T current potentiation increases the occurrence and temporal fidelity of synaptically evoked burst firing in sensory thalamic neurons

A growing number of in vivo experiments shows that high frequency bursts of action potentials can be recorded in thalamocortical neurons of awake animals. The mechanism underlying these bursts, however, remains controversial, because they have been proposed to depend on T-type Ca(2+) channels that are inactivated at the depolarized membrane potentials usually associated with the awake state. Here, we show that the transient potentiation of the T current amplitude, which is induced by neuronal depolarization, drastically increases the probability of occurrence and the temporal precision of T-channel-dependent high frequency bursts. The data, therefore, provides the first biophysical mechanism that might account for the generation of these high frequency bursts of action potentials in the awake state. Remarkably, this regulation finely tunes the response of thalamocortical neurons to the corticofugal excitatory and intrathalamic inhibitory afferents but not to sensory inputs.     

Why inclusion bodies do assume different locations in thalassaemic erythrocytes

Summary The reported scanning (SEM), transmission (TS), and freeze-etching (FE) electron microscopic studies have agreed in confirming that in thalassaemic erythrocytes, previously incubated with brilliant cresyl blue (BCB), the unpaired alpha chains precipitate in the central portions of the cell whereas excess beta chains locate in the submembranous regions. This is due to the fact that beta chains, possessing two thiols instead of only one (as in alpha chains), are more liable to bind to similar groups contained in the inner red cell leaflet. Less soluble alpha chains tend to form inter-chain bridges and thus precipitate centrally. SEM observations have given evidence that on the surface of the affected red cells denaturated alpha chains give rise to large and shallow invaginations whereas denatured beta chains lead to a diffuse wrinkled appearance. The causes of the different SEM aspects have been suggested.     

Gender differences in human cortical synaptic density

Certain cognitive functions differ in men and women, although the anatomical and functional substrates underlying these differences remain unknown. Because neocortical activity is directly related with higher brain function, numerous studies have focused on the cerebral cortex when searching for possible structural correlates of cognitive gender differences. However, there are no studies on possible gender differences at the synaptic level. In the present work we have used stereological and correlative light and electron microscopy to show that men have a significantly higher synaptic density than women in all cortical layers of the temporal neocortex. These differences may represent a microanatomical substrate contributing to the functional gender differences in brain activity.     

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.     

Comparative ultrastructural study of endoplasmic reticulum in colorectal carcinoma cell lines with different degrees of differentiation

The endoplasmic reticulum (ER) consists of a complex system of tubules, lamellae, and flattened vesicles, and has a variety of morphologies in different cells. It is believed to play a central role in the biosynthesis of cholesterol, phospholipids, steroids, prostaglandins, membrane and secretory proteins[1]. Cancer cells have different functions and ultrastmcture from their original cells[2-4]. The studies on ER membrane system of cancer cells are of great significance in understanding their malignant behavior. In the present work, the ultrastructural characteristics of ER in human colorectal carcinoma cell lines with different differentiation degrees were investigated.     

2006-2008年甘肃省高氟水源及不同生态类型区分布调查

目的 掌握甘肃省地方性氟中毒病区所属生态类型区及高氟水源分布现状,为制订防治策略提供科学依据.方法 于2006-2008年,调查项目县(区)村民定居点生态及饮用水源情况.在各项目县(区)病区村开展即时饮用水源水氟筛查,按东、西、南、北、中5个方位选取水源水5份,用氟离子选择电极测定水氟.结果 共在45个县进行了筛查,在41个县筛查出超标水样7174份,占29.84%(7174/20 241),分布在1524个村,占31.62%(1524/4819),其中28个县有>2.0 mg/L的水样检出,9个县的30个村有>4.0 mg/L的水样检出.按超标水样和有超标水样的村比率从低到高排序,均为甘南高原草原草甸区<荒漠区<河西走廊戈壁区<黄土高原丘陵区<黄土高原沟壑区.结论 不同生态类型区内居民所面临的饮用水问题不同,应合理规划和分类指导地方性氟中毒的预防控制工作.     

Cell- and stimulus-dependent heterogeneity of synaptic vesicle endocytic recycling mechanisms revealed by studies of dynamin 1-null neurons

Mice lacking expression of dynamin 1, a GTPase implicated in the fission reaction of synaptic vesicle endocytosis, fail to thrive and exhibit severe activity-dependent endocytic defects at their synapses. Here, we have used electron tomography to investigate the massive increase in clathrin-coated pit abundance that is selectively observed at a subset of synapses in dynamin 1 KO primary neuron cultures under conditions of spontaneous network activity. This increase, leading to branched tubular plasma membrane invaginations capped by clathrin-coated buds, occurs selectively at inhibitory synapses. A similar massive increase of clathrin-coated profiles (in this case, of clathrin-coated vesicles) is observed at inhibitory synapses of neurons that lack expression of synaptojanin 1, a phosphoinositide phosphatase involved in clathrin-coated vesicle uncoating. Thus, although excitatory synapses are largely spared under these conditions, inhibitory synapses are uniquely sensitive to perturbation of endocytic proteins, probably as a result of their higher levels of tonic activity leading to a buildup of clathrin-coated intermediates in these synapses. In contrast, the predominant endocytic structures observed at the majority of dynamin 1 KO synapses after acute stimulation are endosome-like intermediates that originate by a dynamin 1-independent form of endocytosis. These findings reveal a striking heterogeneity in the mode of synaptic vesicle recycling in different synapses and functional states.     

微山湖区放养子代钉螺生殖腺透射电镜观察

目的观察在微山湖区螺笼放养12个冬季、繁殖12代的成年钉螺睾丸、卵巢形态变化。方法以亲代来源于长江扬州段的微山湖子代成年钉螺为实验组,以长江扬州段江滩阴性成年钉螺为对照组,解剖两组钉螺获得完整的睾丸和卵巢标本。以常规方法固定、脱水、包埋标本,烘箱内聚合,用超薄切片机常规切片,取切片在透射电子显微镜下拍照和观察,比较两组钉螺生殖腺超微结构。结果对照组钉螺精子细胞尾部横切面可见"9+2"微管结构,头端可见三角形顶体和呈螺旋形且致密的核质;实验组钉螺精子细胞尾部横切面"9+2"微管结构消失,头端染色质密度稀疏。对照组钉螺卵巢卵细胞核仁、核膜清晰,细胞质内可见卵黄体、脂质体、内质网,核膜为双层曲折,核仁均匀;实验组钉螺卵巢卵细胞核膜光滑,核仁不明显,细胞内含物更加稀疏。结论经过12个冬季放养,钉螺不能完全适应中国北方的自然环境,其生殖腺超微结构发生了明显变化,这可能是微山湖区放养钉螺逐步减少甚至趋于消亡的原因之一。     

Procedure for recording the simultaneous activity of single neurons distributed across cortical areas during sensory discrimination

We report a procedure for recording the simultaneous activity of single neurons distributed across five cortical areas in behaving monkeys. The procedure consists of a commercially available microdrive adapted to a commercially available neural data collection system. The critical advantage of this procedure is that, in each cortical area, a configuration of seven microelectrodes spaced 250-500 mum can be inserted transdurally and each can be moved independently in the z axis. For each microelectrode, the data collection system can record the activity of up to five neurons together with the local field potential (LFP). With this procedure, we normally monitor the simultaneous activity of 70-100 neurons while trained monkeys discriminate the difference in frequency between two vibrotactile stimuli. Approximately 20-60 of these neurons have response properties previously reported in this task. The neuronal recordings show good signal-to-noise ratio, are remarkably stable along a 1-day session, and allow testing several protocols. Microelectrodes are removed from the brain after a 1-day recording session, but are reinserted again the next day by using the same or different x-y microelectrode array configurations. The fact that microelectrodes can be moved in the z axis during the recording session and that the x-y configuration can be changed from day to day maximizes the probability of studying simultaneous interactions, both local and across distant cortical areas, between neurons associated with the different components of this task.     

椭圆食粉螨各发育阶段外部形态扫描电镜观察

目的 观察椭圆食粉螨生活史中各发育阶段外部形态和电镜下超微形态特征。 方法 将椭圆食粉螨洗涤干净后,用2.5%戊二醛固定、酒精洗涤、临界点干燥,置于导电双面胶上,整姿,在电镜下扫描观察。结果 幼螨3对足,且生殖系统尚未发育完全;雌雄成螨大体相似,足Ⅰ基节前方有一格氏器,足Ⅳ跗节有吸盘一对。雄成螨阳茎为直管状,末端分叉。肛门两侧有一对吸盘。3对肛后毛几乎排列在同一直线上;雌成螨体积略大,有2对肛后毛。讨论 对椭圆食粉螨电镜下的超微观察有助于对其进一步的科学分类和进行生活史研究。     

椭圆食粉螨各发育阶段外部形态扫描电镜观察

目的 观察椭圆食粉螨生活史中各发育阶段外部形态和电镜下超微形态特征。 方法 将椭圆食粉螨洗涤干净后，用2.5%戊二醛固定、酒精洗涤、临界点干燥，置于导电双面胶上，整姿，在电镜下扫描观察。结果 幼螨3对足，且生殖系统尚未发育完全；雌雄成螨大体相似，足Ⅰ基节前方有一格氏器，足Ⅳ跗节有吸盘一对。雄成螨阳茎为直管状，末端分叉。肛门两侧有一对吸盘。3对肛后毛几乎排列在同一直线上；雌成螨体积略大，有2对肛后毛。讨论 对椭圆食粉螨电镜下的超微观察有助于对其进一步的科学分类和进行生活史研究。     

粉尘螨各发育阶段外部形态扫描电镜观察

目的　观察粉尘螨（Dermatophagoides farinae）不同发育阶段的形态特征。方法　将纯培养的粉尘螨分离后,应用扫描电子显微镜观察卵、幼螨、若螨、成螨等不同发育阶段外部形态。结果　粉尘螨卵呈长椭圆形。幼螨足3对,若螨足4对,生殖孔不发达,有生殖毛和肛毛。成螨呈长圆形,表皮有细致花纹,足4对;雄成螨足Ⅰ明显加粗,足Ⅲ较足Ⅳ粗长,生殖区位于足Ⅲ、Ⅳ基节间,肛门被一圆形围肛环包围,环内有肛门吸盘和刚毛1对;雌成螨足Ⅲ、Ⅳ较细,长度相等,腹面有1个呈“人”字形的生殖孔,其前端有1个类似月牙形的生殖板,肛门为1个纵形裂缝。结论　对粉尘螨进行扫描电镜观察能够清晰了解其形态结构特征,对粉尘螨分类鉴定具有重要意义。     

Assembly of Weibel-Palade body-like tubules from N-terminal domains of von Willebrand factor

Endothelial cells assemble von Willebrand factor (VWF) multimers into ordered tubules within storage organelles called Weibel-Palade bodies, and tubular packing is necessary for the secretion of VWF filaments that can bind connective tissue and recruit platelets to sites of vascular injury. We now have recreated VWF tubule assembly in vitro, starting with only pure VWF propeptide (domains D1D2) and disulfide-linked dimers of adjacent N-terminal D'D3 domains. Assembly requires low pH and calcium ions and is reversed at neutral pH. Quick-freeze deep-etch electron microscopy and three-dimensional reconstruction of negatively stained images show that tubules contain a repeating unit of one D'D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging.     

Morphological characterization of the squamocolumnar junction of the esophagus in patients with and without Barrett's epithelium

Barrett's esophagus is a metaplastic condition in which the normal stratified squamous epithelium of the distal esophagus is replaced by columnar epithelium. Our group has previously characterized a unique surface cell (the distinctive cell) at the junction of squamous and Barrett's epithelium. This cell is notable for the simultaneous presence on its surface of both squamous and columnar cell features. The aims of our present study were, first, to evaluate prospectively the frequency with which Barrett's patients have the distinctive cell at the squamo-Barrett's junction; second, to further elucidate the characteristics of the distinctive cell; and third, to perform a combined morphological study of the squamo-Barrett's junction using scanning electron microscopy followed by transmission and light microscopy. We divided study patients into two groups: Group I consisted of Barrett's patients and group II of non-Barrett's control patients. Of eight group I Barrett's patients with junctional biopsies, three were noted to have the distinctive cell (37.5%). In contrast, this cell was not observed in any of the group II control patients. Biopsies in control patients as well as Barrett's patients without the distinctive cell revealed abrupt squamogastric or squamo-Barrett's junctions by scanning electron microscopy and light microscopy. In contrast, biopsies from the Barrett's patients with the distinctive cell revealed junctions that were not abrupt and had the distinctive cells overlying normal squamous epithelium. By scanning electron microscopy, the distinctive cells were flattened, polygonal cells with surface microvilli (a columnar cell feature) and were demarcated from one another by shallow depressions, or by intercellular ridges (a squamous cell feature). By transmission electron microscopy, the distinctive cells were cuboidal in shape with abundant apical microvilli and secretory vesicles. We have confirmed that distinctive cells are present in some Barrett's patients. This cell is a morphologic hybrid, sharing features of both squamous and columnar cells, and may be analogous to hybrid cells identified in other locations that undergo metaplasia (eg, the human cervix). Its origin may be the result of transformation of multipotential basal cells of squamous epithelial origin. We hypothesize that the distinctive cells may represent an intermediate stage in the development of Barrett's epithelium.This work was presented in part as an oral presentation at the American Gastroenterological Association's Annual Meeting in Boston in May 1993.     

Nuclear localization and characterization of alkaline phosphatase in neutrophils from normal controls and pregnant women.

There were controversial data concerning localization of alkaline phosphatase (AP) in neutrophil nuclei under physiological conditions. In this context, the AP pattern has been determined on nuclei preparations from normal human neutrophils. Blood cells were isolated from 10 healthy adults and from 3 women in the third trimester of an uncomplicated pregnancy. Purity of nuclear suspension was checked by electron microscopy and assay of organelle marker enzymes. Electron microscope cytochemistry and immunocytochemistry studies were carried out on WBC. Enzyme characterization was performed by the usual biochemical procedures. AP was found in nuclear preparations from four of ten normal controls. When present, AP was detected in approximately two-thirds of the nuclei examined, representing an average of 20% of the total cell activity. Conversely, a large amount of nucleus-bound enzyme (55% of total AP activity) was recognized in all pregnant women samples. Biochemical and immunological characteristics clearly differentiate AP forms in the two groups of subjects. Normal controls have an heterogeneous enzyme pattern. AP positive preparations contain a mixture of isoenzymes: a prominent heat labile form and a relatively heat stable minor component. The heat stable fraction displays some properties similar to those previously described in leukocyte AP. Pregnant women express a unique very heat labile isoenzyme identical in its main characteristics to the early placental type.     

Biochemical and aggregation analysis of Bence Jones proteins from different light chain diseases

Deposition of immunoglobulin light chains is a result of clonal proliferation of monoclonal plasma cells that secrete free immunoglobulin light chains, also called Bence Jones proteins (BJP). These BJP are present in circulation in large amounts and excreted in urine in various light chain diseases such as light chain amyloidosis (AL), light chain deposition disease (LCDD) and multiple myeloma (MM). BJP from patients with AL, LCDD and MM were purified from their urine and studies were performed to determine their secondary structure, thermodynamic stability and aggregate formation kinetics. Our results show that LCDD and MM proteins have the lowest free energy of folding while all proteins show similar melting temperatures. Incubation of the BJP at their melting temperature produced morphologically different aggregates: amyloid fibrils from the AL proteins, amorphous aggregates from the LCDD proteins and large spherical species from the MM proteins. The aggregates formed under in vitro conditions suggested that the various proteins derived from patients with different light chain diseases might follow different aggregation pathways.     

阿奇霉素抗弓形虫感染的体外实验研究

目的　为探索孕期弓形虫感染治疗效果好的药物。方法　本实验采用体外细胞培养技术 ,MTT(四甲基偶氮唑盐 )比色法 ,观察阿齐霉素抗弓形虫侵袭的猴肾细胞的活力及功能状态及通过透射电镜、扫描电镜观察猴肾细胞内弓形虫的形态、体积并超微结构的改变。结果　猴肾细胞的增殖在不同浓度的阿齐霉素加药组比未加药组差异均有显著性 ,P <0 0 1。经透射电镜观察发现 :虫体在药物作用下有的发生破裂、崩解 ,细胞内留下空泡 ,有的呈团块 ,没有明显的细胞器可见。扫描电镜观察显示猴肾细胞内的虫体与未加药组相比明显皱缩。结论　阿齐霉素具有很好的抗弓形虫效应 ,而对猴肾细胞无显著影响。这为阻断孕期弓形虫宫内垂直感染胎儿治疗时提供科学的实验依据     

Primary cilia safeguard cortical neurons in neonatal mouse forebrain from environmental stress-induced dendritic degeneration

The developing brain is under the risk of exposure to a multitude of environmental stressors. While perinatal exposure to excessive levels of environmental stress is responsible for a wide spectrum of neurological and psychiatric conditions, the developing brain is equipped with intrinsic cell protection, the mechanisms of which remain unknown. Here we show, using neonatal mouse as a model system, that primary cilia, hair-like protrusions from the neuronal cell body, play an essential role in protecting immature neurons from the negative impacts of exposure to environmental stress. More specifically, we found that primary cilia prevent the degeneration of dendritic arbors upon exposure to alcohol and ketamine, two major cell stressors, by activating cilia-localized insulin-like growth factor 1 receptor and downstream Akt signaling. We also found that activation of this pathway inhibits Caspase-3 activation and caspase-mediated cleavage/fragmentation of cytoskeletal proteins in stress-exposed neurons. These results indicate that primary cilia play an integral role in mitigating adverse impacts of environmental stressors such as drugs on perinatal brain development.

Exposure to harmful environmental factors during prenatal and perinatal stages of human brain development can disrupt a number of molecular pathways involved in critical steps of brain development, potentially leading to the development of neurodevelopmental disorders or intellectual disabilities (1, 2). Of those factors, alcohol and ketamine represent major drugs that are frequently used by pregnant women (3, 4). These two drugs share γ-minobutyric acid (GABA) mimetic and N-methyl-d-aspartic acid (NMDA) antagonistic properties, which have been shown to mediate strong cell death-promoting effects in the developing brain (5, 6). Exposing mice to alcohol or anesthetics, such as ketamine, at about postnatal day (P) 7 is commonly used to model exposure of human fetuses between the third trimester of gestation and the neonatal stage. During this critical period, a brain undergoes a growth spurt (7), and disruption of developmental processes during this period is particularly harmful, often triggering long-term brain dysfunctions.Alcohol (ethanol) exposure can perturb essential processes in brain development, including neurogenesis, migration, and cell survival (8), leading to a wide range of functional deficits (9–13). Many studies have revealed that immature neurons are particularly vulnerable targets for ethanol-induced apoptotic cell death during the brain growth spurt (14).Ketamine is used as an analgesic and anesthetic for surgery, and has also emerged as an effective antidepressant (15, 16). However, ketamine is also abused as a recreational drug (17). Increasingly widespread use and misuse of ketamine by pregnant and lactating women, raises concerns about its neurotoxicity to the immature perinatal brain of their offspring (18, 19). In a number of animal and human studies, perinatal exposure to ketamine was shown to trigger neuronal degeneration, which subsequently leads to long-term and permanent functional deficits (20–22).Under the risk of exposure to such factors, the developing brain relies on various intrinsic neuroprotective mechanisms including heat shock signaling and endoplasmic reticulum stress pathways (23, 24). Increasing attention has been paid to these pathways as attractive targets for the development of novel interventions and therapies for neuropsychiatric and neurodegenerative disorders (24, 25). Yet, our understanding of the intrinsic protective mechanisms remains limited.Cilia, which consist of motile and primary types, are protuberances that project from the soma of various cell types (26, 27). Primary cilia are known to play a pivotal role in early normal brain development (28) by regulating the signal transduction of key molecular pathways such as Hedgehog and Wnt signaling (29). The elongation (maturation) of primary cilia in the cerebral cortex occurs during early postnatal development in mice (around P0 to P14) (27), and deficits are suspected to be involved in the pathogenesis of neuropsychiatric disorders such as autism and schizophrenia (30–32). Although the risk for these disorders is known to be increased by exposure to various environmental stressors, including alcohol and ketamine (33–35), it is unknown whether cilia play a role in preserving normal brain development in the face of environmental factors.In the present study, we tested whether primary cilia serve a protective role during exposure of the developing cerebral cortex to environmental insults such as ethanol and ketamine. We used transgenic mice in which cilia are genetically lost in cortical excitatory neurons as a model system. Our data provide evidence that primary cilia are an essential organelle for efficient activation of cilia-localized insulin-like growth factor 1 receptor (IGF-1R), and downstream Akt signaling to protect immature neurons from caspase-mediated dendritic degeneration in the developing brain.     

Lateralization of observational fear learning at the cortical but not thalamic level in mice

Major cognitive and emotional faculties are dominantly lateralized in the human cerebral cortex. The mechanism of this lateralization has remained elusive owing to the inaccessibility of human brains to many experimental manipulations. In this study we demonstrate the hemispheric lateralization of observational fear learning in mice. Using unilateral inactivation as well as electrical stimulation of the anterior cingulate cortex (ACC), we show that observational fear learning is controlled by the right but not the left ACC. In contrast to the cortex, inactivation of either left or right thalamic nuclei, both of which are in reciprocal connection to ACC, induced similar impairment of this behavior. The data suggest that lateralization of negative emotions is an evolutionarily conserved trait and mainly involves cortical operations. Lateralization of the observational fear learning behavior in a rodent model will allow detailed analysis of cortical asymmetry in cognitive functions.     

SARS-CoV-2: Ultrastructural Characterization of Morphogenesis in an In Vitro System

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted public health and the world economy and fueled a worldwide race to approve therapeutic and prophylactic agents, but so far there are no specific antiviral drugs. Understanding the biology of the virus is the first step in structuring strategies to combat it, and in this context several studies have been conducted with the aim of understanding the replication mechanism of SARS-CoV-2 in vitro systems. In this work, studies using transmission and scanning electron microscopy and 3D electron microscopy modeling were performed with the goal of characterizing the morphogenesis of SARS-CoV-2 in Vero-E6 cells. Several ultrastructural changes were observed—such as syncytia formation, cytoplasmic membrane projections, lipid droplets accumulation, proliferation of double-membrane vesicles derived from the rough endoplasmic reticulum, and alteration of mitochondria. The entry of the virus into cells occurred through endocytosis. Viral particles were observed attached to the cell membrane and in various cellular compartments, and extrusion of viral progeny took place by exocytosis. These findings allow us to infer that Vero-E6 cells are highly susceptible to SARS-CoV-2 infection as described in the literature and their replication cycle is similar to that described with SARS-CoV and MERS-CoV in vitro models.