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Endocrinology Vol. 143, No. 12 4520-4526
Copyright © 2002 by The Endocrine Society

ARTICLE

Exaggerated Adrenomedullary Response to Immobilization in Mice with Targeted Disruption of the Serotonin Transporter Gene

Olga A. Tjurmina, Ines Armando, Juan M. Saavedra, David S. Goldstein and Dennis L. Murphy

Laboratory of Clinical Science (O.A.T., D.L.M.) and Section on Pharmacology (I.A., J.M.S.), National Institute of Mental Health, and Clinical Neurocardiology Section, National Institute of Neurological Disorders and Stroke (D.S.G.), National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Olga A. Tjurmina, Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Building 10, Room 3D41, 10 Center Drive, MSC-1264, Bethesda, Maryland 20892-1264. E-mail: ot3d{at}nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study examined whether serotonin transporter (5-HTT) gene knockout influences adrenomedullary, sympathoneural, or hypothalamo-pituitary-adrenal responses to acute immobilization. In conscious, cannulated mice, arterial plasma concentrations of catecholamines, ACTH, and corticosterone were measured at baseline and after 15 min of immobilization. Tissue levels of serotonin (5-HT), catecholamines, and hormones were also measured in pituitary and adrenal glands. At baseline, adrenal and pituitary 5-HT concentrations in knockout (5-HTT-/-) mice were markedly lower than those in littermate control (5-HTT+/+) mice, whereas the groups did not differ in levels of catecholamines or hormones in plasma or tissue. Immobilization increased plasma levels of catecholamines, ACTH, and corticosterone in all genotypes. 5-HTT-/- mice had exaggerated responses of plasma epinephrine to immobilization and significant reductions in adrenal epinephrine, norepinephrine, and 5-HT contents compared with values in littermate controls. Pituitary ACTH was significantly reduced after immobilization in 5-HTT-/- mice only, but increases in plasma ACTH and corticosterone levels did not differ between genotypes. The results suggest that one 5-HTT function is to restrain adrenomedullary activation in response to immobilization. Exaggerated adrenomedullary responses seem to be an autonomic correlate of the anxiety-like behaviors in 5-HTT knockout mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SEROTONIN transporter (5-HTT) plays a key role in the regulation of serotonergic neurotransmission, providing the primary mechanism for inactivation and recycling of serotonin (5-HT) after its release into the synaptic cleft. The 5-HTT has been implicated in depression, anxiety, and substance abuse (1, 2) and is the target for antidepressant and antianxiety drugs such as fluoxetine and paroxetine (3, 4, 5). To study the role of 5-HTT in physiology and psychiatry, we developed an animal model with targeted disruption of the 5-HTT gene (6).

5-HTT-deficient mice exhibit heightened anxiety-like behavior in both the elevated zero-maze and light/dark exploration tests; diazepam reverses this abnormality (7). 5-HTT knockout mice also have an enhanced corticosterone response to chronic stress (8) and an enhanced ACTH response to mild acute stress (9). The anxiety-like phenotype could reflect central 5-HT system dysfunction or alterations in the hypothalamo-pituitary-adrenal (HPA) axis or in adrenomedullary activity. A number of observations have been made regarding serotonergic effects on sympathoadrenal regulation and release of catecholamines, both centrally and peripherally. 5-HT neurons stimulate sympathetic nervous system outflow (10). 5-HT1A and 5-HT2 receptor agonists elevate plasma epinephrine (EPI), ACTH, and corticosterone concentrations (11, 12, 13, 14), and 5-HT1A, 5-HT2A, and 5-HT2B/C antagonists (pindolol, ketanserin, and ritanserin) block the adrenal EPI response to these agonists (15, 16, 17). 5-HT may modulate CRH release (18), and CRH contributes to EPI responses to several stimuli (19).

In addition, 5-HT can influence catecholamine release by direct actions in the adrenal medulla. 5-HTT-binding sites have previously been identified by autoradiography in the rat adrenal medulla, and EPI-synthesizing chromaffin cells accumulate and store 5-HT via the 5-HTT and the vesicular monoamine transporter (20). Adrenal catecholamine release can be induced by peripherally administered 5-HT, which acts on 5HT2A receptors in the adrenal glands; this release is blocked by cyproheptadine, methysergide, xylamidine, and ketanserin (21, 22). Thus, loss of adrenal 5-HTT activity might be expected to affect adrenal catecholamines or corticosteroid production.

In the present study we examined whether functional differences in 5-HTT might modulate sympathoneural, adrenomedullary, or HPA activation during immobilization stress, by measuring arterial plasma and tissue concentrations of catecholamines, 5-HT, ACTH, and corticosterone in conscious, cannulated mice of 5-HTT+/+, 5-HTT+/-, and 5-HTT-/- genotypes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The experimental procedures were approved by the animal care and use committee of NIMH, NIH. Mutant C57BL/6 mice with absent (-/-) or diminished (+/-) 5-HT transporters and their littermate controls (+/+) were studied. A genomic segment containing exon 2 of the murine 5-HTT gene was replaced with a neo gene cassette by homologous recombination in embryonic stem cells (6). Because female 5-HTT-deficient mice exhibited a greater increase in anxiety-like behavior than male mice (Wichems, C. H., unpublished observations), and female rats have been shown to be more vulnerable than males in an animal model of depression (23), we chose female mice for our experiments. Mice were 3–6 months old, and they were used randomly in the estrous cycle; therefore, any variation due to the estrous cycle would be included in the overall statistical variations. The mice (F8 generation; weight, 27.4 ± 0.9 g) were housed three to five per cage with room temperature at 20 C, food and water available ad libitum, and a 12-h light, 12-h dark cycle. Experiments were performed during the light period and were started at 0900 h.

Preparation of conscious mice
Under general anesthesia [Avertin: 10 g tribromoethylethanol (Aldrich, Milwaukee, WI) in 10 ml t-amylalcohol (Sigma, St. Louis, MO); 15 ml/kg of 2.5% Avertin in saline, ip], a micro-renathane catheter (MRE-025 connected to MRE-040, Braintree Scientific, Inc., Braintree, MA) was implanted 1 d before the experimental day. The catheter was inserted into the carotid artery, sutured in place, and led sc to the nape, where it was exteriorized and anchored using tape. The inner volume of the catheter was 15–20 µl. After cannulation, the animals recovered overnight separately in plastic cylindrical cages (18-cm diameter, 21-cm height; Instech Laboratories, Inc., Plymouth Meeting, PA), with water and food provided ad libitum. This system enabled repeated blood samples to be obtained from conscious unrestrained mice.

Experimental protocol, and arterial plasma and tissue collection
The morning following surgery, catheters were connected to 25 cm of tubing (PE 20; inner volume, ~25 µl) filled with heparinized saline. After a 2-h equilibration period, blood samples (100 µl) were collected by free flow in heparinized polyethylene tubes twice, at baseline and 2 h later. Heparinized saline (100 µl) was immediately injected through the catheter to provide replacement of fluid. Animals were studied undisturbed or immobilized in a prone position (24, 25, 26) for 15 min immediately before the second sampling. Animals were used for one experimental immobilization or sham period only. A total of 200 µl blood were removed from each mouse. Blood was refrigerated and centrifuged immediately at 3500 rpm for 60 sec, and plasma aliquots were removed and stored at -80 C until assayed. At the end of each experiment animals were killed by cervical dislocation. Pituitary and adrenal glands were collected and stored at -80 C until analyzed.

Analytical methods
Pituitary and adrenal glands, suspended in 1 ml 0.3 N perchloric acid for catecholamines and hormones or in 100 µl 0.1 N perchloric acid for 5-HT, were homogenized using an ultrasonic cell disrupter. For catecholamines, after centrifugation at 14,000 rpm for 10 min, the supernatant was stored at -80 C, and a 1:500 dilution of the supernatant from adrenal glands was used. For 5-HT, the undiluted 100-µl aliquot obtained immediately after centrifugation was used.

Concentrations of catechols were analyzed using reverse phase liquid chromatography with electrochemical detection after partial purification by adsorption on alumina (27). The HPLC system consisted of an ESA model 580 dual piston pump (ESA, Chelmsford, MA), a 717 Plus autosampler (Waters Corp., Milford, MA); 5-µm LUNA C18 150 x 4.6 mm column (Phenomenex, Torrance, CA), a CERA column cooler 250 (set at 19 C; Thompson Instruments, Springfield, VA), and an ESA 5200A coulorimetric detector with ESA 5011 dual electrode analytical cell. The first electrode was set at +150 mV, and the second at +350 mV. The potential of the ESA 5021 conditioning cell, placed between the column and the analytical cell, was set at +300 mV. The mobile phase, delivered at 0.9 ml/min, was 13.8 g/liter sodium monobasic phosphate, 48 mg/liter octanesulfonic acid, 50 mg/liter EDTA, and 1.5% (vol/vol) acetonitrile, pH 3.1, with phosphoric acid. The limit of detection was about 12 fmol for injected norepinephrine (NE), 10 fmol for EPI, 30 fmol for 3,4-dihydroxyphenylglycol (DHPG), about 900 fmol for 3,4-dihydroxyphenylacetic acid (DOPAC), 30 fmol for dopamine (DA), and about 15 fmol for 3,4-dihydroxyphenylalanine (DOPA).

Concentrations of 5-HT were assayed by HPLC using the procedure described by Mefford (28) with some modifications. The HPLC system consisted of an ESA model 580 pump (0.5 ml/min flow rate), a Gilson 231 autosampler (Thompson Instruments), a 5-µm Axxion ODS C18 250 x 4.6-mm column (Thompson Instruments), and an ESA 5200A coulorimetric detector with an ESA 5014 analytical cell. The first electrode was set at -150 mV, and the second at +325 mV. The potential of the ESA 5020 guard cell was set at +200 mV. The mobile phase consisted of 1.85 g/liter heptane sulfonic acid, 0.1 g/liter EDTA, 0.3% (vol/vol) phosphoric acid, 0.27% (vol/vol) triethylamine, and 12% (vol/vol) acetonitrile at pH 3.2 without adjustment. The limit of detection was 10 fmol.

Concentrations of ACTH in pituitary and of corticosterone in plasma and adrenal glands were determined by RIA using commercially available [125I]ACTH and [125I]corticosterone kits (ICN Biomedicals, Inc., Costa Mesa, CA). ACTH concentrations in plasma were measured by a modified RIA procedure (29).

Data analysis and statistics
Concentrations of catechols were calculated using a Dynamax HPLC method manager (Rainin Instruments, Inc., Woburn, MA). Data are presented as the mean ± SEM. The effects of immobilization were evaluated by two-way ANOVAs for repeated measures, followed by Fisher’s protected least significant difference post hoc test using StatView SE+ (Abacus Concepts, Berkeley, CA). The between-group factor was 5-HTT genotype (-/-, +/-, or +/+), and the within-group factor was time. The unpaired t test was used for comparison of two groups. P < 0.05 defined statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Evaluation of the blood-sampling method
We first determined in undisturbed animals the timing and the volume of arterial blood that could be removed in conscious mice without eliciting plasma catecholamine responses. In the present study arterial plasma levels of catechols (Table 1Go) were similar to or lower than those found previously using other methods (in anesthetized mice, carotid artery cannulation or cardiac puncture, or in awake mice, retroorbital sinus phlebotomy) (30, 31, 32, 33). This suggests that the blood-sampling procedure caused minimal distress in the animals.


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  		Table 1. Evaluation of the blood-sampling method in conscious undisturbed mice

 
At 120 min after the baseline blood sampling, levels of catecholamines (Table 1Go) and corticosterone (data not shown) in plasma remained unchanged. Thus, this method enabled us to obtain at least two blood draws from conscious, freely moving mice without eliciting sympathoadrenal activation due to hypovolemia or distress.

Basal levels of 5-HT, catechols, and hormones in conscious 5-HTT mutant mice
Basal adrenal 5-HT concentrations were reduced by approximately 70% in 5-HTT-/- mice compared with 5-HTT+/+ mice (8.1 ± 0.5 vs. 23.6 ± 1.4 pmol/gland; Fig. 1Go). Similarly, basal pituitary 5-HT levels were reduced by approximately 80% (1.0 ± 0.4 and 4.8 ± 0.8 pmol/gland).



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  		Figure 1. Effects of immobilization stress (15 min) on adrenal whole gland (top) and pituitary tissue (bottom) 5-HT levels in 5-HT transporter knockout mice. Black, 5-HTT+/+ genotype; dark gray, 5-HTT+/- genotype; light gray, 5-HTT-/- genotype. Data are the mean ± SEM (n = 4–7). *, P < 0.05, significant difference from baseline of same genotype mice; #, P < 0.05, significant difference from 5-HTT+/+ mice.

 
Despite substantial alterations in 5-HT levels, there were no differences among the three genotypes in levels of catecholamines or hormones either in plasma or tissue under basal conditions (Figs. 2–4GoGoGo). In the three groups average baseline concentrations plasma were: EPI, 2.31 ± 0.32 pmol/ml; NE, 5.29 ± 0.67 pmol/ml; DHPG, 10.53 ± 0.52; DOPAC, 10.68 ± 1.18; DOPA, 3.48 ± 0.34; and DA, 0.67 ± 0.21 pmol/ml (Figs. 2–4GoGoGo). Adrenal tissue concentrations were; EPI, 17.75 ± 0.70; NE, 10.23 ± 0.64 (Figs. 2Go and 3Go); and DA, 0.27 ± 0.01 nmol/gland.



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  		Figure 2. Effects of immobilization stress (15 min) on plasma (top) and adrenal gland tissue (bottom) EPI levels in 5-HT transporter knockout mice. Black, 5-HTT+/+ genotype; dark gray, 5-HTT+/- genotype; light gray, 5-HTT-/- genotype. Data are the mean ± SEM (n = 8–14). *, P < 0.05 significant difference from baseline of same genotype mice; #, P < 0.05, significant difference from 5-HTT+/+ mice.

 


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  		Figure 3. Effects of immobilization stress (15 min) on plasma (top) and adrenal gland (bottom) NE levels in 5-HT transporter knockout mice. Black, 5-HTT+/+ genotype; dark gray, 5-HTT+/- genotype; light gray, 5-HTT-/- genotype. Data are the mean ± SEM (n = 8–14). *, P < 0.05, significant difference from baseline of same genotype mice; #, P < 0.05, significant difference from 5-HTT+/+ mice.

 


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  		Figure 4. Effects of immobilization stress (15 min) on plasma DHPG (top), DOPAC (middle), and DOPA (bottom) levels in 5-HT transporter knockout mice. Black, 5-HTT+/+ genotype; dark gray, 5-HTT+/- genotype; light gray, 5-HTT-/- genotype. Data are the mean ± SEM (n = 8–12). *, P < 0.05, significant difference from baseline of same genotype mice; #, P < 0.05, significant difference from 5-HTT+/+ mice.

 
Plasma ACTH was 148 ± 11 pg/ml and plasma corticosterone was 84 ± 6 pg/ml at baseline (Figs. 5Go and 6Go). The pituitary ACTH concentration was 57 ± 2 ng/pituitary (Fig. 5Go). The adrenal corticosterone concentration was 48 ± 10 ng/gland (Fig. 6Go).



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  		Figure 5. Effects of immobilization stress (15 min) on plasma (top; n = 8–12) and pituitary tissue (bottom; n = 4–7) ACTH levels in 5-HT transporter knockout mice. Black, 5-HTT+/+ genotype; dark gray, 5-HTT+/- genotype; light gray, 5-HTT-/- genotype. Data are the mean ± SEM. *, P < 0.05, significant difference from baseline of same genotype mice.

 


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  		Figure 6. Effects of immobilization stress (15 min) on plasma (top; n = 8–12) and adrenal gland (bottom; n = 6–12) corticosterone levels in 5-HT transporter knockout mice. Black, 5-HTT+/+ genotype; dark gray, 5-HTT+/- genotype; light gray, 5-HTT-/- genotype. Data are the mean ± SEM. *, P < 0.05, significant difference from baseline of same genotype mice.

 
Effects of immobilization on 5-HT levels
In both 5-HTT+/+ and 5-HTT+/- mice, immobilization resulted in increased adrenal tissue 5-HT concentrations by 19% and 30%, whereas in 5-HTT-/- mice a 42% decrease was observed (~80% lower than in 5-HTT+/+; Fig. 1Go). There were no significant effects of immobilization on pituitary 5-HT concentrations in any group.

Effects of immobilization on catecholamine levels
Plasma levels of EPI, NE, DHPG, DOPAC, and DOPA all increased after stress in all genotypes (Figs. 2–4GoGoGo). 5-HTT-/- mice had significantly exaggerated responses of plasma EPI and DOPAC, but not of NE to immobilization. Thus, in response to immobilization, plasma EPI increased 7- and 5-fold in 5-HTT+/+ and 5-HTT+/- mice, whereas in 5-HTT-/- mice a 10-fold increase occurred (~30% higher than in 5-HTT+/+ mice; Fig. 2Go). At the same time, stress effects on plasma NE were similar in -/-, +/-, and in +/+ mice (2.2-, 2.1-, and 2.6-fold increases; Fig. 3Go).

Adrenal tissue levels of both EPI and NE decreased after stress in 5-HTT-/- mice (by 23% and 18%), but were unchanged in 5-HTT+/+ or 5-HTT+/- mice (Figs. 2Go and 3Go), whereas tissue levels of DA increased by approximately 40% in all groups (data not shown).

Hormonal responses to stress
Pituitary levels of ACTH were diminished by 27% after 15 min of immobilization in 5-HTT-/- mice, but were unchanged in 5-HTT+/+ and 5-HTT+/- mice (Fig. 5Go).

Plasma levels of ACTH and corticosterone increased markedly, about 2.7- and 2-fold, respectively, in all genotypes, after 15 min of immobilization (Figs. 5Go and 6Go). Corticosterone concentrations in adrenal glands were elevated after immobilization in all genotypes, 4.9-, 5.0-, and 3.9-fold for 5-HTT-/-, 5-HTT+/-, and 5-HTT+/+ mice respectively, without significant group differences (Fig. 6Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In conscious chronically cannulated mice, we found that genetic inactivation of the 5-HT transporter led to reductions in 5-HT concentrations in mouse adrenal and pituitary glands and resulted in enhanced adrenomedullary responses to acute immobilization.

Chromaffin cells and sympathetic fibers that innervate adrenal gland are known to be sites of 5-HT uptake in the mouse (34). 5-HT immunoreactivity appears to be sequestered in secretory vesicles of frog and rat chromaffin cells (35, 36), and 5-HT is colocalized with phenylethanolamine N-methyltransferase in these vesicles (20), suggesting that 5-HT might be released together with catecholamines under splanchnic nerve stimulation and act as a paracrine factor locally regulating adrenocortical and adrenomedullary secretion. 5-HT present in the adrenal gland can be taken up from the circulation or newly synthesized within the tissue (37, 38). As 5-HTT-deficient mice had low basal 5-HT concentrations in adrenal glands, abolition of 5-HT recycling eventually depletes vesicular 5-HT stores. This is in line with findings that the 5-HTT mediates intracellular 5-HT accumulation by EPI-secreting chromaffin cells (20), and that 5-HTT-deficient mice lack blood 5-HT (39). Adrenal 5-HT synthesis appears unable to compensate for loss of the 5-HTT.

Despite a marked reduction in 5-HT tissue levels and other serotonergic dysfunction (6, 9, 39, 40), mutant mice and their normal littermate controls did not differ in basal plasma or tissue levels of any catechol. Basal levels are maintained via multiple homeostatic mechanisms, and 5-HT appears to play only a small role in tonic adrenomedullary secretion. This may also be a reflection of adaptive changes in 5-HT synthesis and turnover in 5-HTT-deficient mice or of compensatory mechanisms at the receptor level (8, 9, 40).

Altered regulation of catecholamine systems is more likely to be evident during exposure to stressors. During stress, increased 5-HT uptake from blood or enhanced local synthesis may cause the increased adrenal 5-HT content found in control and heterozygote mice. The immobilization- induced increase in plasma EPI was similar in these two genotypes. 5-HTT-/- mice, however, exhibited a further depletion of adrenal 5-HT during stress and an exaggerated release of EPI from the adrenal medulla, demonstrated not only by increased plasma EPI levels but also by a decreased adrenal EPI content. This potentiated EPI release could result from a combination of central and peripheral mechanisms mediated by 5-HT receptors. These mechanisms have been evaluated using 5-HT receptor agonists and antagonists (11, 12, 13, 14, 15, 16, 17, 21, 22), but which one predominates in stress responses has not yet been fully established. Adrenal catecholamine synthesis is most probably not affected by 5-HTT deficiency, as shown by equal stress-induced increases in adrenal DA content in all genotypes.

Increases in plasma NE levels during immobilization reflect release mainly from sympathetic nerve endings and less so from the adrenal medulla by different mechanisms (11, 41, 42, 43). This can explain why plasma NE levels were similar in all genotypes both at baseline and during immobilization, whereas the adrenal NE content was decreased during stress in 5-HTT-/- mice. The findings suggest that 5-HTT dysfunction augments activation of the adrenomedullary axis without affecting activation of the sympathoneural axis during this type of stress.

Plasma DOPAC changes during stress derive at least partly from altered synthesis and metabolism of DA in noradrenergic nerves (44). Enhanced stress-elicited increases in plasma DOPAC levels in mutant mice may represent increased intracellular metabolism of DA as an NE precursor. The relative sympathoneural and adrenomedullary contributions to plasma DOPAC responses in this situation remain unknown.

In addition to changes localized to the adrenal medulla, 5-HTT-/- mice have marked increases in extracellular brain 5-HT levels, demonstrated by microdialysis and voltammetry (45, 46) as well as reduced tissue 5-HT content (6). Different stress-related stimuli increase serotonergic neurotransmission in median and dorsal raphe nuclei and in forebrain structures innervated by both nuclei (47). Bagdy et al. (12) reported serotonergic stimulatory effects on sympathoadrenal regulation and release of catecholamines. Thus, the exaggerated catecholamine response to stress may also result from either increased activation or decreased inhibition of central structures controlling adrenomedullary discharge located in the hypothalamus, midbrain, and brain stem, where serotonergic fibers and receptors have been identified (48, 49). A possible analogous situation might be found in a study of tail pinch stress, in which a potentiated evoked response of extracellular NE was found in the locus coeruleus of rats treated chronically with the 5-HTT inhibitor fluoxetine (50).

Enhanced adrenomedullary activation during acute stress in 5-HTT-/- mice may contribute to or correlate with heightened anxiety. In both animals and humans, increases in plasma catecholamines have been reported as a result of anxiety (51, 52, 53, 54). In mice, genetic disruption of the 5-HTT results in anxiety-like behavioral abnormalities (7, 55); likewise, in humans, genetic variation in 5-HTT promoter region regulatory elements is associated with anxiety-related traits (1, 56).

The association between 5-HT depletion and augmented adrenomedullary responsiveness might reflect alterations in autonomic outflow from the central nervous system or local processes in adrenomedullary chromaffin cells. For instance, although we found decreased pituitary 5-HT concentrations in 5-HTT-/- mice both at baseline and under stress, probably reflecting both lack of 5-HT uptake and altered local synthesis (57), the effects of these abnormalities, if any, are unknown in terms of both adrenomedullary and adrenocortical function. Because serotonergic mechanisms are involved in local regulation of pituitary hormones (38, 58), one might hypothesize alterations in the HPA axis in 5-HTT-/- mice. In our study basal plasma and tissue ACTH and corticosterone levels were unaltered, and the stress-induced increase in plasma levels of both hormones did not differ significantly between genotypes, although pituitary ACTH content substantially decreased after stress in 5-HTT-/- mice, but not in their littermate controls. Previously it was reported that basal plasma corticosterone levels in 5-HTT-/- mice were lower than those in 5-HTT+/- or 5-HTT+/+ mice, whereas basal plasma ACTH levels were similar in all genotypes (8, 9). 5-HTT-/- mice have enhanced plasma ACTH responses to handling during ip saline injection (9). The discrepancy between our data and those published previously may be due to different genetic backgrounds for the mutant mice (C57BL6 vs. CD1), producing different sensitivities to stress (59, 60). Moreover, different stressors produce different effects on extracellular 5-HT levels in different brain regions (47), and these effects do not correlate with the magnitude of HPA activation (61). In addition, somewhat higher than expected (8, 9, 62) basal ACTH and corticosterone levels in our experiments, which could be related to postsurgical recovery, could have influenced the HPA response to stress that were similar to or less than those reported by others in mice (31, 63, 64, 65, 66, 67). Exaggerated HPA responses in 5-HTT-deficient mice (9) could be due to the alterations in pituitary 5-HT levels reported here or in the hypothalamic 5-HT system regulating the HPA axis. Hypothalamic 5-HT is thought to mediate CRH release (18), and both EPI and ACTH responses might be mediated by CRH (12). The increased availability of extracellular 5-HT resulting from loss of 5-HT reuptake (45) may potentiate central excitatory effects on EPI and possible ACTH release in 5-HTT-deficient mice.

Results from our volume and time course studies indicate that conscious chronically cannulated knockout mice provide a good model for studying sympathoneural, adrenomedullary, and HPA function in genetically modified mice. Previous studies involving anesthetized animals or retroorbital puncture to obtain blood have necessarily entailed large artifactual effects of the experimental procedures themselves that would obscure or prevent altered adrenomedullary responsiveness. Plasma catecholamine levels reported in anesthetized mice (33, 68) were lower than those observed in our experiments and may not accurately reflect the true basal levels expected in conscious rodents, as anesthetics are known to alter catecholaminergic activity (69, 70). Basal catecholamine levels of conscious retroorbital phlebotomized mice (30, 31, 32) were not only higher than basal levels in our mice, but also 2-fold higher than those found after 15 min of immobilization. We suggest that the data obtained in unrestrained and unhandled cannulated mice represent the most accurate value of basal catecholamine levels in arterial plasma.

In summary, our data provide evidence that in 5-HTT-/- mutant mice, the enhanced EPI release from adrenal medulla may be attributable to a 5-HT deficiency directly at the level of the adrenal gland, confirming an important role of the 5-HTT in the modulation of adrenomedullary activation and suggesting the existence of an intraadrenal serotonergic regulatory system. Likewise, altered pituitary ACTH responses to acute stress in 5-HTT-deficient mice may result from 5-HT depletion at the level of pituitary. Impaired homeostatic regulation at the local adrenal and pituitary levels may interact with altered central nervous system dynamics of 5-HT and 5-HT receptors present in 5-HTT-deficient mice (9, 45). Further studies investigating the distribution and function of 5-HT receptors in adrenal glands in these mice will help to clarify the role of peripheral serotonergic system alterations in adrenal catecholamine and hormone release. The results of the present study further suggest that 5-HTT function restrains adrenomedullary, but not sympathoneural, activation in response to immobilization. Exaggerated adrenomedullary responses in 5-HTT-/- mice may contribute to or be correlated with previously reported anxiety-like behavioral abnormalities in this animal model.


   Acknowledgments
 
We thank Dr. Graeme Eisenhofer for fruitful discussion and support, and Ms. Theresa B. DeGuzman, Ms. Su-Jan Huang, Ms. Theresa Tolliver, Ms. Suzanne Cheplo, and Ms. Courtney Holmes for their technical assistance.


   Footnotes
 
Abbreviations: DA, Dopamine; DHPG, 3,4-dihydroxyphenylglycol; DOPA, 3,4-dihydroxyphenylalanine; DOPAC, 3,4-dihydroxyphenylacetic acid; EPI, epinephrine; HPA, hypothalamo-pituitary-adrenal; 5-HT, serotonin; 5-HTT, serotonin transporter; NE, norepinephrine.

Received April 18, 2002.

Accepted for publication August 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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