Role of the purinergic P2Y2 receptor in hippocampal function in mice.

OBJECTIVE: The aim of this study is to investigate the role of the purinergic P2Y2 receptor in learning and memory processes. MATERIALS AND METHODS: Behavioral, electrophysiological, and biochemical tests of memory function were conducted in P2Y2 receptor knockout (P2Y2R-KO) mice, and the findings were compared to those of wild-type mice with the help of unpaired Student's t-test. RESULTS: The findings of the behavioral Y-maze test showed that the P2Y2R-KO mice had impaired memory and cognitive function. Electrophysiological studies on paired-pulse facilitation showed that glutamate release was higher in the P2Y2R-KO mice than in the WT mice. Furthermore, PCR and Western blot analysis revealed that the mRNA and protein expression of acetylcholinesterase E (AChE) and alpha-7 nicotinic acetylcholine receptors (α7 nAChRs) were increased in the hippocampus of P2Y2R-KO mice. CONCLUSIONS: The findings of this study indicate that P2Y2 receptors are important regulators of both glutamatergic and cholinergic systems in the hippocampus.

Benzylpiperazine: "A messy drug".

Designer drugs are synthetic structural analogues/congeners of controlled substances with slightly modified chemical structures intended to mimic the pharmacological effects of known drugs of abuse so as to evade drug classification. Benzylpiperazine (BZP), a piperazine derivative, elevates synaptic dopamine and serotonin levels producing stimulatory and hallucinogenic effects, respectively, similar to the well-known drug of abuse, methylenedioxymethamphetamine (MDMA). Furthermore, BZP augments the release of norepinephrine by inhibiting presynaptic autoreceptors, therefore, BZP is a "messy drug" due to its multifaceted regulation of synaptic monoamine neurotransmitters. Initially, pharmaceutical companies used BZP as a therapeutic drug for the treatment of various disease states, but due to its contraindications and abuse potential it was withdrawn from the market. BZP imparts predominately sympathomimetic effects accompanied by serious cardiovascular implications. Addictive properties of BZP include behavioral sensitization, cross sensitization, conditioned place preference and repeated self-administration. Additional testing of piperazine derived drugs is needed due to a scarcity of toxicological data and widely abuse worldwide.

Impaired ILK Function Is Associated with Deficits in Hippocampal Based Memory and Synaptic Plasticity in a FASD Rat Model.

Fetal Alcohol Spectrum Disorder (FASD) is an umbrella term that encompasses a wide range of anatomical and behavioral problems in children who are exposed to alcohol during the prenatal period. There is no effective treatment for FASD, because of lack of complete characterization of the cellular and molecular mechanisms underlying this condition. Alcohol has been previously characterized to affect integrins and growth factor signaling receptors. Integrin Linked Kinase (ILK) is an effector of integrin and growth-factor signaling which regulates various signaling processes. In FASD, a downstream effector of ILK, Glycogen Synthase Kinase 3ß (GSK3ß) remains highly active (reduced Ser9 phosphorylation). GSK3ß has been known to modulate glutamate receptor trafficking and channel properties. Therefore, we hypothesize that the cognitive deficits accompanying FASD are associated with impairments in the ILK signaling pathway. Pregnant Sprague Dawley rats consumed a "moderate" amount of alcohol throughout gestation, or a calorie-equivalent sucrose solution. Contextual fear conditioning was used to evaluate memory performance in 32-33-day-old pups. Synaptic plasticity was assessed in the Schaffer Collateral pathway, and hippocampal protein lysates were used to evaluate ILK signaling. Alcohol exposed pups showed impaired contextual fear conditioning, as compared to control pups. This reduced memory performance was consistent with decrease in LTP as compared to controls. Hippocampal ILK activity and GSK3ß Ser21/9 phosphorylation were significantly lower in alcohol-exposed pups than controls. Increased synaptic expression of GluR2 AMPA receptors was observed with immunoprecipitation of post-synaptic density protein 95 (PSD95). Furthermore, immunoprecipitation of ILK revealed a decreased interaction with GluR2. The ILK pathway appears to play a significant role in memory and synaptic plasticity impairments in FASD rats. These impairments appear to be mediated by reduced GSK3ß regulation and increased synaptic stabilization of the calcium-impermeable GluR2 AMPA receptors.

Long-lasting teratogenic effects of nicotine on cognition: gender specificity and role of AMPA receptor function.

Nicotine, the main psychoactive ingredient in tobacco, readily crosses the placental barrier to cause growth and neurobehavioral abnormalities in the offspring. The current study was designed to assess whether nicotinic action causes long lasting teratogenic effects and synaptic dysfunctions. Pregnant Sprague-Dawley rats were infused with nicotine via osmotic minipumps at a dose of 6 mg/kg/day corresponding to the dose receiving during heavy smoking. A battery of behavioral tests and electrophysiological experiments were performed during specific postnatal periods. A spectrum of developmental and behavioral modifications in adolescent, young-adult and aged animals resulted after prenatal nicotine exposure. The potentially teratogenic effect of nicotine was clearly demonstrated in both genders by changes in developmental reflexes, exploratory and novelty seeking behavior, as well as a higher level of anxiety, and changes in individual and group responses in learning and memory. Most of the behavioral abnormalities were transitional with advancing age (6 months), although cognitive deficits measured by a two-way active avoidance task were long-lasting for male rats. Electrophysiological studies show decreased excitatory postsynaptic responses (mEPSCs) mediated by AMPA receptors in the hippocampus. These results suggest that teratogenic effect of nicotine on cognition is age and gender-specific, long-lasting and associated with AMPA receptor function.

Member of the Ampakine class of memory enhancers prolongs the single channel open time of reconstituted AMPA receptors.

Ampakines are small benzamide compounds that allosterically produce the positive modulation of AMPA receptors and improve performance on a variety of behavioral tasks. To test if the native synaptic membrane is necessary for the effects of such positive modulators, the mechanism of action of the Ampakine 1-(1,3-benzodioxol-5-ylcarbonyl)-1,2,3,6-tetrahydropyridine (CX509) was investigated in isolated rat brain AMPA receptors reconstituted in lipid bilayers. The drug increased the open time of AMPA-induced single channel current fluctuations with an EC(50) of 4 microM. The action of CX509 was highly selective since it had no effect on the amplitude or close time of channel events. The open time effect had a maximum enhancement of 70-fold and the modulated currents were blocked by CNQX. It is concluded that the synaptic membrane environment is not necessary for Ampakine effects. In fact, CX509 was about 100 times more potent on the reconstituted AMPA receptors than on receptors in their native membrane. These findings indicate that centrally active Ampakines modulate specific kinetic properties of AMPA currents. They also raise the possibility that AMPA receptors are regulated by factors present in situ, thus explaining the more efficient modulatory effects of CX509 when acting on receptors removed from their synaptic location.

Supplementation with Lactobacillus reuteri or L. acidophilus reduced intestinal shedding of cryptosporidium parvum oocysts in immunodeficient C57BL/6 mice.

The effect of L. acidophilus supplementation to reduce fecal shedding of Cryptosporidium parvum oocysts was compared to L. reuteri using C57BL/6 female mice immunosuppressed by murine leukemia virus (strain LP-BM5) inoculation. After 12 weeks post LP-BM5 inoculation, 15 immunosuppressed mice each were randomly assinged to one of the following treatment groups: historical control (group A), LP-BM5 control (group B), C. parvum (group C), L. reuteri plus C. parvum (group D) or L. acidophilus plus C. parvum (group E). Mice were pre-fed the L. reuteri or L. acidophilus bacteria strains daily for 13 days, challenged with C. parvum oocysts and thereafter fed the specified Lactobacillus regimens daily during the experimental period. Animals supplemented with L. reuteri shed fewer (p<0.05) oocysts on day-7 post C. parvum challenge compared to controls. Mice supplemented with L. acidophilus also shed fewer (p<0.05) oocysts on days 7 and 14 post-challenge compared to controls. Overall, Lactobacillus supplementation reduced C. parvum shedding in the feces but failed to suppress the production of T-helper type 2 cytokines [interleukin-4 (IL-4), IL-8)] which are associated with immunosuppression. Additionally, Lactobacillus supplementation did not restore T-helper type 1 cytokines (interleukin-2 (IL-2) and gamma interferon (IFN-gamma), which are required for recovery from parasitic infections. Altered T-helper types 1 and 2 cytokine production as a consequence of immunodysfunction permitted the development of persistent cryptosporidiosis while mice with intact immune system were refractory to infection with C. parvum. Reduction in shedding of oocysts observed in the Lactobacillus supplemented mice during deminished IL-2 and IFN-gamma production may be mediated by factors released into the intestinal lumen by the Lactobacillus and possibly other host cellular mechanisms. These observations suggest that L. reuteri or L. acidophilus can reduce C. parvum parasite burdens in the intestinal epithelium during cryptosporidiosis and may serve potential benefits as probiotics for host resistance to intestinal parasitic infections. L. acidophilus was more efficacious in reducing fecal shedding than L. reuteri and therefore may also have implication in the therapy of cryptosporidiosis during immunosuppressive states including human AIDS.

Heparin modulates the single channel kinetics of reconstituted AMPA receptors from rat brain.

Glutamate receptors specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have been reported to interact with the highly sulfated glycosaminoglycan, heparin, and to subsequently express lower binding affinity for [3H]AMPA. The present study examined whether heparin also modifies the kinetic properties of single channel activity expressed by isolated AMPA receptors from rat forebrain. Upon application of 280 nM AMPA, the partially purified receptors reconstituted in lipid bilayers expressed bursting channel activity that was inhibited by dinitroquinoxaline-2-3,-dione (DNQX). Treating the receptors with heparin (10 microg/ml) produced no change in conductance but the mean burst length for 280 nM AMPA was nearly doubled. Heparin also prolonged the lifetime of open states of the individual ion channels 3-5-fold, perhaps by causing a decrease in the closing rate constant for channel gating. Heparin had no effect on the lifetime of the closed state or on the amplitude of currents. The single channel open time was voltage-dependent and an increase of applied voltage caused a decrease in the heparin effect on channel open times. While the lifetime of the open channel was increased 3-4 times by heparin at 20 mV, there was no significant change induced at 43 mV. The equivalent electric charge of the channel gate was increased by 40%. The heparin effects were specific as another polysaccharide, dextran, and a monomeric constituent of heparin, glucosamine 2,3-disulfate, failed to have any effect on the receptors. These findings suggest that heparin-containing extracellular matrix components can interact with AMPA receptors and influence their functional properties.

Effects of heparin on the properties of solubilized and reconstituted rat brain AMPA receptors.

Heparin was found to bind to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and to alter their functional properties. AMPA receptors solubilized in 0.4% Triton X-100 bound to a heparin-agarose column and were eluted by 0.4 M NaCl. Soluble heparin inhibited 10 nM [3H]AMPA binding to detergent-solubilized receptors by 75% (IC50 = 10 micrograms/ml), but had little effect on binding to membrane-associated receptors. The inhibition of [3H]AMPA binding to detergent-solubilized receptors was not observed when binding was measured in the presence of 0.4 M NaCl, and no effect of heparin was observed on binding of the AMPA receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Scatchard analyses of [3H]AMPA binding to solubilized receptors revealed that the inhibition induced by heparin was caused by a decrease in the apparent affinity of a portion of the total binding sites. Studies on AMPA receptors reconstituted in artificial lipid bilayers indicated that 10 micrograms/ml heparin enhanced cooperativity between channels and prolonged the lifetime of the open channel, but did not affect the amplitude of single channel currents. Thus, heparin may be added to the list of compounds known to modulate AMPA receptor function. These data also raise the possibility that heparin-containing proteoglycans, which are known to be concentrated at synaptic junctions, might be able to bind AMPA receptors and influence their functional characteristics.

Single channel recordings of reconstituted AMPA receptors reveal low and high conductance states.

Glutamate receptors belonging to the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subclass were partially purified 30- to 60-fold from forebrain of adult rats and incorporated into planar bimolecular lipid membranes. The channel conductance associated with the reconstituted receptors was activated by kainate and AMPA in a manner that suggests cooperative binding of two to three agonist molecules is required to induce channel opening. This conductance was blocked by the specific antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). When the partially purified AMPA receptors were reconstituted by the tip-dipping method in asymmetric saline conditions ('outside-out configuration'), the addition of 300 nM AMPA to the pseudo-extracellular solution elicited single channel current fluctuations that were also inhibited by DNQX. Analyses of the currents revealed that the ion channels of reconstituted AMPA receptors have two distinct conductance levels of 12 and 60 pS with the great majority of receptors belonging to the former variety. These results suggest that reconstitution may be useful in identifying factors that regulate the binding and conductance properties of AMPA receptors.

Functional reconstitution of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors from rat brain.

Glutamate receptors belonging to the subclass specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were solubilized from rat forebrain membranes with Triton X-100 and partially purified through a series of three chromatographic steps. Specific [3H]AMPA binding increased 30-60-fold during the isolation procedure. A protein band recognized by antibodies against specific amino acid sequences of the glutamate receptor-A subunit was enriched with each purification step; the molecular mass of this band (105 kDa) corresponded to that of cloned AMPA receptor subunits. Photoaffinity labeling of forebrain membranes with 6-cyano-7-[3H]nitroquinoxaline-2,3-dione, a specific antagonist of the AMPA receptor, labeled a single band that comigrated with the immunolabeled protein. On reconstitution of the partially purified material into bilayer patches, single-channel current fluctuations were elicited by 300 nM AMPA and blocked by 1 microM 6,7-dinitroquinoxaline-2,3-dione.