Kimon J. Angelides

Effect of phosphorylation on 68 KDa neurofilament subunit protein assembly by the cyclic AMP dependent protein kinase in vitro

The effect of phosphorylation by cyclic AMP dependent protein kinase on the assembly of the core-forming 68 KDa neurofilament subunit protein (NF-L) was studied in vitro by fluorescence energy transfer and electron microscopy. Phosphorylation of unassembled NF-L in a low ionic strength buffer by cyclic AMP dependent protein kinase led to the incorporation of 1-2 phosphate groups/mole protein. Assembly of this phosphorylated NF-L was inhibited significantly; compared to non-phosphorylated NF-L, the critical concentration of phosphorylated NF-L was raised by greater than 30-fold. Assembled NF-L filaments could also be phosphorylated by cyclic AMP dependent protein kinase indicating that the sites were accessible. Phosphorylation of NF-L in the filamentous state induced their disassembly. The results suggest that phosphorylation by cyclic AMP dependent protein kinase is a possible means to modulate the assembly state of NF-L.

Immuno-ultrastructural localization of sodium channels at nodes of Ranvier and perinodal astrocytes in rat optic nerve

Immuno-electron microscopic localization of sodium channels at nodes of Ranvier within adult optic nerve was demonstrated with polyclonal antibody 7493. The 7493 antisera, which is directed against purified sodium channels from rat brain, recognizes a 260 kDa protein in immunoblots of the crude glycoprotein fraction from adult rat optic nerve. Intense immunoreactivity with 7493 antisera was observed at nodes of Ranvier. Axon membrane at the node was densely stained, whereas paranodal and internodal axon membrane did not exhibit immunoreactivity. The axoplasm beneath the nodal membrane displayed variable immunostaining. Neither terminal paranodal oligodendroglial loops nor oligodendrocyte plasmalemma were immunoreactive with 7493 antisera. However, perinodal astrocyte processes exhibited intense immunoreactivity with the anti-sodium channel antisera. Optic nerves incubated with pre-immune sera, or with 7493 antisera that had been pre-adsorbed with purified sodium channel protein, displayed no immunoreactivity. These results demonstrate localization of sodium channels at high density at mammalian nodes of Ranvier and in some perinodal astrocyte processes. The latter observation offers support for an active role for perinodal astrocyte processes in the aggregation of sodium channels within the axon membrane at the node of Ranvier.

The voltage-dependent sodium channel in mammalian CNS and PNS: antibody characterization and immunocytochemical localization

Monoclonal and polyclonal antibodies were generated against the voltage-dependent sodium channel purified from rat brain, and were used to characterize and localize sodium channels within mammalian central nervous system (CNS) and peripheral nervous system (PNS). These antibodies immunoblot and immunoprecipitate from labeled membrane proteins a 260-kDa polypeptide, as well as immunoprecipitate sodium channels saturated with [3H]saxitoxin. These monoclonal and polyclonal antibodies do not, however, recognize sodium channels in cardiac or skeletal muscle. Immunocytochemical analyses of cultured CNS and PNS neurons and immuno-ultrastructural localization of sodium channel reactivity within CNS tissue in situ indicate that these probes provide a unique tool for studying the level of expression, organization and turnover of sodium channels within the CNS and PNS.

Adenovirus‐mediated gene transfer into dissociated and explant cultures of rat hippocampal neurons

Genetic manipulation offers great potential for studying the molecular and cellular processes which control or regulate the complex developmental properties of neurons. Gene transfer into neurons, however, is notoriously difficult. In this study we have used a replication‐defective adenovirus (Adv/RSVβgal), expressing β‐galactosidase (β‐gal) as a reporter gene, to infect dissociated cultures of rat hippocampal neurons and hippocampal slice cultures. Because future studies will require either long‐term (e.g., developmental) or short‐term (e.g., electrophysiological) expression of recombinant genes in neuronal cultures, we have optimized infection conditions for each situation. The Adv/RSVβgal construct infects neurons and glial cells equally well, with no apparent alterations in cellular morphology. In slice cultures, the same efficiency and temporal control of β‐gal expression following Adv/RSVβgal infection was achieved. Focal application of the adenoviruses, by microinjection, permitted infection of discrete subregions within the hippocampal explants. Whole cell recordings of dissociated hippocampal neurons and field recordings from the explant cultures, infected with Adv/RSVβgal at low multiplicities of infection, indicated no significant alteration in the electrophysiological profiles of neurons in these cultures. The results demonstrate the utility of adenoviruses as gene transfer vectors for primary cultures of neurons. Adenovirus‐mediated gene transfer into slice cultures also provides an opportunity to study development or plasticity in an environment where the circuitry and cytoarchitecture of the tissue are preserved and the areas of genetic manipulation can be spatially isolated.

Addition of tetrodotoxin alters the morphology of thalamocortical axons in organotypic cocultures

Living organotypic cocultures of rat thalamic and cortical explants were used to examine the effects of blocking action potential activity on the morphological development of axons in the mammalian neocortex. Studies in vivo have suggested that blocking sodium channel‐dependent activity influences the growth characteristics of thalamocortical axons during development. We have extended these observations by using an in vitro system that affords more direct observational analysis of the early events of axonal growth in an accessible cellular environment DiI‐labeled thalamocortical axons grow exuberantly into the target cortex and establish axonal connections that reflect the events of early thalamocortical afferent development. Within these cocultures, the morphological features of DiI‐labeled axons can be readily distinguished. Tracings of thalamocortical axons were quantitated with respect to number, length, and termination pattern of axonal branches, as well as number of varicosities. Addition of the voltage‐dependent sodium channel blocker, tetrodotoxin, to cocultures did not change the general pattern of thalamocortical axonal ingrowth or the average length of collateral branches of these axons. However, in the presence of tetrodotoxin, axons were more highly branched, with an increased number of varicosities as compared to untreated cocultures. This pattern of axonal growth and branching may reflect the activity‐dependent fine‐tuning and trimming of collaterals that occur as thalamic afferents begin to refine their cortical territory. Our observations in thalamocortical cocultures are consistent with the view that neuronal activity modulates the pattern of axonal growth and development.

Voltage-dependent sodium channel alpha subunit immunoreactivity is expressed by distinct cell types of the cat and monkey retina

Polyclonal (7493 and 7317) and monoclonal (mAb3) antibodies, generated to the alpha subunit of the voltage-gated sodium channel (alpha NaCh), were employed to assess the cell types containing alpha NaCh-like immunoreactivity in the mature cat and monkey retina. Immunoblot analyses of retinal proteins in the cat revealed that the polyclonal and monoclonal antibodies we employed labeled a band in the 260-kDa region which corresponds to the molecular mass of the alpha subunit of the NaCh. In both the cat and monkey, these antibodies immunolabeled several distinct types of retinal cells. With the polyclonal antibodies immunoreactivity was observed in ganglion cells and their intraretinal axons, in horizontal cells, and unexpectedly, in cones. In addition, in both species, a limited number of heavily labeled profiles, presumed to be bipolar cells, were seen in the inner nuclear layer. In cat and monkey the monoclonal antibody labeled axons in the fiber layer, ganglion cell somata, and a continuous band of immunoreactive cell bodies (presumed bipolar cells) situated in the outer half of the inner nuclear layer. By immunolabeling isolated cells dissociated from the cat retina, it was possible to demonstrate unequivocally that a population of bipolar cells was labeled by the monoclonal and the polyclonal antibodies we employed. The differences in the labeling observed with the monoclonal antibody as compared to the polyclonal antibodies were interpreted as reflecting the presence of different alpha-subunit subtypes in the mammalian retina. Collectively, our findings suggest that alpha NaCh-like proteins are expressed by a more diverse population of retinal cells than expected on the basis of previous physiological and immunohistochemical studies.

GABAA receptor immunoreactivity in the white matter.

USING immunohistochemical methods with polyclonal antibodies directed against a specific sequence of the β1-subunit of the GABAA receptor, we found strong immunoreactivity in the white matter of cat brain. The immunopositive products were present primarily on processes of glial cells, especially astrocytes. Immunoreactivity appeared also on the cell bodies of astrocytes and on the cytoplasmic membranes of neurons. The abundant immunostaining in the white matter suggests that (1) GABAA receptors are present on glial cells in vivo, (2) GABAA receptors may be localized on non-synaptic membranes in the white matter and (3) activation of GABAA receptors may have some trophic effects on preservation of the structure and functional properties of the white matter.

Distinct Temporal Patterns of Expression of Sodium Channel-like Immunoreactivity During the Prenatal Development of the Monkey and Cat Retina

Polyclonal and monoclonal antibodies prepared against the α‐subunit of the voltage‐gated sodium channel (αNaCh) were used to examine the distribution of sodium channel‐like immunoreactivity during the prenatal development of the cat and rhesus monkey (Macaca mulatta) retina. At all prenatal ages studied, beginning on embryonic day 29 (E29) in the cat and E52 in the monkey, both antibodies labelled optic axons. With the polyclonal antibodies, the appearance of positive cells largely mirrored the onset of their morphological maturation. Immunoreactivity appeared first in the somata of ganglion cells, and subsequently the inner plexiform layer could be distinguished by its intense immunolabelling. A few weeks later horizontal cells displayed immunolabelling that extended to their dendrites in the developing outer plexiform layer. This was followed by immunoreactive cones, with bipolar cells labelled only postnatally. By contrast, with the monoclonal antibody some cells were found to be immunoreactive while their somata were still in the ventricular layer (E33 in cat and E52 in monkey). Many of these cells appeared to migrate to the outer portion of the prospective inner nuclear layer, where they gradually acquired the morphological appearance of bipolar cells. Transient expression of immunolabelling with monoclonal sodium channel antibody was found in the cones of the cat and cones and rods of the monkey. These results indicate that different types of αNaCh‐like proteins are expressed in the mammalian retina at distinct developmental periods. Their presence at very early stages during development suggests that these proteins could play a specific role in the commitment and/or differentiation of specific retinal cell types.

Chapter 3 Fluorescent Analogs of Toxins

Publisher Summary This chapter describes the different aspects of fluorescent analogs of toxins. The isolation, chemical characterization, and use of toxins in cell biology have increased significantly. Pharmacological dissection of the ionic currents responsible for the action potential showed several classes of toxins that act specifically on sodium channels. Fluorescent derivatives of these sodium channel toxins have been prepared to map the molecular structure, the cellular topography, and lateral mobility of sodium channels on nerve and muscle cell surfaces. The labeling of sodium channels on cultured neurons by several fluorescent toxin analogs has shown that, on cultured neurons, voltage-dependent sodium channels are localized to morphologically distinct regions of these cells. The P-scorpion toxins, or toxins from North American scorpions, elicit their action by modifying the activation kinetics of channel opening, inducing repetitive firing due to abnormal activation but have no effects on the activation kinetics. Each class of scorpion toxins binds to the channel at different sites. These reagents that elicit distinct pharmacological modifications of sodium channels have been extremely useful in the elucidation of the molecular structure, biochemistry, development, and cellular dynamics of the voltage-dependent Na + channel.

Chapter 7 Development, Maintenance, and Modulation of Voltage-Dependent Sodium Channel Topography in Nerve Cells

Publisher Summary Neurons are extremely polarized cells, characterized by a cell surface organization, where proteins are segregated and maintained in discrete functional domains. A neurons electrical fingerprint arises from how voltage-sensitive ion channels and receptors are placed in specific regions of the cell surface and are maintained. In myelinated nerve, clustering of voltage-dependent sodium channels (NaChs) to nodes of Ranvier creates sites of large inward sodium current and enables the conduction to jump from node-to-node, thereby facilitating conduction down the axon. The segregation of NaChs to the nodes of Ranvier is an example of how specific cell surface components are localized and segregated to the compartments and even within the local domains of the axon. The distribution of NaCh channel is critical in determining some of the differential electrical properties of one part versus the other part of the neuron. This chapter explains some of the mechanisms by which voltage-dependent NaChs are distributed and maintained on the nerve cell surface. The chapter discusses the development of the node of Ranvier, where NaChs are clustered, and a hypothesis based on current information to stimulate further experimentation.