Microtubule-associated smooth endoplasmic reticulum in the frog's brain

Summary Electron microscopic studies of neural processes in the cerebellum, optic tectum, and cerebral hemisphere of the frog reveal a distinctive system of SER cisternae lying at intervals (commonly 1–2 m apart) perpendicular to the long axis of axons and dendrites, interconnected by tubular, longitudinally orientated SER elements, and in direct continuity with the outer membrane of mitochondria. The transverse cisternae are fenestrated, with a single mierotubule (or rarely, two) passing through the centre of each 50–75 nm fenestration. Extensions of the SER-microtubule complex may be located parasynaptically in axon terminals and dendrites. The SER of dendritic spines also appears to be continuous with the fenestrated cisternae.Possible roles for the specialized SER (particularly of the parasynaptic extensions), such as calcium ion sequestration and ATP or monoamine oxidase transport, are discussed.Thanks are due to Profs. E. G. Gray and J. Z. Young for helpful discussion and to Mrs. N. Morgan and Mr. R. Boddy for technical assistance.     

Mitochondrial potassium channels and uncoupling proteins in synaptic plasticity and neuronal cell death

The function of the nervous system relies upon synaptic transmission, a process in which a neurotransmitter released from pre-synaptic terminals of one neuron (in response to membrane depolarization and calcium influx) activates post-synaptic receptors on dendrites of another neuron. Synapses are subjected to repeated bouts of oxidative and metabolic stress as the result of changing ion gradients and ATP usage. Mitochondria play central roles in meeting the demands of synapses for ATP and in regulating calcium homeostasis, and mitochondrial dysfunction can cause dysfunction and degeneration of synapses, and can trigger cell death. We have identified two types of mitochondrial proteins that serve the function of protecting synapses and neurons against dysfunction and death. Mitochondrial ATP-sensitive potassium (MitoKATP) channels modulate inner membrane potential and oxyradical production; mitochondrial potassium fluxes can affect cytochrome c release and caspase activation and may determine whether neurons live or die in experimental models of stroke and Alzheimer's disease. Uncoupling proteins (UCPs) are a family of mitochondrial membrane proteins that uncouple electron transport from ATP production by transporting protons across the inner membrane. Neurons express at least three UCPs including the widely expressed UCP-2 and the neuron-specific UCP-4 and UCP-5 (BMCP-1). We have found that UCP-4 protects neurons against apoptosis by a mechanism involving suppression of oxyradical production and stabilization of cellular calcium homeostasis. The expression of UCP-4 is itself regulated by changes in energy metabolism. In addition to their roles in neuronal cell survival and death, MitoKATP channels and UCPs may play roles in regulating neuronal differentiation during development and synaptic plasticity in the adult.     

Branching dendrites with resonant membrane: a “sum-over-trips” approach

Dendrites form the major components of neurons. They are complex branching structures that receive and process thousands of synaptic inputs from other neurons. It is well known that dendritic morphology plays an important role in the function of dendrites. Another important contribution to the response characteristics of a single neuron comes from the intrinsic resonant properties of dendritic membrane. In this paper we combine the effects of dendritic branching and resonant membrane dynamics by generalising the “sum-over-trips” approach (Abbott et al. in Biol Cybernetics 66, 49–60 1991). To illustrate how this formalism can shed light on the role of architecture and resonances in determining neuronal output we consider dual recording and reconstruction data from a rat CA1 hippocampal pyramidal cell. Specifically we explore the way in which an I _h current contributes to a voltage overshoot at the soma.     

Reduction in Microtubule Dynamics In Vitro by Brain Microtubule-Associated Proteins and by a Microtubule-Associated Protein-2 Second Repeated Sequence Analogue

Abstract: Microtubule-associated protein (MAP) binding to assembled microtubules (MTs) can be reduced by the addition of polyglutamate without significant MT depolymerization or interference with MT elongation reactions. Ensuing polymer length redistribution in MAP-depleted MTs occurs on a time scale characteristic of that observed with MAP-free MTs. The redistribution phase occurs even in the absence of mechanical shearing and without appreciable effects from end-to-end annealing, as indicated by the time course of incremental changes in polymer length and MT number concentration. We also observed higher rates of MT length redistribution when the [MAP]/[tubulin] ratio was decreased. Together, these results demonstrate that MT length redistribution rates are greatly influenced by MAP content, and the data are compatible with the dynamic instability model. We also found that a peptide analogue corresponding to the second repeated sequence in the MT-binding region of MAP-2 can also markedly retard MT length redistribution kinetics, a finding that accords with the ability of this peptide to promote tubulin polymerization in the absence of MAPs and to displace MAP-2 from MTs. These results provide further evidence that MAPs can modulate MT assembly/disassembly dynamics and that peptide analogues can mimic the action of intact MAPs without the need for three contiguous repeated sequences in the MT-binding region.     

Distribution of acetylated α-tubulin in brain

Summary We studied the solubility properties of brain acetylated -tubulin, as well as the localization of this tubulin in brain tissue. Endogenous unpolymerized tubulin and cytoskeletal tubulin were fractionated after brain Triton-solubilization. Using the immunoblotting technique, we found that acetylated -tubulin was recovered in the cytoskeletal fraction, and that most (92%) of the acetylated microtubules of this fraction were depolymerized by cold/Ca²⁺ treatment. In another set of experiments, axonal and soma-dendritic preparations were found to have equivalent amounts of acetylated -tubulin. By immunogold electron microscopy, we established that acetylated microtubules are widely distributed in dendrites of the central nervous system.     

Special dendritic and axonal endings formed by the cerebrospinal fluid contacting neurons of the spinal cord

Summary The cerebrospinal fluid (CSF) contacting neurons have a dendritic process which protrudes into the central canal, and is provided with one long kinocilium and many shorter stereocilia (about 80 in the turtle) as revealed by scanning electron microscopy. The shape, number and arrangement of the cilia are similar to those of known receptor endings.The silver impregnated axons of these cells converge to a paired centrosuperficial tract forming terminal enlargements at the ventrolateral surface of the spinal cord. Lying among glial endfeet these terminals are ultrastructurally similar to those present in known neurosecretory areas. The nerve endings are attached to the basal lamina, and they comprise many synaptic vesicles (200 to 400 Å in diameter), as well as granular vesicles of different sizes (diameter 600 to 1800 Å). The axons may lie within finger-like protrusions on the surface of the spinal cord, or they may terminate around vessels.Morphological evidence suggests that these nerve terminals and the corresponding CSF contacting perikarya represent a spinal neurosecretory system possibly influenced by information taken up by its special dendrites protruding into the inner CSF space.     

Functional organization of dendritic Ca2+ signals in midbrain dopamine neurons

Dendritic Ca²⁺ plays an important role not only in synaptic integration and synaptic plasticity, but also in dendritic excitability in midbrain dopamine neurons. However, the functional organization of dendritic Ca²⁺ signals in the dopamine neurons remains largely unknown. We therefore investigated dendritic Ca²⁺ signals by measuring glutamate-induced Ca²⁺ increases along the dendrites of acutely isolated midbrain dopamine neurons.Maximal doses of glutamate induced a [Ca²⁺]_c rise with similar amplitudes in proximal and distal dendritic regions of a dopamine neuron. Glutamate receptors contributed incrementally to the [Ca²⁺]_c rise according to their distance from the soma, with a reciprocal decrement in the contribution of voltage-operated Ca²⁺ channels (VOCCs). The contribution of AMPA and NMDA receptors increased with dendritic length, but that of metabotropic glutamate receptors decreased. At low doses of glutamate at which spontaneous firing was sustained, the [Ca²⁺]_c rise was higher in the distal than the proximal regions of a dendrite, possibly due to the increased spontaneous firing rate.These results indicate that functional organization of Ca²⁺ signals in the dendrites of dopamine neurons requires different combination of VOCCs and glutamate receptors according to dendritic length, and that regional Ca²⁺ rises in dendrites respond differently to applied glutamate concentration.     

Protein synthesis within dendrites: Ionic and neurotransmitter modulation of synthesis of particular polypeptides characterized by gel electrophoresis

This study evaluates whether physiological variables differentially affect the local synthesis of protein constituents of synapses in subcellular fractions containing pinched-off dendrites (synaptodendrosomes). Synaptodendrosomes were pulse-labeled in a medium containing³⁵S-methionine with 3 or 25 mM KCl and in the presence or absence of 0.5 mM EGTA or 10 μM glutamate. Synaptodendrosomes were then subfractionated to prepare synaptic plasma membranes and synaptic junctional complexes. The protein constituents of the synaptic plasma membrane and synaptic junctional complex fractions that were locally synthesized were identified using SDS-PAGE and two-dimensional gel electrophoresis and the extent of labeling of individual bands was analyzed using a Phosphorimager. Analysis of incorporation into individual bands resolved by SDS-PAGE revealed that depolarizing conditions (25 mM KCl) increased the extent of labeling of different bands to a different extent (ranging from 10–70% increases in labeling). Addition of 0.5 mM EGTA decreased the extent of labeling of the same group of bands in both 3 mM KCl and 25 mM KCl conditions. Addition of 10 μM glutamate reduced incorporation especially in the synaptodendrosomes incubated in 25 mM KCl. Two-dimensional gel electrophoresis analyses revealed that the labeled spots that showed differential labeling under the different conditions did not correspond to the most prominent Coomassie-stained spots. These results indicate that the proteins that are synthesized in synaptodendrosomes and regulated by physiological variables are not amongst the more abundant protein constituents of the fractions. Taken together, these results are consistent with the idea that protein synthesis within dendrites may be regulated by synaptic activity.     

Developmental Regulation of Microtubule-Associated Protein 2 Expression in Regions of Mouse Brain

The relative levels of microtubule-associated protein 2(MAP2) were determined during postnatal development of the mouse in six different discrete brain regions: cerebellum, cortex, hippocampus, olfactory bulb, brainstem, and hypothalamus. Brain homogenates were electrophoresed on sodium dodecyl sulfate-containing gels and analyzed by immunoblotting with MAP2-specific antibodies. The levels of MAP2 in each region were determined using radiolabeled secondary antibodies and densitometric quantification of the autoradiograms over a range that was determined to have a linear response. The results indicated that in all regions and at all ages there was only one high-molecular-weight polypeptide of MAP2, which did not change in electrophoretic mobility after dephosphorylation. In most regions, the levels of MAP2 increased during the first 2 postnatal weeks. However, there were differences in the time course and relative levels of MAP2 between regions. In addition, all regions of the brain expressed the low-molecular-weight form of MAP2 (MAP2c) that was present at birth as a heterogeneous group of polypeptides with an apparent molecular weight of 70K. Most of the heterogeneity of MAP2c, however, was eliminated after dephosphorylation. The levels of MAP2c decreased dramatically after 2 weeks postnatally, except for the olfactory bulb, where the levels of MAP2c remained relatively high even in adults.