Voltage-gated potassium (K+) channels display a wide variety of conductances and gating properties in vivo. This diversity can be attributed not only to the presence of many K(+)-channel gene products, but also to the possibility that different K(+)-channel subunits co-assemble to form heteromultimeric channels in vivo. When expressed in Xenopus oocytes or transfected cells, K(+)-channel polypeptides assemble to form tetramers. Certain combinations of Shaker-like subunits have been shown to co-assemble, forming heteromultimeric channels with distinct properties. It is not known, however, whether K(+)-channel polypeptides form heteromultimeric channels in vivo. Here we describe the co-localization of two Shaker-like voltage-gated K(+)-channel proteins, mKv1.1 and mKv1.2, in the juxtaparanodal regions of nodes of Ranvier in myelinated axons, and in terminal fields of basket cells in mouse cerebellum. We also show that mKv1.1 and mKv1.2 can be coimmunoprecipitated with specific antibodies that recognize only one of them. These data indicate that the two polypeptides occur in subcellular regions where rapid membrane repolarization may be important and that they form heteromultimeric channels in vivo.

Mice lacking the voltage-gated potassium channel alpha subunit, K(V)1.1, display frequent spontaneous seizures throughout adult life. In hippocampal slices from homozygous K(V)1.1 null animals, intrinsic passive properties of CA3 pyramidal cells are normal. However, antidromic action potentials are recruited at lower thresholds in K(V)1.1 null slices. Furthermore, in a subset of slices, mossy fiber stimulation triggers synaptically mediated long-latency epileptiform burst discharges. These data indicate that loss of K(V)1.1 from its normal localization in axons and terminals of the CA3 region results in increased excitability in the CA3 recurrent axon collateral system, perhaps contributing to the limbic and tonic-clonic components of the observed epileptic phenotype. Axonal action potential conduction was altered as well in the sciatic nerve--a deficit potentially related to the pathophysiology of episodic ataxia/myokymia, a disease associated with missense mutations of the human K(V)1.1 gene.

Previous studies have shown that estradiol induces new dendritic spines and synapses on hippocampal CA1 pyramidal cells. We have assessed the consequences of estradiolinduced dendritic spines on CA1 pyramidal cell intrinsic and synaptic electrophysiological properties. Hippocampal slices were prepared from ovariectomized rats treated with either estradiol or oil vehicle. CA1 pyramidal cells were recorded and injected with biocytin to visualize spines. The association of dendritic spine density and electrophysiological parameters for each cell was then tested using linear regression analysis. We found a negative relationship between spine density and input resistance; however, no other intrinsic property measured was significantly associated with dendritic spine density. Glutamate receptor autoradiography demonstrated an estradiol-induced increase in binding to NMDA, but not AMPA, receptors. We then used input/output (I/O) curves (EPSP slope vs stimulus intensity) to determine whether the sensitivity of CA1 pyramidal cells to synaptic input is correlated with dendritic spine density. Consistent with the lack of an estradiol effect on AMPA receptor binding, we observed no relationship between the slope of an I/O curve generated under standard recording conditions, in which the AMPA receptor dominates the EPSP, and spine density. However, recording the pharmacologically isolated NMDA receptor-mediated component of the EPSP revealed a significant correlation between I/O slope and spine density. These results indicate that, in parallel with estradiol-induced increases in spine/synapse density and NMDA receptor binding, estradiol treatment increases sensitivity of CA1 pyramidal cells to NMDA receptor-mediated synaptic input; further, sensitivity to NMDA receptor-mediated synaptic input is well correlated with dendritic spine density. Key words: estradiol; dendritic spines; hippocampal slice; CA1 pyramidal cells; biocytin; autoradiography; NMDA receptorSince the initial anatomical description of dendritic spines by Ramon y Cajal in the late 1800s, many investigators have speculated about dendritic spine function. Spines have been proposed to play primarily connective, electrical, and /or biochemical roles in neuronal physiology (for review, see Koch and Zador, 1992;Horner, 1993;Harris and Kater, 1994). Recent efforts to understand dendritic spine function based on imaging of dye-labeled spiny dendrites have yielded valuable information, particularly with regard to spines' potential for Ca 2ϩ compartmentalization (Guthrie et al., 1991;Muller et al., 1991). Such findings have been incorporated into proposals for dendritic spine function in synaptic integration (see, for example, Yuste and Denk, 1995) and neuroprotection (Harris and Kater, 1994;Segal, 1995). An additional approach to exploring dendritic spine function, which has not been used to date, is to assess the functional consequences of adding dendritic spines to the dendrites of spiny neurons.Hippocampal CA1 pyramidal cells are an ideal population of neurons in w...

The mammalian protein ZnT3 resides on synaptic vesicle membranes of zinc-containing neurons, suggesting its possible role in vesicular zinc transport. We show here that histochemically reactive zinc, corresponding to the zinc found within synaptic vesicles, was undetectable in the brains of mice with targeted disruption of the ZnT3 gene. Total zinc levels in the hippocampus and cortex of these mice were reduced by about 20%. The ultrastructure of mossy fiber boutons, which normally store the highest levels of vesicular zinc, was unaffected. Mice with one normal ZnT3 allele had reduced levels of ZnT3 protein on synaptic vesicle membranes and had intermediate amounts of vesicular zinc. These results demonstrate that ZnT3 is required for transport of zinc into synaptic vesicles and suggest that vesicular zinc concentration is determined by the abundance of ZnT3.

Multiple voltage-gated potassium (K) channel gene products are likely to be involved in regulating neuronal excitability of any single neuron in the mammalian brain. Here we show that two closely related voltage-gated K channel proteins, mKv1.1 and mKv1.2, are present in multiple subcellular locations including cell somata, juxta-paranodal regions of myelinated axons, synaptic terminals, unmyelinated axons, specialized junctions among axons, and proximal dendrites. Staining patterns of the two channel polypeptides overlap in some areas of the brain, yet each has a unique pattern of expression. For example, in the hippocampus, both mKv1.1 and mKv1.2 proteins are present in axons, often near or at synaptic terminals in the middle molecular layer of the dentate gyrus, while only mKv1.1 is detected in axons and synaptic terminals in the hilar/CA3 region. In the cerebellum, both channel proteins are localized to axon terminals and specialized junctions among axons in the plexus region of basket cells. Strong differential staining is observed in the olfactory bulb, where mKv1.2 is localized to cell somata and axons, as well as to proximal dendrites of the mitral cells. This overlapping yet differential pattern of expression and specific subcellular localization may contribute to the unique profile of excitability displayed by a particular neuron.

Electrophysiological and anatomical techniques were used to determine the role, in the hippocampal circuitry, of local circuit neurons located at the oriens/alveus border (O/A interneurons). Intracellular recording from these cells showed that their response characteristics were clearly nonpyramidal: high input resistance, short membrane time constant, short-duration action potential, pronounced, brief afterhyperpolarizations (AHP), and nondecremental firing during intrasomatic depolarizing current pulses. Intracellular Lucifer yellow (LY) injection and subsequent fluorescence microscopy confirmed their nonpyramidal nature. O/A interneuron somata were bipolar or multipolar; their dendrites projected mostly parallel to the alveus, except for 1 or 2 processes that turned perpendicularly, and ascended through stratum oriens and pyramidale and into radiatum. Their axons were seen to branch profusely in stratum oriens and pyramidale. Simultaneous intracellular recordings from O/A interneurons and CA 1 pyramidal cells showed that pyramidal cells directly excite these interneurons. Major hippocampal afferents also directly excited the O/A interneurons. In a small number of interneuron-pyramidal pairs, stimulation of the O/A interneuron directly inhibited pyramidal cells. In one case, reciprocal connections were observed: The pyramidal cell excited the interneuron, and the interneuron inhibited the pyramidal cell. In 1 interneuron-to-interneuron pair, an inhibitory connection from O/A interneuron to stratum pyramidale interneuron was also observed. With intracellular HRP injections into O/A interneurons and subsequent electron microscopy, we observed that O/A interneuron axons made contacts with pyramidal and nonpyramidal cells. HRP-filled symmetric synaptic contacts were found on pyramidal cell dendrites and somata. HRP-filled axons also made contacts with pyramidal cell initial segments. HRP-filled O/A interneuron axon contacts were also found on nonpyramidal cell dendrites in stratum oriens. These electrophysiological and anatomical results suggest that O/A interneurons make synaptic contact with pyramidal cells and may mediate feedforward and feedback inhibition onto CA 1 pyramidal cells.

The tumor suppressor gene p53 has been implicated in the loss of neuronal viability, but the signaling events associated with p53-mediated cell death in cortical and hippocampal neurons are not understood. Previous work has shown that adenovirus-mediated delivery of the p53 gene causes cortical and hippocampal neuronal cell death with some features typical of apoptosis. In the present study we determined whether p53-initiated changes in neuronal viability were dependent on members of the Bcl-2 family of cell death regulators. Primary cultures of cortical neurons were derived from animals containing Bax (+/+ and +/-) or those deficient in Bax (-/-). Cell damage was assessed by direct cell counting and by measurements of MTT activity. Neurons containing at least one copy of the Bax gene were damaged severely by exposure to excitotoxins or by the induction of DNA damage. In contrast, Bax-deficient neurons (-/-) exhibited significant protection from both types of injury. Bax protein expression was elevated significantly by glutamate exposure, but not by camptothecin-induced DNA damage in wild-type neurons. The glutamate-induced increase in Bax protein was dependent on the presence of the p53 gene. However, increased p53 expression, using adenovirus-mediated transduction, was not sufficient by itself to elevate Bax protein levels. These results demonstrate that Bax is required for neuronal cell death in response to some forms of cytotoxic injury and further support the key role for p53 activation in response to excitotoxic and genotoxic injury.

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