The maintenance of a healthy mitochondrial network and the ability to adjust organelle population in response to internal or external stimuli are essential for the function and the survival of eukaryotic cells. Over the last two decades several studies have demonstrated the paramount importance of mitophagy, a selective form of autophagy that removes damaged and/or superfluous organelles, in organismal physiology. Post-mitotic neuronal cells are particularly vulnerable to mitochondrial damage, and mitophagy impairment has emerged as a causative factor in multiple neurodegenerative pathologies, including Alzheimer's disease and Parkinson's disease among others. Although mitochondrial turnover is a multifaceted process, neurons have to tackle additional complications, arising from their pronounced bioenergetic demands and their unique architecture and cellular polarisation that render the degradation of distal organelles challenging. Mounting evidence indicates that despite the functional conservation of mitophagy pathways, the unique features of neuronal physiology have led to the adaptation of compartmentalised solutions, which serve to ensure seamless mitochondrial removal in every part of the cell. In this review, we summarise the current knowledge concerning the molecular mechanisms that mediate mitophagy compartmentalisation and discuss their implications in various human pathologies.

High-throughput immuno-electron microscopy is required to capture the protein–protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell’s plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.

Their unique patterns of size, numbers, and stratification indicate that amacrine cells have diverse functions. These are mostly unknown, as studies using imaging and electrophysiological methods have only recently begun. However, some of the events that occur within the amacrine cell population—and some important unresolved puzzles—can be stated purely from structural reasoning.

Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and ‘spotty’ in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.

Gap junction (GJ) endocytosis appears to be part of a cycle of GJ renewal in horizontal cells of the teleost fish retina. At least three stages of GJ endocytosis in these neurons have been identified using conventional electron microscopy (EM): invagination of GJ membranes (GJ blebs); free GJ vesicles; and GJ vesicle fusion with mature lysosomes (Vaughan & Lasater, 1990a). In the present study, EM-level acid phosphatase (AP) histochemistry of white bass retina was used to determine at what stage enzymatic degradation of endocytosed GJs begins. Electron-dense AP reaction product was observed within the trans face of the Golgi apparatus, mature lysosomes, and occasional, internal GJ vesicles. In contrast, GJ blebs, peripheral GJ vesicles, and most internal GJ vesicles lacked AP reaction product. These results support the idea that at least some of the GJ vesicles observed within these retinal neurons arise from endocytosis, are on a degradative pathway, and can be termed GJ “endosomes.” Furthermore, GJ vesicles appear to be initially free of AP, but some later acquire it (presumably from transport vesicles bearing degradative enzymes). It is still unclear whether our previous report of GJ vesicle fusion with mature lysosomes is a subsequent step in GJ degradation or part of a different degradative pathway altogether.

Oligodendrocyte precursor cells (OPCs) have become the focus of intense research, not only because they generate myelin-forming oligodendrocytes in the normal CNS, but because they may be suitable for transplantation to treat disorders in which myelin does not form or is damaged, and because they have stem-cell-like properties in that they can generate astrocytes and neurons as well as oligodendrocytes. In this article we review the electrical signalling properties of OPCs, including the synaptic inputs they receive and their use of voltage-gated channels to generate action potentials, and we describe experiments attempting to detect output signalling from OPCs. We discuss controversy over the existence of different classes of OPC with different electrical signalling properties, and speculate on the lineage relationship and myelination potential of these different classes of OPC. Finally, we point out that, since OPCs are the main proliferating cell type in the mature brain, the discovery that they can develop into neurons raises the question of whether more neurons are generated in the mature brain from the classical sites of neurogenesis in the subventricular zone of the lateral ventricle and the hippocampal dentate gyrus or from the far more widely distributed OPCs.

NG2-glia are a distinct class of CNS glial cells that are generally classed as oligodendrocyte progenitor cells. However, in the adult CNS a large fraction of NG2 cells does not appear to divide or generate oligodendrocytes. The functions of these adult NG2-glia, which we have termed synantocytes, are unknown. NG2-glia (synantocytes) form interactive domains with astrocytes and neurons. Within their domains, NG2-glia and astrocytes contact the same neurons, form multiple heterologous contacts with each other, and contact pericytes which regulate cerebral blood flow. NG2-glia receive presynaptic input from neurons and respond to neurotransmitters released at synapses. In addition, NG2-glia are intimately associated with astroglia and respond to astroglial signals, a hitherto neglected aspect of NG2-glial cell physiology. The non-overlapping domain organisation of astrocytes is believed to be important in isolating and integrating activity at the synapses and blood vessels within their domains. The domains of NG2-glia overlap with astrocytes, suggesting they could play a role in integrating non-overlapping astrocyte domains.

Second-harmonic generation (SHG) can be used for imaging membrane potential in neurons, but poor signal-to-noise (S/N) limits accurate measurements of small voltage transients. We use photon counting to improve the S/N of weak SHG signal detection. Photon counting generates shot-noise limited and integrable signals, eliminates pulse-to-pulse variation, and built-in discriminators reduces the background to practically zero. In single trials, by using photon counting, we obtain a more than a twofold S/N increase over analog voltage detection. Trial-to-trial variability is also reduced by 50%. Finally, we show that, using photon counting, the kinetics of fast events such as action potentials can be recorded more accurately.

Interactions between neurons and glial cells are crucial for nervous system development and function in all complex organisms, and many functional, morphological and molecular features of glia are well conserved among species. Here we review studies of the longitudinal glia (LG) in the Drosophila CNS. The LG envelop the neuropil in a membrane sheath, and have features resembling both oligodendrocytes and astrocytes. Because of their unique lineage, morphology and molecular features, the LG provide an excellent model to study the genetic mechanisms underlying glial subtype differentiation and diversity, glial morphogenesis and neuron–glial interactions during development. In addition, they are proving useful in understanding how glial cells maintain ion and neurotransmitter homeostasis and protect neurons from environmental insult.

Stem cells and progenitor cells derived from the developing human brain have been shown to differentiate into neurons and astrocytes. However, few studies have examined the functional, physiological properties of these differentiated neurons and astrocytes. In this study we have used immunocytochemistry in combination with electrophysiology to examine protein machinery and functional properties of neurons and astrocytes differentiated from human brain progenitor cells (hBPCs). Our results show that serum induces mainly astrocytic phenotype cells that express GFAP and have physiological properties that are typical of astrocytes. hBPCs differentiated with BDNF and PDGF develop mainly into neurons expressing mature neuronal proteins MAP-2, synaptobrevin II and vesicular glutamate transporter I in the process, plus a small population of GFAP-positive radial cells. Based on electrophysiology of BDNF/PDGF-treated cells two classes of cell were identified. Class I cells have functional neuronal properties, including functional voltage-gated Na+ and K+ currents, functional AMPA receptors and the ability to generate action potentials. A smaller subpopulation of cells (Class II cells) expresses GFAP and exhibit functional properties of astrocytes, including linear current–voltage relationship and dye-coupling.

A number of plasma membrane receptor types originally thought to be specific to neurons have been found in other somatic cells. More surprisingly, the mammalian sperm and neuron appear to share many of these ‘neuronal’ receptors. The morphology, chromosome number, genomic activity, and functions of those two cell types are as unlike as any two cells in the body, but they both achieve their highly disparate goals with the aid of a number of the same receptors. Exocytosis in neurons and sperm is essential to the functions of these cells and is strongly influenced by similar receptors. ‘Neuronal’ receptor types in sperm may also play a role in the control of sperm motility (a function of course not shared by neurons).

This review will consider the evidence for the presence of sperm plasma membrane ‘neuronal’ receptors and for their significance to mammalian sperm function. The persuasiveness of the evidence varies depending on the receptor being considered, but there is strong experimental support for the presence and importance of a number of ‘neuronal’ receptors in sperm.

Brain injury following cerebral ischaemia (stroke) involves a complex combination of pathological processes, including excitotoxicity and inflammation leading to necrotic and apoptotic forms of cell death. At the cellular level, excitotoxicity is mediated by glutamate and its cognate receptors, resulting in increased intracellular calcium and free radical production, and eventual cell death. Recent evidence suggests that scaffolding molecules that associate with glutamate receptors at the postsynaptic density allow coupling of receptor activity to specific second messengers capable of mediating excitotoxicity. These findings have important implications in the search for effective neuroprotective therapies in treating stroke.

We identified a novel gene, Ks-1, which is expressed preferentially in the small-type Kenyon cells of the honeybee brain. This gene is also expressed in some of the large soma neurons in the brain and in the suboesophageal ganglion. Reverse transcription–polymerase chain reaction experiments indicated that Ks-1 transcripts are enriched in the honeybee brain. cDNA cloning revealed that the consensus Ks-1 cDNA is over 17 kbp and contains no significant open reading frames. Furthermore, fluorescent in situ hybridization revealed that Ks-1 transcripts are located in the nuclei of the neural cells, accumulating in some scattered spots. These findings demonstrate that Ks-1 encodes a novel class of noncoding nuclear RNA and is possibly involved in the regulation of neural functions.

The mouse c-src gene contains a short neuron-specific exon, N1. N1 exon splicing is partly controlled by an intronic splicing enhancer sequence that activates splicing of a heterologous reporter exon in both neural and nonneural cells. Here we attempt to dissect all of the regulatory elements controlling the N1 exon and examine how these multiple elements work in combination. We show that the 3′ splice site sequence upstream of exon N1 represses the activation of splicing by the downstream intronic enhancer. This repression is stronger in nonneural cells and these two regulatory sequences combine to make a reporter exon highly cell-type specific. Substitution of the 3′ splice site of this test exon with sites from other exons indicates that activation by the enhancer is very dependent on the nature of the upstream 3′ splice site. In addition, we identify a previously uncharacterized purine-rich sequence within exon N1 that cooperates with the downstream intronic enhancer to increase exon inclusion. Finally, different regulatory elements were tested in multiple cell lines of both neuronal and nonneuronal origin. The individual splicing regulatory sequences from the src gene vary widely in their activity between different cell lines. These results demonstrate how a simple cassette exon is controlled by a variety of regulatory elements that only in combination will produce the correct tissue specificity of splicing.

A simple model of a neuron is proposed, not intended to be biologically faithful, but to incorporate dynamic and stochastic features which seem to be realistic for both the natural and the artificial case. It is shown that the use of feedback for assemblies of such neurons can produce bistable behaviour and sharpen the discrimination of the assembly to the level of input. Particular attention is paid to bistable devices which are to serve as bit-stores (and so constitute components of a memory) and which suffer a disturbing input due to mutual interference. Approximate expressions are obtained for the equilibrium distribution of the excitation level for such assemblies and for the expected escape time of such an assembly from a metastable excitation level.

Using an integral equation of Darling and Siegert in conjunction with the backward Kolmogorov equation for the transition probability density function, recurrence relations are derived for the moments of the time of first exit of a temporally homogeneous Markov process from a set in the phase space. The results, which are similar to those for diffusion processes, are used to find the expectation of the time between impulses of a Stein model neuron.