A transgenic mouse line for molecular genetic analysis of excitatory glutamatergic neurons

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Abstract

Excitatory glutamatergic neurons are part of most of the neuronal circuits in the mammalian nervous system. We have used BAC-technology to generate a BAC-Vglut2::Cre mouse line where Cre expression is driven by the vesicular glutamate transporter 2 (Vglut2) promotor. This BAC-Vglut2::Cre mouse line showed specific expression of Cre in Vglut2 positive cells in the spinal cord with no ectopic expression in GABAergic or glycinergic neurons. This mouse line also showed specific Cre expression in Vglut2 positive structures in the brain such as thalamus, hypothalamus, superior colliculi, inferior colliculi and deep cerebellar nuclei together with nuclei in the midbrain and hindbrain. Cre-mediated recombination was restricted to Cre expressing cells in the spinal cord and brain and occurred as early as E 12.5. Known Vglut2 positive neurons showed normal electrophysiological properties in the BAC-Vglut2::Cre transgenic mice. Altogether, this BAC-Vglut2::Cre mouse line provides a valuable tool for molecular genetic analysis of excitatory neuronal populations throughout the mouse nervous system.

Introduction

Excitatory glutamatergic neurons are located throughout the mammalian brain and spinal cord and are important components in the majority of neuronal circuits in the nervous system. Vesicular glutamate transporters are responsible for transport of glutamate into presynaptic vesicles and so far three different vesicular glutamate transporters – Vglut1-3 – have been described (Aihara et al., 2000, Bellocchio et al., 2000, Fremeau et al., 2002, Herzog et al., 2001, Takamori et al., 2002, Takamori et al., 2000, Takamori et al., 2001, Varoqui et al., 2002). Today these transporters are used as tools for functional studies of glutamatergic neurons, since the combined expression of the Vglut isoforms seems to cover all known glutamatergic neurons (Fremeau et al., 2004a, Fremeau et al., 2004b, Moriyama & Yamamoto, 2004) with a distribution of expression that to a large extent is complementary. Vglut1 expression is high in the cerebral cortex and hippocampus, while Vglut2 is expressed in thalamus, hypothalamus, inferior and superior colliculi, many nuclei in the mid- and hindbrain, and in the spinal cord (Bai et al., 2001, Fremeau et al., 2001, Varoqui et al., 2002). Vglut3 is more sparsely expressed in selected nuclei in the midbrain (Fremeau et al., 2002, Herzog et al., 2004, Schafer et al., 2002). In addition glutamate transporters are expressed in certain non-glutamatergic neurons that may use glutamate as a co-transmitter such as dopaminergic, GABAergic, serotoninergic and peptidergic neurons (Dal Bo et al., 2004, Gras et al., 2002, Kawano et al., 2006, Ponzio et al., 2006). All three isoforms have also been detected in astrocytes (Montana et al., 2006). Concordant with the expression pattern, previous studies have shown that Vglut1 is important for glutamatergic transmission in hippocampus (Fremeau et al., 2004b, Wojcik et al., 2004), while studies of Vglut2 knockouts have shown impaired thalamic glutamatergic transmission (Moechars et al., 2006). In addition the Vglut2 knockout mice die at birth due to the disruption of the respiratory local network in the brainstem (Moechars et al., 2006, Wallen-Mackenzie et al., 2006).

Genetically modified mice have become a powerful tool in the analysis of neuronal networks (Callaway, 2005, Crone et al., 2008, Crone et al., 2009, Dougherty & Kiehn, 2010, Gosgnach et al., 2006, Hinckley et al., 2005, Kullander et al., 2003, Lanuza et al., 2004, Luo et al., 2008, Sohal et al., 2009, Tan et al., 2006, Tan et al., 2008, Thoby-Brisson et al., 2009, Tsai et al., 2009, Wilson et al., 2005, Zhang et al., 2007, Zhang et al., 2008). An important approach in such analyses is the use of cell specific driven expression of Cre recombinase in combination with conditional mouse lines. This strategy can provide both cell specific labeling and inactivation/ablation or activation of cells (Callaway, 2005, Crone et al., 2008, Gosgnach et al., 2006, Luo et al., 2008, Sohal et al., 2009, Tan et al., 2006, Tan et al., 2008, Thoby-Brisson et al., 2009, Tsai et al., 2009, Zhang et al., 2007, Zhang et al., 2008).

Local neuronal networks called central pattern generators, or CPGs control rhythmic motor behaviors. For instance, respiration and locomotion are two biological functions known to be controlled by such specialized networks located in the brainstem and spinal cord, respectively (Feldman & Del Negro, 2006, Grillner, 2003, Kiehn, 2006, Kiehn & Butt, 2003). These networks are organized by a mixture of excitatory and inhibitory neurons. In the mammalian spinal locomotor CPG, the main excitatory drive appears to be glutamatergic and the majority of the spinal excitatory glutamatergic neurons express Vglut2 (Kiehn et al., 2008).

Here we report the generation and characterization of a transgenic mouse line that shows specific expression of Cre in Vglut2 positive neurons in the spinal cord and in brain areas known to express Vglut2. This transgenic mouse line provides a valuable tool by itself or in combination with other conditional alleles containing loxP sites, to identify and analyze the functional role of excitatory glutamatergic components in neuronal circuits throughout the nervous system including the locomotor networks in the spinal cord.

Section snippets

Generation of the BAC-Vglut2::cre transgenic mouse line

We have used bacterial artificial chromosomal (BAC) clone RP23-84M15 to direct the expression of the codon-improved Cre recombinase, iCre (Shimshek et al., 2002). This clone contained the complete slc17a6 gene (Vglut2) including a 96 kb sequence upstream and a 56 kb sequence downstream of the gene. BAC-technology was used to increase the probability for a temporal and cell-specific expression that does not interfere with the intrinsic gene function. The BAC clone was modified using a targeting

Discussion

The ability to perform genetic manipulations is fundamental for the analysis of neuronal networks, and the most common technique used to target specific cell types involves cell specific expression of Cre recombinase combined with mice carrying conditional targeted alleles.

One strategy to obtain a reproducible and efficient cell-specific gene expression in vivo is to use BAC technology to create transgenic mice. (Giraldo & Montoliu, 2001, Gong et al., 2003, Heintz, 2001). In addition, this

Functional use

The reliable and widespread expression of Cre in known Vglut2 expressing cells in the rodent nervous system makes the present mouse line an asset for functional studies of neuronal networks and cellular properties. Targeting of fluorescent proteins to Vglut2 expressing cells will immediately facilitate electrophysiological and anatomical studies of glutamatergic neurons throughout the brain and spinal cord (Al-Mosawie et al., 2007, Crone et al., 2008). Genetically driven activation and

Generation of the BAC-Vglut2::Cre mouse

The mouse BAC clone (RCPI23-84M15) encompassing the slc17a6 gene from the female C57BL/6 J mouse BAC library was obtained from CHORI-USA and was modified by homologous recombination in EL250 bacteria. A modified pConst plasmid was used as a targeting construct containing the open reading frame of iCre followed by the ampicillin resistance gene (bla), flanked by two FRT-site in the same orientation. The targeting cassette was created by first generating the 5′ and 3′ Vglut2 homology arms by PCR

Acknowledgments

This work was supported by NIH R01NS040795-09 (OK), EU-grant (SPINAL CORD REPAIR, OK), Swedish Medical Research Council (OK), Söderbergs Foundation (OK), Friends of Karolinska Institutet (OK) and Hjärnfonden (OK). RNL was supported by a Long Term Fellowship from the International Human Frontier Science Program. The Vglut2flox/flox mice were kindly provided by Drs. T. Hnasko and R. Palmiter. The GAD67::eGFP mice were a kind gift from Dr. N. Tamamaki. We thank Drs. M. Watanabe and T. Hökfelt for

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