Basic Neuroscience
Use of vivo-morpholinos for control of protein expression in the adult rat brain

https://doi.org/10.1016/j.jneumeth.2011.10.009Get rights and content

Abstract

Vivo-morpholinos are commercially available morpholino oligomers with a terminal octa-guanidinium dendrimer for enhanced cell-permeability. Existing evidence from systemically delivered vivo-morpholinos indicate that genetic suppression can last from days to weeks without evidence of cellular toxicity. However, intravenously delivered vivo-morpholinos are ineffective at protein suppression in the brain, and no evidence is available regarding whether intracranially delivered vivo-morpholinos effectively reduce target protein levels, or do so without inducing neurotoxicity. Here we report examples in which in vivo microinjection of antisense vivo-morpholinos directed against three different targets (xCT, GLT1, orexin) in two different brain regions resulted in significant suppression of protein expression without neurotoxicity. Expression was significantly suppressed at six to seven days post-administration, but returned to baseline levels within fourteen days. These results indicate that direct intracranial administration of vivo-morpholinos provides an effective means by which to suppress protein expression in the brain for one to two weeks.

Highlights

► Here we test use of vivo-morpholinos for protein suppression in the rat brain. ► We show effective suppression of GLT-1, xCT, and orexin proteins. ► We provide evidence that vivo-morpholinos can be used without evidence of toxicity. ► However, high dose vivo-morpholinos are neurotoxic.

Introduction

Effective methods for temporally controlled genetic suppression in the developed brain are critical for investigating the contribution of specific gene products to adult nervous system function. Although current transgenic methods provide sophisticated tools in this regard, behavioral methods that are optimized for use in rat models are limited, in large part due totechnical hurdles in generating transgenic rat models (Aitman et al., 2008, Tesson et al., 2005). Nonetheless, direct brain microinjection of pharmacological agents, antisense oligonucleotides, and RNAi allow complementary approaches to genetic suppression that are useful in rat models (Self, 2005). However, acute administration of RNAi typically leads to transient suppression, and extended suppression requires the use of continual administration by mini-pump, or delivery via a viral vector (Morris, 2008).

In parallel with the development of transgenic and oligonucleotide (DNA and RNA) based methods, morpholino oligomers have emerged as an alternative and promising approach. Morpholino antisense technologies are widely used in a number of systems including rodent embryos, Xenopus, zebrafish, sea urchin and others (Corey and Abrams, 2001, Heasman, 2002). A morpholino antisense approach offers comparable or superior suppression to DNA oligonucleotides and RNAi, but with enhanced structural stability and without many of the described non-specific, off-target effects (Summerton, 2007). This is due in part to the six-membered morpholino backbone and non-ionic phosphorodiamidate linkage, which limits interactions with the extracellular matrix and renders oligomers resistant to nuclease degradation (Summerton, 2007). Morpholinos suppress protein expression by binding to mRNA with high affinity and thus block translation or splicing, depending on the sequence location at translation initiation start sites or intron/exon junctions (Li and Morcos, 2008, Moulton and Jiang, 2009, Moulton and Yan, 2008).

A recent derivative of morpholinos are vivo-morpholinos. Vivo-morpholinos are designed from the same structure as morpholinos described above, with an additional octa-guandinium dendrimer conjugated at the terminal 3′ end (Moulton and Jiang, 2009). The dendrimer (derived from Greek for “tree”) is a branched molecule, generally symmetrical and overall spherical in shape. In this case, the dendrimer is formed by the presence of either octa-guanidinium groups. This terminal attachment allows for facilitated cell permeability, via chemistry similar to that employed by TAT and other related peptide membrane permeability domains (Wadia and Dowdy, 2005). While vivo-morpholinos are superior to non-conjugated morpholinos for achieving suppression following i.v. administration, brain tissue is not significantly accessed following systemic delivery (Li and Morcos, 2008, Morcos et al., 2008, Parra et al., 2011). A few examples of successful knockdown with non-conjugated morpholinos administered directly in the brain have been reported (Hiroi et al., 2011, Oh et al., 2006). However, evidence is not available on the efficacy or toxicity of vivo-morpholinos administered intracranially.

Here we demonstrate use of vivo-morpholinos for genetic suppression in the adult rat brain, using three separate targets: the high-affinity glutamate transporter GLT-1/EAAT2, the catalytic subunit of the cystine glutamate exchanger xCT, and orexin/hypocretin. Important roles for glutamate transport by GLT-1 and xCT have been described in addiction, affective disorders, and protection against neurotoxicity and ischemia (Albrecht et al., 2010, Reissner and Kalivas, 2010, Valentine and Sanacora, 2009). The orexins are implicated in mechanisms of sleep as well as in motivation to obtain both natural rewards and drugs of abuse (Aston-Jones et al., 2010, Berridge et al., 2010, Cason et al., 2010, Thompson and Borgland, 2011). Because these targets are all of interest in the mechanisms of reward learning, we wished to determine applicability of vivo-morpholinos for temporal and regional control of protein expression. These results indicate that vivo-morpholinos can be effectively used to suppress protein expression in the brain in vivo, in a region-specific manner for a period of days to weeks, thus providing a genetic tool for the use of nervous system function and behavioral analysis without the necessity of transgenic or viral-mediated strategies.

Section snippets

Animal housing and procedures

Adult male Sprague-Dawley rats (300–350 g, Charles River) were used for all studies. Animals were individually housed on a 12-h reverse-light cycle and provided rat chow and water ad libitum. Animals were anesthetized with ketamine/xylazine (56.5 mg/kg: 8.7 mg/kg, i.p.) and placed in the stereotaxic apparatus.

For injections into nucleus accumbens core (NAc), cannulae (Plastics One, 26G) were surgically implanted bilaterally into NAcat coordinates relative to Bregma +1.5 A/P, +1.7 M/L, −5.5 D/V (

Three consecutive microinjections of vivo-morpholinos are sufficient to suppress either GLT-1 or xCT in NAc

Initial experiments were used to assess knockdown of glutamate transporter GLT-1. Three daily microinjections of GLT-1 AS morpholinos (10 μM, 30 nmol total) were made contralaterally to the reverse control sequence, and protein levels were assessed in NAc separately in each hemisphere seven days later. Expression of xCT and GLT-1 was normalized to PSD-95 because PSD-95 is enriched in the membrane subfraction preparation, should be unaffected by either antisense sequence, and is of a molecular

Discussion

Conventional non-transgenic methods for suppression of protein expression in adult rat brain include antisense oligonucleotides and RNAi. Although these techniques are effective, the time course of suppression is generally limited in the absence of continual infusion via osmotic minipump or viral vector. Vivo-morpholinos provide an alternative means by which expression may be controlled, utilizing different chemistry from canonical nucleic acid based technology. In this report, we provide

Acknowledgements

This work is supported by NIH grants DA026254 (KJR), DA007288 (GCS), DA06214 (GAJ), and DA015369/DA003906 (PWK). The authors thank members of the Kalivas lab for constructive comments on a previous version of the manuscript.

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