Faculty Bibliography

The high molecular weight subunit of neurofilaments (NF-H) in mouse NB2a/d1 neuroblastoma cells is extensively phosphorylated and exhibits an apparent molecular weight of 200 kDa by SDS gel electrophoresis. In this study, we observed that extensively phosphorylated NF-H variants exist as both Triton-soluble and -insoluble forms, which display different cellular distributions. Perikarya and neurites of differentiated NB2a/d1 cells were immunostained by a polyclonal antiserum (anti-NF-H) that specifically recognizes the extensively phosphorylated NF-H forms and a monoclonal antibody (SMI-31) that recognizes phosphorylated epitopes of neurofilament proteins (NFPs). When cells were extracted with Triton X-100 to remove soluble proteins, however, only axonal neurites remained immunoreactive. Immunoblot analyses established the specificity of anti-NF-H and SMI-31 and demonstrated that both Triton-soluble and -insoluble NF-H subunits exhibit an apparent molecular weight of 200 kDa. Incorporation of radiolabeled phosphate into Triton-soluble NF-H following incubation of intact NB2a/d1 cells with 32P-orthophosphate confirmed that the Triton-soluble form of NF-H is a phosphoprotein. Most NF-H subunits in the Triton-soluble fraction sedimented after centrifugation at 100,000 g for 1 h, indicating that they may be present as oligomers. The implications of these data for the development of neurofibrillary pathology are discussed

Five depressed patients who had shown no improvement with trials of antidepressants from several chemical families, including fluoxetine, responded when lithium was given in conjunction with fluoxetine. Lithium augmentation of fluoxetine may represent a useful strategy in refractory depression

Induction of axonal neuritogenesis in NB2a/d1 cells was associated with an increased content of neurofilament proteins (NFPs) by immunoblot analysis. The major NFP subunits in differentiated [NB2a(+)] cells included microheterogenous forms with apparent molecular weights of 200-190 kDa (NFP-H), 143-142 kDa (NFP-M) and 70 kDa (NFP-L) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only NFP-L was detected in cytoskeletal preparations of undifferentiated [NB2a(-)] cells. All three NFPs of NB2a(+) cells incorporated 32P-orthophosphate in intact cells. A 160/155 kDa NFP-H immunoreactive polypeptide in NB2a(-) and NB2a(+) cells represented a relatively unmodified form of the 200 kDa NFP-H, since dephosphorylation of the 200 kDa NFP-H in vitro with alkaline phosphatase generated the 160/155 kDa forms. Triton-extracted NB2a(+) cells displayed NFP-H immunoreactivity in neurites and occasionally in perikaryal regions at the base of neurites. NFP-M was present throughout the neurites and somata of NB2a(+) cells, and was regularly detected in portions of perikarya in NB2a(-) cells. NFP-L immunoreactivity was distributed throughout the Triton-insoluble cytoskeleton of NB2a(-) and NB2a(+) cells. Immunocytochemical analyses revealed that extensively phosphorylated forms of NFP-H were largely restricted to the neurites of NB2a(+) cells, and less modified forms predominated throughout both perikarya and neurites of NB2a(-) and NB2a(+) cells

The low molecular mass (70 kDa) subunit of neurofilaments (NF-L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site-specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two-dimensional alpha-chymotryptic phosphopeptide map analyses of the NF-L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse-radiolabeled with [32P]orthophosphate in vivo

Mouse NB2a/d1 cells assemble all 3 neurofilament protein subunits (NFPs) into the detergent-insoluble cytoskeleton and segregate phosphorylated forms of the 200-kDa subunit (NFP-H) within neurites when differentiation is induced with dibutyryl cyclic AMP (dbcAMP). Before and after differentiation, these cells also incorporate vimentin into both the perikaryal and neuritic cytoskeleton (Shea et al., 1988, Dev. Brain Res., submitted). To determine whether NFPs and vimentin constitute separate intermediate filament systems or exist as heteropolymers, we perturbed cytoskeletal architecture by inducing the retraction of neurites with colchicine. After cells were exposed to colchicine, vimentin immunoreactivity partitioned into perikarya in the form of fibrous whorls that did not cross-react with antisera to NFPs. By contrast, NFP immunoreactivity remained dispersed throughout the cell body following neurite retraction. We interpret these different responses to colchicine to indicate that NFPs and vimentin are assembled into separate intermediate filaments in NB2a/d1 cells

A specific population of cells located in the hilus of the hippocampal fascia dentata was studied in guinea pig hippocampal slices using standard intracellular recording techniques. Twenty-one such cells were characterized using electrophysiological techniques and were identified morphologically as mossy cells following intracellular injection of the fluorescent dye Lucifer yellow. These cells had a resting membrane potential (mean, -64.6 mV), action potential amplitude (mean, 78.6 mV), action potential duration (mean, 2.2 msec), and time constant (mean, 24.2 msec) similar to those of hippocampal pyramidal cells of area CA3. Rectification seen in their I-V curves, and their ability to fire action potentials in accommodating trains or bursts in response to injected current pulses, were also similar to those of area CA3 pyramidal cells. However, these cells could be distinguished from area CA3 pyramidal cells by their higher input resistance (mean, 97.4 M omega) and higher level of spontaneous activity. The synaptic responses of mossy cells were also different from those of CA3 pyramidal cells. First, mossy cells responded to low levels of stimulation in all areas of the hippocampal slice that were tested, even areas as remote as area CA1. Second, the responses of mossy cells to stimulation consisted primarily of EPSPs. Hyperpolarizing IPSP-like events followed EPSPs in some cells, but the hyperpolarizations were small and monophasic, even after the cell was depolarized with current injection. This response contrasts with the smaller EPSP and the prominent, biphasic IPSP elicited by afferent stimulation of area CA3 pyramidal cells. The physiological and morphological characteristics of these cells suggest that they could play an important role in the integration of electrical activity in the hippocampus

1. In slice studies of mature and immature CA1 hippocampal pyramidal cells from rabbit, somatostatin 14 (SS14), the related peptide somatostatin 28(1-12) [SS(1-12)], and the synthetic analogue of somatostatin 14, SMS-201995 (SMS), had similar effects. When pressure-ejected onto cell somata, these peptides elicited depolarizations, often accompanied by action potential discharge. When applied to dendrites, the peptides produced depolarizations or hyperpolarizations. 2. When a large amount of one of the three somatostatin-related (SS) peptides was applied to the slice at some distance from the impaled cell, hyperpolarizations were observed that were not always blocked by tetrodotoxin (TTX) or low Ca2+. Since SS peptides were also found to depolarize interneurons in area CA1, it seems likely that the hyperpolarizations that were blocked by TTX or low Ca2+ were mediated via excitation of interneurons that in turn hyperpolarized pyramidal cells. 3. All SS peptides also had long-lasting effects on CA1 pyramidal cells that led to spontaneous firing of action potentials and an increase in the number of action potentials discharged in response to a given depolarizing current pulse; the spontaneous discharge effect was blocked by TTX or low Ca2+ plus Mn2+ and, thus, appeared to have a presynaptic mechanism. However, the increase in discharge in response to a constant depolarizing current pulse was not dependent on intact synaptic transmission and, therefore, was attributable to a direct postsynaptic effect of the SS peptides

Responses to focal application of gamma-aminobutyric acid (GABA) were compared to synaptic potentials elicited by afferent stimulation of rat visual cortical neurons, using a slice preparation and conventional intracellular recording techniques. GABA produced three types of responses: a brief hyperpolarization (mean reversal potential, -72 mV), brief depolarization (mean reversal potential, -50 mV), or a prolonged hyperpolarization (mean reversal potential, -80 mV). Synaptic potentials included simple or complex EPSPs and EPSPs followed by mono- or biphasic IPSPs. A comparison of the characteristics of the GABA responses and synaptic potentials indicated that GABA may mediate both phases of the IPSP in these cells. Our results suggest that despite differences in the circuitry of the visual cortex as opposed to other neocortical and allocortical (hippocampal) areas (Mountcastle and Poggio, 1968; Colonnier and Rossignol, 1969; Creutzfeldt, 1978; Kuhlenbeck, 1978), the inhibitory control of cortical pyramidal and nonpyramidal neurons by GABA is quite similar

The progressive modification of newly synthesized neurofilament proteins (NFPs) during axoplasmic transport in mouse retinal ganglion cell (RGC) neurons was studied after RGC perikarya were pulse-labeled with 32P-orthophosphate or radiolabeled amino acids. The 3 NFP subunits, H(igh), M(iddle), and L(ow), were among a group of axonally transported proteins that incorporated high levels of 32P. Covalent addition of phosphate slowed the electrophoretic mobility of H and M on SDS polyacrylamide gels and shifted the charge of all 3 subunits toward more acidic pH values, thereby providing an index of the phosphorylation state of this radiolabeled population of NFPs. NFPs were extensively phosphorylated before they entered axons at the optic nerve level, and continued to be modified during transport along RGC axons at the optic nerve and tract level. H and M exhibited charge shifts of 0.2-0.6 units toward a more acidic pH during axoplasmic transport. The charge modifications became more prominent when NFPs reached distal axonal levels, which may indicate regional differences in the activity of this modification process along axons. By contrast, the L subunit became more basic in charge, consistent with decreases in the phosphorylation state during transport. Additional observations (Nixon and Lewis, 1986) that a considerable proportion of phosphate groups initially added to L and M were later removed as neurofilaments advanced along RGC axons support the notion that the changing phosphorylation state of NFP subunits during axoplasmic transport reflects a dynamic equilibrium between phosphorylation and dephosphorylation events. Topographical remodeling of phosphate groups on NFPs during axoplasmic transport is proposed as a possible mechanism for coordinating interactions between neurofilaments and other constituents, as these elements are transported and integrated into the axonal cytoskeleton