Christiaan P. J. de Kock

Spatial Organization of Neuronal Population Responses in Layer 2/3 of Rat Barrel Cortex

Individual pyramidal neurons of neocortex show sparse and variable responses to sensory stimuli in vivo. It has remained unclear how this variability extends to population responses on a trial-to-trial basis. Here, we characterized single-neuron and population responses to whisker stimulation in layer 2/3 (L2/3) of identified columns in rat barrel cortex using in vivo two-photon calcium imaging. Optical detection of single action potentials from evoked calcium transients revealed low spontaneous firing rates (0.25 Hz), variable response probabilities (range, 0–0.5; mean, 0.2 inside barrel column), and weak angular tuning of L2/3 neurons. On average, both the single-neuron response probability and the percentage of the local population activated were higher in the barrel column than above septa or in neighboring columns. Within the barrel column, mean response probability was highest in the center (0.4) and declined toward the barrel border. Neuronal pairs showed correlations in both spontaneous and sensory-evoked activity that depended on the location of the neurons. Correlation decreased with increasing distance between neurons and, for neuronal pairs the same distance apart, with distance of the pair from the barrel column center. Although neurons are therefore not activated independently from each other, we did not observe precisely repeating spatial activation patterns. Instead, population responses showed large trial-to-trial variability. Nevertheless, the accuracy of decoding stimulus onset times from local population activity increased with population size and depended on anatomical location. We conclude that, despite their sparseness and variability, L2/3 population responses show a clear spatial organization on the columnar scale.

Cell Type–Specific Three-Dimensional Structure of Thalamocortical Circuits in a Column of Rat Vibrissal Cortex

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.

Dimensions of a projection column and architecture of VPM and POm axons in rat vibrissal cortex.

This is the first article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). S1 receives 2 major types of TC inputs, lemiscal and paralemniscal. Lemiscal axons arise from the ventral posteromedial nucleus (VPM) of the thalamus, whereas paralemniscal fibers originate in the posteromedial nucleus (POm). While these 2 TC projections are largely complementary in L4, overlap in other cortical layers is still a matter of debate. VPM and POm axons were specifically labeled in the same rat by virus-mediated expression of different fluorescent proteins. We show that columnar and septal projection patterns are maintained throughout most of the cortical depth with a lower degree of separation in infragranular layers, where TC axons form bands along rows. Finally, we present anatomical dimensions of “TC projection domains” for a standard column in S1.

Number and Laminar Distribution of Neurons in a Thalamocortical Projection Column of Rat Vibrissal Cortex

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 mm3 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.

Cell Type–Specific Thalamic Innervation in a Column of Rat Vibrissal Cortex

This is the concluding article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). We used viral synaptophysin-enhanced green fluorescent protein expression in thalamic neurons and reconstructions of biocytin-labeled cortical neurons in TC slices to quantify the number and distribution of boutons from the ventral posterior medial (VPM) and posteromedial (POm) nuclei potentially innervating dendritic arbors of excitatory neurons located in layers (L)2–6 of a cortical column in rat somatosensory cortex. We found that 1) all types of excitatory neurons potentially receive substantial TC input (90–580 boutons per neuron); 2) pyramidal neurons in L3–L6 receive dual TC input from both VPM and POm that is potentially of equal magnitude for thick-tufted L5 pyramidal neurons (ca. 300 boutons each from VPM and POm); 3) L3, L4, and L5 pyramidal neurons have multiple (2–4) subcellular TC innervation domains that match the dendritic compartments of pyramidal cells; and 4) a subtype of thick-tufted L5 pyramidal neurons has an additional VPM innervation domain in L4. The multiple subcellular TC innervation domains of L5 pyramidal neurons may partly explain their specific action potential patterns observed in vivo. We conclude that the substantial potential TC innervation of all excitatory neuron types in a cortical column constitutes an anatomical basis for the initial near-simultaneous representation of a sensory stimulus in different neuron types.

Layer-Specific Modulation of the Prefrontal Cortex by Nicotinic Acetylcholine Receptors

Acetylcholine signaling through nicotinic receptors (nAChRs) in the prefrontal cortex (PFC) is crucial for attention. Nicotinic AChRs are expressed on glutamatergic inputs to layer V (LV) cells and on LV interneurons and LVI pyramidal neurons. Whether PFC layers are activated by nAChRs to a similar extent or whether there is layer-specific activation is not known. Here, we investigate nAChR modulation of all PFC layers and find marked layer specificity for pyramidal neurons: LII/III pyramidal neurons and glutamatergic inputs to these cells do not contain nAChRs, LV and LVI pyramidal neurons are modulated by α7 and β2* nAChRs, respectively. Interneurons across layers contain mixed combinations of nAChRs. We then tested the hypothesis that nAChRs activate the PFC in a layer-specific manner using 2-photon population imaging. In all layers, nAChR-induced neuronal firing was dominated by β2* nAChRs. In LII/III, only interneurons were activated. In LV and LVI, both interneurons and pyramidal neurons were activated, the latter most strongly in LVI. Together, these results suggest that in the PFC nAChR activation results in inhibition of LII/III pyramidal neurons. In LV and LVI, nAChR-induced activation of inhibitory and excitatory neurons results in a net augmentation of output neuron activity.

Three-dimensional axon morphologies of individual layer 5 neurons indicate cell type-specific intracortical pathways for whisker motion and touch

The cortical output layer 5 contains two excitatory cell types, slender- and thick-tufted neurons. In rat vibrissal cortex, slender-tufted neurons carry motion and phase information during active whisking, but remain inactive after passive whisker touch. In contrast, thick-tufted neurons reliably increase spiking preferably after passive touch. By reconstructing the 3D patterns of intracortical axon projections from individual slender- and thick-tufted neurons, filled in vivo with biocytin, we were able to identify cell type-specific intracortical circuits that may encode whisker motion and touch. Individual slender-tufted neurons showed elaborate and dense innervation of supragranular layers of large portions of the vibrissal area (total length, 86.8 ± 5.5 mm). During active whisking, these long-range projections may modulate and phase-lock the membrane potential of dendrites in layers 2 and 3 to the whisking cycle. Thick-tufted neurons with soma locations intermingling with those of slender-tufted ones display less dense intracortical axon projections (total length, 31.6 ± 14.3 mm) that are primarily confined to infragranular layers. Based on anatomical reconstructions and previous measurements of spiking, we put forward the hypothesis that thick-tufted neurons in rat vibrissal cortex receive input of whisker motion from slender-tufted neurons onto their apical tuft dendrites and input of whisker touch from thalamic neurons onto their basal dendrites. During tactile-driven behavior, such as object location, near-coincident input from these two pathways may result in increased spiking activity of thick-tufted neurons and thus enhanced signaling to their subcortical targets.

Label-free live brain imaging and targeted patching with third-harmonic generation microscopy

The ability to visualize neurons inside living brain tissue is a fundamental requirement in neuroscience and neurosurgery. Especially the development of a noninvasive probe of brain morphology with micrometer-scale resolution is highly desirable, as it would provide a noninvasive approach to optical biopsies in diagnostic medicine. Two-photon laser-scanning microscopy (2PLSM) is a powerful tool in this regard, and has become the standard for minimally invasive high-resolution imaging of living biological samples. However, while 2PLSM-based optical methods provide sufficient resolution, they have been hampered by the requirement for fluorescent dyes to provide image contrast. Here we demonstrate high-contrast imaging of live brain tissue at cellular resolution, without the need for fluorescent probes, using optical third-harmonic generation (THG). We exploit the specific geometry and lipid content of brain tissue at the cellular level to achieve partial phase matching of THG, providing an alternative contrast mechanism to fluorescence. We find that THG brain imaging allows rapid, noninvasive label-free imaging of neurons, white-matter structures, and blood vessels simultaneously. Furthermore, we exploit THG-based imaging to guide micropipettes towards designated neurons inside live tissue. This work is a major step towards label-free microscopic live brain imaging, and opens up possibilities for the development of laser-guided microsurgery techniques in the living brain.

Driver or Coincidence Detector: Modal Switch of a Corticothalamic Giant Synapse Controlled by Spontaneous Activity and Short-Term Depression

Giant synapses between layer 5B (L5B) neurons of somatosensory (barrel) cortex and neurons of the posteromedial nucleus (POm) of thalamus reside in a key position of the cortico-thalamo-cortical (CTC) loop, yet their synaptic properties and contribution to CTC information processing remain poorly understood. Fluorescence-guided local stimulation of terminals were combined with postsynaptic whole-cell recordings in thalamus to study synaptic transmission at an identified giant synapse. We found large EPSCs mediated by Ca2+-permeable AMPA and NMDA receptors. A single presynaptic electrical stimulus evoked a train of postsynaptic action potentials, indicating that a single L5B input can effectively drive the thalamic neuron. Repetitive stimulation caused strong short-term depression (STD) with fast recovery. To examine how these synaptic properties affect information transfer, spontaneous and evoked activity of L5B neurons was recorded in vivo and played back to giant terminals in vitro. We found that suprathreshold synaptic transmission was suppressed because of spontaneous activity causing strong STD of the L5B–POm giant synapse. Thalamic neurons only spiked after intervals of presynaptic silence or when costimulating two giant terminals. Therefore, STD caused by spontaneous activity of L5B neurons can switch the synapse from a “driver mode” to a “coincidence mode.” Mechanisms decreasing spontaneous activity in L5B neurons and inputs synchronized by a sensory stimulus may thus gate the cortico-thalamo-cortical loop.

Hyperconnectivity and Slow Synapses during Early Development of Medial Prefrontal Cortex in a Mouse Model for Mental Retardation and Autism

Neuronal theories of neurodevelopmental disorders (NDDs) of autism and mental retardation propose that abnormal connectivity underlies deficits in attentional processing. We tested this theory by studying unitary synaptic connections between layer 5 pyramidal neurons within medial prefrontal cortex (mPFC) networks in the Fmr1-KO mouse model for mental retardation and autism. In line with predictions from neurocognitive theory, we found that neighboring pyramidal neurons were hyperconnected during a critical period in early mPFC development. Surprisingly, excitatory synaptic connections between Fmr1-KO pyramidal neurons were significantly slower and failed to recover from short-term depression as quickly as wild type (WT) synapses. By 4-5 weeks of mPFC development, connectivity rates were identical for both KO and WT pyramidal neurons and synapse dynamics changed from depressing to facilitating responses with similar properties in both groups. We propose that the early alteration in connectivity and synaptic recovery are tightly linked: using a network model, we show that slower synapses are essential to counterbalance hyperconnectivity in order to maintain a dynamic range of excitatory activity. However, the slow synaptic time constants induce decreased responsiveness to low-frequency stimulation, which may explain deficits in integration and early information processing in attentional neuronal networks in NDDs.