Ultrastructural evidence for synaptic contacts between cortical noradrenergic afferents and endocannabinoid-synthesizing post-synaptic neurons.

Endocannabinoids (eCBs) are involved in a myriad of physiological processes that are mediated through the activation of cannabinoid receptors, which are ubiquitously distributed within the nervous system. One neurochemical target at which cannabinoids interact to have global effects on behavior is brain noradrenergic circuitry. We, and others, have previously shown that CB type 1 receptors (CB1r) are positioned to pre-synaptically modulate norepinephrine (NE) release in the rat frontal cortex (FC). Diacylglycerol lipase (DGL) is a key enzyme in the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). While DGL-α is expressed in the FC in the rat brain, it is not known whether noradrenergic afferents target neurons expressing synthesizing enzymes for the endocannabinoid, 2-AG. In the present study, we employed high-resolution neuroanatomical approaches to better define cellular sites for interactions between noradrenergic afferents and FC neurons expressing DGL-α. Immunofluorescence microscopy showed close appositions between processes containing the norepinephrine transporter (NET) or dopamine-ß-hydroxylase (DßH) and cortical neurons expressing DGL-α-immunoreactivity. Ultrastructural analysis using immunogold-silver labeling for DGL-α and immunoperoxidase labeling for NET or DßH confirmed that NET-labeled axon terminals were directly apposed to FC somata and dendritic processes that exhibited DGL-α-immunoreactivity. Finally, tissue sections were processed for immunohistochemical detection of DGL-α, CB1r and DßH. Triple label immunofluorescence revealed that CB1r and DßH were co-localized in common cellular profiles and these were in close association with DGL-α. Taken together, these data provide anatomical evidence for direct synaptic associations between noradrenergic afferents and cortical neurons exhibiting endocannabinoid synthesizing machinery.

Spine morphogenesis in newborn granule cells is differentially regulated in the outer and middle molecular layers.

New neurons are continuously added to the hippocampus of adult mammals. Their survival and integration into the circuitry are highly dependent on experience. Here we show that mushroom spine formation in newborn granule cells was modulated by experience and that dendritic segments in different areas of the molecular layer were differentially regulated. Specifically, spines of new neurons in the outer molecular layer of the dentate gyrus were more readily influenced by nonspatial features in the living environment. Those in the middle molecular layer were more likely to be influenced by the size of the living environment. Therefore, the activity of cortical inputs into newborn granule cells may be reflected in the formation of mushroom spines in different dendritic segments in the molecular layer.

A novel CaM kinase II pathway controls the location of neuropeptide release from Caenorhabditis elegans motor neurons.

Neurons release neuropeptides via the regulated exocytosis of dense core vesicles (DCVs) to evoke or modulate behaviors. We found that Caenorhabditis elegans motor neurons send most of their DCVs to axons, leaving very few in the cell somas. How neurons maintain this skewed distribution and the extent to which it can be altered to control DCV numbers in axons or to drive release from somas for different behavioral impacts is unknown. Using a forward genetic screen, we identified loss-of-function mutations in UNC-43 (CaM kinase II) that reduce axonal DCV levels by ∼90% and cell soma/dendrite DCV levels by ∼80%, leaving small synaptic vesicles largely unaffected. Blocking regulated secretion in unc-43 mutants restored near wild-type axonal levels of DCVs. Time-lapse video microscopy showed no role for CaM kinase II in the transport of DCVs from cell somas to axons. In vivo secretion assays revealed that much of the missing neuropeptide in unc-43 mutants is secreted via a regulated secretory pathway requiring UNC-31 (CAPS) and UNC-18 (nSec1). DCV cargo levels in unc-43 mutants are similarly low in cell somas and the axon initial segment, indicating that the secretion occurs prior to axonal transport. Genetic pathway analysis suggests that abnormal neuropeptide function contributes to the sluggish basal locomotion rate of unc-43 mutants. These results reveal a novel pathway controlling the location of DCV exocytosis and describe a major new function for CaM kinase II.

Stereological and ultrastructural quantification of the afferent synaptome of individual neurons.

Determining the number and placement of synaptic inputs along the distinct plasma membrane domains of neurons is essential for explaining the basis of neuronal activity and function. We detail a strategy that combines juxtacellular labeling, neuronal reconstructions and stereological sampling of inputs at the ultrastructural level to define key elements of the afferent 'synaptome' of a given neuron. This approach provides unbiased estimates of the total number and somato-dendritic distribution of synapses made with individual neurons. These organizational properties can be related to the activity of the same neurons previously recorded in vivo, for direct structure-function correlations at the single-cell level. The approach also provides the quantitative data required to develop biologically realistic models that simulate and predict neuronal activity and function.

Intracortical excitatory and thalamocortical boutons are intact in primary auditory cortex in schizophrenia.

Schizophrenia is associated with auditory processing impairments that could arise as a result of primary auditory cortex excitatory circuit pathology. We have previously reported a deficit in dendritic spine density in deep layer 3 of primary auditory cortex in subjects with schizophrenia. As boutons and spines can be structurally and functionally co-regulated, we asked whether the densities of intracortical excitatory or thalamocortical presynaptic boutons are also reduced. We studied 2 cohorts of subjects with schizophrenia and matched controls, comprising 27 subject pairs, and assessed the density, number, and within-bouton vesicular glutamate transporter (VGluT) protein level of intracortical excitatory (VGluT1-immunoreactive) and thalamocortical (VGluT2-immunoreactive) boutons in deep layer 3 of primary auditory cortex using quantitative confocal microscopy and stereologic sampling methods. We found that VGluT1- and VGluT2-immunoreactive puncta densities and numbers were not altered in deep layer 3 of primary auditory cortex of subjects with schizophrenia. Our results indicate that reduced dendritic spine density in primary auditory cortex of subjects with schizophrenia is not matched by a corresponding reduction in excitatory bouton density. This suggests excitatory boutons in primary auditory cortex in schizophrenia may synapse with structures other than spines, such as dendritic shafts, with greater frequency. The discrepancy between dendritic spine reduction and excitatory bouton preservation may contribute to functional impairments of the primary auditory cortex in subjects with schizophrenia.

Altered branching patterns of Purkinje cells in mouse model for cortical development disorder.

Disrupted cortical cytoarchitecture in cerebellum is a typical pathology in reeler. Particularly interesting are structural problems at the cellular level: dendritic morphology has important functional implication in signal processing. Here we describe a combinatorial imaging method of synchrotron X-ray microtomography with Golgi staining, which can deliver 3-dimensional(3-D) micro-architectures of Purkinje cell(PC) dendrites, and give access to quantitative information in 3-D geometry. In reeler, we visualized in 3-D geometry the shape alterations of planar PC dendrites (i.e., abnormal 3-D arborization). Despite these alterations, the 3-D quantitative analysis of the branching patterns showed no significant changes of the 77 ± 8° branch angle, whereas the branch segment length strongly increased with large fluctuations, comparing to control. The 3-D fractal dimension of the PCs decreased from 1.723 to 1.254, indicating a significant reduction of dendritic complexity. This study provides insights into etiologies and further potential treatment options for lissencephaly and various neurodevelopmental disorders.

Recurrent spontaneous spreading depolarizations facilitate acute dendritic injury in the ischemic penumbra.

Spontaneous spreading depolarizations (SDs) occur in the penumbra surrounding ischemic core. These SDs, often referred to as peri-infarct depolarizations, cause vasoconstriction and recruitment of the penumbra into the ischemic core in the critical first hours after focal ischemic stroke; however, the real-time spatiotemporal dynamics of SD-induced injury to synaptic circuitry in the penumbra remain unknown. A modified cortical photothrombosis model was used to produce a square-shaped lesion surrounding a penumbra-like "area at risk" in middle cerebral artery territory of mouse somatosensory cortex. Lesioning resulted in recurrent spontaneous SDs. In vivo two-photon microscopy of green fluorescent protein-expressing neurons in this penumbra-like area at risk revealed that SDs were temporally correlated with rapid (<6 s) dendritic beading. Dendrites quickly (<3 min) recovered between SDs to near-control morphology until the occurrence of SD-induced terminal dendritic injury, signifying acute synaptic damage. SDs are characterized by a breakdown of ion homeostasis that can be recovered by ion pumps if the energy supply is adequate. Indeed, the likelihood of rapid dendritic recovery between SDs was correlated with the presence of nearby flowing blood vessels, but the presence of such vessels was not always sufficient for rapid dendritic recovery, suggesting that energy needs for recovery exceeded energy supply of compromised blood flow. We propose that metabolic stress resulting from recurring SDs facilitates acute injury at the level of dendrites and dendritic spines in metabolically compromised tissue, expediting penumbral recruitment into the ischemic core.

Novel processes invaginate the pre-synaptic terminal of retinal bipolar cells.

Mixed-rod cone bipolar (Mb) cells of goldfish retina have large synaptic terminals (10 microm in diameter) that make 60-90 ribbon synapses mostly onto amacrine cells and rarely onto ganglion cells and, in return, receive 300-400 synapses from gamma-aminobutyric acid (GABA)-ergic amacrine cells. Tissue viewed by electron microscopy revealed the presence of double-membrane-bound processes deep within Mb terminals. No membrane specializations were apparent on these invaginating processes, although rare vesicular fusion was observed. These invaginating dendrites were termed "InDents". Mb bipolar cells were identified by their immunoreactivity for protein kinase C. Double-label immunofluorescence with other cell-type-specific labels eliminated Müller cells, efferent fibers, other Mb bipolar cells, dopaminergic interplexiform cells, and somatostatin amacrine cells as a source of the InDents. Confocal analysis of double-labeled tissue clearly showed dendrites of GABA amacrine cells, backfilled ganglion cells, and dendrites containing PanNa immunoreactivity extending into and passing through Mb terminals. Nearly all Mb terminals showed evidence for the presence of InDents, indicating their common presence in goldfish retina. No PanNa immunoreactivity was found on GABA or ganglion cell InDents, suggesting that a subtype of glycine amacrine cell contained voltage-gated Na channels. Thus, potassium and calcium voltage-gated channels might be present on the InDents and on the Mb terminal membrane opposed to the InDents. In addition to synaptic signaling at ribbon and conventional synapses, Mb bipolar cells may exchange information with InDents by an alternative signaling mechanism.

Dendritic spine detection using curvilinear structure detector and LDA classifier.

Dendritic spines are small, bulbous cellular compartments that carry synapses. Biologists have been studying the biochemical pathways by examining the morphological and statistical changes of the dendritic spines at the intracellular level. In this paper a novel approach is presented for automated detection of dendritic spines in neuron images. The dendritic spines are recognized as small objects of variable shape attached or detached to multiple dendritic backbones in the 2D projection of the image stack along the optical direction. We extend the curvilinear structure detector to extract the boundaries as well as the centerlines for the dendritic backbones and spines. We further build a classifier using Linear Discriminate Analysis (LDA) to classify the attached spines into valid and invalid types to improve the accuracy of the spine detection. We evaluate the proposed approach by comparing with the manual results in terms of backbone length, spine number, spine length, and spine density.

Dendritic architecture of the von Economo neurons.

The von Economo neurons are one of the few known specializations to hominoid cortical microcircuitry. Here, using a Golgi preparation of a human postmortem brain, we describe the dendritic architecture of this unique population of neurons. We have found that, in contrast to layer 5 pyramidal neurons, the von Economo neurons have sparse dendritic trees and symmetric apical and basal components. This result provides the first detailed anatomical description of a neuron type unique to great apes and humans.

New methods for the computer-assisted 3-D reconstruction of neurons from confocal image stacks.

Exact geometrical reconstructions of neuronal architecture are indispensable for the investigation of neuronal function. Neuronal shape is important for the wiring of networks, and dendritic architecture strongly affects neuronal integration and firing properties as demonstrated by modeling approaches. Confocal microscopy allows to scan neurons with submicron resolution. However, it is still a tedious task to reconstruct complex dendritic trees with fine structures just above voxel resolution. We present a framework assisting the reconstruction. User time investment is strongly reduced by automatic methods, which fit a skeleton and a surface to the data, while the user can interact and thus keeps full control to ensure a high quality reconstruction. The reconstruction process composes a successive gain of metric parameters. First, a structural description of the neuron is built, including the topology and the exact dendritic lengths and diameters. We use generalized cylinders with circular cross sections. The user provides a rough initialization by marking the branching points. The axes and radii are fitted to the data by minimizing an energy functional, which is regularized by a smoothness constraint. The investigation of proximity to other structures throughout dendritic trees requires a precise surface reconstruction. In order to achieve accuracy of 0.1 microm and below, we additionally implemented a segmentation algorithm based on geodesic active contours that allow for arbitrary cross sections and uses locally adapted thresholds. In summary, this new reconstruction tool saves time and increases quality as compared to other methods, which have previously been applied to real neurons.

Correlates across the structural, functional, and molecular phenotypes of fragile X syndrome.

Fragile X syndrome (FXS) is characterized by a pattern of morphological, functional, and molecular characteristics with, in at least some cases, apparent relationships among phenotypic features at different levels. Gross morphology differences in the sizes of some human brain regions are accompanied by fine structural alterations in the shapes and in the numbers of dendritic spines in both humans and the knockout mouse model. The excess number of spines, their immature appearance, and the impaired withdrawal of inappropriately oriented dendrites in FXS or the mouse model suggest impairment of neuronal maturation, including dendritic and spine pruning. It is not clear how these differences arise, although regionally or globally impaired translation of the mRNAs that interact with the Fmr1 protein product, FMRP, in the vicinity of the synapse, including genes involved in synapse development and plasticity and dendritic retraction, is certainly plausible. FMRP binds mRNA and may be involved in both transport and translation of the mRNAs it binds. The mRNAs it binds belong to multiple functional classes, apparently indicating that FMRP may impact multiple cellular processes. In one example, the glucocorticoid receptor, whose mRNA binds FMRP, regulates the stress-sensitive glucocorticosteroids. Both human FXS and the mouse model exhibit a protracted elevation in glucocorticosteroids after stress. Possible relationships of other genes to morphological and functional characteristics of FXS are also discussed.

Environmental enrichment: effects on stereotyped behavior and dendritic morphology.

We evaluated whether environmental enrichment-related effects on the development of stereotyped behavior in deer mice were associated with alterations in dendritic morphology. Deer mice were reared under enriched or standard housing conditions and then tested in automated photocell detectors and classified as stereotypic or nonstereotypic. Dendritic morphology was assessed in layer V pyramidal neurons of the motor cortex, medium spiny neurons of the dorsolateral striatum, and granule cells of the dentate gyrus using Golgi-Cox histochemistry. Enriched nonstereotypic mice exhibited significantly higher dendritic spine densities in the motor cortex and the striatum than enriched stereotypic or standard-cage mice. Significant increases in dendritic arborization following environmental enrichment also were observed. These results suggest that the enrichment-related prevention of stereotyped behavior is associated with increased dendritic spine density.

Dendritic spine pathology: cause or consequence of neurological disorders?

Altered dendritic spines are characteristic of traumatized or diseased brain. Two general categories of spine pathology can be distinguished: pathologies of distribution and pathologies of ultrastructure. Pathologies of spine distribution affect many spines along the dendrites of a neuron and include altered spine numbers, distorted spine shapes, and abnormal loci of spine origin on the neuron. Pathologies of spine ultrastructure involve distortion of subcellular organelles within dendritic spines. Spine distributions are altered on mature neurons following traumatic lesions, and in progressive neurodegeneration involving substantial neuronal loss such as in Alzheimer's disease and in Creutzfeldt-Jakob disease. Similarly, spine distributions are altered in the developing brain following malnutrition, alcohol or toxin exposure, infection, and in a large number of genetic disorders that result in mental retardation, such as Down's and fragile-X syndromes. An important question is whether altered dendritic spines are the intrinsic cause of the accompanying neurological disturbances. The data suggest that many categories of spine pathology may result not from intrinsic pathologies of the spiny neurons, but from a compensatory response of these neurons to the loss of excitatory input to dendritic spines. More detailed studies are needed to determine the cause of spine pathology in most disorders and relationship between spine pathology and cognitive deficits.

[Histomorphometric assessment of spiral ganglion neurite outgrowth in vitro]. / Histomorphometrische Bestimmung des Wachstumsverhaltens von Spiralganglienzellneuriten in vitro.

BACKGROUND: During the last years, various groups of growth factors have been identified to influence spiral ganglion cell survival and neurite extension in the mammalian cochlea. To evaluate and compare the effects of different growth factors, a precise histomorphometrical analysis of neurite outgrowth patterns has to be applied. The here presented technique is compared to already published methods, that only approximately estimate the neurite length. METHOD: A software has been developed to analyse digitalised scans of spiral ganglion cells and to measure the length of the neurites. Therefore, the neurites are being separated in any given number of straight lines. The totals of these lines can then be added like a polygon. This polygon method was compared to a semi-quantitative procedure in which the neurite length was determined by concentric circles that were crossed by the neurites in a certain distance. The accuracy of both methods was analysed. Both methods were performed in 20 specimen of neonatal rat spiral ganglion cells after in vitro stimulation with neurotrophic factors. RESULTS: The semi-quantitative method has shown to involve a systematic error between +/- 10 to 15 %. The polygon method, on the contrary, has a systematic error of around +/- 1 %, which admits much more accurate measurement of spiral ganglion neurite outgrowth. CONCLUSION: With the described polygon method, spiral ganglion neurite growth patterns in cell culture studies can be characterised more precisely and, thus, helps to better differentiate the action domain of neurotrophic factors.

Functional recovery and dendritic hypertrophy after posterior and complete cingulate lesions on postnatal day 10.

Rats received lesions of the posterior cingulate cortex or both the anterior and posterior cingulate cortex (total cingulate), or sham procedures, on postnatal Day 10. As adults, animals were trained in the Morris water task. Both the cingulate lesion groups showed substantial functional recovery relative to our previous studies of adult operates or animals with perinatal cingulate lesions. A Golgi analyis of layer III pyramidal cells in parietal cortex showed an increase in dendritic length in the lesion animals relative to sham controls, which is similar to previous findings for rats with anterior cingulate but not motor or parietal lesions. In addition, there was a partial regeneration of the anterior tissue in the total cingulates, which in some cases extended into the posterior region. This is consistent with earlier findings that anterior cingulate lesions around Day 10 stimulate neurogenesis. It appears that there is something special about the reparative processes and subsequent functional recovery that follow midline neocortical lesions.