Co-release of glutamate and aspartate from cholinergic and GABAergic synaptosomes

Glutamate and aspartate are known to be released in a calcium-dependent fashion by depolarizing stimulation of mammalian brain synaptosomes (isolated nerve endings), an observation which strengthens their claims to be neurotransmitter candidates. The source of these compounds has been interpreted as the exclusively glutamatergic or aspartatergic synaptosome sub-populations assumed to be present in the standard heterogeneous preparations from mammalian brain. Several neurotransmitter-specific synaptosomal surface markers have recently been identified by immunolysis studies and these have allowed separation of subpopulations of synaptosomes by an affinity purification method. These markers appear to be closely related to the biosynthetic enzyme for the principal neurotransmitter released by each sub-category of synaptosome. We have isolated highly purified, metabolically active, GABAergic and cholinergic synaptosomes from cerebral cortex using antisera recognizing either glutamate decarboxylase (GAD) or choline acetyltransferase (ChAT), in conjunction with magnetic microspheres covalently coupled to Protein A (ref. 8), and now report that these synaptosomes release both glutamate and aspartate, in addition to their principal neurotransmitter, when treated with chemical depolarizing agents.     

Synaptosomes possess an exocytotic pool of glutamate

There is increasing evidence that L-glutamate is a major excitatory neurotransmitter in the central nervous system. Immunocytochemical studies indicate that glutamate within nerve terminals may be concentrated in vesicles and glutamate-accumulating vesicles have recently been isolated. Exocytotic release of glutamate from synaptosomes (isolated nerve terminals) has not been convincingly demonstrated, however, and remains highly controversial. In order to study the kinetics of release of endogenous L-glutamate from guinea pig cerebral cortical synaptosomes we have devised a continuous enzymatic assay. This has enabled us to identify a pool, equivalent to 15-20% of the total synaptosomal glutamate, which is capable of rapid Ca2+-dependent exocytotic release.     

A small GTP-binding protein dissociates from synaptic vesicles during exocytosis.

Low-molecular-weight GTP-binding proteins are strong candidates for regulators of membrane traffic. In yeast, mutations in the sec4 or ypt1 genes encoding small GTP-binding proteins inhibit constitutive membrane flow at the plasma membrane or Golgi complex, respectively. It has been suggested that membrane fusion-fission events are regulated by cycling of small GTP-binding proteins between a membrane-bound and free state, but although most of these small proteins are found in both soluble and tightly membrane-bound forms, there is no direct evidence to support such cycling. In rat brain a small GTP-binding protein, rab3A, is exclusively associated with synaptic vesicles, the secretory organelles of nerve terminals. Here we use isolated nerve terminals to study the fate of rab3A during synaptic vesicle exocytosis. We find that rab3A dissociates quantitatively from the vesicle membrane after Ca2(+)-dependent exocytosis and that this dissociation is partially reversible during recovery after stimulation. These results are direct evidence for an association-dissociation cycle of a small GTP-binding protein during traffic of its host membrane.     

Correlation between the anaesthetic effect of halothane and saturable binding in brain

Two theories of the molecular mechanism of volatile anaesthetic action suggest either that anaesthetics cause a generalized perturbation of neuronal membrane structure, probably through a nonspecific interaction with membrane lipids, or that anaesthetics bind to sets of sites of appropriate molecular dimension on membrane proteins. Based on the recent finding that fluorinated anaesthetics can be observed in animal tissue by 19F nuclear magnetic resonance (19F-NMR) spectroscopy, we have used 19F-NMR to quantify the interaction between the volatile anaesthetic halothane and rat brain tissue. Steady-state brain halothane concentration was found to be a non-linear function of inspired concentration, with apparent saturation of brain occurring at inspired halothane concentrations above 2.5% by volume. Using a spin-echo pulse sequence it was found that halothane exists in two distinct chemical environments in brain, characterized by different spin-spin relaxation times (T2), chemical shifts and kinetics of occupancy. Halothane concentration in one of these environments (T2 = 3.6 ms) was saturated at approximately 2.5% inspired halothane; occupancy of this environment was found to correlate with the anaesthetic effect of the drug. In the other environment (T2 = 43 ms), brain halothane concentration was a linear function of inspired concentration. These data suggest the existence of a saturable anaesthetic site for halothane in brain and do not support the concept that anaesthetics act by nonspecific membrane perturbation.     

Calcium/calmodulin-dependent protein kinase II increases glutamate and noradrenaline release from synaptosomes

A variety of evidence indicates that calcium-dependent protein phosphorylation modulates the release of neurotransmitter from nerve terminals. For instance, the injection of rat calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-dependent PK II) into the preterminal digit of the squid giant synapse leads to an increase in the release of a so-far unidentified neurotransmitter induced by presynaptic depolarization. But until now, it has not been demonstrated that Ca2+/CaM-dependent PK II can also regulate neurotransmitter release in the vertebrate nervous system. Here we report that the introduction of Ca2+/CaM-dependent PK II, autoactivated by thiophosphorylation, into rat brain synaptosomes (isolated nerve terminals) increases the initial rate of induced release of two neurotransmitters, glutamate and noradrenaline. We also show that introduction of a selective peptidergic inhibitor of Ca2+/CaM-dependent PK II inhibits the initial rate of induced glutamate release. These results support the hypothesis that activation of Ca2+/CaM-dependent PK II in the nerve terminal removes a constraint on neurotransmitter release.     

Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells

Lymph nodes prevent the systemic dissemination of pathogens such as viruses that infect peripheral tissues after penetrating the body's surface barriers. They are also the staging ground of adaptive immune responses to pathogen-derived antigens. It is unclear how virus particles are cleared from afferent lymph and presented to cognate B cells to induce antibody responses. Here we identify a population of CD11b+CD169+MHCII+ macrophages on the floor of the subcapsular sinus (SCS) and in the medulla of lymph nodes that capture viral particles within minutes after subcutaneous injection. Macrophages in the SCS translocated surface-bound viral particles across the SCS floor and presented them to migrating B cells in the underlying follicles. Selective depletion of these macrophages compromised local viral retention, exacerbated viraemia of the host, and impaired local B-cell activation. These findings indicate that CD169+ macrophages have a dual physiological function. They act as innate 'flypaper' by preventing the systemic spread of lymph-borne pathogens and as critical gatekeepers at the lymph-tissue interface that facilitate the recognition of particulate antigens by B cells and initiate humoral immune responses.     

Release of somatostatin-28(1-12) from rat hypothalamus in vitro

Following the discovery of the growth hormone release-inhibiting factor somatostatin from extracts of ovine hypothalamus, an N-terminally extended somatostatin of 28 amino acids has been identified in mammalian tissue. The original peptide, somatostatin-14 (SS14), corresponds to the C-terminus of somatostatin-28 (SS28). Both SS28 and SS14 have biological activity, occur in several rat brain regions, are present in cell bodies and nerve terminals and can be released in vitro upon depolarization in a calcium-dependent manner. Further, high-affinity binding sites were described for SS14, which also bind SS28 (refs 20-23). Recently, a dodecapeptide which corresponds to the N-terminus of somatostatin-28, somatostatin-28(1-12), has been characterized in rat hypothalamus. Radioimmunological and immunohistochemical studies have indicated the presence of SS28(1-12)-like immunoreactivity in several cortical and subcortical regions of the rat brain. This peptide was found to be unevenly distributed with the highest concentration in the hypothalamus, and preferentially localized to dendritic and axonal processes and terminals. These observations suggest that SS28(1-12) may be a neurotransmitter. In this study, we describe a calcium-dependent release of a SS28(1-12)-like peptide from hypothalamic slices in vitro. This finding supports a neurotransmitter function for this peptide.     

Dynamic molecular processes mediate cellular mechanotransduction

Cellular responses to mechanical forces are crucial in embryonic development and adult physiology, and are involved in numerous diseases, including atherosclerosis, hypertension, osteoporosis, muscular dystrophy, myopathies and cancer. These responses are mediated by load-bearing subcellular structures, such as the plasma membrane, cell-adhesion complexes and the cytoskeleton. Recent work has demonstrated that these structures are dynamic, undergoing assembly, disassembly and movement, even when ostensibly stable. An emerging insight is that transduction of forces into biochemical signals occurs within the context of these processes. This framework helps to explain how forces of varying strengths or dynamic characteristics regulate distinct signalling pathways.     

Calcitic microlenses as part of the photoreceptor system in brittlestars

Photosensitivity in most echinoderms has been attributed to 'diffuse' dermal receptors. Here we report that certain single calcite crystals used by brittlestars for skeletal construction are also a component of specialized photosensory organs, conceivably with the function of a compound eye. The analysis of arm ossicles in Ophiocoma showed that in light-sensitive species, the periphery of the labyrinthic calcitic skeleton extends into a regular array of spherical microstructures that have a characteristic double-lens design. These structures are absent in light-indifferent species. Photolithographic experiments in which a photoresist film was illuminated through the lens array showed selective exposure of the photoresist under the lens centres. These results provide experimental evidence that the microlenses are optical elements that guide and focus the light inside the tissue. The estimated focal distance (4-7 micrometer below the lenses) coincides with the location of nerve bundles-the presumed primary photoreceptors. The lens array is designed to minimize spherical aberration and birefringence and to detect light from a particular direction. The optical performance is further optimized by phototropic chromatophores that regulate the dose of illumination reaching the receptors. These structures represent an example of a multifunctional biomaterial that fulfills both mechanical and optical functions.     

Immunocytochemical localization in rat brain of a prolactin release-inhibiting sequence of gonadotropin-releasing hormone prohormone

The structure of a precursor protein for gonadotropin-releasing hormone (GnRH) of relative molecular mass 10,000 has recently been deduced from cloned complementary DNA sequences derived from human placental messenger RNA. The 56-amino-acid peptide representing residues 14-69 of this prohormone exhibits potent inhibition of prolactin secretion. To investigate whether the same prohormone is synthesized in mammalian brain and describe the anatomical distribution of the prolactin-inhibiting region of this molecule, we have generated antiserum to a synthetic peptide containing residues 40-53 of the human placental precursor. We report here that a substance recognized by this antibody is present in GnRH-containing neurones of the rat brain and appears to coexist with GnRH in secretory granules of nerve terminals in the median eminence. These results indicate homology between hypothalamic and placental prohormones for GnRH and are consistent with the suggestion elsewhere in this issue that a prolactin-inhibiting factor (PIF) is generated from this prohormone and cosecreted with GnRH by nerve terminals in the median eminence.     

Neuronal-type Na+ and K+ channels in rabbit cultured Schwann cells

Nerve axons in the central and peripheral nervous system are normally surrounded by satellite cells. These cells, known as Schwann cells in the peripheral nervous system, interact with axons to form a myelin sheath, so allowing nerve impulses to proceed at high speed. Schwann cells are thought to differ from neurones in their membrane properties in one important aspect: they lack excitability. Using the patch-clamp technique we have now measured directly the ionic currents across the membrane of single Schwann cells cultured from newborn rabbits. Surprisingly, we found that these Schwann cells possess voltage-gated sodium and potassium channels that are similar to those present in neuronal membranes.     

Evidence for two populations of excitatory receptors for noradrenaline on arteriolar smooth muscle

We have recorded the responses of arteriolar smooth muscle cells to iontophoretically applied noradrenaline. Records of both muscle movement and muscle membrane potential were made. We found that two distinct types of response could be detected, depending on the position of the noradrenaline micropipette. One type of response consisted of a localised constriction near the noradrenaline source: this effect could be abolished by the alpha-antagonist phentolamine and was not associated with a change in arteriolar membrane potential. The other type of response was a depolarisation similar to the excitatory junction potentials (e.j.ps) produced by sumpathetic nerve stimulation. These observations suggest that there are two populations of receptors for noradrenaline on arterioles, and could explain the paradoxical failure of alpha-antagonists to block neuromuscular transmission at some sutonomic end organs such as the vas deferens, arteries and arterioles.     

Restoration of grasp following paralysis through brain-controlled stimulation of muscles

Patients with spinal cord injury lack the connections between brain and spinal cord circuits that are essential for voluntary movement. Clinical systems that achieve muscle contraction through functional electrical stimulation (FES) have proven to be effective in allowing patients with tetraplegia to regain control of hand movements and to achieve a greater measure of independence in daily activities. In existing clinical systems, the patient uses residual proximal limb movements to trigger pre-programmed stimulation that causes the paralysed muscles to contract, allowing use of one or two basic grasps. Instead, we have developed an FES system in primates that is controlled by recordings made from microelectrodes permanently implanted in the brain. We simulated some of the effects of the paralysis caused by C5 or C6 spinal cord injury by injecting rhesus monkeys with a local anaesthetic to block the median and ulnar nerves at the elbow. Then, using recordings from approximately 100 neurons in the motor cortex, we predicted the intended activity of several of the paralysed muscles, and used these predictions to control the intensity of stimulation of the same muscles. This process essentially bypassed the spinal cord, restoring to the monkeys voluntary control of their paralysed muscles. This achievement is a major advance towards similar restoration of hand function in human patients through brain-controlled FES. We anticipate that in human patients, this neuroprosthesis would allow much more flexible and dexterous use of the hand than is possible with existing FES systems.     

A novel class (H3) of histamine receptors on perivascular nerve terminals

Two types of histamine receptor, the H1- and H2-receptors, are found not only on vascular smooth muscle cells but on the perivascular autonomic nerve terminals. Activation of the prejunctional histamine receptors modifies transmitter release from the nerve terminals. Recently, histamine was shown to inhibit its own release from depolarized slices of rat cerebral cortex. This phenomenon was found to be mediated by a novel class of histamine receptor, the H3-receptor, that was pharmacologically distinct from the H1- and H2-receptors. Up to now, there has been no indication whether this third class of histamine receptor is present in any tissue other than the brain. We report here that histamine depresses sympathetic neurotransmission in the guinea-pig mesenteric artery by interacting with histamine H3-receptors on the perivascular nerve terminals. The pharmacological properties of these receptors are similar to those reported for the H3-receptors in the brain. Our data provide evidence for the existence of H3-receptors in the autonomic nervous system.     

Cell-surface labelling reveals no evidence for membrane assembly and disassembly during fibroblast locomotion.

As a cultured fibroblast moves, particles or pieces of debris attached to its surface move backwards with respect to both the cell and the substrate1,2, as also do concanavalin A (Con A) receptors in the membrane of the leading lamella3. One suggested explanation is that membrane components from the cytoplasm are assembled and introduced into the surface membrane of the moving cell close to its leading edge and flow backwards to a 'sink'; there the membrane is disassembled, and its components enter the cytoplasm and flow forward within the cell, to be re-introduced later into the surface membrane1,4. However, because cell-surface receptors can be redistributed as a result of cross-linking by external ligands5,6 it has been proposed that the backward flow of Con A receptors and particles may result from such a cross-linking rather than from a flow of intact cell membrane7,8. To investigate these alternatives, I have studied moving fibroblasts by means of a cell membrane label that does not induce the redistribution of its receptors. My results do not seem compatible with the membrane flow model.     

Order in the initial retinotectal map in Xenopus: a new technique for labelling growing nerve fibres

Retinal nerve fibres form an orderly map of visual space in several centres in the vertebrate brain. Such topographic maps are a common feature of central nervous system organization, yet the way in which they develop is poorly understood. Early nerve projections in the fetal and neonatal mammalian brain have been found in several cases to be less restricted than those in the adult, suggesting that nerve fibres may initially form a diffuse set of connections in their target structure from which the adult map is sculpted by the elimination of terminals. Indeed, previous electrophysiological data indicate that the retinotectal map in Xenopus laevis might be initially disorganized. We report here, however, that the retinotectal projection is ordered from the beginning of tectal innervation (stage 39/40). We demonstrate this first autoradiographically by tracing groups of growing ganglion cell axons which we labelled by incubating sectors of eye rudiments, before axonal outgrowth, in 3H-proline and replacing them orthotopically. Separate labelling of dorsal and ventral parts of the initial projection showed that retinal fibres are organized topographically, as in the adult, in the tectal rudiment and throughout much of the pathway. Second, we show that visual responses are ordered in the tectum from the first stage that they can be mapped (stage 40). We conclude that the topographic ordering of retinotectal connections develops as a result of directed axonal outgrowth.     

Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome)

There have been many reports that eukaryotic cells contain ring-shaped 19S or 20S particles which are composed of numerous polypeptide subunits ranging in size between 25 and 35 kilodaltons. Because these particles seemed to copurify with inactive mRNA, they were assumed to function in regulating mRNA translation and hence were named 'prosomes' (for 'programmed-o-some'). A number of properties have been reported for these structures, including an association with specific RNA species or with certain heat-shock proteins and involvement in tRNA processing or aminoacyl tRNA synthesis. However, these proposed activities have not been supported by definitive evidence. During studies of the proteolytic systems in mammalian tissues, we noted many similarities between these 19S particles and the high molecular weight protease complexes that are present in most or all eukaryotic cells. This (700 kilodalton) enzyme complex, designated here as LAMP for 'large alkaline multi-functional protease', contains three distinct endoproteolytic sites which function at neutral or alkaline pH and are specific for hydrolysis of proteins, hydrophobic peptides, or basic peptides. This protease also exists in a latent form which can be activated by polylysine, fatty acids, or ATP. In this report, we show that the prosomes and these protease complexes are very similar or identical with respect to their size, polypeptide composition, immunological cross-reactivity, appearance in the electron microscope, radial symmetry of subunits, subcellular localization, and proteolytic activities. Therefore, the 'prosome' probably plays a critical role in intracellular protein breakdown, and we propose that it be renamed 'proteasome'.     

Outer hair cells in the mammalian cochlea and noise-induced hearing loss

Hair cells in the mammalian cochlea transduce mechanical stimuli into electrical signals leading to excitation of auditory nerve fibres. Because of their important role in hearing, these cells are a possible site for the loss of cochlear sensitivity that follows acoustic overstimulation. We have recorded from inner and outer hair cells (IHC, OHC) in the guinea pig cochlea during and after exposure to intense tones. Our results show functional changes in the hair cells that may explain the origin of noise-induced hearing loss. Both populations of hair cells show a reduction in amplitude and an increase in the symmetry of their acoustically evoked receptor potentials. In addition, the OHCs also suffer a sustained depolarization of the membrane potential. Significantly, the membrane and receptor potentials of the OHCs recover in parallel with cochlear sensitivity as measured by the IHC receptor potential amplitude and the auditory nerve threshold. Current theories of acoustic transduction suggest that the mechanical input to IHCs may be regulated by the OHCs. Consequently, the modified function of OHCs after acoustic overstimulation may determine the extent of the hearing loss following loud sound.     

Subthreshold Na+-dependent theta-like rhythmicity in stellate cells of entorhinal cortex layer II

The oscillation of membrane potential in mammalian central neurons is of interest because it relates to the role of oscillations in brain function. It has been proposed that the entorhinal cortex (EC), particularly the stellate cells of layer II (ECIIscs), plays an important part in the genesis of the theta rhythm. These neurons occupy a key position in the neocortex-hippocampus-neocortex circuit, a crucial crossroad in memory functions. Neuronal oscillations typically rely on the activation of voltage-dependent Ca2+ conductances and the Ca2+ -dependent K+ conductance that usually follows, as seen in other limbic subcortical structures generating theta rhythmicity. Here we report, however, that similar oscillations are generated in ECIIscs by a Na+ conductance. The finding of a subthreshold, voltage-gated, Na+ -dependent rhythmic membrane oscillation in mammalian neurons indicates that rhythmicity in heterogeneous neuronal networks may be supported by different sets of intrinsic ionic mechanisms in each of the neuronal elements involved.     

Pathological changes in olfactory neurons in patients with Alzheimer's disease

Alzheimer's disease is a central nervous system disorder characterized by the presence of neurofibrillary tangles, neuritic plaques and dystrophic neurites in susceptible areas of the brain. Investigation of the mechanism and development of the disease has been hampered by the lack of an animal model and the inaccessibility of neural tissue during the illness. Deficits in odour detection and discrimination are among the signs of Alzheimer's and previous anatomical studies suggest that olfactory pathways may be involved early in the illness. Neurons in the olfactory epithelium, which are of central origin, are relatively accessible for biopsy and could be used as a source of living nerve cells for the study of Alzheimer's disease if they can be shown to have characteristics of this disease. As these neurons have the unusual property of arising from stem cells throughout the life of the organism, they are good candidates for the development of cell cultures or cell lines which may express the disorder from living patients. We report here that nasal epithelium tissue taken at autopsy shows unique pathological changes in morphology, distribution and immunoreactivity of neuronal structures in patients with Alzheimer's disease.