ABSTRACT

Calbindin D28k is a high-affinity Ca2+-binding protein (CABP) found to be widely distributed in the nervous system where it has been shown to modulate Ca2+ signaling (1). Light and electron microscopy have clearly demonstrated the existence of calbindin D28k in presynaptic nerve terminals, where it exists in up to millimolar concentrations (2-4). It is therefore tempting to speculate that calbindin D28k also plays a role in modulating synaptic physiology. However, no direct experimental evidence is presently available to validate such a contention.Several techniques have been developed for the overexpression of proteins in cells. Unfortunately, many of these suffer from very low efficiency or require the exogenous DNA to integrate into the genome of a replicating target cell. Recently, several groups have used adenoviruses as expression vectors because these vectors do not require host cell replication and give rise to highly efficient gene expression in host cells (5, 6). Several other advantages of using adenoviruses include the capacity to propagate the virus to high titers and the large amount of exogenous DNA that may be inserted into the viral vector (-7 kb if the El and E3 regions of the viral genome are deleted).In vivo injection of a recombinant adenovirus expressing 13-galactosidase (LacZ gene) into the rat hippocampus and substantia nigra resulted in widespread expression of LacZ in neuronal and nonneuronal cells (5). In addition, infecting cultured neurons with a recombinant adenovirus containing the ,B-galactosidase gene resulted in 80-100% of the cells expressing the protein. Moreover, f3-galactosidase expression appeared to be sustained for at least 1 month after infection without any indication of morphological damage (7).In the present study, we have constructed a replicationdefective adenovirus containing the calbindin D28k cDNAThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.under the control of the elongation factor la (EF-la) promoter and the 4F2HC enhancer (8). We Louis). A blunt-endedBan II/Spe I fragment of calbindin D28k cDNA was inserted into the EcoRV site of transfer vector pAdKN-downstream from EF-la promoter and upstream from the cellular heavychain enhancer (4F2) and bovine growth hormone polyadenylation site (pA+). The pAdKN plasmid also contained 0-1 and 9-16 map units of DNA sequence of adenovirus 5.To construct the recombinant adenovirus AdCABP-1, the plasmid pAdKN-CABP-1 was first linearized by digesting it with Nhe I, while Xba I and Cla I were used to digest adenovirus 5 (sub 360) DNA. The linear DNA was then cotransfected into human embryonic kidney cells (HEK 293 cells)-a trans-complementing cell line for El function (9)-by the calcium phosphate precipitation method. The recombinant AdCABP-1 was plaque purified three times to isolate a homogeneous population of the virus. The virus was further purified...

. Role of persistent sodium current in bursting activity of mouse neocortical networks in vitro. J Neurophysiol 96: 2564 -2577, 2006. First published July 26, 2006 doi:10.1152/jn.00446.2006. Most types of electrographic epileptiform activity can be characterized by isolated or repetitive bursts in brain electrical activity. This observation is our motivation to determine mechanisms that underlie bursting behavior of neuronal networks. Here we show that the persistent sodium (Na P ) current in mouse neocortical slices is associated with cellular bursting and our data suggest that these cells are capable of driving networks into a bursting state. This conclusion is supported by the following observations. 1) Both low concentrations of tetrodotoxin (TTX) and riluzole reduce and eventually stop network bursting while they simultaneously abolish intrinsic bursting properties and sensitivity levels to electrical stimulation in individual intrinsically bursting cells.2) The sensitivity levels of regular spiking neurons are not significantly affected by riluzole or TTX at the termination of network bursting. 3) Propagation of cellular bursting in a neuronal network depended on excitatory connectivity and disappeared on bath application of CNQX (20 M) ϩ CPP (10 M). 4) Voltage-clamp measurements show that riluzole (20 M) and very low concentrations of TTX (50 nM) attenuate Na P currents in the neural membrane within a 1-min interval after bath application of the drug. 5) Recordings of synaptic activity demonstrate that riluzole at this concentration does not affect synaptic properties. 6) Simulations with a neocortical network model including different types of pyramidal cells, inhibitory interneurons, neurons with and without Na P currents, and recurrent excitation confirm the essence of our experimental observations that Na P conductance can be a critical factor sustaining slow population bursting.

Although a neurotoxic role has been postulated for the beta-amyloid protein (beta AP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (beta AP25-35), in particular, has been reported to be toxic in cultured neurons. We report that beta AP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca2+ concentration ([Ca2+]i), as measured by fura 2 fluorimetry. Furthermore, beta AP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The beta AP25-35-induced [Ca2+]i elevations and electrical activity were enhanced by removal of extracellular Mg2+, and they could be blocked by tetrodotoxin, by non-N-methyl-D-aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca2+ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca2+]i increases were evoked by beta AP1-40. Responses to beta AP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca2+]i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of beta AP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by beta AP25-35 in voltage-clamp studies. We conclude that beta AP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca2+ influx. This property may explain the toxicity of beta AP25-35.