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Differential Responsiveness of Dopamine-ß-Hydroxylase Gene Expression to Glucoprivation in Different Catecholamine Cell Groups

Programs in Neuroscience, Washington State University, Pullman, Washington 99164

Address all correspondence and requests for reprints to: Dr. A-J. Li, Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6520. E-mail: aijunli{at}vetmed.wsu.edu.

	Abstract

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Hindbrain catecholaminergic neurons are key participants in systemic glucoregulation. However, the specific subpopulations critical for glucoregulatory function have not been fully identified. Here we used in situ hybridization and immunohistochemistry to investigate effects of glucoprivation on expression of the gene for the catecholamine biosynthetic enzyme, dopamine-ß-hydroxylase (DBH), to further localize the critical cell populations. Glucoprivation induced by the glycolytic inhibitor, 2-deoxy-D-glucose (2DG) (250 mg/kg) increased total DBH mRNA expression in caudal ventrolateral medullary cell groups (namely A1, the A1/C1 overlap, and the middle portion of C1) from six to 49 times control levels. In retrofacial C1, no enhancement was observed. In the dorsomedial medulla, hybridization signal was modestly increased (tripled) in A2 but was not increased in the area postrema. Previous microinjection of the retrogradely transported catecholamine immunotoxin (anti-DBH-saporin, or DSAP) into the paraventricular nucleus of the hypothalamus reduced the number of DBH-immunoreactive cells in cell groups known to project to the paraventricular nucleus of the hypothalamus as well as reducing the 2DG-stimulated increases in total DBH mRNA expression in the caudal ventrolateral medulla and A2. The strong enhancement of DBH gene expression by glucoprivation is consistent with the demonstrated importance of catecholaminergic neurons for glucoregulation. The differential sensitivity of these neurons to glucoprivation is evidence of functional specialization within the total population. The pattern of 2DG-induced gene expression indicates that the ventrolateral medulla contains the vast majority of catecholamine neurons responsive to glucoprivation.

	Introduction

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A CONTINUOUS SUPPLY of glucose is essential for the function and survival of the brain. Glucoprivation, a condition of reduced brain glucose availability, evokes glucoregulatory responses adapted to restore the availability of this essential fuel. Administration of 2-deoxy-D-glucose (2DG), a competitive inhibitor of intracellular glucose use (1), induces glucoprivation and triggers a number of glucoregulatory responses, including hyperphagia (2, 3, 4), increased adrenal medullary secretion (5, 6, 7), and inhibition of the estrous cycle (8, 9, 10). Although the specific location and phenotype of these hindbrain glucoreceptive cells are still unknown, studies using the retrogradely transported catecholamine immunotoxin, anti-dopamine-ß-hydroxylase (anti-DBH)-saporin (DSAP), have shown that hindbrain norepinephrine (NE) and/or epinephrine (E) neurons are required for elicitation of major glucoregulatory responses, including increased feeding, corticosterone secretion, and adrenal medullary secretion (10, 11, 12).

In addition to glucoregulation, hindbrain catecholamine neurons are involved in a variety of homeostatic controls. For example, NE neurons in cell group A1 are strongly implicated in control of vasopressin secretion in response to hypovolemia (13, 14, 15). E neurons in rostral C1 are involved in cardiovascular function and respond to baroreceptor unloading (16, 17, 18, 19). Cell group A2 contains neurons that respond to gonadal steroid in the control of reproductive activity (20, 21, 22, 23) and that respond to cholecystokinin octapeptide to inhibit food intake (24). Given the variety of functions associated with these hindbrain cell groups, the localization of cells with specific functions is an important objective. Indeed, the extent to which different catecholamine subgroups are specifically targeted by particular homeostatic challenges is not completely resolved. In the present experiment, we analyze DBH mRNA expression to further investigate the response of hindbrain catecholamine neurons to glucoprivation and to localize a particular subset of neurons in which expression is differentially increased by glucose deficit.

DBH is a catecholamine biosynthetic enzyme uniquely expressed by NE and E neurons, where it is responsible for conversion of dopamine to NE. Although DBH is not the rate-limiting enzyme for catecholamine synthesis, certainly enhanced availability of DBH enzyme is a mechanism for increasing vesicular NE and E for synaptic transmission. Both levels and activity of DBH enzyme have been shown to be increased by neural activation. For example, social stress or repeated immobilization stress enhances the expression of DBH mRNA in rat adrenal medulla (25, 26). In cell group A2, as well as in A1 and C1, the expression of DBH mRNA, but not tyrosine hydroxylase (TH, rate-limiting enzyme for NE and E synthesis) mRNA, is enhanced during gonadal steroid-induced LH surges (23).

In the present study, we used in situ hybridization and immunohistochemistry to test the hypothesis that DBH mRNA is selectively increased by glucoprivation in subgroups of catecholamine neurons shown by other methods to be critical for elicitation of glucoregulatory responses. We also examined the effect of hypothalamic DSAP injections on basal and glucoprivation-induced levels of DBH mRNA expression. Results suggest that glucoprivation-responsive DBH mRNA is a marker for NE and E neurons involved in glucoregulation.

	Materials and Methods

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Animals
Male Sprague Dawley rats were purchased from Simonsen Laboratories (Gilroy, CA) and housed individually in an animal care facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Rats were maintained on a 12-h light, 12-h dark cycle with ad libitum access to pelleted rodent food and tap water. All experimental procedures were approved by Washington State University Institutional Animal Care and Use Committee, which conforms to National Institutes of Health Guidelines.

Immunotoxin microinjection
DSAP (42 ng/200 nl; Chemicon International, Temecula, CA) or unconjugated saporin (SAP) control solution (8.82 ng/200 nl; Advanced Targeting Systems, Carlsbad, CA), dissolved in 0.1 M phosphate buffer (PBS, pH 7.4) was infused bilaterally using a Picospritzer through a pulled glass capillary pipette (30-µm tip diameter) positioned just dorsal to the targeted site in the paraventricular nucleus of the hypothalamus (PVH), 1.8 mm caudal to bregma, 0.4 mm lateral to midline, and 7.4 mm ventral to the dura mater. The amount of unconjugated SAP in the control solution approximated the amount of SAP present in the DSAP conjugate (21%), as indicated in the manufacturer’s product information. Previous work comparing SAP and uninjected controls demonstrated that SAP did not produce behavioral or histological signs of toxicity (11). A 3-wk interval was allowed to elapse between the DSAP injections and additional experimentation to permit retrograde transport of the toxin and complete degeneration of lesioned neurons (11).

Tissue preparation
Rats (weighing 320–450 g) were injected (1 ml/kg, sc) with 0.9% saline (Sal) or 2DG (250 mg/kg; Sigma Chemical Co., St. Louis, MO) and returned to their home cages without food. Ninety minutes later they were anesthetized deeply with halothane (Halocarbon Laboratories, River Edge, NJ). As indicated previously, corticosterone levels and hyperglycemia peak at approximately 90 min after 2DG (11, 27). In addition, c-Fos and neuropeptide genes are strongly expressed in the hindbrain and hypothalamus between 90 and 120 min after 2DG injection (28, 29, 30). Transcardial perfusion was initiated just before cessation of the heartbeat using PBS followed by fresh cold 4% paraformaldehyde in PBS. After perfusion, brains were rapidly removed and placed in 4% paraformaldehyde/PBS at 4 C for overnight, then into a series of sucrose (10–25%) in PBS for a total of 20 h. The hindbrains were sectioned at 16 µm thickness and collected into five serial sets that were mounted onto SuperFrost Plus slides (Fisher Scientific, Los Angeles, CA). After drying, sections were stored in desiccated slide boxes at –80 C until processed for in situ hybridization or immunohistochemistry.

In situ hybridization
Plasmid containing a 535-bp fragment of rat DBH (gift of Dr. Lothar Jennes, Department of Anatomy and Neurobiology, University of Kentucky) was linearized using either HindIII (antisense) or XbaI (sense), purified with phenol/chloroform, and then ethanol precipitated. Linearized plasmids were transcribed for antisense riboprobe with T7 RNA polymerase and for sense riboprobe with SP6 RNA polymerase in the presence of [³³P]UTP (PerkinElmer, Indianapolis, IN), using MAXIscript kit (Ambion, Austin, TX). Unincorporated isotope was removed using spin columns (Roche, Indianapolis, IN). Probes were quantified in a scintillation counter. Before use, the probe was combined with 1/20 vol Torula RNA (Ambion) and 0.1 M Tris/0.01 M EDTA (pH 8.0) and then mixed with hybridization buffer, containing 6.25% deionized formamide, 12.5% dextran sulfate, 0.375 M NaCl, 10 mM Tris (pH 8.0), 1.6 mM EDTA, 1.25x Denhardt’s solution, and 10 mM dithiothreitol, at a ratio of 1:3. Probe mix was heat denatured at 65 C for 15 min. The final volume of probe mix plus hybridization buffer was equal to 150 µl/slide (or 1.5 x 10⁶ cpm/slide) (23, 30).

For hybridization, hindbrain sections were first treated in protein kinase K (5 µg/ml) in PBS at 37 C for 5 min. After washes in PBS, sections were placed in 0.1 M triethanolamine with 250 µl/ml acetic anhydride for 10 min. Sections were then rinsed in 2x sodium citrate-sodium chloride buffer (SSC), dehydrated in graded ethanol solutions (70, 90, and 100%; 3 min each), and allowed to air dry before the hybridization procedure. After adding the hybridization mix, the sections were covered with Parafilm (Pechiney Plastic Packaging, Inc., Neenah, WI) and were placed in humidity chambers and placed into an oven at calculated hybridization temperatures (50 C) for overnight (17 h). After hybridization, the slides were washed twice in 2x SSC for 30 min each at room temperature and then incubated for 30 min at 37 C in RNase buffer (10 mM Tris, 0.5 M NaCl, 1 mM EDTA, pH 8.0) containing RNase-A (0.02 mg/ml) followed by incubation for 10 min at 37 C in the buffer without RNase-A. Next, after a wash in 2x SSC at room temperature, slides were incubated twice in 0.1x SSC at 60 C for 30 min, then rinsed again in fresh 0.1x SSC at room temperature. Sections were then dehydrated in graded ethanol (50, 70, 90, and 100%; 3 min each) and allowed to air dry. Slides were dipped in K5 emulsion (Polysciences, Warrington, PA) and placed in light-tight boxes containing desiccant and stored at 4 C for 10 wk. The slides were then removed under safe light conditions and developed and counterstained in 0.5% cresyl violet in acetate buffer, dehydrated in graded ethanol, and cleared in Citrasol (VWR International, West Chester, PA) and coverslipped with DPX (EM Sciences, Fort Washington, PA). Hybridization controls, using the sense probe, showed no hybridization signal.

Immunohistochemistry of DBH
Immunohistochemical staining of DBH was performed using standard avidin-biotin-peroxidase techniques as described previously (11). Briefly, sections were treated with 50% ethanol for 30 min. After incubation in 1% BSA in PBS for 30 min, the sections were incubated for 2 d in mouse monoclonal anti-DBH (1:100,000; Chemicon International) in 1% BSA/PBS. After washing with PBS, sections were incubated in biotinylated donkey antimouse IgG (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 d. The tissue was washed and reacted for visualization of DBH immunoreactivity in a peroxidase reaction using the ABC Elite kit (Vector Laboratories, Burlingame, CA) and associated protocol.

Quantification and analysis
NE/E cell groups are referred to using conventional terminology (31), except that subdivisions of the ventrolateral medullary cell column were distinguished, as noted below (11). To quantify DBH mRNA expression levels in each cell group, three consecutive coronal sections at the following anatomical levels were used [distance in mm from Bregma, according to Paxinos and Watson (31)]: A1 (14.5–14.2), at the rostral terminus of the pyramidal decussation; the overlap of A1 and C1 (A1/C1; 13.4–13.1), just rostral to the calamus scriptorius [defined as the most caudal extent of the area postrema (AP)]; the middle portion of C1 (C1m; 12.6–12.3), immediately rostral to the obex (defined as the most rostral extent of the AP); the retrofacial portion of C1 (C1r; 11.6–11.3), at the rostral terminus of the inferior olive; C2 (12.8–12.5), within the nucleus of the solitary tract rostral to the obex; C3 (as C1r), at the rostral terminus of the inferior olive; A2 (14.3–14.0), within the nucleus of the solitary tract at the level of the calamus scriptorius; A5 (10.0–9.7), just rostral to the nucleus of cranial nerve 6 and the genu of cranial nerve 7; A7 (8.8–8.5), at the level of the Kölliker-Fuse nucleus; AP at 13.8 –13.5; and A6 cells were not quantified.

Quantification of gene expression was done from digitized images of the DBH mRNA hybridization signal, obtained with a high-resolution digital camera (MicroPublisher 5.0; QImaging Co., Burnaby, British Columbia, Canada). The cresyl violet counterstaining was subtracted by use of Kodak Wratten filter (Edmund Scientifics, Tonawanda, NY). Quantification was conducted using Image-Pro Plus software (Cybernetics, Silver Spring, MD). Two quantification methods were used. 1)The total silver grain pixel number, representing the DBH mRNA hybridization signal in the following hindbrain catecholamine cell regions, was counted on three consecutive sections for each cell group. A total of six (or three for AP) images representing an area of 438 x 328 µm (or 2560 x 1920 pixels) on each side of the section were captured bilaterally and counted. The size and location of these quantification sites were determined from adjacent sections immunoreacted to detect DBH protein and were confirmed to include all of the DBH-immunoreactive (DBH-ir) cells in each cell group of interest. This means of quantification was selected because our protocol only weakly detected DBH mRNA under basal conditions, even after a 10-wk exposure, such that individual cells could not be identified in some areas. 2) In the stimulated condition, where DBH was highly expressed, DBH mRNA-positive cells were counted using cluster analysis from the same digitalized images as above. A cluster of hybridization signals with more than 1000 pixels in total, which represents approximately 15–20 silver grains in average size, was considered as one DBH mRNA-positive cell. The percentage of the DBH-ir cells expressing DBH mRNA was also calculated.

The DBH-ir-positive cell numbers in each hindbrain population were counted from the same areas described for in situ hybridization. For each section, the number of DBH-ir-positive cells was counted bilaterally. For each rat, two consecutive coronal sections were counted. The number of DBH-ir cells in A5 or A7 was not quantified because only weak basal or 2DG-induced DBH mRNA signal was detected among all treatment groups in these regions. In previous work using the same protocol as in the present study, PVH DSAP injections did not appreciably alter the DBH-ir cell number in A5 or A7 (11).

Statistical analysis
All results are presented as mean ± SEM. For statistical analysis of data, we used a one-way ANOVA or Kruskal-Wallis ranks. After the above, multiple comparisons between individual groups were carried out using a post hoc test [Fisher least-significant difference (LSD) method or Dunn’s test]. P < 0.05 was considered to be statistically significant.

	Results

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DBH immunoreactivity in hindbrain and hypothalamus in SAP- and DSAP-injected rats
Consistent with our previous report (11), PVH DSAP injection caused significant loss of DBH-ir neurons in caudal ventrolateral medulla, most prominently in A1, A1/C1, and C1m, but not in C1r (Fig. 1 and Table 1). In the dorsomedial medulla cell groups (A2, C2, and C3), where a smaller proportion of the NE/E neurons project to the PVH, the DSAP injection had a smaller impact on cell numbers (Table 1). In addition, DSAP had no effect on the DBH-ir cells in AP (Table 1) or the pontine lateral tegmental NE cell groups (A5 and A7; data not shown). Associated with their loss of hypothalamically projecting catecholamine cell bodies, DSAP rats also had greatly reduced DBH fiber and terminal staining in the PVH area, compared with SAP rats (Fig. 1).

View larger version (74K): [in this window] [in a new window]   FIG. 1. Coronal brain sections showing DBH immunoreactivity. Rats were microinjected previously with the retrogradely transported immunotoxin DSAP or with the unconjugated SAP control. A and C, A1/C1 and PVH from SAP controls, respectively; B and D, same areas in DSAP-treated rats. DSAP profoundly reduced DBH-ir terminals in the PVH and cell bodies in A1/C1. V, Third ventricle. Bar, 50 µm.

View larger version (107K): [in this window] [in a new window]   FIG. 2. Representative bright-field images of A1, A1/C1, C1m, and C1r regions 90 min after Sal or 2DG (250 mg/kg, sc) in rats previously microinjected into the PVH with the immunotoxin DSAP or SAP control. Cresyl violet counterstaining allowed visualization of cell bodies. Dark grains represent DBH mRNA expression. The basal level of DBH mRNA expression was low in all of these groups and was strongly enhanced in A1, A1/C1, and C1m by 2DG in the SAP controls. DSAP reduced the effect of 2DG. Bar, 5 µm.

View larger version (79K): [in this window] [in a new window]   FIG. 4. Representative bright-field images of the A2 cell group and AP showing expression of DBH mRNA in A2 and AP 90 min after 2DG (250 mg/kg, sc) or Sal injection from rats treated previously by microinjection of DSAP or SAP control into the PVH. Cresyl violet counterstaining allowed visualization of cell bodies. Dark grains represent DBH mRNA expression. Higher basal expression of DBH mRNA hybridization signal was present in these groups than was observed in the ventrolateral medullary groups (Fig. 2), but 2DG was less effective in increasing expression level in the SAP controls. In addition, DSAP produced a smaller reduction (A2) or no reduction of expression (AP). Bar, 5 µm.

View larger version (10K): [in this window] [in a new window]   FIG. 3. Quantitative analysis of DBH mRNA expression in regions of the ventrolateral medulla containing catecholamine cell groups. Data show mean DBH mRNA hybridization signal (number of silver grains per area in pixels) in A1, A1/C1, C1m, and C1r in SAP- and DSAP-treated rats 90 min after saline (SAP-Sal or DSAP-Sal) or 2DG (SAP-2DG or DSAP-2DG) injection. ***, P < 0.001 vs. SAP-Sal rats, one-way ANOVA followed by a post hoc test of Fisher LSD.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Quantitative data of DBH mRNA expressions in A2 and AP. Data show mean DBH mRNA hybridization signal (number of silver grains per area in pixels) in A2 and AP in SAP- and DSAP-treated rats 90 min after injection of Sal (SAP-Sal or DSAP-Sal) or 2DG (SAP-2DG or DSAP-2DG). *, P < 0.05 vs. SAP-Sal rats, one-way ANOVA followed by a post hoc test of Fisher LSD.

	Discussion

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Previous studies have shown that 2DG-induced glucoprivation enhances the expression of c-Fos (27), a neuronal activation marker protein, and neuropeptide Y (NPY) mRNA (30), a peptide neurotransmitter that is coexpressed in many NE and E neurons that innervate the PVH (35). Systemic glucoprivation produced selective increases in Fos protein in a large percentage of TH-ir neurons in the ventrolateral medulla; i.e. 42, 44, and 18% of TH-ir-positive neurons in A1, A1/C1, and in C1, rostral to the area of overlap with A1, respectively (27). In the present study, glucoprivation increased the expression of DBH mRNA in a similar percentage of cell bodies in these same regions (21.3, 39.6, and 13.1% in A1, A1/C1, and C1m, respectively). In the present study, no significant changes of DBH mRNA by glucoprivation were observed in C2 and C3 regions. However, previous studies have suggested activation of E neurons in the C2 and C3 by glucoregulation. A large population (30%) of E neurons in C2 and C3 was activated by 2DG (27). In addition, the expression of NPY mRNA, which is coexpressed with E in C2 and C3 neurons, was also enhanced by glucoprivation in these groups, as it was in A1, A1/C1, and C1 (30). Failure to detect changes in DBH mRNA in C2 and C3 may be because of the low basal and 2DG-induced expression levels and the ability of our method to detect them. An important question, not addressed by this study, is whether glucoprivation-induced DBH mRNA enhancement is accompanied by increased DBH protein expression. Although DBH protein is detected by immunohistochemistry, a more sensitive quantitative method for determination of protein levels will be required to answer this question. Taken together, however, the highly consistent distribution of increased Fos protein, NPY mRNA, and DBH mRNA expression indicate that catecholamine neurons responsive to glucose deficit are heavily concentrated in the caudal and middle regions of the ventrolateral medullary cell column and that many of these neurons coexpress NPY mRNA.

A number of glucoregulatory responses have been shown to be dependent on hindbrain catecholamine neurons. Of these, there is currently very strong evidence that the glucoprivic feeding response requires the DBH/NPY-coexpressing phenotype. Both catecholamines and NPY are potent orexigens (36, 37, 38, 39). Acute pharmacological blockade of catecholamine neurotransmission or injection of anti-NPY antibody into the PVH impairs glucoprivic feeding (40, 41). Knockout of the DBH gene renders mice less sensitive to glucoprivation (42), and knockout of the NPY gene blocks glucoprivic feeding (43). In addition, DSAP lesions that abolish glucoprivic feeding destroy all or nearly all catecholamine/NPY-coexpressing neurons (30). Glucoregulatory responses other than feeding that require rostrally projecting catecholamine neurons may also depend on the DBH/NPY phenotype, but this possibility requires additional investigation. However, the spinally projecting catecholamine neurons required for the adrenal medullary response to glucoprivation (11) appear to be a different phenotype, because spinally projecting catecholamine neurons do not express NPY (35). This anatomical deduction is consistent with studies of mutant mice with deletions of the NPY gene, showing that this deletion does not impair the adrenal medullary response to glucoprivation (43).

In the retrofacial region of C1, as well as in A5 and A7, expression of DBH mRNA was below the level of detection for our hybridization protocol both under basal and glucoprivic conditions. The most likely explanation of this finding is that E and NE neurons in these regions are not activated by, or involved in, glucoprivation. In this light, it is interesting that the spinally projecting neurons in rostral C1 have been strongly implicated in the baroreceptor reflex (16, 17, 18, 19). A5 and A7 are not likely to have glucoregulatory function because they do not appear to innervate sympathetic or adrenal medullary preganglionic neurons (44, 45, 46, 47). However, it is also possible that these particular catecholamine neurons are more heavily reliant on a coexpressed substance than on E or NE during neurotransmission. If so, then DBH might not be a sensitive indicator of their responsiveness to glucoprivation or other forms of activation. For example, both cocaine- and amphetamine-regulated transcript (48, 49) and glutamate (50) are coexpressed in rostral C1 neurons with vasomotor function. Finally, cell bodies not activated by glucoprivation might be inhibited and could contribute to elicitation of glucoregulatory responses by disinhibiting the essential neural circuits. The present approach would not reveal this possibility.

Previous studies have shown that the hindbrain catecholamine neurons are involved in a variety of homeostatic controls, including glucoregulatory responses. In the present study, glucoprivation selectively enhanced DBH gene expression in the caudal ventrolateral medulla. This result is consistent with the demonstrated importance of catecholaminergic neurons for glucoregulation and indicates the presence of functionally specialized subgroups within the total population of catecholaminergic neurons in the hindbrain.

	Acknowledgments

 
We thank Dr. Lothar Jennes, University of Kentucky, for DBH plasmid.

	Footnotes

 
This work was supported by Public Health Service Grants NS 045520 and DK 40498 (to S.R.).

A.-J.L., Q.W., and S.R. have nothing to declare.

First Published Online April 13, 2006

Abbreviations: AP, Area postrema; DBH, dopamine-ß-hydroxylase; 2DG, 2-deoxy-D-glucose; DSAP, anti-DBH-saporin; E, epinephrine; ir, immunoreactive; LSD, least-significant difference; NE, norepinephrine; NPY, neuropeptide Y; PVH, paraventricular nucleus of the hypothalamus; Sal, 0.9% sodium chloride; SAP, saporin; SSC, sodium citrate-sodium chloride buffer; TH, tyrosine hydroxylase.

Received February 22, 2006.

Accepted for publication April 3, 2006.

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