Altered expression and localization of hippocampal A-type potassium channel subunits in the pilocarpine-induced model of temporal lobe epilepsy.

Altered ion channel expression and/or function may contribute to the development of certain human epilepsies. In rats, systemic administration of pilocarpine induces a model of human temporal lobe epilepsy, wherein a brief period of status epilepticus (SE) triggers development of spontaneous recurrent seizures that appear after a latency of 2-3 weeks. Here we investigate changes in expression of A-type voltage-gated potassium (Kv) channels, which control neuronal excitability and regulate action potential propagation and neurotransmitter release, in the pilocarpine model of epilepsy. Using immunohistochemistry, we examined the expression of component subunits of somatodendritic (Kv4.2, Kv4.3, KChIPl and KChIP2) and axonal (Kv1.4) A-type Kv channels in hippocampi of pilocarpine-treated rats that entered SE. We found that Kv4.2, Kv4.3 and KChIP2 staining in the molecular layer of the dentate gyrus changes from being uniformly distributed across the molecular layer to concentrated in just the outer two-thirds. We also observed a loss of KChIP1 immunoreactive interneurons, and a reduction of Kv4.2 and KChIP2 staining in stratum radiatum of CA1. These changes begin to appear 1 week after pilocarpine treatment and persist or are enhanced at 4 and 12 weeks. As such, these changes in Kv channel distribution parallel the acquisition of recurrent spontaneous seizures as observed in this model. We also found temporal changes in Kv1.4 immunoreactivity matching those in Timm's stain, being expanded in stratum lucidum of CA3 and in the inner third of the dentate molecular layer. Among pilocarpine-treated rats, changes were only observed in those that entered SE. These changes in A-type Kv channel expression may contribute to hyperexcitability of dendrites in the associated hippocampal circuits as observed in previous studies of the effects of pilocarpine-induced SE.

Experimental localization of Kv1 family voltage-gated K+ channel alpha and beta subunits in rat hippocampal formation.

In the mammalian hippocampal formation, dendrotoxin-sensitive voltage-gated K(+) (Kv) channels modulate action potential propagation and neurotransmitter release. To explore the neuroanatomical basis for this modulation, we used in situ hybridization, coimmunoprecipitation, and immunohistochemistry to determine the subcellular localization of the Kv channel subunits Kv1.1, Kv1.2, Kv1.4, and Kvbeta2 within the adult rat hippocampus. Although mRNAs encoding all four of these Kv channel subunits are expressed in the cells of origin of each major hippocampal afferent and intrinsic pathway, immunohistochemical staining suggests that the encoded subunits are associated with the axons and terminal fields of these cells. Using an excitotoxin lesion strategy, we explored the subcellular localization of these subunits in detail. We found that ibotenic acid lesions of the entorhinal cortex eliminated Kv1.1 and Kv1.4 immunoreactivity and dramatically reduced Kv1.2 and Kvbeta2 immunoreactivity in the middle third of the dentate molecular layer, indicating that these subunits are located on axons and terminals of entorhinal afferents. Similarly, ibotenic acid lesions of the dentate gyrus eliminated Kv1.1 and Kv1.4 immunoreactivity in the stratum lucidum of CA3, indicating that these subunits are located on mossy fiber axons. Kainic acid lesions of CA3 dramatically reduced Kv1.1 immunoreactivity in the stratum radiatum of CA1-CA3, indicating that Kv1.1 immunoreactivity in these subfields is associated with the axons and terminals of the Schaffer collaterals. Together with the results of coimmunoprecipitation analyses, these data suggest that action potential propagation and glutamate release at excitatory hippocampal synapses are directly modulated by Kv1 channel complexes predominantly localized on axons and nerve terminals.

Association and colocalization of the Kvbeta1 and Kvbeta2 beta-subunits with Kv1 alpha-subunits in mammalian brain K+ channel complexes.

The differential expression and association of cytoplasmic beta-subunits with pore-forming alpha-subunits may contribute significantly to the complexity and heterogeneity of voltage-gated K+ channels in excitable cells. Here we examined the association and colocalization of two mammalian beta-subunits, Kvbeta1 and Kvbeta2, with the K+ channel alpha-subunits Kv1.1, Kv1.2, Kv1.4, Kv1.6, and Kv2.1 in adult rat brain. Reciprocal coimmunoprecipitation experiments using subunit-specific antibodies indicated that Kvbeta1 and Kvbeta2 associate with all the Kv1 alpha-subunits examined, and with each other, but not with Kv2.1. A much larger portion of the total brain pool of Kv1-containing channel complexes was found associated with Kvbeta2 than with Kvbeta1. Single- and multiple-label immunohistochemical staining indicated that Kvbeta1 codistributes extensively with Kv1.1 and Kv1.4 in cortical interneurons, in the hippocampal perforant path and mossy fiber pathways, and in the globus pallidus and substantia nigra. Kvbeta2 codistributes extensively with Kv1.1 and Kv1.2 in all brain regions examined and was strikingly colocalized with these alpha-subunits in the juxtaparanodal region of nodes of Ranvier as well as in the axons and terminals of cerebellar basket cells. Taken together, these data provide a direct demonstration that Kvbeta1 and Kvbeta2 associate and colocalize with Kv1 alpha-subunits in native tissues and provide a biochemical and neuroanatomical basis for the differential contribution of Kv1 alpha- and beta-subunits to electrophysiologically diverse neuronal K+ currents.

Voltage-gated K+ channel beta subunits: expression and distribution of Kv beta 1 and Kv beta 2 in adult rat brain.

Recent cloning of K+ channel beta subunits revealed that these cytoplasmic polypeptides can dramatically alter the kinetics of current inactivation and promote efficient glycosylation and surface expression of the channel-forming alpha subunits. Here, we examined the expression, distribution, and association of two of these beta subunits, Kv beta 1 and Kv beta 2, in adult rat brain. In situ hybridization using cRNA probes revealed that these beta-subunit genes are heterogeneously expressed, with high densities of Kv beta 1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kv beta 2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kv beta 1 and Kv beta 2 polypeptides frequently co-localize and are concentrated in neuronal perikarya, dendrites, and terminal fields, and in the juxtaparanodal region of myelinated axons. Immunoblot and reciprocal co-immunoprecipitation analyses indicated that Kv beta 2 is the major beta subunit present in rat brain membranes, and that most K+ channel complexes containing Kv beta 1 also contain Kv beta 2. Taken together, these data suggest that Kv beta 2 is a component of almost all K+ channel complexes containing Kv 1 alpha subunits, and that individual channels may contain two or more biochemically and functionally distinct beta-subunit polypeptides.

Generation and characterization of subtype-specific monoclonal antibodies to K+ channel alpha- and beta-subunit polypeptides.

Molecular characterization of mammalian voltage-sensitive K+ channel genes and their expression became possible with the cloning of the Shaker locus of Drosophila. However, analysis of the expression patterns and subunit composition of native K+ channel protein complexes requires immunological probes specific for the individual K+ channel gene products expressed in excitable tissue. Here, we describe the generation and characterization of monoclonal antibodies (mAbs) against eight distinct mammalian K+ channel polypeptides; the Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 Shaker-related alpha-subunits, the Kv2.1 Shab-related alpha-subunit, and the Kv beta 1 and Kv beta 2 beta-subunits. We characterized the subtype-specificity of these mAbs against native K+ channels in mammalian brain and against recombinant K+ channels expressed in transfected mammalian cells. In addition, we used these mAbs to investigate the cellular and subcellular distribution of the corresponding polypeptides in rat cerebral cortex, as well as their expression levels across brain regions.

Reinforcer magnitude (sucrose concentration) and the matching law theory of response strength.

This experiment investigated the relationship between reinforcer magnitude (sucrose concentration) and response rate. The purpose was to evaluate the behavior of two parameters of an equation that predicts absolute response rate as a function of reinforcement rate and two free parameters. According to Herrnstein's (1970) theory of reinforced behavior, one parameter of this "response-strength equation" measures the efficacy of the reinforcer maintaining responding and the other parameter measures motoric components of response rate, such as response duration. Seven rats served as subjects. Experimental sessions consisted of a series of five different variable-interval schedules of reinforcement, each in effect for 5 minutes. Within each session, obtained reinforcement rates varied over more than a 30-fold range, from about 20 per hour to 700 per hour. The reinforcer was sucrose solution, and, between sessions, its concentration was varied from 0.0 to 0.64 molar (0 to 21.9%). For sucrose concentrations of 0.16 to 0.64 m, response rate was a negatively accelerated function of reinforcement rate. Increases in sucrose concentration increased response rates maintained by low but not high reinforcement rates. This pattern of changes corresponds to a change in the reinforcement-efficacy parameter of the response-strength equation. In contrast, the motor-performance parameter did not change as a function of sucrose concentration. These findings are inconsistent with the results of a similar study (Bradshaw, Szabadi, & Bevan, 1978) but support Herrnstein's theory of reinforced behavior.

Low doses of cis-flupentixol attenuate motor performance.

We evaluated the effects of cis-flupentixol on reinforced responding. The experimental subjects were rats and the reinforced response was a lever press. The procedure was a five-component multiple schedule that provided five different reinforcement rates. Cis-flupentixol produced dose-dependent decreases in reinforced responding. An equation, the matching law, was fitted to the results. One parameter of this equation represents the estimated response rate asymptote. Cis-flupentixol produced dose-dependent decreases in the asymptotes. A second parameter of the equation represents the rate of reinforcement that maintains a one-half asymptotic response rate. Cis-flupentixol did not appear to affect this measure. There is evidence that the response rate asymptote measures motor components of response rate and that the reinforcement parameter measures the efficacy of the reinforcement maintaining the response. According to these results, cis-flupentixol systematically affected the motor-component of reinforced responding-it slowed down lever pressing-without affecting the subject's sensitivity to the reinforcer maintaining the response. In contrast, other neuroleptics have decreased the subjects' sensitivity to reinforcement, according to the matching law measures.