Robert S. Lasher

The uptake of [3H]GABA and differentiation of stellate neurons in cultures of dissociated postnatal rat cerebellum

Abstract Two-day rat cerebellum cultures of various ages were labeled by incubation with [3H]GABA in saline G, and examined by light microscope radioautography. Uptake of [3H]GABA was restricted to stellate neurons, Purkinje cells and interstitial neurons. It was Na+-dependent, very rapid, and had some of the characteristics of a high affinity, highly specific process. The cell components responsible for [3H]-GABA uptake appeared to be distributed throughout the plasma membrane of the neuron soma and processes. Membrane affinity for GABA developed concomitant with the earliest stages of stellate neuron differentiation: a doubling in size of the germinal cell and development of one process; and was not dependent on any progressive increase in neuron-neuron interactions.

Association of Calcium/Calmodulin‐Dependent Kinase with Cytoskeletal Preparations: Phosphorylation of Tubulin, Neurofilament, and Microtubule‐Associated Proteins

Calcium and calmodulin have been implicated in the regulation of cytoskeletal function. In this report, we demonstrate that microtubule preparations from rat brain contain a calcium/calmodulin-dependent protein kinase that phosphorylates endogenous MAP-2, tubulin, synapsin I, and neurofilament proteins. This cytoskeletal-associated kinase has been biochemically characterized and shown to be identical to Type II calcium/calmodulin-dependent protein kinase (CaM kinase II). The subunits of CaM kinase II represented major calmodulin-binding proteins in cytoskeletal preparations. A monoclonal antibody against the 52000 Da subunit of CaM kinase II specifically labeled cytoskeletal elements in cortical neurons. These results indicate that CaM kinase II is associated with the neuronal cytoskeleton and may play a role in mediating some of the effects of calcium on cytoskeletal function.

Electron microscopic autoradiography of the uptake of [3H]GABA in dispersed cell cultures of rat cerebellums. I. The morphology of the GABAergic synapse.

Dispersed cell cultures of rat cerebellums 21 days in vitro were incubated in saline containing 0.5 micrometer [3H]GABA for 5 min, fixed and prepared for electron microscopic autoradiography. Quantitative methods for analyzing the resulting autoradiographs were developed to distinguish lightly labeled neurons from heavily labeled (i.e. GABAergic) neurons. After a 3 week exposure, 2% of the presynaptic elements were found to be GABAergic. A longer incubation time of 15 min resulted in a greatly increased grain density over lightly labeled neurons, making it more difficult to distinguish them from the GABAergic neurons. When the concentration of [3H]GABA was increased to 1.0 micrometer, the grain densities over both GABAergic and other neurons increased to a similar extent. The morphology of the GABAergic synapses differed from that of the total population synapses. The GABAergic synapses had larger cross-sectional areas, lower densities of vesicles, greater numbers of vesicles, and thinner postsynaptic densities. Both the GABAergic and total population had similar lengths of the postsynaptic densities, cleft widths, vesicle sizes and types of postsynaptic elements. The morphology of the GABAergic synapse reported here is similar to that described for basket and stellate presynaptic elements in vivo.

Calmodulin Kinase II in Pure Cultured Astrocytes

Abstract: Calcium‐ and calmodulin‐dependent protein kinase activity was studied in pure neuronal and glial cultures. The addition of calcium and calmodulin stimulated 32P incorporation into several neuronal proteins including two in the 50‐ and 60‐kilodalton (kD) region which comigrated with purified forebrain calmodulin kinase II sub‐units (CaM kinase II). In mature astrocytes, CaM kinase activity was also present, and was inhibited by trifluoroperazine and diazepam. Again in homogenates of these cells, two phosphoproteins of apparent molecular masses of 50 and 60 kD comigrated with purified CaM kinase. CaM kinase activity was absent in immature mixed glia and oligodendrocytes. The presence of CaM kinase in neurons and mature astrocytes was confirmed using monoclonal antibodies specific for the 50‐kD subunit of the enzyme. No immunoreactivity was observed in oligodendrocytes. The presence of CaM kinase in astrocytes suggests a more ubiquitous role of this enzyme in regulating cellular processes than was previously recognized.

Axonal transport of the Ca2+-dependent protein modulator of 3':5'-cyclic-AMP phosphodiesterase in the rabbit visual system.

Abstract: Water‐soluble proteins were extracted from individual retinas, optic nerves, combined optic tracts and lateral geniculate bodies, and superior colliculi of rabbits at 1, 3, and 18 days after injection of [3H]leucine into the right eye. The Ca2+‐dependent protein modulator of 3′:5′‐cyclic‐AMP phos‐phodiesterase (calmodulin) was isolated from these samples by a two‐step polyacrylamide gel electrophoresis procedure. An analysis of the radioactivity incorporated into the total soluble proteins and the calmodulin revealed that most of the calmodulin was axonally transported at a slow rate (2–4 mm/day) and represented about 0.45% of the total transported soluble protein.

Electron microscopic autoradiography of the uptake of [3H]GABA in dispersed cell cultures of rat cerebellums. II. The development of GABAergic synapses.

Abstract The formation of GABAergic synapses in dispersed cell cultures of the rat cerebellum was followed from 7 to 21 days in vitro (DIV). The majority of GABAergic synapses appeared between 10 and 14 DIV, and apparently no additional GABAergic synapses formed after 14 DIV. The first step in the development of a GABAergic synapse appeared to be the formation of a large diameter swelling in a GABAergic neuronal process. After the initial contact between the pre- and postsynaptic elements was established, both the number of synaptic vesicles and the thickness of the postsynaptic density increased, while the cross-sectional area of the presynaptic element decreased. The length of the postsynaptic density showed some increase, but no changes were noted in the synaptic cleft thickness, size of the synaptic vesicles or the shape of the synaptic vesicles. Our findings indicate that the formation of GABAergic synapses was not preceded by the formation of other types of junctions or preformed synaptic elements. In addition, the timing and the rate of formation of GABAergic synapses appears not to depend on contact with a single type of postsynaptic neuron, but rather to depend upon intrinsic properties of the development of the GABAergic neuron.

Dark-induced changes in activity and compartmentalization of retinal calmodulin kinase in the rat

Calmodulin kinase (CaM kinase) activity and immunoreactivity were measured in the cytosol and crude synaptic membranes of light- and dark-adapted rat retinas. Dark adaptation increased the calcium-independent CaM kinase activity 2.7 times and calcium-stimulated activity 3.9 times in membrane fractions. Dark adaptation also increased membrane-bound CaM kinase immunoreactivity 2.4 times. In the cytosol, dark adaptation increased calcium- and calmodulin-independent kinase activity 3.3-fold but did not enhance calcium- and calmodulin-dependent activity. Soluble CaM kinase immunoreactivity was decreased by 13% by dark exposure. These changes in enzyme activity and immunoreactivity are likely due to changes in the endogenous state of autophosphorylation and compartmental concentrations of CaM kinase and may represent translocation of CaM kinase from cytosol to membranes. CaM kinase may have an important role in modulating visual processes.

Association of calmodulin-dependent kinase II and its substrate proteins with neuronal cytoskeleton.

Publisher Summary The neuronal cytoplasm contains a diverse array of interconnecting filamentous and tubular elements. Increasing evidence suggests that the second messenger-dependent phosphorylation systems activated by calcium ions and cyclic adenosine monophosphate (cAMP) are important regulators of the neuronal cytoskeleton. This chapter reviews the work that indicates that the phosphorylation of cytoskeletal proteins may mediate many of the physiological responses to calcium influx. The importance of calmodulin-dependent kinase activities in regulating cytoskeletal dynamics has been recognized for some time in the well-characterized case of myosin light chain kinase in smooth muscle. In the smooth muscle enzyme system, the phosphorylation of myosin light chains following the influx of calcium ions into the cell stimulates actin–myosin interactions, and thereby induces contraction. No such contractile process has been observed with calmodulin-dependent kinase activity in the brain, but recent evidence has demonstrated the association of brain calmodulin-dependent kinases with the neuronal cytoskeleton. The chapter presents evidence for calmodulin kinase II (CAMK II) phosphorylation of both core and projection proteins. The main cytoskeletally associated projection protein, microtubule-associated protein 2, is phosphorylated by both cyclic AMP-dependent and calmodulin-dependent kinases.

A modification of the Bodian technique for embedded, frozen, and cultured nervous tissue.

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Immunocytochemical localization of endopeptidase-24.11 in the nucleus tractus solitarius of the rat brain

Recently, it has been hypothesized that the N-terminal portion of substance P (SP), SP(1-7), which results from the action of endopeptidase 24.11 (EC3.4.24.11), could be involved in mediating the depressor effects of baroreceptor afferent activation via its action on cells in the nucleus tractus solitarius (NTS). In this study, the binding of a monoclonal antibody to endopeptidase 24.11 was examined immunohistochemically at the level of the caudal medulla of the rat brain. By light microscopy, intense immunoreactivity was seen in the NTS, in fibers bordering the area postrema, and in the area postrema itself. After electron microscopy, endopeptidase 24.11-like immunoreactivity was seen to be associated with the cytoskeleton and plasma membrane in axons, dendrites and glial processes. Antigen was also associated with synaptic vesicles and plasma membranes in presynaptic terminals forming mainly axo-dendritic synapses typical of vagal afferent terminals involved in the baroreceptor reflex. Thus, endopeptidase 24.11 appears to be localized at sites where it could effectively process SP prior to its binding to postsynaptic receptors.