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Endocrinology Vol. 145, No. 10 4470-4479
Copyright © 2004 by The Endocrine Society

Site of Action of Acute Alcohol Administration in Stimulating the Rat Hypothalamic-Pituitary-Adrenal Axis: Comparison between the Effect of Systemic and Intracerebroventricular Injection of this Drug on Pituitary and Hypothalamic Responses

Soon Lee, Daniel Selvage, Keith Hansen and Catherine Rivier

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Catherine Rivier, Ph.D., The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier{at}salk.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The peripheral injection of alcohol stimulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis, but the ready penetration of this drug in most bodily compartments has made it difficult to identify its specific sites of action. Here we determined whether alcohol can directly influence the corticotropes. We first determined whether alcohol acted within the brain to stimulate neurons in the paraventricular nucleus (PVN) of the hypothalamus, which synthesizes corticotropin-releasing factor (CRF) and vasopressin (VP). To test this hypothesis, we injected alcohol intracerebroventricularly (icv; 5 µl of 200-proof; 86 µmol) and compared these results with those obtained after its ip administration (3.0 g/kg). Although not causing neuronal damage and not leading to detectable levels of the drug in the general circulation, icv alcohol significantly up-regulated PVN CRF heteronuclear RNA levels and increased plasma ACTH levels, a change comparable to the one observed in the ip model. To determine whether alcohol stimulated the corticotropes independently of CRF and/or VP, we injected the drug ip or icv and measured changes in anterior pituitary proopiomelanocortin (POMC) transcripts and ACTH release in the presence or absence of endogenous CRF and/or VP. Intracerebroventricular and ip alcohol significantly increased POMC primary transcript levels, measured by ribonuclease protection assay, over a time-course that corresponded to ACTH release. Both the POMC and the ACTH responses were completely abolished by removal of CRF and VP. Collectively, these results indicate that alcohol-induced activation of the corticotropes does not represent a direct influence of the drug on the pituitary but requires CRF and VP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ABILITY OF acute, investigator-controlled alcohol (EtOH, ethanol) exposure to activate the hypothalamic-pituitary-adrenal (HPA) axis is well known (see Ref. 1). We have shown that the ip or intragastric injection of this drug rapidly and significantly increases plasma ACTH levels as well as the neuronal activity of the paraventricular nucleus (PVN) of the hypothalamus, particularly in cell bodies that express corticotropin-releasing factor (CRF) and vasopressin (VP) (2, 3, 4). The importance of these peptides in modulating ACTH release caused by the peripheral (systemic) administration of EtOH is supported by the finding that their respective immunoneutralization nearly abolishes (in the case of CRF) or significantly blunts (in the case of VP) this response (5, 6, 7, 8), whereas concomitant immunoneutralization of both peptides or blockade of their respective receptors completely abolishes ACTH release (9, 10). However, it is often very difficult to ascertain that ACTH levels have been fully brought back to basal levels, particularly when these levels fall outside the reliable portion of the immunoradiometric assay standard curve. Consequently, at least in our view, it has not been possible to reliably establish that CRF antibodies completely prevent the pituitary’s response to EtOH. Also, the possibility that EtOH directly stimulates adrenal corticosterone secretion (11) is incompatible with measuring levels of this steroid as an index of altered pituitary activity in vivo. Finally, whereas some investigators have suggested a stimulatory effect of acute EtOH treatment on isolated corticotropes (12, 13), studies carried out in our laboratory did not lead us toward a similar conclusion (5). Thus, we do not believe that presently available in vivo experiments have satisfactorily determined whether the acute systemic injection of EtOH influences pituitary activity independently of CRF and/or VP, or not. This unresolved issue remains a major obstacle in the elucidation of the mechanisms through which EtOH stimulates the HPA axis.

Another problem in the characterization of EtOH’s precise sites of action is the ability of this drug to readily diffuse throughout the periphery and the brain (14). We therefore decided to differentiate between the importance of these two regions with the following approaches. First, we developed a model in which EtOH would stimulate the HPA axis by strictly acting within the brain, rather than by reaching other levels of the HPA axis (i.e. corticotropes in the pituitary and the adrenals). This was done by establishing the conditions under which the intracerebroventricular (icv) injection of this drug would release ACTH and stimulate PVN neuronal activity. Second, we tested the hypothesis that the peripheral administration of EtOH influenced corticotropes’ responses independently of endogenous CRF and/or VP, and compared these results to those obtained in the icv model. To circumvent the problems linked to ACTH measurement, we focused on changes in pituitary transcripts of the ACTH precursor proopiomelanocortin (POMC), and determined whether pretreatment with CRF and/or VP antibodies would interfere with the POMC response to EtOH. Coupled with our previous report of the effect of these antibodies on EtOH-induced ACTH release (1, 7), the present results provide novel information regarding the site of action of EtOH in activating the HPA axis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley rats (200–220 g) obtained from Harlan (Indianapolis, IN) were kept under standard light (lights on 0630–1830 h) and feeding (rat chow and water ad libitum) regimens. Aseptic insertion of an ip cannulae was done under isoflurane anesthesia 9–10 d before the assay (3). Intracerebroventricular cannulae were implanted in the left lateral ventricle under sc administration of ketamine (100 mg/kg)/acepromazine (4 mg/kg)/xylazine (10 mg/kg) also 9–10 d before the assay. Intraventricular cannulae were inserted into the jugular vein 48–72 h before the assay as previously described (2). All animals were singly housed to prevent chewing of the cannulae. All protocols were approved by The Salk Institute IACUC.

Protocols
On the day of the experiment, the animals were removed to a soundproof room, singly housed in opaque buckets and their iv, ip, and/or icv cannulae were connected to injection syringes. The rats were then left undisturbed for 3 h. After injection of the various treatments; blood samples (0.3 ml) were taken through the iv cannula in undisturbed rats, and immediately replaced with an equivalent volume of apyrogenic isotonic saline. Bloods were drawn into tubes that contained EDTA (10 µl of a 60 mg/ml solution) and placed on ice. They were centrifuged at 4 C, and plasma was stored at –20 C until assayed.

Reagents
Antibodies raised in our laboratory against CRF or VP were administered iv at doses (CRF antibodies: 0.4 ml/kg body weight; VP antibodies, 1.0 ml/kg body weight) and over a time frame (2–3 min before EtOH) that we previously showed sufficient to completely block the ACTH response to exogenous injections of large doses of the corresponding peptide (2, 15), and to significantly reduce ACTH release due to the ip injection of EtOH (5, 16). EtOH for ip injections was diluted to a less than 18% (vol/vol) final concentration in saline containing 10% anthocaine (Anthony Products Co., Arcadia, CA), a local anesthetic that we found useful in reducing possible discomfort. The dose chosen, 3.0 g/kg, causes moderate intoxication (3, 4) and was administered through the ip cannula to otherwise undisturbed and unhandled rats. Intracerebroventricular EtOH (USP grade, 200-proof, Aaper Alcohol and Chemical Co., Shelbyville, KY) was injected in a 5-µl volume at the rate of 1 µl/10 sec. Control rats received the appropriate vehicle(s).

ACTH and blood EtOH levels
Plasma ACTH levels were determined by a commercially available two-site immunoradiometric assay (Allegro kit, Nichols Institute, San Juan Capistrano, CA), which has been validated for the measurement of rat ACTH (17). The lower detection limit of this assay is 15 pg/ml, and samples in which ACTH levels were less than 15 pg/ml were assigned that value for statistical analysis. The intra- and interassay coefficients of variation are 7 and less than 15%, respectively. For measurement of EtOH levels in the cerebrospinal fluid (CSF), we used a 25-µl Hamilton syringe to withdraw 10 µl fluid from the cisterna magna of rats anesthetized with isoflurane. For measurement of blood EtOH levels (BALs), blood was withdrawn through the iv cannulae. EtOH concentrations were measured with an Analox AM 1 analyzer available from Analox Instruments Ltd. (Lunenburg, MA) (18). The reaction is based on the oxidation of EtOH by EtOH oxidase in the presence of molecular oxygen (EtOH + O2 —-> acetaldehyde + H2O2). Under the conditions of the assay, the rate of oxygen consumption is directly proportional to the EtOH concentration. Single point calibration is done for each set of samples with reagents, provided by Analox Instruments, that read 100 mg/dl (21.7 mmol/liter). The sensitivity of the assay is 1 mg/dl, the precision is 1–2% and the curve is linear up to 400 mg/dl.

cRNA probe synthesis and preparation
The pGEM3 vector containing an EcoRI fragment of rat CRF intron (provided by Dr. S. Watson, University of Michigan, Ann Arbor, MI) and the pGEM3 vector containing a rat VP gene fragment of intron I (provided by Dr. T. Sherman, University of Michigan) was linearized with HindIII. Radioactive cRNA copies were synthesized by incubation of 250 ng linearized plasmid in 6 mM MgCl2, 36 mM Tris (pH 7.5), 2 mM spermidine, 8 mM dithiothreitol, 25 mM ATP/GTP/CTP, [{alpha}-33P]-uridine triphosphate, 1 U RNasin (Promega, Madison, WI) and 10 U T7 RNA polymerase for 60 min at 37 C. Unincorporated nucleotides were removed using MicroSpin G-50 columns (Amersham Biosciences, Piscataway, NJ). A sense probe was used as a control for nonspecific signal in some adjacent sections for in situ hybridization. No signal was detected after pretreatment of sections with ribonuclease or hybridization with labeled sense-strand probes.

Brain preparation
Rats were deeply anesthetized with chloral hydrate, a drug that does not increase immediate early genes/peptides mRNA levels (Lee, S., and C. Rivier, unpublished observations). They were then perfused transcardially with saline followed by 4% paraformaldehyde/0.1 M borate buffer (pH 9.5). The brains were removed and postfixed in 4% paraformaldehyde for 4–5 d, then placed overnight in 10% sucrose/4% paraformaldehyde/0.1 M borate buffer. They were cut into 30-µm coronal slices obtained at 120-µm intervals throughout the hypothalamus, and stored at –20 C in a cryoprotectant solution (50% 0.1 M PBS, 30% ethylene glycol and 20% glycerol) until histochemical analysis. Brains from control and experimental animals belonging to the same experiment were always analyzed in the same hybridization experiment. Hybridization histochemical localization of each transcript was carried out using 33P-labeled cRNA probes prepared as previously described (19, 20). A sense probe was used as a control for nonspecific signal in some adjacent sections for in situ hybridization.

Quantitative analysis of in situ hybridization results
Semiquantitative densitometric analysis of hybridization signals for RNAs of interest was carried out in nuclear emulsion-dipped slides. Brain paste standards containing serial dilutions of 33P-uridine triphosphate, used for quantification of heteronuclear (hn) RNA signal, were prepared concurrently to ensure that OD was found within the linear range of the standard curve (21). In addition, analyses with emulsion-coated slides were carried out with two to three different exposure times to confirm that signals were not saturated. Densitometric analyses of autoradiographic signals were done over the confines of cells within the PVN using a Leitz optical system coupled to a Macintosh II computer and Image software (version 1.61, W. Rasband, National Institutes of Health). Dark-field measurements for the parvo- and magnocellular divisions of the PVN were obtained separately, as previously reported (2, 22). Gray level measurements (OD) were taken under dark-field illumination of hybridized sections over the medial parvo-PVN or magnocellular PVN, as defined by redirected sampling from the corresponding Nissl-stained sections under bright-field images. Data were expressed in gray-scale values of 1–256. All gray-level measurements were corrected for background, which corresponded to the areas immediately adjacent to those under study. Signals were measured in both sides of the brain, and mean values for all animals (4/group) were determined in three sections for each rat throughout the PVN.

POMC subcloning
A 589-bp POMC IAEX2IB fragment, which is made up of a 391-bp intron B and a 198 bp exon 2 of POMC (23), was isolated from its parent plasmid pBluescript M13+ vector (Dr. S. Watson) by a double digest with PstI and SpeI and subcloned into pBluescript SKII. POMC IAEX21B is an original clone that contains an 1100 bp of both intron A, exon 2 and intron B in a pBluescript M13+ vector. To generate a cloning site, a T4 polymerase treatment was done with the IAEX fragment to create blunt ends at the PstI 3'-overhang and SpeI 5'-overhang. pBluescript SKII was digested with EcoRV creating blunt ends and treated with calf intestine alkaline phosphatase enzyme for 1 h at 37 C. The IAEX insert size was verified by a double digestion of pBluescript SKII with BamHI and SalI. The fragment was sequenced to check the orientation. The pBluescript SKII vector containing POMC IAEX cDNA was linearized with HindIII, and the resulting T3 antisense transcripts were used as a cRNA probe for ribonuclease (RNase) protection assay.

RNase protection assay
Total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Valencia, CA). Rat POMC primary transcript, mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured simultaneously by RNase protection, using rat GAPDH as an internal loading control. A 615-nucleotide (nt) POMC antisense riboprobe specific to the rat POMC primary transcript and mRNA was synthesized using T3 RNA polymerase. A 200-nt antisense riboprobe specific to rat GAPDH mRNA was synthesized using T7 RNA polymerase. All riboprobes were synthesized, and RNase protection assays were carried out in our laboratory as described previously (24). The fragment sizes protected by POMC primary transcripts, mRNA and GAPDH riboprobes are 589, 165, and 135 nt, respectively. RNA samples (15 µg of anterior pituitary tissues) were hybridized in 30 µl of hybridization buffer containing 24 µl of deionized Formamide (Shelton Scientific, Shelton, CT), 3 µl of 10x hybridization buffer [4 M NaCl, 400 mM piperazine-N,N’-bis[2-ethanesulfonic acid] (pH 6.4), 10 mM EDTA, in diethyl pyrocarbonate water], 6.5 x 105 cpm POMC probe, 1.25 x 105 GAPDH probe, then brought to final volume with diethyl pyrocarbonate water. After heating at 85 C for 5 min, the samples were hybridized at 42 C for 16–18 h. After hybridization, 350 µl of RNase solution [0.375 M NaCl, 5 mM EDTA, 10 mM Tris-HCl (pH 7.5), 45.5 µg RNase A (Sigma, St. Louis, MO), 175 U RNase T1 (Ambion, Austin, TX)] was added to the hybridization solution and incubated for 1 h at room temperature. To stop RNases, 10 µl of 20% sodium dodecyl sulfate and 2 µl of Proteinase K (20 mg/ml) were added, then samples were vortexed and incubated for 15 min at 37 C. Samples were cleaned and lyophilized. Six microliters of loading dye were added, and samples were resolved on 6% polyacrylamide urea gels. Image analysis was performed using the PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) and the IMAGEQUANT 5.2 software package.

Fluoro-Jade detection of neuronal damage
Fluoro-Jade B (FJ; Histo-Chem Inc., Jefferson, AR) is an anionic tribasic fluorescein derivative that stains the cell bodies, dentrites, axons, and axon terminals of degenerating neurons but does not detect healthy neurons, myelin, vascular elements, or neuropil (25, 26). Brains, obtained over a time-frame relevant for the neuroendocrine responses induced by EtOH, were perfused and prepared as described above. They were cut into 30-µm coronal slices obtained at 120-µm intervals throughout the hypothalamus. Brain sections were then mounted onto gelatin-coated slides, dried, and immersed in 100% EtOH for 3 min, followed by a 1-min change to 70% EtOH and a 1-min change in distilled water. The slides were then transferred to a solution of 0.06% potassium permanganate and gently shaken for 15 min. They were then transferred to the FJ staining solution for 30 min, rinsed three times for 1 min each in distilled water, and air dried. The coverslipped slides were examined with a 450- to 490-nm excitation filter.

Statistical analysis
Data were analyzed by ANOVA followed by Fisher’s protected least significant difference test and Bonferroni/Dunn as a post hoc test. Each value was expressed as the mean ± SEM, and statistical significance was accepted for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of the icv injection of EtOH on neuronal integrity, and assessment of the parameters of icv injection
We recently provided evidence that neuronal damage, as assessed by FJ staining, was not detected in the PVN or any other brain regions studied over the course of the experiments (up to 3 h after the icv injection of 5 µl 200-proof EtOH) or when we extended the survival time up to 4 d after this treatment (27). By comparison, significant staining-related neuronal damage could be detected in the thalamus (but not the PVN) of rats injected with kainic acid (2 µl, icv) 2 d earlier. Similarly, in the present study 5 µl of 200-proof EtOH-injected icv did not result in FJ staining (not shown). We then conducted a series of preliminary experiments to identify the most appropriate volume and amount of EtOH necessary to elicit the endocrine responses in which we were interested. Our past experience, based on the appearance of nonspecific (i.e. vehicle induced) cellular responses in terms of c-fos (Lee, S., and C. Rivier, unpublished), argues against the injection of volumes larger than 5 µl. Consequently, our preliminary experiments were conducted with various dilutions of EtOH administered at that volume. Although this somewhat restricted our approach, we believe that not using larger volumes was experimentally justified (with one exception mentioned below). The icv injection of 2.5 µl 200-proof EtOH induced significant (P < 0.05) increases in the blood ACTH levels of some, but not all rats (not shown). On the basis of these inconsistent results, all subsequent studies were performed with a 5 µl dose of undiluted EtOH (200-proof; 86 µmol), which provided the most reliable and consistent activation of the HPA axis.

BALs of both vehicle- and EtOH-injected rats remained below the limit of detection of the assay in all experiments. CSF EtOH concentrations of rats injected with 5 µl of 200-proof EtOH (86 µmol) were within 1–5 mg% (21.25–106.25 mM) during the first 15 sec, then became undetectable. To determine whether this was due to the low amount of EtOH that was administered or represented an artifact, we also injected 7.5 or 10 µl EtOH (200-proof). Under these conditions, CSF concentrations of the drug were 23.6 ± 2.7 and 24.35 ± 2.55 mg% over the following 15–30 sec. Thus, the very transient presence of EtOH in the CSF of rats injected with a small dose of the drug icv appears to be a real finding, and this agrees with previously reported observations (28). By comparison, CSF EtOH levels induced by the peripheral injection of EtOH remain significant and detectable for much longer (29). Interestingly, however, and despite the very transient presence of EtOH in the CSF, both icv and intragastric-administered EtOH cause a very significant inhibition of the T response to human chorionic gonadotropin that is independent of LH release (29).

Having established the parameters necessary for our experiments, we then determined whether the icv injection of 5 µl 200-proof EtOH could stimulate HPA axis activity and if it did, to identify the site(s) at which this stimulatory effect took place. These results were compared with those obtained in rats injected with EtOH ip (3.0 g/kg) (see below).

Effect of the ip injection of EtOH on ACTH release, pituitary POMC synthesis, and PVN CRF and VP transcripts
ACTH.
The ip injection of EtOH, but not the vehicle, significantly (P < 0.01) increased plasma ACTH levels over a 60-min time frame, with a peak response measured at 15 min (Fig. 1Go).



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  		FIG. 1. Effect of the ip injection of alcohol (3.0 g/kg) on plasma ACTH levels. Each point represents the mean ± SEM of six rats. **, P < 0.01 vs. vehicle.

 
POMC.
We then determined whether this response was accompanied by increases in pituitary POMC transcripts. As a first step, we conducted preliminary studies to identify 1) the molecular tools best suited to detect significant changes in pituitary POMC transcripts, and 2) the time-course of changes in the levels of these transcripts. POMC mRNA primary transcripts levels in rats injected with EtOH ip (3.0 g/kg) increased only modestly over a time-course known to elevate plasma ACTH levels after EtOH treatment (mean percentage of control value ± SEM, 100.0 ± 15.6; EtOH 15 min, 114.4 ± 14.0; EtOH 30 min, 94.7 ± 8.8; EtOH 60 min, 115.0 ± 13.7; P > 0.05). Because we considered the magnitude of this response insufficient for future studies, we turned to the measurement of primary transcripts by RNase protection assay. EtOH (3.0 g/kg, ip) induced a significant (P < 0.01) increase in pituitary POMC primary transcript levels 15–60 min later (Fig. 2Go).



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  		FIG. 2. A, Time-related effect of the ip injection of alcohol (3.0 g/kg) on pituitary POMC primary transcript levels. Each bar represents the mean ± SEM of three to four rats. **, P < 0.01 vs. vehicle. Fifteen micrograms of total RNA were loaded in each lane. Values were calculated as POMC primary transcripts per GAPDH mRNA, then expressed as the percentage of the control value (vehicle-treated group). B, Representative autoradiogram of POMC primary transcripts, mRNA and GAPDH mRNA of vehicle- and alcohol-injected rats.

 
PVN CRF and VP transcripts.
We have already reported that ip-injected EtOH (3.0 g/kg) significantly up-regulated PVN CRF hnRNA levels measured 20–40 min after drug administration, whereas changes in VP transcripts did not reach statistical significance over this time frame (10). These measurements were not repeated here.

Effect of the icv injection of EtOH on ACTH release, pituitary POMC synthesis, and PVN CRF and VP transcripts
ACTH.
The icv injection of EtOH, but not the vehicle, significantly (P < 0.01) increased plasma ACTH levels over a 60 min time-frame, with a peak response measured at 15 min (Fig. 3Go).



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  		FIG. 3. Effect of the icv injection of alcohol (5 µl of 200-proof; 86 µmol) on plasma ACTH levels. Each point represents the mean ± SEM of five to seven rats. **, P < 0.01 vs. vehicle.

 
POMC.
Anterior pituitary POMC primary transcript levels responses were significantly (P < 0.01) increased 15, but not 30, min after icv EtOH administration (Fig. 4Go). In contrast, there were no significant effects on POMC mRNA levels (Table 1Go).



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  		FIG. 4. A, Time-related effect of the icv injection of alcohol (5 µl of 200-proof; 86 µmol) on pituitary POMC primary transcript levels. Each bar represents the mean ± SEM of five to six rats. **, P < 0.01 vs. vehicle. B, Representative autoradiogram of POMC primary transcripts, mRNA and GAPDH mRNA of vehicle- and alcohol-injected rats.

 

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  		TABLE 1. Changes in POMC mRNA levels in response to alcohol

 
PVN CRF and VP transcripts.
The icv injection of 5 µl 100-proof EtOH significantly (P < 0.01) up-regulated CRF hnRNA levels in the parvocellular portion of the PVN (Fig. 5Go). In contrast, EtOH did not alter VP hnRNA in the magnocellular division of the PVN (vehicle, 15.6 ± 1.1 arbitrary units; 15 min after EtOH treatment, 13.1 ± 1.4 arbitrary units; 30 min after EtOH, 14.9 ± 3.7 arbitrary units, P > 0.05).



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  		FIG. 5. A, Effect of the icv injection of alcohol (5 µl of 200-proof; 86 µmol) on PVN CRF hnRNA levels. Dark-field photomicrographs show coronal sections through the PVN 30 min after vehicle or alcohol injection. This time point was chosen because, although significant increases could already be detected at 15 min (not shown), they were inconsistent between animals. III, Third ventricle. B, Statistical analysis of the data presented in Fig. 5AGo. Each bar represents the mean ± SEM of four rats. *, P < 0.05.

 
Role of endogenous CRF and VP in modulating the ACTH and pituitary POMC response to the ip or icv injection of EtOH
ACTH.
Although we obtained a full time-course of the ACTH response to ip or icv administered EtOH in the presence or absence of CRF and/or VP antibodies, we only illustrate here data obtained at the time of peak ACTH levels (i.e. 15 min) in order not to repeat data presented in Figs. 1Go and 3Go. CRF or VP antibodies were injected 2–3 min before EtOH, a time-frame sufficient to completely block the effect of these peptides on ACTH release (1, 16). As expected (1, 10), the absence of endogenous CRF drive reduced the ACTH response to ip-injected EtOH by more than 90% (Fig. 6AGo). It exerted a similar influence in the icv model (Fig. 6BGo). VP antibodies also blunted these responses, and removal of both CRF and VP totally abolished them.



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  		FIG. 6. Effect of the immunoneutralization of endogenous CRF, VP or both peptides on the ACTH response to the ip (A, 3.0 g/kg) or icv (B, 5 µl of 200-proof; 86 µmol) injection of alcohol. Plasma ACTH levels were measured 15 min after alcohol administration. Antibodies or their vehicle were injected iv 2–3 min before alcohol. Each bar represents the mean ± SEM of six rats. a, P < 0.01 vs. vehicle/vehicle; **, P < 0.01 vs. corresponding vehicle/EtOH.

 
POMC.
To further investigate the physiological role played by CRF and VP in modulating the pituitary response to the icv injection of EtOH, we measured pituitary POMC levels in rats pretreated with the vehicle or antibodies to CRF, VP, or both peptides. In our hands, the influence of CRF antibodies on basal POMC and ACTH levels can be variable, and this is reflected for example by the slight (but not significant, P > 0.05) decrease in POMC primary transcripts measured in the experiments illustrated in Figs. 7Go and 9Go, but not the one illustrated in Fig. 8Go. EtOH-injected ip or icv produced the expected increase in POMC levels (Figs. 7–9GoGoGo). In the ip model, separate removal of endogenous CRF or VP reduced this response, but this effect did not reach statistical significance (Fig. 7Go). In contrast, simultaneously depriving the corticotropes of both peptides completely abolished EtOH-induced increases in POMC hnRNA levels (Fig. 8Go). In rats injected with EtOH icv, in which only concomitant pretreatment with CRF and VP antibodies was tested, the pituitary POMC response was also abolished by removal of these peptides (Fig. 9Go). Measurement of POMC mRNA levels showed only slight changes that only reached statistical significance in pituitaries obtained from rats injected with CRF and VP antibodies before icv EtOH (Table 1Go).



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  		FIG. 7. A, Effect of the immunoneutralization of endogenous CRF or VP on the pituitary POMC response to the ip (3.0 g/kg) injection of alcohol. Antibodies or their vehicle were injected iv 2–3 min before alcohol and anterior pituitaries were collected and extracted for total RNA after alcohol injection. Each bar represents the mean ± SEM of three to four rats. a, P < 0.01 vehicle/vehicle. B, Representative autoradiogram of POMC primary transcripts, mRNA and GAPDH mRNA of vehicle- and alcohol-injected rats. For details, see Fig. 2Go.

 


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  		FIG. 9. A, Effect of the immunoneutralization of both endogenous CRF and VP on the pituitary POMC response to the icv (5 µl of 200-proof; 86 µmol) injection of alcohol. Antibodies or their vehicle were injected iv 3–5 min before alcohol, and anterior pituitaries were collected and extracted for total RNA 15 min after alcohol injection. Each bar represents the mean ± SEM of four to five rats. a, P < 0.05 vs. vehicle/vehicle; **, P < 0.01 vs. vehicle/icv EtOH. B, Representative autoradiogram of POMC primary transcripts, mRNA and GAPDH mRNA of vehicle- and alcohol-injected rats. For details, see Fig. 2Go.

 


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  		FIG. 8. A, Effect of the immunoneutralization of both endogenous CRF and VP on the pituitary POMC response to the ip (3.0 g/kg) injection of alcohol. Antibodies or their vehicle were injected iv 3–5 min before alcohol, and anterior pituitaries were collected and extracted for total RNA 15 min after alcohol injection. Each bar represents the mean ± SEM of four to five rats. a, P < 0.01 vs. vehicle/vehicle; *, P < 0.05 vs. vehicle/ip EtOH. B, Representative autoradiogram of POMC primary transcripts, mRNA and GAPDH mRNA of vehicle- and alcohol-injected rats. For details, see Fig. 2Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because the peripheral injection of EtOH results in its diffusion throughout the body, including the endocrine hypothalamus, the sites of action of this drug in releasing ACTH have remained controversial. The first part of our work focused on testing the hypothesis that EtOH could up-regulate HPA axis activity through mechanisms that primarily targeted the brain, and not the median eminence-pituitary-adrenal axis. This was done by determining whether the icv injection of EtOH at doses that did not cause neuronal damage over the time-course relevant for our experiments (Ref. 27 and present work) and was not accompanied by measurable levels of the drug in the circulation, stimulated PVN CRF transcripts, and released ACTH. We show here that this was the case, and indeed, results pertaining to PVN neuronal activity extend a recently published article regarding the influence of icv-injected EtOH on the central nervous system (30). These results, which complement very similar ones that we previously obtained with a model of ip EtOH injection (1, 10, 16), convincingly demonstrated that EtOH can activate the HPA axis through primarily central mechanisms, i.e. those that involve the PVN and/or its afferents. However, it is well known that the icv injection of compounds leads to their rapid appearance in the general circulation (31, 32, 33, 34). We therefore measured BALs in rats injected with EtOH icv. Although these levels remained undetectable in the general circulation, we could not be absolutely certain that EtOH had not reached nerve terminals in the median eminence and/or the corticotropes via the portal circulation. Thus, although of interest, our results did not unequivocally prove that EtOH acted strictly on the PVN and/or its afferents to activate the HPA axis. Microdialysis of EtOH directly into potential target regions such as the PVN, an approach that has been used for other brain areas potentially involved in a variety of neuronal responses and behaviors (see, for example, Refs. 35 and 36), will undoubtedly provide important information in this regard. At this point, it may also be useful to make a comment regarding the functional relationship between PVN CRF and pituitary POMC/ACTH. We show here that peak increases in these latter parameters were measured 15 min after EtOH injection, but 30 min for CRF. This should not be regarded as inconsistent because this apparent discrepancy most probably pertains to methodological considerations rather than to the lack of a direct correlation between CRF and ACTH/POMC. Indeed, up-regulated PVN CRF synthesis, as reflected by increased hnRNA levels, was already detectable by 15 min in most rats (see legend of Fig. 2Go), but we chose to illustrate data at the 30 min time point because changes were more consistent.

In view of these considerations, and to provide further support of the involvement of the PVN in the ability of icv-injected EtOH to activate the HPA axis, the second part of our work was aimed at determining whether the drug could stimulate the corticotropes in the absence of endogenous CRF and/or VP. CRF is known to up-regulate pituitary POMC synthesis in normal rats (37, 38) as well as in AtT20 cells (39, 40). The acute systemic injection of EtOH increases hypothalamic CRF synthesis in the PVN and release to the pituitary (4, 5, 10), and it was therefore reasonable to expect that this peptide would also increase pituitary POMC synthesis. However, to our knowledge this had not been demonstrated in intact animals. We show here that indeed, the ip injection of EtOH significantly up-regulated pituitary POMC primary transcripts. Many studies have shown that there is a parallel relationship between primary transcript levels and protein release (41), including in the case of plasma ACTH levels and anterior pituitary POMC primary transcript (42). In this latter study, the increase in POMC primary transcript was followed by an increase in POMC intermediate processing RNA without alterations in POMC mRNA levels. These observations suggest that stimulation of POMC gene transcription accompanies maturation of the primary transcripts. Thus, our finding that the time-course of changes in pituitary POMC transcripts corresponded to the known pattern of ACTH release into the general circulation (1), is further evidence that measurement of POMC levels was an excellent index of activation of the corticotropes. Nevertheless, the stimulatory effect of peripherally injected EtOH on PVN CRF and VP (1), coupled with the above-mentioned ability of CRF to stimulate pituitary POMC synthesis, did not allow us to distinguish between a direct influence of EtOH on the corticotropes, and an effect mediated through CRF and/or VP. Furthermore, because endogenous CRF plays such a major role in ACTH release (43, 44), it has been difficult to convincingly rule out that EtOH might not act directly on the corticotropes even though immunoneutralization of this peptide blocked its stimulatory effect. To address this issue, we therefore determined whether the pituitary POMC response to EtOH would be retained in the absence of endogenous CRF and/or VP, i.e. in rats pretreated with CRF or VP antibodies. We show here that removal of either CRF or VP only slightly decreased the POMC response to ip-injected EtOH. In contrast, the absence of both peptides completely abolished the ability of the drug to stimulate corticotropes activity regardless of whether EtOH was injected ip or icv. Whereas these results unequivocally demonstrate the role concomitantly played by CRF and VP in mediating the stimulatory influence of blood-borne EtOH on pituitary POMC synthesis and ACTH release, the lack of efficacy of the CRF or VP antibodies alone on EtOH-induced POMC synthesis (at least in the ip model, the only one we studied with this protocol) was surprising in view of the known ability of these peptides to significantly decrease ACTH secretion in the ip as well as icv model (Refs. 5, 6, 7, 8 and present data). However, because we obtained comparable results in two different experiments, we feel confident that our observations were valid.

These findings not only demonstrate, as indicated above, that endogenous CRF and VP play obligatory roles in the influence of systemic EtOH on pituitary corticotropes—i.e. EtOH does not act directly on the pituitary to release ACTH, they also suggest that EtOH can target PVN CRF and VP neurons through an influence that is strictly mediated at the central level. This being said, a still unresolved question pertains to the site(s) of action of EtOH: does EtOH primarily act on the PVN or does it also target PVN afferents? Redei et al. (45) had reported that EtOH increased radioimmunoassayable CRF levels by superfused rat hypothalami. Although of interest, these results do not resolve the relative importance of the PVN and other hypothalamic structures. Our own studies in rats injected with EtOH icv failed to indicate significant increases in levels of the early genes often used to mark neuronal activation, such as Fos and NGFI-B, in regions known to have direct inputs to the PVN. However, it is unlikely that such an approach would permit us to rule out phenomena of disinhibition such as those linked to the GABA innervation of the PVN (see for example Refs. 46 and 47). Crankshaw et al. (30) reported that icv-injected EtOH up-regulated Fos signals in brain sites associated with feeding and reward, including the bed nucleus of the stria terminalis, the lateral dorsal area, the nucleus accumbens, and the lateral septum. However, none of these regions directly innervate the PVN. On the other hand, we sometimes observed increased neuronal activity in the amygdala of rats injected with EtOH systemically (Lee, S., and C. Rivier, unpublished observations), a finding also recently reported by others (48). These signals tended to be slightly delayed, compared with those observed in the PVN, and at present it is difficult to determine whether this response is primarily linked to some emotional charge linked, for example, to the fear of losing balance, and whether it is functionally linked or not to the activation of the HPA axis. Consequently, our results suggest, although they certainly do not prove, that the PVN represents the primary site of EtOH of the drug when it is administered icv.

In conclusion, the present work confirms and extends results previously reported by us and others showing that EtOH can stimulate PVN CRF and VP neurons. Using a newly developed model that allowed us to isolate EtOH influence in the brain from its peripheral effect, we now show that this hypothalamic response does not require the presence of circulating levels of the drug. It was of interest to note, as did others who used a model of icv EtOH injection (30, 49), how despite its remarkably ephemeral presence in the brain ventricles, EtOH can nevertheless induce physiologically relevant biological responses. A still unresolved question, however, regards the potential role of EtOH metabolites such as acetaldehyde in mediating the influence of EtOH. Studies conducted with the injection of this compound and/or in rats pretreated with blockers of EtOH metabolism are likely to resolve this issue. Also deserving further work is the identification of the mechanisms through which icv EtOH exerts its endocrine effects, such as the involvement of ligand-gated ion channels and/or second messengers. In the meantime, our work provides the first unambiguous evidence that the stimulatory effect exerted by blood-borne EtOH on the corticotropes does not involve a direct pituitary influence of this drug, but requires the release of endogenous CRF and VP.


   Acknowledgments
 
The authors are indebted to Elaine Law, Melissa Herman, Yaira Haas, Ahn-Khoi Nguyen, Raymond Chan, and Michael Rothwell for excellent technical support, and to Debbie Doan for manuscript preparation.


   Footnotes
 
This work was supported by National Institutes of Health Grant AA-06420 and the Foundation for Research.

Abbreviations: BAL, Blood EtOH level; CRF, corticotropin-releasing factor; EtOH, ethanol; FJ, Fluoro-Jade; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hn, heteronuclear; HPA, hypothalamic-pituitary-adrenal; icv, intracerebroventricular(ly); nt, nucleotides; POMC, proopiomelanocortin; PVN, paraventricular nucleus; RNase, ribonuclease; VP, vasopressin.

Received January 30, 2004.

Accepted for publication June 10, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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