Brain Research 945 (2002) 50–59www.elsevier.com/ locate/bres

Research report

I ntracerebroventricular administration ofa-melanocyte stimulatinghormone increases phosphorylation of CREB in TRH- and

CRH-producing neurons of the hypothalamic paraventricular nucleusa a a,b ,*´ ´ ´Sumit Sarkar , Gabor Legradi , Ronald M. Lechan

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

bDepartment of Neuroscience, Tufts University School of Medicine, Boston, MA 02111,USA

Accepted 21 February 2002

Abstract

Changes in circulating leptin levels, as determined by nutritional status, are important for the central regulation of neuroendocrine axes.Among these effects, fasting reduces TRH gene expression selectively in the hypothalamic paraventricular nucleus (PVN), which can bereversed by leptin administration. Intracerebroventricular (i.c.v.) infusion ofa-MSH recapitulates the effects of leptin on hypophysiotropicTRH neurons, completely restoring proTRH mRNA to levels in fed animals despite continuation of the fast, makinga-MSH a candidatefor mediating the central effects of leptin. Asa-MSH binds to a G-protein coupled receptor that activates cAMP anda-MSH stimulatesthe TRH promoter through the phosphorylation of the transcription factor CREB in vitro, we determined whether i.c.v. injection ofa-MSH to rats regulates phosphorylation of CREB, specifically in hypophysiotropic TRH neurons of PVN. Asa-MSH also induces theactivation of CRH gene expression in the PVN, we further determined whether i.c.v. injection ofa-MSH regulates the phosphorylation ofCREB in hypophysiotropic CRH neurons. In vehicle-treated animals, only rare neurons contained nuclear phospho-CREB (PCREB)immunoreactivity in the parvocellular PVN. I.c.v. injection of 10mg a-MSH dramatically increased the number of PCREB-immunolabeled cell nuclei in the PVN in fasted groups at 10 min postinjection, particularly in the medial, periventricular, anterior andventral parvocellular subdivisions, whereas a moderate increase of PCREB immunoreactivity was observed at 30 min and PCREBimmunoreactivity was lowest at 1 h postinfusion. Double immunolabeling with proTRH antiserum revealed that following i.c.v.a-MSHinfusion at 10 min, the majority of TRH neurons contained PCREB in the anterior (71%), medial (83%) and periventricular (63%)parvocellular subdivisions. The percentage of double-labeled TRH neurons declined at 30 min and 1 h posta-MSH infusion. Similarly,only 16% of CRH neurons of the medial parvocellular neurons contained PCREB nuclei in vehicle treated animals, whereas 10 minfollowing a-MSH infusion the percentage of CRH neurons colocalizing with the PCREB rose to 54%, then fell to 37 and 17% at 30 and60 min postinfusion, respectively. These data demonstrate that i.c.v.a-MSH administration increases the phosphorylation of CREB inseveral subdivisions of the PVN including TRH and CRH neurons in the medial and periventricular parvocellular subdivisions, suggestingthat phosphorylation of CREB may be necessary fora-MSH-induced activation of the TRH and CRH genes. The increase in PCREB inthe anterior and ventral parvocellular subdivisions of the PVN, regions linked to nonhypophysiotropic functions such as autonomicregulation, would also imply a role for these neurons in anorectic and energy wasting responses of melanocortin signaling. 2002Elsevier Science B.V. All rights reserved.

1 . Introduction neuroendocrine axes [2,9]. For example, while the bio-synthesis and secretion of thyrotropin-releasing hormone

Responses to starvation not only include the shift from a (TRH) in hypophysiotropic neurons of the paraventricularcarbohydrate-based to a fat-based metabolism, but also nucleus (PVN) are regulated by a thyroid hormone-depen-result in profound functional reorganization of several dent negative feedback control mechanism [16,39], these

neurons are also powerfully affected by nutritional status.During fasting, a significant reduction of TRH gene

*Corresponding author. Division of Endocrinology, Box No. 268, Newexpression occurs selectively in the PVN [4], the seat ofEngland Medical Center, 750 Washington St., Boston, MA 02111, USA.hypophysiotropic TRH neurons, an effect that is complete-Tel.: 11-617-636-8517; fax:11-617-636-4719.

E-mail address: rlechan@lifespan.org(R.M. Lechan). ly reversed by systemic administration of the adipostatic

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )02619-7

S. Sarkar et al. / Brain Research 945 (2002) 50–59 51

hormone, leptin [21]. The arcuate nucleus of the mediobas- Animal Research Committee at the New England Medicalal hypothalamus has been recognized as a primary target Center and Tufts University School of Medicine.site for leptin’s actions [9] which may be transmitted byextensive monosynaptic and parallel multisynaptic path-ways to hypophysiotropic neurons of the PVN [5,27,46]. 2 .2. Animal preparation and a-MSH infusion

An intact arcuate nucleus appears to be a prerequisite forthe ability of leptin to regulate the hypothalamic–pitui- Ten days prior to experimentation, a 22-gauge stainlesstary–thyroid axis [22]. Moreover, intracerebroventricular steel guide cannula (Plastic One, Roanke, VA, USA) was(i.c.v.) infusion ofa-MSH recapitulates the effect of leptin placed into the lateral cerebral ventricle under stereotaxicon hypophysiotropic TRH neurons, completely restoring control (coordinates from bregma, antero-posterior,20.8proTRH mRNA levels, despite continuation of the fast [6], mm; lateral, 1.2 mm; and ventral, 3.2 mm) through a burrmaking this arcuate nucleus-derived peptide an important hole in the skull. The cannula was secured to the skull withcandidate to mediate the effects of leptin on hypophysiot- three stainless steel screws and dental cement and tempo-ropic TRH neurons. I.c.v. administration ofa-MSH also rarily occluded with a dummy cannula. The rats were madereactivates corticotropin-releasing hormone (CRH) gene accustomed to handling by mock injections consisting ofexpression in the PVN of fasting animals [7], indicating removal of the dummy cannula and connecting to anthata-MSH is capable of acting simultaneously on diverse empty cannula connector daily for at least 1 week prior toneuroendocrine axes. experimentation to reduce stress. In addition, 5 days before

Characterization of melanocortin receptors has revealed the experiments, all rats were jugular vein-cannulated tothat all couple in a stimulatory fashion to cAMP and ensure rapid and stress-free anesthesia at the termination ofinduce the transcription of genes by activation of protein the experiment. Animals were either given free access tokinase A (PKA) [30]. The TRH as well as CRH promoters food (Fed Group,n54) or fasted for 65 h (Fasted Group,contain a consensus cAMP response element (CRE)n524). The Fed Group was administered 6ml artificial[20,38], suggesting that these genes are regulated by CSF (aCSF) (140 mM NaCl, 3.35 mM KCl, 1.15 mMbinding of the cAMP response element binding protein or MgCl , 1.26 mM CaCl , 1.2 mM Na HPO , and 0.3 mM2 2 2 4

CREB. CREB is a constitutively expressed transcription NaH PO , pH 7.4) containing 0.05% bovine serum al-2 4

factor whose phosphorylation by PKA at serine-133 acti- bumin. All i.c.v. injections were made in freely movingvates a number of well-characterized neuropeptide genes animals through a 28-gauge needle that extended 1 mm[28,38]. Indeed, recent in vitro evidence in a heterologous below the guide cannula, connected by polyethylene tubingcell system indicates that phosphorylated CREB (PCREB) to a 1-cc GlasPak syringe over 2 min by a microprocessorcan bind to the CRE in the TRH promoter and activate the controlled infusion pump (Bee Electronic Minipump; BAS,gene [13]. West Lafayette, IN, USA). The fasted group was sub-

To determine whether a similar mechanism of cell divided into additional six groups. The first three groupssignaling bya-MSH is observed in vivo, we examined the were administered aCSF i.c.v. and studied 10, 30 and 60presence of PCREB in the nucleus TRH and CRH produc- min later (n54 in each group). The remaining three groupsing neurons in the PVN following the i.c.v. administration were administered 10mg of a-MSH (Peninsula Labs.,of a-MSH. Further, we hypothesized that ifa-MSH Belmont, CA, USA) in 6ml of aCSF and studied 10, 30induces the phosphorylation of CREB in target neurons in and 60 min later. After 10, 30 or 60 min, the animals werethe PVN, then the immunocytochemical delineation of deeply anesthetized with sodium pentobarbital (50 mg/kg)PCREB would identify other neuronal populations in the through the jugular catheters. Within 2 min of initiation ofPVN affected by melanocortin signaling including the anesthesia, the rats were perfused transcardially with 10 mllocus of those neurons involved in anorexia and energy of heparinized saline for 10–20 s followed by a mixture ofdisposal. 1% acrolein and 3% paraformaldehyde in 0.1 M phosphate

buffer (pH 7.4) for 10–15 min. Following perfusion, thebrains were dissected and postfixed by immersion in 3%

2 . Materials and methods paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) forseveral hours at 48C. The hypothalamus was cut out using

2 .1. Animals a coronal rat brain matrix (Ted Pella, Redding, CA, USA)and the block transferred into 20% sucrose in 0.01 M

These experiments were performed on adult male phosphate buffered saline (PBS) at 48C for 2 days.Sprague–Dawley rats (Taconic Farms, Germantown, NY,USA) weighing 210–230 g. The animals were housedindividually in cages under standard environmental con- 2 .3. Immunohistochemical detection of PCREBditions (light between 0600 and 1800 h; temperature,2261 8C; rat chow and water available ad libitum). All To determine the extent to which the population of cellsexperimental protocols were reviewed and approved by the within the parvocellular division of the PVN expressa-

52 S. Sarkar et al. / Brain Research 945 (2002) 50–59

MSH induced phosphorylation of CREB, series of sections 2 .5. Quantitative analysis of PCREB labeled nuclei andwere cut on cryostat at 25mm through the PVN from proTRH and CRH immunoreactive perikarya in the PVNanimals killed at different time points. Sections were firstwashed in PBS, treated with 1% sodium borohydride in Double-labeled sections containing PCREB and proTRHdeionized water for 30 min, and then rinsed several times as well as CRH immunoreactivities were used for quantita-in deionized water and PBS until the sections became free tive analysis. ProTRH immunostaining was confined ex-of bubbles. The sections were placed in 0.5% H O in clusively to perikarya and proximal dendrites, developed2 2

PBS for 15 min to remove the endogenous peroxidase with a light-brown chromogen, allowing the determinationactivity from the tissues, washed in PBS, and treated in whether the nucleus contained the previously developed0.5% Triton-X in PBS for 1 h to improve antibody dark-purple label for PCREB. Only cells with intense orpenetration. Following preincubation in 10% normal horse medium density nuclear labeling and distinct cytoplasmicserum for 30–60 min, the sections were incubated in the reaction product were counted as double labeled. Theprimary antiserum against phosphorylated CREB 128-141, percentage of proTRH neurons containing PCREB-labeleddiluted 1:12 000 (gift of Dr. Marc R. Montminy, Salk nucleus was determined from three distinct rostro-caudalInstitute, La Jolla, CA, USA) for 3 days at 48C with levels of the PVN placed at 300-mm intervals (rostral,continuous agitation on a rotary shaker. All primary mid-level, and caudal PVN) in all animal groups. On theantisera dilutions were made in 1% normal horse serum in basis of this analysis, counts of labeled nuclei for PCREBPBS containing 0.08% sodium azide and 0.2% Kodak were made in single sections of aCSF treated and 10mg ofPhoto-Flo. After thorough rinsing in PBS, sections were a-MSH treated animals. The subdivisions of the PVN wereincubated in biotinylated goat anti-rabbit IgG (1:200, identified based on the rat brain atlas of Paxinos andVector Labs., Burlingame, CA, USA) for 2 h. The sections Watson [34] and projected from a Zeiss microscopewere then washed three times in PBS and incubated in equipped with a COHU video camera (San Diego, CA,avidin–peroxidase complex (1:100, ABC Elite Kit, Vector USA) onto the monitor of a Macintosh computer usingLabs.) for 1 h. After three washes in PBS and a rinse in IMAGE 1.54 software (NIH). All nuclei with intense or0.05 M Tris buffer (pH 7.8), the color reaction was medium-intensity PCREB labeling in the parvocellulardeveloped in 0.025% diaminobenzidine (DAB) containing subdivision of the PVN were counted on each side of the0.06% nickel ammonium sulfate and 0.0027% H O for 7 PVN and the two sides averaged to yield the representative2 2

min to yield a dark-purple labeling in the cell nucleus. A number of PCREB-labeled nuclei for each animal per side.rinse in Tris buffer (pH 7.6) was used to stop the reaction. The mean and standard errors of the total number ofSets of PCREB labeled sections were then mounted onto PCREB-labeled nuclei per side in the parvocellular PVNslides, dehydrated in graded series of ethanol followed by from animals in the corresponding treatment groups andthree changes of Histosol and coverslipped in DPX (Fluka) the percentage of proTRH immunostained neurons con-for light microscopy. taining PCREB-labeling were determined and statistically

analyzed. The percentages of CRH neurons containingPCREB-labeled nuclei were determined from the mid-level

2 .4. Light microscopic double-labeling of the PVN.immunocytochemistry of PCREB IR nuclei and proTRHand CRH neurons in the PVN 2 .6. Antisera characterization

Sets of PCREB-labeled PVN sections were rinsed in 2 .6.1. PCREB antiserumTris buffered saline (TBS) and then incubated in either Antiserum to PCREB was raised in rabbits againstrabbit antiserum recognizing rat preproTRH 53–74 (gift of synthetic CREB 128–141, containing a phosphorylated

133Dr. Ivor Jackson, Rhode Island Hospital, Providence, RI, Ser residue, and purified by removing antibodies to theUSA) at a dilution 1:20 000 or in rabbit antiserum raised unphosphorylated peptide using affinity chromatographyagainst rat CRH (gift of Dr. Paul E. Sawchenko, Salk with phosphopeptide resin [11]. Immunoblot analysis ofInstitute) at a dilution of 1:20 000 for 2 days at 48C. After nuclear extracts from the hypothalamus showed a singlewashing in Tris buffer saline (TBS), the tissue sections band corresponding to PCREB and no cross reactivity withwere incubated in donkey anti-rabbit IgG (1:400, Jackson unphosphorylated CREB [11]. Immunostaining in the PVNImmunoResearch) and the ABC elite complex (1:100). The is completely abolished when the antiserum is preabsorbedimmunolabeling was visualized by 0.025% DAB and with excess of the corresponding phosphopetide (10mm)0.03% H O in Tris buffer (pH 7.6) alone, to yield a [23].2 2

contrasting brown cytoplasmic labeling. After develop-ment, the sections were rinsed in Tris buffer (pH 7.6), 2 .6.2. ProTRH antiserummounted into slides, air-dried, dehydrated in ethanol Antiserum to rat prepro TRH 53–74 was raised in Newfollowed by Histosol and coverslipped. Zealand White rabbits and its specificity characterized both

S. Sarkar et al. / Brain Research 945 (2002) 50–59 53

by RIA and immunocytochemistry [19,49]. Preabsorption further investigated only in fasted animals at various26of this antiserum with excess (10 M) preproTRH 53–74 postinjection time intervals.

(Peninsula) results in the complete loss of immunostainingin the PVN [49]. 3 .2. Effect of i.c.v. a-MSH administration to fasting

animals on PCREB immunoreactivity in the PVN2 .6.3. CRH antiserum

Antiserum to rat CRH (code no. C70) was generated in Within 10 min ofa-MSH infusion into the lateralrabbits against synthetic CRH and has been characterized ventricle, a dramatic increase in PCREB immunoreactivein detail previously [36]. Preabsorption of the CRH nuclei was observed in the anterior, periventricular, medialantiserum by 10mM of synthetic rat CRH (American and ventral parvocellular subdivisions of the paraventricu-Peptide, Sunnyvale, CA, USA) results in the complete loss lar nucleus (Fig. 2D–F). Substantial elevation was stillof immunolabelling in the PVN [23]. present at 30 min (Fig. 2G–I), but the activation di-

minished progressively after 60 min (Fig. 2J–L). In2 .7. Statistical analysis contrast, fasted aCSF-treated animals showed sparse

PCREB immunoreactivity in the parvocellular neurons ofThe results are presented as mean6S.E.M. Statistical the paraventricular nucleus (Fig. 2A–C).

significance was determined by analysis of variance fol-lowed by posthoc Newman–Keuls test. Differences were 3 .3. Double labeling PCREB–proTRH immunostainingconsidered to be significant atP,0.05. in the PVN

Mid and caudal levels of PVN sections, which contain3 . Results the highest numbers of proTRH neurons, were used for

detailed analysis. Only 3% of the proTRH neurons of the3 .1. Effect of fasting on PCREB immunoreactivity in the medial parvocellular neurons contained PCREB followingPVN aCSF administration (Fig. 3A and B). Within 10 min of

a-MSH infusion, the number of proTRH neurons in theIn the fed state, the PCREB immunolabeling was PVN that contained PCREB was dramatically increased in

confined to the nuclei of magnocellular neurosecretory the anterior parvocellular subdivision (71.461.1%), medialneurons, with limited immunostaining in the parvocellular parvocellular subdivision (83.261.8%), and periventricularsubdivision (Fig. 1A). In the fasting state, PCREB im- parvocellular subdivision (63.162.4%) (Fig. 3C and D).munolabeling was even further reduced with fewer nuclei The percentage of proTRH neurons that contained PCREBin the magnocellular subdivision and nearly complete was diminished progressively at 30 min (34.764.7%) (Fig.absence of immunoreactivity in the parvocellular subdivi- 3F and G) and 1 h posta-MSH infused groupssion (Fig. 1B). Thus, presence of PCREB in TRH and (27.461.9%) (Fig. 3G and H). In comparison to the PVN,CRH neurons of the PVN in response toa-MSH was only few TRH neurons in other regions of the hypo-

Fig. 1. Distribution of PCREB immunoreactivity in the PVN in (A) fed and (B) fasted animals. Note the reduction in the PCREB immunolabeling in thefasted state throughout the parvocellular subdivision (arrows). 3V, third ventricle; PVN , magnocellular division of the paraventricular nucleus.m

54 S. Sarkar et al. / Brain Research 945 (2002) 50–59

Fig. 2. Distribution of PCREB-labeled nuclei in the anterior, mid and Fig. 3. Medium- (A,C,E,G) and high-power magnification (B,D,F,H)caudal levels of the PVN in fasted animals. Animals were treated with light microscopic photomicrographs showing double immunolabeling foraCSF (A–C) ora-MSH (D–L) and studied at 10 min (D–F), 30 min proTRH neurons (brown cytoplasm) and PCREB-positive cells (black(G–I), and 60 min (J–L) post-a-MSH infusion. Note the dramatic nuclei) in the medial parvocellular subdivision of the PVN in controlincrease of PCREB immunolabeling in animals killed at 10 min post-a- fasted animals (A,B) and 10 min (C,D), 30 min (E,F) and 60 min (G,H)MSH infusion and progressively diminished PCREB immunolabeling at post-a-MSH infusion. Double-labeled neurons are rarely found in the30 and 60 min post-a-MSH infusion. PVN , anterior parvocellular aCSF treated group (A,B). The maximum amount of double labeling isa

subdivision of the PVN; PVN , medial parvocellular subdivision of the observed at 10 min post-a-MSH infusion, where the vast majority ofmp

PVN; PVN , periventricular parvocellular subdivision of the PVN; proTRH neurons contain PCREB in their nucleus (C,D). The percentagepv

PVN , ventral parvocellular subdivision of the PVN; PVN , magnocel- of double-labeled neurons decreases at 30 and 60 min (E–H); 3V, thirdvp m

lular division of the PVN. ventricle.

thalamus contained PCREB followinga-MSH infusion(Fig. 4). The average percentage of proTRH neurons in allparvocellular subdivisions of the PVN that contain PCREBnuclei in aCSF anda-MSH infused groups are representedgraphically in Fig. 5.

3 .4. Double labeling PCREB–CRH immunostaining inthe PVN

Midlevel PVN sections that contain the highest numbersof CRH neurons were used for detailed analysis. InPCREB-labeled sections of the PVN, sequentially labeledwith CRH antiserum, approximately 35–40 neurons wereobserved in each animal. Only 15.960.9% of CRH

Fig. 4. Medium power magnification of proTRH neurons (brown cyto-neurons of the medial parvocellular neurons containedplasm) and PCREB-positive cells (black nuclei) showing that in the PVN,

PCREB following aCSF administration (Fig. 6A and B). the majority of TRH neurons (arrows) contain of PCREB immuno-Ten min following a-MSH infusion, the percentage of reactivity, whereas in the perifornical region, the majority of proTRHCRH neurons containing PCREB rose to 53.664.6% (Fig. neurons (arrows) are not double labeled.

S. Sarkar et al. / Brain Research 945 (2002) 50–59 55

Fig. 5. Percentage of TRH neurons containing PCREB nuclei in vehicle treated anda-MSH infused rats. *,P,0.05.

6C and D) and then fell to 36.362.2% and 17.160.9% at30 min (Fig. 6E and F) and 60 min (Fig. 6G and H)postinfusion, respectively.Values at the 10 and 30 min timepoints were statistically different from the aCSF treatedcontrol animals. The percentage of CRH neurons con-taining PCREB in aCSF treated control anda-MSHinfused groups are represented in the graph in Fig. 7.

4 . Discussion

The mechanisms by which animals adapt to inadequatenutrient availability has been vastly expanded by thediscovery of the white fat-derived hormone, leptin [50].During fasting, circulating levels of leptin are decreased,orchestrating a series of central responses that result inenergy conservation [2]. Included among these is suppres-sion of the hypothalamic–pituitary–thyroid axis, largelydue to inhibition of proTRH gene expression selectively inhypophysiotropic neurons of the hypothalamic PVN [4,21].We have proposed that at least one of the central mediatorsresponsible for fasting-induced inhibition of proTRHmRNA is suppression of POMC gene expression, simulta-neous with the upregulation of neuropeptide Y and AGRPgene expression in hypothalamic arcuate nucleus neurons[6,22]. This understanding is partly based on the observa-tion thata-MSH, a posttranslational end product of POMCFig. 6. Medium- (A,C,E,G) and high-power magnification (B,D,F,H)

light microscopic photomicrographs showing double immunolabeling for processing [32], can fully restore proTRH mRNA levels inCRH neurons (brown cytoplasm) and PCREB-positive cells (black the PVN when administered i.c.v. to fasting animals, evennuclei) in the medial parvocellular subdivision of the PVN in control though AGRP-gene expression remains elevated [6]. Infasted animals (A,B) and 10 min (C,D), 30 min (E,F) and 60 min (G,H)

addition, a-MSH-containing axons in the PVN establishpost-a-MSH infusion. Note numerous CRH neurons that contain PCREBsynaptic contacts with both the soma and first-orderin the10 min postinfusion group (C,D). The percentage of doubly-labeled

neurons decreases at 30 and 60 min (E–H); 3V, third ventricle. dendrites of proTRH-IR neurons, providing an anatomical

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Fig. 7. Percentage of CRH neurons containing PCREB nuclei in vehicle treated anda-MSH infused rats. *,P,0.05.

basis to propose an important physiological role fora- CREB in parvocellular CRH neurons of the PVN, whichMSH in the regulation of these neurons [6]. can be avoided by rapidly anesthetizing the animals

Since melanocortin receptors couple in a stimulatory through an indwelling jugular cannula before perfusionfashion to cAMP [45] and induce the transcription of genes fixation of the brain.by the activation of PKA which could then result in the The administration of aCSF i.c.v. to fasting animals hadphosphorylation of CREB, and both the TRH and CRH no effect on the phosphorylation of CREB in the PVN.genes contain functional CREs in their promoter [20,38], Following the i.c.v. administration ofa-MSH, however, awe hypothesized that the mechanism ofa-MSH activation marked increase in both the number of cells containingof these genes in vivo is mediated by the transcription PCREB and the intensity of its nuclear immunostaining infactor, PCREB. This concept has been recently demon- the medial, periventricular, ventral and anterior parvocellu-strated in vitro by Harris et al. [13], showing that deletion lar subdivisions was seen. PCREB-IR was maximal withinor mutation of the CRE in the human TRH promoter 10 min ofa-MSH administration, and progressively di-lowers basal activity and reduces stimulation of the TRH minished over 60 min. Similar observations have beengene bya-MSH by more than 50%. To test this hypothesis made on the time course of CREB phosphorylation inin vivo, we administereda-MSH i.c.v. in a dose expected CRH neurons in the PVN following ether stress [17].to induce proTRH mRNA [6] and CRH mRNA [7] in The presence of PCREB in the nucleus of medial andhypophysiotropic neurons in the PVN. periventricular parvocellular subdivision neurons, which

As previously recognized [23], ad lib fed, unstressed have known projections to the neural–hemal contact zoneanimals showed PCREB-IR primarily in the nucleus of in the median eminence [18] and the presence ofmagnocellular neurons in the PVN with minimal immuno- melanocortin 4 receptors (MC4) in the medial and periven-reactivity in parvocellular neurons. With fasting, however, tricular parvocellular part of the paraventricular nucleusPCREB-IR was even more profoundly suppressed, with [29,44], suggest a role fora-MSH in the regulation ofminimal to no immunoreactivity in the parvocellular anterior pituitary function. By double-labeling immuno-division and reduced immunoreactivity in the magnocellu- cytochemistry, the majority of PCREB-positive cells inlar division. While these observations are contrary to those these PVN subdivisions were comprised of TRH and CRHreported by Sheriff et al. [40], we presume that the neurons. Ten minutes followinga-MSH administration,increase in CREB phosphorylation in the PVN observed in PCREB was observed in approximately 83% of medialtheir study may have been due to acute handling stress. parvocellular and 63% of periventricular parvocellularEarlier observations by Legradi et al. [23], have shown that TRH neurons, and 54% of medial parvocellular CRHeven mild handling stress can induce phosphorylation of neurons. In contrast, adjacent populations of TRH neurons

S. Sarkar et al. / Brain Research 945 (2002) 50–59 57

outside of the PVN such as the perifornical group that do AGRP, independently derived from neurons in the arcuatenot subserve a hypophysiotropic function, were largely nucleus [12,41], suggesting a major action of these pep-devoid of PCREB immunoreactivity. These data support tides on neurons in these regions. In addition, focalthe concept that the phosphorylation of CREB is an injections ofa-MSH into the PVN reduces feeding andimportant initial step in the activation of the TRH and fully replicates the reduced feeding responses observedCRH genes in vivo bya-MSH, but in a selective popula- following i.c.v. administration [48]. Conversely, focaltion of TRH and CRH neurons in the brain that regulate injection of thea-MSH antagonist, SHU9119, into theanterior pituitary function. Sincea-MSH is capable of PVN has an extremely potent effect to increase feedingmaintaining the activation of the proTRH gene with [10]. Thus, it is conceivable that in addition to regulationcontinued administration in fasting animals over at least a of hypophysiotropic function bya-MSH through effects on65-h fast [6], however, these data further indicate that the medial and periventricular parvocellular PVN neurons,phosphorylation of CREB may be required only initially some of the other functions ofa-MSH in the regulation ofafter a-MSH, and that other factors may be recruited to feeding and energy disposal are mediated by the anteriorsustain its activating response. and ventral parvocellular subdivisions.

While the majority of TRH neurons in the medial Approximately 71% of anterior parvocellular TRHparvocellular PVN showed CREB phosphorylation and an neurons contained PCREB-IR following the i.c.v. adminis-increase in TRH mRNA following the i.c.v. administration tration ofa-MSH, similar to the percentage in the medialof a-MSH to fasting animals, we have previously observed and periventricular parvocellular subdivisions. However,that only approximately 34% of medial parvocellular PVN these neurons are not only functionally distinct from TRHneurons actually receive contacts by axon terminals con- neurons in the medial and periventricular parvocellulartaining a-MSH [6]. This discrepancy suggests that al- subdivisions with respect to their lack of projections to thethough some TRH-producing neurons in the medial par- median eminence [18] and response to hypothyroidismvocellular subdivision of the PVN are not innervated by [16,39], but are also neurochemically distinct in that theya-MSH-containing axons, they may still possess do not co-contain CART as do the majority of medial andmelanocortin receptors. As practically all medial parvocel- periventricular TRH neurons [8]. Nevertheless, as notedlular subdivision TRH neurons are innervated by axons above, anterior parvocellular TRH neurons are closelycontaining AGRP [25], that acts as an endogenous antago- integrated into the arcuate nucleus regulatory system, andnist toa-MSH at melanocortin 3 and 4 receptors [33], the heavily innervated by axons containinga-MSH (approxi-alternative possibility that AGRP acts alone as an inverse mately 70% of anterior parvocellular subdivision TRHagonist at melanocortin receptors on hypophysiotropic neurons [6]), as well as by AGRP and NPY fibers [24,25].PVN neurons must be considered. This possibility is In addition, recent studies by Takatsu et al. [43] havesupported by recent reports by Nijenhuis et al. [31] and elucidated a unique leptin-regulated population of arcuateHaskell-Luevano and Monck [14], showing that the nucleus neurons that produce galanin-like peptide (GALP)melanocortin 4 receptor can be constitutively active under that within the hypothalamic PVN, projects almost exclu-some circumstances, and that its activity can be suppressed sively to the anterior parvocellular subdivision. Since theby AGRP. Yet, were this the case, one might have expected injection of the anterogradely transported marker sub-that constitutively active melanocortin receptors would stance, PHA-L, into a region of the PVN that includes theupregulate TRH gene expression following ablation of the anterior parvocellular subdivision labels the lateral septumAGRP neurons in the arcuate nucleus, but this is not and medial nucleus of the amygdala [26,35], it is possibleobserved [22]. Other mechanisms by whicha-MSH could that this region of the PVN is integrated into the limbicactivate hypophysiotropic TRH neurons have been pro- system and has a role in the behavioral manifestations ofposed by Mihaly et al. [27], suggesting an indirect action feeding induced by melanocortin signaling.through an arcuate–dorsomedial nucleus–PVN mul- Following i.c.v.a-MSH administration, the ventraltisynaptic pathway. parvocellular subdivision of the PVN contains an even

In addition to the medial and periventricular parvocellu- more substantial concentration of PCREB-IR cells than thelar subdivisions of the PVN, i.c.v. administration ofa- anterior parvocellular subdivision, comparable to the num-MSH markedly increased PCREB-IR in the nucleus of ber of PCREB-IR neurons observed in the medial andanterior and ventral parvocellular subdivision PVN neu- periventricular parvocellular subdivisions. This subdivisionrons. While melanocortin receptors are concentrated in of the PVN is involved in the regulation of the autonomicseveral, distinct regions within the hypothalamus and nervous system through descending projections to brain-brainstem that could mediate the effects of melanocortin stem and spinal cord targets [35,37,42]. Since the i.c.v.signaling on energy balance [1,47], there is compelling administration of melanocortin receptor agonists stimulatesevidence that the action of melanocortin signaling system sympathetic outflow to brown adipose tissue (BAT) toon energy disposal is primarily exerted in the PVN. Both increase uncoupling protein-1 (UCP-1) activation, therebythe anterior and ventral parvocellular subdivisions receive increasing thermogenesis [15], it is possible that thea particularly high density of axons containinga-MSH and PCREB-responsive neurons in the ventral parvocellular

58 S. Sarkar et al. / Brain Research 945 (2002) 50–59

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