BACKGROUND:Parkinson's disease (PD) is a progressive neurodegenerative condition characterized by an increasing loss of dopaminergic neurons resulting in motor dysfunction. However, cognitive impairments in PD patients are a common clinical feature that has gained increased attention.OBJECTIVE:The purpose of the current study was to evaluate the effects of an MPTP-induced dopaminergic lesion in mice on social odor recognition (SOR) memory.METHODS:Mice were acutely treated with MPTP and evaluated for memory impairments in the SOR assay and characterized using biochemical and immunohistochemical methods approximately 2 weeks later.RESULTS:Here we demonstrate that SOR memory is sensitive to MPTP treatment and that it correlates with multiple measures of nigrostriatal integrity. MPTP treatment of C57BL/6N mice produced a profound decrease in dopamine levels, dopamine transporter binding and tyrosine hydroxylase immunoreactivity in the striatum. These impairments in stratial dopaminergic function were blocked by pretreatment with the MAO-B inhibitor deprenyl. Changes in the dopaminergic system parallel those observed in SOR with MPTP treatment impairing recognition memory in the absence of a deficit in odor discrimination during learning. Deprenyl pretreatment blocked the MPTP-induced impairment of SOR memory.CONCLUSION:The use of the SOR memory model may provide a preclinical method for evaluating cognitive therapies for PD.

The c-Jun N-terminal kinase (JNK) pathway potentially links together the three major pathological hallmarks of Alzheimer's disease (AD): development of amyloid plaques, neurofibrillary tangles, and brain atrophy. As activation of the JNK pathway has been observed in amyloid models of AD in association with peri-plaque regions and neuritic dystrophy, as we confirm here for Tg2576/PSM146L transgenic mice, we directly tested whether JNK inhibition could provide neuroprotection in a novel brain slice model for amyloid precursor protein (APP)-induced neurodegeneration. We found that APP/amyloid β (Aβ)-induced neurodegeneration is blocked by both small molecule and peptide inhibitors of JNK, and provide evidence that this neuroprotection occurs downstream of APP/Aβ production and processing. Our findings demonstrate that Aβ can induce neurodegeneration, at least in part, through the JNK pathway and suggest that inhibition of JNK may be of therapeutic utility in the treatment of AD.

The diuretic amiloride has recently proven neuroprotective in models of cerebral ischemia, a property attributable to the drug's inhibition of central acid-sensing ion channels (ASICs). Given that Parkinson's disease (PD), like ischemia, is associated with cerebral lactic acidosis, we tested amiloride in the MPTP-treated mouse, a model of PD also manifesting lactic acidosis. Amiloride was found to protect substantia nigra (SNc) neurons from MPTP-induced degeneration, as determined by attenuated reductions in striatal tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry, as well as smaller declines in striatal DAT radioligand binding and dopamine levels. More significantly, amiloride also preserved dopaminergic cell bodies in the SNc. Administration of psalmotoxin venom (PcTX), an ASIC1a blocker, resulted in a much more modest effect, attenuating only the deficits in striatal DAT binding and dopamine. These findings represent the first experimental evidence of a potential role for ASICs in the pathogenesis of Parkinson's disease.

The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice produces a reliable and selective degeneration of the nigrostriatal pathway, a hallmark feature of Parkinson's disease (PD). Determining the brain concentrations of 1-methyl-4-phenyl pyridium (MPP+), the neurotoxic metabolite of MPTP, is critical for evaluating drugs designed to potentially treat PD. We have developed sensitive and specific quantitative methods for the determination of MPP+ in mouse striatal tissue by liquid chromatography/tandem mass spectrometry. The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer. Neutralizing the brain sample with 0.2M phosphate buffer successfully solved a high-performance liquid chromatography (HPLC) peak tailing of MPP+ in brain extracts with 0.4M perchloric acid (HClO4) under the reversed phase HPLC conditions, which significantly improved the sensitivity of the method. The HPLC peak shape of MPP+ using cation exchange chromatography was not affected by the pH of the samples. Optimization of electrospray ionization (ESI) conditions for the quaternary ammonium compound MPP+ established the limits of detection (LOD) (S/N=3) at 0.34pg/mg tissue and 0.007pg/mg tissue (5microl of injection) using the reversed phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the cation exchange LC/MS/MS, respectively. Both methods were selective, precise (%R.S.D.<6%), and sensitive over a range of 0.001-1ng/mg tissue. The cation exchange method showed greater sensitivity and tolerance to low pH samples than the reversed phase method. The developed methods were applied to monitoring changes in MPP+ concentrations in vivo. Two reference agents, R-(-) Deprenyl and MK-801, known to alter the concentration of MPP+ in MPTP treated mice were evaluated.

Kv1.1 channels are expressed in many regions of the brain and spinal cord [Monaghan, M. M.; Trimmer, J. S.; Rhodes, K. J. J. Neurosci.2001, 21, 5973; Rasband, M. N.; Trimmer, J. S. J. Comp. Neurol.2001, 429, 166; Trimmer, J. S.; Rhodes, K. J. Ann. Rev. Physiol.2004, 66, 477]. When expressed alone, they produce a delayed rectifier slowly inactivating type current that contributes to hyperpolarizing the neuron following depolarization. In the hippocampus Kv1.1 is co-expressed with Kvbeta1 (and other beta subunits), which converts Kv1.1 into a transient, fast inactivating current, reducing its ability to hyperpolarize the cell and thus increasing neuronal excitability. To reduce neuronal excitability, screening for compounds that prevent inactivation of Kv1.1 channels by Kvbeta1 was performed using a yeast two-hybrid screen. A variety of compounds were discovered in this assay and subsequently determined to disrupt inactivation of the ionic currents, and hence were termed 'disinactivators'. Several of these disinactivators also inhibited pentylenetetrazole-induced seizures (PTZ) in mice. Compounds were found to act by several mechanisms to prevent Kvbeta1 inactivation of Kv1.1 channels, including enhancement of Ca(2+) release/influx and by direct mechanisms. Two structural classes were identified that act on a Kvbeta1N70-Kv1.1 chimera where the N-terminal 70 amino acids of Kvbeta1 were attached to the N-terminus of Kv1.1. It is likely that these disinactivators act directly on the Kvbeta1 N-terminus or its receptor site on Kv1.1, thus preventing it from blocking Kv1.1 channels. Compounds acting by this mechanism may be useful for reducing neuronal hyperexcitability in diseases such as epilepsy and neuropathic pain.

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.

Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs ( Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family alpha subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation ( An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family alpha subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 alpha subunits in brain membranes, indicating that KChIPs 1 - 4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.

Kenneth J. Rhodes,1 Karen I. Carroll,1 M. Amy Sung,1 Lisa C. Doliveira,1 Michael M. Monaghan,1 Sharon L. Burke,1 Brian W. Strassle,1 Lynn Buchwalder,2 Milena Menegola,4 Jie Cao,3 W. Frank An,3 and James S. Trimmer2,4 1Neuroscience, Wyeth Discovery Research, Princeton, New Jersey 08543, 2Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794, 3Millennium Pharmaceuticals, Cambridge, Massachusetts 02139, and 4Department of Pharmacology, School of Medicine, University of California, Davis, California 95616

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.

The differential expression and association of cytoplasmic β-subunits with pore-forming α-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 β-subunits, Kvβ1 and Kvβ2, with the K+ channel α-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 Kvβ1 and Kvβ2 associate with all the Kv1 α-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 Kvβ2 than with Kvβ1. Single- and multiple-label immunohistochemical staining indicated that Kvβ1 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. Kvβ2 codistributes extensively with Kv1.1 and Kv1.2 in all brain regions examined and was strikingly colocalized with these α-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 Kvβ1 and Kvβ2 associate and colocalize with Kv1 α-subunits in native tissues and provide a biochemical and neuroanatomical basis for the differential contribution of Kv1 α- and β-subunits to electrophysiologically diverse neuronal K+ currents.

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 α-subunits, the Kv2.1 Shab-related α-subunit, and the kvβ1 and Kvβ2 β-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. Copyright © 1996 Elsevier Science Ltd

Recent cloning of K+channel β 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 α subunits. Here, we examined the expression, distribution, and association of two of these β subunits, Kvβ1 and Kvβ2, in adult rat brain.In situhybridization using cRNA probes revealed that these β-subunit genes are heterogeneously expressed, with high densities of Kvβ1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kvβ2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kvβ1 and Kvβ2 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 Kvβ2 is the major β subunit present in rat brain membranes, and that most K+channel complexes containing Kvβ1 also contain Kvβ2. Taken together, these data suggest that Kvβ2 is a component of almost all K+channel complexes containing Kv1 α subunits, and that individual channels may contain two or more biochemically and functionally distinct β-subunit polypeptides.

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.

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.

The relation between response rate and reinforcement rate is described by the matching law equation. For an experiment in which there is just one explicit source of reinforcement, the equation has two parameters. The magnitude of one is equal to the response rate asymptote; the magnitude of the other is equal to the rate of reinforcement that maintains a one-half asymptotic response rate. This report describes experimental manipulations that affect these two parameters. Rats were trained on a series of variable-interval reinforcement schedules that provided reinforcement rates ranging from about 20 to 700 reinforcements per hour. The response was a lever press, and the reinforcer was water In Experiment I, the duration of the deprivation period was varied. Response rates maintained by the lower reinforcement rates showed the largest changes, and, accordingly, the parameter that is equal to the reinforcement rate for a one-half asymptotic response rate changed. In Experiment 2, the weight of the lever was varied. Response rates changed independently of reinforcement rate, and, as a result, the parameter that is equal to the asymptotic response rate changed. In Experiment 3, manipulations from Experiments 1 and 2 were combined. The results replicated those of Experiments 1 and 2, and there was no evidence of interactions. Our interpretation is that the asymptote of the matching law equation is a measure of motor performance and that the reinforcement parameter is a measure of the efficacy of the reinforcer maintaining the response. The matching law describes the relation between measures of reinforcement, such as amount and delay, and measures of behavior, such as rate and latency. The relations are described mathematically, with the terms and operations depending on such factors as the number of reinforcement sources, whether reinforcers are available simultaneously or sequentially, and the delay from response to reinforcement . Applications have varied, and they include social psychology experiments in which the frequency of conversations was the dependent variable (Conger & Killeen, 1974) and ethological studies in which the amount of time spent foraging was the measure of interest (Houston, 1986). The most elementary matching law equation applies to a situation in which there is just one measured reinforcement source, just one measured behavior, and no delay. This equation was introduced by Herrnstein (1970), and it is written as follows: