Proc. Natl. Acad. Sci. USA Vol. 96, pp. 3217–3222, March 1999 Neurobiology Cortical bitufted, horizontal, and Martinotti cells preferentially express and secrete reelin into perineuronal nets, nonsynaptically modulating gene expression (GABAergic interneuronsyCa21-binding proteinsyneuropeptidesychandelier cellsybasket cells) CHRISTINE PESOLD*†,WEN SHENG LIU*, ALESSANDRO GUIDOTTI*, ERMINIO COSTA*, AND HECTOR J. CARUNCHO*‡ *Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois at Chicago, 1601 West Taylor Street, MyC 912, Chicago IL, 60612; and ‡Department of Fundamental Biology, University of Santiago de Compostela, Galicia, Spain 15706 Contributed by Erminio Costa, December 28, 1998 ABSTRACT Reelin (Reln) is a protein with some struc- mouse, shows a normal Reln expression in Cajal–Retzius cells tural analogies with other extracellular matrix proteins that and secretion in the extracellular matrix in embryonic devel- functions in the regulation of neuronal migration during the opment but neuroanatomical abnormalities reminiscent of development of cortical laminated structures. In the cortex of those in the reeler mouse (4, 5). It is currently believed that the adult animals, Reln is expressed primarily in g-aminobutyric protein encoded by this gene acts downstream of Reln in a acid (GABA)ergic neurons and is secreted into perineuronal signaling pathway that controls laminar corticogenesis and the nets. However, only 50–60% of GABAergic interneurons ex- hippocampal and cerebellar development in mammalian brain press Reln. We have characterized this subpopulation of (6). cortical GABAergic neurons that expresses Reln by using two In adult telencephalon, Reln is preferentially expressed in strategies: (i) a double immunolabeling procedure to deter- GABAergic neurons (7, 8), and Dab1 is expressed predomi- mine the colocalization of Reln with neuropeptides and Ca21- nantly in pyramidal cells, as well as in a small population of binding proteins and (ii) a combination of Golgi staining and non-pyramidal cells (9). Indeed, there is a complementary Reln immunolabeling to determine the morphology of the rat pattern in the distribution of Reln and Dab1 in the brain that cortical cells that store Reln. Many interneurons that express suggests a functional link between these two proteins in the Neuropeptide Y (NPY) or somatostatin (but none of those that signaling pathway triggered extracellularly by Reln in adult express parvalbumin) are Reln-immunopositive. A small pop- mammalian brain. ulation of calbindin-positive interneurons and very few cal- Before addressing the functional significance of the pathway retinin-positive cells express Reln immunopositivity. Golgi triggered by Reln extracellularly in adult brain, it is important staining revealed that layer I horizontal cells, layer II–V to consider that perineuronal nets, which include extracellular bitufted neurons, and some deep cortical layer Martinotti matrix proteins (10, 11), may communicate with neurons via cells express Reln. Basket and chandelier cells are often substrate adhesion molecules and integrins (for a review, see immunopositive to parvalbumin, but never to Reln. Although ref. 12). Hence, although the binding site of the released Reln Reln is secreted by GABAergic neurons, its target are not the may be extracellular in the perineuronal nets, it may influence GABA receptors, but rather may be extrasynaptically located neuronal trophic activity indirectly via an interaction with the in perineuronal nets and concerned with the modulation of Dab1 protein. This interaction requires phosphorylation, neuronal plasticity. Dab1, the target adapter protein that which allows this protein to act as an adapter protein operative presumably mediates transcription regulation via the extra- in the translocation of Src kinases from the cytosol to the synaptic actions of Reln, is expressed predominantly in pyra- nucleus (13). Because a high percentage of Reln in the adult midal neurons, but it can also be detected in a small popu- cerebral cortex is located in GABAergic interneurons, whose lation of GABAergic neurons that are neither horizontal nor function in the various subtypes differs in a manner related to bitufted neurons. their morphology, it is important to characterize whether a morphologically specialized set of these interneurons ex- The notation ‘‘reelin’’ (Reln) refers to a protein secreted into presses Reln. In an effort to further characterize which sub- the extracellular matrix during embryonic development that is types of GABAergic interneurons express Reln in the adult structurally related to other extracellular matrix proteins (1). cortex, we have used a combination of immunocytochemical In early development, Reln is selectively synthesized and and Golgi-staining techniques. This study will allow us to secreted by specialized neurons (termed Cajal–Retzius) lo- suggest a possible interaction of Reln–Dab1 among specific cated in the marginal zone of the developing brain (1). A populations of cortical GABAergic interneurons, or among mutant mouse that fails to synthesize or secrete this protein these interneurons and pyramidal neurons. This work will also shows a characteristic reeling gate and a disruption of the allow us to address the functional role of Reln released by typical laminated pattern of telencephalic cortex (for review, GABAergic neurons into the extracellular matrix of perineu- see refs. 2 and 3). In these mice, the structure of the hip- ronal nets (10, 11). pocampus and cerebellum is also disrupted, and the cerebellar size is grossly reduced. Another protein, which in embryos is MATERIALS AND METHODS normally expressed by the migrating cortical and hippocampal neurons and cerebellar Purkinje cells, is encoded by a gene A total of 12 adult Fisher rats (180–240 g) were used in this termed Dab1, which is mutated in the scrambler mouse (4–6). study. They were anaesthetized with equithesin and perfused intracardially with saline followed by a cold solution of 4% This neurological phenotype, which is similar to the reeler paraformaldehyde in PBS (pH 7.4). Brains were removed, left The publication costs of this article were defrayed in part by page charge Abbreviations: GABA, g-aminobutyric acid; NPY, neuropeptide Y; payment. This article must therefore be hereby marked ‘‘advertisement’’ in GAD, glutamic acid decarboxylase. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. e-mail: cpesold@ PNAS is available online at www.pnas.org. psych.uic.edu. 3217 Downloaded by guest on September 28, 2021 3218 Neurobiology: Pesold et al. Proc. Natl. Acad. Sci. USA 96 (1999) in fresh fixative for 24 hours at 4°C, and washed in PBS. Brains because of space limitations) were immunostained for Reln to be processed for light microscopy immunolabeling were according to a previously described protocol (7). After dia- infiltrated in 30% sucrose, and 20-mm thick sections were minobenzidine immunostaining, 80-mm sections were embed- obtained with a cryostat. For Golgi staining, blocks of cortex ded in LR-white resin. Semithin sections (0.5–1 mm in thick- about 1–2 mm thick were cut with a razor blade. ness) containing the neurons of interest were taken with an Double Immunolabeling for Confocal Microscopy. The co- LKB-Nova Biochem ultramicrotome, mounted on slides, and localization of Reln and either somatostatin, neuropeptide Y examined under the light microscope for Reln-immunoreac- (NPY), or calretinin was determined by using a double im- tivity. Photomicrographs of these same cells, now immunola- munofluorescence approach. Because the antibodies against beled for Reln, were taken and deemed Reln-immunopositive parvalbumin and calbindin were raised in the same species as if their cytoplasm was immunostained. the Reln antibody, and similarly the antibodies against Dab1 It is important to note here that Golgi staining does not and GAD67 were raised in the same species, a combination readily infiltrate the axon myelin sheaths, therefore in adult immunogoldyimmunofluorescence was applied. This proce- brain it is sometimes difficult to trace the axonal arborizations. dure allows the silver shell that amplifies the gold particles to It is therefore necessary to impregnate many cells to make an completely engulf the antigenic site, preventing any cross- accurate identification based on their axonal arborization. reactivity. Quantification of the Coexistence of Neuropeptides and For double immunofluorescent labeling, sections were Proteins in GABAergic Interneurons Epressing Reln. To washed in PBS, blocked for 30 minutes in each of RPMI determine which percentage of frontoparietal cortex cells that 21 medium 1640 (GIBCO), 2% normal serum (Chemicon), and express either a given neuropeptide or a Ca -binding protein 1% BSA (Sigma) in PBS before incubation in a solution also express Reln, three consecutive coronal sections from containing both the G-10 (14) primary antibody for Reln each of three animals were studied by using a Leica TCS-NT (1:1,000) and either rat anti-somatostatin (1:500; Chemicon), laser confocal microscope. For each neurochemical marker rabbit anti-calretinin (1:2,000; Chemicon) or rabbit anti-NPY (e.g., calretinin, calbindin, parvalbumin, NPY, and somatosta- (1:5,000; Sigma). After several washes, sections were incubated tin), every cell that contained that marker in the frontoparietal for 1 hour with an anti-mouse secondary antibody conjugated cortex was identified