Green-fluorescent-protein-expressing mice as models for the study of axonal growth and regeneration in vitro

B RA I N R E SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s r e v

Review

Green-fluorescent-protein-expressing mice as models for the study of axonal growth and regeneration in vitro Daniel Hechler, Robert Nitsch, Sven Hendrix⁎ Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité-Universitätsmedizin Berlin, Schumannstr. 20-21, D-10098 Berlin, Germany

A R T I C LE I N FO

AB S T R A C T

Article history:

The culture of hippocampal–entorhinal brain slices is a widely used model for studying

Accepted 17 January 2006

neuronal differentiation, axon growth and pathfinding in vitro. The application of tracers

Available online 23 February 2006

(e.g. biocytin) is a well-established method for studying single or multiple neurons and their extensions in this model. For quantifying the growth of high numbers of axons after lesion,

Keywords:

however, genetically expressed enhanced green fluorescent protein (EGFP) has proven

Tracing

particularly useful for labeling living axons in vivo and in vitro. Here, we introduce several

β-actin-EGFP

EGFP-expressing mouse lines which improve the organotypic brain slice model. The

Tau-EGFP

questions addressed determine which mouse line to use: β-actin-EGFP mice for labeling all

Thy-1.2-EGFP

cells and their extensions; Tau-EGFP mice for labeling the axoplasma; or Thy-1.2-EGFP mice

Organotypic

for labeling the axonal membrane. Cocultures of EGFP-positive entorhinal cortex explants

hippocampal–entorhinal brain slices

with EGFP-negative hippocampal explants allow the monitoring of fluorescent axons growing into the hippocampus in an easily quantifiable manner.

Abbreviations:

© 2006 Elsevier B.V. All rights reserved.

CNS, central nervous system EGFP, enhanced green fluorescent protein GFP, green fluorescent protein IL, interleukin

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Green fluorescent protein: an established marker in living cells of the nervous system. . . . . . . . . . . . The EGFP/wildtype coculture model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The β-actin-EGFP mouse — a source for cells with cytoplasmic EGFP . . . . . . . . . . . . . . . . . . 3.2. The Tau-EGFP mouse — a source for neuronal cells that express EGFP in the axoplasma. . . . . . . 3.3. The Thy-1.2-EGFP mouse — a source for neuronal cells that express EGFP on the axonal membrane Outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Fax: +49 30 450 528902. E-mail address: [email protected] (S. Hendrix). 0165-0173/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainresrev.2006.01.005

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

161 161 162 163 164 164 165

B RA I N RE SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

Introduction

A particular feature of higher vertebrates is the suppression of neuroregeneration in the mature central nervous system (CNS). Much research has focused on inhibitory signals and the injury-induced loss of trophic stimuli needed to promote the survival and regeneration of axotomized neurons (Goldberg and Barres, 2000). However, effective in vitro screening methods designed to search for factors which promote neuroregeneration in the CNS are also needed. Organotypic hippocampal–entorhinal brain slices are useful in vitro tools for analyzing basic regenerative mechanisms such as axonal outgrowth, pathfinding and synaptogenesis. They can be used to dissect these mechanisms in the brain tissue of genetically modified animals and to screen for pharmaceutical agents which may support regenerative processes (Prang et al., 2001). Organotypic hippocampal–entorhinal brain slices are widely used due to the differentiated termination of distinct synaptic inputs in the hippocampus and dentate gyrus (Fig. 1A). One major excitatory afferent input to the hippocampus, the perforant path, is of particular interest in neuroscience because it is easy to characterize after labeling and preserves many of its in vivo features. The perforant path derives from neuronal cells located in the upper layers of the entorhinal cortex, which terminate in the marginal zones of the hippocampus, the stratum lacunosum-moleculare and the outer two-thirds of the molecular layers of the dentate gyrus (Amaral and Witter, 1995; Blackstad, 1958). These projections in the outer molecular layer are clearly separated from axons of the commissural/associational pathway terminating in the inner molecular layer. In organotypic brain slices, the perforant path fibers terminate in a lamina-specific manner in the dentate gyrus, similar to the in vivo situation (Frotscher and Heimrich, 1993; Li et al., 1993, 1994; Prang et al., 2001). In vitro, entorhinal axons preserve the capacity to regenerate the lamina-specific projection after lesion (Frotscher et al., 1997). This regenerative ability is dependent on the age of the donor animal (Li et al., 1994) and the time in culture (Woodhams et al., 1993). Due to the target-specific termination of the perforant path fibers, it is possible to study their growth and remodeling and to quantify the number of reinnervating axons after axotomy in vitro (Heimrich and Frotscher, 1993; Prang et al., 2001; Stoppini et al., 1991). The application of tracers such as biotinylated dextrane amine (BDA) is an excellent method for studying single or multiple neurons and their extensions in this model (Kobbert et al., 2000; Prang et al., 2001). However, the application of tracers to higher numbers of axons needs considerable expertise to avoid unintentional mechanical damage to neurons or imprecise labeling which would result in falsepositive or false-negative fiber tracts. For the study of high numbers of growing axons, genetically expressed green fluorescent protein (GFP) has proven particularly useful for labeling living axons in vivo and in vitro. GFP has started to revolutionize many fields of science as a marker in living cells

161 166 167

(Schmid and Neumeier, 2005; Tsien, 1998) and appears to be a convenient tool for investigating axonal outgrowth, sprouting and re-innervation (Glumm et al., 2002). Here, we will introduce several mouse lines which express enhanced GFP on neuronal cells as excellent sources of prelabeled axons.

2. Green fluorescent protein: an established marker in living cells of the nervous system The native source of GFP is the jellyfish Aequorea victoria. GFP is a monomer with an Mr of 27,000 to 30,000 Da encoded by 238 amino acids (Prendergast and Mann, 1978) with an extremely stable conformation (Shimomura and Shimomura, 1981; Ward and Bokman, 1982). The in vivo luminescence of green light in A. victoria is the result of a Ca2+-dependent energy transfer from the light emitter of the bioluminescent photoprotein aequorin to GFP (Morin and Hastings, 1971; Shimomura and Johnson, 1967; Shimomura et al., 1962). The process of energy transfer is independent of exogenous substrates or cofactors, leading to a relatively bright and particularly stable fluorescent signal (Ellenberg et al., 1999; Shimomura, 1979; Ward and Bokman, 1982). GFP has become a very useful tool for labeling selected structures in vivo because it can be monitored non-invasively in living cells by macroscopic observation with an adequate light source, fluorescence microscopy or flow cytometry (Cheng et al., 1996; Cubitt et al., 1995; Kain et al., 1995; Prasher, 1995). At present, a variety of GFPs exist with differences in brightness and stability (Andersen et al., 1998; Corish and Tyler-Smith, 1999; Li et al., 1998). Here, we present four mouse lines which express on their axons a particularly bright and stable GFP that has been termed “enhanced GFP” (EGFP). The high stability of the protein (a half-life of ∼26 h in most cells (Corish and Tyler-Smith, 1999; Cubitt et al., 1995)) can be useful for identifying labeled structures but can also be a limiting factor for its application, particularly for the study of axonal degeneration. After axotomy or nerve crush, stable GFP might be disadvantageous because the regenerating GFP-positive axons of skin nerves, the ischiadic nerve or the spinal cord cannot be distinguished from degenerating fibers in the distal end, which are still GFP-positive (Witzel et al., 2005; Hendrix, unpublished observation). In order to use GFP as a marker in living neural cells and/or their extensions, it is necessary to consider toxic side effects of the protein. The toxic effects of GFP in transfected or transgenic cells have been controversial: some authors have reported no toxicity (Alexander et al., 1997), while others have reported considerable toxicity of excited GFP (Clontech Ltd., 1996; Liu et al., 1999). Similarly, the immunogenicity of GFP has also been a matter of debate: while some authors could not demonstrate a general immunogenic effect of GFP, others have shown strong immune response against the protein (Rosenzweig et al., 2001; Stripecke et al., 1999). The immunogenic effect seems to be

162

B RA I N R E SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

dependent on the genetic background of the animals used as an immune response could be elicited in BALB/c mice but not in C57BL/6 mice (Denaro et al., 2001; Gambotto et al., 2000; Skelton et al., 2001). Therefore, in the context of neuroimmunological questions, potential toxic or immunogenic effects of GFP have to be considered to avoid artifacts.

3.

The EGFP/wildtype coculture model

The culture of hippocampal–entorhinal brain slices is an excellent, well-established method for studying the morphology, growth and remodeling of axons (Feldmeyer et al., 2005; Heimrich and Frotscher, 1993; Stoppini et al., 1991). This method has been improved by combining the entorhinal cortex of a β-actin-EGFP mouse and the hippocampus of a

wildtype mouse (here referred to as the EGFP/wildtype coculture model, Figs. 1A–D; Glumm et al., 2002). The entorhinal–hippocampal and commissural/associational projections are easily detectable by the content of intra-axonal GFP in the non-fluorescent wildtype hippocampus (Figs. 1A–D; Glumm et al., 2002). The EGFP/wildtype coculture model is particularly useful for studying high numbers of axons, which grow into and start to re-innervate the target tissue (Fig. 1B). The potential problems of applying a tracer such as BDA or biocytin to high numbers of axons are incomplete labeling or toxic damage. These problems can be easily avoided by using the EGFP/wildtype coculture model. Due to the overlay of many GFP-positive nerve fibers (Figs. 1C, D), the GFP-positive area in the GFP-negative hippocampus can be easily determined using an image analysis system, e.g. after 2 days (Figs. 1C, D) or 14 days (Fig. 1B) in culture.

163

B RA I N RE SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

Table 1 – Features of four selected EGFP-expressing mouse lines used as sources of pre-labeled axons for organotypic hippocampal–entorhinal brain slice co-cultures β-actin-EGFP mouse model Cells/ structures labeled Special breeding features Fluorescence intensity on axons Phenotype separation

Tau-EGFP mouse model

Thy-1.2 L17 mouse model

Thy-1.2 L21 mouse model

Cytoplasm of most cells including neurons, astrocytes, immune cells, etc.

Axoplasma of most axons

Axonal membrane of most axons

Axonal membrane of selected axons

Only homozygous mice can be used (heterozygous show low fluorescence intensity) High in homozygous mice

Only heterozygous mice can be used Only heterozygous (homozygous mice are TAU-KO mice) mice have been analyzed Moderate in heterozygous mice High in heterozygous mice

Only heterozygous mice have been analyzed High in heterozygous mice

Optic differentiation with UV light

PCR

PCR

PCR

Comparison of four EGFP-expressing mouse lines for the study of axonal outgrowth in vitro.

Here, we will review the advantages and disadvantages of different EGFP-expressing mouse lines which can be used in the EGFP/wildtype coculture model. Table 1 summarizes the most important features of the introduced EGFP-expressing mouse lines to facilitate choosing the best model for the questions addressed.

3.1. The β-actin-EGFP mouse — a source for cells with cytoplasmic EGFP The β-actin-EGFP mice are particularly easy to use because the littermates can be easily distinguished macroscopically (Figs. 1E–F) and no time-consuming genotyping is necessary. The common methods for detecting transgenic mice are PCR analysis or Southern blotting after DNA extraction, usually from tails of 3- to 4-week-old pups (Mülhardt, 2000). These methods require skill and are time-consuming, especially when applied to a larger number of animals. By using EGFP as a reporter protein, it is possible to distinguish wildtype littermates from heterozygous and homozygous transgenic littermates directly after birth by simple eye examination under hand-held 254 nm/366 nm UV light (Figs. 1E–F). Homozygous mice display more intense and brighter fluores-

cence in the skin than heterozygous mice, while wildtype mice show no fluorescence (Figs. 1E–F). A first line of transgenic mice, which expressed wildtype GFP under the control of the β-actin promoter, displayed green fluorescence only in muscle, pancreas and kidney but not in other organs such as the brain (Ikawa et al., 1995). Since βactin is expressed almost ubiquitously in mammalian cells, these expression patterns suggest that the GFP signal might be too weak to be detected in the other tissues. Using the same vector construct with the cDNA for EGFP instead of wildtype GFP, it was possible to generate a mouse strain with EGFP expression in most cells and organs including the brain and peripheral nerves (Table 2). In all β-actin-EGFP transgenic mouse strains generated, most investigated organs and cells show a high expression rate of EGFP when excited with UV light of 254 nm and 366 nm, with just a few exceptions such as red blood cells (Table 2; Okabe et al., 1997). The application of organs or cells derived from the β-actin-EGFP mouse makes it easier to distinguish and follow single cells in host tissue during transplantation experiments, e.g. in bone marrow, nerve and stem cell experiments (Furuya et al., 2003; Lu et al., 2002; Maurer et al., 2003; Simard and Rivest, 2004; Tanaka et al., 2003; van den Pol and Santarelli, 2003).

Fig. 1 – The EGFP/wildtype slice coculture model for the analysis of axon growth into the hippocampus. (A) Schematic representation of the EGFP/wildtype slice coculture model. An EGFP-positive entorhinal cortex (EC) of a β-actin-EGFP-transgenic mouse, which expresses EGFP on all axons, is cocultured with a hippocampus from a wildtype littermate. The axons of the entorhinal–hippocampal axon projection originate in the EGFP-positive entorhinal cortex (EC), grow as EGFP-positive perforant path (PP) fibers into the EGFP-negative hippocampus and terminate in the dentate gyrus (DG). (B) Fourteen days after cultivation, EGFP-positive axons (arrows) have grown into the wildtype hippocampus and terminate in the dentate gyrus (DG), which is labeled by red ethidium bromide Nissl staining. Note: the EGFP fluorescence of the ingrowing perforant path axons is reduced due to the overlay of the Nissl staining photomicrograph. Exposure time: 2500 ms. (C) Two days after cultivation, EGFP-positive perforant path axons (PP) have grown into the wildtype hippocampus. The box indicates the area that is shown at higher magnification in panel D. Exposure time: 2500 ms. (D) Bundles of multiple perforant path axons (PP), which express EGFP in the axoplasma, are present in the wildtype hippocampus. Due to the higher magnification, an exposure time of 1500 ms is sufficient to visualize the strong EGFP fluorescence of the axons. Single axons cannot be differentiated because the photomicrograph shows an overlay of high numbers of axons, which express EGFP in the axoplasma. (E) Homozygote, heterozygote and wildtype (wt) mouse littermates by daylight. (F) Under hand-held 254 nm/366 nm UV light, the fluorescence intensity is maximal in homozygous littermates, moderate in heterozygous littermates and absent in wildtype (wt) littermates. CA: cornu ammonis; DG: dentate gyrus; EC: entorhinal cortex; FF: fimbria-fornix; PP: perforant path. Black bar: border between recombined EGFP-positive entorhinal cortex and EGFP-negative hippocampus. Scale bars: B,C, D = 100 μm.

164

B RA I N R E SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

Table 2 – Expression of EGFP in various tissues of β-actinGFP (Ikawa et al., 1995; Okabe et al., 1997) and Thy-1.2EGFP mice (Barker et al., 2004; Caroni, 1997; Morris, 1985; Schlamp et al., 2001; Vidal et al., 1990) Tissues/cells Brain/neuronal cells Liver Kidney Pancreas Adrenal gland Lung Muscle Heart Intestine Adipose tissue Spleen/splenocytes thymus/thymocytes Peripheral T cells Red blood cells Skin Blood vessels Testis Hair

Expression of β-actin EGFP

Expression of Thy-1.2-EGFP

+ + + + + + + + + + + + +b − + + + −

+a ni ni ni ni (+) ni ni ni ni ? − − ni ni ni ni ni

The examination of the cells and tissues of β-actin-GFP mice was performed on groups of wildtype and transgenic organs under hand-held 360 nm UV light. EGFP expression in selected cells and tissues of Thy-1.2-EGFP mice was analyzed by immunohistochemistry, in situ hybridization, RNase protection analysis, RT-PCR and radioimmunoassay (Barker et al., 2004; Caroni, 1997; Morris, 1985; Schlamp et al., 2001; Vidal et al., 1990). Due to the different detection methods, the degree of protein expression cannot easily be compared. +, high level of protein expression, (+), low level of protein expression, −, no detection of protein, ?, no data available. a Depending on the Thy-1.2-EGFP mouse line, multiple (L17 mice) or single (L21 mice) axons display strong EGFP expression. b FACS analysis (Okabe et al., 1997); ni: not investigated because wildtype Thy-1.2 is not expressed in these cells or tissues.

It is important to note that invading cells (e.g. T cells, microglial cells) and astrocytic processes are also EGFPpositive. In the context of neuroimmunological questions, this fact can be useful as the EGFP-positive immune cells of these mice can easily be tracked in vivo and in vitro. On the other hand, additional immunohistochemistry might be needed to exclude non-neuronal structures for quantifying axonal growth into the GFP-negative hippocampus.

3.2. The Tau-EGFP mouse — a source for neuronal cells that express EGFP in the axoplasma The Tau-EGFP mouse model (Fig. 2) is another new tool for the analysis of axon growth into EGFP-negative tissues. Compared to the β-actin-EGFP mouse model, it has the advantage of axon-specific GFP labeling; thus, no other structures are GFPpositive and no additional immunohistochemistry has to be applied to exclude non-neuronal structures. Tau is a member of the microtubule-associated protein (MAP) family and is primarily expressed in neurons and, to a lesser degree, oligodendrocytes and astrocytes (Gu et al., 1996; LoPresti et al., 1995; Vanier et al., 1998). Improved

sensitivity in detection methods has revealed high levels in the cerebral cortex, spinal cord, dorsal root ganglia and retina, medium levels in skeletal muscle, heart, testis, lung and kidney, and very low levels in stomach, adrenal gland, liver, aorta and spleen (Gu et al., 1996). No Tau protein could be detected in thymus and white blood cells (Gu et al., 1996). The Tau-EGFP transgenic mouse line was generated using a targeting vector containing an EGFP cDNA, a Pgk-1 polyadenylation signal and the G418-selection marker cloned into the exon 1 of the Tau gene (Tucker et al., 2001). This construct leads to a fusion protein with the first 31 amino acids of Tau followed by EGFP. Since the microtubulebinding domain is located at the carboxyl terminus, the resulting EGFP protein is located in the cytoplasm of the axons. Heterozygous and homozygous Tau-EGFP mice are both viable and fertile; phenotype analysis revealed no significant loss in the development or function of the brain or other organs compared to wildtype C57BL/6 mice (Tucker et al., 2001). However, due to the knock-in construct, it cannot be excluded that homozygous Tau-EGFP mice may have characteristics of TAU knock-out mice, which have not been characterized, yet. The use of heterozygous Tau-EGFP mice is an alternative since they express Tau albeit at lower levels. A disadvantage of using heterozygous Tau-EGFP mice is the reduced EGFP fluorescence intensity.

3.3. The Thy-1.2-EGFP mouse — a source for neuronal cells that express EGFP on the axonal membrane Thy-1.2-EGFP mice express EGFP on the cell membrane of either most axons (L17 mice) or selected axons (L21 mice) (Fig. 3; Caroni, 1997). The membranous localization of GFP makes it easier to distinguish single axons compared to β-actin-EGFP mice or Tau-EGFP mice, which both express EGFP in the axoplasma only. Thus, for analyzing selected axons, Thy-1.2EGFP mice might be the most appropriate. Thy-1 exists in two allelic forms in the different mouse strains, Thy-1.1 (e.g. AKR/J) and Thy-1.2 (e.g. C75BL/6) (Barclay et al., 1975; Letarte-Muirhead et al., 1975; Morris, 1985). Thy-1 is a member of the immunoglobulin-like superfamily and is linked via glycosyl-phosphatidylinositol (GPI) to the plasma membrane of several cell types such as neuronal cells (Barclay et al., 1975; Morris, 1985, 1992), thymocytes (Letarte-Muirhead et al., 1975; Morris, 1985; Reif and Allen, 1966a,b; Tokugawa et al., 1997; Vidal et al., 1990; Williams and Barclay, 1988), peripheral T cells (Page et al., 1997; Vidal et al., 1990), lung fibroblasts (Barker et al., 2004; Caroni, 1997; Penney et al., 1992) and retinal ganglion cells (Morris, 1985; Schlamp et al., 2001; Simon et al., 1999). The construct for generating the Thy-1.2-EGFP mouse contains the Thy-1.2 gene sequence and the EGFP cDNA from the vector pEGFP-N2 MCS (Clontech). The EGFP cDNA was integrated into the Thy-1.2 expression cassette over a XhoI linker, resulting in a deletion. The deletion encompasses the coding sequence of the Thy-1.2 protein together with exon 3 and both flanking introns, including the translation initiation site (Caroni, 1997). While the generated construct still contains parts of the 3′-untranslated Thy-1.2 sequence

B RA I N RE SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

165

Fig. 2 – Expression patterns of Tau-EGFP in adult mouse brains and in the organotypic hippocampal–entorhinal brain slice coculture model. (A) Hippocampus and entorhinal cortex (EC) in a 50-μm-thick horizontal slice of an adult Tau-EGFP mouse brain fixed immediately after harvesting with 4% paraformaldehyde. Exposure time: 1500 ms. CA: cornu ammonis; EC: entorhinal cortex; FF: fimbria fornix; PCL: pyramidal cell layer; in the dentate gyrus, ML: molecular layer; GL: granule cell layer; PL: polymorphic layer. (B) After 14 days in culture, Tau-EGFP-positive axons of the perforant path (PP) are present in the wildtype hippocampus. The Tau-EGFP fluorescence is weaker than the β-actin-EGFP fluorescence because heterozygous animals have to be used as homozygous mice may have characteristics of Tau knock-out mice. To visualize Tau-EGFP-positive axons, an exposure time of 2500 ms is appropriate. Overexposure of the entorhinal cortex (EC) is irrelevant for the evaluation of fibers growing into the hippocampus. DG: dentate gyrus. Scale bars: A, B = 100 μm.

including the mRNA polyadenylation site, the transgene insertion is needed to include its own translation initiation and Kozak consensus sites for improving the expression rate. The Thy-1.2 expression cassette was used for homologous recombination. In the context of neuroimmunological questions, the deletion of the entire exon 3 is an important feature of this particular Thy-1.2-EGFP expression cassette because it erases the expression of Thy-1.2 in the mouse thymus and on peripheral T cells (Vidal et al., 1990). The expression of Thy1.2 in the nervous system is not affected by the deletion nor is the faint expression in lung tissue (Table 2; Barker et al., 2004; Caroni, 1997; Morris, 1985; Schlamp et al., 2001; Vidal et al., 1990). Thy-1.2-EGFP transgenic mice display no reduction in vitality or fertility compared to wildtype mice. Furthermore, the expression of the fluorescent proteins did not influence or harm the synaptic structure or show any detectable toxicity even 9 months after birth (Feng et al., 2000). Interestingly, there was striking variability in EGFP expression patterns among mice generated with the same construct. Different mouse lines with a comparable genetic background but differences in integration site and/or copy number have been generated (Feng et al., 2000). Thus, there are Thy-1.2EGFP mice which express GFP on most axons (L17 mice, Figs. 3A–D), as well as Thy-1.2-EGFP mice with only a few labeled axons (L21 mice, Figs. 3E–H) (Caroni, 1997). Due to the expression of EGFP on the axonal membrane in Thy-1.2-EGFP mice, it is easier to detect single axons and to

analyze their morphology and growth compared to the other mice, which express EGFP in the axoplasma. In particular, L21 mice might be helpful for studying selected axons, e.g. during pathfinding and synaptogenesis in the wildtype hippocampus. However, the subtypes of the nerve fibers which express EGFP in these mice have not yet been comprehensively analyzed. In contrast, L17 mice may be used to study the growth of higher numbers of EGFP-positive axons into EGFP-negative tissues — similar to β-actin-EGFP or Tau-EGFP mice.

4.

Outlook

Using these different mouse lines in the EGFP/wildtype coculture model, it is possible to monitor in vitro the growth of living EGFP-positive axons into GFP-negative tissue after mechanical lesion, as well as the re-innervation of target structures. The EGFP/wildtype coculture model offers a simple and instructive tool for analyzing the growth of high numbers of axons in an easily quantifiable manner (e.g. when using β-actin-EGFP mice) or for investigating the morphology and growth of many or selected axons in wildtype target tissue (e.g. when using Thy-1.2-L17 or-L21 mice, respectively). Moreover, the biological effects of cocultured cells on axonal growth, re-innervation and synaptogenesis can be studied by cocultivating the cells under a membrane to investigate their secreted factors. In addition, cells may be

166

B RA I N R E SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

applied directly to the slice to investigate, for example, neuroimmune interactions such as the invasion of T cells or monocytes. Finally, the effects of pharmacological agents on neuroregenerative processes can easily be quantified in vitro.

Acknowledgments The studies in this review were supported in part by grants from the Deutsche Forschungsgemeinschaft to RN and SH

B RA I N RE SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

(SFB507B11). The authors wish to thank Kimberly Rosegger for editing the manuscript, Sabine Lewandowski for help with digital image processing, Bernd Heimrich and Robert Glumm for introducing us to the coculture model, as well as Eva Peters and Stefan Schumacher for helpful discussions. The authors also wish to thank the following persons for generously providing EGFP-mice: Masaru Okabe (β-actin-EGFP mice), Kerry Tucker (TAU-EGFP mice) and Pico Caroni (Thy-1.2-EGFP mice).

REFERENCES

Alexander, L., Lee, H., Rosenzweig, M., Jung, J.U., Desrosiers, R. C., 1997. EGFP-containing vector system that facilitates stable and transient expression assays. BioTechniques 23, 64–66. Amaral, D.G., Witter, M.P., 1995. Hippocampal formation. The Rat Nervous System. Academic Press. Andersen, J.B., Sternberg, C., Poulsen, L.K., Bjorn, S.P., Givskov, M., Molin, S., 1998. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240–2246. Barclay, A.N., Letarte-Muirhead, M., Williams, A.F., 1975. Purification of the Thy-1 molecule from rat brain. Biochem. J. 151, 699–706. Barker, T.H., Grenett, H.E., MacEwen, M.W., Tilden, S.G., Fuller, G.M., Settleman, J., Woods, A., Murphy-Ullrich, J., Hagood, J.S., 2004. Thy-1 regulates fibroblast focal adhesions, cytoskeletal organization and migration through modulation of p190 RhoGAP and Rho GTPase activity. Exp. Cell Res. 295, 488–496. Blackstad, T.W., 1958. On the termination of some afferents to the hippocampus and fascia dentata; an experimental study in the rat. Acta Anat. (Basel) 35, 202–214. Caroni, P., 1997. Overexpression of growth-associated proteins in the neurons of adult transgenic mice. J. Neurosci. Methods 71, 3–9. Cheng, L., Fu, J., Tsukamoto, A., Hawley, R.G., 1996. Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nat. Biotechnol. 14, 606–609. Clontech Ltd., 1996. Living Color GFP Application Notes. CLONTECH Lab., Palo Alto, CA.

167

Corish, P., Tyler-Smith, C., 1999. Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 12, 1035–1040. Cubitt, A.B., Heim, R., Adams, S.R., Boyd, A.E., Gross, L.A., Tsien, R.Y., 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455. Denaro, M., Oldmixon, B., Patience, C., Andersson, G., Down, J., 2001. EGFP-transduced EL-4 cells from tumors in C57BL/6 mice. Gene Ther. 8, 1814–1815. Ellenberg, J., Lippincott-Schwartz, J., Presley, J.F., 1999. Dual-colour imaging with GFP variants. Trends Cell Biol. 9, 52–56. Feldmeyer, D., Roth, A., Sakmann, B., 2005. Monosynaptic connections between pairs of spiny stellate cells in layer 4 and pyramidal cells in layer 5A indicate that lemniscal and paralemniscal afferent pathways converge in the infragranular somatosensory cortex. J. Neurosci. 25, 3423–3431. Feng, G., Mellor, R.H., Bernstein, M., Keller-Peck, C., Nguyen, Q.T., Wallace, M., Nerbonne, J.M., Lichtman, J.W., Sanes, J.R., 2000. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51. Frotscher, M., Heimrich, B., 1993. Formation of layer-specific fiber projections to the hippocampus in vitro. Proc. Natl. Acad. Sci. U. S. A. 90, 10400–10403. Frotscher, M., Heimrich, B., Deller, T., 1997. Sprouting in the hippocampus is layer-specific. Trends Neurosci. 20, 218–223. Furuya, T., Tanaka, R., Urabe, T., Hayakawa, J., Migita, M., Shimada, T., Mizuno, Y., Mochizuki, H., 2003. Establishment of modified chimeric mice using GFP bone marrow as a model for neurological disorders. NeuroReport 14, 629–631. Gambotto, A., Dworacki, G., Cicinnati, V., Kenniston, T., Steitz, J., Tuting, T., Robbins, P.D., DeLeo, A.B., 2000. Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope. Gene Ther. 7, 2036–2040. Glumm, R., Kloting, A., Heimrich, B., 2002. Development of the hippocampal formation visualized with the β-actin-GFP transgenic slices. Neuroembryology 1, 17–22. Goldberg, J.L., Barres, B.A., 2000. The relationship between neuronal survival and regeneration. Annu. Rev. Neurosci. 23, 579–612. Gu, Y., Oyama, F., Ihara, Y., 1996. Tau is widely expressed in rat tissues. J. Neurochem. 67, 1235–1244. Heimrich, B., Frotscher, M., 1993. Slice cultures as a model to study entorhinal–hippocampal interaction. Hippocampus 3 Spec No, 11–17.

Fig. 3 – Expression patterns of Thy-1.2-EGFP in adult mouse brains and in the organotypic hippocampal–entorhinal brain slice coculture model. (A) Hippocampal–entorhinal formation in a 50-μm-thick horizontal slice of an adult L17 mouse brain expressing Thy1-EGFP on most axons. The brain was fixed immediately after harvesting with 4% paraformaldehyde. The white box indicates the area enlarged in panel B. (B) Enlargement of the hippocampal region (white box in panel A). Note the homogenous expression of EGFP in each layer due to the overlay of multiple EGFP-positive fibers. (C) After 14 days in culture, multiple Thy1-EGFP-positive perforant path fibers (PP) originate from the entorhinal cortex (EC) of Thy1-EGFP-L17 mice, which express EGFP on most axons. Single axons (arrow) are easily detectable due to the membranous EGFP expression. The white box indicates the area enlarged in panel D. (D) Enlargement of the hippocampal region (white box in panel C). Arrows indicate single axons which have grown into the wildtype hippocampus. (E) Hippocampal–entorhinal formation in a 50-μm-thick horizontal slice of an adult L21 mouse brain expressing Thy1-EGFP only on low numbers of axons. The brain was fixed immediately after harvesting with 4% paraformaldehyde. The white box indicates the area enlarged in panel E. (F) Enlargement of the hippocampal region (white box in panel A). Note the non-homogenous expression of EGFP due to the irregular overlay of selected EGFP-positive fibers. (G) After 14 days in culture, in Thy1-EGFPL21 mice only, low numbers of axons express EGFP and no perforant path-like structure is visible. Single axons (arrow) are easily detectable due to the membranous EGFP expression. The white box indicates the area enlarged in panel H. (H) Enlargement of the hippocampal region (white box in panel G). Arrows indicate single axons which have grown into the wildtype hippocampus. CA: cornu ammonis; DG: dentate gyrus; EC: entorhinal cortex; HC: hippocampus; FF: fimbria fornix; PCL: pyramidal cell layer; in the dentate gyrus ML: molecular layer; GL: granule cell layer; PL: polymorphic layer; PP: perforant path; white arrows indicate single-labeled axons. Scale bars: A–C, E, F: 100 μm; D, G, H: 50 μm.

168

B RA I N R E SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

Ikawa, M., Kominami, K., Yoshimura, Y., Tanaka, K., Nishimune, Y., Okabe, M., 1995. Green fluorescent protein as a marker in transgenic mice. Dev. Growth Differ. 37, 455–459. Kain, S.R., Adams, M., Kondepudi, A., Yang, T.T., Ward, W.W., Kitts, P., 1995. Green fluorescent protein as a reporter of gene expression and protein localization. BioTechniques 19, 650–655. Kobbert, C., Apps, R., Bechmann, I., Lanciego, J.L., Mey, J., Thanos, S., 2000. Current concepts in neuroanatomical tracing. Prog. Neurobiol. 62, 327–351. Letarte-Muirhead, M., Barclay, A.N., Williams, A.F., 1975. Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes. Biochem. J. 151, 685–697. Li, D., Field, P.M., Starega, U., Li, Y., Raisman, G., 1993. Entorhinal axons project to dentate gyrus in organotypic slice co-culture. Neuroscience 52, 799–813. Li, D., Field, P.M., Yoshioka, N., Raisman, G., 1994. Axons regenerate with correct specificity in horizontal slice culture of the postnatal rat entorhino–hippocampal system. Eur J. Neurosci. 6, 1026–1037. Li, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., Fan, C., Huang, C.C., Kain, S.R., 1998. Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273, 34970–34975. Liu, H.S., Jan, M.S., Chou, C.K., Chen, P.H., Ke, N.J., 1999. Is green fluorescent protein toxic to the living cells? Biochem. Biophys. Res. Commun. 260, 712–717. LoPresti, P., Szuchet, S., Papasozomenos, S.C., Zinkowski, R.P., Binder, L.I., 1995. Functional implications for the microtubuleassociated protein tau: localization in oligodendrocytes. Proc. Natl. Acad. Sci. U. S. A. 92, 10369–10373. Lu, B., Kwan, T., Kurimoto, Y., Shatos, M., Lund, R.D., Young, M.J., 2002. Transplantation of EGF-responsive neurospheres from GFP transgenic mice into the eyes of rd mice. Brain Res. 943, 292–300. Maurer, M., Muller, M., Kobsar, I., Leonhard, C., Martini, R., Kiefer, R., 2003. Origin of pathogenic macrophages and endoneurial fibroblast-like cells in an animal model of inherited neuropathy. Mol. Cell. Neurosci. 23, 351–359. Morin, J.G., Hastings, J.W., 1971. Energy transfer in a bioluminescent system. J. Cell. Physiol. 77, 313–318. Morris, R., 1985. Thy-1 in developing nervous tissue. Dev. Neurosci. 7, 133–160. Morris, R., 1992. Thy-1, the enigmatic extrovert on the neuronal surface. BioEssays 14, 715–722. C. Mülhardt, Der Experimentator: Molekularbiologie. In, Spektrum Akademischer Verlag, Gustav Fischer, 2000, pp. 36, 59. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., Nishimune, Y., 1997. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–319. Page, D.M., Tokugawa, Y., Silver, J., Stewart, C.L., 1997. Role of Thy-1 in T cell development. J. Immunol. 159, 5285–5292. Penney, D.P., Keng, P.C., Derdak, S., Phipps, R.P., 1992. Morphologic and functional characteristics of subpopulations of murine lung fibroblasts grown in vitro. Anat. Rec. 232, 432–443. Prang, P., Del Turco, D., Kapfhammer, J.P., 2001. Regeneration of entorhinal fibers in mouse slice cultures is age dependent and can be stimulated by NT-4, GDNF, and modulators of G-proteins and protein kinase C. Exp. Neurol. 169, 135–147. Prasher, D.C., 1995. Using GFP to see the light. Trends Genet. 11, 320–323. Prendergast, F.G., Mann, K.G., 1978. Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskalea. Biochemistry 17, 3448–3453. Reif, A.E., Allen, J.M., 1966a. Mouse nervous tissue iso-antigens. Nature 209, 523.

Reif, A.E., Allen, J.M., 1966b. Mouse thymic iso-antigens. Nature 209, 521–523. Rosenzweig, M., Connole, M., Glickman, R., Yue, S.P., Noren, B., DeMaria, M., Johnson, R.P., 2001. Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34(+) hematopoietic cells. Blood 97, 1951–1959. Schlamp, C.L., Johnson, E.C., Li, Y., Morrison, J.C., Nickells, R.W., 2001. Changes in Thy1 gene expression associated with damaged retinal ganglion cells. Mol. Vis. 7, 192–201. Schmid, J.A., Neumeier, H., 2005. Evolutions in science triggered by green fluorescent protein (GFP). ChemBioChem 6 (7), 1149–1156. Shimomura, A., 1979. Structure of the chromophore of Aequorea green fluorescent protein. FEBS Lett. 104, 220–222. Shimomura, O., Johnson, F.H., 1967. Extraction, purification, and properties of the bioluminescence system of the euphausid shrimp Meganyctiphanes norvegica. Biochemistry 6, 2293–2306. Shimomura, O., Shimomura, A., 1981. Resistivity to denaturation of the apoprotein of aequorin and reconstitution of the luminescent photoprotein from the partially denatured apoprotein. Biochem. J. 199, 825–828. Shimomura, O., Johnson, F.H., Saiga, Y., 1962. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell Comp. Physiol. 59, 223–239. Simard, A.R., Rivest, S., 2004. Bone marrow stem cells have the ability to populate the entire central nervous system into fully differentiated parenchymal microglia. FASEB J. 18, 998–1000. Simon, P.D., McConnell, J., Zurakowski, D., Vorwerk, C.K., Naskar, R., Grosskreutz, C.L., Dreyer, E.B., 1999. Thy-1 is critical for normal retinal development. Brain Res. Dev. Brain Res. 117, 219–223. Skelton, D., Satake, N., Kohn, D.B., 2001. The enhanced green fluorescent protein (eGFP) is minimally immunogenic in C57BL/6 mice. Gene Ther. 8, 1813–1814. Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182. Stripecke, R., Carmen Villacres, M., Skelton, D., Satake, N., Halene, S., Kohn, D., 1999. Immune response to green fluorescent protein: implications for gene therapy. Gene Ther. 6, 1305–1312. Tanaka, R., Komine-Kobayashi, M., Mochizuki, H., Yamada, M., Furuya, T., Migita, M., Shimada, T., Mizuno, Y., Urabe, T., 2003. Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia. Neuroscience 117, 531–539. Tokugawa, Y., Koyama, M., Silver, J., 1997. A molecular basis for species differences in Thy-1 expression patterns. Mol. Immunol. 34, 1263–1272. Tsien, R.Y., 1998. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544. Tucker, K.L., Meyer, M., Barde, Y.A., 2001. Neurotrophins are required for nerve growth during development. Nat. Neurosci. 4, 29–37. van den Pol, A.N., Santarelli, J.G., 2003. Olfactory ensheathing cells: time lapse imaging of cellular interactions, axonal support, rapid morphologic shifts, and mitosis. J. Comp. Neurol. 458, 175–194. Vanier, M.T., Neuville, P., Michalik, L., Launay, J.F., 1998. Expression of specific tau exons in normal and tumoral pancreatic acinar cells. J. Cell Sci. 111 (Pt 10), 1419–1432. Vidal, M., Morris, R., Grosveld, F., Spanopoulou, E., 1990. Tissuespecific control elements of the Thy-1 gene. EMBO J. 9, 833–840.

B RA I N RE SE A R CH RE V I EW S 52 ( 20 0 6 ) 1 6 0–1 6 9

Ward, W.W., Bokman, S.H., 1982. Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry 21, 4535–4540. Williams, A.F., Barclay, A.N., 1988. The immunoglobulin superfamily-domains for cell surface recognition. Annu. Rev. Immunol. 6, 381–405.

169

Witzel, C., Rohde, C., Brushart, T.M., 2005. Pathway sampling by regenerating peripheral axons. J. Comp. Neurol. 485, 183–190. Woodhams, P.L., Atkinson, D.J., Raisman, G., 1993. Rapid decline in the ability of entorhinal axons to innervate the dentate gyrus with increasing time in organotypic co-culture. Eur. J. Neurosci. 5, 1596–1609.