Analysis of organelles within the nervous system: impact on brain and organelle functions

Currently, neuroproteomic approaches aimed at the profiling of total brain areas generally mirror the expression of the most abundant proteins, but fail to uncover less abundant proteins. By contrast, the focus on typical brain subproteomes, (e.g., synaptic vesicles, synaptic terminal membranes or the postsynaptic density), may give a more specific insight into brain function. Subproteomes are accessible via several strategies, including subcellular fractionation or affinity-based pull-down approaches. Combined with mass spectrometric quantification approaches, subcellular proteomics is expected to reveal differences in the protein constitution of related cellular organelles. Focusing on novel functions and mechanistic models, we review recent data on the analysis of brain-derived organelles and subproteomes, including presynaptic termini, synaptic vesicles, neuronal plasma membranes, postsynaptic density and neuromelanin granules, which were identified as novel lysosome-related organelles within the human brain.     

Analysis of organelles within the nervous system: impact on brain and organelle functions

Currently, neuroproteomic approaches aimed at the profiling of total brain areas generally mirror the expression of the most abundant proteins, but fail to uncover less abundant proteins. By contrast, the focus on typical brain subproteomes, (e.g., synaptic vesicles, synaptic terminal membranes or the postsynaptic density), may give a more specific insight into brain function. Subproteomes are accessible via several strategies, including subcellular fractionation or affinity-based pull-down approaches. Combined with mass spectrometric quantification approaches, subcellular proteomics is expected to reveal differences in the protein constitution of related cellular organelles. Focusing on novel functions and mechanistic models, we review recent data on the analysis of brain-derived organelles and subproteomes, including presynaptic termini, synaptic vesicles, neuronal plasma membranes, postsynaptic density and neuromelanin granules, which were identified as novel lysosome-related organelles within the human brain.     

Changes in the spectrum of LDH isoenzymes in foci of epileptic activity induced by penicillin in the cerebral cortex of rats

The correlation between electrophysiological changes and isozymes of LDH of the rat brain cortex was studied in seizure foci induced by application of sodium penicillin. It was discovered that activity of LDH1 was suppressed, and that of LDH5 fraction was elevated in the determinant focus, which indicates the enhanced glucose anaerobic transformation. The spectrum of LDH isozymes did not practically differ from the indicators in control animals in a homotopic region of the contralateral hemisphere prior to creation of the mirror focus. The anaerobic processes were found to be increased in the mirror focus and in the determinant one as well. Similar pattern of changes in electrophysiological and neurochemical characteristics in the determinant and dependent mirror foci attests to the formation of a pathological system out of the two epileptic foci.     

What is the function of neuronal AP‐3?

Neurotransmission requires the proper organization and rapid recycling of synaptic vesicles. Rapid retrieval has been suggested to occur either by kiss-and-stay or kiss-and-run mechanisms, whereas classical recycling is mediated by clathrin-dependent endocytosis. Molecular coats are key components in the selection of cargos, AP-2 (adaptor protein 2) playing a prominent role in synaptic vesicle endocytosis. Another coat protein, AP-3, has been implicated in synaptic vesicle biogenesis and in the generation of secretory and lysosomal-related organelles. In the present review, we will particularly focus on the recent data concerning the recycling of synaptic vesicles and the function of AP-3 and the v-SNARE (vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein) in these processes. We propose that AP-3 plays an important regulatory role in neurons which contributes to the basal and stimulated exocytosis of synaptic vesicles.     

Transmission,Development, and Plasticity of Synapses

Chemical synapses are sites of contact and information transfer between a neuron and its partner cell. Each synapse is a specialized junction, where the presynaptic cell assembles machinery for the release of neurotransmitter, and the postsynaptic cell assembles components to receive and integrate this signal. Synapses also exhibit plasticity, during which synaptic function and/or structure are modified in response to activity. With a robust panel of genetic, imaging, and electrophysiology approaches, and strong evolutionary conservation of molecular components, Drosophila has emerged as an essential model system for investigating the mechanisms underlying synaptic assembly, function, and plasticity. We will discuss techniques for studying synapses in Drosophila, with a focus on the larval neuromuscular junction (NMJ), a well-established model glutamatergic synapse. Vesicle fusion, which underlies synaptic release of neurotransmitters, has been well characterized at this synapse. In addition, studies of synaptic assembly and organization of active zones and postsynaptic densities have revealed pathways that coordinate those events across the synaptic cleft. We will also review modes of synaptic growth and plasticity at the fly NMJ, and discuss how pre- and postsynaptic cells communicate to regulate plasticity in response to activity.     

Changes in the activity of gamma-amino butyric acid transaminase and succinic semialdehyde dehydrogenase in the cobalt and iron experimental epileptogenic foci in the rat brain

GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSA-DH) activities were measured in the mitochondrial fractions from the cobalt- and FeCl3-induced chronic epileptogenic foci in the rat brain. Electroencephalographically, the FeCl3 epileptogenic focus remained active for a duration longer than that of the cobalt focus. In both the foci SSA-DH activity showed significant increases which were concomitant with the EEG epileptiform activity. In cobalt focus, the GABA-T activity fell whereas, in the FeCl3 focus it was unchanged. In cobalt focus fall in GABA-T activity seemed to be concomitant with EEG epileptiform discharge. The measurements of the enzyme activities in the mirror (secondary) foci showed that, except for a brief stimulation of SSA-DH activity in the mirror focus in FeCl3 epileptic animals, the enzyme activities remained unchanged. Possible significance of the observed enzymatic changes in the physiology of epileptogenic focus is discussed.     

Effect of damage to the zone of the medial forebrain bundle and preoptic area on the activiy of a penicillin-induced epileptiform focus (phenomenon of the hyperactive determinant dispatch station)]

It was shown in experiments on cats under nembutal anesthesia that a lesion of the medial forebrain bundle (MFB) and partly of the preoptic region at the side of local penicillin application on the cerebral cortex (g. suprasylvius medius) results in depression of the epileptiform activity in the penicillin-induced focus, as well as in the secondary "mirror" focus, which appeared in the symmetrical cortex area of the other hemisphere. The MFB lesion at the "mirror" focus side led to depression of the seizure spike potentials in this focus only and did not change the activity in the primary epileptiform one. The described effects are considered from the aspect of the conception on the role of the determinant dispatch station (DDS) in the central nervous system: the primary epileptiform focus plays the role of the hyperactive DDS which induces the secondary focus and determines the character of its activity. The results of the study substantiate a suggestion that the MFB can take part in the modulation of the cortical epileptiform activity.     

Calcium and Protein Kinase C Regulate the Actin Cytoskeleton in the Synaptic Terminal of Retinal Bipolar Cells

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 ± 0.1 in the dark to 2.1 ± 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark.Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K⁺, the actin network in the synaptic pedicle extended up to 2.5 μm from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca²⁺ influx through L-type Ca²⁺ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca²⁺ influx and accelerated F-actin breakdown on removal of Ca²⁺.To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca²⁺ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.     

Tyrosinated alpha-tubulin changes following epileptogenic and non-epileptogenic lesions in rat brain cortex

The expression of the tyrosinated isoform of alpha-tubulin was monitored in rat frontal cortex, in order to investigate the neuronal plasticity changes occurring either in a mirror focus or in a deafferented area. A mirror focus was triggered by epidural implantation of a cobalt gelatin disk in the contralateral left somatosensory area (group one). A deafferented area was obtained by surgical removal of the left frontal cortex (group two). All animals including controls underwent EcoG recordings immediately before killing (45, 60, 90 days post surgery). The right frontal cortex was removed from all the animals and processed with Western blot method. EcoG recordings revealed a paroxysmal activity in epileptic rats, whereas in rats with frontal deafferentation and controls, EcoG activity was normal. A significant increase in tyrosinated alpha-tubulin expression was detected both in the mirror focus (group one) and the "non-epileptic" deafferented frontal cortex (group two) in comparison with controls (group three). The transcallosal deafferentation, which is involved in both epileptogenic and non-epileptogenic lesions, is supposed to play a role in the mechanism responsible for the plasticity responses recorded in the cortical areas studied.     

Amygdala: Neuroanatomy and neurophysiology of fear

This review is devoted to neuroanatomical and neurophysiological mechanisms of Pavlovian fear conditioning with a focus on the amydgalae, two subcortical nuclear groups, as primary structures responsible for controlling conditioned fear responses, and synaptic plasticity at their afferent and efferent projections as a cellular mechanism to mediate the formation and retention of fear memory. We survey current data on anatomical organization of the amygdaloid complex, as well as on its afferent and efferent projections and their functional significance. A special consideration is given to auditory inputs to the amygdala to analyze the mechanisms of aversive conditioning to sensory (acoustic) stimuli.     

A presynaptic giant ankyrin stabilizes the NMJ through regulation of presynaptic microtubules and transsynaptic cell adhesion

In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.     

Rho GTPase signaling at the synapse: Implications for intellectual disability

Intellectual disability (ID) imposes a major medical and social–economical problem in our society. It is defined as a global reduction in cognitive and intellectual abilities, associated with impaired social adaptation. The causes of ID are extremely heterogeneous and include non-genetic and genetic changes. Great progress has been made over recent years towards the identification of ID-related genes, resulting in a list of approximately 450 genes. A prominent neuropathological feature of patients with ID is altered dendritic spine morphogenesis. These structural abnormalities, in part, reflect impaired cytoskeleton remodeling and are associated with synaptic dysfunction. The dynamic, actin-rich nature of dendritic spines points to the Rho GTPase family as a central contributor, since they are key regulators of actin dynamics and organization. It is therefore not surprising that mutations in genes encoding regulators and effectors of the Rho GTPases have been associated with ID. This review will focus on the role of Rho GTPase signaling in synaptic structure/function and ID.     

A survey of the cytology and synaptic organization of the insular trigeminal-cuneatus lateralis nucleus in the rat, including an identification of spinal afferent inputs

The cytology and synaptic organization of the insular trigeminal-cuneatus lateralis (iV-Cul) nucleus was examined in the rat. In addition, the ultrastructural morphology and synaptic connectivity of anterogradely labeled spinal afferent axons terminating in iV-Cul were examined following injection of horseradish peroxidase (HRP) into the cervical spinal cord. The uniformity of the ultrastructural features of iV-Cul neurons supports the presence of a homogeneous neuronal population. The most prominent feature of the iV-Cul neuropil is the presence of numerous interdigitating astrocytic processes, which extensively isolate neuronal somata and processes. iV-Cul contains a heterogeneous population of axonal endings that can be separated into three categories, depending upon whether they contain predominantly spherical-shaped agranular synaptic vesicles (R endings), predominantly pleomorphic-shaped agranular synaptic vesicles (P endings), or a heterogeneous population of dense-core vesicles (DC endings). The R endings represent the majority of axonal endings in iV-Cul and establish asymmetrical axodendritic and axospinous synaptic contacts, primarily along the distal portions of the dendritic tree. P endings establish symmetrical axosomatic, axodendritic, and axospinous synaptic contacts and exhibit a more generalized distribution along the somadendritic tree. DC terminals establish asymmetrical axodendritic synaptic contacts with distal dendritic processes and are the least frequently observed endings in the iV-Cul neuropil. Numerous synaptic glomeruli, exhibiting a single large central R bouton that establishes multiple axodendritic or axospinous synapses, characterize the iV-Cul neuropil. Axoaxonic synapses are conspicuously absent from the iV-Cul neuropil and glomeruli. The anterograde HRP labeling of spinal afferent axons that terminate in iV-Cul indicates that the terminals along these fibers are of the R type and that they are engaged predominantly in synaptic glomeruli. The results of this study indicate that the synaptic organization of iV-Cul is distinctly different from that of neighboring somatosensory nuclei, and supports the recent suggestion that this nucleus should be considered a separate precerebellar spinal relay nucleus in the lateral medulla.     

Molecular architecture of postsynaptic Interactomes

The postsynaptic density (PSD) plays an essential role in the organization of the synaptic signaling machinery. It contains a set of core scaffolding proteins that provide the backbone to PSD protein-protein interaction networks (PINs). These core scaffolding proteins can be seen as three principal layers classified by protein family, with DLG proteins being at the top, SHANKs along the bottom, and DLGAPs connecting the two layers. Early studies utilizing yeast two hybrid enabled the identification of direct protein-protein interactions (PPIs) within the multiple layers of scaffolding proteins. More recently, mass-spectrometry has allowed the characterization of whole interactomes within the PSD. This expansion of knowledge has further solidified the centrality of core scaffolding family members within synaptic PINs and provided context for their role in neuronal development and synaptic function. Here, we discuss the scaffolding machinery of the PSD, their essential functions in the organization of synaptic PINs, along with their relationship to neuronal processes found to be impaired in complex brain disorders.     

Location matters: the endoplasmic reticulum and protein trafficking in dendrites

Neurons are highly polarized, but the trafficking mechanisms that operate in these cells and the topological organization of their secretory organelles are still poorly understood. Particularly incipient is our knowledge of the role of the neuronal endoplasmic reticulum. Here we review the current understanding of the endoplasmic reticulum in neurons, its structure, composition, dendritic distribution and dynamics. We also focus on the trafficking of proteins through the dendritic endoplasmic reticulum, emphasizing the relevance of transport, retention, assembly of multi-subunit protein complexes and export. We additionally discuss the roles of the dendritic endoplasmic reticulum in synaptic plasticity.     

Presynaptic CK2 promotes synapse organization and stability by targeting Ankyrin2

The precise regulation of synapse maintenance is critical to the development and function of neuronal circuits. Using an in vivo RNAi screen targeting the Drosophila kinome and phosphatome, we identify 11 kinases and phosphatases controlling synapse stability by regulating cytoskeletal, phospholipid, or metabolic signaling. We focus on casein kinase 2 (CK2) and demonstrate that the regulatory (β) and catalytic (α) subunits of CK2 are essential for synapse maintenance. CK2α kinase activity is required in the presynaptic motoneuron, and its interaction with CK2β, mediated cooperatively by two N-terminal residues of CK2α, is essential for CK2 holoenzyme complex stability and function in vivo. Using genetic and biochemical approaches we identify Ankyrin2 as a key presynaptic target of CK2 to maintain synapse stability. In addition, CK2 activity controls the subcellular organization of individual synaptic release sites within the presynaptic nerve terminal. Our study identifies phosphorylation of structural synaptic components as a compelling mechanism to actively control the development and longevity of synaptic connections.     

Regulation of motor circuit assembly by spatial and temporal mechanisms

Precision of synaptic connections in neuronal circuits is the product of an orderly assembly process during development. Circuits controlling motor behavior have been studied extensively in many animal species, allowing an assessment of evolutionarily conserved organizational principles that underlie neuronal subtype specification, connectivity and function. Across phylogenetically distant species, motor circuit assembly is based on spatial organization of interconnected circuit elements. Developmental molecular coordinate systems demarcate dendritic and axonal targeting territories, thereby regulating convergence of synaptic partners. Additional mechanisms subsequently control fine synaptic connection specificity within these domains. Spatial organization often correlates with the orderly sequence of neurogenesis contributing to the generation of distinct postmitotic neuronal subpopulations, a developmental strategy implemented far beyond motor circuits.     

Network Evolution Induced by Asynchronous Stimuli through Spike-Timing-Dependent Plasticity

In sensory neural system, external asynchronous stimuli play an important role in perceptual learning, associative memory and map development. However, the organization of structure and dynamics of neural networks induced by external asynchronous stimuli are not well understood. Spike-timing-dependent plasticity (STDP) is a typical synaptic plasticity that has been extensively found in the sensory systems and that has received much theoretical attention. This synaptic plasticity is highly sensitive to correlations between pre- and postsynaptic firings. Thus, STDP is expected to play an important role in response to external asynchronous stimuli, which can induce segregative pre- and postsynaptic firings. In this paper, we study the impact of external asynchronous stimuli on the organization of structure and dynamics of neural networks through STDP. We construct a two-dimensional spatial neural network model with local connectivity and sparseness, and use external currents to stimulate alternately on different spatial layers. The adopted external currents imposed alternately on spatial layers can be here regarded as external asynchronous stimuli. Through extensive numerical simulations, we focus on the effects of stimulus number and inter-stimulus timing on synaptic connecting weights and the property of propagation dynamics in the resulting network structure. Interestingly, the resulting feedforward structure induced by stimulus-dependent asynchronous firings and its propagation dynamics reflect both the underlying property of STDP. The results imply a possible important role of STDP in generating feedforward structure and collective propagation activity required for experience-dependent map plasticity in developing in vivo sensory pathways and cortices. The relevance of the results to cue-triggered recall of learned temporal sequences, an important cognitive function, is briefly discussed as well. Furthermore, this finding suggests a potential application for examining STDP by measuring neural population activity in a cultured neural network.