RNA interference of Xenopus NMDAR NR1 in vitro and in vivo
Introduction
The rapid rise in use of RNA interference (RNAi) to silence or reduce specific proteins (Bernstein et al., 2001, Hannon, 2002) has facilitated applications in vertebrate neurons (Scherr et al., 2003). Twenty to 32 nucleotide double stranded RNAs (dsRNAs) produced by the enzyme Dicer from long, dsRNAs are the effectors of RNAi in all organisms (Elbashir et al., 2001). Dicer can also produce RNAi from short hairpin RNAs coded for by DNA plasmids using the RNA polymerase III (Pol III) promoter U6 to drive the expression of the hairpin (Brummelkamp et al., 2002).
We focus here on the short interfering RNA (siRNA) knockdown of N-methyl-d-aspartate subtype of glutamate receptor (NMDAR) in developing vertebrate neurons because this receptor is critical to early glutamate neurotransmission, its complete knockout causes neonate death in mice, and the NMDAR is crucial to the synaptic competition that refines pathways in the early brain and is widely employed in human cognition, learning and memory (Constantine-Paton and Cline, 1998). For example, a 95% global reduction of NMDAR levels in mice causes symptoms similar to schizophrenia (Mohn et al., 1999) and decreased mental function in the elderly may also be related to decreased NMDAR levels in aged human brains (Lu et al., 2004).
There have been several studies in which NMDAR function is eliminated in the entire nervous system or in classes of neurons in one or scattered brain regions. In these cases excitatory neurons with normal NMDARs are seldom interacting with other NMDAR deficient excitatory cells in the same circuitry. To rigorously test the several suggested roles of the NMDAR in synaptic competition it would be very useful to reduce or eliminate its function in a small population of neurons situated among normal neurons of the same class to determine whether neurons can survive to participate in an otherwise normal circuit without the NMDAR to initiate the activity-dependent potentiation or the depression of their developing inputs.
To knockdown the NMDAR we targeted NR1 the obligate subunit of NMDAR. (Schorge and Colquhoun, 2003). Mice lacking the NR1 subunit show disrupted local refinement of pathways formed in utero (Li et al., 1994), significant cell loss in the somatosensory thalamus (Adams et al., 2004) and they die shortly after birth probably because the NMDAR is necessary for normal breathing (Forrest et al., 1994). NR1 knockouts specific to hippocampal area CA1 show defects in spatial learning (Tsien et al., 1996), and NR1 KO's targeted to neocortical pyramidal neurons show a disruption of barrel organization in somatosensory cortex (Iwasato et al., 2000). Chimeric mice containing large blocks of normal neurons and large blocks of NR1 KO neurons have also been produced (Maskos et al., 2001, Maskos and McKay, 2003), but in these studies individual KO neurons were not followed over time to determine whether their survival or differentiation differed from adjacent normal neurons.
Here, we use two different siRNA sequences against NR1 mRNA (iNR1) introduced into Xenopus laevis embryos or neurons as DNA plasmids coding for shRNA to knockdown the expression of the NR1 subunit and NMDAR function in identifiable cells. Compared to controls the siRNA animals have significantly reduced NR1 transcript and protein levels, cultured iNR-expressing neurons have significantly reduced NMDAR mediated Ca2+ influx, NMDAR synaptic currents appear to be eliminated in these neurons, while AMPAR currents remain. In addition, live imaging of small, low density groups of neurons expressing the iNRs were compared to controls expressing siRNA against the mammalian NR2B subunit of the NMDAR. The NR1 “silenced” neurons showed normal morphologies and the same low levels of cell death seen in the controls. Thus, vertebrates with a low density of iNR silenced neurons should be powerful and readily available models with which to study the role of the NMDAR in establishing connectivity of single neurons in the competitive environment of a predominantly normal brain.
Section snippets
Knockdown reagents
Two pairs of RNA oligos with 2 base pair 3′ overhangs were constructed (Dharmicon). Oligo target sequences met the following criteria: a GC content between 35 and 55%, in a well-conserved region, and sequences unable to hybridize with other Xenopus genes. Sequences were from the NR1 Genbank accession number X94081. The nucleotide position of the oligos in that sequence is given in the subscript of the oligo name, as follows: Xenopus iNR2524 sense strand: 5′-GAGCUGGAUAAGACCUGGGUC; antisense
In vivo suppression of NR1 protein in young embryos
To test the effectiveness of inhibitory oligos designed against the NR1 transcript, approximately 100 pmol of chemically synthesized double-stranded RNAs (iNR2524 and iNR2700) were directly injected into one cell Xenopus embryos. Levels of NR1 transcript in the injected embryos were examined using quantitative RT-PCR. The transcript of an unrelated protein (the transcription factor EF1α) was used to normalize RNA amounts in the PCR products from each embryo for each age examined. Sample gels of
Discussion
These findings demonstrate a rapid and versatile method for controlling the activity of a crucial neural gene product in an identified subset of neurons or neuron progenitors throughout development while the co-localized reporter allows assays of the morphology or connectivity of the cells within a wild type background. In Xenopus laevis embryos, RNAi reduces the mRNA levels of the NR1 NMDAR subunit up to 88%, the protein levels by 93% and functionally reduces or eliminates the NMDAR current as
References (35)
- et al.
LTP and activity-dependent synaptogenesis: the more alike they are, the more different they become
Curr Opin Neurobiol
(1998) Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting
Neuron
(2000)- et al.
Targeted disruption of NMDAR1 gene abolishes NMDA response and results in neonatal death
Neuron
(1994) - et al.
Subunit rules governing the sorting of internalized AMPA receptors in hippocampal neurons
Neuron
(2004) - et al.
Whisker-related neuronal patterns fail to develop in the trigeminal brainstem nuclei of NMDAR1 knockout mice
Cell
(1994) - et al.
Activation of synaptic NMDARs induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons
Neuron
(2001) - et al.
Long-term survival, migration, and differentiation of neural cells without functional NMDARs in vivo
Dev Biol
(2001) - et al.
Neural cells without functional N-methyl-d-aspartate (NMDA) receptors contribute extensively to normal postnatal brain development in efficiently generated chimaeric NMDA R1 −/− ↔ +/+ mice
Dev Biol
(2003) - et al.
Mice with reduced NMDAR expression display behaviors related to schizophrenia
Cell
(1999) - et al.
Low access resistance perforated patch recordings using amphotericin B
J Neurosci Methods
(1991)
The projection of the accessory olfactory bulb in the frog
Brain Res
The essential role of hippocampal CA1 NMDAR-dependent synaptic plasticity in spatial memory
Cell
Ras and Rap control AMPA receptor trafficking during synaptic plasticity
Cell
Pronounced cell death in the absence of NMDARs in the developing somatosensory thalamus
J Neurosci
The rest is silence
RNA
A system for stable expression of short interfering RNAs in mammalian cells
Science
Development of the prosencephalon
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2011, Mutation Research - Reviews in Mutation ResearchCitation Excerpt :After these findings, several articles were published which used siRNAs to successfully reduce the expression of endogenous genes, among them, a Xenopus MinK-related peptide K+ channel β subunits (xMiRP2) involved in ion channel function in oocytes [114]. Similarly in vitro and in vivo experiments using microinjection in cultured Xenopus tectal neurons and tadpoles reduced the expression of the NR1 gene that encodes a subunit of the glutamate NMDA receptor that controls synaptic plasticity [115]. Interestingly, by that time, there were still reports of successful knock-downs using dsRNA, for instance, Evaul et al. [116] reduced the expression of the androgen receptor (AR) by dsRNA injection in oocytes.
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