Brain Research 985 (2003) 163–168www.elsevier.com/ locate/brainres

Short communication

G lucagon like peptide-1 (7-36) amide (GLP-1) nerve terminalsdensely innervate corticotropin-releasing hormone neurons in the

hypothalamic paraventricular nucleusa b a a,c ,*´ ´ ´Sumit Sarkar , Csaba Fekete , Gabor Legradi , Ronald M. Lechan

aTupper Research Institute and Department of Medicine, Division of Endocrinology, Diabetes, Metabolism, and Molecular Medicine, New EnglandMedical Center, Boston, MA 02111,USA

bDepartment of Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest 1083, HungarycDepartment of Neuroscience, Tufts University School of Medicine, Boston, MA 02111,USA

Accepted 5 June 2003

Abstract

Glucagon like peptide-1 (7-36) amide (GLP-1), a potent regulator of glucose homeostasis, is also produced in the central nervoussystem and has been implicated in the control of hypothalamic–pituitary function and food intake. GLP-1 immunoreactive (IR) fibers andterminals are widely distributed in the septum, hypothalamus, thalamus and brainstem, likely originating from GLP-1-IR neuronal cellbodies from the nucleus of the solitary tract of the medulla oblongata. Central administration of GLP-1 increases plasma corticosteronelevels and elicits c-fos expression in corticotropin releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN).To identify the endogenous neurocircuitry that may underlie this response, the present study determined whether there is an innervation ofPVN CRH neurons by GLP-1-containing nerve terminals. GLP-1-IR fibers and nerve terminals were found in the parvocellular parts ofthe PVN, with highest concentrations in the anterior and medial parvocellular subdivisions. The magnocellular divisions of the PVN alsoshowed moderate numbers of GLP-1-IR nerve fibers. Double immunolabelling revealed numerous GLP-1-IR nerve fibers in closeapposition to|65% of detectable CRH neurons in the medial parvocellular subdivision of the rat PVN. At the ultrastructural level,GLP-1-IR terminals were observed to establish synapses on both perikarya and dendrites of CRH neurons. These findings support thehypothesis that the GLP-1-induced activation of CRH neurons and the associated pituitary–adrenocortical activation may be accomplishedby GLP-1’s direct action on hypophysiotropic CRH neurons. Since central CRH is also thought to be an anorexigenic factor and GLP-1neurons contain leptin receptors, activation of CRH neurons in the PVN by GLP-1 may contribute to the complex neuroendocrine andmetabolic actions by the adipostatic hormone, leptin. 2003 Elsevier B.V. All rights reserved.

Theme: Endocrine and autonomic regulation

Topic: Hypothalamic-pituitary-adrenal regulation

Keywords: Glucagon-like peptide; Corticotropin-releasing hormone; Paraventricular nucleus; Immunocytochemistry; Hypothalamic-pituitary-adrenal axis

Glucagon-like-peptide-1 (7-36) amide is a major splic- aminergic neurons of the nucleus of the solitary tracting product of the preproglucagon peptide precursor in the (NTS) and the reticular nucleus of the medulla oblongatabrain and may serve as a central regulator for a number of [14,20]. GLP-1 immunoreactive (IR) nerve fibers areneuroendocrine and autonomic functions[12,14]. In the rat present in discrete regions of the hypothalamus such as thebrain, GLP-1 is exclusively synthesized in non-catechol- paraventricular (PVN), dorsomedial and arcuate nuclei

[10,13,14,27].In the rat, central but not peripheral administration of

*Corresponding author. Present address: Division of Endocrinology,GLP-1 significantly inhibits feeding behavior, whereasBox No. 268, New England Medical Center, 750 Washington St., Boston,both central and peripheral administration of GLP-1 inhib-MA 02111, USA. Tel.:11-617-636-8517; fax:11-617-636-4719.

E-mail address: rlechan@tufts-nemc.org(R.M. Lechan). its drinking behavior[29]. Intracerebroventricular injection

0006-8993/03/$ – see front matter 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0006-8993(03)03117-2

164 S. Sarkar et al. / Brain Research 985 (2003) 163–168

of GLP-1 also induces c-fos expression in oxytocin and Sawchenko, Salk Institute, La Jolla, CA, USA) at acorticotropin-releasing hormone (CRH) neurons of the dilution of 1:20,000 for 2 days at 48C. After washing inPVN and results in elevated plasma corticosterone levels Tris-buffered saline (TBS), the tissue sections were incu-[23,31]. ‘Interoceptive stressors’ such as intraperitoneal bated sequentially in biotinylated donkey anti-rabbit IgGinjections of lithium chloride, cholecystokinin or bacterial (1:400, Jackson ImmunoResearch) and the ABC Elitelipopolysaccharide, activate GLP-1 neurons in the NTS as Complex (1:100). Immunolabeling was visualized bywell as a large number of PVN neurons, especially in those 0.025% DAB and 0.0036% H O in Tris buffer (pH 7.6).2 2

PVN subdivisions that are also innervated by GLP nerve Thus, the black, silver-intensified Ni-DAB labeled GLP-1fibers [22]. Collectively, these effects appear to be me- fibers, and the brown, DAB labeled CRH immunoreactivediated by a neuronal GLP-1 system projecting from the elements could be easily distinguished in the same section.caudal NTS to the hypothalamus through interactions with The frequency of close appositions between GLP-1 fibersspecific GLP-1 receptors[20,22,27].Thus, to identify the and terminals and CRH neurons was then counted byendogenous neurocircuitry that may underlie the central direct light microscopic examination with 633 oil immer-effects of GLP-1 on the hypothalamic–pituitary–adrenal sion objective from PVN sections taken at 150-mm inter-(HPA) axis, the present study determined whether there is vals.an innervation of PVN CRH neurons by GLP-1-containing Sections processed for electron microscopy were treatednerve terminals. with 1% sodium borohydride in 0.1 M PB for 30 min

Adult, male Sprague–Dawley rats were used for this followed by 0.5% H O in PBS for 15 min. After2 2

study. All procedures involving animals were reviewed cryoprotection in 15% sucrose in PBS for 15 min at roomand approved by the Animal Research Committee at New temperature and 30% sucrose in PBS overnight at 48C, theEngland Medical Center and Tufts University School of sections were snap frozen on liquid nitrogen to improveMedicine. To enhance CRH immunoreactivity in perikarya antibody penetration. Sections were then preincubated withof the PVN, rats were injected intracerebroventricularly 10% normal horse serum for 10 min and incubated inwith 50–60 mg colchicine. After 20 h of survival, the rabbit anti-GLP-1 antiserum (1:4000) for 4 days at 48Canimals were perfused transcardially with 20 ml of saline followed by biotinylated donkey anti-rabbit IgG (1:500)followed by 100 ml of 2% paraformaldehyde and 3.75% overnight at 48C and the ABC Elite complex (1:100).acrolein in 0.1 M phosphate buffer (PB, pH 7.4). Brains GLP-1 immunolabeling was visualized by DAB. Thewere rapidly removed and stored in phosphate buffer saline sections were then incubated in the rabbit antiserum to rat(PBS) for 24 h at 48C. Serial 25-mm-thick coronal CRH (1:3000 for 2 days at 48C), and detected by gold-sections were cut with a vibratome and collected in PBS. silver labeling using IntenSE kit (Amersham) as describedLight microscopic immunohistochemistry of GLP-1 was previously[4,17,32].Sections were then osmicated, treatedperformed using the avidin–biotin–peroxidase method with 2% uranyl acetate in 70% ethanol for 30 min,with a commercial rabbit antiserum to GLP-1 (Peninsula dehydrated in an ascending series of ethanol followed byLaboratories). Briefly, the vibratome sections were treated propylene oxide, and flat embedded in Durcupan ACMwith 1% sodium borohydride in distilled water for 30 min epoxy resin (Fluka) on liquid release agent (Electronfollowed by 0.5% H O in PBS for 15 min, then 0.5% Microscopy Sciences)-coated slides, polymerized at 568C2 2

Triton X-100 in PBS for 30 min. Sections were preincu- for 2 days. Ultrathin sections were cut with a Leica ultracutbated with 10% normal horse serum for 30 min and UCT ultramicrotome (Leica Microsystems, Wetzlar, Ger-incubated in the GLP-1 antiserum diluted at 1:12,000 in many), collected onto formvar coated single-slot grids, and1% normal horse serum containing 0.08% sodium azide examined using a Philips CM-10 electron microscope.and 0.2% Kodak Photo-Flo for 3 days at 48C. Specificity At the light microscopic level, GLP-1-IR fibers andof the immunolabeling has been described elsewhere[22]. nerve terminals were found in the parvocellular parts of theAfter rinses in PBS, sections were incubated in PVN, with highest concentrations in the anterior andbiotinylated goat anti-rabbit-IgG for 2 h (1:400), followed medial and ventral parvocellular subdivisions. The mag-by avidin–biotin–peroxidase complex (ABC Elite 1:100; nocellular divisions of the PVN also showed moderateVector, CA, USA) in PBS for 2 h at room temperature. The numbers of GLP-1-IR nerve fibers. No GLP-1 immuno-immunoreaction product was visualized with 0.025% reactivity was found in the perikarya and dendrites intrin-diaminobenzidine (DAB) containing 0.06% nickel am- sic to the parvocellular areas of the PVN. Sequentialmonium sulfate and 0.0027% H O in 0.05 M Tris buffer, immunolabelling for GLP-1 and CRH revealed that2 2

pH 7.6 (TB) for 6 min. A modification of the Gallyas 6561% of the CRH-immunolabeled perikarya in the PVNsilver intensification technique[18] was then used without were juxtaposed by numerous GLP-1 labeled nerve termi-thioglycolic acid treatment[6] to yield a stable black nals (Fig. 1A–C). By ultrastructural analysis of the medialprecipitate. parvocellular subdivision, DAB labeled GLP-1-IR termi-

After visualizing GLP-1 immunoreactivity in nerve nals containing numerous small clear vesicles and someterminals in the PVN, sections were incubated in rabbit dense core vesicles were seen to establish synapses onantiserum against rat CRH (code C-70, gift of Dr Paul E. CRH neurons, the latter identified by the presence of the

S. Sarkar et al. / Brain Research 985 (2003) 163–168 165

highly electron-dense silver particles (Figs. 2 and 3). Bothaxodendritic and axosomatic synapses between GLP-1terminals and CRH neurons were apparent.

The present results establish that hypophysiotropic CRH

neurons in the PVN receive substantial innervation byaxons containing GLP-1-IR. Since GLP-1 derives primari-ly from the caudal portion of the NTS and adjacentreticular formation in the brainstem[14], it is likely thataxons containing GLP-1 in synaptic relationship with CRHneurons also derive from these neurons. Indeed, studies byLarsen et al.[14] have shown that following the adminis-tration of the retrogradely transported marker substance,Flurorgold, into the PVN, greater than 90% of GLP-1neurons in the NTS concentrated the tracer. Taken togetherwith the observations that GLP-1 can induce early geneexpression in the majority of CRH perikarya in medialparvocellular neurons in the PVN[15] and increasecirculating levels of corticosterone[15], we presume thatGLP-1 neurons in the NTS have a direct activating effecton the HPA axis through monosynaptic projections to CRHneurons in the PVN. Since GLP-1 binds to G-proteincoupled receptors[30], resulting in a potent stimulatoryaction on the cyclic AMP-protein kinase A (PKA) intracel-lular signaling pathway[16], activation of CRH neuronsby GLP-1 is likely to occur as a result of PKA-inducedphosphorylation of the nuclear transcription factor, CREB.Increasing evidence suggests that phosphoCREB is im-portant for activation of the CRH gene by binding to acAMP response element (CRE) in the promoter of theCRH gene in all mammalian species studied[1,26].

The functional significance for the GLP-1/CRH mono-synaptic pathway is suggested by the observation byRinaman [22] that in a model of interoceptive stress,intraperitonial administration of bacterial lipopolysac-charide induces c-fos in all GLP-1-containing neurons inthe NTS. Endotoxins and proinflammatory cytokines arewell known to activate the HPA axis[3,33], commensuratewith activation of the CRH gene in PVN neurons[11,28].Although several mechanisms have been proposed[25],disconnection of ascending catecholamine pathways frombrainstem to the PVN results in a reduction in CRHmRNA [5] in the PVN, indicating that catecholamines maycontribute to the activating effects of endotoxin on theHPA axis. While it has been established that GLP-1-containing neurons do not coexist with epinephrine/nor-epinephrine neurons of the dorsal medulla[14], the exacttrajectory of ascending GLP-1 efferents throughout thebrainstem to reach hypothalamic targets is unknown.

Fig. 1. Medium (A) and high-power (B, C) magnification light micro-scopic photomicrographs showing double immunolabeling for CRHneurons (brown) and GLP-1 nerve terminals (black) in the medialparvocellular subdivision of the PVN. Note that the majority of theCRH-IR neurons are contacted by GLP-1-IR nerve terminals (arrows). III,third ventricle; PVN, paraventricular nucleus. Scale bars: A, 100mm; B,50 mm; C, 25mm.

166 S. Sarkar et al. / Brain Research 985 (2003) 163–168

Fig. 2. (A) Electron micrograph showing axodendritic associations between CRH containing neurons and GLP-1 containing axon terminals (arrows). (Band C) High-power magnification of regions of (A) showing synaptic associations. CRH neurons are labeled with highly electron dense silver granules,whereas GLP-1 terminals are labeled with electron dense DAB. Original magnification33300 in (A) and313,000 in (B) and (C).

Nevertheless, significant lesions produced by hemisectionsor medullary knife cuts [5,21,24] would have likelyproduced the interruption of ascending GLP-1 fibers to thePVN. Accordingly, attenuation of stress induced CRHgene expression following unilateral knife cuts in thebrainstem may have been a result of a combined effect byinterruption of both the catecholaminergic fibers andascending GLP-1 projections, as well as potentially otherimportant efferents originating from the lower brainstem.

One of the best recognized central effect of GLP-1 is itspotent ability to induce anorexia[31] that can be main-tained over 5 days using the long acting GLP-1 analogue,exendin-4 [2]. GLP-1-producing neurons in the NTScontain leptin receptors[7] and are activated by leptin[8],suggesting that GLP-1 may contribute to the anorexigenicFig. 3. Electron micrograph view of an axosomatic synapse (arrowhead)effects of leptin. NPY-producing neurons in the hypo-between a CRH containing neuron in the PVN and a GLP-1 containing

axon terminal (arrow). Original magnification313,000. thalamic arcuate nucleus have been proposed as the

S. Sarkar et al. / Brain Research 985 (2003) 163–168 167

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