sensory-evoked synaptic and action potential responses of these major cortical output neurons are compared with responses in the afferent pathway, viz. the neurons in primary somatosensory thalamus and in one of their efferent targets, the secondary somatosensory thalamus. Coincidence-detection mechanisms appear to be implemented in vivo as judged from the occurrence of AP bursts. Three-dimensional reconstructions of anatomical projections suggest that inputs of several combinations of thalamocortical projections and intra- and transcolumnar connections, specifically those from infragranular layers, could trigger active dendritic mechanisms that generate AP bursts. Finally, recordings from target cells of a column reveal the importance of AP bursts for signal transfer to these cells. The observations lead to the hypothesis that in vS1 cortex, the sensory afferent sensory code is transformed, at least in part, from a rate to an interval (burst) code that broadcasts the occurrence of whisker touch to different targets of L5tt cells. In addition, the occurrence of pre- and postsynaptic AP bursts may, in the long run, alter touch representation in cortex.

In the vibrissal area of rodent somatosensory cortex, information on whisker stimulation is processed by neuronal networks in a corresponding cortical column. To understand how sensory stimuli are represented in a column, it is essential to identify cell types constituting these networks. Layer 6 (L6) comprises 25% of all neurons in a column. In rats, 430 of these are inhibitory interneurons (INs). Little is known about the axon projection of L6 INs with reference to columnar and laminar organization. We quantified axonal projections of L6 INs (n = 68) with reference to columns and layers in somatosensory cortex of rats. We found distinct projection types differentially targeting layers of a cortical column. The majority of L6 INs did not show a column-specific innervation, densely projecting to neighboring columns as well as the home column. However, a small fraction targeted granular and supragranular layers, where axon projections were confined to the home column. We also quantified putative innervation of pyramidal cells as a functional correlate of axonal distribution. Electrophysiological properties were not correlated to axon projection. The quantitative data on axonal projections and electrophysiological properties of L6 INs can guide future studies investigating cortical processing of sensory information at the single cell level.

Soma location, dendrite morphology, and synaptic innervation may represent key determinants of functional responses of individual neurons, such as sensory-evoked spiking. Here, we reconstruct the 3D circuits formed by thalamocortical afferents from the lemniscal pathway and excitatory neurons of an anatomically defined cortical column in rat vibrissal cortex. We objectively classify 9 cortical cell types and estimate the number and distribution of their somata, dendrites, and thalamocortical synapses. Somata and dendrites of most cell types intermingle, while thalamocortical connectivity depends strongly upon the cell type and the 3D soma location of the postsynaptic neuron. Correlating dendrite morphology and thalamocortical connectivity to functional responses revealed that the lemniscal afferents can account for some of the cell type- and location-specific subthreshold and spiking responses after passive whisker touch (e.g., in layer 4, but not for other cell types, e.g., in layer 5). Our data provides a quantitative 3D prediction of the cell type-specific lemniscal synaptic wiring diagram and elucidates structure-function relationships of this physiologically releVant pathway at single-cell resolution. © The Author 2011. Published by Oxford University Press. All rights reserved.

Single cortical neurons in the mammalian brain receive signals arising from multiple sensory input channels. Dendritic integration of these afferent signals is critical in determining the amplitude and time course of the neurons' output signals. As of yet, little is known about the spatial and temporal organization of converging sensory inputs. Here, we combined in vivo two-photon imaging with whole-cell recordings in layer 2 neurons of the mouse vibrissal cortex as a means to analyze the spatial pattern of subthreshold dendritic calcium signals evoked by the stimulation of different whiskers. We show that the principle whisker and the surrounding whiskers can evoke dendritic calcium transients in the same neuron. Distance-dependent attenuation of dendritic calcium transients and the corresponding subthreshold depolarization suggest feed-forward activation. We found that stimulation of different whiskers produced multiple calcium hotspots on the same dendrite. Individual hotspots were activated with low probability in a stochastic manner. We show that these hotspots are generated by calcium signals arising in dendritic spines. Some spines were activated uniquely by single whiskers, but many spines were activated by multiple whiskers. These shared spines indicate the existence of presynaptic feeder neurons that integrate and transmit activity arising from multiple whiskers. Despite the dendritic overlap of whisker-specific and shared inputs, different whiskers are represented by a unique set of activation patterns within the dendritic field of each neuron.

Neuron morphology is frequently used to classify cell-types in the mammalian cortex. Apart from the shape of the soma and the axonal projections, morphological classification is largely defined by the dendrites of a neuron and their subcellular compartments, referred to as dendritic spines. The dimensions of a neuron’s dendritic compartment, including its spines, is also a major determinant of the passive and active electrical excitability of dendrites. Furthermore, the dimensions of dendritic branches and spines change during postnatal development and, possibly, following some types of neuronal activity patterns, changes depending on the activity of a neuron. Due to their small size, accurate quantitation of spine number and structure is difficult to achieve (Larkman, J Comp Neurol 306:332, 1991). Here we follow an analysis approach using high-resolution EM techniques. Serial block-face scanning electron microscopy (SBFSEM) enables automated imaging of large specimen volumes at high resolution. The large data sets generated by this technique make manual reconstruction of neuronal structure laborious. Here we present NeuroStruct, a reconstruction environment developed for fast and automated analysis of large SBFSEM data sets containing individual stained neurons using optimized algorithms for CPU and GPU hardware. NeuroStruct is based on 3D operators and integrates image information from image stacks of individual neurons filled with biocytin and stained with osmium tetroxide. The focus of the presented work is the reconstruction of dendritic branches with detailed representation of spines. NeuroStruct delivers both a 3D surface model of the reconstructed structures and a 1D geometrical model corresponding to the skeleton of the reconstructed structures. Both representations are a prerequisite for analysis of morphological characteristics and simulation signalling within a neuron that capture the influence of spines. Electronic supplementary material The online version of this article (doi:10.1007/s10827-011-0316-1) contains supplementary material, which is available to authorized users.

This is the second article in a series of three studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). Here, we report the number and distribution of NeuN-positive neurons within the C2, D2, and D3 TC projection columns in P27 rat somatosensory barrel cortex based on an exhaustive identification of 89,834 somata in a 1.15 mm(3) volume of cortex. A single column contained 19,109 ± 444 neurons (17,560 ± 399 when normalized to a standard-size projection column). Neuron density differences along the vertical column axis delineated "cytoarchitectonic" layers. The resulting neuron numbers per layer in the average column were 63 ± 10 (L1), 2039 ± 524 (L2), 3735 ± 905 (L3), 4447 ± 439 (L4), 1737 ± 251 (L5A), 2235 ± 99 (L5B), 3786 ± 168 (L6A), and 1066 ± 170 (L6B). These data were then used to derive the layer-specific action potential (AP) output of a projection column. The estimates confirmed previous reports suggesting that the ensembles of spiny L4 and thick-tufted pyramidal neurons emit the major fraction of APs of a column. The number of APs evoked in a column by a sensory stimulus (principal whisker deflection) was estimated as 4441 within 100 ms post-stimulus.

Sensory experience can, over the course of days to weeks, produce long-lasting changes in brain function. Recent studies suggest that functional plasticity is mediated by alterations of the strengths of existing synapses or dynamics of dendritic spines. Alterations of cortical axons could also contribute to functional changes, but little is known about the effects of experience at the level of individual corticocortical axons. We reconstructed individual layer (L) 2/3 pyramidal neurons filled in vivo in developing barrel cortex of control and partially sensory-deprived rats. L2 axons had larger field spans than L3 axons but were otherwise equivalently affected by deprivation. Whisker trimming over ∼2 weeks markedly reduced overall length of axonal branches in L2/3, but individual horizontal axons were as likely to innervate deprived areas as spared ones. The largest effect of deprivation was instead to reduce the length of those axonal branches in L2/3 oriented toward deprived regions. Thus, the location of a branch relative to its originating soma, rather than its own location within any specific cortical column, was the strongest determinant of axonal organization. Individual axons from L2/3 into L5/6 were similarly altered by whisker trimming although to a lesser extent. Thus, sensory experience over relatively short timescales may change the patterning of specific axonal branches within as well as between cortical columns during development. Copyright © 2009 Society for Neuroscience.

High frequency (≥ 100 Hz) bursts of action potentials (APs) generated by neocortical neurons are thought to increase information content and, through back-propagation, to influence synaptic integration and efficacy in distal dendritic compartments. It was recently shown in acute slice experiments that intrinsic bursting properties differ between neocortical L2/3 and L5B (thick tufted) neurons. In L2/3 neurons for instance, dendritic APs were brief and generated only one additional AP after the initial somatic AP. In L5B neurons, dendritic plateau potentials facilitated the generation of trains of three or more APs. We recently showed in vivo that spiking frequencies are very different for L2/3 and L5B thick tufted neurons under anaesthesia. Here, we addressed the question whether in vivo the bursting properties are different for these two cell types. We recorded from L2/3 and L5B thick tufted neurons of rat primary somatosensory (barrel) cortex under anaesthetized and awake conditions and found that AP activity is dominated by single APs. In addition, we found that in the anaesthetized animal also bursts of two APs were observed in L2/3 neurons but the relative occurrence of these bursts was low. In L5B thick tufted neurons, bursts consisting of up to six APs were recorded and their relative occurrence was significantly higher. Frequencies within bursts were also significantly higher in L5B thick tufted neurons than in L2/3 neurons. In awake (head-restrained) animals, average spike frequencies of L2/3 and L5B thick tufted neurons were surprisingly similar to spike rates under anaesthesia. However, bursting behaviour in L2/3 neurons was comparable to L5B thick tufted neurons. Thus, the distribution of interspike intervals was changed in L2/3 neurons without affecting the average spiking rate. We observed bursts consisting of up to five APs in both cell types and both probability of bursts and AP frequency within bursts were similar for L2/3 and L5B thick tufted neurons. Our analysis shows that most cortical APs occur as single APs, although a minor fraction of APs in L2/3 and L5B thick tufted neurons are part of high frequency bursts (15%). This AP bursting is dependent on the behavioural state of the animal in a cell-type dependent manner.

The probability of synaptic transmitter release determines the spread of excitation and the possible range of computations at unitary connections. To investigate whether synaptic properties between neocortical pyramidal neurons change during the assembly period of cortical circuits, whole-cell voltage recordings were made simultaneously from two layer 5A (L5A) pyramidal neurons within the cortical columns of rat barrel cortex. We found that synaptic transmission between L5A pyramidal neurons is very reliable between 2 and 3 weeks of postnatal development with a mean unitary EPSP amplitude of approximately 1.2 mV, but becomes less efficient and fails more frequently in the more mature cortex of approximately 4 weeks of age with a mean unitary EPSP amplitude of 0.65 mV. Coefficient of variation and failure rate increase as the unitary EPSP amplitude decreases during development. The paired-pulse ratio (PPR) of synaptic efficacy at 10 Hz changes from 0.7 to 1.04. Despite the overall increase in PPR, short-term plasticity displays a large variability at 4 weeks, ranging from strong depression to strong facilitation (PPR, range 0.6-2.1), suggesting the potential for use-dependent modifications at this intracortical synapse. In conclusion, the transmitter release probability at the L5A-L5A connection is developmentally regulated in such a way that in juvenile animals excitation by single action potentials is efficiently transmitted, whereas in the more mature cortex synapses might be endowed with a diversity of filtering characteristics.

Sensory stimuli are encoded differently across cortical layers and it is unknown how response characteristics relate to the morphological identity of responding cells. We therefore juxtasomally recorded action potential (AP) patterns from excitatory cells in layer (L) 2/3, L4, L5 and L6 of rat barrel cortex in response to a standard stimulus (e.g. repeated deflection of single whiskers in the caudal direction). Subsequent single-cell filling with biocytin allowed for post hoc identification of recorded cells. We report three major conclusions. First, sensory-evoked responses were layer- and cell-type-specific but always < 1 AP per stimulus, indicating low AP rates for the entire cortical column. Second, response latencies from L4, L5B and L6 were comparable and thus a whisker deflection is initially represented simultaneously in these layers. Finally, L5 thick-tufted cells dominated the cortical AP output following sensory stimulation, suggesting that these cells could direct sensory guided behaviours.

The connectivity of the hippocampal trisynaptic circuit, formed by the dentate gyrus, the CA3 and the CA1 region, is well characterized anatomically and functionally in vitro . The functional connectivity of this circuit in vivo remains to be understood. Toward this goal, we investigated the influence of the spontaneous, synchronized oscillations in the neocortical local field potential, reflecting up–down states (UDS) of cortical neurons, on the hippocampus. We simultaneously measured the extracellular local field potential in association cortex and the membrane potential of identified hippocampal excitatory neurons in anesthetized mice. Dentate gyrus granule cells showed clear UDS modulation that was phase locked to cortical UDS with a short delay. In contrast, CA3 pyramidal neurons showed mixed UDS modulation, such that some cells were depolarized during the cortical up state and others were hyperpolarized. CA1 pyramidal neurons, located farther downstream, showed consistent UDS modulation, such that when the cortical and dentate gyrus neurons were depolarized, the CA1 pyramidal cells were hyperpolarized. These results demonstrate the differential functional connectivity between neocortex and hippocampal subfields during UDS oscillations.

Voltage-gated potassium channels effectively regulate dendritic excitability in neurones. It has been suggested that in the distal apical dendrite of layer 5B (L5B) neocortical pyramidal neurones, K+ conductances participate in active dendritic synaptic integration and control regenerative dendritic potentials. The ionic mechanism for triggering these regenerative potentials has yet to be elucidated. Here we used two-electrode voltage clamp (TEVC) to quantitatively record K+ conductance densities of a sustained K+ conductance in the soma and apical dendrite of L5B neurones of adult rats. We report that the somatic and proximal dendritic sustained voltage-gated K+ conductance density is more than 10-fold larger than previous estimates. The results obtained using TEVC were corroborated using current-clamp experiments in combination with compartmental modelling. Possible error sources, including inaccurate measurement of the passive membrane parameters and unknown axonal and basal dendritic conductance distributions, were shown not to distort the density estimation considerably. The sustained voltage-gated K+ conductance density was found to decrease steeply along the apical dendrite. The steep negative K+ conductance density gradient along the apical dendrite may help to define a distal, low-threshold region for amplification of distal synaptic input in L5B pyramidal neurones.

Sir Bernard Katz established the cellular basis of synaptic transmission at the neuromuscular junction, the contact point between nerve and muscle. With his death, we lost one of the most distinguished biophysicists of our time. He laid the foundations for our understanding of almost every aspect of synaptic transmission. Bernard Katz revealed the existence of key molecules and formally described their interaction. With the benefit of his almost magical intuition, he formulated hypotheses that are now recognized as facts.

Vertical thalamocortical afferents give rise to the elementary functional units of sensory cortex, cortical columns. Principles that underlie communication between columns remain however unknown. Here we unravel these by reconstructing in vivo-labeled neurons from all excitatory cell types in the vibrissal part of rat primary somatosensory cortex (vS1). Integrating the morphologies into an exact 3D model of vS1 revealed that the majority of intracortical (IC) axons project far beyond the borders of the principal column. We defined the corresponding innervation volume as the IC-unit. Deconstructing this structural cortical unit into its cell type-specific components, we found asymmetric projections that innervate columns of either the same whisker row or arc, and which subdivide vS1 into 2 orthogonal [supra-]granular and infragranular strata. We show that such organization could be most effective for encoding multi whisker inputs. Communication between columns is thus organized by multiple highly specific horizontal projection patterns, rendering IC-units as the primary structural entities for processing complex sensory stimuli.

Cortical inhibitory interneurons (INs) are subdivided into a variety of morphologically and functionally specialized cell types. How the respective specific properties translate into mechanisms that regulate sensory-evoked responses of pyramidal neurons (PNs) remains unknown. Here, we investigated how INs located in cortical layer 1 (L1) of rat barrel cortex affect whisker-evoked responses of L2 PNs. To do so we combined in vivo electrophysiology and morphological reconstructions with computational modeling. We show that whisker-evoked membrane depolarization in L2 PNs arises from highly specialized spatiotemporal synaptic input patterns. Temporally L1 INs and L2–5 PNs provide near synchronous synaptic input. Spatially synaptic contacts from L1 INs target distal apical tuft dendrites, whereas PNs primarily innervate basal and proximal apical dendrites. Simulations of such constrained synaptic input patterns predicted that inactivation of L1 INs increases trial-to-trial variability of whisker-evoked responses in L2 PNs. The in silico predictions were confirmed in vivo by L1-specific pharmacological manipulations. We present a mechanism—consistent with the theory of distal dendritic shunting—that can regulate the robustness of sensory-evoked responses in PNs without affecting response amplitude or latency.

Synaptically coupled layer 2/3 (L2/3) pyramidal neurones located above the same layer 4 barrel ('barrel-related') were investigated using dual whole-cell voltage recordings in acute slices of rat somatosensory cortex. Recordings were followed by reconstructions of biocytin-filled neurones. The onset latency of unitary EPSPs was 1.1 +/- 0.4 ms, the 20-80% rise time was 0.7 +/- 0.2 ms, the average amplitude was 1.0 +/- 0.7 mV and the decay time constant was 15.7 +/- 4.5 ms. The coefficient of variation (c.v.) of unitary EPSP amplitudes decreased with increasing EPSP peak and was 0.33 +/- 0.18. Bursts of APs in the presynaptic pyramidal cell resulted in EPSPs that, over a wide range of frequencies (5-100 Hz), displayed amplitude depression. Anatomically the barrel-related pyramidal cells in the lower half of layer 2/3 have a long apical dendrite with a small terminal tuft, while pyramidal cells in the upper half of layer 2/3 have shorter and often more 'irregularly' shaped apical dendrites that branch profusely in layer 1. The number of putative excitatory synaptic contacts established by the axonal collaterals of a L2/3 pyramidal cell with a postsynaptic pyramidal cell in the same column varied between 2 and 4, with an average of 2.8 +/- 0.7 (n = 8 pairs). Synaptic contacts were established predominantly on the basal dendrites at a mean geometric distance of 91 +/- 47 mum from the pyramidal cell soma. L2/3-to-L2/3 connections formed a blob-like innervation domain containing 2.8 mm of the presynaptic axon collaterals with a bouton density of 0.3 boutons per mum axon. Within the supragranular layers of its home column a single L2/3 pyramidal cell established about 900 boutons suggesting that 270 pyramidal cells in layer 2/3 are innervated by an individual pyramidal cell. In turn, a single pyramidal cell received synaptic inputs from 270 other L2/3 pyramidal cells. The innervation domain of L2/3-to-L2/3 connections superimposes almost exactly with that of L4-to-L2/3 connections. This suggests that synchronous feed-forward excitation of L2/3 pyramidal cells arriving from layer 4 could be potentially amplified in layer 2/3 by feedback excitation within a column and then relayed to the neighbouring columns.

Sensory stimuli reach the brain via the thalamocortical projection, a group of axons thought to be among the most powerful in the neocortex. Surprisingly, these axons account for only ∼15% of synapses onto cortical neurons. The thalamocortical pathway might thus achieve its effectiveness via high-efficacy thalamocortical synapses or via amplification within cortical layer 4. In rat somatosensory cortex, we measured in vivo the excitatory postsynaptic potential evoked by a single synaptic connection and found that thalamocortical synapses have low efficacy. Convergent inputs, however, are both numerous and synchronous, and intracortical amplification is not required. Our results suggest a mechanism of cortical activation by which thalamic input alone can drive cortex.

Intracellular recording, which allows direct measurement of the membrane potential and currents of individual neurons, requires a very mechanically stable preparation and has thus been limited to in vitro and head-immobilized in vivo experiments. This restriction constitutes a major obstacle for linking cellular and synaptic physiology with animal behavior. To overcome this limitation we have developed a method for performing whole-cell recordings in freely moving rats. We constructed a miniature head-mountable recording device, with mechanical stabilization achieved by anchoring the recording pipette rigidly in place after the whole-cell configuration is established. We obtain long-duration recordings (mean of approximately 20 min, maximum 60 min) in freely moving animals that are remarkably insensitive to mechanical disturbances, then reconstruct the anatomy of the recorded cells. This head-anchored whole-cell recording technique will enable a wide range of new studies involving detailed measurement and manipulation of the physiological properties of identified cells during natural behaviors.

Transmitter release is triggered by highly localized, transient increases in the presynaptic Ca2+ concentration ([Ca2+]). Rapidly decaying [Ca2+] elevations were generated using Ca2+ uncaging techniques, and [Ca2+] was measured with a low-affinity Ca2+ indicator in a giant presynaptic terminal, the calyx of Held, in rat brain slices. The rise time and amplitude of evoked excitatory postsynaptic currents (EPSCs) depended on the half-width of the fluorescence transient, which was predicted by a five-binding site model of a Ca2+ sensor having relatively high affinity (Kd approx13 muM). Very fast [Ca2+] transients (half-width 2+] transients of variable amplitudes demonstrated the supralinear transfer function of the sensor. The sensitivity of release to the time course of the [Ca2+] transient may contribute to mechanisms by which the presynaptic AP waveform controls synaptic strength.

Layer 5 (L5) pyramidal neurones constitute a major sub- and intracortical output of the somatosensory cortex. This layer 5 is segregated into layers 5A and 5B which receive and distribute relatively independent afferent and efferent pathways. We performed in vivo whole-cell recordings from L5 neurones of the somatosensory (barrel) cortex of urethane-anaesthetized rats (aged 27-31 days). By delivering 6 deg single whisker deflections, whisker pad receptive fields were mapped for 16 L5A and 11 L5B neurones located below the layer 4 whisker-barrels. Average resting membrane potentials were -75.6 +/- 1.1 mV, and spontaneous action potential (AP) rates were 0.54 +/- 0.14 APs s(-1). Principal whisker (PW) evoked responses were similar in L5A and L5B neurones, with an average 5.0 +/- 0.6 mV postsynaptic potential (PSP) and 0.12 +/- 0.03 APs per stimulus. The layer 5A sub- and suprathreshold receptive fields (RFs) were more confined to the principle whisker than those of layer 5B. The basal dendritic arbors of layer 5A and 5B cells were located below both layer 4 barrels and septa, and the cell bodies were biased towards the barrel walls. Responses in both L5A and L5B developed slowly, with onset latencies of 10.1 +/- 0.5 ms and peak latencies of 33.9 +/- 3.3 ms. Contralateral multi-whisker stimulation evoked PSPs similar in amplitude to those of PW deflections; whereas, ipsilateral stimulation evoked smaller and longer latency PSPs. We conclude that in L5 a whisker deflection is represented in two ways: focally by L5A pyramids and more diffusely by L5B pyramids as a result of combining different inputs from lemniscal and paralemniscal pathways. The relevant output evoked by a whisker deflection could be the ensemble activity in the anatomically defined cortical modules associated with a single or a few barrel-columns.

Pyramidal neurons in layer 2/3 (L2/3) of the rat somatosensory cortex excite somatostatin-positive inhibitory bitufted interneurons located in the same cortical layer via glutamatergic synapses. A rise in volume-averaged dendritic [Ca2+]ievoked by backpropagating action potentials (APs) reduces glutamatergic excitation via a retrograde signal, presumably dendritic GABA. To measure the rise in local [Ca2+]iat synaptic contacts during suprathreshold excitation, we identified single synaptic contacts in the acute slice preparation in pairs of pyramidal and bitufted cells each loaded with a Ca2+indicator dye. Repetitive APs (10-15 APs at 50 Hz) evoked in a L2/3 pyramidal neuron gave rise to facilitating unitary EPSPs in the bitufted cell. Subthreshold EPSPs evoked a transient rise in [Ca2+]iof 80-250 nmpeak amplitude at the postsynaptic dendritic site. The local postsynaptic [Ca2+]itransient was restricted to ∼10 μm of dendritic length, lasted for ∼200 msec, and was mediated predominantly by NMDA receptor channels. When EPSPs were suprathreshold, the evoked AP backpropagated into the apical and basal dendritic arbor and increased the local [Ca2+]itransient at active contacts by approximately twofold, with a peak amplitude reaching 130-450 nm. This value is in the range of the half-maximal dendritic [Ca2+]i, evoking retrograde inhibition of glutamate release from boutons of pyramids. The localized enhancement of dendritic Ca2+influx at synaptic contacts by synaptically evoked backpropagating APs could represent one mechanism by which a retrograde signal can limit the excitation of bitufted interneurons by L2/3 pyramids when these are repetitively active.

A major aim of sensory physiology is to identify those synaptic connections in cortical representational areas (functional maps) by which sensory stimuli are transformed into a specific pattern of sub- (PSPs) and suprathreshold (APs) electrical activity. In the neocortex such maps consist of functional units, referred to as columns (Mountcastle, 1957; Hubel & Wiesel, 1962). These comprise the cells in different cortical layers that respond to a particular sensory stimulus. To understand sensory maps mechanistically and at a subcellular resolution, firstly the synaptic connections between cells that constitute a column and also those between different columns have to be identified in a layer-specific manner. Secondly the spatial and temporal transformations of PSP and AP patterns along sensory pathways and in the different cortical layers have to be understood. The coarse layout of sensory information flow within a column is comparable across different sensory cortices. Afferent signals arrive in cortical layer 4 (L4) from thalamic nuclei. They are relayed from L4 to supragranular layers 3 (L3) and 2 (L2) as well as to infragranular layers (L5 and L6). Extracellular unit recording and anatomical work have compiled a detailed picture of the columnar cytoarchitecture and AP activity in columns of some sensory cortices. The detailed anatomy and synaptic mechanisms of the connections that generate specific patterns of PSPs and APs are, however, largely unclear. Few studies have determined both the soma location and the dendritic and axonal morphology of cortical cells as well as their sub- and suprathreshold RFs (e.g. Ito, 1992; Brecht & Sakmann, 2002a,b). Such measurements are, however, a prerequisite if one wants to infer how PSPs or APs represent a sensory stimulus in the different layers of the cortex. L4 of the rodent somatosensory cortex contains aggregates of neuronal somata referred to as barrels, which are innervated in a strict topographical order by inputs representing individual facial whiskers (Woolsey & Van der Loos, 1970). Anatomical studies have demonstrated that barrel cells are targeted by thalamic inputs from the ventral posterior medial nucleus (VPM), which are part of the lemniscal pathway (Diamond, 1995), while the septa between barrels are innervated by thalamic afferents projecting from the posterior medial nucleus (POM), which belong to the paralemniscal pathway (Koralek et al. 1988; Lu & Lin, 1993). While most lemniscal afferents innervate the barrels, some VPM inputs also target the L5B/L6 border and paralemniscal POM afferents densely innervate L5A (Koralek et al. 1988; Lu & Lin, 1993). Barrel borders and the morphology of a cortical cell can be visualised simultaneously (Ito, 1992), such that the laminar position of a cell and its position relative to barrel column borders as well as its detailed dendritic and axonal morphology can be measured. Such techniques provided physiological evidence that lemniscal (the VPM/barrel projection) and paralemniscal (the POM/septum projection) pathways are largely segregated in L4 (Brecht & Sakmann, 2002a). Furthermore the RFs of barrel and septum cells are dynamic but are narrow and restricted to a PW and at most the first order SuWs. The homogeneous appearance of L3 and L2 in the horizontal plane may indicate merging of the whisker-specific anatomical pathways, whose strict separation in L4 gives rise to the discontinuous appearance of barrels (Woolsey & Van der Loos, 1970). The projection pattern of L4 spiny neuron axons suggests, however, that selectively connected barrel columns also exist (Petersen & Sakmann, 2000; Petersen et al. 2003; Lubke et al. 2003). The convergence of whisker-evoked responses between columns is also suggested by unit recordings from unidentified cells (Simons, 1978, 1995; Armstrong-James & Fox, 1987; Armstrong-James et al. 1992; Armstrong-James, 1995). They show that suprathreshold RFs in L3 and L2 cells are larger in size than those of L4 cells. The work of Ahissar and colleagues on the representation of temporal frequencies in L2/3 cell spike trains suggests a merging of barrel and septum inputs in supragranular layers (Ahissar et al. 2001). Anatomical data, however, suggest that barrel and septal pathways also remain separate in L3 and L2 (Kim & Ebner, 1999). We report here in vivo whole-cell voltage recordings of whisker-evoked PSPs and APs from cells in L2/3, combined with reconstruction of their dendritic and axonal arbors. We determined the horizontal and vertical position of these cells with reference to the barrel map to establish relationships between individual cell classes in L3 and L2 located above barrel and septa, and their sub- and suprathreshold RFs. The aim is to construct relationships between anatomical cell classes and their functional properties. Comparison with similar data from L4 cells (Brecht & Sakmann, 2002b) and incorporation of in vitro data (Feldmeyer et al. 2002; Lubke et al. 2003) on connectivity should allow a quantitative description of the flow of excitation through and between cortical barrel columns.

Pyramidal neurons in layer 2/3 of the neocortex are central to cortical circuitry, but the intrinsic properties of their dendrites are poorly understood. Here we study layer 2/3 apical dendrites in parallel experiments in acute brain slices and in anesthetized rats using whole-cell recordings and Ca2+imaging. We find that backpropagation of action potentials into the dendritic arbor is actively supported by Na+channels bothin vitroandin vivo. Single action potentials evoke substantial Ca2+influx in the apical trunk but little or none in the dendritic tuft. Supralinear Ca2+influx is produced in the tuft, however, when an action potential is paired with synaptic input. This dendritic supralinearity enables layer 2/3 neurons to integrate ascending sensory input from layer 4 and associative input to layer 1.

During the last decade, advances in experimental techniques and quantitative modelling have resulted in the development of the calyx of Held as one of the best preparations in which to study synaptic transmission. Here we review some of these advances, including simultaneous recording of pre- and postsynaptic currents, measuring the Ca2+ sensitivity of transmitter release, reconstructing the 3-D anatomy at the electron microscope (EM) level, and modelling the buffered diffusion of Ca2+ in the nerve terminal. An important outcome of these studies is an improved understanding of the Ca2+ signal that controls phasic transmitter release. This article illustrates the spatial and temporal aspects of the three main steps in the presynaptic signalling cascade: Ca2+ influx through voltage-gated calcium channels, buffered Ca2+ diffusion from the channels to releasable vesicles, and activation of the Ca2+ sensor for release. Particular emphasis is placed on how presynaptic Ca2+ buffers affect the Ca2+ signal and thus the amplitude and time course of the release probability. Since many aspects of the signalling cascade were first conceived with reference to the squid giant presynaptic terminal, we include comparisons with the squid model and revisit some of its implications. Whilst the characteristics of buffered Ca2+ diffusion presented here are based on the calyx of Held, we demonstrate the circumstances under which they may be valid for other nerve terminals at mammalian CNS synapses.

During the last decade, advances in experimental techniques and quantitative modelling have resulted in the development of the calyx of Held as one of the best preparations in which to study synaptic transmission. Here we review some of these advances, including simultaneous recording of pre- and postsynaptic currents, measuring the Ca2+ sensitivity of transmitter release, reconstructing the 3-D anatomy at the electron microscope (EM) level, and modelling the buffered diffusion of Ca2+ in the nerve terminal. An important outcome of these studies is an improved understanding of the Ca2+ signal that controls phasic transmitter release. This article illustrates the spatial and temporal aspects of the three main steps in the presynaptic signalling cascade: Ca2+ influx through voltage-gated calcium channels, buffered Ca2+ diffusion from the channels to releasable vesicles, and activation of the Ca2+ sensor for release. Particular emphasis is placed on how presynaptic Ca2+ buffers affect the Ca2+ signal and thus the amplitude and time course of the release probability. Since many aspects of the signalling cascade were first conceived with reference to the squid giant presynaptic terminal, we include comparisons with the squid model and revisit some of its implications. Whilst the characteristics of buffered Ca2+ diffusion presented here are based on the calyx of Held, we demonstrate the circumstances under which they may be valid for other nerve terminals at mammalian CNS synapses.

Cerebral cortex 25(4), 849-858 (2015). doi:10.1093/cercor/bht268, Published by Oxford Univ. Press, Oxford

Stimulation of a principal whisker yields sparse action potential (AP) spiking in layer 2/3 (L2/3) pyramidal neurons in a cortical column of rat barrel cortex. The low AP rates in pyramidal neurons could be explained by activation of interneurons in L2/3 providing inhibition onto L2/3 pyramidal neurons. L2/3 interneurons classified as local inhibitors based on their axonal projection in the same column were reported to receive strong excitatory input from spiny neurons in L4, which are also the main source of the excitatory input to L2/3 pyramidal neurons. Here, we investigated the remaining synaptic connection in this intracolumnar microcircuit. We found strong and reliable inhibitory synaptic transmission between intracolumnar L2/3 local-inhibitor-to-L2/3 pyramidal neuron pairs [inhibitory postsynaptic potential (IPSP) amplitude -0.88 ± 0.67 mV]. On average, 6.2 ± 2 synaptic contacts were made by L2/3 local inhibitors onto L2/3 pyramidal neurons at 107 ± 64 µm path distance from the pyramidal neuron soma, thus overlapping with the distribution of synaptic contacts from L4 spiny neurons onto L2/3 pyramidal neurons (67 ± 34 µm). Finally, using compartmental simulations, we determined the synaptic conductance per synaptic contact to be 0.77 ± 0.4 nS. We conclude that the synaptic circuit from L4 to L2/3 can provide efficient shunting inhibition that is temporally and spatially aligned with the excitatory input from L4 to L2/3.

The three-dimensional morphology of the axosomatic synaptic structures between a calyx of Held and a principal neuron in the medial nucleus of the trapezoid body (MNTB) in the brainstem of young postnatal day 9 rats was reconstructed from serial ultrathin sections. In the apposition zone between the calyx and the principal neuron two types of membrane specializations were identified: synaptic contacts (SCs) with active zones (AZs) and their associated postsynaptic densities (PSDs) constituted approximately 35% (n = 554) of the specializations; the remaining 65% (n = 1010) were puncta adherentia (PA). Synaptic contacts comprised approximately 5% of the apposition area of presynaptic and postsynaptic membranes. A SC had an average area of 0.100 microm(2), and the nearest neighbors were separated, on average, by 0.59 microm. Approximately one-half of the synaptic vesicles in the calyx were clustered within a distance of 200 nm of the AZ membrane area, a cluster consisting of approximately 60 synaptic vesicles (n = 52 SCs). Approximately two synaptic vesicles per SC were "anatomically docked." Comparing the geometry of the synaptic structure with its previously studied functional properties, we find that during a single presynaptic action potential (AP) (1) approximately 35% of the AZs release a transmitter quantum, (2) the number of SCs and anatomically docked vesicles is comparable with the low estimates of the readily releasable pool (RRP) of quanta, and (3) the broad distribution of PSD areas [coefficient of variation (CV) = 0.9] is likely to contribute to the large variability of miniature EPSC peaks. The geometry of the reconstructed synapse suggests that each of the hundreds of SCs is likely to contribute independently to the size and rising phase of the EPSC during a single AP.

The elaborate morphology of cortical neurons has been established for a long time (Ramon y Cajal, 1893), yet the functional significance of the differences in the architecture of the dendritic and axonal arbors of cells located in the same or different cortical layers is still unclear. Layer 4 of rodent somatosensory cortex is divided cytoarchitectonically into barrels with a high density of neurons, and septa between barrels with a lower density (Woolsey & Van der Loos, 1970). Barrel cells are targeted by thalamic inputs from the ventral posterior medial nucleus (VPM; for review see Diamond, 1995) while septum cells are innervated by thalamic afferents projecting from the posterior medial nucleus (PoM; for review see Kim & Ebner, 1999). A functional equivalent of the cytoarchitectonically defined barrels are the barrel-columns, ensembles of cells in the different cortical layers which share functional properties such as a response preference for the deflection of a particular whisker. The receptive fields (RFs) of barrel-column cells are characterised by a dominant input from a principal whisker (PW) and weaker inputs from surround whiskers (SuW). In L4 the barrel-columns correspond in their dimensions roughly to barrels (Welker, 1976). Barrel borders can be visualised simultaneously with the dendritic morphology of individual cells (Ito, 1992), and both the laminar location of a cell's soma and the spread of dendrites and axon collaterals can be determined relative to the barrel borders. Thus possible anatomical determinants of RF structure, such as the geometry of the dendritic and axonal arbor can be delineated. Spiny stellate cells are confined to the borders of barrels and their dendritic arbor is asymmetric (Woolsey et al. 1975; Simons & Woolsey 1984; Feldmeyer et al. 1999; Lubke et al. 2000). They relay thalamic output to other cortical layers via axon collaterals projecting to L2/3 and to L5 or L6. Although most anatomical studies on L4 neurons have focused on spiny stellate cells, pyramidal neurons have also been described in somatosensory (Lorente de No, 1922; Elston et al. 1997; Lubke et al. 2000) and in visual cortex (Martin & Whitteridge, 1984). In the somatosensory cortex neurons in layer 4 are selective in their responses to the direction of whisker deflection and they respond with short latency. Their RF structure is somewhat controversial, however. Intracellular recordings with microelectrodes (Carvel & Simons, 1988) and more recently, whole-cell voltage recordings have demonstrated afferent inputs from several whiskers and large subthreshold RFs (Moore & Nelson, 1998; Zhu & Connors, 1999). Most (Simons, 1995) but not all (Armstrong-James, 1995) extracellular unit-recording studies report small, often single-whisker RFs. In addition multielectrode unit recordings indicate that RF properties are time dependent (Petersen & Diamond, 2000). We report in vivo whole-cell voltage recordings combined with morphological reconstruction of the recorded neurons and determination of their columnar position. The aim was to establish firstly the dependency of RF structure on the geometry of dendritic and axonal arborisation of the different classes of neurons to identify possible constraints of RF structure given by cell morphology. Secondly we wanted to determine possible relationships between a cell's location and morphology and the time dependent structure of sub- and suprathreshold RFs. Such relations are essential to elucidate how different tactile object cues are represented at the input (PSPs) and the output stage (APs) of specific ensembles of cells in the cortical input layer.

A blind patch-clamp technique for in vivo whole-cell recordings in the intact brain is described. Recordings were obtained from various neuronal cell types located 100-5,000 microm from the cortical surface. Access resistance of recordings was as low as 10 M Omega but increased with recording depth and animal age. Recordings were remarkably stable and it was therefore possible to obtain whole-cell recordings in awake, head-fixed animals. The whole-cell configuration permitted rapid dialysis of cells with a calcium buffer. In most neurons very little ongoing action potential (AP) activity was observed and the spontaneous firing rates were up to 50-fold less than what has been reported by extracellular unit recordings. AP firing in the brain might therefore be far sparser than previously thought.

How patterns of odour-evoked glomerular activity are transformed into patterns of mitral cell action potentials (APs) in the olfactory bulb is determined by the functional connectivity of the cell populations in the bulb. We have used paired whole-cell voltage recordings from olfactory bulb slices to compare the functional connectivity of mitral cells to the known anatomy of the mitral cell network. Both inhibitory and excitatory coupling were observed between pairs of mitral cells. Inhibitory coupling was seen as an increased frequency of small, asynchronous GABAergic IPSPs following APs in the presynaptic cell. Excitatory coupling was short in latency, beginning about 1.3 ms after the presynaptic AP and was mediated by both NMDA and AMPA receptors. Mitral cell pairs were coupled by excitation if and only if their apical dendrites terminated in the same glomerulus. The excitatory coupling between mitral cells resembles conventional fast synaptic transmission in its time course, amplitude and latency, despite the absence of evidence for anatomically defined synapses between mitral cells.

Whole-cell voltage recordings were obtained from 64 synaptically coupled excitatory layer 4 (L4) spiny neurones and L2/3 pyramidal cells in acute slices of the somatosensory cortex ('barrel' cortex) of 17- to 23-days-old rats. Single action potentials (APs) in the L4 spiny neurone evoked single unitary EPSPs in the L2/3 pyramidal cell with a peak amplitude of 0.7 +/- 0.6 mV. The average latency was 2.1 +/- 0.6 ms, the rise time was 0.8 +/- 0.3 ms and the decay time constant was 12.7 +/- 3.5 ms. The percentage of failures of an AP in a L4 spiny neurone to evoke a unitary EPSP in the L2/3 pyramidal cell was 4.9 +/- 8.8 % and the coefficient of variation (c.v.) of the unitary EPSP amplitude was 0.27 +/- 0.13. Both c.v. and percentage of failures decreased with increased average EPSP amplitude. Postsynaptic glutamate receptors (GluRs) in L2/3 pyramidal cells were of the N-methyl-D-aspartate (NMDA) receptor (NMDAR) and the non-NMDAR type. At -60 mV in the presence of extracellular Mg2+ (1 mM), 29 +/- 15 % of the EPSP voltage-time integral was blocked by NMDAR antagonists. In 0 Mg2+, the NMDAR/AMPAR ratio of the EPSC was 0.50 +/- 0.29, about half the value obtained for L4 spiny neurone connections. Burst stimulation of L4 spiny neurones showed that EPSPs in L2/3 pyramidal cells depressed over a wide range of frequencies (1-100 s(-1) ). However, at higher frequencies (30 s(-1)) EPSP summation overcame synaptic depression so that the summed EPSP was larger than the first EPSP amplitude in the train. The number of putative synaptic contacts established by the axonal collaterals of the L4 projection neurone with the target neurone in layer 2/3 varied between 4 and 5, with an average of 4.5 +/- 0.5 (n = 13 pairs). Synapses were established on basal dendrites of the pyramidal cell. Their mean geometric distance from the pyramidal cell soma was 67 +/- 34 microm (range, 16-196 microm). The results suggest that each connected L4 spiny neurone produces a weak but reliable EPSP in the pyramidal cell. Therefore transmission of signals to layer 2/3 is likely to have a high threshold requiring simultaneous activation of many L4 neurons, implying that L4 spiny neurone to L2/3 pyramidal cell synapses act as a gate for the lateral spread of excitation in layer 2/3.

1. Bitufted interneurons in layer 2/3 of the rat (P14) somatosensory cortex have elongated apical and basal dendritic arbors that can span the entire depth of the cortex. Simultaneous dendritic and somatic whole-cell voltage recordings combined with Ca2+ fluorescence measurements were made to quantify voltage and Ca2+ signalling in dendritic arbors of bitufted neurons. 2. Action potentials (APs) initiated close to the soma by brief current injection back-propagated into the apical and basal dendritic arbors and evoked a transient increase in volume-averaged dendritic Ca2+ concentration (Delta[Ca(2+)](i)) of about 140 nM peak amplitude per AP. The AP evoked Ca2+ signal decayed with a time constant of about 200 ms. 3. A relatively high endogenous Ca(2+) binding ratio of approximately 285 determines the comparatively small rise in [Ca(2+)](i) of bitufted cell dendrites evoked by a back-propagating AP. 4. The [Ca(2+)](i) transient evoked by back-propagating dendritic APs decreased with distance (< or = 50 microm) from the soma in some neurons. At distances greater than 50 microm transients did not show a spatial gradient between the proximal and distal dendritic branches. 5. During trains of APs the mean amplitude of the steady-state increase in dendritic [Ca(2+)](i) encoded the AP frequency linearly up to 40 Hz with a slope of 20 nM Hz(-1). 6. The results suggest that APs initiated in the axon of bitufted neurons back-propagate and 'copy' the pattern of the axon's electrical activity also to the dendritic arbor. The AP pattern is transduced into a transient rise of dendritic [Ca(2+)](i) which, presumably, can regulate the receptive properties of the dendritic arbor for synaptic input. 7. Bitufted interneurons in layer 2/3 of the rat (P14) somatosensory cortex have elongated apical and basal dendritic arbors that can span the entire depth of the cortex. Simultaneous dendritic and somatic whole-cell voltage recordings combined with Ca2+ fluorescence measurements were made to quantify voltage and Ca2+ signalling in dendritic arbors of bitufted neurons. 8. Action potentials (APs) initiated close to the soma by brief current injection back-propagated into the apical and basal dendritic arbors and evoked a transient increase in volume-averaged dendritic Ca2+ concentration (Delta[Ca(2+)](i)) of about 140 nM peak amplitude per AP. The AP evoked Ca2+ signal decayed with a time constant of about 200 ms. 9. A relatively high endogenous Ca2+ binding ratio of approximately 285 determines the comparatively small rise in [Ca(2+)](i) of bitufted cell dendrites evoked by a back-propagating AP. 10. The [Ca(2+)](i) transient evoked by back-propagating dendritic APs decreased with distance (< or = 50 microm) from the soma in some neurons. At distances greater than 50 microm transients did not show a spatial gradient between the proximal and distal dendritic branches. 11. During trains of APs the mean amplitude of the steady-state increase in dendritic [Ca(2+)](i) encoded the AP frequency linearly up to 40 Hz with a slope of 20 nM Hz(-1). 12. The results suggest that APs initiated in the axon of bitufted neurons back-propagate and also 'copy' the pattern of the axon's electrical activity to the dendritic arbor. The AP pattern is transduced into a transient rise of dendritic [Ca(2+)](i) which, presumably, can regulate the receptive properties of the dendritic arbor for synaptic input.

1. Double, triple and quadruple whole-cell voltage recordings were made simultaneously from different parts of the apical dendritic arbor and the soma of adult layer 5 (L5) pyramidal neurons. We investigated the membrane mechanisms that support the conduction of dendritic action potentials (APs) between the dendritic and axonal AP initiation zones and their influence on the subsequent AP pattern. 2. The duration of the current injection to the distal dendritic initiation zone controlled the degree of coupling with the axonal initiation zone and the AP pattern. 3. Two components of the distally evoked regenerative potential were pharmacologically distinguished: a rapidly rising peak potential that was TTX sensitive and a slowly rising plateau-like potential that was Cd(2+) and Ni(2+) sensitive and present only with longer-duration current injection. 4. The amplitude of the faster forward-propagating Na(+)-dependent component and the amplitude of the back-propagating AP fell into two classes (more distinctly in the forward-propagating case). Current injection into the dendrite altered propagation in both directions. 5. Somatic current injections that elicited single Na(+) APs evoked bursts of Na(+) APs when current was injected simultaneously into the proximal apical dendrite. The mechanism did not depend on dendritic Na(+)-Ca(2+) APs. 6. A three-compartment model of a L5 pyramidal neuron is proposed. It comprises the distal dendritic and axonal AP initiation zones and the proximal apical dendrite. Each compartment contributes to the initiation and to the pattern of AP discharge in a distinct manner. Input to the three main dendritic arbors (tuft dendrites, apical oblique dendrites and basal dendrites) has a dominant influence on only one of these compartments. Thus, the AP pattern of L5 pyramids reflects the laminar distribution of synaptic activity in a cortical column.

Amplitudes of EPSPs evoked by repetitive presynaptic action potentials can either decrease (synaptic depression) or increase (synaptic facilitation). To determine whether facilitation and depression in the connections between neocortical pyramidal cells varied with the identity of the pre- or the postsynaptic cell and whether they changed during postnatal development, whole-cell voltage recordings were made simultaneously from two or three pyramidal cells in layers 2/3 and 5 of the rat sensorimotor cortex. Unitary EPSPs were evoked when pre- and postsynaptic neurons were in the same and in different layers. In young [postnatal day 14 (P14)] cortex, EPSPs evoked in all connected neurons depressed. The degree of depression was layer specific and was determined by the identity of the presynaptic cell. EPSPs evoked by stimulation of presynaptic layer 5 neurons depressed significantly more than did those evoked by stimulation of layer 2/3 neurons. In mature cortex (P28), however, the EPSPs evoked in these connected neurons facilitated to a comparable degree regardless of the layer in which pre- and postsynaptic neurons were located. The results suggest that in young cortex the degree of synaptic depression in connected pyramidal cells is determined primarily by whether the presynaptic cell was in layer 2/3 or 5 and that maturation of the cortex involves a developmental switch from depression to facilitation between P14 and P28 that eliminates the layer-specific differences. A functional consequence of this switch is that in mature cortex the spread of excitation between neocortical pyramidal neurons is enhanced when action potentials occur in bursts.

1. A new form of synaptic depression of excitatory synaptic transmission was observed when making voltage-clamp recordings from large presynaptic terminals, the calyces of Held and postsynaptic cells, the principal cells of the medial nucleus of the trapezoid body (MNTB), in slices of the rat auditory brainstem. 2. A short (100 ms) depolarization of the postsynaptic cell to 0 mV reduced the amplitude of the EPSCs by 35 +/- 5 % (n = 7), measured at 10 ms following the depolarization. Recovery occurred within 0.5 s. 3. The reduction of the EPSCs was most probably due to reduced presynaptic calcium influx, since postsynaptic depolarization reduced presynaptic calcium or barium currents. Conversely, presynaptic depolarization also reduced postsynaptic calcium or barium influx, under conditions where transmitter release was minimal. 4. The calcium currents and the postsynaptic depolarization-induced suppression of synaptic transmission recovered with a similar time course, suggesting that this form of synaptic depression was, most probably, due to depletion of Ca2+ in the synaptic cleft. 5. We conclude that when the Ca2+ influx into the pre- or postsynaptic cell is large, extracellular Ca2+ is depleted. Under these conditions, the Ca2+ concentration in the synaptic cleft is a sensitive indicator of the level of synaptic activity. However, the synaptic cleft is less sensitive to Ca2+ depletion than predicted from its estimated volume.