Brain Mediation of Anolis Social Dominance Displays III. Differential Forebrain 3H-Sumatriptan Binding in Dominant vs. Submissive Males

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Brain Mediation of Anolis Social Dominance Displays III. Differential Forebrain 3H-Sumatriptan Binding in Dominant vs. Submissive Males Powered By Docstoc
					Original Paper
Brain Behav Evol 2001;57:202–213

Brain Mediation of Anolis Social Dominance Displays
III. Differential Forebrain 3H-Sumatriptan Binding in Dominant vs. Submissive Males

Lewis R. Baxter, Jr.a–f
Department of Psychiatry and Behavioral Neurobiology, bDepartment of Neurobiology, Department of Psychology, University of Alabama at Birmingham, Ala. (UAB), dDepartment of Molecular and Medical Pharmacology, eDepartment of Psychiatry and Biobehavioral Sciences, University of California at Los Angeles, Calif. (UCLA), and fYerkes Primate Center, Atlanta, Ga., USA
c a

Key Words Serotonin • 5-HT1B • 5-HT1D • Territoriality • Dominance Submission • Anolis


anisms might influence the likelihood of dominance in both mammals and reptiles.
Copyright © 2001 S. Karger AG, Basel

Abstract Measures of serotonin (5-HT) turnover in A. carolinensis forebrain increase acutely when males exhibit dominant social/territorial display routines, but decrease during submissive displays [Baxter et al., 2001a, b]. The present investigation sought to determine whether a difference in presynaptic regulatory receptors – one that might affect 5-HT flux – distinguish dominant vs. submissive anoles. Both 5-HT1B and 5-HT1D receptors are presynaptic regulators of output; this role is prominent at 5-HT terminals, where stimulation inhibits 5-HT release. Here, 3 H-sumatriptan binding at sites similar to mammalian 5-HT1B/D receptors was significantly higher in forebrain regions of submissive anoles than in dominant cagemates; this receptor site seemed pharmacologically more like a 5-HT1B than a 5-HT1D receptor. Higher densities of presynaptic 5-HT1B receptors in subordinates than in dominants might account for differences in 5-HT flux (lower in subordinates than in dominants) observed in displaying anoles of different status. Knockout mice missing the 5-HT1B receptor show heightened male territorial aggressiveness, thus similar 5-HT regulatory mech-


In prior experiments with Anolis carolinensis, my colleagues and I found evidence that forebrain serotonin (5-HT) turnover increases acutely during the territorial displays of dominant males, but decreases in submissively displaying subordinates [Baxter et al., 2001b]. The question remained, however, regarding what might account for such differences in 5-HT-flux. Here, studies of 3H-sumatriptan binding at 5-HT1B and/or 5-HT1D-like receptor sites – receptors that in mammals are presynaptic autoreceptors regulating 5-HT release – suggest one possibility. Background Although now recognized as genetically distinct, 5-HT1B and 5-HT1D receptors (formerly 5-HT1D beta and 5-HT1D alpha, respectively) share many similarities [Gerhardt and van Heerikhuizen, 1997; Pauwels, 1997; Barnes and Sharp, 1999]. Both receptors are thought to be primarily presynaptic (axonal) – 5-HT1B apparently exclusively so [Maroteaux et al., 1992; Sari et al., 1999; Riad et al., 2000] – although a minority of 5-HT1D receptors may be postsynaptic [Pau-

© 2001 S. KargerAG, Basel 0006–8977/01/0574–0202$17.50/0 Fax + 41 61 306 12 34 E-Mail Accessible online at:

Lewis R. Baxter, Jr., MD Ireland Professor of Psychiatric Research, 1001 Sparks Building 1720 Seventh Avenue South, Birmingham, AL 35294-0017 (USA) Tel. (205) 934-5216, Fax (205) 934-3709 E-Mail

Abbreviations 5-HT aDVR BG Ctx Sept DL-BG dl-St Hypo Th LFB nA1 nAcc Raph nRot Sept SN tec VM-BG 5-hydroxytryptamine; serotonin anterior dorsalventricular ridge complex forebrain basal ganglia complex cortex forebrain septum dorsolateral aspect of forebrain basal ganglia; dorsolateral striatum and lateral pallidum dorsolateral aspect of striatum hypothalamus lateral forebrain bundle nucleus A1 nucleus accumbens raphe nuclear complex nucleus rotundus of the thalamus forebrain septum substantia nigra midbrain (optic) tectum ventromedial aspect of forebrain basal ganglia; nucleus accumbens, olfactory tubercle and medial pallidum

wels, 1997; Barnes and Sharp, 1999]. These receptors are extensively (but not totally) co-localized in brain, and are known to exist not only as monomers and homodimers, but also bound together as heterodimers [Xie et al., 1999]. Presynaptically, both 5-HT1B and 5-HT1D receptors reside on axons, where their stimulation inhibits neurotransmitter output. There they function both as ‘autoreceptors’ on 5-HT neurons, and as ‘heteroreceptors’ on the axons of regionally specific subpopulations of some neurons that release other transmitters, such as acetylcholine, glutamate, dopamine, norepinephrine and gamma-aminobutyric acid (GABA) [Pauwels, 1997]. Thus, 5-HT1B and 1D receptors are believed to have important [although not identical; Schlicker et al., 1997] roles regulating local neurotransmitter release [Pauwels, 1997; Knobelman et al., 2000]. Widely phylogenetically conserved [Peroutka and Howel, 1994; Saudou and Hen, 1994; Gerhardt and van Heerikhuizen, 1997], homologues of both have been identified across several vertebrate groups [Gerhardt and van Heerikhuizen, 1997; Barnes and Sharp, 1999]; similarly functioning 5-HT1-like receptors exist even in diverse invertebrate classes [Sun and Schacher, 1996; Gerhardt and van Heerikhuizen, 1997; Barnes and Sharp, 1999]. Importantly for the present work, the drug ligand binding properties of 5-HT1B and 5-HT1D receptors are so similar in most mammals that they are difficult to distinguish pharmacologically; sites identified with classic ligands such as 3H-sumatriptan [Saudou and Hen, 1994; Waeber and

Moskowitz, 1995; Bonaventure et al., 1997] are, therefore, usually referred to generically as ‘5-HT1B/D receptors’, as if the two were a single binding site. Although the 5-HT1B receptors of rodents and opossum do have high affinity for some beta-adrenergic drugs (e.g., cyanopindolol) that do not have high affinity for 5-HT1D (or even non-rodent 5-HT1B) sites [Gerhardt and van Heerikhuizen, 1997], unfortunately even in rodents these same beta-adrenergic drugs have high affinity for adrenergic, other serotonergic, and non-specific sites [Waeber and Palacios, 1993]. As a result, even when masking agents are employed to reduce retention at these other sites, the classic rodent 5-HT1B receptor ligand, 125 I-cyanopindolol, often shows high levels of non-specific binding in ex vivo slice preparations. The only truly selective (in mammals) ligands reported to date, SB-216641 (high 5-HT1B 5-HT1D affinity, with little if any binding at other sites) and BRL-15572 (5-HT1D 5-HT1B) [Price et al., 1997; Schlicker et al., 1997], are not commercialy available as radioligands. Despite the inherent inability of 3H-sumatriptan to distinguish 5-HT1B from 5-HT1D receptors, this ligand shows low affinity for most other sites, making it the generally preferable commercial choice for identifying 5-HT1B/D receptors [Saudou and Hen, 1994; Waeber and Moskowitz, 1995]. It does have some affinity for the 5-HT1F site, however [Mengod et al., 1996]. Most important for the work here, 3 H-sumatriptan binding in A. carolinensis is similar to that of mammals [Clark and Baxter, 2000; and below]. Rationale for Investigation In the context of prior work examining the binding properties of several radioligands at potential 5-HT1A, 5-HT1B/D, 5-HT2A, 5-HT2C, 5-HT3 receptor and 5-HT reuptake sites in A. carolinensis [Clark and Baxter, 2000], it was noted that forebrain 3H-sumatriptan binding appeared greater in subordinate than in dominant males (fig. 1). If true, and if the site so marked is a 5-HT1B/D receptor, it was recognized that such a difference might indicate a mechanism accounting for why subordinate anoles have reduced forebrain 5-HT output compared to dominants during their respective acute territorial displays [Baxter et al., 2001b]. Therefore, 3H-sumatriptan was employed in new, prospective experiments to determine whether the density of 5-HT1B/D receptors is in fact higher in the forebrains of subordinate male anoles than in their dominant cagemates. Related studies of the ability of SB-216641 vs. other ligands to displace 3H-sumatriptan, as well as studies of 125 I-cyanopindolol binding, were also undertaken to provide information regarding whether the site of interest here in the anole might be 5-HT1B-like.

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Fig. 1. 3H-Sumatriptan binding in forebrains of one pair of dominant and submissive Anolis carolinensis cagemates. Coronal section autoradiographs at basal ganglia level; left to right, more rostral to more caudal. To right, Nissl stains of same section. Sections were incubated in 3 nM 3H-sumatriptan. Dom. = dominant animal; Sub. = submissive animal radioactivity scale: nCi = nano Curi; Bq. = Bequerel.

Materials and Methods
Animals All experiments were approved by UAB animal and radiation experimental use committees. Male A. carolinensis were obtained and maintained as described previously [Baxter et al., 2001a], as were smaller numbers of the closely related A. sagrei. Animals were housed and tested in glass terraria, and as before acutely introduced mirrors served as a territorial challenge by which to determine whether an animal displayed in a dominant or submissive manner to their own image. Animals were judged 1) dominant if they responded to mirror image with stereotypic strutting, vertical enlargement and up-anddown movements (= ‘push-ups’), 2) submissive if they responded with vertical flattening in a ‘body down’ position and holding still (= ‘flattening and freezing’), 3) non-territorial if they did neither; all experiments were 30–50 min. in duration [Baxter et al., 2001a, b]. Pair-housed animals were submitted to experiments three days after a stable, peaceful, dominant/subordinate hierarchy had been established [Baxter et al., 2001a, b], but unlike in the prior experiments, these animals did not have a patch applied over one eye.

Ex vivo in situ Receptor Autoradiography The autoradiographic receptor binding equilibrium methods employed were similar to those described previously [Clark and Baxter, 2000]. Animals were sacrificed with i.p. pentobarbital 2–4 hours after a final mirror challenge to check dominance status [Baxter et al., 2001a]. Brains were then frozen and 20 µm sections mounted on SuperFrost Plus Gold® (Fisher Scientific) slides and frozen at –80 °C until use. After incubation in Tris-buffered saline (pH 7.4) to remove endogenous ligands, sections were incubated with similarly buffered radioligand (concentrations, below) in great excess (> 4 ml per slide) for 90 min. then rinsed in 4 °C buffer, followed by 4 °C deionized water and dried quickly in a stream of cool, dry air. Brain sections from paired dominant and subordinate cagemates were incubated, washed, and dried together in the same rack so that they were always adjacent and facing each other throughout processing; the rack had the capacity for brain sections from 5–6 animal pairs at a time. Liquid scintillation counting of radioactivity in a 100 µl aliquot of incubation medium at the end of experiments measured radioligand concentration at equilibrium. Brain sections incubated with all 3H ligands were then apposed to Hyperfilm-3H® (Amersham) together with 3H radiation standards, and developed after an appropriate time interval (2–12 weeks, depending


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Fig. 2. 3H-Sumatriptan competitive ligand characteristics in male anole brain. a Above, 3 nM 3H-sumatriptan (SUM) was displaced by 300 nM serotonin, and to a considerable extent by 50 nM SB-216641, a 5-HT1B ligand; it was not displaced by the 5-HT1A ligand, WAY-100635, 100 nM. Below, sagittal brain sections showing WAY-100635 displaces the 5-HT1A ligand, 3H-8-OH-DPAT (DPAT). b 1 nM 3H-sumatriptan binding in representative sections throughout male anole brain. Sections numbered 1–4, rostral to caudal; Nissl stains below autoradiographs. Note that at this concentration, 3H-sumatriptan binding is reduced to background by 50 nM SB-21664. # = consecutive sections incubated with and without SB-21664.

on radioligand and concentration). Sections from paired dominant/ subordinate animals were mounted next to each other on the same film sheet. H-Sumatriptan Dimethylamino-N-methyl-3H-sumatriptan (3H-sumatriptan; specific activity 83 Ci/mmol.; Amersham) [Saudou and Hen, 1994; Waeber and Moskowitz, 1995], was employed. The specific 5-HT1A receptor ligand in mammals, WAY-100635 (Sigma-RBI; 100 nM) [Forster et al., 1995], SB-216641 (5-HT1B 5-HT1D ligand; Tocris, Cookson, Ltd.; 50 nM) [Price et al., 1997; Schlicker et al., 1997; Roberts et al., 1997], and 5-HT itself (Sigma-RBI; 300 nM) were used as competitive masking ligands in other incubations with 3H-sumatriptan to identify nonspecific vs. specific binding. H-8-OH-DPAT Similar brain sections were also incubated with 3 nM 3H-(±)-8hydroxydipropyl-amino-tetralin hydrobromide (3H-8-OH-DPAT; specific activity 127 Ci/mmol; NEN Life Sciences) which in mammals
3 3

binds to 5-HT1A, and to a lesser extent other 5-HT1 subtypes [Glennon, 1987; Waeber and Palacios, 1993] and the 5-HT7 receptor [Wood et al., 2000]. In the current experiment, WAY-100635 (100 nM) was used as a masking ligand to verify that it competes with 3H-8-OH-DPAT in the anole brain as a 5-HT1A ligand should. Previous work in A. carolinensis had established that 5-HT displaces 3H-8-OH-DPAT [Clark and Baxter, 2000]. I-Cyanopindolol To gain more information about this apparent 5-HT1B/D receptor in Anolis, other brain sections were incubated in the same manner with 0.5 nM 125I-cyanopindolol [Waeber and Palacios, 1993]. Here, WAY-100635 or 8-OH-DPAT (100 nM, each) and the highly specific beta-adrenergic agonist, isoproterenol (Sigma/RBI, 400 nM) [Weiner, 1985] were used together as masking agents [Waeber and Palacios, 1993]. This ligand cocktail, with and without SB-216641 (50 nM), was then used to reveal 5-HT1B-specific binding. After incubation, tissue sections were apposed along with 125I-radiation standards (Amersham) to Kodak EctaScan® C-EC-1 film.

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(For legend, see p. 205.)

Analyses Methods The resulting autoradiographs were analyzed for regional photographic optical densities as previously described [Baxter et al., 2001a] to determine regional radioligand retention. The ratio of photographic density to radiation exposure, as calculated via the applied 3H standards, was highly log/log-linear within the Hyperfilm-3H® exposures used for all ligand retention quantification (r ≥ .98). Calculations of concentrations of free vs. tissue-bound radioligand were then used for Scatchard plot analyses [Wharton and Polak, 1993; Davies and Dunn, 1999]. Statistical analyses have been described previously [Baxter et al., 2001a]. In cases where data distributions were not normal, non-parametric statistics are reported; otherwise parametric statistics were used. A priori significance was set at p < .05, one-tailed (subordinates would have higher 3H-sumatriptan binding values than dominants) for each test.


H-Sumatriptan Binding Properties H-sumatriptan binding throughout the anole brain closely resembled 5-HT1B/D receptor site distribution identified via several radiologands [Glennon, 1987; Waeber et al., 1989; Palacios et al., 1990; Saudou and Hen, 1994; Mengod et al., 1996, Bonaventure et al., 1997; Sari et al., 1999] – as well as the distribution of antibodies specific to 5-HT1B receptors [Sari et al., 1999] – in homologous mammalian brain structures [Clark and Baxter, 2000; fig. 2]. Even at a high concentration (3 nM), 3H-sumatriptan was displaced from forebrain by 300 nM 5-HT and to a considerable extent by SB-216641 (5-HT1B 5-HT1D ligand), despite the latter having a low solubility that limited the concentration (50 nM) that could be used to compete for binding. At



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Fig. 3. 125I-Cyanopindolol competitive ligand characteristics in male anole brain. Representative sections demonstrating that 125I-cyanopropranolol binds at a pharmacologically rodent-like 5-HT1B site. 0.5 nM 125I-cyanopindolol (CYP) alone, with addition of: 400 nM isoproterenol (Iso; beta adrenergic ligand); Iso + 100 nM WAY-100635 (WAY; 5-HT1A ligand); Iso + WAY + 50 nM SB-216641 (SB; 5-HT1B ligand). Below is use of another 5-HT1A ligand, 100 nM 8-OHDPAT (DPAT), in place of WAY.

a lower 3H-sumatriptan concentration (1 nM), 50 nM SB216641 reduced the radiosignal to non-specific background levels. In contrast, WAY-100635 (5-HT1A ligand) even at 100 nM did not displace 3H-sumatriptan despite having the capacity to displace appreciable amounts of 3H-8-OHDPAT. These results suggest that 3H-sumatriptan is probably binding to a pharmacologically 5-HT1B -like receptor site in anole forebrain. I-Cyanopindolol Binding Properties Results of the 125I-cyanopindolol binding experiments showed that this ligand had appreciable binding at sites remaining after isoproterenol (beta adrenergic receptor ligand) + WAY-100635 (5-HT1A ligand) masking. The resultant binding could be reduced to a considerable extent by 50 nM SB-216641 (fig. 3). Thus, the receptor of interest here seems to have a pharmacological profile similar to the rodent 5-HT1B receptor.

Dominants vs. Subordinates After these pharmacological specificity studies, 3Hsumatriptan binding was conducted with forebrain coronal sections from pairs of dominantly and submissively displaying cagemates; posterior brain was not examined. Two separate experimental runs were performed with tissue from different animal pairs incubated at a 3H-sumatriptan concentration of 1 nM (fig. 4). Both times submissives had higher ventromedial basal ganglia (VM-BG) 3H-sumatriptan binding than did their dominant cagemates; this was confirmed by statistical analyses of local radioactivity concentrations. Further, in the first experimental run (6 animal pairs) it also appeared that 3H-sumatriptan retention in dorsolateral BG (DL-BG) and the anterior dorsoventricular ridge (aDVR) of non-dominants might be higher than that of dominants, but tissue retention was not adequate in those regions to make a firm judgment. The second experimental run (5 different animal pairs), where tissue retention was good, confirmed

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exposure signal relative to background film noise, at these two concentrations allowed an estimate of ligand binding affinity (Kd) and binding site density (Bmax), via the standard Skatchard method [Wharton and Polak, 1993; Davies and Dunn, 1999; fig. 6]. 3H-sumatriptan affinity was similar in dominants (Kd = 7.6 ± 1.2) and submissives (Kd = 7.2 ± 1.1) (z = 1.28, n = 5 pairs, p = 0.20), comparable to that reported for 3H-sumatriptan and related compounds at 5-HT1B/D sites in mouse, rat and guinea pig brain (Kd = 8.0–9.3) [Maroteaux et al., 1992; Waeber and Moskowitz, 1995]. The Bmax in submissives (517 ± 95 fmol/mg tissue), however, was significantly greater than that in dominants (442 ± 90 fmol/mg tissue) (z = 2.16, n = 5 pairs, p = 0.03).


Fig. 4. 3H-Sumatriptan binding in ventro-medial basal ganglia (VMBG) of two separate groups of dominant vs. submissive male A. carolinensis cagemates. a and b are two separate but identical experimental runs. a was completed before b, which was designed as a replication.

these differences in aDVR and DL-BG (fig. 5). Three additional pairs of dominant/subordinate A. sagrei also gave qualitatively similar results (fig. 5c). As part of the extensive second experimental run, consecutive brain sections were also incubated in high (8.4 nM) vs. low (0.8 nM) concentrations of 3H-sumatriptan. Calculations of the resultant radioactivity retained in VM-BG, the brain region with best tissue retention, as well as a strong

Submissive male anoles had higher forebrain 3H-sumatriptan binding than their dominant cagemates. Even though the Scatchard method has many inherent limitations [Davies and Dunn, 1999] – and the use of only two data points per animal in this experiment certainly is problematic – the statistical results suggest that the differences observed in 3 H-sumatriptan binding between dominant and submissive anoles is probably due to differences in Bmax, rather than Kd. Given the very small volume of the anole brain, methods other than those used here will be needed for definitive tracer-kinetic binding studies [Davies and Dunn, 1999]. An increase/decrease in either Bmax or Kd, however, would increase/decrease the over-all effect of native 5-HT at this site. Although not rigorously established, drug competition studies suggest that the binding site of interest in the anole resembles a 5-HT1B receptor. Besides the 5-HT1B/D receptors, in mammals sumatriptan also has some affinity for the 5-HT1F [Mengod et al., 1996]; studies similar to these but employing a more specific 5-HT1B radiolabel such as SB216641 (no 5-HT1F affinity) might prove more definitive. It is acknowledged, however, that the pharmacological ligand displacement profile, and similarities in binding distributions among homologous structures, are the only evidence offered that the site identified in A. carolinensis is similar to mammalian 5-HT1B/D receptors. Further, there certainly is no proof that this receptor is presynaptic as in mammals. Nevertheless, given extensive evidence for evolutionary and functional homologies for sites having the properties identified here [Gerhardt and van Heerikhuizen, 1997], for purposes of further discussion it will be assumed that this is a presynaptic 5-HT1B site.


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Fig. 5. 3H-Sumatriptan binding in dorsolateral basal ganglia (DL-BG) and anterior dorsal ventricular ridge (aDVR) of dominant vs. submissive male cagemates. a and b are results from the same animals as in fig. 4b. c 3H-sumatriptan autoradiographs from a representative dominant/subordinate pair of the closely related A. sagrei shows same forebrain effects as observed in A. carolinensis.

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Fig. 6. 3H-Sumatriptan binding site density is increased in dominant (a) vs. submissive (b) male A. carolinensis cagemates. a and b Scatchard plot analyses of binding in ventromedial basal ganglia. Animals are the same as in fig. 4b.

(See text.). X-axis intercepts = binding site density (Bmax) (p = 0.03); Slopes of lines = receptor affinity for ligand (Kd) (p = n.s).

As mentioned in the Introduction, although the 5-HT1B receptor is pharmacologically similar in most mammals, rodents and opossum have a well-established difference, in that the receptor has high affinity for certain beta-adrenergic drugs; this difference has been traced to a substitution at a single amino acid site [Parker et al., 1993]. Results here with 125I-cyanopindolol binding in the anole suggest that this reptile has a 5-HT1B receptor in some ways more similar to that of rodents than other eutherian mammals. Similar studies do not seem to have been done in birds, other reptiles, or amphibians. It is very unlikely, however, that the 5-HT1B-like receptor studied here in the anole is identical to that of rodents because the other common mammalian 5-HT1B/D radioligand, 3H-GR-127935 (Amersham) [Audinot et al., 1997; Pauwels, 1997], shows no specificity vis a vis 5-HT in anole brain using similar methods, nor does the related GR-127935 displace 3H-sumatriptan [L.R. Baxter and E.C. Clark, unpublished data]. Indeed, the apparent high affinity of the site of interest here in the anole for 3H-sumatriptan is less like the 5-HT1B receptors of rodents than of non-rodent eutherians [Gerhardt and van Heerikhuizen, 1997]. Given the evolutionary distance between mammals

and reptiles, such a mixed comparative receptor/ligand pharmacology is to be expected [Peroutka and Howell, 1994; Gerhardt and Heerikhuizen, 1997]. Elucidation of the exact relationship of this receptor site to various mammalian 5-HT1B/D receptors will require its cloning and molecular sequence determination. Relevance to 5-HT Function in Social Dominance Behavior My colleagues and I have demonstrated that aDVR and striatum are brain regions where differences in both acute activation and 5-HT flux distinguish dominant from submissive anoles during territorial displays [Baxter et al., 2001a, b]. In mice, blocking 5-HT1B receptors has been shown to elevate striatal 5-HT [Knobelman et al., 2000]. The major afferents to both striatum and aDVR are glutamatergic [ten Donkelaar, 1998; Fowler et al., 1999], which is also true in the homologous striatum and isocortex of mammals [Voogd et al., 1998]. In rats, local glutamatergic stimulation of striatum has been shown to raise 5-HT levels acutely from baseline [Ohta et al., 1994; Tao and Auerbach, 2000]. If glutamate has a similar effect in the anole, the initial stimulation


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to aDVR and striatum on sighting a competitor might result in an acute local burst of 5-HT output. In the case of a subordinate, however, high levels of 5-HT1B autoreceptors could restrain further 5-HT release, whereas lower autoreceptor function in dominants would allow continued 5-HT output, facilitating the expression of dominant behavioral routines, as outlined previously [Baxter et al., 2001b]. The other well known class of 5-HT-output-regulating receptors is the somatodendritic 5-HT1A receptor of the raphe nuclei [Gerhardt and van Heerikhuizen, 1997; Knobelman et al., 2000; Riad et al., 2000]. The more prevalent location for 5-HT1A receptors throughout the brain, however, is postsynaptic. Differences in 3H-8-OH-DPAT binding (classic 5-HT1A ligand) between dominant and subordinate anole males have not been apparent on visual inspection of the raphe during our group’s prior work leading to the experiments of this report [Clark and Baxter, 2000; and unpublished observations on 6 further anole pairs]. However, there are problems with specificity and signal-to-noise in anole hindbrain with this ligand in our laboratory. Given its higher specificity, it is possible that differences between dominants and subordinates would be apparent if 14C-WAY-100635 were the 5-HT1A ligand used for future studies. Although the emphasis here is on the role of 5-HT1B receptors as 5-HT output-regulating autoreceptors, there are 5-HT1B heteroreceptor functions that might be relevant in the mediation of anole dominant vs. submissive behavior. For instance, among the 5-HT1B heteroreceptor locations mentioned in the Introduction are those found on GABA neurons. There is clear evidence that 5-HT1B receptors not only reside on striatal GABA efferents, but preferentially on those of the direct (as opposed to the indirect) BG system [Compan et al., 1998; Sari et al., 1999]. Following a mechanism proposed by our group for the mediation of anole dominant display behaviors [Baxter et al., 2001b], a reduction of these inhibitory 5-HT1B receptors at this location would increase tone in these direct BG system GABA elements, and thereby tend to facilitate dominant displays. Given the same proposed mechanism, there are also ways in which differences in 5-HT1B heteroreceptor densities on neurons of other transmitter types [e.g., dopamine projections to striatum; Sarhan et al., 1999, 2000] could vector dominant vs. submissive displays, but detailing such speculations does not seem appropriate now. In situ hybridization studies of 5-HT1B gene expression [Maroteaux et al., 1992; Alberts et al., 2000], when suitable Anolis probes are developed might not only establish whether differences between dominants and subordinates are found at this receptor, rather than the 5-HT1D, but also whether the relevant differences are at 5-HT1B autoreceptors; if the latter is so, differences

should be apparent in the raphe nuclei, rather than elsewhere [e.g., Neumaier et al., 1996; Anthony et al., 2000]. If differences in the density of 5-HT1B receptors mediate the expression of dominant vs. submissive behavior in Anolis as hypothesized, one would expect that administration of a 5-HT1B antagonist into the appropriate brain region(s) might elicit dominant displays from otherwise submissive males. My colleagues and I have, in fact, presented evidence that systemically administered SB-216641 elicits such behavior in Anolis sagrei. Studies of 3H-sumatriptan binding in three A. sagrei male pairs gave quantitative results similar to those in A. carolinensis (fig. 5C). At this stage it is not known whether differences apparent in 5-HT1B receptor density observed in our study between dominant and subordinate males were present before the pairing of antagonist animals – and thereby helped determine which would win or lose – or whether they were a consequence of the competition’s outcome. The latter possibility is favored, however, as it is possible to reverse dominance status in paired anoles by removing the dominant for several days to the cage of a hyperaggressive male, then reintroducing him into a cage in which the former nondominant is now ascendant [L.R. Baxter and E.C. Clark, unpubl. observ.]. If the density of forebrain 5-HT1B receptors is statedependent, it could be that elevation of their density results in a defeated animal showing deferential behaviors to the dominant, secondary to changes in 5-HT-flux [Baxter et al., 2001b]. However, given that a confrontation between newly paired dominants (both vigorously displaying as such), can result in a clear hierarchy (one changing to submissive behavior, then and thereafter) in less than one minute [L.R. Baxter and E.C. Clark, videotaped data available], it seems unlikely that a change in gene expression-regulated 5-HT1B density could happen so quickly as to affect the submissive behaviors (attempts to escape, as well as submissive postures) that are seen in acute defeat. However, other alterations of 5-HT1B receptor function, perhaps via 5-HT-moduline [a tetrapeptide neurotransmitter that interacts with local 5-HT1B receptors to rapidly change their functional activity and that is also implicated in anxiety modulation; Massot et al., 1998; Grimaldi et al., 1999] could effect such changes quickly. Subsequent alterations in the actual density of 5-HT1B receptors might operate later (as here), when peaceful co-existence has been established, and the submissive animal not only does not attempt to flee, but seems unafraid of the dominant, who now tolerates him in his deference [Baxter et al., 2001a]. Time-course correlation of 5-HT1B density changes with changes in dominance status display would be of great interest.

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Commonalties with Other Amniotes? Of particular relevance are ‘gene knockout’ mice missing the 5-HT1B receptor, as they show heightened male aggressiveness on territorial challenge and exhibit behaviors thought to indicate reduced threat anxiety on a number of other measures [Saudou et al., 1994; Brunner et al., 1999; Zhuang et al., 1999]. Also, specific 5-HT1B agonist drugs reduce normal male mouse offensive territorial aggression, and the effect is blocked by 5-HT1B antagonists [Fish et al., 1999]. In other experiments estrogens restrict, whereas androgens amplify, the magnitude of the effect of 5-HT1B agonists on such offensive inter-male territorial aggression [Simon et al., 1998; Cologer-Clifford et al., 1999]. Thus,

noting this rodent literature in the context of findings reported here, it is suggested that 5-HT1B receptors might have a similar role across amniote classes in regulating male territorial dominance behaviors.

Supported by the W.M. Keck Foundation, donations from Mr. and Mrs. Brian Harvey, Mr. and Mrs. Albert Levenson, Baxter family trusts, the Judson Braun Chair of Psychiatry at UCLA, and the Kathy Ireland Chair for Psychiatric Research at UAB. Edward C. Clark gave important technical aid, and reviewed the manuscript. Dr. Robert F. Ackermann provided key constructive criticisms.

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5-HT1B Receptors in Social Dominance

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