Trends in Neurosciences
ReviewLooking BAC at striatal signaling: cell-specific analysis in new transgenic mice
Introduction
Since the pioneering work of Santiago Ramón y Cajal, neuroscientists have been fascinated by the beauty and variety of brain neurons. This diversity is daunting because it implies that to fully understand the brain function, it is necessary to decipher the molecular events that take place in each type of neuron. The task is particularly difficult not only because there are hundreds of distinct neuronal populations but also because these populations are not always easy to distinguish. A combination of anatomical, physiological and molecular features is often necessary for classifying neurons, as recently exemplified in the case of cortical interneurons [1]. The pattern of gene expression, arguably the ultimate consequence of cell differentiation, is a great help, when studied by single cell RT-PCR [2]. Its applicability, however, is limited to studies in which one or a few neurons are recorded using whole cell patch-clamp.
Until recently, the expression of reporter genes driven by specific promoters was not sufficiently precise to reliably identify cell populations, because transgenes are subject to positional effects, resulting in variable expression patterns that vary depending on their insertion site. This limitation was overcome to a large extent by the use of transgenic mice carrying bacterila artificial chromosomes (Box 1), pioneered by N. Heintz and his collegues at the Rockefeller university and Gensat. BACs contain several hundreds of thousands of base pairs of regulatory sequences, which recapitulate the regulation of endogenous genes much better than shorter transgenes [3]. Furthermore, they can be easily manipulated by homologous recombination in bacteria [3]. BAC-driven expression of tagged proteins allows an easy and reproducible identification of specific neuronal populations ([4], Figure 1). The usefulness of these approaches is illustrated by recent work in the striatum, a region of the brain that receives abundant dopamine innervation and is involved in several pathological conditions including Parkinson's disease and Huntington's disease, drug addiction, Tourette syndrome and obsessive–compulsive disorder. We review anatomical evidence supporting the validity of BAC transgenic mice to distinguish neurons expressing D1 and D2 dopamine receptors (D1R and D2R) (Figure 2). The main focus of this review is on signaling mechanisms and we show how the distinction of these neurons allowed for untangling the regulation of the extracellular signal-regulated kinase (ERK) pathway, dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) and gene expression. We also summarize the main in vivo methods developed so far based on BAC transgenesis and describe the results brought about by some of these approaches concerning the morphology and physiology of principal striatal neurons.
Section snippets
A simple view of the striatum output pathways
The dorsal striatum and its ventral extension, the nucleus accumbens (NAc), constitute the primary input structure of the basal ganglia, an ensemble of interconnected subcortical nuclei involved in the selection and execution of action through interactions with sensorimotor and associative brain areas 5, 6, 7, 8, 9. The striatum is unique in its complete lack of intrinsic glutamatergic neurons. Instead, most of the cells (∼95% of striatal neurons in rodents) are GABAergic medium-sized spiny
Selective activation of the ERK pathway in striatonigral MSNs by psychostimulants and l-DOPA
The striatum is perhaps the most extensively studied region of the brain in terms of pharmacology and signaling pathways, because of its involvement in many diseases and because of its size, which makes it accessible to biochemical studies. However, most studies used methods examining mixed populations of cells, whereas the use of BAC transgenic mice allows the identification of the actual cell populations in which the signaling events take place. This is well illustrated by the case of the ERK
Selective activation of signaling pathways by D2R antagonists in striatopallidal neurons
A striking observation in naive mice treated with psychostimulants or in 6-OHDA-lesioned mice receiving l-DOPA is that the ERK pathway is not activated in D2R-expressing neurons, although the degree of colocalization with D1R (Table 1) suggests that it should be the case in a fraction of these neurons. One possible explanation is that D2Rs exert a strong tonic inhibition on the activation of the ERK pathway. Indeed, the pharmacological blockade of D2R by haloperidol, raclopride or eticlopride
Untangling the complexity of DARPP-32 phosphorylation in vivo
DARPP-32 is a dual-function protein that plays a key role in striatal signaling [42]. When phosphorylated on Thr34 by protein kinase A (PKA), DARPP-32 becomes an inhibitor of serine/threonine protein phosphatase-1 (PP-1), thereby enhancing phosphorylation of proteins targeted by PP-1. In contrast, when DARPP-32 is phosphorylated on Thr75 by cyclin-dependent kinase 5 (Cdk5), it is converted into an inhibitor of PKA. However, although the regulation of the phosphorylation state of these two sites
Differential morphological plasticity of striatonigral and striatopallidal MSNs
The dendritic spines of MSNs integrate information from glutamate afferents from cortical, limbic and thalamic areas and dopamine fibers from the SNc or the VTA [45]. The number of dendritic spines correlates with the number of excitatory synapses, whereas the size of the spine heads reflects synaptic strength 46, 47, 48. Therefore, a change in spine number and spine head diameter is thought to indicate functionally important neuronal plasticity. Long-term exposure to cocaine or amphetamine
Cell population-specific gene profiling in the striatum
Microarray analysis is a powerful method to understand the changes in gene expression underlying normal and dysfunctional biological processes. Many studies have started to characterize the gene expression profiles in various brain regions in response to a variety of therapeutic agents or drugs of abuse. However, the exploitation of these results is limited by the cellular heterogeneity of brain structures. As outlined below, new and exciting strategies taking advantage of BAC transgenic mice
Perspectives
The BAC transgenic mice expressing fluorescent proteins provide an invaluable resource to easily identify specific neuronal types in vivo and in fixed tissues, and thus study specific cellular functions including signaling pathways and electrophysiological properties in identified neuronal populations. These mouse models revealed a profound, functional dichotomy of striatal neurons in physiological and pathological conditions, far beyond expectations. This dichotomy strongly suggests that the
Acknowledgements
This work was supported by Inserm and grants from Fondation pour la Recherche Médicale (FRM) to DH and JAG, from Agence Nationale de la Recherche to JAG (ANR-05-NEUR-020-03, ANR-BLAN08-1_346422), from Neuropôle de Recherche Francilien (NeRF), Région Ile de France, from Swedish Research Grants 20175, 13482 and 14862, the Wenner-Gren Foundations, the Swedish Brain Foundation and the Parkinson Foundation in Sweden (GF).
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