Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

BALB/cJ and C57BL/6J mice, a sequence variation in the Rpe65 gene cosegregated with the low LDS of C57BL/6J mice (Danciger et al., 2000). We have typed the six albino strains of mice with ARRD identified in our screening study for sequence variation in the Rpe65 gene and found that two of them (RIII/DmMobJ and NZW/LacJ) have the same Rpe65 genotype as C57BL/6J. The other four carry the susceptibility allele.

Cataracts in human and mouse eyes

Early in life the lens is transparent, and incoming light encounters no difficulty in its passage through the eye. With aging, the lens becomes less clear, incoming light is scattered to an increasing degree, and, if loss of lens clarity is severe enough, vision is affected. When clouding of the lens impairs vision, a clinically significant cataract is present. More than half of Americans over age 65 have a cataract ( Jacques et al., 1997). Cataracts account for 42% of all vision loss, making the disorder the leading cause of blindness worldwide (National Eye Institute report, Vision Research: A National Plan: 1999–2003). Based on our preliminary studies, agerelated cataracts are also very common in mice. At TJL, when we screened aged mice from 35 strains for models of human age-related ocular diseases, we identified 21 strains with a high incidence of late-onset cataracts. Onset ranged from 8 to 22 months, and 14 strains of mice had clear lenses throughout their lives (see table 48.2).

There is increasing epidemiological evidence that agerelated cataracts are a multifactorial disease, with environmental and genetic components interacting (West and Valmadrid, 1995). Epidemiological studies also suggest a major inherited genetic component in the etiology of both anterior cortical and nuclear cataracts (Heiba et al., 1993, 1995), and twin studies suggest that 50% of age-related cataracts can be accounted for by inheritance (Hammond et al., 2000). Inheritance is already known to be the major contributor to congenital cataract occurring in childhood, and several mutated genes causing human congenital cataracts have already been identified (Francis et al., 2000).

Because cataracts are easily detected by external clinical examination, the number of described mouse cataract models has grown rapidly. A 1982 review listed 18 spontaneous and induced mutations that cause cataracts as part of the resulting phenotype (Foster et al., 1982). By 1997 the number had grown to 46 (Smith et al., 1997). Research by our group at TJL has identified the spontaneous mutation lop18 in the mouse Cryaa (α-crystallin) gene (Chang et al., 1999), the spontaneous mutation Lop12 in the Crygd (γDcrystallin) gene (Smith et al., 2000), and the spontaneous mutation Lop10 in the Gja8 (connexin 50) gene (Chang et al., 2002). Currently, human mutations have been identified in the genes encoding some lens-specific crystallins,

connexins, aquaporin, and beaded filament protein, BFSP2 (Sheils et al., 1998; Heon et al., 1999; Mackay et al., 1999; Berry et al., 2000; Jakobs et al., 2000; Rees et al., 2000). In most cases the genetic mutations causing cataracts have been identified using a candidate gene approach once the chromosomal location of the mutation has been determined. It has been estimated that there might be as many as 40 genes contributing to congenital cataracts in the mouse, and it would be reasonable to assume a similar number in humans (Hejtmancik and Kantorow, 2004). Thus, the concept of genetic mechanisms causing earlyonset cataracts is well established, but to date, very little is known concerning the identity of genes that, either alone or together with environmental factors, confer susceptibility to age-related cataracts.

Discovery of Age-Related Cataracts in Mouse In our search for late-onset ocular diseases in mice, we have found that the majority of eye diseases occur in aged mice, just as they do in humans. In the study described earlier in which we evaluated aged mice of 35 selected, genetically diverse inbred strains at greater than 2 years of age for age-related eye disorders, we also screened for lens opacity. As part of this program, we have found seven strains of mice with lens opacity starting at eight months of age (RIII/DmMobJ, CBA/CaJ, CASA/RkJ, CAST/EiJ, LEWES/EiJ, PERA/ EiJ, TIRANO/EiJ), eight strains of mice with cataract starting at 14 months of age (C57BL/6J, C57BR/cdJ, AU/ SsJ, CZECHII/EiJ, MOLC/RkJ, MOLD/RkJ, PERC/EiJ, SKIVE/EiJ), and six strains of mice with a mild lens opacity starting at 22 months of age (DBA/1J, LP/J, CE/J, PANCEVO/EiJ, SPRET/EiJ, WSB/EiJ). Mice of another 14 strains have clear lenses throughout their lives (see table 48.2).

Summary

Because the 35 strains of mice in our age-related vision loss study were all inbred, the individuals in a strain are genetically identical (like monozygotic twins), and all mice were maintained in the same standard environment, our findings demonstrate that genetic mechanisms contribute to the etiology and pathogenesis of ARRD and cataracts. No matter what the mode of inheritance or number of genes involved, the strains themselves provide reproducible models for research on similar human eye disorders.

acknowledgments Work was supported by the Foundation Fighting Blindness. I am grateful to Norm Hawes, Ron Hurd, Jieping Wang, Muriel Davisson, Tom Roderick, and John Heckenlively for their excellent help on the mouse aging project, and Melissa Berry for her critical reading of the manuscript.

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Despite its comparatively reduced visual abilities, the mouse is becoming one of the most important organisms for investigating the physiology, organization, and development of the mammalian visual system. This trend reflects the ease with which genetic manipulations can be performed in the whole animal, including manipulation of defined subsets of neurons. Mouse studies have made significant contributions to the three main and interrelated areas of vision research: the molecular mechanisms of neuronal function and development, circuits and information coding, and pathological mechanisms of disease. In each of these areas, the modern mouse geneticist’s approach consists largely in introducing or removing various genetic elements and asking how neuronal or glial structure, function, or development has been modified.

This chapter surveys current genetic approaches used to visualize subpopulations of cells or to alter their function or development. The emphasis is on retinal neurons, although the experimental approaches we discuss apply equally to glia and to visual pathways beyond the retina. Rather than providing an exhaustive summary, we highlight examples that illustrate general principles.

Gene transfer and gene targeting methods

In some situations, it is feasible to directly deliver DNA or RNA to target cells in the living animal, for example by injecting them into the eye as recombinant viruses or by using electroporation (Matsuda and Cepko, 2004; Kachi et al., 2005; Bi et al., 2006). With these approaches the researcher can achieve a decrement in gene expression by RNA interference (RNAi), or the ectopic production of reporters, light-activated channels, or other proteins. This is especially attractive for animals in which germ-line manipulation is difficult or impossible or in which the eye is particularly accessible to injection. In addition, ex vivo experiments,

in which isolated retinas are maintained in culture, allow gene delivery by the aforementioned approaches, as well as by particle-mediated gene transfer (the “gene gun”) (Wellmann et al., 1999; Gan et al., 2000; O’Brien and Lummis, 2004). Individual neurons can also be labeled using particlemediated transfer of lipophilic dyes (Sun et al., 2002; Pignatelli and Strettoi, 2004). Applications of somatic cell gene transfer technologies include visualizing cell lineages and neuronal morphology by expressing histochemical reporters (Price et al., 1987; Turner et al., 1990), and modifying or monitoring function, for example, by RNAi or expression of a fluorescent calcium indicator. In some cases of somatic cell gene transfer, gene expression can be limited to a defined subset of neurons with a cell type–specific promoter. More commonly, the inefficient gene delivery process produces a sparse population of transduced cells, permitting the identities of individual cells to be determined later based on morphology and position, a strategy that was used in the retroviral marking of neuronal lineages (Turner et al., 1990). Because somatic cell gene transfer approaches are not specific to the mouse, they are not the focus of this review. However, they represent valuable alternatives to germ-line manipulation and can also be used in conjunction with the germ-line approaches discussed in this chapter.

Among techniques for altering the germ line, the insertion of transgenes (figure 49.1) is conceptually the simplest. Classically, this has involved the random insertion of relatively small constructs in which promoters with more or less restricted expression patterns control reporter genes (such as E. coli β-galactosidase; human placental alkaline phosphatase [PLAP], or fluorescent proteins such as green fluorescent protein [GFP]), genetic modulators (such as Cre recombinase or the ligand-controlled reverse tetracycline transactivator [rtTA]), or, more recently, modulators of physiological function (such as drugor light-activated ion channels). Reporter genes are usually enzymes with

B

Reporter Gene

Figure 49.1 Transgenic approaches. Transgenic constructs are stably inherited genetic elements that integrate at random chromosomal locations and with a variable number of copies at the site of integration. A, Typical transgenes are composed of promoter elements that can direct transcription in more or less restricted cell populations and various cDNAs encoding reporter genes (such as PLAP or GFP), genetic regulators (represented here by the Cre recombinase), or other functional effectors (represented here by the α subunit of diphtheria toxin, which ablates the cell in which it is expressed). B, Bacterial artificial chromosome (BAC) transgenes are also randomly integrated in the genome; however, they carry larger segments of DNA and often reproduce more faithfully the expression pattern of the endogenous gene. The desired genetic elements can be placed under the control of the transgene by means of an internal ribosomal entry site (IRES) as a second translation unit (top), or they can replace the transgene coding region (bottom).

well-established histochemical stains, such as β-galactosidase or PLAP, or fluorescent proteins such as GFP. Transgene constructs are injected into the pronuclei of fertilized eggs and the resulting founders are screened by polymerase chain reaction (PCR) or Southern blotting for the presence of the transgene (Hogan et al., 1986). These founders will transmit the stably integrated transgene through the germ line to their offspring, which can then be analyzed to determine the pattern of transgene expression. In general, only a fraction of the lines express the injected transgene, and the expression pattern may vary substantially from line to line. In addition, transgene expression patterns are often variegated (i.e., expressed in only a subset of the expected cells), an effect that appears to reflect the chromatin structure at the site of integration. Finally, many transgenes are expressed in subsets of cells distinct from the ones in which the promoter segment used to drive expression is normally active (Xiang

et al., 1996). In some situations, variegation or misexpression has proved useful, as described later in the chapter.

The utility of the classic transgenic approach depends on the availability of promoter segments that can drive expression in defined subsets of neurons. So far, this has been the case in only a few instances, most notably in primary sensory neurons that express receptor proteins in a cell type–specific manner. In the mouse retina, rhodopsin is produced in rod photoreceptors, short-wavelength-sensitive (S) photopigment is produced in S cones, middle-wavelength-sensitive (M) photopigment is produced in M cones, and melanopsin is produced in a special class of light-responsive retinal ganglion cells (RGCs). Even in this relatively simple system, however, there is an imperfect correlation between cell type and molecular marker: in mice and other rodents, some cones coexpress both S and M photopigment genes (Rohlich et al., 1994; Applebury et al., 2000). A second route to cell type–specific promoters for transgene expression in the retina is through the use of genes that are expressed by many neuronal populations elsewhere in the CNS but are restricted in the retina to one or a few cell types. Two examples are the genes encoding choline acetyltransferase and tyrosine hydroxylase, which are expressed in starburst and dopaminergic amacrine cells, respectively (Tauchi and Masland, 1984; Gustincich et al., 1997). Many other promoters are available for broad classes of neurons; for example, the Crx promoter is expressed in all photoreceptors (Furukawa et al., 2002).

Bacterial artificial chromosome (BAC) transgenesis (figure 49.1B) represents a variant strategy in which a large chromosomal fragment containing the gene of interest is typically engineered to direct the expression of a foreign protein (Heintz, 2001). The rationale for this strategy is that a large (100–200 kb) fragment, often containing the entire gene of interest, will more faithfully reproduce the endogenous expression pattern than a small promoter fragment, and will therefore direct reporter expression more precisely to that cell population. In some instances in which a transgene is normally expressed in multiple cell types or at multiple stages of development, it is desirable to activate the transgene in a subset of these cells or only during a limited temporal window. This can be achieved by using conditional expression systems, which are discussed at the end of the chapter.

The second major class of germ-line genetic manipulation involves targeted alterations using embryonic stem (ES) cells. This typically involves reporter insertion (knock-in), target gene deletion (knockout), or a combination of the two (figure 49.2). Briefly, in this approach, the construct of interest is introduced into ES cells, where it recombines with the target locus by homologous recombination. The potentially difficult steps are (1) construction of the targeting plasmid,

(2) screening for the correct homologous recombination event among many ES clones, and (3) the subsequent generation

Figure 49.2 Knock-in and knockout strategies. In the example shown here, the endogenous exons of the target gene are flanked by loxP sites, and the endogenous transcription unit is followed by a reporter gene that is transcriptionally silent prior to Cre-mediated recombination. Recombination induced by Cre deletes the endogenous gene’s exons and thereby allows the expression of the reporter gene under the same 5′ transcriptional regulatory elements as the

of the mutant mouse by injection of ES lines into early embryos. However, the advantages are substantial. Knock-in lines generally exhibit less variegation and more faithful patterns of reporter gene expression than transgenic lines, the dosage of the modified gene is well defined, and the line-to- line variability in expression that is seen in transgenic lines (reflecting the different locations and copy numbers of the transgene) is eliminated. As a result of this last characteristic, the number of lines to be screened is substantially reduced.

Targeted gene manipulations are increasingly being used in combination with site-specific DNA rearrangement catalyzed by sequence-specific Cre or Flp recombinases (Branda and Dymecki, 2004), which can be expressed (from a second locus) in defined subsets of cells and, in the case of the CreER(T) fusion protein, can also be controlled pharmacologically. This approach has been used to circumvent problems of lethality or other severe phenotypes associated with the ubiquitous loss of gene function that in many cases is observed with the standard knockout allele. With the availability of new mouse lines that express the recombinases under cell type–specific control, this approach is increasingly being used to produce changes in gene function in defined subsets of cells and to map the cell types and time windows in which a particular gene acts.

In the following sections we describe specific examples of these approaches as they have been applied to different retinal cell types. At the end of the chapter we discuss techniques for temporal control of gene expression.

Photoreceptors

Promoter sequences of bovine (Zack et al., 1991), human (Wang et al., 1992; Chen et al., 1994; Chiu and Nathans, 1994a), and murine (Chiu and Nathans, 1994b) photopigment genes have been used to drive expression of β-

endogenous gene. We note here that any regulatory elements located within introns would be deleted. This technique can be used to guide functional and morphological analyses of the cells that normally express the target gene. These cells can be studied in the heterozygous or homozygous mutant state, depending on whether the other allele of the gene is wild type or mutant (assuming that the gene is autosomal).

galactosidase or PLAP reporters in transgenic mice. These experiments showed that photopigment promoter transgenes from various mammals generally recapitulate the expression patterns of the corresponding endogenous genes. However, the expression of these transgenes exhibits substantial variegation, and, in the case of the human S cone photopigment promoter, misexpression was also observed in a subset of cone bipolar cells (Chen et al., 1994; Chiu and Nathans, 1994a). Interestingly, the human long-wavelength

(L) pigment promoter and 5′ enhancer drives transgene expression in both M and S cones in the mouse (Wang et al., 1992). With larger photopigment promoter segments, expression is generally observed in a larger fraction of the expected target cells (Zack et al., 1991). For example, transgenic constructs carrying 11 kb of the mouse rhodopsin locus, including 4.5 kb of 5′ flanking sequences and all of the introns and exons, efficiently express rhodopsin in rod photoreceptors, which facilitated the study of a rhodopsin mutant responsible for retinitis pigmentosa (Sung et al., 1994).

One of the most informative genetic approaches is the targeted ablation of a specific cell type to assess its role in the circuitry of the system. Soucy et al. (1998) used this strategy to assess the role of cones in the scotopic (dim light) visual pathway. Prior studies had shown that rods transmit their signals via metabotropic glutamate receptors on rod bipolar cells, all of which are of the ON type; these bipolar cells then synapse onto AII amacrine cells in the innermost division of the inner plexiform layer (IPL). The AII amacrine cells in turn feed the rod signals into the cone pathway by means of gap junctions with ON cone bipolar cells and by sign-inverting glycinergic synapses with OFF cone bipolar cells. From this circuitry, it would be predicted that the glutamate analogue L-(+)-2-amino-4-phosphonobutyric acid (APB), which selectively activates metabotropic glutamate receptors, and therefore blocks both rod-to-rod bipolar and

cone-to-ON-cone bipolar synaptic signals, should inhibit both ON and OFF responses of RGCs under scotopic conditions. However, Soucy and colleagues observed that applying APB to the retina ex vivo effectively abolished only ON responses under scotopic conditions; OFF responses remained largely intact. Soucy and colleagues also observed that strychnine, which blocks glycinergic signaling from AII amacrine cells to OFF cone bipolar cells, failed to inhibit the scotopic OFF RGC response. A second, APB-resistant pathway involving electrical coupling between rods and cones had previously been identified in the rabbit retina and could, in theory, have accounted for these data (DeVries and Baylor, 1995). To address this possibility, a human red pigment gene promoter was used to drive the expression of the α subunit of diphtheria toxin in cones, but not in rods or other cell types (Soucy et al., 1998). This transgene selectively eliminates nearly all cones, leaving only a few survivors in the inferior retina. Not surprisingly, retinas prepared from these animals failed to respond to light stimuli in the photopic (high light level) range. Under scotopic conditions, the RGC responses were nearly normal, as assayed by multielectrode array recordings. Interestingly, the scotopic APBresistant OFF pathway responses were comparable to those in wild-type mice, indicating that, under these experimental conditions, cones are not required for the transmission of rod signals to OFF RGCs. These data implicated a third and previously unknown pathway leading from rod photoreceptors to OFF RGCs, which has since been characterized as a direct synaptic contact between rods and OFF cone bipolar cells (Hack et al., 1999; Tsukamoto et al., 2001; FykKolodziej et al., 2003; Li et al., 2004).

Bipolar cells

The role of gap junctions in the bipolar-AII amacrine cell circuit has been investigated with the use of transgenic and knockout/knock-in mice. Connexins are the building blocks of gap junctions, providing a direct electrical connection between neurons. In the mouse retina, connexin 36 and connexin 45 are expressed in distinct subsets of neurons. For example, an ON cone bipolar cell type specifically labeled by GFP in the BPGus-GFP transgenic mouse line expresses connexin 36 but not connexin 45 (Han and Massey, 2005; Lin and Masland, 2005). Replacing the open reading frame of the connexin 36 gene with a bicistronic gene encoding both PLAP and β-galactosidase revealed connexin 36 gene expression in photoreceptors, several classes of bipolar cells, and AII amacrine cells (Deans et al., 2001, 2002). In retinas from a connexin 36 knockout mouse, scotopic responses for both ONand OFF-center RGCs were abolished, and there was a defect in gap junctional coupling between AII amacrine cells and cone bipolar cells. Although the RGC defect might well be a direct consequence of the AII amacrine-

bipolar cell defect, connexin 36 is also expressed at other points within this circuit, such as the rod-to-cone gap junction, which might contribute to the phenotype (Deans et al., 2002; Völgyi et al., 2004). Conditionally knocking out connexin 36 in defined subsets of neurons should resolve this issue.

More recently, the role of connexin 45 has been investigated using a conditional knock-in strategy. Since a knockout of connexin 45 is embryonic lethal, a knock-in allele was constructed in which exon 3 (encompassing the entire open reading frame) was flanked by loxP sites and followed by the coding region for GFP (see figure 49.2 for an analogous knock-in strategy). After crossing in a Nestin-Cre transgene, which leads to Cre-mediated recombination principally in the CNS and is compatible with viability, several types of OFF and ON cone bipolar cells, but not rod bipolar cells, were seen to express this connexin gene, as judged by the localization of GFP. In addition, in the conditional mutant mouse, the electroretinogram (ERG) b-wave, which is thought to largely represent bipolar cell depolarization, was greatly reduced under scotopic but not photopic conditions, suggesting an involvement of connexin 45 in the pathway leading from rods to cone bipolar cells (Maxeiner et al., 2005; Schubert et al., 2005).

In the retina, expression of the metabotropic glutamate receptor mGluR6 is restricted to rod and ON cone bipolar cells. In mGluR6 knockout mice, the ERG b-wave is abolished, and visually evoked potentials in the superior colliculus are severely reduced. Nevertheless, the general architecture of the retina is preserved, and the mice can be trained to respond to light stimuli (Masu et al., 1995; Tagawa et al., 1999).

Signaling through mGluR6 appears to require the Goα subunit of heterotrimeric G proteins, since the Goα gene knockout phenocopies the mGluR6 phenotype in the retina (Dhingra et al., 2000, 2002). A 9.5 kb upstream region encompassing the promoter of the mGluR6 gene is sufficient to drive β-galactosidase expression in the correct cell types (Ueda et al., 1997). This information was used to construct the corresponding GFP line, and by imaging the GFPexpressing retinas ex vivo, Morgan et al. (2006) were able to observe the development of axonal and dendritic processes of bipolar cells and to show that they derive from basal and apical processes of the postmitotic precursor. This example nicely illustrates the utility of ex vivo imaging with a fluorescent reporter that is limited to one or a few cell types.

Several other useful genetic tools have been generated for the study of bipolar cells. A BAC transgenic line has been constructed in which the Pcp-2 gene (Purkinje cell protein-2, a protein/gene of unknown function, also known as L7) was modified to carry an internal ribosome entry site (IRES)-Cre coding region in the 3′ untranslated region, resulting in Cre expression specifically in rod bipolar cells (Zhang et al.,

2005). To trace synaptic connections and projection patterns, wheat germ agglutinin (WGA), a plant lectin known to be transported anterogradely along axons (Yoshihara et al., 1999), has been introduced as a transgene under the control of the L7 promoter, which drives expression in rod bipolar cells (Hanno et al., 2003). The level of WGA production is high enough to observe retrograde transsynaptic transport to photoreceptors, as well as anterograde transsynaptic transport of the protein to RGCs, revealing their axonal projections into the brain.

The intracellular concentrations of ions associated with signaling and membrane voltage changes can be visualized with subcellular spatial resolution using genetically encoded fluorescent reporters. Kuner and Augustine in 2000 reported that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) is dependent on chloride concentration. If a CFP-YFP fusion protein (Clomeleon) is excited with a wavelength suitable for CFP, the energy can be efficiently transferred to YFP, but the efficiency of energy transfer decreases with increasing chloride concentration. By expressing this construct in neurons one can monitor the opening of chloride channels such as GABA receptors. In one application of this technique, the Thy-1 promoter, which is discussed in detail in the next section, was used to drive the expression of transgenic Clomeleon in a diverse population of retinal neurons to study the ionic basis of ON versus OFF bipolar cell responses (Duebel et al., 2006). At the synapse between photoreceptors and bipolar cells, ON bipolar cells invert the polarity of the membrane potential (by using mGluRs) and OFF bipolar cells conserve the polarity (by using ionotropic glutamate receptors). It was previously inferred that ON and OFF bipolar cells have different dendrite:soma internal chloride (iCl) ratios. The YFP : CFP emission ratio of the transgenic Clomeleon showed that ON bipolar neurons have a high iCl in their dendrites, and therefore the opening of chloride channels (following GABA release from horizontal cells) leads to an outward chloride flux and membrane depolarization. By contrast, OFF bipolar cells have a low dendritic iCl concentration and respond to chloride channel opening with chloride influx and membrane hyperpolarization. By this simple mechanism, GABA release from horizontal cells produces receptive field antagonism in both the ON and OFF channels.

In cone bipolar cells, the transgenic Clomeleon was used to measure iCl in various cell compartments at resting membrane potential. Even though the iCl at the resting potential varies among different cells, the dendritic iCl was consistently higher and the axonal (i.e., output) arbor iCl was consistently lower than that of the soma. Moreover, two morphologically distinct bipolar cell types, type 7 and type 9, had distinct dendrite:soma iCl ratios, with type 9 having significantly higher dendritic iCl. Application of GABA to

these two cell types had different effects, inducing chloride influx in type 7 and efflux in type 9 cells. Interestingly, one of the Thy-1–Clomeleon lines expresses the transgene in a bipolar cell subpopulation in which the dendritic arbors exclusively contact S cones (Haverkamp et al., 2005).

Amacrine cells

As noted earlier in this chapter, transgene expression patterns can vary considerably among different mouse lines carrying the same construct, depending on the location of transgene integration. Although this is not always a desirable characteristic, sometimes the “wrong” expression pattern turns out to be useful. As an example, expression of a β- galactosidase transgene driven by the vasoactive intestinal peptide (VIP) promoter was observed in a subpopulation of amacrine cells that do not normally express VIP (Nirenberg and Meister, 1997). Since these cells appeared quite uniform with respect to their distribution across the retina and dendritic arborization, they were considered to be members of one specific amacrine cell class, V-amacrine cells. The V- amacrine cells were photoablated with approximately 95% efficiency by incubating with the cell-permeant β-galactosi- dase substrate fluorescein-d-galactopyranoside, followed by intense illumination in the presence of aminoethyl carbazole (Nirenberg and Cepko, 1993). Multielectrode array recordings revealed a significant increase in response latency and response duration in ON-transient RGCs in the photoablated retinas (Nirenberg and Meister, 1997). Immunohistochemical characterization indicated that V-amacrine cells are GABA-ergic, consistent with the observation that picrotoxin, a GABA receptor antagonist, has effects on response latency and duration that resemble the effects of photoablation. These data suggest that V-amacrine cells shape RGC responses via GABA release, although their exact place in the inner retinal circuitry remains to be elucidated.

A second subpopulation of amacrine cells that have been labeled genetically consists of dopaminergic amacrine cells. Gustincich et al. (1997) isolated a transgenic mouse line that expresses PLAP under the control of the tyrosine hydroxylase (TH) promoter in dopaminergic amacrine cells. Interestingly, PLAP expression was also observed in a second class of wide-field amacrine cells, with dendritic arbors stratifying in the middle of the IPL; the dendritic arbors of dopaminergic amacrine cells stratify only in the outermost strata of the IPL. After enzymatic dissociation of the transgenic retina, live dopaminergic amacrine cells were identified by staining with fluorescent anti-PLAP antibody (PLAP is a glyosyl phosphatidyl inositol [GPI]-anchored protein that accumulates on the extracellular face of the plasma membrane) and studied electrophysiologically. With this approach, Gustincich et al. showed that these cells fire spontaneous action potentials that are repressed by GABA. Moreover, by