The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and γ-aminobutyric acid receptors.
Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors’ function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and γ-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.
Neurodegenerative disorders affect a large percentage of the population. Although their devastating consequences are well known, the link between the functional alterations of neurotransmitter receptors, which are critical for brain function, and their symptomatology is still poorly understood. Inter-individual variability, chronic nature of the disease, and insidious onset of symptoms are just some of the reasons that have delayed the understanding of the many brain disorders where chemical imbalances are well documented1,2. Animal models have generated invaluable information and expanded our knowledge about the mechanisms underlying physiology and pathophysiology in evolutionary conserved systems; however, several interspecies differences between rodents and humans preclude the direct extrapolation of receptor function from animal models to the human brain3. Thus, initial efforts to study native human receptors were developed by Ricardo Miledi's lab using surgically removed tissue and frozen samples. These initial experiments used whole membranes that include neuronal synaptic and extra synaptic receptors as well as non-neuronal neurotransmitter receptors, and although they provide important information about diseased states, there is a concern that the mix of receptors complicates the interpretation of data4,5,6,7. Importantly, synapses are the major target in many neurodegenerative disorders8,9; therefore, assays to test the functional properties of affected synapses are fundamental to obtain information about disease-relevant changes affecting synaptic communication. Here, a modification of the original method is described: microtransplantation of synaptic membranes (MSM), which focuses on the physiological characterization of enriched synaptic protein preparations and has been successfully applied to study rat and human synaptosomes10,11,12,13,14,15. With this methodology, it is possible to transplant synaptic receptors that were once working in the human brain, embedded in their own native lipids and with their own cohort of associated proteins. Moreover, because MSM data is quantitative, it is possible to use this data to integrate with large proteomic or sequencing datasets10.
It is important to note that many pharmacological and biophysical analyses of synaptic receptors are done on recombinant proteins16,17. While this approach provides better insight into the structure-function relationships of receptors, it cannot provide information about complex multimeric receptor complexes found in neurons and their changes in disease. Therefore, a combination of native and recombinant proteins should provide a more comprehensive analysis of synaptic receptors.
There are many methods to prepare synaptosomes10,11,12,13,14,15 which can be adjusted for the requirements of a lab. The protocol begins with the assumption that synaptosomal enriched preparations were isolated and are ready to be processed for microtransplantation experiments. In the lab, the Syn-Per method is used following the manufacturer instructions. This is done because of high reproducibility in electrophysiological experiments10,11. There is also abundant literature explaining how to isolate Xenopus oocytes18,19, which can also be purchased ready for injection20.
All research is performed in compliance with institutional guidelines and approved by the institutional Animal Care and Use Committee of the University of California Irvine (IACUC-1998-1388) and the University of Texas Medical Branch (IACUC-1803024). Temporal cortex from a non-Alzheimer's disease (AD) brain (female, 74 years old, postmortem interval 2.8 h) and an AD-brain (female, 74 years old, postmortem interval 4.5 h) were provided by the University of California Irvine Alzheimer's disease research center (UCI-ADRC). Informed consent for brain donation was obtained by UCI-ADRC.
NOTE: Unfixed human brain tissue should be treated as a source of blood borne pathogens (BBP). Accordingly, BBP training is needed prior to start experiments. This protocol is performed in a biosafety level 2 (BSL2) laboratory under BSL2 requirements. Guidelines and precautions within the laboratory include: no food or drinks allowed in the laboratory, good laboratory practices must be followed, personal protection equipment (gloves, gown, no open-toed shoes) is required, and the door must be closed at all times.
1. Xenopus oocyte microinjection preparation
2. Loading the sample
3. Injecting the oocytes
NOTE: Standardized rat cortex synaptosomal membranes are also injected in all experiments into a set of oocytes to measure changes in fusion capacity between different batches of oocytes.
4. Recording of ion currents using a two electrode voltage clamp
5. Analyzing TEVC recordings
Within a few hours after injection, the synaptic membranes, carrying their neurotransmitter receptors and ion channels, begin to fuse with the oocyte plasma membrane. Figure 1 shows recordings of AMPA and GABAA receptors microtransplanted into Xenopus oocytes. For most of the analysis, the responses from two or three oocytes per sample were measured, using two or three batches of oocytes from different frogs, for a total of six to nine oocytes per sample. This is done for a large cohort of human subjects to observe group differences. The analysis is straightforward and measures amplitude of responses. It is important to note that the fusion of the transplanted membranes is a polarized process, thus selection of the injection site is very important. Injection into the vegetal hemisphere or into the equator gives consistent results, with all injected oocytes successfully inserting receptors and generating ion currents that follow a unimodal distribution22. Interestingly, when the membranes were initially injected into the animal pole, the oocyte responses followed a bimodal distribution: one group of oocytes had none or very low responses while the other group had large responses, even larger than those from oocytes injected in the vegetal pole or in the equator. To determine if some of the oocytes injected in the animal pole had low responses because the human membranes were being injected and trapped within the nucleus of the oocyte, a mix of membranes isolated from the electrical organ of Torpedo and cDNA coding for the GABAρ1 subunit was injected into the pole of the animal side. The nicotinic receptors from the Torpedo membranes showed a fast activation and fast desensitization during perfusion of acetylcholine23, while the ρ1 subunit forms homomeric GABAρ1 receptors that do not desensitize during continuous perfusion of GABA24,25,26,27. If the co-injected sample was accidentally being delivered into the nucleus and not the cytoplasm, then the cDNA would be transcribed into RNA and translated into functional GABAρ1 receptors. Figure 2 shows that when the injection was aimed at the nucleus, oocytes with high responses to acetylcholine did not express GABAρ1 receptors; conversely, oocytes with zero or low response to acetylcholine expressed GABA receptors. Results of this experiment indicate, first, that in low-response oocytes, membranes were accidentally deposited into the nucleus of the oocyte; second, the injection of torpedo membranes into the nucleus does not interfere greatly with the transcription of ρ1 cDNA. A few co-injected oocytes had large responses to both acetylcholine and GABA, suggesting rupture of the nucleus during the injection. The enhanced insertion of transplanted membranes into the animal hemisphere of the oocyte mirrors the polarized localization of the heterologously-expressed neurotransmitter receptors 26,28. Therefore, by injecting into the animal hemisphere without targeting the nucleus, it is possible to obtain larger responses. This is important to study specimens with low density of receptors, for example from Alzheimer's disease (AD) tissue with low numbers of receptors (Figure 3).
Figure 1: Representative ion currents of oocytes. Ion currents from oocytes injected with membranes from human synaptic receptors were recorded. AMPA receptors were activated with 100 mM Kainate, and GABAA receptors with 1 mM GABA. VH = -80 mV. Please click here to view a larger version of this figure.
Figure 2: Co-injection of Torpedo and GABAρ1. Co-injection of filtered membranes (0.1 µm) from the electric organ of Torpedo, rich in acetylcholine receptors, and cDNA coding for the GABAρ1 receptor into the animal pole produced mainly two groups of oocytes based on their responses: one group of oocytes (A) had large responses to acetylcholine (Ach; 1 mM) but no responses to 1 µM GABA (voltage clamped to -80 mV), and oocytes in group (B) had null or low responses to ACh but large responses to GABA. (C) Graph shows mean ± standard error of mean (SEM) of peak current in group 1 (n = 5 oocytes) and group 2 (n = 11 oocytes). One oocyte in group 2 had large GABA and ACh responses suggesting rupture of the nucleus; consequently the distribution of the responses was skewed to low values, as noted by the difference between mean and median of the distribution of membrane current (22 nA vs 6 nA). This result indicates that one of the causes of low-response oocytes is that the membranes are being injected and trapped into the nucleus of the oocyte. Please click here to view a larger version of this figure.
Figure 3: Polarized insertion of cell membranes in Xenopus oocytes. GABA and kainate responses of oocytes injected into the animal or vegetal poles, with unfiltered membranes obtained from a non-AD brain (female, 74 years old, postmortem interval 2.8 h) and an AD-brain (female, 74 years old, postmortem interval 4.5 h). Oocytes injected near the animal pole, without targeting the nucleus, gave larger responses than oocytes injected into the vegetal pole, thus allowing the study of tissue samples with very low numbers of receptors. Student's t-test between vegetal and animal poles: ** p < 0.01, *** p < 0.001, Bars indicate mean ± SEM of peak current, n = 5 oocytes each column. Please click here to view a larger version of this figure.
Analysis of native protein complexes from human brains is needed to understand homeostatic and pathological processes in brain disorders and develop therapeutic strategies to prevent or treat diseases. Thus, brain banks containing snap frozen samples are an invaluable source of a large and mostly untapped wealth of physiological information29,30. An initial concern to use postmortem tissue is the clear possibility of mRNA or protein degradation that may confound the interpretation of data. For example, the pH of the brain consistently shows a positive correlation with the quantification of mRNA by quantitative real time-PCR, and this correlation is independent of the pathology31. Inter-subject differences of pH may lead to differences of mRNA quantification with large variances that may obscure the interpretation of results32. Interestingly, despite the lower quantification of mRNA transcripts as the pH acidifies, the covariances among the different transcripts are maintained33, providing a framework for obtaining reliable transcriptome profiling of pathological states. Importantly, while mRNA is highly degradable, transmembrane proteins in the postmortem tissue are very resistant to degradation34. Previous experiments in the lab have demonstrated that GABA and glutamate receptors of the human brain are functional after extremely large postmortem intervals35. Moreover, the MSM method allows to directly measure the effects of potential confounding factors like pH at moment of death, agonal state, and mRNA and protein degradation directly on the function of receptors, thus providing ways to use this information in the analysis and interpretation of data.
Modifications and troubleshooting of the technique
As mentioned before, the MSM is a slight modification of the original transplantation of total membranes, which is a method that has been widely validated and confirmed by many labs in academics and the pharmaceutical industry12,36,37,38,39,40. For example, microtransplantation of receptors was important in the development of Eli Lilly's LY3130481, which is a TARP gamma-8-selective antagonist of AMPA receptors which shows brain region selectivity12. MSM follows the same microtransplantation principle using synaptosomal enriched preparations; therefore, having good synaptosomal preparations is important. We use the Syn-Per method because the results that are obtained with this procedure are very consistent and the amplitude of the ion currents correlate very well with the levels of synaptic proteins in proteomic experiments10. However, the microtransplantation or the MSM can be adapted to study receptors from many sources (e.g., insect membranes38,39, neurolemma40) with excellent results. There are some disorders where synapses are strongly affected like Alzheimer's disease, or are available in low quantities; in this case, injection of the membranes in the animal side helps in getting larger currents. Increasing the amount of protein injected also increases the fusion of membranes and the size of the responses. Although there is a negative correlation between the concentration of membranes injected and the survival of oocytes, it is possible to find a suitable concentration of membranes that allows the given experimental analysis.
Limitations of MSM
The MSM is a quantitative method that was developed for the study of human receptors or ion channels; therefore, it is well suited to study tissue with limited availability. Because Xenopus oocytes do not have endogenous glutamate or GABA receptors, any response to GABA or glutamate in microtransplanted oocytes comes from the injected receptors that fused with the membrane of the oocytes. However, an important limitation of MSM is the study of ion channels or transporters that are also endogenously expressed in oocytes, as the separation of ion currents from microtransplanted and endogenous channels/transporters is difficult. Future studies silencing endogenous genes may be helpful with this limitation.
The significance to existing methods is that MSM incorporates the native membrane with its corresponding proteins and receptors, and thus, provides a more physiological environment for the analysis. It has been found that the properties of the receptors in their native membrane are retained after transplantation to the oocytes, as observed in neurotransmitter receptors AMPA-type GluR1, α7-AcChoRs, and α4β2-AcChoRs from cultured cells41.
Future applications of the technique include the study of the electrophysiological properties of different proteins and receptors of interest in the membranes of cultured and non-cultured cells and tissues from healthy and diseased human brains.
The authors have nothing to disclose.
This work was supported by NIA/NIH grants R01AG070255 and R01AG073133 to AL. We also thank University of California Irvine Alzheimer's disease research center (UCI-ADRC) for providing the human tissue shown in this manuscript. The UCI-ADRC is funded by NIH/NIA grant P30 AG066519.
For Microinjection | |||
3.5" Glass Capillaries | Drummond | 3-000-203-G/X | |
24 well, flat bottom Tissue Culture Plate | Thermofisher | FB012929 | |
Flaming/Brown type micropipette puller | Sutter | P-1000 | |
Injection Dish | Thermofisher | 08-772B | |
Microcentrifuge Tubes | Thermofisher | 02-682-002 | |
Mineral Oil | Thermofisher | O121-1 | |
Nanoject II | Drummond | 3-000-204 | |
Nylon mesh | Industrial Netting | WN0800 | |
Parafilm | Thermofisher | S37440 | |
Stereoscope | Fisher Scientific | 03-000-037 | |
Syringe | Thermofisher | 14-841-31 | |
Ultrasonic cleaning bath | Thermofisher | FS20D | |
Xenopus laevis frogs | Xenopus 1 | 4217 | |
For Two Electrode Voltage clamp | |||
15 cm long fire polished borosilicate glass capillaries | Sutter | B200-116-15 | |
Any PC computer or laptop | |||
Low-pass Bessel Filter | Warner Instruments | LPF-8 | |
Stereoscope | Fisher Scientific | 03-000-037 | |
Two electrode voltage clamp workstation | Warner Instruments | TEV-700 | |
ValveLink 8.2 Perfusion Controller | Automate Scientific | SKU:01-18 | |
WInEDR Free software | University of Strathclyde Glasgow | https://spider.science.strath.ac.uk/sipbs/software_ses.htm | |
X Series Multifunction DAQ | National Instruments | NI USB-6341 | |
Reagents | |||
Calcium dichloride | Thermofisher | C79 | |
Calcium nitrate tetrahydrate | Thermofisher | C109 | |
Collagenase | Sigma-Aldrich | C0130 | |
GABA | Sigma-Aldrich | A2129 | |
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) | Thermofisher | BP310 | |
Kainic acid | Tocris | 0222 | |
Magnesium sulfate heptahydrate | Thermofisher | M63 | |
Potassium chloride | Thermofisher | P217 | |
Sodium bicarbonate | Thermofisher | S233 | |
Sodium chloride | Thermofisher | S271-1 | |
Ultrafree-0.1 µm MC filter, | Amicon |