Structure of intraglomerular dendritic tufts of mitral cells and their contacts with olfactory nerve terminals and calbindin-immunoreactive type 2 periglomerular neurons.

Intraglomerular dendritic tufts of Golgi-impregnated and biotinylated dextran amine (BDA)-labeled mitral cells in the rat main olfactory bulb were analyzed in detail. In particular, the relationships of BDA-labeled tufts with olfactory nerve (ON) terminals and processes of calbindin D-28K-immunoreactive (CB-IR) cells were investigated with confocal laser-scanning light microscopic (CLSM) and electron microscopic (EM) analyses. CB-IR cells were type 2 periglomerular cells that restricted their processes in the ON-free (non-ON) zone of the glomerulus and received few synapses from ON terminals. The mitral tufts varied in complexity, but individual branches were rather simple, smooth processes that bore some branchlets and spines and extended more or less in a straight line or a gentle curve rather than winding tortuously within glomeruli as though they did not consider the compartmental organization, which consisted of ON and non-ON zones that interdigitated in a complex manner with one another. Conventional EM analysis revealed that both thin and thick, presumed proximal branches of mitral/tufted cell dendritic tufts received asymmetrical synapses from ON terminals. Correlated CLSM-EM analysis confirmed direct contacts between the BDA- and CB-labeled processes detected in the CLSM examinations, and synapses were recognized at some of those sites. Furthermore, ON terminals and CB-IR processes were distributed on both proximal and distal dendritic branches in a more or less mosaic pattern. These findings revealed that, on the mitral dendritic tufts, ON terminals and processes of type 2 periglomerular neurons were not clearly segregated proximodistally but, rather, were arranged in a mosaic pattern, which may be important in fine tuning the output from individual glomeruli.     

石杉碱甲对血管性痴呆小鼠模型海马神经细胞静息态[Ca~(2+)]i的影响

目的　观察血管性痴呆 (VD)小鼠海马神经细胞内静息态游离 Ca2 +浓度 ([Ca2 + ]i)变化 ,以及石杉碱甲 (哈伯因 )对 VD的治疗效果和 [Ca2 + ]i的影响 ,进而探讨 [Ca2 + ]i的改变在 VD发病机制中的作用。方法　采用双侧颈总动脉线结、反复缺血 -再灌注法 ,制作小鼠血管性痴呆动物模型 ,并设假手术组作为对照 ,服用石杉碱甲者为治疗组。分别于术后第 2 9天、第 30天进行学习、记忆和行为学测试 ;然后快速制取海马活细胞 ,以 Fluo- 3/ AM为荧光探针 ,在激光扫描共聚焦显微镜下观察各组海马神经细胞静息态 [Ca2 + ]i变化。结果　 (1)模型组的学习和记忆成绩低于假手术组及治疗组 (P<0 .0 5 ) ,治疗组及假手术组间无显著性差别 ;(2 )模型组神经细胞静息态 [Ca2 + ]i显著高于假手术组、治疗组 (P<0 .0 5 ) ,治疗组及假手术组间无显著性差别。结论　石杉碱甲作为中枢神经系统特异性胆碱酯酶抑制剂和 NMDA受体拮抗剂 ,可降低血管性痴呆小鼠海马静息态 [Ca2 + ]i,并改善其临床症状 ;因此提示 :海马神经细胞静息态 [Ca2 + ]i过高参与了血管性痴呆的发病过程。     

Drebrin A is a postsynaptic protein that localizes in vivo to the submembranous surface of dendritic sites forming excitatory synapses

Drebrin A is a neuron-specific, actin binding protein. Evidence to date is from in vitro studies, consistently supporting the involvement of drebrin A in spinogenesis and synaptogenesis. We sought to determine whether drebrin A arrives at the plasma membrane of neurons, in vivo, in time to orchestrate spinogenesis and synaptogenesis. To this end, a new antibody was used to locate drebrin A in relation to electron microscopically imaged synapses during early postnatal days. Western blotting showed that drebrin A emerges at postnatal day (PNd) 6 and becomes progressively more associated with F-actin in the pellet fraction. Light microscopy showed high concentrations of drebrin A in the synaptic layers of the hippocampus and cortex. Electron microscopy revealed that drebrin A in these regions is located exclusively in dendrites both neonatally and in adulthood. In adulthood, nearly all of the synaptic drebrin A is within spines forming asymmetric excitatory synapses, verified by gamma-aminobutyric acid (GABA) negativity. At PNd7, patches of drebrin A immunoreactivity were discretely localized to the submembranous surfaces of dendrites forming slight protrusions-protospines. The drebrin A sites exhibited only thin postsynaptic densities and lacked axonal associations or were contacted by axons that contained only a few vesicles. Yet, because of their immunoreactivity to the NR2B subunit of N-methyl-D-aspartate receptors and immunonegativity of axon terminals to GABA, these could be presumed to be nascent, excitatory synapses. Thus, drebrin A may be involved in organizing the dendritic pool of actin for the formation of spines and of axospinous excitatory synapses during early postnatal periods.     

Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses

Choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme and a definitive marker for cholinergic neurons, was localized immunocytochemically in the motor and somatic sensory regions of rat cerebral cortex with monoclonal antibodies. ChAT-positive (ChAT+) varicose fibers and terminal-like structures were distributed in a loose network throughout the cortex. Some immunoreactive cortical fibers were continuous with those in the white matter underlying the cortex, and many of these fibers presumably originated from subcortical cholinergic neurons. ChAT+ fibers appeared to be rather evenly distributed throughout all layers of the motor cortex, but a subtle laminar pattern was evident in the somatic sensory cortex, where lower concentrations of fibers in layer IV contrasted with higher concentrations in layer V. Electron microscopy demonstrated that immunoreaction product was concentrated in synaptic vesicle-filled profiles and that many of these structures formed synaptic contacts. ChAT+ synapses were present in all cortical layers, and the majority were of the symmetric type, although a few asymmetric ones were also observed. The most common postsynaptic elements were small to medium-sized dendritic shafts of unidentified origin. In addition, ChAT+ terminals formed synaptic contacts with apical and, probably, basilar dendrites of pyramidal neurons, as well as with the somata of ChAT-negative nonpyramidal neurons. ChAT+ cell bodies were present throughout cortical layers II-VI, but were most concentrated in layers II-III. The somata were small in size, and the majority of ChAT+ neurons were bipolar in form, displaying vertically oriented dendrites that often extended across several cortical layers. Electron microscopy confirmed the presence of immunoreaction product within the cytoplasm of small neurons and revealed that they received both symmetric and asymmetric synapses on their somata and proximal dendrites. These observations support an identification of ChAT+ cells as nonpyramidal intrinsic neurons and thus indicate that there is an intrinsic source of cholinergic innervation of the rat cerebral cortex, as well as the previously described extrinsic sources.     

Synaptic connections of first‐stage visual neurons in the locust Schistocerca gregaria extend evolution of tetrad synapses back 200 million years

The small size of some insects, and the crystalline regularity of their eyes, have made them ideal for large‐scale reconstructions of visual circuits. In phylogenetically recent muscomorph flies, like Drosophila, precisely coordinated output to different motion‐processing pathways is delivered by photoreceptors (R cells), targeting four different postsynaptic cells at each synapse (tetrad). Tetrads were linked to the evolution of aerial agility. To reconstruct circuits for vision in the larger brain of a locust, a phylogenetically old, flying insect, we adapted serial block‐face scanning electron microscopy (SBEM). Locust lamina monopolar cells, L1 and L2, were the main targets of the R cell pathway, L1 and L2 each fed a different circuit, only L1 providing feedback onto R cells. Unexpectedly, 40% of all locust R cell synapses onto both L1 and L2 were tetrads, revealing the emergence of tetrads in an arthropod group present 200 million years before muscomorph flies appeared, coinciding with the early evolution of flight. J. Comp. Neurol. 523:298–312, 2015. © 2014 Wiley Periodicals, Inc.     

α4βδ‐GABAA receptors in dorsal hippocampal CA1 of adolescent female rats traffic to the plasma membrane of dendritic spines following voluntary exercise and contribute to protection of animals from activity‐based anorexia through localization at excitatory synapses

In hippocampal CA1 of adolescent female rodents, α4βδ‐GABA_A receptors (α4βδ‐GABA_ARs) suppress excitability of pyramidal neurons through shunting inhibition at excitatory synapses. This contributes to anxiolysis of stressed animals. Socially isolated adolescent female rats with 8 days of wheel access, the last 4 days of which entail restricted food access, have been shown to exhibit excessive exercise, choosing to run instead of eat (activity‐based anorexia [ABA]). Upregulation of α4βδ‐GABA_ARs in the dorsal hippocampal CA1 (DH), seen among some ABA animals, correlates with suppression of excessive exercise. We used electron microscopic immunocytochemistry to show that exercise alone (EX), but not food restriction alone (FR), also augments α4βδ‐GABA_AR expression at axospinous excitatory synapses of the DH (67%, P = 0.027), relative to socially isolated controls without exercise or food restriction (CON). Relative to CON, ABA animals' synaptic α4βδ‐GABA_AR elevation was modestly elevated (37%), but this level correlated strongly and negatively with individual differences in ABA vulnerability—i.e., food restriction–evoked hyperactivity (Pearson R = ?0.902, P = 0.002) and weight changes (R = 0.822, P = 0.012). These correlations were absent from FR and EX brains or ventral hippocampus of ABA brains. Comparison to CON of α4βδ‐GABA_AR location in the DH indicated that ABA induces trafficking of α4βδ‐GABA_AR from reserve pools in spine cytoplasm to excitatory synapses. Pair‐housing CON animals reduced cytoplasmic α4βδ‐GABA_AR without reducing synaptic α4βδ‐GABA_AR. Thus, exercise induces trafficking of α4βδ‐GABA_ARs to excitatory synapses, while individual differences in ABA vulnerability are linked most strongly to trafficking of α4βδ‐GABA_ARs in the reverse direction—from excitatory synapses to the reserve pool during co‐occurring food restriction. © 2017 Wiley Periodicals, Inc.     

Protein kinase C gamma interneurons in the rat medullary dorsal horn: Distribution and synaptic inputs to these neurons,and subcellular localization of the enzyme

The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (II_i) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina II_i PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A‐ and C‐fibers low threshold mechanoreceptors (LTMRs) terminate in lamina II_i raise the general issue of synaptic inputs to lamina II_i PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ‐immunoreactivity is mostly restricted to interneurons in lamina II_i of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ‐immunoreactive interneurons are asymmetric (likely excitatory). PKCγ‐immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3‐immunoreactive bouton, specific to C‐LTMRs, on PKCγ‐immunoreactive interneurons. PKCγ‐immunoreactive interneurons contain GABA_Aergic and glycinergic receptors. At the subcellular level, PKCγ‐immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ‐immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia. J. Comp. Neurol. 522:393–413, 2014. © 2013 Wiley Periodicals, Inc.     

Hormone complement of the Cancer productus sinus gland and pericardial organ: an anatomical and mass spectrometric investigation

In crustaceans, circulating hormones influence many physiological processes. Two neuroendocrine organs, the sinus gland (SG) and the pericardial organ (PO), are the sources of many of these compounds. As a first step in determining the roles played by hemolymph-borne agents in the crab Cancer productus, we characterized the hormone complement of its SG and PO. We show via transmission electron microscopy that the nerve terminals making up each site possess dense-core and/or electron-lucent vesicles, suggesting diverse complements of bioactive molecules for both structures. By using immunohistochemistry, we show that small molecule transmitters, amines and peptides, are among the hormones present in these tissues, with many differentially distributed between the two sites (e.g., serotonin in the PO but not the SG). With several mass spectrometric (MS) methods, we identified many of the peptides responsible for the immunolabeling and surveyed the SG and PO for peptides for which no antibodies exist. By using MS, we characterized 39 known peptides [e.g., beta-pigment-dispersing hormone (beta-PDH), crustacean cardioactive peptide, and red pigment-concentrating hormone] and de novo sequenced 23 novel ones (e.g., a new beta-PDH isoform and the first B-type allatostatins identified from a non-insect species). Collectively, our results show that diverse and unique complements of hormones, including many previously unknown peptides, are present in the SG and PO of C. productus. Moreover, our study sets the stage for future biochemical and physiological studies of these molecules and ultimately the elucidation of the role(s) they play in hormonal control in C. productus.