Regulation of excitatory transmission at hippocampal synapses by calbindin D 28 k ( calcium / adenovirus / synaptic plasticity )

Distinct subpopulations of neurons in the brain contain one or more of the Ca2+-binding proteins calbindin D28k calretinin, and parvalbumin. Although it has been shown that these high-afflnity Ca2+-binding proteins can increase neuronal Ca2+ buffering capacity, it is not clear which aspects of neuronal physiology they normally regulate. To investigate this problem, we used a recently developed method for expressing calbindin D28k in the somatic and synaptic regions of cultured hippocampal pyramidal neurons. Ninety-six hours after infection with a replication-defective adenovirus containing the calbindin D28k gene, essentially all cultured hippocampal pyramidal neurons robustly expressed calbindin D28k. Our results demonstrate that while calbindin D28k does not alter evoked neurotransmitter release at excitatory pyramidal cell synapses, this protein has a profound effect on synaptic plasticity. In particular, we show that calbindin D28k expression suppresses posttetanic potentiation. 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 cDNA The 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 have used this vector to express calbindin D28k in cultured hippocampal pyramidal neurons and have studied the protein's effects on synaptic