Role Played by Brainstem Neurons in Regulating Testosterone Secretion via a Direct Neural Pathway between the Hypothalamus and

the Testes
Daniel J. Selvage, Loren Parsons and Catherine Rivier Endocrinology 2006 147:3070-3075 originally published online Mar 23, 2006; , doi: 10.1210/en.2005-1358

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Endocrinology 147(6):3070 3075 Copyright 2006 by The Endocrine Society doi: 10.1210/en.2005-1358

Role Played by Brainstem Neurons in Regulating Testosterone Secretion via a Direct Neural Pathway between the Hypothalamus and the Testes
Daniel J. Selvage, Loren Parsons, and Catherine Rivier
The Clayton Foundation Laboratories for Peptide Biology (D.J.S., C.R.), The Salk Institute; and The Scripps Research Institute (L.P.), Department of Neuropharmacology, La Jolla, California 92037
We previously reported anatomical and functional evidence for a direct, inhibitory neural pathway that regulates testosterone (T) secretion independently of the pituitary. This pathway is activated by the intracerebroventricular (icv) administration of agents that stimulate stress responses, such as IL-1 , corticotropin-releasing factor (CRF), and norepinephrine (NE), which results in a blunted T response to the administration of human chorionic gonadotropin (hCG). Blunting of the T response is mediated by central -adrenergic receptor stimulation. CRF, but not ethanol (EtOH) or IL-1 , acts directly on the paraventricular nucleus of the hypothalamus to activate the pathway. Here we explored the role played by brain areas hypothesized to be part of this pathway, such as neurons in the dorsal pons [including the locus coeruleus (LC) of the brainstem], where NE is produced. Microinfusion of EtOH or IL-1 , but not CRF, into these neurons activated the pathway. Electrolytic lesions of this region significantly reversed the inhibitory effect of icv-administered EtOH on hCG-induced T release, while having no effect on the ability of IL-1 or CRF to do so. However, the icv administration of IL-1 , EtOH, or CRF, in doses that rapidly inhibit the T response to hCG, all caused a significant depletion of NE from the LC. Collectively, these results indicate that in addition to the paraventricular nucleus, the brainstem area containing the LC is part of a neural pathway that connects the brain to the testes independently of the pituitary. We also speculate that EtOH may stimulate this pathway through NEdependent activation of the dorsal pons. (Endocrinology 147: 3070 3075, 2006)

ESTOSTERONE (T) SECRETION by Leydig cells is regulated by the pituitary gonadotropin LH, which itself is under the regulatory influence of the hypothalamic peptide LHRH (13). We recently reported anatomical and functional evidence for an additional means by which T secretion is controlled: a direct, multisynaptic neural pathway between the brain and the testes that provides a rapid inhibitory regulation of T release that is independent of the LHRH-LH axis (4). Support for this novel view of the rapid regulation of T release stems from the fact that the intracerebroventricular (icv) injection of agents associated with stress responses, such as corticotropin-releasing factor (CRF) (5), IL-1 (6, 7), and ethanol (EtOH) (8, 9) all rapidly inhibited the T response to human chorionic gonadotropin (hCG) independently of LH, corticosteroids, or prolactin release, and in the absence of detectable changes in testicular blood flow (10). The influence of CRF, IL-1 , or EtOH was mimicked by the icv injection of -adrenergic agonists, whereas specific blockade of -adrenergic receptors restored normal Leydig cell activity in rats injected icv with CRF, IL-1 , or EtOH (7, 8). The rapidity of the effect of these icv-administered agents
First Published Online March 23, 2006 Abbreviations: CRF, Corticotropin-releasing factor; EtOH, ethanol; hCG, human chorionic gonadotropin; icv, intracerebroventricular; LC, locus coeruleus; NE, norepinephrine; NTS, nucleus of the solitary tract; 6-OHDA, neurotoxin 6-hydroxydopamine; PRV, pseudorabies virus; PVN, paraventricular nucleus; T, testosterone. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

on T secretion suggested that the responses were dependent on a neural, rather than hormonal, mechanism. Anatomically, this pathway has been mapped by the injection of the transganglionic retrograde tracer pseudorabies virus (PRV) into the rat testes, followed by the monitoring of the virus progression to various brain regions (12, 13). The fact that spinal cord transection at the T7/T8 level prevented the progression of the virus from the testes to the central nervous system (13) suggested that this inhibitory neural circuit traveled through the spine, at least from the midthoracic region upwards. The main areas labeled by PRV included the paraventricular nucleus (PVN) of the hypothalamus, the central amygdala, the nucleus of the solitary tract (NTS), the locus coeruleus (LC) of the brainstem, and the cortex (12). Of note, the Barringtons nucleus only displays very modest labeling, which makes it an unlikely candidate as an important part of the proposed pathway. In investigating the roles of these nuclei, we found that although the PVN represented an important functional site of action for CRF and the -adrenergic agonist isoproterenol (5, 10, 13), the main site of action of EtOH was located outside this nucleus. Because of this, we chose to focus on the role of noradrenergic neurons in the dorsal pons because 1) the icv injection of -adrenergic agonists and antagonists had suggested the importance of catecholamines as regulators of the proposed pathway; 2) catecholamine depletion by the neurotoxin 6-hydroxydopamine (6-OHDA) fully reversed the ability of icv EtOH to stimulate this circuit, and partially did so in the case of icv IL-1 (10); and 3) the LC displayed a more intensive labeling than the NTS during viral tracing (12).

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Materials and Methods Animals

Adult male Sprague Dawley rats (Harlan Laboratories, Indianapolis, IN), weighing 200 220 g on arrival, were housed individually after any surgical treatment under controlled lighting conditions (12-h light, 12-h dark) with food and water available ad libitum. All protocols were approved by The Salk Institute Institutional Animal Care and Use Committee.

Lesions and surgeries

Bilateral, electrolytic lesions of the region of the dorsal pons that includes the LC (LCx, illustrated in Fig. 1) were made by passing a 0.8 mA current through a tungsten-tipped electrode (A-M Systems, Inc., Carlsborg, WA) for 10 sec. Animals anesthetized with ketamine (100 mg/kg)/acepromazine (4 mg/kg)/xylazine (10 mg/kg) were placed in a stereotaxic device, with the electrode arm held at a 15-degree angle. Lesions were made using the following measurements from : anteriorposterior, 3.4 mm; lateral, 1.2 mm; dorsoventral, 6.8 mm. Sham electrolytic lesions were performed using the same coordinates, but the electrode was lowered 5 mm dorsoventral, and no current passed through it. Intracerebroventricular cannulae (coordinates from bregma: anteriorposterior, 0.4 mm, lateral, 1.4 mm; dorsoventral, 3.8 mm) were inserted 7 8 d after LC lesions or, in animals without these lesions, 7 8 d before experimentation (5). Placement of LC lesions and icv cannula for each animal was checked by examining Nissl-stained sections of brains cut on a cryostat. Only animals with appropriately placed cannula and lesions determined to have destroyed the LC with none, or only very minimal damage to the adjoining Barringtons nucleus were used in the statistical analysis of the data. Intravenous cannulae were inserted 48 h before experimentation under isoflurane anesthesia as previously described (10). On the day of the assay, EtOH (86 m), CRF (2 g), IL-1 (80 ng), or vehicle was infused icv to a total volume of 5 l. hCG (1 IU/kg) was injected iv 20 min later. Blood samples were drawn before icv infusion, at the time of hCG injection, and 15, 30, and 60 min after injection, or as noted.

terior-posterior, 3.2 mm; lateral, 0.8 mm; dorso-ventral, 4.2 mm. The cannulae were held in place using plastic dental cement and small machine screws, and kept patent by insertion of indwelling stylets. After 710 d recovery time, animals were fitted with iv catheters and allowed to recover a further 23 d. One microliter of IL-1 (10 ng/side), CRF (0.5 g/side), EtOH (17 m/side) or vehicle (apyrogenic water) was bilaterally infused directly into the LC over 5 min, using an automated microinfusion pump (Harvard Instruments, Cambridge MA). We previously showed that none of these treatments induced tissue damage over the time-course of the experiment when given icv, as indicated by the absence of FluoroJade staining (14). Twenty minutes after microinfusion, hCG (1 IU/ kg, purchased from Sigma Corp., St. Louis, MO) was injected iv. Blood samples for T measurement were taken before microinfusion of agents, at the time of hCG injection, and 15, 30, and 60 min later, or as depicted in Results.

Experimental protocol
On the day of experimentation, animals were removed to a soundproof room and housed individually in opaque buckets. Their iv catheters and icv cannulae were connected by polyethylene tubing to injection sites outside the buckets, such that blood could be withdrawn, and treatments administered, without disturbing the freely moving animals. All injections were given at least 2 h after rehousing, a procedure we previously found necessary to allow a return of all stress-related hormone levels to normal (Lee, S., and C. Rivier, unpublished).

LC monoamine measurements
After the icv administration of IL-1 (80 ng), CRF (2 g), EtOH (86 m), or vehicle, animals were decapitated and brains quickly removed, quick-frozen with cold isopentane in dry ice, and stored at 70 C until assayed. LC tissue samples were collected using 2-mm diameter neuropunches (Fine Science Tools Inc., Foster City, CA). Briefly, using the rostral paraflocculus as a landmark for the first incision, horizontal brain sections were made. A second horizontal incision was made 1 mm caudal to this, using the vestibule cochlear ganglion and caudal flocculus as external landmarks. This resulted in a brain section of 1 mm in thickness, from which bilateral LC punches were taken just ventral to the most lateral extent of the fourth ventricle in these sections. Because we wanted to include as much of the LC as possible in our analysis, and because it is a somewhat irregular nucleus, it is possible that bordering areas were included in the punches. The mean wet weight of the bilateral tissue samples was 4.1 0.23 mg, and tissue samples not within one sd ( 1.25 mg) of this weight were not used in the final analysis. Monoamine measurement from these punches by HPLC was based on previously

Microinfusion studies
Deeply anesthetized animals were fitted with bilateral guide cannulae inserted directly above the LC or the cortex. The coordinates from used for the LC were: anterior-posterior, 3.4 mm; lateral, 1.2 mm, dorsoventral, 5.8 mm. The injection needle projected 1 mm from the end of the guide cannula. Coordinates for the frontal cortex were: an-

FIG. 1. Representative illustrations (coronal sections after Nissl stain) of a control and a lesioned brain from rats undergoing sham or electrolytic lesions. To include the entire region of interest on each side of the brain, we show a separate picture for each side of the same animal (L, left; R, right).

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published methodology (15). Briefly, preweighed tissue samples were homogenized by ultrasonic disruption in 250 l of chilled 0.1 n perchloric acid containing 100 nm 3,4-dihydroxybenzylamine as an internal standard. After centrifugation, 30 l of the supernate were injected onto an HPLC column (2 150 mm BetaBasic C18, 3 m particles 150 pore size; Keystone Scientific, Bellefonte, PA) and monoamines and their metabolites were eluted using a mobile phase consisting of 150 mm citric acid, 15 mm sodium acetate, 1.4 mm sodium octyl sulfate, 100 m EDTA, 29 mm triethylamine and 5% (vol/vol) methanol (apparent pH of 2.5) delivered at 0.1 ml/min by an 1100 series HPLC pump from Agilent Technologies (Wilmington, DE). The column eluent was delivered directly to a standard electrochemical cell containing two glassy carbon working electrodes (model MF-1000; BioAnalytical Systems, Lafayette, IN) arranged in series and maintained at 700 mV against an Ag/AgCl reference electrode (Model RE4; BioAnalytical Systems). The electrode potential and current analyses were controlled by an LC-4B amperometric detector (BioAnalytical Systems). External calibration curves were generated daily from fresh standard solutions, and the limit of detection was approximately 4 nm for all analyses.

FIG. 2. The icv administration of EtOH (86 M), CRF (2 g), or IL-1 (80 ng) causes depletion of NE stores in the LC. Tissue was taken 20 30 min after icv injection, and processed for catecholamine content using HPLC as described in Materials and Methods. Each bar corresponds to mean NE levels for seven to eight rats SEM. *, P 0.05; **, P 0.01.

Reagents
Absolute, reagent grade EtOH (USP, 200 proof) was purchased from Aaper EtOH and Chemical Co. (Shelbyville, KY). It is free of any additives. We conducted preliminary studies to determine the optimum dose, which was chosen as that producing maximum biological responses but no neuronal damage (Selvage, D. J., and C. Rivier, unpublished; also see Results). Recombinant human IL-1 was generously provided by Otsuka Pharmaceutical Co. (Japan). Rat/human CRF was synthesized by solid phase methodology (16) and generously provided by Dr. Jean Rivier (The Salk Institute). IL-1 and CRF were diluted in apyrogenic water. hCG was purchased from Sigma Corp. and diluted in apyrogenic saline. The doses of IL-1 (80 ng, icv), CRF (2 g, icv), and 1 IU hCG (1 IU/kg, iv) were chosen on the basis of our previous experience (57). Vehicle treatments consisted of apyrogenic water alone.

dorsal pons containing the LC mimicked the influence of their icv injection (Fig. 3). Microinfusion of these agents into the frontal cortex was used as a control. hCG (1 U/kg, iv) was injected 20 min after the end of the microinfusions. A first blood sample was obtained immediately before hCG, and subsequent samples were taken 15, 30, and 60 min later. Compared with vehicle, the microinfusion into the LC of 10 ng IL-1 [F (14, 20) 7.323, P 0.001] or 17 m EtOH/side [F (17. 20) 1.33, P 0.05], but not 0.5 g CRF/side, blunted the T response to hCG). Microinfusion of CRF, IL-1 , or EtOH into the cortex had no effect.
Effect of dorsal pons lesions on hCG-induced T

Testosterone measurement
T was measured in 50 l duplicate unextracted plasma samples, using a commercially available, solid-phase RIA kit (Diagnostic Products Corp., Los Angeles, CA). The characteristics of this assay have been published (7). Data are presented as a time-course release or as cumulative T levels, measured by adding values measured at all time points after hCG injection.

Statistical analysis
Data were analyzed by one-way ANOVA with repeated measures, and by Students t tests or Bonferroni/Dunn.

Results LC catecholamine levels

The purpose of these experiments was to investigate the ability of icv-injected EtOH, IL-1 , or CRF to alter norepinephrine (NE) levels in the LC. Compared with vehicle, the icv administration of 86 m EtOH [F (7, 7) 10.66, P 0.001], 2 g CRF [F (7, 7) 12.82, P 0.001] or 80 ng IL-1 [F (7, 7) 2.025, P 0.05], all significantly lowered NE content in the LC (Fig. 2). We previously reported that the icv administration of these agents also altered NE levels in the cortex (10), indicating that they probably induce a generalized release of this amine in the brain.
Microinfusion of CRF, IL-1 , or EtOH into the dorsal pons or frontal cortex

These experiments were performed to determine whether lesions of adrenergic neurons of the dorsal pons would interfere with the ability of icv-injected EtOH, IL-1 , or CRF to blunt the T response to hCG. hCG (1 U/kg, iv) was injected 20 min after icv treatments. A first blood sample was obtained immediately before hCG, and subsequent samples were taken 15, 30, and 60 min later. Electrolytic lesions did not alter the ability of the rats to secrete T in response to hCG, which represented an essential control (Fig. 4A). In LCx animals, the T response to hCG was significantly blunted by the icv administration of 80 ng IL-1 , injected 20 min before the gonadotropin [F (11, 17) 3.061, P 0.01] (Fig. 4B) or 2 g CRF [F (19, 20) 12.82, P 0.01] (Fig. 4C), and the magnitude of this decrease was comparable with that measured in sham-lesioned animals. In contrast, LC-lesioned animals receiving icv EtOH (86 m) showed no blunting of hCG-induced T release, compared with sham (Fig. 4D).
Discussion

These experiments were performed to determine whether microinfusion of CRF, IL-1 , or EtOH into neurons of the

Our aim was to further characterize the CNS area(s) and mechanisms involved in the inhibitory effect exerted by brain-infused IL-1 , CRF, or EtOH on testicular activity via a neural, pituitary-independent mechanism. Although we previously reported evidence showing that CRF acted directly on the PVN to activate the pathway and blunt the T response to hCG (5), our experiments indicated that IL-1 or EtOH did not (4, 10), and PVN lesions only partially affected the ability of EtOH to do so (10). This suggested the potential involvement of one or more other brain regions in the path-

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FIG. 3. Effect of the microinfusion of EtOH (17 M/1 l/side), IL-1 (10 ng/1 l/side) or CRF (0.5 g/1 l/side) into the dorsal pons/LC (A) or the frontal cortex (B), on the T response to hCG (1 U/kg). hCG was injected 20 min after the end of the microinfusions. The zero time corresponds to T levels immediately before hCG administration. EtOH and IL-1 microinjected into the dorsal pons blunted hCG-induced T release, but CRF did not. There was no effect of EtOH, IL-1 or CRF microinfused into the cortex. Panels on the left show the time course for T release. Panels on the right show cumulative T release. Each point/bar represents the mean of seven to eight rats SEM. *, P 0.05; **, P 0.01.

way. Because the proposed circuit is stimulated by NE (7), we focused on adrenergic neurons in the dorsal pons, including the LC, which represent the site of many brain catecholamine neurons (17) and was identified by PRV studies as a possible part of the circuit under study (12, 13). We show here that the icv administration of IL-1 , CRF, or EtOH all significantly decreased NE levels in the adrenergic dorsal pons. This finding, combined with our previous observation that the icv injection of these compounds increased NE levels in the cortex (10), suggests that stimulation of the pathway may cause diffuse NE secretion throughout the brain. The effects on dorsal pons noradrenergic content were most dramatic in response to the icv injection of EtOH, indicating that this drug, when injected in an amount small enough that it cannot be detected in the blood stream or in the cerebrospinal fluid of the fourth ventricle of the brain (9, 10), can nonetheless have a potent effect on catecholamine release and brain function. Interestingly, the same dose (86 m) of EtOH injected icv also rapidly stimulates the activity of PVN CRF cell bodies and induces subsequent ACTH release from the pituitary (14), further indicating that even a very brief exposure to alcohol can have significant biological effects. These observations corroborate those of others who reported that the icv

injection of even smaller doses of EtOH were able to modify ingestive behaviors and increase locomotor activity (18, 19). Finally, because alcohol delivered to the brain ventricles up-regulates PVN CRF mRNA levels (10), the mechanisms mediating the influence of this drug may involve CRF, a hypothesis that we are currently testing. CRF itself, on the other hand, exerts its primary influence directly on the PVN via, at least in part, -adrenergic receptors. This may explain why the influence of alcohol also depends on catecholamines. In both cases, this results in a rapid loss of Leydig cell response to LH-like molecules such as hCG. To further explore the role of dorsal pons neurons and the potential participation of catecholamines in the pathway, this region was either microinfused with CRF, IL-1 , or EtOH, or lesioned in animals receiving these agents icv. Microinfusion of IL-1 or EtOH, but not CRF, significantly blunted the T response to hCG. These results indicate that both IL-1 and EtOH were able to stimulate the pathway by acting directly on the dorsal pons, including the LC. On the other hand, lesions of this area, which in themselves did not modify the T response to hCG, significantly reversed the ability of icvinjected EtOH, but not CRF or IL-1 , to blunt this response. This finding agrees with the fact that brain catecholamine

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FIG. 4. Effect of electrolytic lesions of the dorsal pons/locus coeruleus (LCx) on T release in rats preinjected icv with vehicle, EtOH (86 M), IL-1 (80 ng), or CRF (2 g) 20 min before hCG (1U/kg, iv). hCG was injected 20 min after icv treatments. The zero time corresponds to T levels immediately before hCG administration. In themselves, LC lesions did not alter testicular activity, compared with sham-operated rats (A), nor did they reverse the effect of IL-1 (B) or CRF (C). In contrast, they prevented the inhibitory effect of EtOH (D). Panels on the left show the time course for T release. Panels on the right show cumulative T release. Each point/bar represents the mean of six to eight rats SEM. *, P 0.05; **, P 0.01 vs. sham/vehicle.

depletion by 6-OHDA fully reverses the ability of icv EtOH to blunt hCG-induced T release (10). In these studies, 6-OHDA lesions of brain noradrenergic neurons only modestly, though significantly, modified the effect of icv IL-1 on hCG-induced T release and did not affect the ability of CRF to do so (10). Furthermore, pretreatment with the -adren-

ergic receptor antagonist propranolol significantly, but not entirely, reversed the ability of IL-1 to activate the pathway (7). Collectively, these results support the concept that the noradrenergic dorsal pons represents an important component of the proposed pathway between the brain and the testes, but does so only for selective compounds. By pointing to the potential role of NE in this brainstem region, our findings also provide an explanation for the fact that catecholamines modulate the influence exerted by icv-injected IL-1 and EtOH (7, 8) as well as CRF (Selvage, D. J., and C. Rivier, unpublished) on testicular activity. Before we proceed with the discussion, we must emphasize two potential caveats. First, although we used standard and well-accepted techniques for LC microinjections and lesions (see for example Refs. 20 and 21), the lesions that were made and the compounds administered may reach Barringtons nucleus, which lies just ventromedial to the rostral pole of the LC. This nucleus contains CRF neurons and indirectly (through parasympathetic preganglionic neurons that innervate pelvic viscera) projects to the testes (2225). However, the only modest PRV labeling of the Barringtons nucleus after injection of the virus into the testes, when compared with the LC (12), makes it an unlikely component of the brain-testicular pathway under study. Also, Barringtons nucleus contains few, if any, adrenergic neurons or receptors, which are the focus of our studies. In addition, of the agents we microinfused into the LC, CRF would presumably be the most likely to activate Barringtons nucleus, should leakage to this area occur. However, this treatment did not alter T release, indicating that if microinfused agents did leak to Barringtons nucleus, they did not activate it in a way that influenced the brain-testes pathway. Consequently, the presence of CRF and its receptors in this nucleus is not relevant for our results. A second caveat pertains to the possibility that substances injected icv may have reached the NTS, which lies immediately adjacent to the fourth ventricle, projects densely to the PVN (see for example Refs. 26 and 27) and is part of PRV-labeled structures (12) and the A1/A2 or A5 noradrenergic groups in the ventral brainstem, which also send projections to the PVN. Of note, all of these structures are downstream of the PVN in the pathway under investigation, as mapped by PRV studies (12, 13, 28). However, the NTS does get activated after icv injection of CRF, IL-1 , or EtOH, as measured by increases in levels of the immediate early genes c-fos and NGFI- (Lee, S., and C. Rivier, unpublished). Furthermore, we obtained preliminary data showing that DSP-4 injections, which cause highly specific lesions of noradrenergic neurons in the LC (14, 15), also blocked the ability of icv EtOH to blunt hCG-induced T secretion (Selvage, D. J., and C. Rivier, unpublished). This strengthens the conclusion that our lesion and microinfusion procedures most likely had there effect at the level of the LC. Nonetheless, future studies specifically focused on various areas closely related to the LC are needed and will establish their respective importance vis-a-vis this area. ` In conclusion, the icv injection of EtOH, IL-1 , or CRF, which stimulates a brain-testes neural circuit that bypasses the pituitary to regulate T release (4), causes a rapid depletion of LC NE levels. Thus, the well-documented bioamine release that takes place from the LC under many stressful

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conditions (11), also occurred in our model and plays an important role in modulating the ability of stress-related signals to activate the brain-testicular pathway under study. We propose that, within the descending neural pathway that connects the brain to the testes, alcohol or IL-1 stimulate noradrenergic neurons of the dorsal pons, which then causes a widespread release of NE, including within the PVN. We know that this hypothesis may be at odds with the current literature on catecholamine circuitry, which shows that the adrenergic innervation of the PVN mostly derives from catecholamine neurons in the NTS, not the LC. However, the LC does send projections to other nuclei that directly influence PVN function, such as the dorsomedial nucleus, as well as to direct descending projections in the brainstem and pons (26). Thus, our results raise the possibility that the LC-mediated influences of IL-1 and EtOH on the inhibitory pathway that we report may be due to indirect LC-PVN projections, or due to downstream projections from the LC. Although this hypothesis is at least in part supported by the only partial effectiveness of PVN lesions in blocking the inhibitory influence of EtOH on Leydig cell activity (10), we also need to take into consideration the fact, discussed in the introductory text, that the order of PRV progression along the pathway shows the A1/C1/A5 adrenergic nuclei are labeled early, followed by the PVN and the LC, whereas labeling in the NTS is significantly more modest (12). Consequently, more work is obviously required to begin to fully elucidate the neural components of the pathway. This possibly involves testing the effect of lesions of the PVN in rats microinfused with alcohol in the LC, to sort out (23) potential differences.
Acknowledgments
We are indebted to Annie MacDougall, Melissa Herman, Elaine Law, Yaira Haas, and Richard Schroeder for excellent technical assistance. We also thank Drs. Pete James and Soon Lee for their help in obtaining the results presented in Fig. 1A. Received October 25, 2005. Accepted March 10, 2006. Address all correspondence and requests for reprints to: Catherine Rivier, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier@salk.edu. Research was supported by National Institutes of Health Grants AA-12810 (to C.R.) and AA-12294 (to L.P.).

6. 7.

8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18.

19. 20.

21. 22.

23. 24.

References
1. Kalra SP, Kalra PS 1983 Neural regulation of luteinizing hormone secretion in the rat. Endocr Rev 4:311351 2. Saez J 1994 Leydig cells: endocrine, paracrine and autocrine regulation. Endocr Rev 15:574 626 3. McCann SM, Marubayashi U, Sun H-Q, Yu WH 1993 Control of folliclestimulating hormone and luteinizing hormone release by hypothalamic peptides. Ann NY Acad Sci 687:5558 4. Selvage DJ, Rivier C 2004 Leydig cell activity is regulated by a neural pathway between the hypothalamus and the testes that does not include the pituitary. In: Recent research developments in endocrinology. Kerala, India: Transworld Research Network; 97114 5. Selvage D, Rivier C 2003 Importance of the paraventricular nucleus of the hypothalamus as a component of a neural pathway between the brain and the 25. 26.

27.

28.

testes that modulates testosterone secretion independently of the pituitary. Endocrinology 144:594 598 Turnbull AV, Rivier C 1997 Inhibition of gonadotropin-induced testosterone secretion by the intracerebroventricular injection of interleukin-1 in the male rat. Endocrinology 138:1008 1013 Ogilvie KM, Rivier C 1998 The intracerebroventricular injection of interleukin-1 blunts the testosterone response to human chorionic gonadotropin: role of corticotropin-releasing factor and adrenergic-dependent pathways. Endocrinology 139:3088 3095 Rivier C 1999 Alcohol rapidly lowers plasma testosterone levels in the rat: evidence that a neural brain-gonadal pathway may be important for decreased testicular responsiveness to gonadotropin. Alcohol Clin Exp Res 23:38 45 Selvage D, Hales D, Rivier C 2004 Comparison between the influence of the systemic and central injection of alcohol on Leydig cell activity. Alcohol Clin Exp Res 28:480 488 Selvage D, Lee S, Parsons L, Seo D, Rivier C 2004 A hypothalamic-testicular neural pathway is influenced by brain catecholamines, but not testicular blood flow. Endocrinology 145:1750 1759 Carrasco GA, Van de Kar LD 2003 Neuroendocrine pharmacology of stress. Eur J Pharmacol 463:235272 Gerendai I, Toth IE, Boldogkoi Z, Medveczky I, Halasz B 2000 Central nervous system structures labelled from the testis using the transsynaptic viral tracing technique. J Neuroendocrinol 12:10871095 Lee S, Miselis R, Rivier C 2002 Anatomical and functional evidence for a neural hypothalamic-testicular pathway that is independent of the pituitary. Endocrinology 143:4447 4454 Lee S, Selvage D, Rivier C 2004 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. Endocrinology 145:4470 4479 Taffe M, Davis S, Yuan J, Schroeder R, Hatzidimitriou G, Parsons L, Ricaurte G, Gold L 2002 Cognitive performance of MDMA-treated rhesus monkeys: sensitivity to serotonergic challenge. Neuropsychopharmacology 27:9931005 Rivier J, Gulyas J, Corrigan A, Martinez V, Craig A, Tache Y, Vale W, Rivier C 1998 Astressin analogues (corticotropin-releasing factor antagonists) with extended duration of action in the rat. J Med Chem 41:50125019 Hwang KR, Chan SH, Chan JY 1998 Noradrenergic neurotransmission at PVN in locus ceruleus-induced baroreflex suppression in rats. Am J Physiol 274: H1284 H1292 Crankshaw D, Briggs J, Olszewski P, Shi Q, Grace M, Billington C, Levine A 2003 Effects of intracerebroventricular ethanol on ingestive behavior and induction of c-Fos immunoreactivity in selected brain regions. Physiol Behav 79:113120 Correa M, Arizzi MN, Betz A, Mingote S, Salamone JD 2003 Open field locomotor effects in rats after intraventricular injections of ethanol and the ethanol metabolites acetaldehyde and acetate. Brain Res Bull 62:197202 Mitchell JC, Li XF, Breen L, Thalabard JC, OByrne KT 2005 The role of the locus coeruleus in corticotropin-releasing hormone and stress-induced suppression of pulsatile luteinizing hormone secretion in the female rat. Endocrinology 146:323331 Sved AF, Cano G, Passerin AM, Rabin BS 2002 The locus coeruleus, Barringtons nucleus, and neural circuits of stress. Physiol Behav 77:737742 Valentino RJ, Pavcovich LA, Hirata H 1995 Evidence for corticotropin-releasing hormone projections from Barringtons nucleus to the periaqueductal gray and dorsal motor nucleus of the vagus in the rat. J Comp Neurol 363: 402 422 Valentino RJ, Chen S, Zhu Y, Aston-Jones G 1996 Evidence for divergent projections to the brain noradrenergic system and the spinal parasympathetic system from Barringtons nucleus. Brain Res 732:115 Keegan CE, Herman JP, Karolyi IJ, OShea KS, Camper SA, Seasholtz AF 1994 Differential expression of corticotropin-releasing hormone in developing mouse embryos and adult brain. Endocrinology 134:25472455 Pavcovich LA, Valentino RJ 1995 Central regulation of micturition in the rat the corticotropin-releasing hormone from Barringtons nucleus. Neurosci Lett 196:185188 Sawchenko PE, Cunningham ET, Bittencourt JC 1992 Aminergic and peptidergic pathways subserving the stress response. In: Kvetnansky R, McCarty R, Axelrod J, eds. Stress: neuroendocrine and molecular approaches. New York: Gordon and Breach; 1527 Herman JP, Figueiredo H, Mueller NK, Ulrich-Lai Y, Ostrander MM, Choi DC, Cullinan WE 2003 Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Front Neuroendocrinol 24:151180 Gerendai I 2004 Supraspinal connections of the reproductive organs: structural and functional aspects. Acta Physiol Hung 91:121

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