Construction Of Forebrain-specific VGLUT1 Overexpression Transgenic Mice And Its Preliminary Phenotypes

Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system(CNS).Up to now,three isoforms of type-ⅠNa~+-dependent vesicular glutamate transporters,termed VGLUTs(VGLUT1,VGLUT2 and VGLUT3),have been cloned.VGLUTs mediate the packaging of the glutamate into synaptic vesicles. The driving force for glutamate transport into synaptic vesicles is provided by a proton electrochemical gradient generated by the vacuolar H~+-ATPase.Studies on VGLUT1-deficient mice have led various models to be postulated concerning the possible roles of VGLUTs in synaptic physiology,such as presynaptic regulation of quantal size and activity-dependent short-term plasticity.In the present study,VGLUT1 gene was cloned from the cDNA library of the brain of C57BL/6 mouse or Sprague Dawley rat.First,the cDNA of VGLUT1 was cloned to p265 vector.Then,the sequences together with intron and SV40 polyA were inserted into p279 vector containingα-CaMKⅡpromoter.Two transgenic founders were produced by pronuclear injection of the linearized DNA into B6/CBAF1 inbred zygotes,and then intercrossed with C57BL/6 for various analyses.The genotype of the transgenic mice was analyzed by PCR and Southern blot.The size ofpresynaptic vesicles in the hippocampus and cerebral cortex was detected by electron microscopy. Electrochemical detection of four neurotransmitters,including dopamine(DA), serotonin(5-HT),epinephrine(E),norepinephrine(NE),the outcome showed that there was no significant change in neurotransmitters from brain tissues.To test whether the VGLUT1 is crucial for presynaptic regulation or refinement which involves in learning and memory,we divided the transgenic mice and the controls into 4 groups dependent on the ages.Behavior tests demonstrated that increased locomotor activity was apparent in the transgenic mice at 12 months of age. Elevated plus maze and novel object recognition task results showed that there was no significant abnormality between transgenic mice and wildtype.6 months aged transgenic mice exhibited a deficiency in long-term memory of contextual fear condition,whereas 3 months transgenic mice showed no obvious impairment.These studies demonstrate that forebrain specific overexpression of VGLUT1 possibly involve in learning impairments in an age-dependent manner.