Microtransplantation of neurotransmitter receptors from postmortem autistic brains to Xenopus oocytes

Abstract

Autism is a complex disorder that arises from the pervasive action of genetic and epigenetic factors that alter synaptic connectivity of the brain. Although GABA and glutamate receptors seem to be two of those factors, very little is known about the functional properties of the autistic receptors. Autistic tissue samples stored in brain banks usually have relatively long postmortem times, and it is highly desirable to know whether neurotransmitter receptors in such tissues are still functional. Here we demonstrate that native receptors microtransplanted from autistic brains, as well as de novo mRNA-expressed receptors, are still functional and susceptible to detailed electrophysiological characterization even after long postmortem intervals. The opportunity to study the properties of human receptors present in diseased brains not only opens new avenues toward understanding autism and other neurological disorders, but it also makes the microtransplantation method a useful translational system to evaluate and develop novel medicinal drugs.autism ͉ human brain ͉ GABA receptors ͉ glutamate receptors

GABA and Glutamate Receptors of the Autistic Brain

“…The other two cases generated larger responses. In contrast, when the receptors of the temporal cortex were microtransplanted, the amplitudes of the currents induced by kainate, glutamate, and GABA by the receptors from the autistic membranes were larger than their controls in two of the three autistic cases (Limon et al, 2008). Interestingly, even though the number of patients tested is low, deviations from the controls were already found, supporting the hypothesis of an altered GABAergic signalling in the autistic brain, thus encouraging further studies.…”

“…1 mg/mL) into the equator of the oocyte ReyesRuiz et al, 2010;Limon et al, 2010). Besides its use for the expression of recombinant proteins, Xenopus oocytes can also express membrane proteins after injection of mRNAs directly isolated from human biopsy tissue or from post-mortem brains (Gundersen et al, 1984;Palma et al, 2002;Limon et al, 2008). For the expression of mRNA, the quality of the starting material is important, specially making sure the tissue is not fixed, but snap frozen in liquid nitrogen or CO 2 .…”

“…For the expression of mRNA, the quality of the starting material is important, specially making sure the tissue is not fixed, but snap frozen in liquid nitrogen or CO 2 . We have isolated mRNAs that were able to express functional receptors and ion channels from tissue that was obtained several hours post-mortem and was stored frozen for several years (Limon et al, 2008). For the mRNA isolation procedure, it is preferable to start with at least one gram of tissue.…”

“…The evidence that single mutations of glutamate-and calcium channels can lead to autism indicates that abnormal network excitability and intracellular signalling are critical factors in the generation of autistic phenotypes; and highlights the importance of understanding the electrophysiological properties of the receptors and channels, even in cases where no mutations have been reported. The expression of mRNA or cloned human receptors in Xenopus oocytes is a potent tool to evaluate the functional properties of GABA-, glutamateand calcium-channels in the autistic brain (Limon et al, 2008). Next we describe the method we use to express human receptors in Xenopus oocytes.…”

“…Voltage-clamp recording is then used to study the functional characteristics of the transplanted receptors. Oocytes with transplanted receptors can be studied up to several days post-injection (Miledi et al, 2004;Limon et al, 2008). We have assessed whether autistic brains with long post-mortem intervals still contain functional neurotransmitter receptors and voltage-operated ion channels that could be microtransplanted into Xenopus oocytes.…”