This laboratory investigates the signal transduction in synaptic transmission and plasticity by biochemical, behavioral, and electrophysiological approaches using a strain of genetically modified mice. The mutant mouse was generated by deleting a gene coding for neural-specific protein, neurogranin (Ng). This protein is expressed at high levels in hippocampus, cerebral cortex, and amygdala, and has been implicated in the modulation of synaptic plasticity. Ng is a small molecular weight protein, which binds calmodulin (CaM) under basal physiological conditions when calcium level is low. The binding of Ng and CaM is weakened upon neuronal stimulation that leads to calcium influx. When dissociated from CaM, Ng is phosphorylated by PKC and/or oxidized by nitric oxide and other oxidants. The phosphorylated and oxidized Ngs are poor binding partners of CaM. These multifaceted regulations of Ng provide a fine tune mechanism to control the calcium transients and availability of CaM depending on the strength of stimulation to the neurons and, thus, gate the output response. Deletion of Ng gene in mice causes deficits in learning the hippocampus- and amygdala-dependent behavioral tasks, and the high-frequency stimulation (HFS)-induced long-term potentiation (LTP). In human, mutation of Ng gene has been linked to cognitive deficits, schizophrenia, and bipolar disorder. Deletion of Ng in mice (NgKO) caused deficits in calcium-mediated signaling reactions in hippocampus, impairment in learning and memory, and high frequency stimulation (HFS)-induced long-term potentiation (LTP) in hippocampal slices. Further characterization revealed that these animals also exhibited other behavioral abnormalities, including hyperactivity and deficiency in social interaction. These behavioral deficits could not be improved by environmental enrichment (EE) alone. We have treated these NgKO mice with Ritalin while they were also kept under EE conditions. Ritalin is a psychostimulant drug known to increase the extracellular neurotransmitters. Four groups of animals (including control and drug-treated wild type and NgKO mice) were injected with Ritalin (10 mg/kg/day, i.p.) or saline for three weeks and, afterward, subjected to a battery of behavioral tests. The drug-treated NgKO mice exhibited improvement in their cognitive functions as evidenced by a reduction of the latency time to locate the hidden platform in the water maze and an increase in the freezing time after fear-conditioning. Ritalin also reduced the hyperactivity of NgKO in the open field and an increase in the immobility time in the forced-swim chamber. The drug-treated mutant mice also exhibited improvement of their social interaction with other subjects and recognition of novel ones. The drug treatment, however, only had a marginal effect on the performances of the wild type mice. Measurement of the HFS-induced LTP in the hippocampal CA1 region in vitro showed a positive effect of the drug on the NgKO. At the cellular level, treatment of NgKO with Ritalin increased glial fibrillary acidic protein (GFAP)-positive astrocytes in the hippocampus, especially prominent in the hilus of dentate gyrus and stratum radiatum of the CA1 region. In the hilus, a large number of astrocytes were congregated at the subgranular zone, where subpopulations of these cells are known to be neural precursor cells. Indeed, the drug-treated NgKO exhibited an increase in the number of proliferative Ki-67-positive cells and doublecortin-positive neuronal precursor cells. This structural remodeling may underlie drug-mediated neurobehavioral responses. These results indicate that Ritalin, a drug commonly used for the treatment of attention-deficit hyperactivity disorder (ADHD), can exert beneficial effects on the NgKO. These studies also suggest that NgKO mice will be useful for the development of new treatment strategy for certain behavioral deficits related to ADHD. In neurons, stimulation of PKC is known to enhance transmitter release and facilitate postsynaptic responses by insertion of AMPA receptors. Short term treatment of hippocampal slices from NgKO mice with the PKC-activating phorbol ester caused synaptic facilitation at the hippocampal CA1 region that lasted for several hours. These treatments also promoted the redistribution of PKCs from soma to dendrites. The phorbol ester-mediated effects were most prominent among those tissue slices from dorsal hippocampus, which exhibited positive responses in the field excitatory postsynaptic potential (fEPSP) and amplitude of population spike (POPS). In contrast, for tissue slices from ventral hippocampus, phorbol ester only enhanced the amplitude of POPS without significantly affecting fEPSP. For the dorsal hippocampal slices, the phorbol ester-induced stimulation in fEPSP was inhibited by PKC inhibitor chelerythrine but not by CaMKII inhibitor, KN93, MEK inhibitor, U0126, protein synthesis inhibitor, anisomycin, nor NMDA receptor antagonist, APV. Following a maximal stimulation by phorbol ester, application of theta-burst stimulation (TBS) caused no additional response. However, TBS followed by phorbol ester caused additional potentiation of fEPSP, suggesting that the phorbol ester-mediated responses also overlap with those by TBS. It is intriguing that for the tissue slices from ventral hippocampus, application of phorbol ester followed by TBS induced depotentiation. These findings clearly differentiate the physiological responses of the dorsal versus ventral hippocampus to stimulation by PKC. The positive responses of dorsal hippocampus of NgKO mice to PKC-mediated long-term facilitation suggests that treatment of these animals with activator of PKC may improve the synaptic efficacy of this area, which is thought to associate with cognitive functions.

Project Start
Project End
Budget Start
Budget End
Support Year
33
Fiscal Year
2012
Total Cost
$633,790
Indirect Cost
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Huang, F L; Huang, K-P (2012) Methylphenidate improves the behavioral and cognitive deficits of neurogranin knockout mice. Genes Brain Behav 11:794-805
Huang, Kuo-Ping; Huang, Freesia L (2011) Calcium-Sensitive Translocation of Calmodulin and Neurogranin between Soma and Dendrites of Mouse Hippocampal CA1 Neurons. ACS Chem Neurosci 2:223-230
Shetty, Pavan K; Huang, Freesia L; Huang, Kuo-Ping (2008) Ischemia-elicited oxidative modulation of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 283:5389-401