Alpha2-adrenergic receptor activation regulates cortical interneuron migration

MOLECULAR AND DEVELOPMENTAL NEUROSCIENCE

Alpha2-adrenergic receptor activation regulatescortical interneuron migration

Orbicia Riccio,1,2,3 Nicolas Hurni,1,2,3 Sahana Murthy,1,2,3 Laszlo Vutskits,2,3,4 Lutz Hein5 and Alexandre Dayer1,2,3

1Department of Mental Health and Psychiatry, University of Geneva Medical School, Geneva, Switzerland2Department of Basic Neurosciences, University of Geneva Medical School, Geneva, Switzerland3Geneva Neuroscience Center, University of Geneva Medical School, Geneva, Switzerland4Department of Anesthesiology Pharmacology and Intensive Care, University Hospital of Geneva, Geneva, Switzerland5Institute of Experimental and Clinical Pharmacology and Toxicology and BIOSS Centre for Biological Signalling Studies, Universityof Freiburg, Freiburg, Germany

Keywords: cortex, development, monoamine, noradrenaline

Abstract

Monoamines such as serotonin and dopamine have been shown to regulate cortical interneuron migration but very little is knownregarding noradrenaline. Similarly to other monoamines, noradrenaline is detected during embryonic cortical development andadrenergic receptors are expressed in transient embryonic zones of the pallium that contain migrating neurons. Evidence of afunctional role for the adrenergic system in interneuron migration is lacking. In this study we first investigated the expression patternof adrenergic receptors in mouse cortical interneuron subtypes preferentially derived from the caudal ganglionic eminences, andfound that they expressed different subtypes of adrenergic receptors. To directly monitor the effects of adrenergic receptorstimulation on interneuron migration we used time-lapse recordings in cortical slices and observed that alpha2 adrenergic receptors(adra2) receptor activation inhibits the migration of cortical interneurons in a concentration-dependent and reversible manner.Furthermore, we observed that following adra2 activation the directionality of migrating interneurons was significantly modified,suggesting that adra2 stimulation could modulate their responsiveness to guidance cues. Finally the distribution of corticalinterneurons was altered in vivo in adra2a ⁄ 2c-knockout mice. These results support the general hypothesis that adrenergicdysregulation occurring during embryonic development alters cellular processes involved in the formation of cortical circuits.

Introduction

In rodents, cortical interneurons are mainly generated in the medialand caudal ganglionic eminences of the subpallium and migratetangentially to reach the developing cortex (Wonders & Anderson,2006; Gelman & Marin, 2010; Rudy et al., 2011). The specificationand migration of cortical interneurons is controlled by a combinatorialcascade of transcription factors which regulates a variety of receptorsand effectors required for their proper response to cell-extrinsic cues(Flames & Marin, 2005; Chedotal & Rijli, 2009). Among theseexternal cues, monoamines such as serotonin and dopamine have beenshown to regulate cortical interneuron migration (Crandall et al.,2007; Riccio et al., 2009). Similarly to serotonin and dopamine,noradrenaline is another monoamine which is detected during corticaldevelopment and has been suggested as modulating cellular processesinvolved in the formation of cortical circuits (Lidow & Rakic, 1994).Support for a role for noradrenaline in this process comes from the factthat noradrenergic fibres reach the rodent developing cortex at late

embryonic day (E)16 (Levitt & Moore, 1979), at a time period whencortical interneurons are in the process of actively invading theintermediate zone and cortical plate. A potential role for noradrenalinein neuronal migration is not restricted to rodents. In humans and non-human primates, noradrenaline fibres have been shown to reach theearly developing cortex during a period of intense neuronal migration(Lidow & Rakic, 1994; Zecevic & Verney, 1995; Wang & Lidow,1997). Further support for a developmental role of noradrenalinecomes from studies demonstrating that adrenergic receptors arestrongly expressed during embryonic cortical development (Lidow& Rakic, 1994; Wang & Lidow, 1997; Winzer-Serhan & Leslie,1999). Alpha1 adrenergic receptors (adra1), alpha2 adrenergic recep-tors (adra2) and beta adrenergic receptors (adrb) display distinct andrestricted temporospatial expression throughout the transient embry-onic zones of the macaque and rodent pallium (Lidow & Rakic, 1994;Wang & Lidow, 1997; Winzer-Serhan & Leslie, 1999). The expres-sion pattern of adrenergic receptors in the developing pallium has ledto the hypothesis that these receptors could regulate differentdevelopmental processes including neuronal migration (Wang &Lidow, 1997). Interestingly, in non-neuronal systems, adrenergicmodulation regulates the migration of different cell types includinghematopoietic progenitor cells (Spiegel et al., 2007), corneal epithelial

Correspondence: Dr Alexandre G. Dayer, 1Department of Mental Health and Psychiatry,as above.E-mail: [email protected]

Received 3 May 2012, revised 20 June 2012, accepted 22 June 2012

European Journal of Neuroscience, Vol. 36, pp. 2879–2887, 2012 doi:10.1111/j.1460-9568.2012.08231.x

ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd

European Journal of Neuroscience

cells (Pullar et al., 2007), keratinocytes (Pullar et al., 2006), vascularsmooth muscle cells (Johnson et al., 2006) and different types ofcancer cells (Masur et al., 2001; Bastian et al., 2009). In theneocortex, evidence of a functional role for the adrenergic system inthe migration of cortical neurons is lacking. Early studies suggestedthat the destruction of noradrenergic innervation during the earlypostnatal period could affect the maturation of the cerebral cortex(Maeda et al., 1974; Felten et al., 1982; Brenner et al., 1985).However, no studies have directly tested the effects of adrenergicstimulation on cortical interneuron migration. In this study weinvestigated the expression pattern of adrenergic receptors in embry-onic cortical interneuron subtypes preferentially derived from thecaudal ganglionic eminences, and used time-lapse recordings todirectly monitor the consequences of adrenergic receptor pharmaco-logical manipulation on interneuron migration in control andadra2a ⁄ 2c-knockout (ko) mice. Finally we investigated the position-ing of cortical interneurons in adra2a ⁄ 2c-ko mice in vivo at postnatalday 21.

Materials and methods

Animals

All animal experiments were conducted according to relevant nationaland international guidelines and approved by the local Geneva animalcare committee. The day of the vaginal plug detection was counted asE0.5. To monitor cortical interneurons, we used transgenic miceexpressing GFP under the control of the GAD65 promoter (GAD65-GFP mice; Riccio et al., 2009). Previously characterised adra2a-,adra2c- and adra2a ⁄ 2c-ko mice (Hein et al., 1999) were crossed toGAD65-GFP mice to generate adra2a-ko GAD65-GFP, adra2c-koGAD65-GFP, adra2a ⁄ 2c-ko GAD65-GFP mice.

In utero electroporation, cortical slice preparation and drugs

To label pyramidal neurons and interneurons, GAD65-GFP+ embryosfrom timed pregnant E14.5 dams were electroporated with a pRIXplasmid expressing a red fluorochrome (TOM+) under the regulation ofthe ubiquitin promoter in the ventricular zone (VZ) of the lateral pallium.For details of the construct see Dayer et al., 2007. After in uteroelectroporation, dams were killed at E17.5 by intraperitoneal (i.p.)pentobarbital injection (50 mg ⁄ kg), pups were killed by decapitationand brains were dissected. Cortical slices (200 lm thick) were cut on aVibratome (LeicaVT100S;Nussloch,Germany), washed in a dissectionmedium (minimum essential medium, 1·; Tris, 5 mm; and penicillin–streptomycin, 0.5%) for 5 min, placed on porous nitrocellulose filters(Millicell-CM; Millipore. Zug, Switzerland) in 60-mm Falcon Petridishes and kept in neurobasal medium (Invitrogen, Lucerne, Switzer-land) supplemented with B27 (Invitrogen), 2%; glutamine, 2 mm;sodium pyruvate, 1 mm; N-acetyl-cysteine, 2 mm; and penicillin–streptomycin, 1%. Drugs were obtained from Tocris (Abingdon, UK):medetomidine, cirazoline, guanfacine and isoproterenol hydrochloride(all diluted in H2O; stock 100 mm) and (R)-(+)-m-nitrobiphenylineoxalate (diluted in DMSO; stock 50 mm).

Tissue processing and immunohistochemistry

Animals were deeply anesthetised with pentobarbital injected i.p(50 mg ⁄ kg), and killed by intracardiac perfusion of 0.9% salinefollowed by cold 4% paraformaldehyde (PFA; pH 7.4). Brains werepost-fixed over-night in PFA at 4 �C and coronal sections were cut ona Vibratome (Leica VT100S; Nussloch, Germany; 60-lm-thick

sections) and stored at 4 �C in 0.1 m phosphate-buffered saline(PBS). For free-floating immunohistochemistry, sections were washedthree times with 0.1 m PBS, incubated overnight at 4 �C with aprimary antibody diluted in PBS with 0.5% bovine serum albumin(BSA) and 0.3% Triton X-100, washed in PBS, incubated with theappropriate secondary antibody for 2 h at room temperature, count-erstained in Hoechst 33258 (1 : 10 000) for 10 min and then mountedon glass slides with Immu-Mount� (Thermo Scientific, Erembode-gem, Belgium). Primary antibodies were the following: rabbit anti-calretinin (1 : 1000; Swant, Switzerland), mouse anti-parvalbumin(1 : 5000; Swant), rat anti-somatostatin (1 : 100; Millipore, Zug,Switzerland), rabbit anti-NPY (1 : 1000; Immunostar, Losone, Swit-zerland), rabbit anti-VIP (1 : 1000; Immunostar) and mouse anti-reelin (1 : 1000; Medical Biological Laboratories, Nagoya, Japan).Secondary Alexa-568 antibodies (Molecular Probes, Invitrogen,Lucerne, Switzerland) raised against the appropriate species wereused at a dilution of 1 : 1000.

cDNA synthesis and quantitative polymerase chain reaction(PCR)

E17.5 cortical slices from GAD65-GFP+ pups electroporated at E14.5were prepared and kept in vitro for 24 h. The lateral cortex containingTOM+ pyramidal neurons and GAD65-GFP+ interneurons weretrypsinised in Hanks’ medium for 10 min at 37 �C. After centrifuga-tion the pellet was filtered using 40-lm-pore filters (Falcon). GFP+and TOM+ cells were sorted using fluorescence-activated cell sorting(FACS). Total RNA from the sorted cells was extracted, amplified(MessageAMP� II aRNA Amplification kit; Ambion, Zug, Switzer-land) in order to obtain at least 50 ng of RNA, and converted intocDNA. PCR was done using a REDtaq Ready-Mix (Sigma, Buchs,Switzerland) and PCR products were electrophoresed in a 2% agarosegel. For quantitative PCR, PCR reactions were performed in triplicateon cDNA from TOM+ cells and GAD65-GFP+ cells using SYBRgreen PCR Master Mix (Applied Biosystems, Rotkreuz, Switzerland)in an ABI Prism 7900 Sequence Detection system (Applied Biosys-tems). Four genes were used as internal controls: beta-actin (actb),gamma-actin (actg1), eukaryotic elongation factor-1 (eef1a1) and beta-glucuronidase (Gusb). Primers for the different adrenergic receptorswere designed using the Ensembl database and the Primer3 software.Primer sequences were as follows: adra1a forward, 5¢-CTGCCATTCTTCCTCGTGAT-3¢ and reverse 5¢-GCTTGGAAGACTGCCTTCTG-3¢, adra1b, forward, 5¢-AACCTTGGGCATTGTAGTCG-3¢and reverse 5¢-CTGGAGCACGGGTAGATGAT-3¢ adra1d forward,5¢-TCCGTAAGGCTGCTCAAGTT-3¢ and reverse, 5¢-CTGGAGCAGGGGTAGATGAG-3¢, adra2a forward, 5¢ TGCTGGTTGTTGTGGTTGTT-3¢ and reverse, 5¢-GGGGGTGTGGAGGAGATAAT-3¢,adra2b, forward 5¢-GCCACTTGTGGTGGTTTTCT-3¢, reverse, 5¢-TTCCCCAGCATCAGGTAAAC-3¢, adra2c forward, 5¢-TCATCGTTTTCACCGTGGTA-3¢ and reverse, 5¢-GCTCATTGGCCAGAGAAAAG-3¢, adrb1 forward, 5¢-TCGCTACCAGAGTTTGCTGA-3¢ andreverse, 5¢-GGCACGTAGAAGGAGACGAC-3¢, adrb2, forward. 5¢-GACTACACAGGGGAGCCAAA-3¢, and reverse, 5¢-TGTCACAGCAGAAAGGTCCA-3¢, adrb3 forward, 5¢-TGAAACAGCAGACAGGGACA-3¢, reverse 5¢-TCAGCTTCCCTCCATCTCAC-3¢.

Image acquisition and data analysis

Cortical slices were imaged in a thermoregulated chamber maintainedat 37 �C and CO2 at 5% as previously described (Riccio et al., 2009).Time-lapse movies were acquired in parallel using two fluorescentmicroscopes (Eclipse TE2000; Nikon, Egg, Switzerland) equipped

2880 O. Riccio et al.

ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 36, 2879–2887

with a Nikon Plan 10· ⁄ 0.30 objective connected to a digital camera(Retiga EX). Time-lapse imaging was performed 3–4 h after slicepreparation over a period of 24 h. Images were acquired using theOpen-lab software (version 5.0; Schwerzenbach, Switzerland) every5 min for 200 min in short time-lapse sequences and for 600 min inwashout experiments. A control time-lapse sequence of 95 min wasacquired in each condition before the treatment condition. Time-lapsestacks were generated and analysed using Metamorph software(version 7.4; Visitron, Puchheim, Germany). GAD65-GFP+ cells(n = 40 cells per slice in at least three independent experiments)located in the intermediate zone and the cortical plate of theprospective somatosensory cortex were randomly selected in the con-trol condition and single-cell tracking was performed blind to thetreatment condition on migrating cells (velocity of at least 15 lm ⁄ h).The mean velocity over a 90-min recording period was calculated inthe control and treatment condition. To measure a change inthe directionality of migrating interneurons after treatment conditions,the angle change between the track path of the control condition and ofthe wash condition was calculated.

For quantification of the distribution of GAD65-GFP+ interneurons,sections from GAD65-GFP mice and adra2a ⁄ 2c-ko GAD65-GFP micewere obtained at P21 and quantified in the somatosensory cortex(bregma -1.34; mouse brain atlas, Paxinos and Franklin, 2001).Composite epifluorescent images (Nikon Plan 10· objective) wereobtained with GAD65-GFP+ and Hoechst labelling, a grid was apposedon the corresponding somatosensory cortex using the Metamorphsoftware (version 7.4) and GAD65-GFP+ cells were manually countedin the different cortical layers (n = 6 GAD65-GFP+ brains, total of 881cells; n = 6 adra2a-ko GAD65-GFP+ brains, total of 1015 cells).

Epifluorescent images (Nikon Plan 10· objective) were taken at thelevel of the somatosensory cortex to quantify the percentage ofGAD65-GFP+ interneurons located in upper (I–IV) and lower (V andVI) cortical layers and expressing VIP (n = 3, 529 cells), reelin (n = 3,685 cells), NPY (n = 3, 644 cells), calretinin (n = 3, 673 cells),parvalbumin (n = 3, 726 cells) and somatostatin (n = 3, 623 cells).

Statistical analysis (GraphPad prism software, version 4.0) wasdone using unpaired Student’s t-test, one-way anova with Tukey’smultiple comparison test, or v2 test. Statistical significance wasdefined at *P < 0.05, **P < 0.01. Values given are means ± SEM.

Results

Expression of adrenergic receptors in migrating GAD65-GFP+cortical interneurons

Transgenic mice expressing GFP under the control of the GAD65promoter were used to study cortical interneuron migration aspreviously described (Riccio et al., 2009). Given the high subtypediversity of cortical interneurons, we first characterised the identity ofGAD65-GFP interneurons using molecular markers. As previouslyreported (Lopez-Bendito et al., 2004; Riccio et al., 2011), we found thatGAD65-GFP+ interneurons preferentially express markers that labelcortical interneurons derived from the caudal ganglionic eminences butnot the medial ganglionic eminences (Fig. S1). Quantification atpostnatal day 21 in the somatosensory cortex revealed that GAD65-GFP+ cortical interneurons hardly expressed parvalbumin or somato-statin (Fig. S1), which are classical markers of cortical interneuronsubtypes derived from the medial ganglionic eminences (Rudy et al.2011). In contrast, GAD65-GFP+ interneurons expressed markers suchas reelin, NPY, VIP and calretinin, which preferentially label corticalinterneuron subtypes derived from the caudal ganglionic eminences(Fig. S1; Rudy et al. 2011). Migration of GAD65-GFP+ interneurons

was monitored between E17.5 and E18.5, a developmental time periodwhen GAD65-GFP+ cortical interneurons exit the tangential stream ofthe subventricular zone (SVZ) and invade the intermediate zone andcortical plate. To first determine whether migrating GAD65-GFP+interneurons expressed adrenergic receptors, we used FACS to isolate apopulation of GAD65-GFP+ cortical interneurons from cortical slices.To label and isolate excitatory pyramidal precursors using FACS,in utero electroporation of a TOM+-expressing plasmid in the ventric-ular zone of the dorsal pallium was performed at E14.5 (Fig. 1A). Thismethod is widely used to specifically label excitatory pyramidal neuronsin vivo (Chen et al., 2008). Electroporation in the GAD65-GFP+ miceconfirmed that TOM+ cells did not overlap with cortical interneurons(Fig. 1A). Real-time PCR performed on amplified mRNA extractedfrom FACS-isolated GAD65-GFP+ cells revealed that GAD65-GFP+cells expressed a pattern of adrenergic receptors: the alpha1d adrenergicreceptor (adra1d), the alpha2a adrenergic receptor (adra2a), the alpha2cadrenergic receptor (adra2c) and the beta1 adrenergic receptor (adrb1;Fig. 1B). None of the other adrenergic receptor subtypes were detectedan the mRNA level (data not shown). Quantitative PCR did not revealanymajor differences between the expressions of adrenergic receptors inFACS-isolated GAD65-GFP+ interneurons and TOM+ pyramidalneurons (Fig. 1C), indicating that adrenergic receptor numbers are notspecifically raised in GAD65-GFP+ cortical interneurons. Among thefour adrenergic receptors expressed in GAD65-GFP+ cells, adra2a,adra2c and adrb1 were expressed at higher levels than adra1d (Fig. 1D).

Activation of adrenergic receptors decreased corticalinterneuron migration

To determine whether migrating interneurons could respond toadrenergic stimulation, we used time-lapse imaging of GAD65-GFPinterneurons in cortical slices combined with pharmacological drugapplications. Imaging of cortical interneurons was performed betweenE17.5 and E18.5. Migrating cortical interneurons located in thecortical plate and intermediate zone were randomly selected andtracked initially during a control period of 95 min. After 95 min oftime-lapse imaging, drugs targeting adrenergic receptors expressed inGAD65-GFP+ cells were applied to the bath medium and effects onmigration were analysed. Using this slice assay, application of an adrbagonist (isoproterenol, 500 lm) did not significantly modify the meanspeed of neuronal migration whereas application of an adra1 agonist(cirazoline 500 lm) and an adra2 agonist (medetomidine 500 lm)significantly reduced the mean migratory speed of GAD65-GFPinterneurons (P < 0.01 for both drugs vs. control, one-way anova,Tukey multiple comparison test; Fig. 1, E1, E2–G and Movies S1 andS2). Application of cirazoline and medetomidine shifted the speeddistribution of GAD65-GFP+ interneurons to lower migratory speedsand a greater proportion of cells migrating at < 15 lm ⁄ h wereobserved during exposure to medetomidine and cirazoline than duringcontrol conditions (Fig. 1G).

Activation of alpha2 adrenergic receptors decreased corticalinterneuron migration in a concentration-dependent andreversible manner

To further investigate the effects of adra2 agonist stimulation oninterneuron migration, we applied the agonists medetomidine orguanfacine at different concentrations (P < 0.01 for all concentrationstested vs. control, one-way anova, Tukey’s multiple comparison test;Fig. 2A–C). A concentration-dependent effect of medetomidine onmigratory speed was observed (Fig. 2B). This concentration-dependent

Adrenergic stimulation in cortical interneurons 2881


effect could be detected after application of guanfacine, an agonistwith some selectivity for the adra2a subtype (P < 0.01 for allconcentrations tested vs. control, one-way anova, Tukey’s multiplecomparison test; Fig. 2A–C, Movies S3) and (+)-m-nitrobiphenylineoxalate, a more specific adra2c agonist (P < 0.01 for all concentrationstested vs. control, one-way anova, Tukey’s multiple comparison test;Fig. 2D), further confirming that activation of adra2a and adra2caffects the migratory speed of GAD65-GFP+ cortical interneurons. Totest whether these drugs altered cortical interneuron migration byspecifically acting on adra2a and adra2c receptors, time-lapse imagingwas performed on cortical slices of adra2a ⁄ 2c-ko GAD65-GFP mice(Hein et al., 1999). No basal differences in the mean migratory speedswere observed in adra2a ⁄ 2c-ko GAD65-GFP cells compared tocontrol GAD65-GFP+ cells. Single-cell tracking revealed that guan-facine (300 lm) and medetomidine (300 lm) significantly decreasedthe migration speed of GAD65-GFP+ interneurons compared toadra2a ⁄ 2c-ko GAD65-GFP+ interneurons (P < 0.01 for guanfacine in

controls vs. guanfacine in adra2a ⁄ 2c-ko and P < 0.01 for medetom-idine in controls vs. medetomidine in adra2a ⁄ 2c-ko, one-way anova,Tukey’s multiple comparison test; Fig. 2E and F), indicating that theeffects of these drugs on GAD65-GFP+ migrating interneurons aredependent on the activation of adra2a and adra2c receptors. It shouldbe noted, however, that guanfacine decreased the migratory speedof adra2a ⁄ 2c-ko GAD65-GFP+ cells (P < 0.05, one-way anova,Tukey’s multiple comparison test), suggesting that guanfacine couldpartially act independently of adra2a ⁄ 2c receptor activation.To test whether adra2 agonist stimulation produced persistent effects

on interneuron migration, medetomidine (500 lm) was applied in thebath medium for > 6 h. Using this protocol, we observed that long-termapplication of medetomidine (> 6 h) almost completely halted themigration of cortical interneurons without inducing toxic effects such ascell death (Fig. 3A and C,Movies S4). In contrast, when medetomidinewas washed out of the medium after a shorter time period of drugapplication (95 min), the effects of adra2 activation on the speed of

A

E1

E2 F G

B C D

Fig. 1. Activation of adrenergic receptors expressed in cortical interneurons affected their migration. (A) Image of the developing cortex at E18.5 showing twodistinct non-overlapping populations of GAD65-GFP+ interneurons and TOM+ pyramidal neurons. TOM+ cells were labelled after an E14.5 electroporationtargeting the dorsal pallium. (B) Agarose gel showing PCR bands of adrenergic receptors expressed in E18.5 GAD65-GFP+ cortical interneurons. (C) QuantitativePCR graph showing no major changes in the expression of adrenergic receptors in GAD65-GFP+ cells compared to TOM+ cells. (D) Quantitative PCR graphshowing increases in the expression of adra2a, adra2c and adrb1 compared to adra1d in GAD65-GFP+ cells. (E1) Time-lapse sequence showing that after a95-minute control sequence, application of an adra2 agonist (medetomidine, 500 lm) decreased the migratory speed of GAD65-GFP+ cortical interneurons.Superposed colour lines represent migratory tracks. (E2) Graph showing the migratory distances travelled by GAD65-GFP+ cells shown in E1. (F) Graph showingthat the mean migratory speed of GAD65-GFP+ cortical interneurons significantly decreased after application of an adra2 agonist (medetomidine, 500 lm; P < 0.01,one-way anova, Tukey’s multiple comparison test), an adra1 agonist (cirazoline, 500 lm; P < 0.01, one-way anova, Tukey’s multiple comparison test), but not anadrb1 agonist (isoproterenol, 500 lm). (G) Graph showing that after application of medetomidine (500 lm) or cirazoline (500 lm) the speed distribution of GAD65-GFP+ interneurons shifted to lower migratory speeds. MED, medetomidine, CIR, cirazoline, ISO, isoproterenol. CP, cortical plate, IZ, intermediate zone.**P < 0.01. Values given are means + SEM. Scale bars, 50 lm.



interneuron migration were reversible (Fig 3B and C, Movies S5).Single-cell tracking revealed that after washing out medetomidine, themigratory speed of GAD65-GFP+ interneurons significantly increasedand gradually reached control values (P < 0.01 at the first time intervalafter the drug washout when comparing medetomidine vs. no-washmedetomidine, one-way anova, Tukey’s multiple comparison test;Fig. 3B and C). These effects were also observed after application ofguanfacine. When guanfacine was washed out from the medium, thespeed of interneuron migration significantly increased and graduallyreached control values (P < 0.01 at the first time interval after the drugwash when comparing guanfacine vs. no-wash medetomidine, one-wayanova, Tukey’s multiple comparison test; Fig. 3C). Interestingly, we

observed that although the migratory speed of GAD65-GFP+ cells wasgradually restored during the removal of either medetomidine orguanfacine, the directionality of GAD65-GFP+ cells was modified byadra2 stimulation (Fig. 3B). Quantification revealed that during thewashout period a significant proportion of GAD65-GFP+ cells modifiedtheir directionality following medetomidine or guanfacine application.The percentage of GAD65-GFP+ interneurons that made directionalitychanges in the range > 120–180� after the medetomidine wash or theguafancine wash was significantly increased compared to controlGAD65-GFP+ interneurons (P < 0.01 for guanfacine compared tocontrol and P < 0.05 for medetomidine vs. control, one-way anova

Tukey’s multiple comparison test; Fig. 3D), suggesting that adrenergic

A1

A2 B C

D E F

Fig. 2. Adra2a and adra2c activation affected cortical interneurons in a concentration-dependent manner. (A1) Time-lapse sequence showing that after a 95-mincontrol sequence, application of an adra2 agonist (guanfacine, 500 lm) decreased the migratory speed of GAD65-GFP+ cortical interneurons. Superposed colourlines represent migratory tracks. (A2) Graph showing the migratory distances travelled by the GAD65-GFP+ cells shown in A1. (B–D) Graphs showing that themean migratory speed of GAD65-GFP+ cortical interneurons significantly decreased in a concentration-dependent manner after application of an adra2 agonist(medetomidine; P < 0.01 at all drug concentrations vs. control, one-way anova, Tukey’s multiple comparison test), a more specific adra2a agonist (guanfacine;P < 0.01 for all drug concentrations vs. control, one-way anova, Tukey’s multiple comparison test), and a more specific adra2c agonist [(R)-(+)-m-nitrobiphenylineoxalate; P < 0.01 for all drug concentrations vs. control, one-way anova, Tukey’s multiple comparison test]. (E and F) Graphs showing that guanfacine (300 lm; E)and medetomidine (300 lm; F) significantly reduced the mean migratory speed of wildtype GAD65-GFP+ interneurons compared to adra2a ⁄ 2c-ko GAD65-GFP+interneurons. (P < 0.01 for guanfacine in controls vs. guanfacine in adra2a ⁄ 2c-ko and P < 0.01 for medetomidine in controls vs. medetomidine in adra2a ⁄ 2c-ko,one-way anova, Tukey’s multiple comparison test). *P < 0.05, **P < 0.01. GF, guanfacine, MED, medetomidine, m-nitro, (R)-(+)-m-nitrobiphenyline oxalate.Values given are means + SEM. Scale bars, 50 lm.



stimulation of cortical interneurons may alter their responsiveness toguidance cues.

Cortical interneuron positioning was altered in adra2a ⁄ 2c-komice

To determine whether cortical interneuron migration is altered inadra2a ⁄ 2c-ko mice, we analysed the cortical distribution of GAD65-GFP+ interneurons at postnatal day 21 in GAD65-GFP mice and inadra2a ⁄ 2c-ko GAD65-GFP mice. Quantification revealed that the

distribution of GAD65-GFP+ cortical interneurons in the somatosen-sory cortex was significantly altered in adra2a ⁄ 2c-ko mice (n = 6)compared to the control mice (n = 6; P < 0.05, v2 test; Fig. 4).A significant increase in the percentage of GAD65-GFP+ cells wasobserved in upper cortical layers II ⁄ III in adra2a ⁄ 2c-ko mice(P < 0.05, unpaired t-test), indicating that adrenergic receptors arenecessary for the proper positioning of cortical interneurons in vivo.Quantification of the distribution of GAD65-GFP+ cells at P21 in thesomatosensory cortex of adra2a-ko or of adra2c-ko mice was notsignificantly different from control GAD65-GFP+ mice (data not

A1

B1

B2 C D

A2

Fig. 3. Adrenergic receptor activation affected cortical interneuron migration in a reversible manner and modifies their directionality. (A1) Time-lapse sequenceshowing that after persistent long-term application of medetomidine (500 lm) GAD65-GFP+ cells persistently halted their migration. Superposed colour linesrepresent migratory tracks. (A2) Graph showing the migratory distances travelled by the GAD65-GFP+ cells shown in A1. (B1) Time-lapse sequence showing thatafter short-term application of medetomidine (500 lm) GAD65-GFP+ cells decreased their migratory speed, which was gradually restored after washing out thedrug. Not that two cells (orange and dark blue traces) completely modified their directionality after the wash. Superposed colour lines represent migratory tracks.(B2) Graph showing the migratory distances travelled by the GAD65-GFP+ cells shown in B1. (C) Graph showing that GAD65-GFP+ cells decreased theirmigratory speed after medetomidine (500 lm) and guanfacine (500 lm) application and that after washing out the drugs their migratory speeds were significantlyrestored (P < 0.01 at the first time interval after the drug wash for medetomidine vs. no-wash medetomidine and for guanfacine vs. no-wash medetomidine, one-wayanova, Tukey’s multiple comparison test). (D) Graph showing that the directionality of migrating GAD65-GFP+ cells during the wash period was significantlymodified after application of medetomidine (500 lm) or guanfacine (500 lm). (P < 0.01 for guanfacine compared to control and P < 0.05 for medetomidine vs.control, one-way anova, Tukey’s multiple comparison test). *P < 0.05; **P < 0.01. MED, medetomidine; GF, guanfacine. Values given are means + SEM. Scalebars, 50 lm.



shown), suggesting that constitutive deletion of adra2a or adra2cduring development may be compensated for by the presence of theother subtype.

Discussion

In this study we found that migrating cortical interneuron subtypespreferentially derived from the caudal ganglionic eminences express aspecific pattern of adrenergic receptors and that pharmacologicalactivation of these receptors affects the dynamic migration of corticalinterneurons as they invade the developing cortical plate. Effects ofadrenergic stimulation were most effective after adra2 stimulation, andthey were concentration-dependent and reversible. Furthermore,effects of adra2 activation on the migration of cortical interneuronswere significantly reduced in adra2a ⁄ 2c-ko mice. Altogether thesedata strongly suggest a role for adrenergic stimulation in corticalinterneuron migration.

A role for noradrenaline during cortical development has beenhypothesised on the basis that noradrenergic fibres originating fromthe locus coeruleus (LC) reach the cortical anlage during theembryonic period in rodents, macaques and humans (Levitt & Moore,1979; Zecevic & Verney, 1995; Wang & Lidow, 1997). Duringembryonic cortical development, fibres from the LC express dopa-mine-beta-hydroxylase, the rate-limiting enzyme for noradrenaline,and are thus likely to release noradrenaline in the extracellular space ofthe cortical anlage (Wang & Lidow, 1997). An alternative sourceof noradrenaline could be the cerebrospinal fluid where high levels ofnoradrenaline have been detected during the embryonic period(Masudi & Gilmore, 1983). Noradrenaline in the CSF could originatefrom the fetal blood by passing through the immature blood–brainbarrier, diffuse from the CSF into the ventricular wall and regulatecellular processes involved in the formation of cortical circuits,including neuronal migration. A role for noradrenaline duringembryonic cortical development is further supported by the fact thatdifferent subtypes of adrenergic receptors are dynamically expressedacross species during cortical development and follow a restricted

temporal and spatial pattern of expression. Initial binding studiesrevealed that adra1, adra2 and adrb1 are highly expressed in thedeveloping cortical plate and transient embryonic zones of the non-human primate brain (Lidow & Rakic, 1994). A more detailed studyon adra2a indicated that this receptor is expressed at E70, E90 andE120 throughout the macaque embryonic wall (Wang & Lidow,1997). Interestingly, this study revealed that migrating neurons in theintermediate zone and cortical plate expressed high levels of adra2a,suggesting that this receptor could play a role in regulating neuronalmigration (Wang & Lidow, 1997). A role for adra2a in neuronalmigration is further supported by the fact that strong adra2a expressionis detected in the cortical plate, intermediate and subventricular zonesof the embryonic rat cortex (Winzer-Serhan & Leslie, 1997; Winzer-Serhan & Leslie, 1999).The group of adra2 receptors is composed of three highly

homologous subtypes (adra2a, adra2b and adra2c). In this study wefound that migrating cortical interneurons expressed adra2a andadra2c but not adra2b, and that activation of adra2a and adra2c affectsneuronal migration. Interestingly, it has been recently reported thatadra2 receptors regulate adult hippocampal neurogenesis, a develop-mental process that persists in the adult brain (Yanpallewar et al.,2010). Progenitor cells in the hippocampus express adra2a, adra2b andadra2c subtypes and adra2 stimulation inhibits the proliferation ofgranule cell progenitors in the dentate gyrus, leading to decreasedlevels of adult hippocampal neurogenesis (Yanpallewar et al., 2010).Knockout mice for adra2a and adra2c have been generated (Heinet al., 1999) and the role of these receptors in the cardiovascularsystem has been studied in detail (Knaus et al., 2007a,b). Briefly,adra2a ⁄ 2c-ko mice display elevated plasma concentrations of cate-cholamines, increased blood pressure and cardiac hypertrophy inadulthood (Hein et al., 1999; Knaus et al., 2007a,b). The develop-mental consequences of constitutive deletions of adra2a, adra2c andadra2a ⁄ 2c in the central nervous system are not striking and the brainsof these animals appear to be grossly normal. Quantification of thedistribution of GAD65-GFP+ interneurons in adra2a-ko or adra2c-komice did not reveal any significant changes in the distribution of

A B

C

Fig. 4. Cortical interneuron positioning was affected in adra2a ⁄ 2c-ko mice. (A and B) Images showing the distribution of GAD65-GFP+ cortical interneurons atpostnatal day 21 in the somatosensory cortex of (A) GAD65-GFP mice and of (B) adra2a ⁄ 2c-ko GAD65-GFP mice. (C) Quantification revealed a significantincrease in the percentage of GAD65-GFP+ cortical interneurons in layer II ⁄ III of adra2a-ko GAD65-GFP mice. **P < 0.01, unpaired t-test. Values given aremeans + SEM. Scale bars, 100 lm.



cortical interneurons at P21, suggesting compensatory regulatorymechanisms following constitutive developmental deletion of either ofthese receptors. Interestingly a significant increase in the percentage ofGAD65-GFP+ cells in upper cortical layers II ⁄ III were detected in thesomatosensory cortex of adra2a ⁄ 2c-ko mice, indicating that combineddeletion of adra2a and adra2c receptors significantly modifies thedistribution of cortical interneurons in vivo.The intracellular mechanism mediating the effects of adra2 stimula-

tion on interneuron migration is likely to involve different transductionpathways. Adra2 are G-protein-coupled receptors negatively coupled toadenylate cyclase, and modifications in the levels of cAMP could thusconstitute a downstream effector of adra2 stimulation. Cyclic AMP is akey molecule regulating growth cone dynamics (Song & Poo, 2001),and experimental manipulation of the ratio of cAMP to cGMPdetermines the responsiveness of axonal growth cones to guidance cues(Nishiyama et al., 2003). In the embryonic brain cAMP is critical forproper axonal pathfinding of olfactory sensory neurons (Chesler et al.,2007). In migrating neurons, alteration in the levels of cAMP decreasesthe migratory speed of cerebellar granule cells (Cuzon et al., 2008) andmodulates the effects of serotonin on migrating cortical interneurons(Riccio et al., 2009). Interestingly, there is a functional pathway linkingadra2a, cAMP and hyperpolarization-activated cyclic nucleotide-gatedcation channels (HCN channels; Wang et al., 2007). HCN channelshave been shown to regulate axonal targeting of olfactory sensoryneurons during development (Mobley et al., 2010) and thus represent anattractive downstream developmental target of cAMP that couldregulate interneuron migration. Calcium could also be another down-stream effector mediating the effects of adra2 activation on migratinginterneurons. In other cellular systems, it has been shown that adra2astimulation regulates intracellular calcium levels through the modula-tion of voltage-gated N-type calcium channels and that this processoccurs independently of cAMP modulation (Lipscombe et al., 1989;Ikeda, 1996). As intracellular calcium levels regulate the migration ofcerebellar granule cells (Komuro & Rakic, 1996), adra2a-inducedcalcium changes could potentially regulate cortical interneuron migra-tion. Finally, an interesting observation in this study is that adra2stimulation affected not only the migratory speed of cortical interneu-rons but also their directionality.When adra2 agonist was removed fromthe bath medium, cortical interneurons resumed a normal migratoryspeed but the directionality of migration was significantly modified in afraction of cells compared to the control situation. These results suggestthat changes in cAMP levels through adra2 stimulation couldmodify theresponsiveness of cortical interneurons to guidance cues. Support forthis possibility comes from the observation that in other systemsmanipulation of cAMP levels can modify the responsiveness ofthalamocortical axons to guidance cues through the monoaminergicactivation of G-protein-coupled receptors negatively linked to adenylatecyclase (Bonnin et al., 2007).In this study the effects of adrenergic stimulation on interneuron

migration were detected using several different drugs at relatively highconcentrations. However, it must be noted that in this slice culturesystem drugs reached the migrating cells by passively diffusingthrough the pores of the Millipore inserts. It is thus likely that thecortical interneurons migrating in the slice are exposed to lower drugconcentrations. Importantly, application of adra2a ⁄ 2c agonists signif-icantly decreased the migratory speed of wildtype cortical interneuronscompared to adra2a ⁄ 2c-ko cortical interneurons. These resultsstrongly indicate that the effects of adra2a ⁄ 2c stimulation on corticalinterneurons are dependent on the activation of these receptors. Itshould be noted, however, that guanfacine slightly affected themigratory speed of GAD65-GFP+ interneurons in adra2a ⁄ 2c-ko mice,suggesting that this drug could also act independently of adra2a ⁄ 2c

activation. Interestingly, a study using adra2a ⁄ 2b ⁄ 2c triple-ko micehas revealed that clonidine, an adra2 agonist, could modulate heartreactivity by directly acting on HCN (Knaus et al., 2007b). Finally,although adrab1 was found to be expressed in GAD65-GFP+ cells,application of an adrb1 agonist at relatively high concentration failedto modify the migration of interneurons, suggesting that this receptormay not be functional at this embryonic timepoint.In conclusion, we report that several adrenergic receptors are

expressed in migrating cortical interneurons, particularly the adra2aand adra2c subtypes. Using time-lapse imaging we have demonstratedthat activation of adra2 affects cortical interneuron migration in areversible manner. Finally, the distribution of cortical interneuronswas altered in vivo in adra2a ⁄ 2c-ko mice. These results support thehypothesis that adrenergic dysregulation induced by exposure duringpregnancy to drugs that block adrenergic receptors may affect cellularprocesses involved in the assembly of cortical circuits. These resultsalso open the possibility that a pathological overstimulation ofadrenergic receptors due to excessive levels of norepinephrine duringdevelopment could lead to alterations in cortical circuit formation.

Supporting Information

Additional supporting information may be found in the online versionof this article:Figure S1. GAD65-GFP+ interneurons preferentially express a varietyof markers expressed in CGE-derived interneurons but not MGE-derived interneurons.Movie S1. Activation of adra1 with cirazoline affects interneuronmigration.Movie S2. Activation of adra2 with medetomidine affects interneuronmigration.Movie S3. Activation of adra2 with guanfacine affects interneuronmigration.Movie S4. Long-term activation of adra2 with medetomidine affectsinterneuron migration.Movie S5. Effects of adra2 activation on interneuron migration arereversible.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organised for onlinedelivery, but are not copy-edited or typeset by Wiley-Blackwell.Technical support issues arising from supporting information (otherthan missing files) should be addressed to the authors.

Acknowledgements

We wish to thank C. Aubry for technical lab assistance and the GenevaGenomics Platform for qPCR assistance (Christelle Barraclough and PatrickDescombes). This work was supported by a Swiss National Foundation grant(PP00P3_128379), the NCCR Synapsy, the Thorn Foundation, the MercierFoundation, NARSAD (The brain and Behaviour Research Fund; A.D.) and byBIOSS Centre for Biological Signalling Studies (EXC 294, in support of L.H.).

Abbreviations

actb, beta-actin; actg1, gamma-actin; adra1, alpha1 adrenergic (receptor);adra1d, alpha1d adrenergic (receptor); adra2, alpha2 adrenergic (receptor);adra2a, alpha2a adrenergic (receptor); adra2c, alpha2c adrenergic (receptor);adrb, beta adrenergic (receptor); adrb1, beta1 adrenergic (receptor); E,embryonic day; eef1a1, eukaryotic elongation factor-1; FACS, fluorescence-activated cell sorting; Gusb, beta-glucuronidase; ko, knockout; PCR, polymer-ase chain reaction; SVZ, subventricular zone; VZ, ventricular zone.



References

Bastian, P., Balcarek, A., Altanis, C., Strell, C., Niggemann, B., Zaenker, K.S.& Entschladen, F. (2009) The inhibitory effect of norepinephrine on themigration of ES-2 ovarian carcinoma cells involves a Rap1-dependentpathway. Cancer Lett., 274, 218–224.

Bonnin, A., Torii, M., Wang, L., Rakic, P. & Levitt, P. (2007) Serotoninmodulates the response of embryonic thalamocortical axons to netrin-1. Nat.Neurosci., 10, 588–597.

Brenner, E., Mirmiran, M., Uylings, H.B. & Van der Gugten, J. (1985) Growthand plasticity of rat cerebral cortex after central noradrenaline depletion. Exp.Neurol., 89, 264–268.

Chedotal, A.&Rijli, F.M. (2009) Transcriptional regulation of tangential neuronalmigration in the developing forebrain. Curr. Opin. Neurobiol., 19, 139–145.

Chen, G., Sima, J., Jin, M., Wang, K.Y., Xue, X.J., Zheng, W., Ding, Y.Q. &Yuan, X.B. (2008) Semaphorin-3A guides radial migration of corticalneurons during development. Nat. Neurosci., 11, 36–44.

Chesler, A.T., Zou, D.J., Le Pichon, C.E., Peterlin, Z.A., Matthews, G.A., Pei,X., Miller, M.C. & Firestein, S. (2007) A G protein ⁄ cAMP signal cascade isrequired for axonal convergence into olfactory glomeruli. Proc. Natl. Acad.Sci. USA, 104, 1039–1044.

Crandall, J.E., McCarthy, D.M., Araki, K.Y., Sims, J.R., Ren, J.Q. & Bhide,P.G. (2007) Dopamine receptor activation modulates GABA neuronmigration from the basal forebrain to the cerebral cortex. J. Neurosci., 27,3813–3822.

Cuzon, V.C., Yeh, P.W., Yanagawa, Y., Obata, K. & Yeh, H.H. (2008) Ethanolconsumption during early pregnancy alters the disposition of tangentiallymigrating GABAergic interneurons in the fetal cortex. J. Neurosci., 28,1854–1864.

Dayer, A.G., Jenny, B., Sauvain, M.O., Potter, G., Salmon, P., Zgraggen, E.,Kanemitsu, M., Gascon, E., Sizonenko, S., Trono, D. & Kiss, J.Z. (2007)Expression of FGF-2 in neural progenitor cells enhances their potential forcellular brain repair in the rodent cortex. Brain, 130, 2962–2976.

Felten, D.L., Hallman, H. & Jonsson, G. (1982) Evidence for a neurotropic roleof noradrenaline neurons in the postnatal development of rat cerebral cortex.J. Neurocytol., 11, 119–135.

Flames, N. & Marin, O. (2005) Developmental mechanisms underlying thegeneration of cortical interneuron diversity. Neuron, 46, 377–381.

Gelman, D.M. & Marin, O. (2010) Generation of interneuron diversity in themouse cerebral cortex. Eur. J. Neurosci., 12, 2136–2141.

Hein, L., Altman, J.D. & Kobilka, B.K. (1999) Two functionally distinctalpha2-adrenergic receptors regulate sympathetic neurotransmission. Nature,402, 181–184.

Ikeda, S.R. (1996) Voltage-dependent modulation of N-type calcium channelsby G-protein beta gamma subunits. Nature, 380, 255–258.

Johnson, R., Webb, J.G., Newman, W.H. & Wang, Z. (2006) Regulation ofhuman vascular smooth muscle cell migration by beta-adrenergic receptors.Am. Surg., 72, 51–54.

Knaus, A., Zong, X., Beetz, N., Jahns, R., Lohse, M.J., Biel, M. & Hein, L.(2007a) Direct inhibition of cardiac hyperpolarization-activated cyclic nucle-otide-gated pacemaker channels by clonidine. Circulation, 115, 872–880.

Knaus, A.E., Muthig, V., Schickinger, S., Moura, E., Beetz, N., Gilsbach, R. &Hein, L. (2007b) Alpha2-adrenoceptor subtypes–unexpected functions forreceptors and ligands derived from gene-targeted mouse models. Neuro-chem. Int., 51, 277–281.

Komuro, H. & Rakic, P. (1996) Intracellular Ca2+ fluctuations modulate therate of neuronal migration. Neuron, 17, 275–285.

Levitt, P. & Moore, R.Y. (1979) Development of the noradrenergic innervationof neocortex. Brain Res., 162, 243–259.

Lidow, M.S. & Rakic, P. (1994) Unique profiles of the alpha 1-, alpha 2-, andbeta-adrenergic receptors in the developing cortical plate and transientembryonic zones of the rhesus monkey. J. Neurosci., 14, 4064–4078.

Lipscombe, D., Kongsamut, S. & Tsien, R.W. (1989) Alpha-adrenergicinhibition of sympathetic neurotransmitter release mediated by modulation ofN-type calcium-channel gating. Nature, 340, 639–642.

Lopez-Bendito, G., Sturgess, K., Erdelyi, F., Szabo, G., Molnar, Z. & Paulsen,O. (2004) Preferential origin and layer destination of GAD65-GFP corticalinterneurons. Cereb. Cortex, 14, 1122–1133.

Maeda, T., Toyama, M. & Shimizu, N. (1974) Modification of postnataldevelopment of neocortex in rat brain with experimental deprivation of locuscoeruleus. Brain Res., 70, 515–520.

Masudi, N.A. & Gilmore, D.P. (1983) Biogenic amine levels in the mid-termhuman fetus. Brain Res., 283, 9–12.

Masur, K., Niggemann, B., Zanker, K.S. & Entschladen, F. (2001) Norepi-nephrine-induced migration of SW 480 colon carcinoma cells is inhibited bybeta-blockers. Cancer Res., 61, 2866–2869.

Mobley, A.S., Miller, A.M., Araneda, R.C., Maurer, L.R., Muller, F. & Greer,C.A. (2010) Hyperpolarization-activated cyclic nucleotide-gated channels inolfactory sensory neurons regulate axon extension and glomerular formation.J. Neurosci., 30, 16498–16508.

Nishiyama, M., Hoshino, A., Tsai, L., Henley, J.R., Goshima, Y., Tessier-Lavigne, M., Poo, M.M. & Hong, K. (2003) Cyclic AMP ⁄ GMP-dependentmodulation of Ca2+ channels sets the polarity of nerve growth-cone turning.Nature, 423, 990–995.

Paxinos, G. & Franklin, K.J. (2001) The mouse brain in stereotaxiccoordinates. Academic Press, San Diego, CA.

Pullar, C.E., Rizzo, A. & Isseroff, R.R. (2006) beta-Adrenergic receptorantagonists accelerate skin wound healing: evidence for a catechol-amine synthesis network in the epidermis. J. Biol. Chem., 281, 21225–21235.

Pullar, C.E., Zhao, M., Song, B., Pu, J., Reid, B., Ghoghawala, S., McCaig, C.& Isseroff, R.R. (2007) Beta-adrenergic receptor agonists delay whileantagonists accelerate epithelial wound healing: evidence of an endogenousadrenergic network within the corneal epithelium. J. Cell. Physiol., 211,261–272.

Riccio, O., Murthy, S., Szabo, G., Vutskits, L., Kiss, J.K., Vitalis, T., Lebrand,C. & Dayer, A.G. (2011) New pool of cortical interneuron precursors in theearly postnatal dorsal white matter. Cereb. Cortex, 22, 86–98.

Riccio, O., Potter, G., Walzer, C., Vallet, P., Szabo, G., Vutskits, L., Kiss, J.Z.& Dayer, A.G. (2009) Excess of serotonin affects embryonic interneuronmigration through activation of the serotonin receptor 6. Mol. Psychiatry.,14, 280–290.

Rudy, B., Fishell, G., Lee, S. & Hjerling-Leffler, J. (2011) Three groups ofinterneurons account for nearly 100% of neocortical GABAergic neurons.Dev. Neurobiol., 71, 45–61.

Song, H. & Poo, M. (2001) The cell biology of neuronal navigation. Nat. CellBiol., 3, E81–E88.

Spiegel, A., Shivtiel, S., Kalinkovich, A., Ludin, A., Netzer, N., Goichberg, P.,Azaria, Y., Resnick, I., Hardan, I. Ben-Hur, H., Nagler, A., Rubinstein, M. &Lapidot, T. (2007) Catecholaminergic neurotransmitters regulate migrationand repopulation of immature human CD34 + cells through Wnt signaling.Nat. Immunol., 8, 1123–1131.

Wang, F. & Lidow, M.S. (1997) Alpha 2A-adrenergic receptors are expressedby diverse cell types in the fetal primate cerebral wall. J. Comp. Neurol., 378,493–507.

Wang, M., Ramos, B.P., Paspalas, C.D., Shu, Y., Simen, A., Duque, A.,Vijayraghavan, S., Brennan, A., Dudley, A., Nou, E., Mazer, J.A.,McCormick, D.A. & Arnsten, A.F. (2007) Alpha2A-adrenoceptors strength-en working memory networks by inhibiting cAMP-HCN channel signalingin prefrontal cortex. Cell, 129, 397–410.

Winzer-Serhan, U.H. & Leslie, F.M. (1997) Alpha2B adrenoceptor mRNAexpression during rat brain development. Brain Res. Dev. Brain Res., 100,90–100.

Winzer-Serhan, U.H. & Leslie, F.M. (1999) Expression of alpha2Aadrenoceptors during rat neocortical development. J. Neurobiol., 38,259–269.

Wonders, C.P. & Anderson, S.A. (2006) The origin and specification of corticalinterneurons. Nat. Rev. Neurosci., 7, 687–696.

Yanpallewar, S.U., Fernandes, K., Marathe, S.V., Vadodaria, K.C., Jhaveri, D.,Rommelfanger, K., Ladiwala, U., Jha, S., Muthig, V., Hein, L., Bartlett, P.,Weinshenker, D. & Vaidya, V.A. (2010) Alpha2-adrenoceptor blockadeaccelerates the neurogenic, neurotrophic, and behavioral effects of chronicantidepressant treatment. J. Neurosci., 30, 1096–1109.

Zecevic, N. & Verney, C. (1995) Development of the catecholamine neurons inhuman embryos and fetuses, with special emphasis on the innervation of thecerebral cortex. J. Comp. Neurol., 351, 509–535.



Documents

Alpha2-adrenergic receptor activation regulates cortical interneuron migration