Identifying the efferent projections of leptin-responsive neurons in the dorsomedial hypothalamus using a novel conditional tracing approach.
Tracing the axonal projections of selected neurons is labor intensive and inherently limited by currently available neuroanatomical methods. We developed an adeno-associated virus (AAV) that can be used for efficiently tracing identified neuronal populations. The virus encodes a humanized Renilla green fluorescent protein (hrGFP) that is transcriptionally silenced by a neo cassette flanked by LoxH/LoxP sites (AAV-lox-Stop-hrGFP). Thus, hrGFP is expressed only in neurons with Cre recombinase activity. To demonstrate the utility of this approach, the virus was injected unilaterally into the dorsomedial hypothalamus (DMH) of mice that express Cre in neurons expressing the leptin receptor. Animals with DMH injections showed robust hrGFP expression in DMH neurons, as visualized by its endogenous fluorescence or following immunolabeling. We found that hrGFP was expressed in approximately one-third to one-half of Cre-expressing neurons at the site of injection, but not in non-Cre-expressing neurons. The expression of GFP allowed us to identify the projection fields of DMH leptin-responsive neurons. Our results show hrGFP-positive axonal projections and terminals in the paraventricular nucleus of the hypothalamus, arcuate nucleus, preoptic area, bed nucleus of the stria terminalis, paraventricular thalamus, periaqueductal gray, and precoeruleus. The aforementioned pattern of projections was similar to DMH projections determined by injections of biotinylated dextran amine in the mouse DMH. Interestingly, some hrGFP-positive terminals were seen contacting the ependymal layer of the third and fourth ventricles. In summary, this approach is an effective tool for tracing axonal projections of chemically identified neurons, including leptin-responsive neurons.
Gautron
Laurent
L
Department of Internal Medicine, Division of Hypothalamic Research, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9077, USA.
Lazarus
Michael
M
Scott
Michael M
MM
Saper
Clifford B
CB
Elmquist
Joel K
JK
D000818:Animals; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism; D004525:Efferent Pathways / Q000033:anatomy & histology* / Q000502:physiology; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007031:Hypothalamus / Q000166:cytology*; D019426:Integrases / Q000378:metabolism; D020738:Leptin / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods*
D020738:Leptin; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases
FEBS Lett. 1996 Jun 3;387(2-3):113-6
8674530
J Neurosci. 2009 Mar 11;29(10):3138-47
19279251
J Comp Neurol. 2009 Apr 20;513(6):597-621
19235223
Cereb Cortex. 2006 Oct;16(10):1474-86
16357338
J Comp Neurol. 2009 Jun 10;514(5):518-32
19350671
Neuron. 1998 Dec;21(6):1375-85
9883730
Nat Protoc. 2009;4(8):1145-56
19617886
Eur J Neurosci. 2008 Apr;27(7):1763-70
18371076
Nat Neurosci. 2006 Mar;9(3):398-407
16491082
J Clin Invest. 2005 Dec;115(12):3564-72
16322794
Exp Brain Res. 1987;66(3):500-6
3609196
Gene Ther. 1999 Jun;6(6):973-85
10455399
J Neurosci. 2008 Oct 22;28(43):11071-8
18945914
Neuron. 2002 Oct 10;36(2):199-211
12383777
J Comp Neurol. 1998 Jun 15;395(4):535-47
9619505
J Neurosci. 2001 Apr 1;21(7):2413-24
11264315
Cell Metab. 2005 Jan;1(1):63-72
16054045
Endocrinology. 2003 May;144(5):2121-31
12697721
Brain Res Bull. 1986 Feb;16(2):231-48
3697791
Neuron. 2005 Apr 21;46(2):297-308
15848807
J Comp Neurol. 2008 Apr 10;507(5):1780-94
18260139
Cell Metab. 2009 Aug;10(2):89-98
19656487
Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):741-6
9435263
Curr Gene Ther. 2007 Oct;7(5):347-60
17979681
Endocrinology. 2008 Apr;149(4):1773-85
18162515
Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10835-40
19541658
Physiol Behav. 2002 Jul;76(3):431-42
12117580
Nature. 2000 Apr 6;404(6778):661-71
10766253
Brain Res. 1998 May 18;793(1-2):169-75
9630611
J Chem Neuroanat. 1997 Jun;13(1):1-21
9271192
J Comp Neurol. 1997 Aug 11;384(4):556-68
9259489
J Clin Invest. 2001 Oct;108(8):1113-21
11602618
Endocrinology. 1997 Feb;138(2):839-42
9003024
Brain Res Rev. 2007 Nov;56(1):79-88
17582506
Endocrinology. 1997 Oct;138(10):4489-92
9322969
Am J Physiol Regul Integr Comp Physiol. 2008 Dec;295(6):R2059-66
18843095
Science. 2001 Mar 30;291(5513):2608-13
11283374
J Neurosci. 2009 Sep 23;29(38):11954-64
19776281
Neuron. 1999 Aug;23(4):775-86
10482243
Science. 1995 Jul 28;269(5223):540-3
7624776
J Neurosci. 2009 Jan 7;29(1):179-90
19129396
J Comp Neurol. 2000 Jul 24;423(2):261-81
10867658
J Neurosci. 2003 Nov 19;23(33):10691-702
14627654
Mol Cell. 1998 Mar;1(4):619-25
9660946
Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):7001-5
9192681
J Comp Neurol. 1996 Dec 02;376(1):143-73
8946289