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Tupper Research Institute and Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, Tufts-New England Medical Center (C.F., P.S.S., S.S., R.M.L.), and Departments of Community Health (W.M.R.) and Neuroscience (R.M.L.), Tufts University School of Medicine, Boston, Massachusetts 02111; and Department of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences (C.F.), Budapest H-1083, Hungary
Address all correspondence and requests for reprints to: Dr. Ronald M. Lechan, Division of Endocrinology, Box 268, Tufts-New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111. E-mail: rlechan{at}tufts-nemc.org.
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Introduction |
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-MSH-producing neurons that coexpress cocaine- and amphetamine-regulated transcript (CART), and neuropeptide Y (NPY)-producing neurons that coexpress agouti gene-related protein (AGRP) (see review in Ref.3). Thus, during fasting, the expression of proopiomelanocortin (POMC), a precursor protein of -MSH, and CART are reduced, whereas there is a marked increase in the genes encoding NPY and AGRP (4, 5). Both NPY and AGRP have a potent inhibitory effect on TRH mRNA in the PVN and result in a substantial reduction in TSH and circulating thyroid hormone levels (6). Conversely, -MSH and CART have potent activating effects and can prevent the fasting-induced reduction in TRH mRNA when given exogenously (7, 8).
Although the suppression of POMC and CART gene expression and the up-regulation of NPY and AGRP gene expression contribute to fasting-induced inhibition of the HPT axis, this mechanism does not appear to participate in inhibiting the HPT axis during infection (3). After the administration of bacterial endotoxin, there is an increase in circulating levels of leptin (9) and increased POMC (10, 11) and CART gene expression (11), but no significant change in NPY gene expression in the arcuate nucleus (11). Nevertheless, the HPT axis and, in particular, TRH mRNA in the PVN is suppressed (12), suggesting that a different set of regulatory controls over hypophysiotropic TRH neurons from that observed during fasting occurs after the administration of endotoxin, overriding the activating effects of increased leptin, -MSH, and CART on the HPT axis.
The PVN receives strong input from autonomic centers in the lower brainstem, including the nucleus tractus solitarius, dorsal motor nucleus of the vagus, and several catecholamine groups in the dorsal and ventrolateral medulla (13, 14, 15). These brainstem nuclei are known to be influenced by endotoxin administration (16, 17) and are involved in the activation of hypophysiotropic CRH-synthesizing neurons of the PVN (18, 19). Because hypophysiotropic TRH neurons receive strong input from brainstem catecholamine-producing neurons (20, 21), we hypothesized that ascending brainstem pathways may mediate the inhibitory effects of endotoxin on the HPT axis. Therefore, we unilaterally transected the ascending brainstem pathways in the posterior hypothalamus and determined whether the lack of brainstem inputs alters the effect of bacterial lipopolysaccharide (LPS) on TRH gene expression in hypophysiotropic neurons.
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Materials and Methods |
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Rats were deeply anesthetized with sodium pentobarbital (35 mg/kg body weight, ip), and under stereotaxic guidance using a stereotaxic apparatus (Cartesian Research, Inc., Sandy, OR), a 3-mm-wide glass knife was lowered into the brain at the level of the posterior hypothalamus parallel with the coronal plane. The coordinates of the medial edge of knife were; anterior-posterior, –4.0 mm from the bregma; lateral, 0 mm; and dorsoventral, –9.0 from the surface of the skull. After 2 wk of survival, the animals were divided into two groups. The first group (n = 9) received an ip injection of sterile saline, whereas animals in the second group (n = 9) received an equal volume of bacterial LPS (Sigma-Aldrich Corp., St. Louis, MO; 0127:B8; 250 µg/100 g body weight, ip, in sterile saline). All injections were given between 0900–1200 h. The rectal temperature of the rats was measured by a digital thermometer before and 2, 4, 6, and 24 h after the injection. The animals were weighed before the injection and before being euthanized. Twenty-four hours after the injections, when TRH mRNA is significantly decreased by LPS (12), the animals were overdosed with pentobarbital and perfused transcardially with 20 ml 0.01 M PBS, pH 7.4, containing 15,000 U/liter heparin sulfate, followed by 150 ml 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). After perfusion, the brains were postfixed by immersion in 4% paraformaldehyde in 0.1 M PB (pH 7.4) for 4 h at room temperature. The hypothalamic blocks were transferred into 20% sucrose in 0.01 M PBS at 4 C overnight to promote cryoprotection. Serial 18-µm-thick coronal sections through the rostrocaudal extent of the PVN were cut on a cryostat (CM3050 S, Leica Microsystems, Nussloch, Germany) and adhered to SuperFrost Plus glass slides (Fisher Scientific Co., Pittsburgh, PA) or collected in glass vials containing PBS (pH 7.4) and stored in freezing solution (30% ethylene glycol, 25% glycerol, 0.05 M PB) at –20 C until used. Three sets of sections containing every fourth section through the PVN were mounted, and one set was stored in freezing solution. Mounted tissue sections were desiccated overnight at 42 C and stored at –80 C until prepared for in situ hybridization histochemistry.
Immunohistochemical detection of phenylethanolamine N-methyltransferase (PNMT)-containing fibers in the PVN
To assess the efficacy of the knife cut, PNMT immunocytochemistry was performed on free-floating sections. Briefly, the sections were treated in 0.5% H2O2 for 15 min to block endogenous peroxidase activity, followed by 0.5% Triton X-100 in PBS for 1 h to improve antibody penetration, and then in 10% normal horse serum in PBS for 30–40 min to reduce nonspecific antibody binding. The sections were incubated in sheep antisera to PNMT (Chemicon International, Temecula, CA) at a 1:5000 dilution in 1% normal horse serum in PBS containing 0.08% sodium azide and 0.2% Kodak Photo Flo (Eastman Kodak Co., Rochester, NY) for 2 d at 4 C. After thorough rinsing in PBS, the sections were incubated in rabbit antisheep IgG (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 2 h at room temperature. The sections were then immersed in avidin-peroxidase complex (1:100; ABC Elite Kit, Vector Laboratories, Inc., Burlingame, CA) for 1 h. The immunoreaction was developed in 0.025% diaminobenzidine containing 0.03% H2O2 in Tris buffer (pH 7.6) for 3–5 min to yield brown labeling. A rinse in Tris buffer (pH 7.6) was used to stop the reaction. PNMT-labeled sections were then mounted onto glass slides, dehydrated in a graded series of ethanol, followed by three changes in Histosol, and then coverslipped in DPX mountant (Sigma-Aldrich Corp.) for light microscopy.
In situ hybridization histochemistry
Every fourth section through the hypothalamus from the region of the PVN was subjected to in situ hybridization histochemistry as previously described (22), using a single-stranded, [35S]UTP-labeled cRNA probe for CRH generated from a 976-bp cDNA (23) or TRH generated from a 1241-bp EcoRI-PstI fragment of pro-TRH cDNA (24), respectively. Hybridization was performed under plastic coverslips in buffer containing 50% formamide, a 2-fold concentration of standard sodium citrate, 10% dextran sulfate, 0.255% BSA, 0.25% Ficoll 400, 0.25% polyvinyl pyrollidine 360, 250 mM Tris (pH 7.5), 0.5% sodium pyrophosphate, 0.5% sodium dodecyl sulfate, 250 µg/ml denatured salmon sperm DNA, and 6 x 105 cpm radiolabeled probes for 16 h at 55 C. Slides were washed in graded solutions in ethanol containing 0.3 M ammonium acetate, and dipped into Kodak NTB2 photographic emulsion (Eastman Kodak Co.). Autoradiographs were developed after exposure for 2–4 d. Specificity of hybridization for both CRH and TRH mRNA in the PVN has been described previously (24, 25).
Image analysis
To study the effects of the knife cut on the PNMT-immunoreactive innervation of the PVN, sections were analyzed under darkfield microscopy, and density values of the PNMT innervation exclusive to each side of the PVN for each animal were measured. The images were captured using a COHU 4910 video camera (COHU, Inc., San Diego, CA) and a color PCI frame grabber board (Scion Corp., Frederick, MD) and were analyzed with a Macintosh G4 computer (Apple Computer, Redmond, WA) using Scion Image software. Background density points were removed by thresholding the image, and integrated density values (density x area) of PNMT-immunoreactive axons in the same region on each side of the PVN were measured for each animal.
In situ hybridization autoradiograms were similarly analyzed. Background density points were removed by thresholding the image, and integrated density values (density x area) of hybridized neurons in the same region of the PVN were measured in three (CRH) or five (TRH) consecutive sections for each animal in both the intact and transected sides. The sum of the integrated density units were calculated for each animal. Nonlinearity of radioactivity in the emulsion was evaluated by comparing density values with a calibration curve created from autoradiograms of known dilutions of the radiolabeled probes, immobilized on glass slides in 1.5% gelatin, fixed with 4% formaldehyde, and exposed and developed simultaneously with the in situ hybridization autoradiograms.
Statistical analysis
Analyses used the natural logs of density of both CRH and TRH mRNA because of the skewness and lack of homogeneity of variance of the data. Paired t tests were used for comparing the intact and tran-sected sides of individual animals, whereas two-sample t tests were used to compare the effects of treating animals with either LPS or vehicle in terms of the density ratios. Temperature data were analyzed using nested ANOVA, and the change in weight was analyzed by t tests. A significance level of P < 0.05 was considered statistically significant. All analyses were run with SPSS version 11 (SPSS, Inc., Chicago, IL). Data are presented as the mean ± SEM.
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Results |
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). Of 18 animals, 14 that had greater than 59% reduction in PNMT fiber density on the transected side of the PVN (range, 59.6–85.6%; mean for vehicle-treated animals, 73.0 ± 3.5%; mean for LPS-treated animals, 74.5 ± 2.2%) were selected for additional study.
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shows the changes in rectal temperature over the course of the experiment. Nested ANOVA showed a significant interaction between treatment and time (P < 0.001). The temperature of control rats was stable during the entire experiment compared with LPS administration, which resulted in an early reduction in temperature 2 h after the injection, followed by a temperature rise 6 and 24 h postinjection. The control group behaved differently from the LPS-treated animals; they had an insignificant weight gain (1.0 ± 1.4 g) at the termination of the experiment, whereas the LPS-treated group lost weight (–29.7 ± 5.7 g; P < 0.001).
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and 3A). Neurons containing CRH mRNA were more laterally located in the medial parvocellular subdivision compared with neurons containing TRH mRNA. In addition, only the latter group extended into the periventricular parvocellular subdivision of the PVN and concentrated in a cluster of densely packed neurons in the caudal aspects of the medial parvocellular subdivision.
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), whereas TRH mRNA was significantly increased on the side of transection (Fig. 3A). By image analysis, mean log integrated density units of CRH mRNA on the intact vs. transected sides were 1.84 ± 0.14 vs. 1.39 ± 0.13 (P < 0.001), respectively, and for TRH mRNA, were 3.78 ± 0.56 vs. 4.36 ± 0.57 (P = 0.006; Figs. 2C and 3C).
Effect of LPS treatment on CRH and TRH mRNA expression in the PVN
LPS resulted in nearly a 400% increase in the accumulation of silver grains over hybridized CRH neurons in the PVN on the intact side compared with that in vehicle-treated controls (P < 0.001; compare Fig. 2, A vs. B). LPS also increased CRH mRNA on the transected side, but the increase was significantly less than that on the intact side (intact vs. transected, mean log integrated density units, 6.88 ± 0.91 vs. 4.03 ± 0.33; P < 0.001; Fig. 2C). In contrast, although there was a tendency for LPS treatment to result in a reduction in TRH mRNA in the PVN on the intact side compared with that in vehicle-treated controls (Fig. 3C), by image analysis the difference was not significant (Table 2). TRH mRNA in LPS-treated animals on the transected side also showed a reduction compared with that in vehicle-treated controls (Fig. 3, A and B), which by image analysis was significant (vehicle tran-sected vs. LPS transected, mean log integrated density units, 4.36 ± 0.57 vs. 2.16 ± 0.39; P < 0.005). In addition, a significant reduction in TRH mRNA was observed in LPS-treated animals on the transected side compared with the intact side (LPS intact vs. LPS transected, mean log integrated density units, 2.65 ± 0.51 vs. 2.16 ± 0.39; P = 0.002).
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. This table also shows the results of the primary analyses comparing intact and transected sides in both vehicle- and LPS-treated animals, and additionally, the magnitude and direction of the differences between the two treatment groups. |
Discussion |
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In vehicle-treated animals, transection of the ascending brainstem pathways to the PVN resulted in a significant reduction in CRH mRNA on the ipsilateral side, consistent with the role of catecholamines to increase CRH secretion (27). These pathways also carry axons containing CART and glucagon like peptide-1 (15, 28), however, that are known to activate the hypothalamic-pituitary-adrenal axis through central effects on CRH neurons (29, 30). Thus, the effect of the transection to diminish the stimulatory action of these peptides in the PVN may have also contributed to the reduction in CRH mRNA on that side.
In contrast to the effects of transection on CRH mRNA, TRH mRNA in the PVN was significantly increased on the transected side, suggesting that contrary to the regulation of hypophysiotropic CRH neurons, under basal conditions the ascending brainstem projections to hypophysiotropic TRH neurons exert a net inhibitory effect. Although the nature of this inhibitory input is yet unknown, the fact that NPY exerts a potent inhibitory effect on the HPT axis when administered intracerebroventricularly (22) and is co-contained in a subset of the catecholamine fiber projection to TRH neurons in the PVN (31), raise the possibility that NPY may contribute to this inhibitory tone. Alternatively, given the evidence for innervation of TRH neurons by CRH-containing fibers (32), and that CRH is capable of inhibiting TRH release from hypothalamic cultures (33), the reduction in CRH gene expression on the side of the knife cut may have contributed to the increase in TRH gene expression on the transected side of the brain.
After systemic administration of LPS, a dramatic increase in CRH mRNA was observed on the intact side of the PVN, similar to earlier observations (18, 25). The absence of the ascending brainstem projections to the PVN on the side of the knife cut substantially reduced the effects of LPS administration on CRH gene expression in the PVN, consistent with previous studies by Ericsson et al. (18). Together with the significant reduction of the adrenergic fibers on the side of transection, these data substantiate that transection of projection pathways from the medulla to the PVN was adequate to prevent LPS from exerting maximal effects on hypophysiotropic CRH neurons. Therefore, were LPS to mediate its inhibitory effects on hypophysiotropic TRH gene expression in the PVN via ascending brainstem pathways, we would have anticipated an increase in TRH mRNA in the tran-sected side of the PVN after LPS administration. This is based on the observation that endotoxin increases circulating levels of leptin (9), CART mRNA in the arcuate nucleus (11), and melanocortin signaling in the PVN (10), whereas it does not increase NPY expression in the arcuate nucleus (11). Each of these alterations would have been expected to activate TRH gene expression in hypophysiotropic neurons rather than result in suppression as observed. Thus, removing an overriding inhibitory effect of the brainstem projections on hypophysiotropic TRH should have allowed the activating effects of each of the above factors induced by LPS to exert its effects on TRH mRNA unopposed.
Nevertheless, LPS reduced TRH mRNA on the transected side of PVN compared with vehicle-treated controls, indicating that the ascending brainstem projections to hypophysiotropic TRH neurons do not mediate the inhibitory effects of LPS on the HPT axis. Presumably, therefore, other mechanisms must be operable. Indeed, the observation that the inhibitory effect of LPS on TRH mRNA in the PVN was greater on the transected than the intact side would indicate that under these circumstances, the net effect of brainstem projections to TRH neurons is stimulatory. This activating effect by LPS may be mediated by an increase in the release of catecholamines (34) similar to that observed for CRH and/or by CART, which is coproduced in adrenergic neurons (15, 35).
Although the mechanism by which LPS inhibits the HPT axis remains unknown, we have observed that immune activation increases type 2 iodothyronine deiodinase (D2) mRNA and D2 enzymatic activity in the mediobasal hypothalamus of the rat (36), recently confirmed by Boelen et al. (37) in mice. This increase in D2 activity in the mediobasal hypothalamus occurs primarily in specialized ependymal cells called tanycytes, which are located in the base and infralateral walls of the third ventricle. These cells extend cytoplasmic processes through the substance of the median eminence to terminate on the portal capillary plexus, creating a cytoplasmic conduit interconnecting the cerebrospinal fluid and vascular compartments (38, 39). Because these cells are the main source of T3 production in the hypothalamus, we have proposed that endotoxin may result in tissue-specific, D2-mediated thyrotoxicosis in the mediobasal hypothalamus (9). The increase in the local T3 level may then suppress the synthesis of TRH in hypophysiotropic neurons either by local feedback inhibition through the release of T3 from tanycyte apical processes into the cerebrospinal fluid or by uptake from hypophysiotropic TRH axonal processes in the median eminence and retrograde transport to the hypothalamic PVN (9). Although it could be hypothesized that elevated corticosterone levels have a direct inhibitory effect on hypophysiotropic TRH neurons (40), studies by Kondo et al. (12) have shown that adrenalectomized animals replaced with corticosterone to simulate normal circulating levels shows similar responses of the HPT axis to LPS as intact controls.
In summary, transection of ascending pathways from the brainstem to the PVN does not prevent LPS from inhibiting TRH gene expression in hypophysiotropic neurons and may even accentuate the inhibitory response. We conclude, therefore, that LPS must exert other regulatory mechanisms over hypophysiotropic TRH neurons that contribute to inhibition of the HPT axis.
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Footnotes |
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First Published Online December 16, 2004
Abbreviations: AGRP, Agouti gene-related protein; CART, cocaine- and amphetamine-regulated transcript; D2, type 2 iodothyronine deiodinase; HPT, hypothalamic-pituitary-thyroid; LPS, lipopolysaccharide; NPY, neuropeptide Y; PB, phosphate buffer; PNMT, phenylethanolamine N-methyltransferase; POMC, proopiomelanocortin; PVN, paraventricular nucleus.
Received November 1, 2004.
Accepted for publication December 6, 2004.
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-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression. J Neurosci 20:1550–1558This article has been cited by other articles:
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T. Fuzesi, G. Wittmann, Z. Liposits, R. M. Lechan, and C. Fekete Contribution of Noradrenergic and Adrenergic Cell Groups of the Brainstem and Agouti-Related Protein-Synthesizing Neurons of the Arcuate Nucleus to Neuropeptide-Y Innervation of Corticotropin-Releasing Hormone Neurons in Hypothalamic Paraventricular Nucleus of the Rat Endocrinology, November 1, 2007; 148(11): 5442 - 5450. [Abstract] [Full Text] [PDF]
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 J. R. Klein The immune system as a regulator of thyroid hormone activity. Experimental Biology and Medicine, March 1, 2006; 231(3): 229 - 236. [Abstract] [Full Text] [PDF]
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G. Wittmann, Z. Liposits, R. M. Lechan, and C. Fekete Origin of Cocaine- and Amphetamine-Regulated Transcript-Containing Axons Innervating Hypophysiotropic Corticotropin-Releasing Hormone-Synthesizing Neurons in the Rat Endocrinology, July 1, 2005; 146(7): 2985 - 2991. [Abstract] [Full Text] [PDF]
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