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This article has been cited by 19 other HighWire hosted articles, the first 5 are:
Sucrose self-administration and CNS activation in the rat
Dianne P. Figlewicz, Jennifer L. Bennett-Jay, Sepideh Kittleson, Alfred J. Sipols and Aryana
Zavosh
Am J Physiol Regul Integr Comp Physiol, April , 2011; 300 (4): R876-R884.
[Abstract] [Full Text] [PDF]
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Am J Physiol Regulatory Integrative Comp Physiol
281: R1426–R1436, 2001.
Evans, Scott B., Charles W. Wilkinson, Kathy Bent- ACTH, glucocorticoids [e.g., corticosterone (Cort)] are
son, Pam Gronbeck, Aryana Zavosh, and Dianne P. released by the adrenal cortex. PVN neurons also
Figlewicz. PVN activation is suppressed by repeated hy- project to autonomic preganglionic cells in the spinal
poglycemia but not antecedent corticosterone in the rat. cord (29, 40, 53, 55) and can directly activate the
Address for reprint requests and other correspondence: S. B. The costs of publication of this article were defrayed in part by the
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lism(151), 1660 South Columbian Way, Seattle, WA 98108 (E- marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
mail: ngevans@u.washington.edu). solely to indicate this fact.
R1426 http://www.ajpregu.org
FOREBRAIN ACTIVATION, HAAF, AND CORTICOSTERONE R1427
The HAAF effect may involve adrenal glucocorti- and 0.2 ml Batryl antibiotic (Provet, Bayer) and were main-
coids, because the feedback inhibitory effects of glu- tained on a circulating-water heating pad until recovery from
cocorticoids can act at the level of protein synthesis (12, anesthesia. Catheter lines were filled with 25–30% polyvi-
19), which would produce a time course consistent with nylpyrrolidone (PVP10, Sigma)-heparin (1,000 U/ml; Elkins-
Sinn, NJ) and kept patent by a heparin (100 U/ml) flush
the HAAF effect (developing within 24 h and lasting at
every 3 days. All animals were allowed to reach their presur-
least several days). Additionally, an HAAF-like hor- gery weights (⬃7 days) before study. In rats with an intra-
monal profile has been demonstrated in normal human cerebroventricular cannula, an ANG II test was performed as
subjects (18) when hypoglycemia was induced 1 day routinely established in our laboratory (e.g., Ref. 51) to con-
after intravenous administration of cortisol and insulin firm cannula placement.
in a hypoglycemic clamp paradigm. To investigate Experimental procedures. Animals were divided into two
whether increases of central nervous system (CNS) groups, one receiving only intravenous catheters and the
Cort are sufficient to induce HAAF-like effects, we other, an intracerebroventricular cannula in addition to in-
compared the neuroendocrine responses to hypoglyce- travenous catheters. All animals were subjected to a 2-day
mia in rats with prior exposure to either Cort or hypo- procedure based on a model of HAAF in humans (18). All
infusions were carried out using a programmable syringe
glycemia. In addition to measuring the neuroendocrine
pump (SP101i, World Precision Instruments).
responses to hypoglycemia, we mapped mid- and fore- On day 1, the intravenous group received either insulin
brain areas for the expression of the immediate early (two 2-h infusions of 0.25 U 䡠 100 g body wt⫺1 䡠 h⫺1) or saline
gene c-fos, a marker of neuronal activation (31). We vehicle. In a separate study (n ⫽ 4), we determined that this
quantified c-Fos immunostaining, allowing us to com- insulin-infusion paradigm resulted in two discreet bouts of
pare hypoglycemia-induced CNS activation alone, af- hypoglycemia (glucose fell from 109 ⫾ 0.6 to 34 ⫾ 3 mg/dl
embedding media (Fisher) until sectioning at 40–50 m. peated measure and treatment (Veh-Veh, Veh-Hypo, Cort-
Tissue sections were stored at ⫺20°C in cryoprotectant [30% Hypo, or Hypo-Hypo) as the between-groups factor. Signifi-
sucrose-ethylene glycol (Sigma), 10% polyvinylpyrrolidone cance for all tests was taken as P ⱕ 0.05. For the Veh-Hypo,
(Sigma) in PBS] until assay. Cort-Hypo, and Hypo-Hypo groups, data were excluded from
Plasma assays. Blood samples were obtained for the mea- the analyses if plasma glucose did not decrease to ⬍50 mg/dl
surement of neuroendocrine responses and stored at ⫺80°C by 90 min after the start of insulin infusion on day 2. This
until assayed. Blood for the catecholamine assays was col- resulted in the exclusion of three rats from the intracerebro-
lected on EGTA-glutathione (2.3:1.5 mg/ml; Sigma). Tubes ventricular groups and five rats from the intravenous groups.
for glucagon assays contained 10 l of 1 M benzamidine
(Sigma) and 1 U heparin. Blood for glucose and Cort assays RESULTS
was collected on EDTA. A radioenzymatic method as de-
scribed in Evans et al. (23) was used for determination of Counterregulatory response to hypoglycemia after
plasma epinephrine and norepinephrine (NE). A radioimmu- previous bouts of hypoglycemia. Catecholamine and
noassay procedure was used for plasma Cort measurement Cort levels were basal at 0 min, indicative of healthy,
as described in van Dijk et al. (58). Plasma glucose was well-habituated (i.e., unstressed) rats (Figs. 1 and 2).
measured spectrophotometrically using a glucose oxidase With insulin infusion, glucose levels dropped to nearly
reaction. Glucagon was assayed by the Linco glucagon RIA 30 mg/dl by the end of insulin infusion for both Veh-
kit (Linco Research). Post hoc measurements of ACTH were Hypo and Hypo-Hypo groups. At all times, the glucose
made using the Nichols Institute Diagnostics immunoradio- levels were significantly lower than at the start of the
metric assay kit (Nichols Institute Diagnostics, San Juan infusion and plasma glucose levels did not differ be-
Capistrano, CA) on plasma samples that were pooled at each
tween the Veh-Hypo and Hypo-Hypo groups (P ⬎ 0.1
DISCUSSION
Fig. 5. Brain regions with significantly elevated c-Fos expression in
response to hypoglycemia (Veh-Hypo). Error bars indicate ⫹SE. Pir, Neuroendocrine response to hypoglycemia. All ani-
piriform cortex; INS, insular cortex; AMCe, AMCo, and AMMe, central,
cortical, and medial, respectively, nuclei of the amygdala; MPO, medial
mals made hypoglycemic in the current study exhibited
preoptic nucleus; LS, lateral septum; BNST, bed nucleus of the stria robust counterregulatory neuroendocrine responses to
terminalis; SCH, suprachiasmatic nucleus; SO, supraoptic nucleus; hypoglycemia: activation of the hypothalamic-pituitary-
RCH, retrochiasmatic nucleus; AH, anterior hypothalamic nucleus; adrenal (HPA) axis resulting in increased plasma ACTH
PVN, paraventricular nucleus of the hypothalamus; ARC, arcuate nu- and Cort, activation of the sympathetic nervous system
Antecedent hypoglycemia and hypoglycemia-induced Antecedent Cort and hypoglycemia-induced brain ac-
brain activation. Two bouts of hypoglycemia on day 1 tivation. Although the animals did not demonstrate
resulted in a blunted neuroendocrine response to hy- altered counterregulatory responses to hypoglycemia
poglycemia on day 2. Quantification of c-Fos-IR dem- with prior Cort treatment, they did demonstrate dif-
onstrated changes in brain activation as well. The ferences in CNS activation. When hypoglycemia on day
activation of three structures that have been shown to 2 was preceded by intracerebroventricular Cort infu-
be permissive or stimulatory for HPA/sympathetic ac- sion on day 1, the Arc and DMH of the hypothalamus
tivity (see discussion below), the PVN, Arc, and DMH, and the ThPVP of the thalamus exhibited blunted
was blunted by prior bouts of hypoglycemia. Inhibition activation. However, both the autonomic and HPA re-
of potentially permissive/excitatory structures should sponses were normal (see above). This net lack of effect
lead to a blunted counterregulatory response to hypo- may be explained by experiments demonstrating the
glycemia, as was observed in our study. The PVN plays excitatory/permissive or inhibitory influence of these
a pivotal role in the counterregulatory response to specific brain regions on the HPA axis. Pharmacologi-
hypoglycemia, and this is suggested by the diminished cal manipulation of the DMH reveals that the DMH
counterregulatory response after a 52% decrease of can facilitate HPA and sympathetic responses (52).
PVN activation. The mechanism(s) of blunted PVN Experiments indicate that the Arc (4, 33, 34) can either
activation with repeated exposure to the same stressor potentiate or inhibit the HPA response. However, the
might involve a decrease in activating input to the evidence for Arc having a negative modulatory influ-
PVN, an increase in inhibitory input to the PVN, or ence on the HPA axis derives chiefly from neonatally
both (36, 57). These inputs to the PVN are both neural monosodium glutamate-lesioned rats (33, 34). This is a
Fig. 9. Summary of the results from the 3 experimental conditions, proposing a pivotal role for the PVN in
orchestrating the counterregulatory response to hypoglycemia. In condition 1, hypoglycemia results in a strong
neuroendocrine counterregulatory response, with strong PVN activation resulting from a balance of activating
(ARC, DMH, other inputs) and inhibiting (ThPVP, other inputs) influences. In condition 2, measured activating
influences are dampened by prior hypoglycemia, while the inhibiting influence of the ThPVP is not. Changes in
other inputs may also occur. The net result is a decrease in the activation of the PVN and the neuroendocrine
response. In condition 3, both measured activating and inhibiting influences are dampened by prior Cort infusion,
changes in other inputs may also occur. The net result is no change in PVN activation or the neuroendocrine
response. The unknowns in this model include the influences of circulating factors as well as neural inputs from
regions not examined in the current study (e.g., brain stem, prefrontal cortex).
ular Cort, although potential HPA/sympathetic excita- assistance with animal care and procedures and M. Higgins for
tory regions were inhibited (e.g., Arc, DMH), which technical assistance with animal surgery. The technical assistance of
E. Colasurdo and C. Sikkema with Cort and ACTH assays is grate-
presumably would lead to a blunted HPA/sympathetic fully acknowledged. The authors thank Dr. G. J. Taborsky for helpful
response, a potential inhibitory region, the ThPVP, discussions regarding the manuscript.
was also inhibited, which would disinhibit or increase Support for these studies was provided by grants from the Amer-
HPA/sympathetic responses. The net result of such a ican Diabetes Association, the Juvenile Diabetes Foundation, and
the Veterans Affairs Merit Review Program. Dr. S. B. Evans is
combination of alterations in regional activation is no supported by a Dick and Julia McAbee Endowed Fellowship in
significant change in HPA/sympathetic reactivity. Of Diabetes Research.
course this is a simplified portrait of very complex
neuroanatomical circuitry: the inputs to the PVN prob- REFERENCES
ably do not sum in a simple algebraic fashion, and the
timing of activation/inhibition of inputs to the PVN 1. American Diabetes Association. Implications of the Diabetes
Control and Complications Trial. Diabetes Care 23: S24–S26,
may be critical as well. 2000.
Figure 9 summarizes the results of the three exper- 2. Babu GN, Bawari M, and Ali MM. Lipid peroxidation poten-
imental conditions: hypoglycemia (Veh-Hypo), hypo- tial and antioxidant status of circumventricular organs of rat
glycemia after preexposure to high Cort (Cort-Hypo), brain following neonatal monosodium glutamate. Neurotoxicol-
and hypoglycemia after preexposure to hypoglycemia ogy 15: 773–777, 1994.
3. Bahjaoui-Bouhaddi M, Fellmann D, and Bugnon C. Induc-
(Hypo-Hypo). Consistent with the critical role of the tion of Fos-immunoreactivity in prolactin-like containing neu-
PVN in the neuroendocrine response to hypoglycemia rons of the rat lateral hypothalamus after insulin treatment.
is the fact that even though the DMH and Arc hypo-
18. Davis SN, Shavers C, Costa F, and Mosqueda-Garcia R. 37. Mistlberger RE and Antle MC. Neonatal monosodium gluta-
Role of cortisol in the pathogenesis of deficient counterregulation mate alters circadian organization of feeding, food anticipatory
after antecedent hypoglycemia in normal humans. J Clin Invest activity and photic masking in the rat. Brain Res 842: 73–83,
98: 680–691, 1996. 1999.
19. Dayanithi G and Antoni FA. Rapid as well as delayed inhib- 38. Miyata S, Ishiyama M, Shibata M, Nakashima T, and Kiyo-
itory effects of glucocorticoid hormones on pituitary adrenocor- hara T. Infant cold exposure changes Fos expression to acute
ticotropic hormone release are mediated by type II glucocorticoid cold stimulation in adult hypothalamic brain regions. Neurosci
receptors and require newly synthesized messenger ribonucleic Res 31: 219–225, 1998.
acid as well as protein. Endocrinology 125: 308–313, 1989. 39. Moriguchi T, Sakurai T, Nambu T, Yanagisawa M, and
20. Del Bel EA, Silveira MC, Graeff FG, Garcia-Cairasco N, Goto K. Neurons containing orexin in the lateral hypothalamic
and Guimaraes FS. Differential expression of c-fos mRNA and area of the adult rat brain are activated by insulin-induced acute
Fos protein in the rat brain after restraint stress or pentylenetet- hypoglycemia. Neurosci Lett 264: 101–104, 1999.
razol-induced seizures. Cell Mol Neurobiol 18: 339–346, 1998. 40. Motawei K, Pyner S, Ranson RN, Kamel M, and Coote JH.
21. Elias CF, Kelly JF, Lee CE, Ahima RS, Drucker DJ, Saper Terminals of paraventricular spinal neurones are closely associ-
CB, and Elmquist JK. Chemical characterization of leptin- ated with adrenal medullary sympathetic preganglionic neu-
activated neurons in the rat brain. J Comp Neurol 423: 261–281, rones: immunocytochemical evidence for vasopressin as a possi-
2000. ble neurotransmitter in this pathway. Exp Brain Res 126: 68–76,
22. Emmert MH and Herman JP. Differential forebrain c-fos
1999.
mRNA induction by ether inhalation and novelty: evidence for
41. Niimi M, Sato M, Tamaki M, Yoshinaru W, Takahara J,
distinctive stress pathways. Brain Res 845: 60–67, 1999.
and Kawanishi K. Induction of Fos protein in the rat hypothal-
23. Evans MI, Halter JB, and Porte D. Comparison of double-
amus elicited by insulin-induced hypoglycemia. Neurosci Res 23:
and single-isotope enzymatic derivative methods for measuring
catecholamines in human plasma. Clin Chem 24: 567–570, 1978. 361–364, 1995.
24. Feldman S, Conforti N, Itzik A, and Weidenfeld J. Differ- 42. Olivo M, Kitahama K, Valatx JL, and Jouvet M. Neonatal
56. Umemoto S, Kawai Y, Ueyama T, and Senba E. Chronic 60. Xu Y, Day TA, and Buller KM. The central amygdala modu-
glucocorticoid administration as well as repeated stress affects lates hypothalamic-pituitary-adrenal axis responses to systemic
the subsequent acute immobilization stress-induced expression interleukin-1beta administration. Neuroscience 94: 175–183,
of immediate early genes but not that of NGFI-A. Neuroscience 1999.
80: 763–773, 1997. 61. Yang XJ, Kow LM, Funabashi T, and Mobbs CV. Hypo-
57. Umemoto S, Noguchi K, Kawai Y, and Senba E. Repeated thalamic glucose sensor: similarities to and differences from
stress reduces the subsequent stress-induced expression of Fos pancreatic beta-cell mechanisms. Diabetes 48: 1763–1772,
in rat brain. Neurosci Lett 167: 101–104, 1994. 1999.
58. Van Dijk G, Donahey JC, Thiele TE, Scheurink AJ, Stef- 62. Yates FE, Russell SM, Dallman MF, Hodge GA, McCann
fens AB, Wilkinson CW, Tenenbaum R, Campfield LA,
SM, and Dhariwal AP. Potentiation by vasopressin of cortico-
Burn P, Seeley RJ, and Woods SC. Central leptin stimulates
tropin release induced by corticotropin-releasing factor. Endo-
corticosterone secretion at the onset of the dark phase. Diabetes
46: 1911–1914, 1997. crinology 88: 3–15, 1971.
59. Viau V and Meaney MJ. The inhibitory effect of testosterone 63. Yokoyama C and Sasaki K. Regional expressions of Fos-like
on hypothalamic-pituitary-adrenal responses to stress is medi- immunoreactivity in rat cerebral cortex after stress: restraint
ated by the medial preoptic area. J Neurosci 16: 1866–1876, and intraperitoneal lipopolysaccharide. Brain Res 816: 267–275,
1996. 1999.