Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A

Although physiological data on microcircuits involving a few inhibitory neurons in the mammalian cerebral cortex are available, data on the quantitative relation between inhibition and excitation in cortical circuits involving thousands of neurons are largely missing. Because the distribution of neurons is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than to rely on sparse sampling methods. Here, we report the comprehensive mapping of interneurons (INs) in cortical columns of rat somatosensory cortex, immunolabeled for neuron-specific nuclear protein and glutamate decarboxylase. We found that a column contains ~2,200 INs (11.5% of ~19,000 neurons), almost a factor of 2 less than previously estimated. The density of GABAergic neurons was inhomogeneous between layers, with peaks in the upper third of L2/3 and in L5A. IN density therefore defines a distinct layer 2 in the sensory neocortex. In addition, immunohistochemical markers of IN subtypes were layer-specific. The "hot zones" of inhibition in L2 and L5A match the reported low stimulus-evoked spiking rates of excitatory neurons in these layers, suggesting that these inhibitory hot zones substantially suppress activity in the neocortex.     

Dendritic coding of multiple sensory inputs in single cortical neurons in vivo

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.     

Linear integration of spine Ca2+ signals in layer 4 cortical neurons in vivo

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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ signals was independent of the activity of neighboring spines, without evidence for cooperativity. Furthermore, we found that spine Ca²⁺ signals evoked by back-propagating APs sum linearly with sensory-evoked synaptic Ca²⁺ 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.In the mammalian brain, sensory information is transmitted to the neocortex primarily through thalamo-cortical projections. Thus, in the vibrissae-related somatosensory cortex of rodents, tactile information arrives through axons of neurons that are located in the ventro-posterio-medial thalamic nucleus (VPM). It is well established that these axons provide direct input to layer 4 (L4), where they contact the dendrites of three types of excitatory neurons, namely the spiny stellate cells, the pyramids, and the star pyramids (1). In addition, there is recent evidence that axons originating in the VPM provide a direct input also to layer 5 (L5) neurons (2). A particularly interesting cytoarchitectonical feature of L4 of the rodent vibrissal cortex is the organization in distinct structures called “barrels” (3). The dendritic arborization of each spiny stellate cell is confined within the border of a barrel and is typically asymmetric, with cell bodies located at the barrel’s periphery and the dendrites facing the center (4, 5). Functionally, most spiny stellate cells and also the other L4 neurons in each barrel respond primarily to a single whisker, referred to as the principal whisker, whereas adjacent, or surround, whiskers provide a much weaker input (6). The fraction of thalamocortical inputs to each L4 neuron is remarkably small (about 10–20%) compared with that of intracortical inputs (7). Nevertheless, thalamocortical inputs are very efficient because of their synchronous activation by sensory stimuli (8, 9).The dendritic processes that are associated with the synaptic activation of L4 neurons were investigated initially in vitro in acute brain slices. For example, a study involving the use of whole-cell recordings in combination with two-photon imaging characterized synaptically evoked N-methyl-D-aspartate (NMDA) receptor-mediated Ca²⁺ influx in dendritic spines (10). The analysis of the spike timing dependence of the Ca²⁺ transient amplitudes showed a supralinearity when the synaptic stimulus preceded the back-propagating action potential (bAP) and a sublinearity when the order was reversed. Recently, a study combining whole-cell recordings in vivo and two-photon imaging in vitro investigated the cellular mechanisms of angular tuning in L4 neurons in the mouse barrel cortex (11). The results indicated that angular tuning of somatic voltage responses involves a complex nonlinear dendritic interplay of thalamo-cortical and cortico-cortical inputs. However, the dendritic Ca²⁺ signals in vivo that are associated with the rapid initial activation through whisker stimulation remained unclear.Advances in two-photon Ca²⁺ imaging techniques have enabled direct observation of single-spine activity in neurons in the upper cortical layers in vivo (12–15). Thus, a study focusing on layer 2/3 cortical neurons in mouse barrel cortex in vivo (15) has shown that, in addition to specific inputs that are primarily activated by distinct single whiskers, neurons in the upper cortical layers also receive “shared” single-spine inputs that can be activated by multiple whiskers. These shared inputs are activated by other cortical “feeder” neurons that receive inputs from multiple whiskers. The precise identity of the feeder neurons remained unclear. Up to now, two-photon Ca²⁺ imaging of spines and dendrites was largely restricted in cortical layers 2/3 at depths of up to about 200–250 µm (12, 16, 17). The general feasibility of recordings in deeper cortical layers was recently indicated by a report that analyzed dendritic Ca²⁺ signaling underlying spontaneous activity in the mouse motor cortex (18). In the present study, we implemented an optimized method of two-photon imaging in vivo (SI Text), which allowed the recordings of sensory-stimulation–evoked Ca²⁺ signals in dendritic spines of L4 cortical neurons at depth of up to 520 µm, nearly two times deeper than what had been achieved in most previous work (12, 13, 15, 19).

NMDA Receptor Dependence of Whisker Stimulation-Evoked Activity in L4 Neurons.

Recordings were performed in a subregion of the vibrissal cortex corresponding to the C2 whisker in anesthetized mice. The corresponding C2 barrel was identified at the beginning of each experiment by intrinsic optical imaging (15) (Fig. 1A). For dendritic Ca²⁺ imaging, neurons in L4 of the C2 barrel were loaded with the Ca²⁺-sensitive fluorescent dye Oregon Green BAPTA-1 by means of either single-cell electroporation or in combination with whole-cell recordings (SI Text). The morphologies of neurons were routinely reconstructed from z-stack fluorescence images of the dendritic tree (Fig. 1 B and C). We restricted our analysis to those neurons that were morphologically identified as L4 spiny stellate cells (20).Open in a separate windowFig. 1.Whisker-stimulation–evoked synchronous synaptic activation of L4 neurons. (A) L4 neurons were targeted in the barrel cortex identified with intrinsic signal optical imaging (SI Text). Single whiskers were deflected for 2 s. (Right, Upper) Circled dark area (arrow) indicates the cortical region activated by whisker stimulation. (Right, Lower) The corresponding blood vessel map. (B) Neuronal morphology was recovered from z-stack projections. (C) The side view reconstruction is from the neuron shown in B. (D) (Upper) Whisker-stimulation–evoked EPSPs in two cells. Gray traces are from five consecutive trials; red traces are the average of trials. Stimulation time is indicated above the traces. (Lower) Spontaneous EPSPs in the same two cells. Notations are the same as above. (E) Normalized distribution of the amplitudes of evoked (red, n = 5 cells) and spontaneous (blue, n = 3 cells) EPSPs. (F) Scatter plot of EPSP amplitudes in function of the EPSP (10–90%) rise times. Red and blue markers represent evoked (n = 5 cells) and spontaneous EPSPs (n = 3 cells), respectively. (G) A sample trace of an evoked EPSP. Dotted line indicates the start of stimulation. (H) Distribution of first evoked EPSP onset latencies (n = 198 EPSPs from nine neurons). (I) The effect of AP5 on the amplitude of evoked EPSPs. Gray traces are from eight consecutive trials; black traces show the average. Dotted lines indicate the start of stimulation. (J) The relative average amplitude (±SEM) of EPSPs under control (“Ctrl.”), drug (“AP5”), and wash-out (“Wash”) conditions (paired t test, P < 0.001); all amplitude values are normalized to the mean value under the control condition (K). A sample trace of an extracellularly recorded action potential (AP) current. Dotted line indicates the start of stimulation. (L). Distribution of the latencies of first evoked APs (n = 92 APs from nine neurons). (M) The effect of AP5 on (first) AP firing. Traces marked with black dots indicate single trials. (N) The relative average probability of AP firing under control (“Ctrl.”), drug (“AP5”), and wash-out (“Wash”) condition (paired t test, P < 0.001); all amplitude values are normalized to the mean value under the control condition.First, we compared the spontaneously occurring and the sensory-stimulation–evoked excitatory postsynaptic potentials (EPSPs) of L4 stellate cells. Sensory stimulation, consisting of a single deflection (SI Text) of the C2 whisker (Fig. 1D), known to produce a synchronous barrage of inputs to L4 neurons (8), led to short latency, large amplitude, and fast-rising EPSPs (Fig. 1 D–G). The mean latency of the EPSPs evoked by whisker stimulation was 9.7 ± 1.4 ms (n = 198 EPSPs from four neurons; Fig. 1H), a value that is similar to that reported in the rat barrel cortex (6). By contrast, spontaneously occurring EPSPs had smaller amplitudes and much slower rise times (Fig. 1 E and F). Thus, there are clear differences between the sensory-evoked EPSPs resulting from the synchronous activation of thalamic afferents (9, 21, 22) and the spontaneous EPSPs involving asynchronous activation of mostly cortico-cortical afferents, which form the majority of synaptic inputs to this cell type (23). The short-latency sensory-stimulation–evoked EPSPs, with their rapid onset and decay (e.g., Fig. 1D and SI Text), were highly similar to the short-latency EPSPs that occur in response to whisker stimulation in the nonpreferred orientation, as recently reported by Lavzin et al. (11).Focal application of the NMDA-receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) to the recorded cell produced a reversible decrease of the peak amplitude of sensory-evoked EPSPs to 62 ± 9% of the value in control conditions (five neurons, paired t test P < 0.001; Fig. 1 I and J) (11). This strong attenuation of the EPSPs caused a virtually complete block of whisker stimulation-evoked spiking, as revealed by a different set of experiments that was performed in the noninvasive cell-attached recording configuration (Fig. 1 K–N). These results indicate that NMDA receptor-dependent depolarization is required not only for whisker direction tuning (11), but also for the rapid thalamo-cortical input-mediated signal transfer from the external world to the cortex. Furthermore, the NMDA receptor dependence opened the possibility of studying the spatial and temporal distribution of the sensory-stimulation–activated synapses in L4 neurons in vivo by using the spine Ca²⁺ signal as a “biomarker” (13, 15).

Dendritic Arrangement of Whisker Stimulation-Activated Single-Spine Inputs.

For mapping whisker stimulation-evoked spine Ca²⁺ signals, we restricted our initial analysis to responses that did not produce action potentials (APs) in the postsynaptic cells to avoid ambiguities that can arise from global dendritic Ca²⁺ entry induced by back-propagating APs (14). Whisker stimulation-responsive spines were identified based on Ca²⁺ transients that were detected in single spines (Fig. 2A). The onset latency of the earliest spine Ca²⁺ transients was around 10 ms (median = 10 ms, n = 361 transients from nine neurons; Fig. 2B), consistent with the short latencies of the sensory-evoked EPSPs (Fig. 1 G and H). A total of 672 spines were visualized (44–165 spines per cell located in 2–11 dendritic segments per cell, n = 9 stellate cells), out of which 112 spines had such short-latency Ca²⁺ transients. Whisker-evoked spine Ca²⁺ transients had rise times (10–90%) of 85 ± 53 ms, decay times (single-exponential fit) of 412 ± 240 ms (n = 361 transients from nine cells ± SD). Similar to the sensory-evoked EPSPs, the spine Ca²⁺ transients were highly sensitive to AP5 (Fig. 2 C and D), indicating their common synaptic origin. Thus, short-latency spine Ca²⁺ transients (6) representing mostly, if not exclusively, thalamo-cortical inputs (see discussion below), can be used for the functional mapping of sensory-evoked synapses in vivo.Open in a separate windowFig. 2.Dendritic organization of short-latency, whisker-stimulation–activated spines. (A) Single-spine Ca²⁺ signals from L4 neurons. (Left) Two-photon image of a spiny dendritic segment (average of 6,000 frames). Green circle indicates the spine from which the Ca²⁺ signal on the right was calculated. Right: rising phase of a single Ca²⁺ trace calculated from the spine indicated on the left. Dotted line indicates the start of stimulation. (B) Distribution of the onset latencies of the first Ca²⁺ transients evoked by whisker stimulation (n = 361 transients from nine neurons). (C) The effect of AP5 on short-latency evoked Ca²⁺ transients. Gray traces are from eight consecutive trials; green traces show the average. Dotted lines indicate the onset of whisker stimulation. (D) The amplitudes of whisker-stimulation–evoked Ca²⁺ transients under control (“Ctrl.”), drug (“AP5”), and wash-out (“Wash”) conditions; all amplitude values are normalized to the mean value under the control condition. (n = 24 spines from four neurons, paired t test P < 0.0001). (E) Top view of a reconstructed neuron. Green boxes indicate the position of field of views in the dendritic field. Red dots indicate the position of spines that responded with short latency (10 ms) Ca²⁺ transients upon whisker stimulation. All other identified spines are marked with blue dots. (F) Superposition of spine locations from nine neurons. Neurons were rotated so that for each the barrel center is located to the left. Red and blue dots indicate spine locations as described in E. Red and gray circles indicate the 70- and 140-µm distances from the soma. (G) The depth distribution of identified spines. Red and blue dots indicate spine locations as described in E. Red and gray rectangles mark the 70- and 140-µm distances from the soma. (H) Bar graph comparing the density of responsive spines within 70 µm (“prox.”) to those at the 70- to 140-µm distance (“dist.”) from the soma (mean ± SD, n = 9 neurons, paired t test P < 0.001).With this mapping approach, we compared spine responses that were evoked by the stimulation of the principal whisker (PW) with those evoked by one of the surround whiskers (SWs) (Fig. S1). In contrast to previous observations that were made in layer 2 neurons, in which PW and SW stimulation was almost equally effective in activating “shared” dendritic spines (15), in L4 spiny stellate cells spine Ca²⁺ transients were almost exclusively evoked by PW stimulation (Fig. S1). Fig. 2E shows a representative L4 spiny stellate cell with the dendritic locations of all spines, including those responding to PW stimulation (i.e., the spines that showed Ca²⁺ transients with 10 ms of latency in response to PW stimulation) as well as the nonresponding ones, in four dendritic subregions (Fig. 2E). The overlay of similar recordings from nine neurons demonstrated that the majority of spines responding to PW stimulation were located in the proximal half of the dendritic field within a radius of about 70 µm around the cells’ somata (Fig. 2 F–H). This result is entirely consistent with recently reported anatomical data (24).

Linear Dendritic Integration of Whisker Stimulation-Evoked Single-Spine Inputs.

To study the dendritic processes occurring during the rapid initial activation of L4 neurons, we combined Ca²⁺ imaging with cell-attached recordings. We did not use the more invasive whole-cell configuration to avoid perturbation in membrane potential, mediated, for example, by leak currents. Fig. 3A shows the reconstruction of a representative L4 neuron and the dendritic segment used for Ca²⁺ imaging that was located at a depth of 415 µm below the pial surface (Fig. 3 B and C). Fig. 3D illustrates the subthreshold Ca²⁺ responses that were obtained during repeated trials of sensory stimulation. The median probability of whisker stimulation-evoked spine Ca²⁺ signals was 0.24 (n = 112 spines from nine neurons; Fig. 3E). In this particular experiment, we observed robust and reliable Ca²⁺ transients in 5/13 spines (s1, s2, s3, s8, and s10). By contrast, the Ca²⁺ transients in the immediately adjacent dendritic shaft were small or absent (on average more than 80% smaller; n = 37 spines from nine neurons, paired t test, P < 0.0001) (Fig. 3 F and G). As in other types of neurons, the residual Ca²⁺ transients in the shafts may result from Ca²⁺ diffusion from the active spines (25).Open in a separate windowFig. 3.Trial-by-trial activation pattern of whisker-stimulation–evoked responses in single spines. (A) Top view of a reconstructed neuron. Green box indicates the location of dendrite shown in B. (B) A spiny dendritic segment from the dendrite shown in A. Red arrows mark spines that were included in the analysis. (C) The schematic representation of the dendritic segment shown in B. Red and blue circles indicate responsive and nonresponsive spines, respectively. Black rectangles mark the dendritic shafts analyzed in D. (D) Ca²⁺ transients calculated from the region of interests shown in C for consecutively selected stimulation trials under the condition of the cell body’s subthreshold response. (E) Response probability distribution of responsive spines under subthreshold response conditions (n = 112 spines from nine neurons). (F) (Upper) The superposition of the trials with responses (n = 12) of spine 3 (s3) from D. Thick line shows the average. (Lower) The corresponding traces from the neighboring dendritic shaft (d3). Thick line shows the average. (G) The relative average amplitudes (±SEM) of spine and shaft Ca²⁺ signals (n = 37 spines from nine neurons). (H) Histogram of the distance between nearest-neighbor active spines (n = 61 spine pairs from nine neurons).In the next step of our analysis, we determined the distance between neighboring spines responding to PW stimulation. For the example illustrated in Fig. 3 B and C, we found that the nearest distance ranged from 1 µm (between s2 and s3) to 14 µm (between s8 and s10). Overall, the median distance between nearest-neighboring active spines was 3.2 µm (Fig. 3H, n = 61 spines pairs from nine neurons). The observation of this short distance between spines responding to sensory stimulation raised the question of whether the activity in a given spine has an impact on the amplitude of the Ca²⁺ transient of its coactive neighboring spines. This issue is important because a cooperativity may facilitate the generation of local dendritic spikes involving the nonlinear properties of NMDA receptors (26). To test this possibility, we compared the amplitude of Ca²⁺ transients that were recorded when a spine was activated alone with Ca²⁺ transients that were recorded when multiple neighboring spines on the same dendrite were active. In contrast to recent evidence indicating cooperativity in hippocampal neurons (27), Fig. 4 A–C (n = 33 spines from nine neurons) demonstrates that under both conditions the amplitudes of the Ca²⁺ transients were not different (paired t test, nonsignificant, P = 0.86), without indications for cooperativity.Open in a separate windowFig. 4.Dendritic integration of noncooperative single-spine sensory inputs. (A) Schematic representation of a spiny dendritic segment. Red arrows indicate the responsive spines. (B) Average Ca²⁺ transients from s1 marked in A, when the spine was active alone (Left, average of n = 5 trials) or simultaneously with other spines (Right, average of n = 5 trials). Dotted lines indicate start of stimulation. (C) Normalized average amplitudes (±SEM) of spine Ca²⁺ transients when the spines were active alone (“single”) or simultaneously with other spines (“multiple”); n = 33 spines from nine neurons; paired t test, P = 0.86. (D) Examples of average (average of n = 5 trials) spine Ca²⁺ transients under different conditions. bAP: single bAP in spontaneous conditions; Syn: evoked subthreshold synaptic event; Syn+bAP: evoked synaptic event coinciding with a single bAP; calculated: the arithmetic sum of bAP and Syn conditions. Arrow indicates the time of AP firing. Dotted lines indicate the start of stimulation. (E) The relative average amplitudes (±SEM) of Ca²⁺ transients of the calculated versus the measured sum of synaptic and backpropagating AP events; n = 73 spines from nine neurons; paired t test, P = 0.005. (F) Examples of stimulus-evoked prolonged firing response of L4 neurons; dashed line indicates stimulation onset. The two cells were recorded separately. (G) The overall firing rate of each recorded L4 neuron with respect to the depth from the cortical surface. (H) Cumulative distribution curve of the overall firing rate for n = 32 neurons. (I) “f0” image of a dendritic segment at 415-µm depth (average image in 400 ms before stimulation onset), and the “Δf” images (average image in 400 ms after stimulation onset) corresponding to responses with no AP, 1 AP, 2 APs, or 3 APs. Red arrows indicated the active input spines identified under subthreshold conditions. (J) Ca²⁺ trasients of an active spine (denoted by s1 in I) and its neighboring dendritic shaft corresponding to responses with no AP, 1 AP, 2 APs, or 3 APs. Ca²⁺ trasients were averaged for all stimulation trials in which the given numbers of APs were evoked. Vertical dotted lines indicate whisker stimulation onset time. (K) Δf/f values in dendritic shaft (Upper) as well as in active spines (Lower) versus the number of APs in response to whisker stimulation for 14 dendritic segments (in four neurons). Error bars: ±SD.We found that the amplitudes of synaptically evoked spine Ca²⁺ transients through sensory stimulation (“Syn,” Fig. 4D) recorded under subthreshold conditions were higher than those Ca²⁺ transients produced by a spontaneous bAP in absence of sensory stimulation (“bAP,” Fig. 4D and Fig. S2; paired t test, n = 73, P < 0.001). Remarkably, it turned out that the amplitudes of the Ca²⁺ transients recorded during suprathreshold sensory stimulation (“Syn + bAP”) were similar to those obtained for the transients generated by the arithmetic sum of the subthreshold stimulation- and bAP-evoked transients (“Calculated,” Fig. 4 D and E; n = 73 spines from nine neurons). The linear summation is similar to what has been previously observed with in vitro recordings in spiny stellate cells, provided that the synaptic activation was temporally coincident (within 1–2 ms) with bAPs (10).Next, we investigated whether the linearity is maintained in the highly active neurons thought to be involved in whisker-object–touching tasks in awake animals (28). We found that, under our recording conditions, fast passive whisker deflection activated L4 neurons in a way that resembled neuronal activation in object-touching tasks (28). Indeed, a similarly small fraction of neurons responded with intense spiking (4/32) and at a similarly high frequency (>10 Hz, Fig. 4 F–H). Fig. 4I shows the averaged images of a dendritic segment for “f0” conditions (baseline fluorescence) as well as the stimulation-evoked increase in fluorescence images, “Δf,” when either one, two, or three spikes were fired, respectively. In the dendritic shafts, the bAP-associated Ca²⁺ signals increased linearly with spike number (Fig. 4 J and K, Pearson’s correlation, r = 0.98, P < 0.001) without evidence for regenerative dendritic processes. Moreover, in the active spines, whisker-evoked Ca²⁺ signals also increased linearly with spike number, except with an offset that corresponds to the major contribution of synaptic component (Fig. 4 J and K, Pearson’s correlation, r = 0.93, P < 0.001). Finally, we took advantage of the fact that spine Ca²⁺ transients associated with a single bAP have much smaller amplitudes than sensory-stimulation–evoked ones (Fig. 4D and Fig. S2). Thus, spine activation by afferent inputs can be reliably resolved even in firing neurons. Fig. S3 A1 and A2 shows that the same set of spines is activated under both subthreshold and suprathreshold conditions. However, the probability of activation of such an input is more than twice as high under suprathreshold (Fig. S3B) as under subthreshold conditions (Fig. 3E). This changed probability leads to a significant increase in the fraction of active inputs per dendrite under suprathreshold compared with subthreshold conditions (Fig. S3C), suggesting that sensory-evoked spiking in L4 spiny stellate cells is controlled by the activity of the up-stream afferent neurons located mostly in the thalamus.     

基于模块化的主观认知减退人群静息态皮质网络连接组学研究

目的:探讨主观认知减退（SCD）人群静息态皮质网络的拓扑特征及与认知水平的相关性。方法:收集2017年6月至2019年11月在南京各社区招募36例自诉有记忆下降的中老年人与32名健康对照（NC）,分析认知量表、T 1加权像（T 1WI）和静息态功能磁共振（rs-fMRI）数据。SCD组男5例、女...     

Development and aging of cortical thickness correspond to genetic organization patterns

There is a growing realization that early life influences have lasting impact on brain function and structure. Recent research has demonstrated that genetic relationships in adults can be used to parcellate the cortex into regions of maximal shared genetic influence, and a major hypothesis is that genetically programmed neurodevelopmental events cause a lasting impact on the organization of the cerebral cortex observable decades later. Here we tested how developmental and lifespan changes in cortical thickness fit the underlying genetic organizational principles of cortical thickness in a longitudinal sample of 974 participants between 4.1 and 88.5 y of age with a total of 1,633 scans, including 773 scans from children below 12 y. Genetic clustering of cortical thickness was based on an independent dataset of 406 adult twins. Developmental and adult age-related changes in cortical thickness followed closely the genetic organization of the cerebral cortex, with change rates varying as a function of genetic similarity between regions. Cortical regions with overlapping genetic architecture showed correlated developmental and adult age change trajectories and vice versa for regions with low genetic overlap. Thus, effects of genes on regional variations in cortical thickness in middle age can be traced to regional differences in neurodevelopmental change rates and extrapolated to further adult aging-related cortical thinning. This finding suggests that genetic factors contribute to cortical changes through life and calls for a lifespan perspective in research aimed at identifying the genetic and environmental determinants of cortical development and aging.There is a growing realization that events during development impact brain and cognition throughout the entire lifespan (1). For instance, the major portion of the relationship between cortical thickness and IQ in old age can be explained by childhood IQ (2), and genotype may explain a substantial part of the lifetime stability in intelligence (3). Effects of genes on the organization of the cortex have been shown in adults (4–6), but it is unknown whether and how regional differences in cortical development correspond to these regional genetic subdivisions.Although consensus has not been reached for the exact trajectories, cortical thickness as measured by MRI appears to decrease in childhood (7–12). The exact foundation for this thinning is not known, as MRI provides merely representations of the underlying neurobiology, and available histological data cannot with certainty be used to guide interpretations of MRI results. Although speculative, apparent thickness decrease may be grounded in factors such as synaptic pruning and intracortical myelination, although the link between established synaptic processes (13–15) and cortical thickness has not been empirically confirmed. After childhood, cortical thinning continues throughout the remainder of the lifespan, speculated to reflect neuronal shrinkage and reductions in number of spines and synapses (16), although similar to development, we lack data to support a direct connection between cortical thinning and specific neurobiological events.It has been demonstrated that genetic correlations between thickness in different surface locations can be used to parcellate the adult cortex into regions of maximal shared genetic influence (4). This result can be interpreted according to the hypothesis that genetically programmed neurodevelopmental events cause lasting impact on the organization of the cerebral cortex detectable decades later (4–6). Here we tested how developmental and lifespan changes fit the genetic organization of cortical thickness in a large longitudinal sample with 1,633 scans from 974 participants between 4.1 and 88.5 y of age, including 773 scans from children below 12 y Genetically based subdivisions of cortical thickness from an independent dataset of 406 twins (4) were applied to the data, yielding 12 separate regions under maximum control of shared genetic influences. We hypothesized that thickness in cortical regions with overlapping genetic architecture would show similar developmental and adult age change trajectories and dissimilar trajectories for regions with low genetic overlap.     

Spiking in primary somatosensory cortex during natural whisking in awake head-restrained rats is cell-type specific

Sensation involves active movement of sensory organs, but it remains unknown how position or movement of sensory organs is encoded in cortex. In the rat whisker system, each whisker is represented by an individual cortical (barrel) column. Here, we quantified in awake, head-fixed rats the impact of natural whisker movements on action potential frequencies of single (identified) neurons located in different layers of somatosensory (barrel) cortex. In all layers, we found only weak correlations between spiking and whisker position or velocity. Conversely, whisking significantly increased spiking rate in a subset of neurons located preferentially in layer 5A. This finding suggests that whisker movement could be encoded by population responses of neurons within all layers and by single slender-tufted pyramids in layer 5A.     

Robustness of sensory-evoked excitation is increased by inhibitory inputs to distal apical tuft dendrites

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.Mechanistic understanding of the principles that underlie sensory-evoked neuronal responses remains a key challenge in neuroscience research. Although electrophysiological and optical imaging techniques provide access to activity patterns of individual and/or populations of neurons in live animals, information about the organization of the underlying synaptic input patterns that drive neuronal activity remains scarce. Here, we investigate the mechanisms underlying whisker deflection-evoked responses in pyramidal neurons (PNs) in the vibrissal part of rat primary somatosensory cortex (vS1, i.e., barrel cortex) (1). Specifically, we wanted to know whether and how L1 interneurons (INs) shape responses of L2 PNs. L1 is densely populated by apical tuft dendrites from multiple types of excitatory PNs and a sparse population of GABAergic INs (2). Recent studies in acute parasagittal (3) and coronal (4) brain slices in vitro have shown that L1 INs have axonal projections largely confined to L1, where they form synaptic connections with the dendrites from PNs located in L2/3 (5) and L5 (4). These connections place L1 INs in a perfect position to manipulate the activity of PNs, for example, by feed-forward inhibition and/or more indirect mechanisms such as disinhibition (4, 6). However, the influence of L1 INs on the sensory-evoked responses of PNs remains unclear.To address this, we performed whole-cell patch-clamp recordings in vivo and reconstructed the 3D morphologies of the recorded L1 INs. These data, acquired under the same experimental conditions as previously used to determine whisker-evoked spiking and 3D morphologies for PN cell types (7), were used to inform and constrain simulation experiments. Specifically, we converted the 3D soma/dendrite morphology of an in vivo-labeled L2 PN into a biophysically detailed full-compartmental model (8) and integrated the neuron model into a recently reported detailed reconstruction of the excitatory circuitry in vS1 (9). This integration enabled us to statistically measure the number and subcellular distribution of cell type-specific synaptic contacts impinging onto the exemplary L2 PN from L1 INs and L2–5 PNs, respectively. These spatially constrained synaptic input patterns were further constrained temporally by using the measured cell type-specific spiking probabilities and latencies (7, 10). Finally, we made in silico experiments (i.e., numerical simulations) and investigated how the interplay between biophysical properties of the dendrites and well-constrained spatiotemporal synaptic input patterns give rise to the whisker-evoked responses measured in vivo (11).     

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.     

Prelimbic cortical BDNF is required for memory of learned fear but not extinction or innate fear

In the medial prefrontal cortex, the prelimbic area is emerging as a major modulator of fear behavior, but the mechanisms remain unclear. Using a selective neocortical knockout mouse, virally mediated prelimbic cortical-specific gene deletion, and pharmacological rescue with a TrkB agonist, we examined the role of a primary candidate mechanism, BDNF, in conditioned fear. We found consistently robust deficits in consolidation of cued fear but no effects on acquisition, expression of unlearned fear, sensorimotor function, and spatial learning. This deficit in learned fear in the BDNF knockout mice was rescued with systemic administration of a TrkB receptor agonist, 7,8-dihydroxyflavone. These data indicate that prelimbic BDNF is critical for consolidation of learned fear memories, but it is not required for innate fear or extinction of fear. Moreover, use of site-specific, inducible BDNF deletions shows a powerful mechanism that may further our understanding of the pathophysiology of fear-related disorders.     

Interareal coordination of columnar architectures during visual cortical development

The formation of cortical columns is often conceptualized as a local process in which synaptic microcircuits confined to the volume of the emerging column are established and selectively refined. Many neurons, however, while wiring up locally are simultaneously building macroscopic circuits spanning widely distributed brain regions, such as different cortical areas or the two brain hemispheres. Thus, it is conceivable that interareal interactions shape the local column layout. Here we show that the columnar architectures of different areas of the cat visual cortex in fact develop in a coordinated manner, not adequately described as a local process. This is revealed by comparing the layouts of orientation columns (i) in left/right pairs of brain hemispheres and (ii) in areas V1 and V2 of individual brain hemispheres. Whereas the size of columns varied strongly within all areas considered, columns in different areas were typically closely matched in size if they were mutually connected. During development, we find that such mutually connected columns progressively become better matched in size as the late phase of the critical period unfolds. Our results suggest that one function of critical-period plasticity is to progressively coordinate the functional architectures of different cortical areas—even across hemispheres.     

Robust but delayed thalamocortical activation of dendritic-targeting inhibitory interneurons

GABA-releasing cortical interneurons are crucial for the neural transformations underlying sensory perception, providing “feedforward” inhibition that constrains the temporal window for synaptic integration. To mediate feedforward inhibition, inhibitory interneurons need to fire in response to ascending thalamocortical inputs, and most previous studies concluded that ascending inputs activate mainly or solely proximally targeting, parvalbumin-containing “fast-spiking” interneurons. However, when thalamocortical axons fire at frequencies that are likely to occur during natural exploratory behavior, activation of fast-spiking interneurons is rapidly and strongly depressed, implying the paradoxical conclusion that feedforward inhibition is absent when it is most needed. To address this issue, we took advantage of lines of transgenic mice in which either parvalbumin- or somatostatin-containing interneurons express GFP and recorded the responses of interneurons from both subtypes to thalamocortical stimulation in vitro. We report that during thalamocortical activation at behaviorally expected frequencies, fast-spiking interneurons were indeed activated only transiently because of rapid depression of their thalamocortical inputs, but a subset of layer 5 somatostatin-containing interneurons were robustly and persistently activated after a delay, due to the facilitation and temporal summation of their thalamocortical excitatory postsynaptic potentials. Somatostatin-containing interneurons are considered distally targeting. Thus, they are likely to provide delayed dendritic inhibition during exploratory behavior, contributing to the maintenance of a balance between cortical excitation and inhibition while leaving a wide temporal window open for synaptic integration and plasticity in distal dendrites.     

Astrocytic regulation of cortical UP states

The synchronization of neuronal assemblies during cortical UP states has been implicated in computational and homeostatic processes, but the mechanisms by which this occurs remain unknown. To investigate potential roles of astrocytes in synchronizing cortical circuits, we electrically activated astrocytes while monitoring the activity of the surrounding network with electrophysiological recordings and calcium imaging. Stimulating a single astrocyte activates other astrocytes in the local circuit and can trigger UP state synchronizations of neighboring neurons. Moreover, interfering with astrocytic activity with intracellular injections of a calcium chelator into individual astrocytes inhibits spontaneous and stimulated UP states. Finally, both astrocytic activity and neuronal UP states are regulated by purinergic signaling in the circuit. These results demonstrate that astroglia can play a causal role in regulating the synchronized activation of neuronal ensembles.     

A conserved folding nucleus sculpts the free energy landscape of bacterial and archaeal orthologs from a divergent TIM barrel family

The amino acid sequences of proteins have evolved over billions of years, preserving their structures and functions while responding to evolutionary forces. Are there conserved sequence and structural elements that preserve the protein folding mechanisms? The functionally diverse and ancient (βα)_1–8 TIM barrel motif may answer this question. We mapped the complex six-state folding free energy surface of a ∼3.6 billion y old, bacterial indole-3-glycerol phosphate synthase (IGPS) TIM barrel enzyme by equilibrium and kinetic hydrogen–deuterium exchange mass spectrometry (HDX-MS). HDX-MS on the intact protein reported exchange in the native basin and the presence of two thermodynamically distinct on- and off-pathway intermediates in slow but dynamic equilibrium with each other. Proteolysis revealed protection in a small (α1β2) and a large cluster (β5α5β6α6β7) and that these clusters form cores of stability in I_a and I_bp. The strongest protection in both states resides in β4α4 with the highest density of branched aliphatic side chain contacts in the folded structure. Similar correlations were observed previously for an evolutionarily distinct archaeal IGPS, emphasizing a key role for hydrophobicity in stabilizing common high-energy folding intermediates. A bioinformatics analysis of IGPS sequences from the three superkingdoms revealed an exceedingly high hydrophobicity and surprising α-helix propensity for β4, preceded by a highly conserved βα-hairpin clamp that links β3 and β4. The conservation of the folding mechanisms for archaeal and bacterial IGPS proteins reflects the conservation of key elements of sequence and structure that first appeared in the last universal common ancestor of these ancient proteins.

Proteins are indispensable workhorses of cellular machinery whose functional diversity is defined by their final folded conformations. The folding pathway of a protein is determined by its energy landscape, whose map is encoded in the amino acid sequence. Partially folded states on the landscape often contain elements of the native topology and connect the nascent unfolded polypeptide chain to the functional folded conformation (1, 2). Proteins and their folding pathways have evolved over billions of years, responding to evolutionary forces such as mutation and natural selection (3–5). Orthologs, proteins that have diverged from a common ancestor but share a common structure and function, provide vehicles for exploring the impact of evolution on folding pathways and the intermediates that guide the folding to the native conformation.The functionally diverse (βα)_1–8 TIM barrel motif is an ideal candidate to decipher evolutionary constraints on protein folding pathways. The motif supports a wide variety of essential enzymatic transformations in all three superkingdoms of life (6–8) and is one of the 10 ancestral protein folds that were instrumental in the transition from RNA–protein world to the last universal common ancestor of life (LUCA) to the present complex DNA–RNA–protein world (9, 10). The βα-repeat architecture produces a cylindrical β-barrel core and an amphipathic α-helical shell whose loops between the β-strands and subsequent α-helices form the canonical active site of this very large family of enzymes. Although the pairwise sequence conservation across the family of TIM barrels is typically ∼30%, their folding mechanisms are complex and highly conserved (11). Folding intermediates, both on the productive folding pathway and as misfolded, kinetic traps have been observed for candidate TIM barrels from several bacterial and archaeal organisms (11–16). The divergence of these two superkingdoms, which occurred ∼4 billion y ago, right after life arose, speaks to the robustness of the TIM barrel folding mechanism across the span of evolutionary time.We have previously examined the relationships between sequence, structure, and fitness in a yeast-based competition assay for three thermophilic indole-3-glycerolphosphate synthase (IGPS) orthologs from the TIM barrel family (17). Significant correlations between the archaeal Sulfolobus solfataricus (SsIGPS) and the bacterial Thermotoga maritima (TmIGPS) and Thermus thermophilus (TtIGPS) proteins revealed that both sequence and structure are critical in defining their fitness landscapes. This observation and the conservation of TIM barrel folding mechanisms motivated the hypothesis that the sequences of TIM barrel orthologs from archaeal and bacterial organisms also conserve the structures of their folding intermediates. If valid, we would obtain detailed insights into the constraints that TIM barrel structure and function impose on the enormous sequence space available in ∼4 billion y of evolution (18, 19). We have previously mapped the structures of the on- and off-pathway intermediates for SsIGPS by hydrogen–deuterium exchange mass spectrometry (HDX-MS) (15, 16), providing an archaeal reference for the present study of a bacterial ortholog (SI Appendix, Fig. S1).Comparison of the structures of the folding intermediates and folding mechanisms for S. solfataricus and T. maritima IGPS confirmed our hypothesis. A bioinformatics analysis of thousands of nonredundant IGPS sequences from the bacterial, archaeal, and eukaryota superkingdoms revealed the conservation of three adjacent structural elements that form a nucleus responsible for defining the folding free energy surface of the IGPS family of TIM barrel proteins. We conclude that the folding mechanism of the IGPS TIM barrel, including the structures of key partially folded states, arose in the LUCA and has persisted for over ∼4 billion y.     

Critical role of calcitonin gene-related peptide receptors in cortical spreading depression

Cortical spreading depression (CSD) is a key pathogenetic step in migraine with aura. Dysfunctions of voltage-dependent and receptor-operated channels have been implicated in the generation of CSD and in the pathophysiology of migraine. Although a known correlation exists between migraine and release of the calcitonin gene-related peptide (CGRP), the possibility that CGRP is involved in CSD has not been examined in detail. We analyzed the pharmacological mechanisms underlying CSD and investigated the possibility that endogenous CGRP contributes to this phenomenon. CSD was analyzed in rat neocortical slices by imaging of the intrinsic optical signal. CSD was measured as the percentage of the maximal surface of a cortical slice covered by the propagation of intrinsic optical signal changes during an induction episode. Reproducible CSD episodes were induced through repetitive elevations of extracellular potassium concentration. AMPA glutamate receptor antagonism did not inhibit CSD, whereas NMDA receptor antagonism did inhibit CSD. Blockade of voltage-dependent sodium channels by TTX also reduced CSD. CSD was also decreased by the antiepileptic drug topiramate, but not by carbamazepine. Interestingly, endogenous CGRP was released in the cortical tissue in a calcium-dependent manner during CSD, and three different CGRP receptor antagonists had a dose-dependent inhibitory effect on CSD, suggesting a critical role of CGRP in this phenomenon. Our findings show that both glutamate NMDA receptors and voltage-dependent sodium channels play roles in CSD. They also demonstrate that CGRP antagonism reduces CSD, supporting the possible use of drugs targeting central CGRP receptors as antimigraine agents.     

Similar patterns of cortical expansion during human development and evolution

The cerebral cortex of the human infant at term is complexly folded in a similar fashion to adult cortex but has only one third the total surface area. By comparing 12 healthy infants born at term with 12 healthy young adults, we demonstrate that postnatal cortical expansion is strikingly nonuniform: regions of lateral temporal, parietal, and frontal cortex expand nearly twice as much as other regions in the insular and medial occipital cortex. This differential postnatal expansion may reflect regional differences in the maturity of dendritic and synaptic architecture at birth and/or in the complexity of dendritic and synaptic architecture in adults. This expression may also be associated with differential sensitivity of cortical circuits to childhood experience and insults. By comparing human and macaque monkey cerebral cortex, we infer that the pattern of human evolutionary expansion is remarkably similar to the pattern of human postnatal expansion. To account for this correspondence, we hypothesize that it is beneficial for regions of recent evolutionary expansion to remain less mature at birth, perhaps to increase the influence of postnatal experience on the development of these regions or to focus prenatal resources on regions most important for early survival.     

Age-related changes in GAD levels in the central auditory system of the rat

Changes in the levels of gamma-aminobutyric acid (GABA) are known to occur in different parts of the brain during aging. In our study we attempted to define the effect that aging has on glutamate decarboxylase (GAD), the key enzyme in the synthesis of GABA, in the central parts of the auditory system. Age-related changes in GAD65 and GAD67 levels were investigated using immunohistochemistry and Western blotting in the inferior colliculus (IC), the auditory cortex (AC) and the visual cortex in Long-Evans rats. The results show that aging is associated with a decrease in the numbers of GAD65- and 67-immunoreactive neurons and the optical density of their somas in both the IC and AC. Western blot analysis revealed a pronounced age-related decline in the levels of GAD65 and 67 proteins in both the IC and AC. For comparison, in the visual cortex the decrease in both proteins was less pronounced than in the IC and AC. A similar pattern of age-related changes was found in Fischer 344 rats, a strain that manifests a rapid loss of hearing function with aging. The observed age-related decline in the levels of GAD65 and 67 may contribute significantly to the deterioration of hearing function that accompanies aging in mammals, including man.     

Stable and dynamic cortical electrophysiology of induction and emergence with propofol anesthesia

The mechanism(s) by which anesthetics reversibly suppress consciousness are incompletely understood. Previous functional imaging studies demonstrated dynamic changes in thalamic and cortical metabolic activity, as well as the maintained presence of metabolically defined functional networks despite the loss of consciousness. However, the invasive electrophysiology associated with these observations has yet to be studied. By recording electrical activity directly from the cortical surface, electrocorticography (ECoG) provides a powerful method to integrate spatial, temporal, and spectral features of cortical electrophysiology not possible with noninvasive approaches. In this study, we report a unique comprehensive recording of invasive human cortical physiology during both induction and emergence from propofol anesthesia. Propofol-induced transitions in and out of consciousness (defined here as responsiveness) were characterized by maintained large-scale functional networks defined by correlated fluctuations of the slow cortical potential (<0.5 Hz) over the somatomotor cortex, present even in the deeply anesthetized state of burst suppression. Similarly, phase-power coupling between θ- and γ-range frequencies persisted throughout the induction and emergence from anesthesia. Superimposed on this preserved functional architecture were alterations in frequency band power, variance, covariance, and phase-power interactions that were distinct to different frequency ranges and occurred in separable phases. These data support that dynamic alterations in cortical and thalamocortical circuit activity occur in the context of a larger stable architecture that is maintained despite anesthetic-induced alterations in consciousness.