Single Pyramidal Cells Research Articles

Functional architecture of intracellular oscillations in hippocampal dendrites

Fast electrical signaling in dendrites is central to neural computations that support adaptive behaviors. Conventional techniques lack temporal and spatial resolution and the ability to track underlying membrane potential dynamics present across the complex three-dimensional dendritic arbor in vivo. Here, we perform fast two-photon imaging of dendritic and somatic membrane potential dynamics in single pyramidal cells in the CA1 region of the mouse hippocampus during awake behavior. We study the dynamics of subthreshold membrane potential and suprathreshold dendritic events throughout the dendritic arbor in vivo by combining voltage imaging with simultaneous local field potential recording, post hoc morphological reconstruction, and a spatial navigation task. We systematically quantify the modulation of local event rates by locomotion in distinct dendritic regions, report an advancing gradient of dendritic theta phase along the basal-tuft axis, and describe a predominant hyperpolarization of the dendritic arbor during sharp-wave ripples. Finally, we find that spatial tuning of dendritic representations dynamically reorganizes following place field formation. Our data reveal how the organization of electrical signaling in dendrites maps onto the anatomy of the dendritic tree across behavior, oscillatory network, and functional cell states.

Long-Term-But Not Short-Term-Plasticity at the Mossy Fiber-CA3 Pyramidal Cell Synapse in Hippocampus Is Altered in M1/M3 Muscarinic Acetylcholine Receptor Double Knockout Mice.

Muscarinic acetylcholine receptors are well-known for their crucial involvement in hippocampus-dependent learning and memory, but the exact roles of the various receptor subtypes (M1-M5) are still not fully understood. Here, we studied how M1 and M3 receptors affect plasticity at the mossy fiber (MF)-CA3 pyramidal cell synapse. In hippocampal slices from M1/M3 receptor double knockout (M1/M3-dKO) mice, the signature short-term plasticity of the MF-CA3 synapse was not significantly affected. However, the rather unique NMDA receptor-independent and presynaptic form of long-term potentiation (LTP) of this synapse was much larger in M1/M3-deficient slices compared to wild-type slices in both field potential and whole-cell recordings. Consistent with its presynaptic origin, induction of MF-LTP strongly enhanced the excitatory drive onto single CA3 pyramidal cells, with the effect being more pronounced in M1/M3-dKO cells. In an earlier study, we found that the deletion of M2 receptors in mice disinhibits MF-LTP in a similar fashion, suggesting that endogenous acetylcholine employs both M1/M3 and M2 receptors to constrain MF-LTP. Importantly, such synergism was not observed for MF long-term depression (LTD). Low-frequency stimulation, which reliably induced LTD of MF synapses in control slices, failed to do so in M1/M3-dKO slices and gave rise to LTP instead. In striking contrast, loss of M2 receptors augmented LTD when compared to control slices. Taken together, our data demonstrate convergence of M1/M3 and M2 receptors on MF-LTP, but functional divergence on MF-LTD, with the net effect resulting in a well-balanced bidirectional plasticity of the MF-CA3 pyramidal cell synapse.

Seeing beyond the spikes: reconstructing the complete spatiotemporal membrane potential distribution from paired intra- and extracellular recordings.

Although electrophysiologists have been recording intracellular neural activity routinely ever since the ground-breaking work of Hodgkin and Huxley, and extracellular multichannel electrodes have also been used frequently and extensively, a practical experimental method to track changes in membrane potential along a complete single neuron is still lacking. Instead of obtaining multiple intracellular measurements on the same neuron, we propose an alternative method by combining single-channel somatic patch-clamp and multichannel extracellular potential recordings. In this work, we show that it is possible to reconstruct the complete spatiotemporal distribution of the membrane potential of a single neuron with the spatial resolution of an extracellular probe during action potential generation. Moreover, the reconstruction of the membrane potential allows us to distinguish between the two major but previously hidden components of the current source density (CSD) distribution: the resistive and the capacitive currents. This distinction provides a clue to the clear interpretation of the CSD analysis, because the resistive component corresponds to transmembrane ionic currents (all the synaptic, voltage-sensitive and passive currents), whereas capacitive currents are considered to be the main contributors of counter-currents. We validate our model-based reconstruction approach on simulations and demonstrate its application to experimental data obtained in vitro via paired extracellular and intracellular recordings from a single pyramidal cell of the rat hippocampus. In perspective, the estimation of the spatial distribution of resistive membrane currents makes it possible to distiguish between active and passive sinks and sources of the CSD map and the localization of the synaptic input currents, which make the neuron fire. KEY POINTS: A new computational method is introduced to calculate the unbiased current source density distribution on a single neuron with known morphology. The relationship between extracellular and intracellular electric potential is determined via mathematical formalism, and a new reconstruction method is applied to reveal the full spatiotemporal distribution of the membrane potential and the resistive and capacitive current components. The new reconstruction method was validated on simulations. Simultaneous and colocalized whole-cell patch-clamp and multichannel silicon probe recordings were performed from the same pyramidal neuron in the rat hippocampal CA1 region, in vitro. The method was applied in experimental measurements and returned precise and distinctive characteristics of various intracellular phenomena, such as action potential generation, signal back-propagation and the initial dendritic depolarization preceding the somatic action potential.

Asynchronous glutamate release is enhanced in low release efficacy synapses and dispersed across the active zone

The balance between fast synchronous and delayed asynchronous release of neurotransmitters has a major role in defining computational properties of neuronal synapses and regulation of neuronal network activity. However, how it is tuned at the single synapse level remains poorly understood. Here, using the fluorescent glutamate sensor SF-iGluSnFR, we image quantal vesicular release in tens to hundreds of individual synaptic outputs from single pyramidal cells with 4 millisecond temporal and 75 nm spatial resolution. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among synapses supplied by the same axon, and that the synchronicity of release is reduced at low release probability synapses. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the release area than synchronous sites. Together, our results reveal a universal relationship between the two major functional properties of synapses – the timing and the overall efficacy of neurotransmitter release.

Enriched Environment Modulates Sharp Wave-Ripple (SPW-R) Activity in Hippocampal Slices.

Behavioral flexibility depends on neuronal plasticity which forms and adapts the central nervous system in an experience-dependent manner. Thus, plasticity depends on interactions between the organism and its environment. A key experimental paradigm for studying this concept is the exposure of rodents to an enriched environment (EE), followed by studying differences to control animals kept under standard conditions (SC). While multiple changes induced by EE have been found at the cellular-molecular and cognitive-behavioral levels, little is known about EE-dependent alterations at the intermediate level of network activity. We, therefore, studied spontaneous network activity in hippocampal slices from mice which had previously experienced EE for 10–15 days. Compared to control animals from standard conditions (SC) and mice with enhanced motor activity (MC) we found several differences in sharp wave-ripple complexes (SPW-R), a memory-related activity pattern. Sharp wave amplitude, unit firing during sharp waves, and the number of superimposed ripple cycles were increased in tissue from the EE group. On the other hand, spiking precision with respect to the ripple oscillations was reduced. Recordings from single pyramidal cells revealed a reduction in synaptic inhibition during SPW-R together with a reduced inhibition-excitation ratio. The number of inhibitory neurons, including parvalbumin-positive interneurons, was unchanged. Altered activation or efficacy of synaptic inhibition may thus underlie changes in memory-related network activity patterns which, in turn, may be important for the cognitive-behavioral effects of EE exposure.

Unique Actions of GABA Arising from Cytoplasmic Chloride Microdomains.

Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported. We found a corresponding variance in the GABAA receptor reversal potential (EGABA) for synapses originating from individual interneurons onto a single pyramidal cell. These findings suggest a similar heterogeneity in the cytoplasmic intracellular concentration of chloride ([Cl-]i) in individual dendrites. We determined [Cl-]i in the murine hippocampus and cerebral cortex of both sexes by (1) two-photon imaging of the Cl--sensitive, ratiometric fluorescent protein SuperClomeleon; (2) Fluorescence Lifetime IMaging (FLIM) of the Cl--sensitive fluorophore MEQ (6-methoxy-N-ethylquinolinium); and (3) electrophysiological measurements of EGABA by pressure application of GABA and RuBi-GABA uncaging. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters, but were progressively modified after inhibiting the polymerization of the anionic biopolymer actin. These methods collectively demonstrated stable [Cl-]i microdomains in individual neurons in vitro and in vivo and the role of immobile anions in its stability. Our results highlight the existence of functionally significant neuronal Cl- microdomains that modify the impact of GABAergic inputs.SIGNIFICANCE STATEMENT Microdomains of varying chloride concentrations in the neuronal cytoplasm are a predictable consequence of the inhomogeneous distribution of anionic polymers such as actin, tubulin, and nucleic acids. Here, we demonstrate the existence and stability of these microdomains, as well as the consequence for GABAergic synaptic signaling: each interneuron produces a postsynaptic GABAA response with a unique reversal potential. In individual hippocampal pyramidal cells, the range of GABAA reversal potentials evoked by stimulating different interneurons was >20 mV. Some interneurons generated postsynaptic responses in pyramidal cells that reversed at potentials beyond what would be considered purely inhibitory. Cytoplasmic chloride microdomains enable each pyramidal cell to maintain a compendium of unique postsynaptic responses to the activity of individual interneurons.

Dendritic integration theory: A thalamo-cortical theory of state and content of consciousness

The idea that the thalamo-cortical system is the crucial constituent of the neurobiological mechanisms of consciousness has a long history. For the last few decades, however, consciousness research has to a large extent overlooked the interplay between the cortex and thalamus. Here we revive an integrated view of the neurobiology of consciousness by presenting and discussing several recent major findings about the role of the thalamocortical interactions in consciousness. Based on these findings we propose a specific cellular mechanism how thalamic nuclei modulate the integration of different processing streams within single cortical pyramidal neurons. This theory is inspired by recent work done in rodents, but it integrates decades of work conducted on various species. We illustrate how this new view readily explains various properties and experimental phenomena associated with conscious experience. We discuss the implications of this idea and some of the experiments that need to be done in order to test it. Our view bridges two long-standing perspectives on the neural mechanisms of consciousness and proposes that cortical and thalamo-cortical processing interact at the level of single pyramidal cells.

Single neurons activate cortical networks

Neuroscience All living reptiles and mammals have a brain cortex derived from a common primordial cortex. Thus, they may retain traces of this common ancestry in their structure, connectivity, and function. Hemberger et al. simultaneously sampled action potentials from hundreds of neurons in intact turtle cortical explants and found that minimal manipulation of single cortical pyramidal cells had detectable effects on other neurons and on the animal's behavior. With only one to three spikes, single pyramidal neurons could trigger distributed patterns of firing that cascaded across neurons, space, and time in the form of reliable firing sequences. These sequences are similar to those produced during replay events in the mammalian hippocampus. These data provide evidence for chained synaptic activation in a cortical structure supporting the synfire chain hypothesis. Neuron 10.1016/j.neuron.2019.07.017 (2019).

Reliable Sequential Activation of Neural Assemblies by Single Pyramidal Cells in a Three-Layered Cortex

Recent studies reveal the occasional impact of single neurons on surround firing statistics and even simple behaviors. Exploiting the advantages of a simple cortex, we examined the influence of single pyramidal neurons on surrounding cortical circuits. Brief activation of single neurons triggered reliable sequences of firing in tens of other excitatory and inhibitory cortical neurons, reflecting cascading activity through local networks, as indicated by delayed yet precisely timed polysynaptic subthreshold potentials. The evoked patterns were specific to the pyramidal cell of origin, extended over hundreds of micrometers from their source, and unfolded over up to 200ms. Simultaneous activation of pyramidal cell pairs indicated balanced control of population activity, preventing paroxysmal amplification. Single cortical pyramidal neurons can thus trigger reliable postsynaptic activity that can propagate in a reliable fashion through cortex, generating rapidly evolving and non-random firing sequences reminiscent of those observed in mammalian hippocampus during "replay" and in avian song circuits.

Regulation of Neuronal Na+/K+-ATPase by Specific Protein Kinases and Protein Phosphatases.

The Na+/K+-ATPase (NKA) is a ubiquitous membrane-bound enzyme responsible for generating and maintaining the Na+ and K+ electrochemical gradients across the plasmalemma of living cells. Numerous studies in non-neuronal tissues have shown that this transport mechanism is reversibly regulated by phosphorylation/dephosphorylation of the catalytic α subunit and/or associated proteins. In neurons, Na+/K+ transport by NKA is essential for almost all neuronal operations, consuming up to two-thirds of the neuron's energy expenditure. However, little is known about its cellular regulatory mechanisms. Here we have used an electrophysiological approach to monitor NKA transport activity in male rat hippocampal neurons in situ We report that this activity is regulated by a balance between serine/threonine phosphorylation and dephosphorylation. Phosphorylation by the protein kinases PKG and PKC inhibits NKA activity, whereas dephosphorylation by the protein phosphatases PP-1 and PP-2B (calcineurin) reverses this effect. Given that these kinases and phosphatases serve as downstream effectors in key neuronal signaling pathways, they may mediate the coupling of primary messengers, such as neurotransmitters, hormones, and growth factors, to the NKAs, through which multiple brain functions can be regulated or dysregulated.SIGNIFICANCE STATEMENT The Na+/K+-ATPase (NKA), known as the "Na+ pump," is a ubiquitous membrane-bound enzyme responsible for generating and maintaining the Na+ and K+ electrochemical gradients across the plasma membrane of living cells. In neurons, as in most types of cells, the NKA generates the negative resting membrane potential, which is the basis for almost all aspects of cellular function. Here we used an electrophysiological approach to monitor physiological NKA transport activity in single hippocampal pyramidal cells in situ We have found that neuronal NKA activity is oppositely regulated by phosphorylation and dephosphorylation, and we have identified the main protein kinases and phosphatases mediating this regulation. This fundamental form of NKA regulation likely plays a role in multiple brain functions.

Extracellular waveforms reveal an axonal origin of spikelets in pyramidal neurons.

Spikelets are small spike-like membrane depolarizations measured at the soma whose origin in pyramidal neurons is still unresolved. We investigated the mechanism of spikelet generation using detailed models of pyramidal neurons. We simulated extracellular waveforms associated with action potentials and spikelets and compared these with experimental data obtained by Chorev and Brecht ( J Neurophysiol 108: 1584-1593, 2012) from hippocampal pyramidal neurons in vivo. We considered spikelets originating in the axon of a single cell as well as spikelets generated in two cells coupled with gap junctions. We found that in both cases the experimental data can be explained by an axonal origin of spikelets: in the single-cell case, action potentials are generated in the axon but fail to activate the soma. Such spikelets can be evoked by dendritic input. Alternatively, spikelets resulting from axoaxonal gap junction coupling with a large (greater than several hundred μm) distance between the somata of the coupled cells are also consistent with the data. Our results demonstrate that a cell firing a somatic spikelet generates a detectable extracellular potential that is different from the action potential-correlated extracellular waveform generated by the same cell and recorded at the same location. This, together with the absence of a refractory period between action potentials and spikelets, implies that spikelets and action potentials generated in one cell may easily get misclassified in extracellular recordings as two different cells, albeit they both constitute the output of a single pyramidal neuron. NEW & NOTEWORTHY We addressed the origin of spikelets, using compartmental models of pyramidal neurons. Comparing our simulation results with published extracellular spikelet recordings revealed an axonal origin of spikelets. Our results imply that action potential- and spikelet-associated extracellular waveforms may easily get misclassified as two different cells, albeit they both constitute the output of a single pyramidal cell.

The Effect of Single Pyramidal Neuron Firing Within Layer 2/3 and Layer 4 in Mouse V1.

The influence of cortical cell spiking activity on nearby cells has been studied extensively in vitro. Less is known, however, about the impact of single cell firing on local cortical networks in vivo. In a pioneering study, Kwan and Dan (Kwan and Dan, 2012) reported that in mouse layer 2/3 (L2/3), under anesthesia, stimulating a single pyramidal cell recruits ~2.1% of neighboring units. Here we employ two-photon calcium imaging in layer 2/3 of mouse V1, in conjunction with single-cell patch clamp stimulation in layer 2/3 or layer 4, to probe, in both the awake and lightly anesthetized states, how (i) activating single L2/3 pyramidal neurons recruits neighboring units within L2/3 and from layer 4 (L4) to L2/3, and whether (ii) activating single pyramidal neurons changes population activity in local circuit. To do this, it was essential to develop an algorithm capable of quantifying how sensitive the calcium signal is at detecting effectively recruited units (“followers”). This algorithm allowed us to estimate the chance of detecting a follower as a function of the probability that an epoch of stimulation elicits one extra action potential (AP) in the follower cell. Using this approach, we found only a small fraction (<0.75%) of L2/3 cells to be significantly activated within a radius of ~200 μm from a stimulated neighboring L2/3 pyramidal cell. This fraction did not change significantly in the awake vs. the lightly anesthetized state, nor when stimulating L2/3 vs. underlying L4 pyramidal neurons. These numbers are in general agreement with, though lower than, the percentage of neighboring cells (2.1% pyramidal cells and interneurons combined) reported by Kwan and Dan to be activated upon stimulating single L2/3 pyramidal neurons under anesthesia (Kwan and Dan, 2012). Interestingly, despite the small number of individual units found to be reliably driven, we did observe a modest but significant elevation in aggregate population responses compared to sham stimulation. This underscores the distributed impact that single cell stimulation has on neighboring microcircuit responses, revealing only a small minority of relatively strongly connected partners.One sentence summaryPatch-clamp stimulation in conjunction with 2-photon imaging shows that activating single layer-2/3 or layer-4 pyramidal neurons produces few (<1% of local units) reliable single-cell followers in L2/3 of mouse area V1, either under light anesthesia or in quiet wakefulness: instead, single cell stimulation was found to elevate aggregate population activity in a weak but highly distributed fashion.

Feasibility and resolution limits of opto-magnetic imaging of neural network activity in brain slices using color centers in diamond

We suggest a novel approach for wide-field imaging of the neural network dynamics of brain slices that uses highly sensitivity magnetometry based on nitrogen-vacancy (NV) centers in diamond. In-vitro recordings in brain slices is a proven method for the characterization of electrical neural activity and has strongly contributed to our understanding of the mechanisms that govern neural information processing. However, this traditional approach only acquires signals from a few positions, which severely limits its ability to characterize the dynamics of the underlying neural networks. We suggest to extend its scope using NV magnetometry-based imaging of the neural magnetic fields across the slice. Employing comprehensive computational simulations and theoretical analyses, we determine the spatiotemporal characteristics of the neural fields and the required key performance parameters of an NV magnetometry-based imaging setup. We investigate how the technical parameters determine the achievable spatial resolution for an optimal 2D reconstruction of neural currents from the measured field distributions. Finally, we compare the imaging of neural slice activity with that of a single planar pyramidal cell. Our results suggest that imaging of slice activity will be possible with the upcoming generation of NV magnetic field sensors, while single-shot imaging of planar cell activity remains challenging.

Circuit-specific and neuronal subcellular-wide E-I balance in cortical pyramidal cells

We used ChR2-assisted circuit mapping (CRACM) to examine neuronal/compartmental excitatory and inhibitory synaptic balance (E-I balance) in pyramidal cells (PCs) located in several brain regions (including both neocortices and paleocortices). Within the vS1, different inputs on the same neurons, or the same inputs formed on different targets, induced different E/I ratios. E/I ratios in PCs from different regions were largely different. Chemogenetic silencing of somatostatin (SOM)- or parvalbumin (PV)-containing interneurons (INs) while optogenetically activating long-range M1 inputs demonstrated differential contribution of PV and SOM INs to the E/I ratios in a layer-specific manner in S1. Our results thus demonstrate that there are both universal subcellular-wide E-I balance within single PC and high specificity in the value of E/I ratios across different circuits (i.e. visual, somatosensory, piriform and hippocampal). Specificity of E/I balance are likely caused by unique glutamatergic innervation of interneurons. The dichotomy of high specificity and generalization of subcellular E-I balance in different circuits forms the basis for further understanding of neuronal computation under physiological conditions and various neuro-psychiatric disease-states.

Target Cell Type-Dependent Differences in Ca2+ Channel Function Underlie Distinct Release Probabilities at Hippocampal Glutamatergic Terminals.

Target cell type-dependent differences in presynaptic release probability (Pr ) and short-term plasticity are intriguing features of cortical microcircuits that increase the computational power of neuronal networks. Here, we tested the hypothesis that different voltage-gated Ca2+ channel densities in presynaptic active zones (AZs) underlie different Pr values. Two-photon Ca2+ imaging, triple immunofluorescent labeling, and 3D electron microscopic (EM) reconstruction of rat CA3 pyramidal cell axon terminals revealed ∼1.7-1.9 times higher Ca2+ inflow per AZ area in high Pr boutons synapsing onto parvalbumin-positive interneurons (INs) than in low Pr boutons synapsing onto mGluR1α-positive INs. EM replica immunogold labeling, however, demonstrated only 1.15 times larger Cav2.1 and Cav2.2 subunit densities in high Pr AZs. Our results indicate target cell type-specific modulation of voltage-gated Ca2+ channel function or different subunit composition as possible mechanisms underlying the functional differences. In addition, high Pr synapses are also characterized by a higher density of docked vesicles, suggesting that a concerted action of these mechanisms underlies the functional differences.SIGNIFICANCE STATEMENT Target cell type-dependent variability in presynaptic properties is an intriguing feature of cortical synapses. When a single cortical pyramidal cell establishes a synapse onto a somatostatin-expressing interneuron (IN), the synapse releases glutamate with low probability, whereas the next bouton of the same axon has high release probability when its postsynaptic target is a parvalbumin-expressing IN. Here, we used combined molecular, imaging, and anatomical approaches to investigate the mechanisms underlying these differences. Our functional experiments implied an approximately twofold larger Ca2+ channel density in high release probability boutons, whereas freeze-fracture immunolocalization demonstrated only a 15% difference in Ca2+ channel subunit densities. Our results point toward a postsynaptic target cell type-dependent regulation of Ca2+ channel function or different subunit composition as the underlying mechanism.

Early born neurons are abnormally positioned in the doublecortin knockout hippocampus.

Human doublecortin (DCX) mutations are associated with severe brain malformations leading to aberrant neuron positioning (heterotopia), intellectual disability and epilepsy. The Dcx protein plays a key role in neuronal migration, and hippocampal pyramidal neurons in Dcx knockout (KO) mice are disorganized. The single CA3 pyramidal cell layer observed in wild type (WT) is present as two abnormal layers in the KO, and CA3 KO pyramidal neurons are more excitable than WT. Dcx KO mice also exhibit spontaneous epileptic activity originating in the hippocampus. It is unknown, however, how hyperexcitability arises and why two CA3 layers are observed.Transcriptome analyses were performed to search for perturbed postnatal gene expression, comparing Dcx KO CA3 pyramidal cell layers with WT. Gene expression changes common to both KO layers indicated mitochondria and Golgi apparatus anomalies, as well as increased cell stress. Intriguingly, gene expression analyses also suggested that the KO layers differ significantly from each other, particularly in terms of maturity. Layer-specific molecular markers and BrdU birthdating to mark the final positions of neurons born at distinct timepoints revealed inverted layering of the CA3 region in Dcx KO animals. Notably, many early-born 'outer boundary' neurons are located in an inner position in the Dcx KO CA3, superficial to other pyramidal neurons. This abnormal positioning likely affects cell morphology and connectivity, influencing network function. Dissecting this Dcx KO phenotype sheds light on coordinated developmental mechanisms of neuronal subpopulations, as well as gene expression patterns contributing to a bi-layered malformation associated with epilepsy.

Plasticity in Single Axon Glutamatergic Connection to GABAergic Interneurons Regulates Complex Events in the Human Neocortex.

In the human neocortex, single excitatory pyramidal cells can elicit very large glutamatergic EPSPs (VLEs) in inhibitory GABAergic interneurons capable of triggering their firing with short (3–5 ms) delay. Similar strong excitatory connections between two individual neurons have not been found in nonhuman cortices, suggesting that these synapses are specific to human interneurons. The VLEs are crucial for generating neocortical complex events, observed as single pyramidal cell spike-evoked discharge of cell assemblies in the frontal and temporal cortices. However, long-term plasticity of the VLE connections and how the plasticity modulates neocortical complex events has not been studied. Using triple and dual whole-cell recordings from synaptically connected human neocortical layers 2–3 neurons, we show that VLEs in fast-spiking GABAergic interneurons exhibit robust activity-induced long-term depression (LTD). The LTD by single pyramidal cell 40 Hz spike bursts is specific to connections with VLEs, requires group I metabotropic glutamate receptors, and has a presynaptic mechanism. The LTD of VLE connections alters suprathreshold activation of interneurons in the complex events suppressing the discharge of fast-spiking GABAergic cells. The VLEs triggering the complex events may contribute to cognitive processes in the human neocortex, and their long-term plasticity can alter the discharging cortical cell assemblies by learning.

Human neuronal changes in brain edema and increased intracranial pressure.

Functional and molecular changes associated with pathophysiological conditions are relatively easily detected based on tissue samples collected from patients. Population specific cellular responses to disease might remain undiscovered in samples taken from organs formed by a multitude of cell types. This is particularly apparent in the human cerebral cortex composed of a yet undefined number of neuron types with a potentially different involvement in disease processes. We combined cellular electrophysiology, anatomy and single cell digital PCR in human neurons identified in situ for the first time to assess mRNA expression and corresponding functional changes in response to edema and increased intracranial pressure. In single pyramidal cells, mRNA copy numbers of AQP1, AQP3, HMOX1, KCNN4, SCN3B and SOD2 increased, while CACNA1B, CRH decreased in edema. In addition, single pyramidal cells increased the copy number of AQP1, HTR5A and KCNS1 mRNAs in response to increased intracranial pressure. In contrast to pyramidal cells, AQP1, HMOX1and KCNN4 remained unchanged in single cell digital PCR performed on fast spiking cells in edema. Corroborating single cell digital PCR results, pharmacological and immunohistochemical results also suggested the presence of KCNN4 encoding the α-subunit of KCa3.1 channels in edema on pyramidal cells, but not on interneurons. We measured the frequency of spontaneous EPSPs on pyramidal cells in both pathophysiological conditions and on fast spiking interneurons in edema and found a significant decrease in each case, which was accompanied by an increase in input resistances on both cell types and by a drop in dendritic spine density on pyramidal cells consistent with a loss of excitatory synapses. Our results identify anatomical and/or physiological changes in human pyramidal and fast spiking cells in edema and increased intracranial pressure revealing cell type specific quantitative changes in gene expression. Some of the edema/increased intracranial pressure modulated and single human pyramidal cell verified gene products identified here might be considered as novel pharmacological targets in cell type specific neuroprotection.

Synchronized gamma-frequency inhibition in neocortex depends on excitatory-inhibitory interactions but not electrical synapses.

Synaptic inhibition plays a crucial role in the precise timing of spiking activity in the cerebral cortex. Synchronized, rhythmic inhibitory activity in the gamma (30-80 Hz) range is thought to be especially important for the active, information-processing neocortex, but the circuit mechanisms that give rise to synchronized inhibition are uncertain. In particular, the relative contributions of reciprocal inhibitory connections, excitatory-inhibitory interactions, and electrical synapses to precise spike synchrony among inhibitory interneurons are not well understood. Here we describe experiments on mouse barrel cortex in vitro as it spontaneously generates slow (<1 Hz) oscillations (Up and Down states). During Up states, inhibitory postsynaptic currents (IPSCs) are generated at gamma frequencies and are more synchronized than excitatory postsynaptic currents (EPSCs) among neighboring pyramidal cells. Furthermore, spikes in homotypic pairs of interneurons are more synchronized than in pairs of pyramidal cells. Comparing connexin36 knockout and wild-type animals, we found that electrical synapses make a minimal contribution to synchronized inhibition during Up states. Estimations of the delays between EPSCs and IPSCs in single pyramidal cells showed that excitation often preceded inhibition by a few milliseconds. Finally, tonic optogenetic activation of different interneuron subtypes in the absence of excitation led to only weak synchrony of IPSCs in pairs of pyramidal neurons. Our results suggest that phasic excitatory inputs are indispensable for synchronized spiking in inhibitory interneurons during Up states and that electrical synapses play a minimal role.

Single CA3 pyramidal cells trigger sharp waves in vitro by exciting interneurones.

The CA3 hippocampal region generates sharp waves (SPW), a population activity associated with neuronal representations. The synaptic mechanisms responsible for the generation of these events still require clarification. Using slices maintained in an interface chamber, we found that the firing of single CA3 pyramidal cells triggers SPW like events at short latencies, similar to those for the induction of firing in interneurons. Multi-electrode records from the CA3 stratum pyramidale showed that pyramidal cells triggered events consisting of putative interneuron spikes followed by field IPSPs. SPW fields consisted of a repetition of these events at intervals of 4-8ms. Although many properties of induced and spontaneous SPWs were similar, the triggered events tended to be initiated close to the stimulated cell. These data show that the initiation of SPWs in vitro is mediated via pyramidal cell synapses that excite interneurons. They do not indicate why interneuron firing is repeated during a SPW. Sharp waves (SPWs) are a hippocampal population activity that has been linked to neuronal representations. We show that SPWs in the CA3 region of rat hippocampal slices can be triggered by the firing of single pyramidal cells. Single action potentials in almost one-third of pyramidal cells initiated SPWs at latencies of 2-5ms with probabilities of 0.07-0.76. Initiating pyramidal cells evoked field IPSPs (fIPSPs) at similar latencies when SPWs were not initiated. Similar spatial profiles for fIPSPs and middle components of SPWs suggested that SPW fields reflect repeated fIPSPs. Multiple extracellular records showed that the initiated SPWs tended to start near the stimulated pyramidal cell, whereas spontaneous SPWs could emerge at multiple sites. Single pyramidal cells could initiate two to six field IPSPs with distinct amplitude distributions, typically preceeded by a short-duration extracellular action potential. Comparison of these initiated fields with spontaneously occurring inhibitory field motifs allowed us to identify firing in different interneurones during the spread of SPWs. Propagation away from an initiating pyramidal cell was typically associated with the recruitment of interneurones and field IPSPs that were not activated by the stimulated pyramidal cell. SPW fields initiated by single cells were less variable than spontaneous events, suggesting that more stereotyped neuronal ensembles were activated, although neither the spatial profiles of fields, nor the identities of interneurone firing were identical for initiated events. The effects of single pyramidal cell on network events are thus mediated by different sequences of interneurone firing.