help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-0539
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khan, A. M.
Right arrow Articles by Watts, A. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khan, A. M.
Right arrow Articles by Watts, A. G.
Endocrinology Vol. 145, No. 1 351-359
Copyright © 2004 by The Endocrine Society

Intravenous 2-Deoxy-D-Glucose Injection Rapidly Elevates Levels of the Phosphorylated Forms of p44/42 Mitogen-Activated Protein Kinases (Extracellularly Regulated Kinases 1/2) in Rat Hypothalamic Parvicellular Paraventricular Neurons

Arshad M. Khan and Alan G. Watts

Program in Neural, Informational and Behavioral Sciences, University of Southern California, Los Angeles, California 90089

Address all correspondence and requests for reprints to: Dr. Arshad M. Khan, Department of Biological Sciences, Hedco Neuroscience Building, MC 2520, 3641 Watt Way, University of Southern California, Los Angeles, California 90089-2520. E-mail: arshadk{at}usc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRH neurons within the medial parvicellular part of the hypothalamic paraventricular nucleus (PVHmp) can respond to afferent inputs encoding stress-related information by initiating peptide synthesis (signaling cascades, transcription, and translation) and/or peptide release. However, understanding these cellular events is hampered by three outstanding issues: 1) neural inputs that activate CRH neurons remain incompletely identified; 2) the identity and temporal dynamics of signaling pathways within CRH neurons are poorly understood; and 3) the precise coupling of the first two issues has not been established. Here, we report that the phosphorylated forms of p44/p42 MAPKs (pERK1/2) are rapidly detected in PVHmp cells after iv infusion of the antimetabolite, 2-deoxy-D-glucose (2-DG). Combined immunocytochemistry and in situ hybridization revealed that pERK1/2 immunoreactivity is detectable 10 min after 2-DG infusion not only within most PVHmp neurons containing CRH mRNA (78.6% of mean total CRH cells counted) but also in many non-CRH neurons (45.5% of mean total sampled cells). In contrast, Fos protein in the PVHmp was not detected within this time period, consistent with the known time course for its translation. Stress associated with halothane exposure also robustly elevated pERK1/2 levels in PVHmp neurons approximately 10 min after exposure. Our results implicate pERK1/2 in stress-induced activation of CRH neurosecretory cells and underscore their utility as indices of rapid cellular activation. Because 2-DG-induced activation of CRH gene transcription in these neurons requires a catecholaminergic input, our data also suggest that pERK1/2 could couple afferent catecholaminergic signals with CRH gene expression in these neurons.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRH NEUROENDOCRINE NEURONS in the medial parvicellular part of the hypothalamic paraventricular nucleus (PVHmp) are critical for the generation of adaptive responses to stress. These neurons constitute the principal integrative hub of the hypothalamic-pituitary-adrenal (HPA) axis and respond to afferent signals encoding stress-related information in two distinct ways: first, by releasing CRH and/or arginine vasopressin into the hypophysial vasculature to stimulate ACTH and, ultimately, glucocorticoid secretion; and second, by synthesizing new gene products (including CRH) through recruitment of intracellular signaling machinery. A critical question for this system is how CRH neuroendocrine neurons integrate multiple afferent signals so that these two cellular processes are appropriately coordinated.

Because peptide synthesis and release are difficult to monitor in real time in the conscious animal, many ways have been sought for identifying when neuroendocrine neurons undergo changes in their physiological state during stress. However, each of these ways is uniquely attended by particular limitations. Thus, circulating ACTH and CORT are well established indices of peptide release but are of limited use as indices of gene expression, whereas CRH neuroendocrine neuronal firing rates reveal more about electrical than metabolic or transcriptional processes. The expression of inducible factors such as Fos in these cells does not invariably correlate with cell firing rate or CRH expression (1, 2, 3, 4, 5), and detection of heterogeneous nuclear (hn)RNA or mRNA tracks the end products of gene expression, but not the signaling intermediates. Ideally, a marker for tracking the initiation of gene expression in CRH neurons should be present within the neurons themselves, be induced very rapidly, and be an upstream participant in signaling pathways leading to such expression. Moreover, it can be reasonably presumed that such a marker should be well suited to the task of funneling multiple incoming signals to appropriate intracellular pathways; i.e. it should itself be an integrator of cellular information.

A critical constraint when studying stimulus-transcription coupling is selecting an appropriate stimulus to activate CRH neuroendocrine neurons, because a particular stressor will likely recruit multiple sets of PVH afferents; each set will, in turn, use different neurotransmitter combinations to alter CRH neuronal function. Using complex stressors makes it difficult to determine whether and how particular neurotransmitter/receptor systems are coupled with intracellular signaling pathways to control gene expression. Ideally, the most useful stressor would be one for which all specific neural afferents are known. We have therefore sought to refine experimental approaches for studying cellular mechanisms underlying gene expression in CRH neurons by seeking experimentally expedient stimuli and markers for stress-induced activation.

Intravenous 2-deoxy-D-glucose (2-DG) was used as a stimulus in the present study because it rapidly triggers both CRH transcription in neuroendocrine neurons and CORT release, and it can be delivered with little nonspecific stress (6, 7, 8). Importantly, the induction of CRH gene expression by 2-DG requires an intact ascending catecholaminergic pathway originating in the hindbrain (8, 9).

Using this stressor we asked whether the phosphorylation of p44/42 MAPKs (also known as ERKs 1 and 2, respectively) could be used to detect rapid cellular activation in CRH neuroendocrine neurons in response to iv 2-DG. Because ERKs 1/2 have been shown in other systems to participate in the phospho-relay cascades that result in the activation of cAMP response element-binding protein (CREB) (10, 11, 12 , but see Ref. 13), it is conceivable that they also participate in the activation of the crh gene. To date, CREB is the transcription factor most directly implicated in crh gene transcription (2, 14, 15).

Thus, we reasoned that using 2-DG as a stimulus and phosphorylated MAPK as a marker may together prove useful for delineating the precise functional links among a particular afferent input, its specific postsynaptic receptor targets, and the receptor-driven signaling machinery inside the target cell that ultimately lead to gene expression. Such links could generalize to other PVH afferent systems as well, perhaps leading to an appreciation of the general principles by which afferent control shapes hypothalamic responses to a variety of stressors. To begin exploring this issue, we also examined the effects of halothane anesthesia exposure upon levels of phosphorylated MAPK in PVHmp neurons.

Some parts of this work have been presented in preliminary form (16).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
For all experiments, adult male Sprague Dawley rats were maintained on a 12-h/12-h light/dark photoperiod (lights on at 0600 h) and ad libitum food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Southern California.

Experiments using iv 2-DG injection
Jugular catheterization.
Rats under halothane anesthesia (n = 14; 249–327 g body weight at time of surgery) were surgically implanted with sterile jugular catheters fitted with Silastic tips that terminated near the atrium (7). The free end of each catheter was threaded under the skin and left to protrude through the skin between the shoulders. Animals were allowed to recover their presurgical body weights; during this time their catheters were flushed daily with 0.9% sterile, heparinized saline.

Induction of glucoprivic stress and preparation of perfusion-fixed tissue.
On the day of testing, rats were given iv infusions of 2-DG (250 mg/kg, dissolved in 0.9% sterile saline), followed approximately 10–11 min later by iv anesthesia (2,2,2-tribromoethanol; mean volume 2.4 ml; bolus delivery). Control animals received only the anesthesia. In earlier experiments, approximately 20 min was allowed between 2-DG and anesthesia delivery, and a control group was included that received the saline vehicle instead of 2-DG. After infusions, rats were perfused through the ascending aorta with 100–150 ml of 0.01 M sodium PBS (pH 7.4), followed by 300–500 ml of ice-cold 4% (wt/vol) p-formaldehyde in 0.1 M sodium phosphate buffer (PB). Brains were then fixed in the p-formaldehyde fixative for 24–48 h at 4 C and then immersed in 0.1–0.2 M PB containing 20% glycerol for 24–48 h at 4 C (17). Brains were then frozen using hexane cooled with powdered dry ice and stored at -70 C until sectioning.

Eight series of one-in-eight, 20-µm-thick frozen coronal sections were cut through the PVH, from brain blocks mounted on the freezing stage of a sliding microtome. Sections kept for immunocytochemistry (ICC) were collected in tissue culture wells filled with cryoprotectant (50% 0.05 M PB, 30% ethylene glycol, 20% glycerol, pH 7.4) and stored at -20 C until further processing. Series kept for in situ hybridization (ISH) only were collected in ice-cold potassium PBS (K-PBS) containing 0.25% (wt/vol) p-formaldehyde. They were then mounted the same day on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), vacuum desiccated overnight, postfixed in K-PBS-4% p-formaldehyde for 1 h at room temperature (RT), rinsed five times for 5 min each in clean K-PBS, air dried, and then stored at -70 C in airtight containers containing silica gel dessicant for hybridization at a later date. Serial sections were saved for thionin staining.

Experiments using halothane anesthesia exposure
Stimulation parameters.
Rats (n = 2) were gently transferred from their home cages to a glass chamber containing paper towels moistened with halothane (Halocarbon Laboratories, River Edge, NJ) for approximately 15–30 sec. They were then removed from the chamber and placed back in again 15–30 sec later. This was repeated two or three times in succession, after which they were returned to their home cages. Ten minutes later, the rats were decapitated and their brains rapidly removed for fixation by immersion (see below). Control animals (n = 2) were simply removed from their home cages and decapitated immediately.

Immersion fixation procedure and tissue preparation.
The low-temperature immersion fixation procedure used here, modified from Berod and colleagues (18), also incorporates a cryoprotectant recipe used by Swank and colleagues (17). Brains removed after decapitation were immediately placed in sodium acetate-buffered p-formaldehyde (pH 6.5) chilled at 1–4 C to form a semifrozen slush. The rate of infiltration of p-formaldehyde into fresh brain tissue, but not its ability to cross-link proteins, is reportedly optimal at this pH (18). Brains remained in the slush for approximately 8 h, during which time the temperature was maintained between 1 and 4 C by keeping the brains/fixative on ice in a refrigerator. After this time, each brain was removed from the pH 6.5 fixative, gently blotted dry, and placed in sodium borate-buffered p-formaldehyde (pH 9.5) at 4 C for 6 d. At this pH, the ability of p-formaldehyde to cross-link proteins in brain tissue, but not rapidly infiltrate such tissue, is reportedly optimal (18). Brains were then transferred to 0.1 M sodium phosphate buffer (pH 7.4) and 20% glycerol (17) and kept on a rotating platform at 4 C for 2 d. They were then frozen in supercooled hexane chilled over a bed of dry ice and kept frozen at -70 C until ready for sectioning. The 20-µm-thick coronal sections were cut through the extent of the PVH using the freezing stage of a sliding microtome and then processed for immunocytochemical detection of pERK1/2 as outlined below.

ICC (free-floating method, based on Ref. 17).
For ICC, sections were brought to RT, passed through five 5-min rinses of Tris-buffered saline (TBS; 0.1 M Tris buffer containing 0.9% saline, pH 7.4) at RT. They were then transferred to a blocking solution for 1 h at RT in TBS containing 3% (wt/vol) BSA and 0.2% Triton X-100 (Triton). Sections were then reacted for 72 h at 4 C with an affinity-purified rabbit polyclonal antibody raised against the phosphorylated forms of ERK1/2 (Cell Signaling Technology, Beverly, MA; http://www.cellsignal.com/). This antibody reportedly detects rat ERKs 1 and 2 only when they are phosphorylated at Thr183 and Tyr185 and does not cross-react with phosphorylated JNK/SAPK or p38 MAPKs (see supplier’s web site for antibody validation data). This antibody was diluted in TBS containing 3% BSA and 0.2% Triton to final concentrations of 1:2000 (dual-label experiments) or 1:8000 (single-label experiments). For animals from 2-DG experiments, subsets of tissue sections were processed using a rabbit antiserum targeting Fos protein (1:40,000 and 1:60,000; Calbiochem/Oncogene, San Diego, CA; http://www.calbiochem.com/). This antiserum was raised against a synthetic immunogen corresponding to amino acid residues 4–17 of human Fos protein and recognizes Fos in rats. Sections were then rinsed five times for 5 min each in fresh TBS washes. To amplify the reaction, sections were next reacted for 2 h at RT with a biotinylated goat antirabbit IgG (heavy + light chains), diluted one part stock to two parts TBS containing 3% BSA and 0.2% Triton [Kirkegaard & Perry Labs (KPL), Gaithersburg, MD; http://www.kpl.com/home.cfm], followed by five 5-min fresh washes in TBS. For 1–1.5 h at RT, the immunocomplexes were then conjugated to streptavidin-horseradish peroxidase, diluted one part stock to one part TBS containing 3% BSA and 0.2% Triton (KPL), and rinsed again with the usual TBS washes. Development of the final reaction was performed by reacting sections for 2–4 min in a solution prepared from a commercial diaminobenzidine kit (HistoMark Enhanced Black solution, KPL), according to the manufacturer’s instructions. Reactions were visualized under bright-field optics to determine optimal levels of staining and were stopped by rinsing sections in cold TBS.

ISH (mounted sections).
Sections were hybridized as previously described (19, 20) with a [35S]UTP-labeled cRNA probe transcribed from a 700-bp cDNA sequence encoding RNA for part of exon 1 and all of exon 2 of the prepro-CRH gene. The probe was synthesized using the Promega Gemini kit (Promega Inc., Madison, WI) and the appropriate RNA polymerase, as described in detail (20). Sections were exposed to Biomax x-ray film for appropriate exposure periods (2–3 d), then dipped in nuclear track emulsion (Kodak NTB-2, diluted 1:1 with distilled water), exposed for 5 d, developed and (for single-label studies) counterstained with thionin.

Dual-label procedures (combined ICC and ISH).
For dual-label experiments, precautions were taken to RNase-protect the tissue during the ICC procedures before ISH, using a methodology modified from Watts and Swanson (21). To this end, the following reagents were added to the blocking solution: 0.5% dithiothreitol, heparin (5 mg/ml), and RNAsin (50 U/ml buffer). Also, fraction V BSA (Sigma) was used during ICC steps for dual-label runs. Because washout of immunostaining was observed after ISH, the final concentration of primary antibody was increased from 1:8000 to 1:2000, and nickel ammonium sulfate was added to the development reaction to help intensify and retain the stain within immunolabeled cells.

Quantitation of pERK1/2 immunoreactivity and [35S]UTP-cRNA hybridization signals.
To quantitate the number of PVHmp neurons singly labeled with either pERK1/2 immunoreactivity or CRH mRNA hybridization signal, or dually labeled with both reaction products, a single section from each 2-DG-injected subject was selected for analysis. This section corresponded in plane and rostrocaudal position most closely to that represented in plate 26 of the Swanson atlas (22) and was generally the one with the highest CRH mRNA signal. This level also reportedly contains the greatest representation of CRH neurons in the PVH (23). Hemifields for each selected tissue section were then visualized under bright-field optics and each digitally photographed as a single image at x16 magnification using a Spot RT Color Camera and associated software (version 3.5.5 for MacOS; Diagnostic Instruments, Inc., Sterling Heights, MI). The images were imported into Adobe Photoshop and contrast-enhanced to highlight neuronal elements within the PVHmp. Digital images of hemifields from six subjects were captured in this fashion, and their prints were overlaid with acetate. Under high-power visualization, the neurons in each hemifield were then examined for labeling, and the corresponding neurons depicted in that hemifield’s printed photomicrograph were then coded accordingly on acetate. The marked neurons in each single-image photomicrograph were then counted and the number of pERK1/2+, CRH mRNA+, and dually positive neurons recorded. Counts from all left hemifields were pooled and compared with pooled counts of the right hemifields, and significance of differences between both hemifield sets was determined using one-way ANOVA (Microsoft Excel), with P < 0.05 being regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PVH levels of phosphorylated ERK1/2 are rapidly increased after 2-DG administration
Figure 1Go shows that iv administration of 2-DG was associated with a rapid increase in detectable immunoreactivity for phosphorylated ERK1/2 in the PVHmp compared with control animals receiving only anesthesia (panels C–F). This increase was detectable within 10 min and remained detectable 20 min after injection. The range of immunostaining displayed by saline controls was similar to that of control animals receiving only anesthesia and was reduced relative to the 2-DG-infused animals (data not shown). The immunoreaction product within the PVHmp was found in multiple cellular compartments, including the cytoplasm, nucleus, and dendrites. This is more clearly evident in magnocellular PVH neurons at the level of the forniceal portion of the PVH (Fig. 1GGo), which also immunostained for pERK1/2, although immunostaining differences between control and treated groups were not consistent. Staining for pERK1/2 was absent in tissue sections of 2-DG-injected animals processed without primary antibody (Fig. 1HGo). In contrast to the robust increase in PVHmp pERK levels observed after 2-DG injection, virtually no Fos protein expression was observed in this region after 10 min (Fig. 1BGo) using two different dilutions of Fos antibody, consistent with the relatively delayed time course of its translation in neuroendocrine neurons (24).



View larger version (122K):
[in this window]
[in a new window]
  		FIG. 1. Bright field photomicrographs of the PVH. A, Nissl-stained section of the PVH, with parcellation scheme of cell groups presented according to Swanson (22 ). Relative to controls (C and E), 2-DG rapidly increases pERK1/2 in medial parvicellular cells (D and F). As shown in B, Fos protein (1:60,000 dilution) is not detectable in PVHmp cells 10 min after 2-DG administration. G, Magnocellular PVH neurons with pERK1/2 immunoreactivity, selected here simply to illustrate how the reaction product filled cell bodies as well as neurites. H, Representative control section processed without primary antibody. Scale bar in D (100 µm) applies to A–D; scale bar in F (50 µm) applies to E and F; scale bars in G and H, 50 and 100 µm, respectively.

 
pERK immunoreactivity colocalizes with most neurons containing CRH mRNA and with many neurons devoid of CRH message
To assess whether the robust increase of pERK 1/2 levels in the PVHmp occurs in CRH neuroendocrine neurons, a combined ICC/ISH protocol was employed. CRH mRNA was reliably detected in the PVHmp using isotopic ISH (Fig. 2BGo). A higher concentration of antibody and an increased metal intensification of the diaminobenzidine reaction product were employed during ICC to ensure the stable retention of the pERK1/2 antigen-antibody complex after the high-temperature and high-stringency ISH washes (see Materials and Methods). These steps successfully preserved the pERK1/2 reaction product within ISH-treated PVHmp (Fig. 2AGo), which again was robustly increased in 2-DG-injected rats relative to their controls.



View larger version (84K):
[in this window]
[in a new window]
  		FIG. 2. A, Bright-field image of the PVH, revealing robust pERK1/2 in the medial parvicellular division after 2-DG injection. B, Dark-field image of the identical section shown in A, illustrating CRH mRNA detected in this same region using a 35S-labeled riboprobe. C, Examples of labeling for singly labeled pERK1/2-positive and dually labeled pERK1/2/CRH-positive neurons. Note that the arrowheads are color-coordinated with the cell counts depicted in the graph in D. D, Analysis of the extent to which pERK1/2 immunoreactivity colocalizes with CRH mRNA+ neurons, summarized with respect to mean total CRH-positive cells, mean total cells counted, and spatial distribution within a representative PVH hemifield. Scale bars in A and B,100 µm; scale bar in C, 50 µm.

 
High-power examination of tissue samples revealed that the majority of neurons containing CRH mRNA within the PVHmp also contained immunoreactivity for pERK1/2 (Fig. 2Go, C and D). Specifically, 78.6% of the mean total number of CRH-labeled PVHmp cells were also immunopositive for pERK1/2 (Fig. 2DGo and Table 1Go). Qualitatively, these dual-labeled cells were not spatially confined to any particular subregion of the PVHmp but were instead interspersed among singly pERK-labeled and singly CRH-labeled cells (Fig. 2DGo). These cells contained a dark immunoreaction product over which dense clusters of silver grain deposits were readily observed (Fig. 2CGo). In addition to being localized within the majority of CRH neurons sampled, robust pERK 1/2 immunoreactivity was also present in a significant number of cells (45.5% of the mean total) devoid of CRH mRNA (Fig. 2DGo and Table 1Go).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Number of labeled PVH cells in sampled plane

 
Plane of section considerations and caveats concerning sampling
A quantitative analysis of single- vs. dual-labeled cells in this study was performed for a small sample of PVHmp cells (a single section per animal), as outlined in Materials and Methods. Ostensibly, slight variations in the plane of section may influence counts of cells by introducing dramatically asymmetric values between two hemifields containing the PVHmp. With this limitation in mind, we compared the counts obtained for each hemifield from the single section sampled from each rat included in this analysis. There were no significant differences in the mean numbers of single pERK1/2-positive, single CRH-positive, dually labeled, or total number of neurons counted from the left vs. right hemifields (P = 0.51, 0.74, 0.62, and 0.36 respectively, by single-factor ANOVA), suggesting that errors introduced by differences in plane of section were negligible.

PVH levels of phosphorylated ERK1/2 are rapidly increased after halothane anesthesia exposure
Figure 3BGo shows that, similar to rats treated with 2-DG infusions, rats exposed to halothane anesthesia displayed robustly elevated levels of pERK1/2 in PVHmp neurons. Although dual localization experiments were not performed for this tissue, it appears that the neuronal population displaying this elevated pERK1/2 immunoreactivity should include most PVHmp neurons containing CRH. In contrast to halothane-exposed rats, control animals displayed little to no pERK1/2 in PVHmp neurons (Fig. 3AGo). Our halothane exposure experiments also involved the use of a rapid immersion fixation protocol that differed from the perfusion fixation protocol used to fix tissue from animals from the 2-DG experiments. The use of this protocol proved successful, with little qualitative difference in pERK1/2 immunostaining in the PVHmp observed between the two methods (unpublished observations, but also compare Fig. 1DGo and Fig. 3BGo).



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 3. Bright-field photomicrographs of the PVH. Relative to the control condition (A), the PVHmp displayed elevated pERK1/2 immunoreactivity in rats, 10 min after they were exposed to halothane anesthesia (B). Scale bar in A, 100 µm (applies to both panels).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our principal finding is that levels of phosphorylated ERKs 1 and 2 (p44/42 MAPKs) are robustly elevated in CRH neuroendocrine neurons of the PVHmp within 10 min of iv 2-DG injection. Although elevated levels of pERK1/2 have been reported in the PVH in response to other stimuli (25, 26, 27, 28), our results constitute the first description of MAPKs in chemically identified PVH parvicellular neuroendocrine neurons following a well defined stimulus in intact animals.

MAPK cascades are organized into modules of at least three sequentially activating protein serine-threonine kinases (29). Within its own three-tiered phospho-relay module, ERKs (phosphotransferase members within EC 2.7.1.37) occupy the most downstream position, being targets of MEK enzymes, which in turn are activated by Raf-1 or B-Raf (30). Sequential activation of Raf, MEK, and ERK enzymes occurs by phosphorylation of each kinase by the preceding one within the module, with Raf activation occurring by way of heptahelical (at times, G protein-coupled) receptor activation (31 , but see Ref. 32). At least one ERK isoenzyme, p42 MAPK (ERK 2) has been localized to the PVH, as well as one of its upstream activators, MEK1 (33), suggesting the presence of these multitiered MAPK modules within hypothalamic paraventricular neurons.

Utility of pERK1/2 as an index of cellular activation
Our results show that iv infusion of 2-DG is associated with a rapid and significant increase of pERK1/2 levels within both CRH and non-CRH parvicellular PVH neurons. Rapid elevations of pERK1/2 were also evident in rats subjected to halothane exposure. The increased levels of pERK1/2 may reflect the recruitment of signaling machinery that mobilizes the coordinated cellular response to these acute but very different challenges. These increases were detected using a highly selective phosphospecific antibody that does not recognize unphosphorylated enzymes already present in the cell and so tracks ERK phosphorylation but not translation. Phosphorylation occurs within seconds to minutes of exposure to a stimulus (e.g. Ref. 34), whereas translation can occur from 30 min to several hours after stimulation; such differences in temporal dynamics have even allowed for the simultaneous tracking of these processes within the same cell (35). Phosphorylated ERKs have the advantage over more traditional markers of activity such as Fos, because they can be detected closer to the time of stimulus presentation (6). Thus, cellular changes that fall within the temporal domain of secretory events within neuroendocrine neurons can be tracked more closely.

It will be of interest to examine whether increases in phosphorylated ERK coincide with the phosphorylation-induced activation of transcription factors such as CREB in CRH neuroendocrine neurons, levels of which have been shown to elevate rapidly in CRH neuroendocrine neurons after ether anesthesia (2). This is significant not only because CREB is implicated in activating the crh gene (14, 15) but also because pERK1/2 activity can be functionally linked to CREB phosphorylation (10, 11, 12) and, in at least one case, has been shown to inhibit CREB phosphorylation (13). It is therefore plausible that pERK1/2 activity could help control the ability of CREB to initiate gene expression in CRH neuroendocrine neurons and that their coincident activation within these neurons may be a useful means to track the mobilization of specific signaling pathways.

Utility of 2-DG as a stressor used to study ERK activation in CRH neuroendocrine neurons
From a neural systems standpoint, a major challenge to studying intracellular mechanisms in vivo is the method employed to activate target neurons. Using a stimulus that activates multiple sets of PVH afferents, each of which likely uses different neurotransmitter combinations to engage CRH neuroendocrine neurons, can lead to confusion with regard to how such afferents are chemically coupled to signaling pathways in these cells to drive gene expression. For example, ether anesthesia temporally correlates with increases in both CREB phosphorylation and CRH transcription in CRH neuroendocrine neurons (2). However, because the PVH afferents encoding ether anesthesia are complex and poorly characterized, it is difficult to identify neurotransmitters and their associated receptor subtypes involved in transducing these stress signals into meaningful transcriptional processes within the cell. If the goal is to explore how extracellularly derived signals are integrated by CRH neurons to drive gene expression, then the routes by which these signals reach CRH neurons must be well characterized and the stimulus to selectively activate these routes selected accordingly.

Intravenous 2-DG administration may circumvent such problems. In addition to its well documented effects as a powerful stimulator of feeding and hyperglycemia (8, 9), 2-DG also robustly stimulates both CORT secretion and CRH transcription in PVHmp cells (8). Specifically, Ritter et al. (8, 9) have recently used an immunotoxin to produce a selective lesion of noradrenergic and adrenergic PVH afferents that originate in the hindbrain. Using this technique, we recently showed that 2-DG-induced increases in feeding, CORT, CRH hnRNA, and c-fos mRNA are profoundly impaired in animals lesioned in this manner. However, basal CRH mRNA expression, circadian CORT release, hyperglycemia, and the response to forced swimming remain intact (8, 9). Thus, ascending catecholaminergic pathways are required for 2-DG-induced activation of CRH neuroendocrine neurons but are dispensable for both the daily rhythms of CORT secretion in unstressed animals and that following forced swimming.

These results (8, 9) provide evidence that a well defined stimulus, 2-DG, requires a single chemically defined neural input to drive the CRH neuroendocrine system. From a signaling standpoint, these results suggest that catecholaminergic neurotransmission may be necessary for driving crh gene expression in response to 2-DG, although the role of neuropeptide Y (NPY), which is colocalized extensively in these terminals (36), cannot be excluded here. The simplest circuit that can explain these findings is one where hindbrain-originating catecholaminergic afferents directly innervate CRH neurons. Alternatively, these catecholaminergic neurons may exert their effects indirectly by way of second-order glutamatergic or GABAergic neurons that in turn innervate the PVHmp (37), or they may work synergistically with NPY to help drive this system. In either case, it is likely that 2-DG-induced catecholaminergic neurotransmission directly or indirectly engages specific postsynaptic receptor subtypes on CRH neurons to drive crh gene expression. We hypothesize that pERK1/2 is an important signaling intermediate linking activation of such receptors to gene expression in CRH neuroendocrine neurons after 2-DG administration. Although we consider this hypothesis below within the context of cellular integration of stress-related signals, we must emphasize that the causal relationships among 2-DG injection, selective activation of PVH afferents, and recruitment of postsynaptic receptors and signaling machinery remain to be rigorously established. However, in the present study, the selective labeling of PVH neurons relative to regions immediately surrounding this nucleus argues against nonspecific humoral influences of 2-DG accounting for pERK immunostaining in these neurons and suggests recruitment of particular neural afferents in response to 2-DG.

MAPKs as potential integrators of afferent signals influencing gene expression in neuroendocrine neurons
CRH neuroendocrine neurons bear many different classes of neurotransmitter receptor, each of which may help convert afferent stress-related signals into meaningful intracellular signals driving gene expression. Many of these receptors have been shown in a variety of other systems to be functionally linked to MAPK pathways within cells. Within the context of the ascending catecholaminergic afferent system, the most relevant receptor subtypes to consider would be the {alpha}1B and {alpha}1D adrenergic receptor subtypes, which have been localized to subsets of PVHmp cells containing CRH mRNA (38, 39). In other cell systems, these receptor subtypes can be functionally coupled to ERK activation (40, 41, 42), albeit with differing agonist dependencies. This suggests the possibility that pERK1/2 may also be functionally linked to these receptor subtypes in CRH neurons to drive gene expression. This possibility is supported by our recent data showing that central administration of norepinephrine rapidly elevates CRH hnRNA, c-fos mRNA, and pERK1/2 in PVHmp neurons (43). Additionally, ERKs can potentially serve as funnels for multiple afferent signals that are transduced by a variety of other receptor systems in CRH neuroendocrine neurons. Thus, ERK1/2 may also be mobilized through activation of receptors for glutamate (44, 45), angiotensin (46), NPY (47), CRH (48), and {alpha}-melanocyte-stimulating hormone/agouti-related protein (28), subtypes for which are known to be present within CRH neurons or, more generally, within the PVHmp (49, 50, 51, 52, 53, 54).

How could ERKs influence gene expression in CRH neuroendocrine neurons? As documented in a variety of systems (55, 56), phosphorylated ERKs (pERK1/2) stimulate transcription by directly phosphorylating transcription factors or by way of phosphorylating other downstream intermediates. Thus, pERK1/2 can phosphorylate members of the Ets family of transcription activators such as Elk-1, which preferentially activate genes containing serum-response elements, such as the c-fos gene (57). Alternatively, pERK1/2 may activate CREB, perhaps by first phosphorylating members of the RSK/MSK family of enzymes (58), which are known signaling intermediates between ERK1/2 and CREB (59). pCREB, in turn, could activate genes that contain Ca2+/cAMP-response elements, which include the crh gene.

Concluding remarks
CRH neuroendocrine neurons in the PVHmp receive inputs from many diverse cell groups (60). An essential question concerning this system is how these neurons integrate multiple afferent signals to produce coordinated responses. A heuristic that has been applied to this question at the level of general HPA axis function (61) is Sherrington’s principle of "the final common path" (62). At a smaller scale, this principle remains useful as a subtext for considering how MAPKs might process signals from multiple receptor subtypes within single CRH cells to help coordinate stereotyped responses (e.g. secretion and gene expression). MAPKs have been broadly, if not explicitly, considered within Sherringtonian contexts as intracellular funnels for signals arriving from a variety of extracellular sources (63), an idea supported by reports of increased PVH pERK1/2 immunoreactivity after various stimuli (25, 26, 27, 28) and by our finding of similar increases after both 2-DG and halothane exposure. Understanding how MAPKs participate within a final common path to influence gene expression in CRH neuroendocrine neurons may provide useful insights (e.g. Ref. 64) regarding how stimulus-transcription coupling is coordinated by the HPA axis. Such coupling may involve the recently postulated role of MAPK acting as a rheostat-like switch that, instead of turning genes on or off, provides a continuously variable rate of gene transcription that is dependent on the strength of the signaling cascades within the cell (65).


   Acknowledgments
 
We thank Graciela Sanchez-Watts for her cheerful help, training and supervision during the ISH runs and Dr. Michael W. Swank for his help with the MAPK immunohistochemistry. We are also grateful to Dr. Joseph A. Majzoub for his generous gift of the cDNA used to prepare the CRH probe.


   Footnotes
 
This study was supported by funds from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, awarded to A.G.W. (NS 29728).

Abbreviations: CORT, Corticosterone; CREB, cAMP response element-binding protein; 2-DG, 2-deoxy-D-glucose; hnRNA, heterogeneous nuclear RNA; HPA, hypothalamic-pituitary-adrenal; ICC, immunocytochemistry; ISH, in situ hybridization; NPY, neuropeptide Y; PB, phosphate buffer; PVHmp, medial parvicellular part of the hypothalamic paraventricular nucleus; RT, room temperature; TBS, Tris-buffered saline.

Received April 30, 2003.

Accepted for publication September 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Luckman SM, Dyball RE, Leng G 1994 Induction of c-fos expression in hypothalamic magnocellular neurons requires synaptic activation and not simply increased spike activity. J Neurosci 14:4825–4830[Abstract]
  2. Kovacs KJ, Sawchenko PE 1996 Sequence of stress-induced alterations in indices of synaptic and transcriptional activation in parvocellular neurosecretory neurons. J Neurosci 16:262–273[Abstract/Free Full Text]
  3. Harbuz MS, Jessop DS 1999 Dissociation between c-fos mRNA in the paraventricular nucleus and corticosterone secretion in rats with adjuvant-induced arthritis. J Endocrinol 163:107–113[Abstract]
  4. Watts AG, Sanchez-Watts G 2002 Interactions between heterotypic stressors and corticosterone reveal integrative mechanisms for controlling corticotropin-releasing hormone gene expression in the rat paraventricular nucleus. J Neurosci 22:6282–6289[Abstract/Free Full Text]
  5. Hoffman GE, Lyo D 2002 Anatomical markers of activity in neuroendocrine systems: are we all ‘fos-ed out’? J Neuroendocrinol 14:259–268[CrossRef][Medline]
  6. Weidenfeld J, Corcos AP, Wohlman A, Feldman S 1994 Characterization of the 2-deoxyglucose effect on the adrenocortical axis. Endocrinology 134:1924–1931[Abstract/Free Full Text]
  7. Salter D, Watts AG 2003 Differential suppression of hyperglycemic, feeding, and neuroendocrine responses in anorexia. Am J Physiol 284:R174–R182
  8. Ritter S, Watts AG, Dinh TT, Sanchez-Watts G, Pedrow C 2003 Immunotoxin lesion of hypothalamically projecting norepinephrine and epinephrine neurons differentially affects circadian and stressor-stimulated corticosterone secretion. Endocrinology 144:1357–1367[Abstract/Free Full Text]
  9. Ritter S, Bugarith K, Dinh TT 2001 Immunotoxic destruction of distinct catecholamine subgroups produces selective impairment of glucoregulatory responses and neuronal activation. J Comp Neurol 432:197–216[CrossRef][Medline]
  10. Davis S, Vanhoutte P, Pages C, Caboche J, Laroche S 2000 The MAPK/ERK cascade targets both Elk-1 and cAMP response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo. J Neurosci 20:4563–4572[Abstract/Free Full Text]
  11. Choe ES, McGinty JF 2001 Cyclic AMP and mitogen-activated protein kinases are required for glutamate-dependent cyclic AMP response element binding protein and Elk-1 phosphorylation in the dorsal striatum in vivo. J Neurochem 76:401–412[CrossRef][Medline]
  12. Impey S, Obrietan K, Wong ST, Poser S, Yano S, Wayman G, Deloulme JC, Chan G, Storm DR 1998 Cross talk between ERK and PKA is required for Ca2+ stimulation of CREB-dependent transcription and ERK nuclear translocation. Neuron 21:869–883[CrossRef][Medline]
  13. Wang Z, Zhang B, Wang M, Carr BI 2003 Persistent ERK phosphorylation negatively regulates cAMP response element-binding protein (CREB) activity via recruitment of CREB-binding protein to pp90RSK. J Biol Chem 278:11138–11144[Abstract/Free Full Text]
  14. Guardiola-Diaz HM, Boswell C, Seasholtz AF 1994 The cAMP-responsive element in the corticotropin-releasing hormone gene mediates transcriptional regulation by depolarization. J Biol Chem 269:14784–14791[Abstract/Free Full Text]
  15. Wolfl S, Martinez C, Majzoub JA 1999 Inducible binding of cyclic adenosine 3',5'-monophosphate (cAMP)-responsive element binding protein (CREB) to a cAMP-responsive promoter in vivo. Mol Endocrinol 13:659–669[Abstract/Free Full Text]
  16. Khan AM, Watts AG, Metabolic stress increases levels of the phosphorylated forms of p42/44 MAP kinase (ERK 1/2) and its downstream effectors in rat hypothalamic paraventricular neurons. Soc Neurosci Abstr [program 865.1], Nov 2–7, 2003, Orlando, FL
  17. Swank MW 2000 Phosphorylation of MAP kinase and CREB in mouse cortex and amygdala during taste aversion learning. Neuroreport 11:1625–1630[Medline]
  18. Berod A, Hartman BK, Pujol JF 1981 Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase. J Histochem Cytochem 29:844–850[Abstract]
  19. Watts AG, Sanchez-Watts G 1995 Physiological regulation of peptide messenger RNA colocalization in rat hypothalamic paraventricular medial parvicellular neurons. J Comp Neurol 352:501–514[CrossRef][Medline]
  20. Tanimura SM, Watts AG 1998 Corticosterone can facilitate as well as inhibit corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus. Endocrinology 139:3830–3836[Abstract/Free Full Text]
  21. Watts AG, Swanson LW 1989 Combination of in situ hybridization with immunohistochemistry and retrograde tract-tracing. Methods Neurosci 1:127–136
  22. Swanson LW 1998 Brain maps: structure of the rat brain. 2nd ed. Amsterdam: Elsevier
  23. Sawchenko PE, Swanson LW, Vale WW 1984 Corticotropin-releasing factor: co-expression within distinct subsets of oxytocin-, vasopressin-, and neurotensin-immunoreactive neurons in the hypothalamus of the male rat. J Neurosci 4:1118–1129[Abstract]
  24. Hoffman GE, Smith MS, Verbalis JG 1993 c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems. Front Neuroendocrinol 14:173–213[CrossRef][Medline]
  25. Reijmers LG, Hernando F, van Ree JM, Spruijt BM, Burbach JP 2000 Differential responses of phosphorylated mitogen-activated protein kinase and phosphorylated cyclic-AMP response element-binding protein immunoreactivity in the rat brain to sub-convulsive pentylenetetrazol. Neuroscience 101:1023–1028[CrossRef][Medline]
  26. Gioia M, Galbiati S, Rigamonti L, Moscheni C, Gagliano N 2001 Extracellular signal-regulated kinases 1 and 2 phosphorylated neurons in the tele- and diencephalon of rat after visceral pain stimulation: an immunocytochemical study. Neurosci Lett 308:177–180[CrossRef][Medline]
  27. Sharp JW, Magrum LJ, Gietzen DW 2002 Role of MAP kinase in signaling indispensable amino acid deficiency in the brain. Mol Brain Res 105:11–18[Medline]
  28. Daniels D, Patten CS, Roth JD, Yee DK, Fluharty SJ 2003 Melanocortin receptor signaling through mitogen-activated protein kinase in vitro and in rat hypothalamus. Brain Res 986:1–11[CrossRef][Medline]
  29. Seger R, Krebs EG 1995 The MAPK signaling cascade. FASEB J 9:726–735[Abstract]
  30. Pearson G, Robinson F, Beers GT, Xu BE, Karandikar M, Berman K, Cobb MH 2001 Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22:153–183[Abstract/Free Full Text]
  31. Johnson GL, Lapadat R 2002 Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298:1911–1912[Abstract/Free Full Text]
  32. Hindley A, Kolch W 2002 Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinase. J Cell Sci 115:1575–1581[Abstract/Free Full Text]
  33. Flood DG, Finn JP, Walton KM, Dionne CA, Contreras PC, Miller MS, Bhat RV 1998 Immunolocalization of the mitogen-activated protein kinases p42MAPK and JNK1, and their regulatory kinases MEK1 and MEK4, in adult rat central nervous system. J Comp Neurol 398:373–392[CrossRef][Medline]
  34. Chawla S, Bading H 2001 CREB/CBP and SRE-interacting transcriptional regulators are fast on-off switches: duration of calcium transients specifies the magnitude of transcriptional responses. J Neurochem 79:849–858[CrossRef][Medline]
  35. Chaudhuri A, Nissanov J, Larocque S, Rious L 1997 Dual activity maps in primate visual cortex produced by different temporal patterns of zif268 mRNA and protein expression. Proc Natl Acad Sci USA 94:2671–2675[Abstract/Free Full Text]
  36. Sawchenko PE, Swanson LW, Grzanna R, Howe PRC, Bloom SR, Polak JM 1985 Colocalization of neuropeptide Y immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus. J Comp Neurol 241:138–153[CrossRef][Medline]
  37. Daftary SS, Boudaba C, Szabo K, Tasker JG 1998 Noradrenergic excitation of magnocellular neurons in the rat hypothalamic paraventricular nucleus via intranuclear glutamatergic circuits. J Neurosci 18:10619–10628[Abstract/Free Full Text]
  38. Day HE, Campeau S, Watson SJ, Jr., Akil H 1999 Expression of {alpha}(1b) adrenoceptor mRNA in corticotropin-releasing hormone-containing cells of the rat hypothalamus and its regulation by corticosterone. J Neurosci 19:10098–10106[Abstract/Free Full Text]
  39. Sands SA, Morilak DA 1999 Expression of {alpha}1D adrenergic receptor messenger RNA in oxytocin- and corticotropin-releasing hormone-synthesizing neurons in the rat paraventricular nucleus. Neuroscience 91:639–649[CrossRef][Medline]
  40. Zhong H, Minneman KP 1999 Differential activation of mitogen-activated protein kinase pathways in PC12 cells by closely related {alpha}1-adrenergic receptor subtypes. J Neurochem 72:2388–2396[CrossRef][Medline]
  41. Chalothorn D, McCune DF, Edelmann SE, Garcia-Cazarin ML, Tsujimoto G, Piascik MT 2002 Differences in the cellular localization and agonist-mediated internalization properties of the {alpha}(1)-adrenoceptor subtypes. Mol Pharmacol 61:1008–1016[Abstract/Free Full Text]
  42. Waldrop BA, Mastalerz D, Piascik MT, Post GR 2002 {alpha}(1B)- and {alpha}(1D)-Adrenergic receptors exhibit different requirements for agonist and mitogen-activated protein kinase activation to regulate growth responses in rat 1 fibroblasts. J Pharmacol Exp Ther 300:83–90[Abstract/Free Full Text]
  43. Khan AM, Watts AG Norepinephrine rapidly elevates CRH hnRNA, c-fos mRNA and levels of phosphorylated MAP kinases (ERK 1 and 2) in hypothalamic parvocellular paraventricular neurons in vivo. Soc Neurosci Abstr [program 397.18], Nov 8–12, 2003, New Orleans, LA
  44. Xia Z, Dudek H, Miranti CK, Greenberg ME 1996 Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. J Neurosci 16:5425–5436[Abstract/Free Full Text]
  45. Thandi S, Blank JL, Challiss RA 2002 Group-I metabotropic glutamate receptors, mGlu1a and mGlu5a, couple to extracellular signal-regulated kinase (ERK) activation via distinct, but overlapping, signalling pathways. J Neurochem 83:1139–1153[CrossRef][Medline]
  46. Yang H, Lu D, Yu K, Raizada MK 1996 Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway. J Neurosci 16:4047–4058[Abstract/Free Full Text]
  47. Mullins DE, Zhang X, Hawes BE 2002 Activation of extracellular signal regulated protein kinase by neuropeptide Y and pancreatic polypeptide in CHO cells expressing the NPY Y(1), Y(2), Y(4) and Y(5) receptor subtypes. Regul Pept 105:65–73[CrossRef][Medline]
  48. Rossant CJ, Pinnock RD, Hughes J, Hall MD, McNulty S 1999 Corticotropin-releasing factor type 1 and type 2 {alpha}-receptors regulate phosphorylation of calcium/cyclic adenosine 3',5'-monophosphate response element-binding protein and activation of p42/p44 mitogen-activated protein kinase. Endocrinology 140:1525–1536[Abstract/Free Full Text]
  49. Aubry JM, Bartanusz V, Pagliusi S, Schulz P, Kiss JZ 1996 Expression of ionotropic glutamate receptor subunit mRNAs by paraventricular corticotropin-releasing factor (CRF) neurons. Neurosci Lett 205:95–98[CrossRef][Medline]
  50. Imaki T, Katsumata H, Miyata M, Naruse M, Imaki J, Minami S 2001 Expression of corticotropin-releasing hormone type 1 receptor in paraventricular nucleus after acute stress. Neuroendocrinology 73:293–301[CrossRef][Medline]
  51. Campbell RE, ffrench-Mullen JMH, Cowley MA, Smith MS, Grove KL 2001 Hypothalamic circuitry of neuropeptide Y regulation of neuroendocrine function and food intake via the Y5 receptor subtype. Neuroendocrinology 74:106–119[CrossRef][Medline]
  52. Lenkei Z, Corvol P, Llorens-Cortes C 1995 Comparative expression of vasopressin and angiotensin type-1 receptor mRNA in rat hypothalamic nuclei: a double in situ hybridization study. Mol Brain Res 34:135–142[Medline]
  53. Mountjoy KG, Mortrud MT, Low MJ, Simerly RB, Cone RD 1994 Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 8:1298–1308[Abstract/Free Full Text]
  54. Kishi T, Aschkenasi CJ, Lee CE, Mountjoy KG, Saper CB, Elmquist JK 2003 Expression of melanocortin 4 receptor mRNA in the central nervous system of the rat. J Comp Neurol 457:213–235[CrossRef][Medline]
  55. Volmat V, Camps M, Arkinstall S, Pouyssegur J, Lenormand P 2001 The nucleus, a site for signal termination by sequestration and inactivation of p42/p44 MAP kinases. J Cell Sci 114:3433–3443[Abstract/Free Full Text]
  56. Pouyssegur J, Volmat V, Lenormand P 2002 Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling. Biochem Pharmacol 64:755–763[CrossRef][Medline]
  57. Janknecht R, Ernst WH, Pingoud V, Nordheim A 1993 Activation of ternary complex factor Elk-1 by MAP kinases. EMBO J 12:5097–5104[Medline]
  58. Blenis J 1993 Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci USA 90:5889–5892[Abstract/Free Full Text]
  59. Xing J, Ginty DD, Greenberg ME 1996 Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 273:959–963[Abstract]
  60. Swanson LW 1991 Biochemical switching in hypothalamic circuits mediating responses to stress. Prog Brain Res 87:181–200[Medline]
  61. Harris GW 1955 The function of the pituitary stalk. Bull Johns Hopkins Hosp 97:358–375[Medline]
  62. Sherrington CS 1904 Correlation of reflexes and the principle of the common path. British Association Report; 728–741
  63. Smith FD, Scott JD 2002 Signaling complexes: junctions on the intracellular information super highway. Curr Biol 12:R32–R40
  64. Naor Z, Benard O, Seger R 2000 Activation of MAPK cascades by G-protein-coupled receptors: The case for gonadotropin-releasing hormone receptor. Trends Endocrinol Metab 11:91–99[CrossRef][Medline]
  65. Hazzalin CA, Mahadevan LC 2002 MAPK-regulated transcription: a continuously variable gene switch? Nat Rev Mol Cell Biol 3:30–34[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
M. M. Arnhold, J. M. Yoder, and W. C. Engeland
Subdiaphragmatic Vagotomy Prevents Drinking-Induced Reduction in Plasma Corticosterone in Water-Restricted Rats
Endocrinology, May 1, 2009; 150(5): 2300 - 2307.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
D. Salter-Venzon and A. G. Watts
The role of hypothalamic ingestive behavior controllers in generating dehydration anorexia: a Fos mapping study
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2008; 295(4): R1009 - R1019.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
P. S. Singru, E. Sanchez, R. Acharya, C. Fekete, and R. M. Lechan
Mitogen-Activated Protein Kinase Contributes to Lipopolysaccharide-Induced Activation of Corticotropin-Releasing Hormone Synthesizing Neurons in the Hypothalamic Paraventricular Nucleus
Endocrinology, May 1, 2008; 149(5): 2283 - 2292.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
L. M. Gorton, A. M. Khan, M. Bohland, G. Sanchez-Watts, C. M. Donovan, and A. G. Watts
A Role for the Forebrain in Mediating Time-of-Day Differences in Glucocorticoid Counterregulatory Responses to Hypoglycemia in Rats
Endocrinology, December 1, 2007; 148(12): 6026 - 6039.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
A. M. Khan, T. A. Ponzio, G. Sanchez-Watts, B. G. Stanley, G. I. Hatton, and A. G. Watts
Catecholaminergic Control of Mitogen-Activated Protein Kinase Signaling in Paraventricular Neuroendocrine Neurons In Vivo and In Vitro: A Proposed Role during Glycemic Challenges
J. Neurosci., July 4, 2007; 27(27): 7344 - 7360.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
C. Wotus, M. M. Arnhold, and W. C. Engeland
Dehydration-induced drinking decreases Fos expression in hypothalamic paraventricular neurons expressing vasopressin but not corticotropin-releasing hormone
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1349 - R1358.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
K. Mukherjee, A. Knisely, and L. Jacobson
Partial Glucocorticoid Agonist-Like Effects of Imipramine on Hypothalamic-Pituitary-Adrenocortical Activity, Thymus Weight, and Hippocampal Glucocorticoid Receptors in Male C57BL/6 Mice
Endocrinology, September 1, 2004; 145(9): 4185 - 4191.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khan, A. M.
Right arrow Articles by Watts, A. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khan, A. M.
Right arrow Articles by Watts, A. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals