Rapid glucocorticoid stimulation and GABAergic inhibition of hippocampal serotonergic response: in vivo dialysis in the lizard anolis carolinensis

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Rapid glucocorticoid stimulation and GABAergic inhibition of hippocampal serotonergic response: in vivo dialysis in the lizard Anolis carolinensis Tangi R. Summers,a John M. Matter,b Jennifer M. McKay,c Patrick J. Ronan,d Earl T. Larson,e Kenneth J. Renner,a and Cliff H. Summersa,* a

Biology and Neuroscience, University of South Dakota, Vermillion, SD 57069, USA b Department of Biology, Juniata College, Huntingdon, PA 16652, USA c Department of Internal Medicine, Washington University, St. Louis, MO 63110, USA d Department of Psychiatry, University of Texas Southwestern Medical Center, and Research Service VA North Texas Health Care System, Dallas, TX 75216, USA e Department of Neuroscience, Uppsala University, Uppsala, SE-75124 Sweden Received 1 March 2002; revised 15 September 2002; accepted 18 September 2002

Abstract Central serotonin (5-HT) is activated during stressful situations and aggressive interactions in a number of species. Glucocorticoids secreted peripherally during stressful events feed back on central systems and may affect 5-HT mediation of stress-induced behavioral events. To test the neuromodulatory effect of stress hormone secretion, serotonin overflow was measured from the hippocampus of the lizard Anolis carolinensis. Microdialysis was used to collect repeated samples from anesthetized lizards, with perfusate measured by HPLC with electrochemical analysis. Following initially high levels of 5-HT, concentrations stabilized to basal levels after approximately 2 h. Intracortical infusion of 200 ng/ml corticosterone evoked transient increases in 5-HT release of approximately 400%. The effect of corticosterone on 5-HT overflow appears to be dose dependent as 20 ng/ml stimulated an increase of 200%, whereas 2 ng/ml stimulated a 50% increase. Administration of 0.1 and 1 ng/ml GABA via the dialysis probe significantly inhibited 5-HT overflow by 20 and 40%, respectively. The duration of GABA inhibition is greater than the stimulatory response for glucocorticoids. Short-lived glucocorticoid stimulation of 5-HT release suggests a possible mechanism for endocrine mediation of continuously changing social behavioral events. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Microdialysis; Serotonin; Corticosterone; ␥-Aminobutyric acid; Lizard; Anolis carolinensis; Hippocampus; Medial cortex

Serotonergic activity is affected by aggressive interactions, with most reports indicating that serotonin (5-HT) inhibits aggression (Raleigh et al., 1991; Deckel, 1996; Larson and Summers, 2001). However, 5-HT is also released during stress (Chaouloff et al., 1999), including aggressive interactions (Matter et al., 1998). The effects of behavior on neurochemical systems may be very rapid, as a single social defeat influences hippocampal serotonergic

* Corresponding author. Department of Biology, University of South Dakota, 414 East Clark Street, Vermillion, SD 57069-2390, USA. Fax: ⫹1-605-677-6557. E-mail address: [email protected] (C.H. Summers).

parameters (Berton et al., 1999). Prolonged serotonergic activity also is consistently associated with subordinate social status (Yodyingyuad et al., 1985; Blanchard et al., 1991; Winberg et al., 1992; Larson and Summers, 2001), but rapid stress-induced increases in serotonergic function are exhibited by dominant animals as well (Matter et al., 1998; Øverli et al., 1999; Summers, 2001, 2002). Glucocorticoids have numerous effects on serotonergic events in the hippocampus (McEwen et al., 1986), which is innervated by serotonergic terminals (Greenberg et al., 1990; Jacobs and Azmitia, 1992). Early studies employing adrenalectomy reported changes in 5-HT concentration in the brain (De Maio, 1959). Stress and circadian elevation of

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plasma corticosterone levels stimulate tryptophan hydroxylase (TH; Azmitia and McEwen, 1969, 1974). In addition, corticotropin (ACTH) and corticosterone stimulate cerebral 5-HT formation from tryptophan (Millard et al., 1972). The mechanism by which adrenalectomy influences TH and serotonergic activity appears to be hormonal, because removing the adrenal causes decreased 5-HT turnover in rat dorsal hippocampus; an effect reversed by corticosterone (de Kloet et al., 1982). Glucocorticoids may act directly on nerve terminals to regulate 5-HT synthesis through modification of tryptophan uptake, and via a permissive role in the induction of tryptophan hydroxylase (Neckers and Sze, 1975). Glucocorticoids delivered peripherally by ingestion or injection and centrally by dialysis have been demonstrated to influence 5-HT levels in amygdala, medial prefrontal cortex, and hippocampus (Luine et al., 1993; Inoue and Koyama, 1996; Summers et al., 1998, 2000). In addition, hippocampal 5-HT1A, 5-HT1B, 5-HT2, 5-HT6, and 5-HT7 receptors are also affected by adrenalectomy, corticosterone, and/or restraint stress (Mendelson and McEwen, 1991, 1992; Yau et al., 1997). Reciprocally, 5-HT is involved in regulation of hypothalmo–pituitary–adrenal axis (HPA) and sympathetic nervous system function in mammals, and may also play a role in limbic regulatory actions (Chaouloff, 1993; Dinan, 1996). Direct stimulation of CRH release by 5-HT has been demonstrated in vitro (Calogero et al., 1989). Serotonin has been demonstrated to stimulate adrenal axis activity in a variety of organisms from fish (Winberg et al., 1997) to humans (O’Keane and Dinan, 1991). ␥-Aminobutyric acid (GABA) also plays a role in limbic and hypothalamic regulation of neuroendocrine stress responses. For example, GABA regulates hippocampal excitability (Freund and Buzsa´ ki, 1996), and glucocorticoids influence GABA release (Ravindran et al., 1994) and binding (Wilson and Biscardi, 1994) and the composition of GABAA receptor subunits (Orchinik et al., 1995, 2001). Plus, GABA and other GABAA modulators inhibit 5-HT release (Balfour, 1980). A wide network of interneurons in the hippocampus are GABAergic in many vertebrates, including lizards (Guirado and Davila, 1999). In addition, there is a direct association between 5-HT3 receptors and GABA interneurons in the rat hippocampus (Morales and Bloom, 1997). Widespread 5-HT innervation in the vertebrate brain explains its potential to influence behavior. Serotonin has been commonly shown to inhibit active behavioral responses like feeding, aggressiveness, and locomotion (Olivier et al., 1989; Øverli et al., 1998), although serotonergic activity is enhanced during such active states as responding to stress (Chaouloff et al., 1999; Summers, 2001), arousal, and locomotor activity (Jacobs and Fornal, 1999). In a recent review, Jacobs and Fornal (1999) suggested that the primary function of serotonergic activity in the brain is to facilitate motor output. They suggested that 5-HT only exerts specialized stress-related effects as a result

of conjunctive activation with hormones like corticosterone, and that this type of interaction may occur in specific brain areas. Selective behavioral influence of 5-HT during social interaction may be suggested by 14 5-HT receptor types, as aggressive behavior may be inhibited by 5-HT1A, 5-HT1B, 5HT2, and 5-HT3 receptor subtypes; and stimulation of the adrenal axis regulated by 5-HT1A, 5-HT1B, and 5-HT2C receptor subtypes (Barnes and Sharp, 1999). In the lizard Anolis carolinensis, aggression appears to be inhibited by 5-HT1A, 5-HT2, and 5-HT3 receptors (Deckel and Fuqua, 1998), whereas motor output of dominant and submissive displays is regulated by the interaction of striatal 5HT2C receptor activity and dopamine (Baxter et al., 2001). Anoline lizards respond to social or physical stressors with rapidly elevated serotonergic activity (Emerson et al., 2000; Summers, 2001). Dominant and subordinate males have amplified hippocampal serotonergic activity after 10 min of social interaction, and the effect is measured in both combatants apparently because they act aggressively and/or observe aggression plus darkened eyespots (eyespots are postorbital chromatic social signals) (Korzan et al., 2000, 2002). Social interaction stimulates increased serotonergic activity in hippocampus, amygdala, striatum, nucleus accumbens, and locus ceruleus, but the hippocampus is of special interest because it is involved in glucocorticoidmediated negative feedback from the adrenal axis in mammals (Sapolsky et al., 1984), the Anolis hippocampus is densely populated with type II glucocorticoid receptors (Summers et al., 1994), and the hippocampus appears to play a prominent role in regulating short-term stress (Summers, 2001, 2002). In the mammalian hippocampus, social stress modifies 5-HT1A receptor binding, 5-HT transporter density, structural elements like dendritic spines, and neurogenesis, the last of which is regulated by NMDA receptor activation (McKittrick et al., 1995, 2000; Gould et al., 1997). In the Anolis hippocampus, NMDA receptor subunits NR2A and NR2B are upregulated in response to social stress (Meyer et al., 2002). As NR2B subunits and hippocampus are important for spatial and temporal learning (Tang et al., 1999; Shors et al., 2001), they may be important in establishing dominant and subordinate social roles. Additionally, corticosterone injections stimulate both increased serotonergic activity and upregulated NR2A and NR2B expression in A. carolinensis hippocampus (Summers et al., 2000; Meyer et al., 2002). Plasma corticosterone is also elevated by 10 min of social interaction in dominant and subordinate animals, but returns to baseline by 20 min in both combatants (Summers, 2001). However, elevated plasma corticosterone and hippocampal serotonergic activity after 20 min occurs only in subordinate males (corticosterone in subordinate males was elevated at 40 min or 3 weeks, as measured by Greenberg et al., 1984; Summers, 2001). Chronically elevated serotonergic activity is measured in the amygdalostriatal region of subordinate male brains (Summers et al., 1998), and when synaptic 5-HT is chronically elevated by the uptake inhibitor sertraline, it is produces submissive

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behavior and can reverse social dominance (Larson and Summers, 2001). Because adrenal axis hormones and serotonin have both been postulated to stimulate submissive behavior, and because there appears to be a circular link between adrenal axis production and serotonergic production, it was hypothesized that glucocorticoids affect serotonin release. Direct and/or rapid stimulation and inhibition of 5-HT in the hippocampus may provide the framework for behavioral adaptations during stressful conditions. Animals must have temporally appropriate and context-dependent plasticity of behavior to cope with a variety of stressors. In short, it has been hypothesized that the foundation for normal adaptive functioning, both behavioral and CNS-HPA, is the timely and efficient initiation and termination of each component of neuroendocrine stress system activity (McEwen et al., 1986). Similarly, faster initial neuroendocrine stress responsiveness increases the probability of becoming dominant during interactions determining social status in the lizard Anolis carolinensis (Summers, 2001, 2002; Larson and Summers, 2001). Based on these studies, we hypothesized that the effect of glucocorticoids and GABA on 5-HT release in the Anolis hippocampus would be rapid, with temporal resolution appropriate for behavioral adaptation.

Materials and methods Animals Adult male lizards, A. carolinensis, were purchased from a commercial supplier (Buck’s Live Animals, La Place, LA) then housed individually in 25 ⫻ 25 ⫻ 25-cm terraria with a 14L32°C:10D20°C photothermal regimen. Surgical implantation of microdialysis probes and dialysis perfusion throughout the experiment were implemented with lizards exposed to methoxyflurane anesthesia (Mallinckrodt). Body temperature (32°C) was maintained with a heating pad. The stereotaxic coordinates for probe implantation, determined from the parietal eye, were, for the medial cortex, 0.6 mm posterior, 0.1 mm lateral, and 1.5 mm below the cortical surface (Greenberg, 1982). The lateral coordinate served to place the probe into the hippocampal cortex and to avoid heavy vascularization. Anterior and lateral coordinates represent the central location of access holes drilled with a 0.4-mm dental bit. The brain of male A. carolinensis lizards weighs 40 –50 mg, and is approximately 8 mm in length (Greenberg, 1982). What is more important with respect to the ⬃1.1-mm-long dialysis surface is that the A. carolinensis brain is about 3.3 mm from dorsal to ventral aspect at the deepest point, and 2.6 mm deep where the probe is placed. The medial and mediodorsal cortices that make up the hippocampus run to 1.5 mm deep from the surface of the head, and are approximately 0.8 mm in total depth. Following removal of bone, the dura was carefully removed under magnification to avoid bleeding. The probe was then slowly

Fig. 1. (A) Probe placement A in the medial, hippocampal cortex of Anolis carolinensis. (B) Typical time course for baseline 5-HT overflow stabilization. (C) Artificial CSF-treated controls (open circles) compared with verification of neuronal source of 5-HT determined by tetrodotoxin (TTx; open diamonds) blocking of Na⫹ ion channels. Values for 5-HT release are expressed as mean (⫹ SEM) percentages of three baseline samples taken prior to treatment. *Samples with 5-HT values significantly different from baseline; **significantly different from control.

lowered into the brain over a 3- to 5-min period (generalized probe placement Fig. 1A). Dialysis A concentric dialysis probe with a surface exchange area of 250-␮m diameter and 1.1- to 1.5-mm length was inserted 1.5 mm into the medial cortex. Artificial cerebrospinal fluid (CSF: NaCl 147 mM, KCl 2.4 mM, CaCl2 1.2 mM, MgCl2

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Concentric dialysis probes were assembled by slipping a 5-mm-length of cellulose dialysis fiber (MW cutoff 5000, Travenol Laboratories) over the beveled end of a 7-cm length of fused silica tubing (i.d. 101 ␮m, Polymicro Technologies, Inc.). The membrane–silica assembly was glued into a 5-cm length of 26G stainless-steel tubing (Small Parts, Inc.) with 2-ton white epoxy (Devcon) so that approximately 4 mm of membrane was exposed. The exposed cellulose surface was cut to the appropriate length and sealed with 2-ton epoxy. Probe tips were cut to lengths ranging from 0.7 to 1.5 mm. After the glue dried, a 5-mm length of PE 50 tubing was slipped over both the distal end of the silica fiber and the cannula. A 1-cm length of 26G stainless-steel cannula was inserted into the PE 50 sleeve and glued in place with 5-min epoxy (Devcon). Neuronal origin of 5-HT in perfusates was tested by evaluating the effects of tetrodotoxin (TTx; Sigma) on the basal levels of serotonin. A concentration 1 ␮M TTx was made in modified Ringer’s solution after being dissolved in acetate buffer. Samples were collected and analyzed until extracellular 5-HT levels were stable for four consecutive measurements. At this point, the perfusion buffer containing 1 ␮M TTx was substituted for the standard modified Ringer’s solution. HPLC Fig. 2. Corticosterone was delivered directly to the lizard hippocampus via microdialysis probes in one of three doses, 2 ng/ml (closed circles A, B) 20 ng/ml (open circles B), or 200 ng/ml (open diamonds A). Hormone delivery time (dark bar) was either 20 min (A) or 120 min (B). Values for 5-HT release are expressed as mean (⫹ SEM) percentages of three baseline samples taken prior to treatment. *Samples with 5-HT values significantly different from baseline; **significantly different from control; ***significantly different from another dose.

1.0 mM, NaH2PO4 0.9 mM, Na2HPO4 1.4 mM, pH 7.4) was circulated through the probe at the rate of 0.42 ␮l/min, and perfusate was collected every 20 min (Figs. 1C, 2A, 3) or 30 min (Figs. 1C, TTx; 2B) for HPLC electrochemical analysis. Perfusion with CSF was changed to perfusion with corticosterone, GABA, or TTx in CSF (N ⫽ 6 – 8 per group) after 4 h of stable 5-HT overflow. Corticosterone was administered to the hippocampal cortex via the probe for 20 min (Fig. 2A) or 120 min (Fig. 2B), and then perfusion with CSF alone was reestablished. Animals were treated with either CSF alone, corticosterone (2, 20, or 200 ng/ml), or GABA (0.1 or 1 ng/ml). Delivery of hormone or drug via the dialysis probe was approximately 1% effective, producing, for example, actual doses of ⬃0.02, 0.2, and 2 ng/ml corticosterone at rates of ⬃8.4, 84, and 840 fg corticosterone/min. Probe recovery was calculated by comparing 5-HT peak heights obtained by dialysis of 0.5 to 1 ⫻ 10⫺7 M 5-HT in CSF through the probe with peak heights from direct analysis of an equivalent volume of the same solution at room temperature. Probes having less than 8% recovery were rejected. Recoveries for most probes ranged from 10 to 15%.

Microdialysis perfusate was analyzed for 5-HT using a 10% acetonitrile (including 483 mg sodium octanesulfonate, 100 mg EDTA, 150 ␮l triethylamine, 4.7 g NaH2PO4 per liter) mobile phase, pH 5.7, and a Sepstick 3-␮m C18 microbore column (Bioanalytical Systems). Serotonin concentrations were analyzed using an HPLC system that maximizes sensitivity by using pneumatic fluid displacement to

Fig. 3. Effect of ␥-aminobutyric acid (GABA) on 5-HT release, expressed as mean (⫹ SEM) percentages of three baseline samples taken prior to treatment. Hippocampal release of 5-HT was inhibited by 100 pg/ml (0.1 ng/ml, open squares) or 1 ng/ml (open triangles) GABA as compared with control (open circles, also Fig. 1c). *Samples with 5-HT values significantly different from baseline; **significantly different from control; ***significantly different from another dose.

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provide a pulseless flow of mobile phase across the detector cell (Bradberry et al., 1991). The pump consists of a regulated tank of pressurized nitrogen pushing mobile phase through a reservoir spiral of 0.21-in. stainless-steel tubing (volume capacity ⬃ 350 ml). Serotonin was detected electrochemically with a potentiostat and a glassy carbon working electrode (Bioanalytical Systems) maintained at a potential of ⫹0.50 V with respect to a Ag/AgCl reference electrode (Bioanalytical Systems). Statistical analyses Serotonin concentrations were calculated by dividing picograms of the 5-HT standard by peak height (pg 5-HT/cm peak height) and multiplying this value by the peak heights obtained from each sample to yield picograms of 5-HT/10 ␮l sample. Moderate variation in basal levels of 5-HT between experiments due to slight differences in probe placement and/or probe recoveries was standardized by calculation of percentage of basal serotonin levels. Basal 5-HT level (100%) was calculated from the mean of three successive samples prior to the initiation of experimental treatments. Serotonin peak heights with less than 2:1 signalto-noise ratio were considered undetectable, and a value based on 2:1 signal-to-noise was used in the calculations. The results from validation studies using TTx were analyzed using a two-way ANOVA with repeated measures. Comparison of TTx effects with pretreatment samples were done using Dunnett’s test where the sample immediately preceding TTx perfusion served as the control. The effects of corticosterone and GABA on 5-HT overflow were analyzed using a two-way analysis of variance (treatment ⫻ time) with repeated measures, where the repeated factor was time. The effect of treatment over time was compared with pretreatment values using Dunnett’s test where the sample immediately preceding the treatment served as the control value.

Results Basal levels Serotonergic overflow stabilized approximately 2 h following probe placement (Fig. 1B). Position of the probe in the hippocampal cortex was verified histologically for each animal. Stable unstimulated levels of 5-HT in dialysate from lizard hippocampus were ⬃500 fg. TTx verification The majority (F ⫽ 18.7, P ⬍ 0.003; two-way rm compared with baseline control) of the 5-HT measured from the medial cortex of the lizard A. carolinensis was inhibited by the Na⫹ channel blocker tetrodotoxin (Fig. 1C). Approximately 60% reduction (interaction effect: treatment ⫻ time:

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F ⫽ 9.5, P ⬍ 0.001) in 5-HT overflow directly followed TTx administration. Significant (F ⫽ 54.1, P ⬍ 0.001) inhibition was measured in the first perfusate sample following TTx (Dunnett’s q ⫽ 6.6, P ⬍ 0.001) and persisted for more than 120 min. Glucocorticoid Corticosterone delivered directly to A. carolinensis hippocampus via a microdialysis probe had a significant (F ⫽ 30.6, P ⬍ 0.001; two-way rm) stimulatory effect on 5-HT overflow, which was detected by the first sampling period (Figs. 2A, 2B; time effect: F ⫽ 12.4, P ⬍ 0.001) regardless of dose or duration (dose ⫻ time interaction: F ⫽ 12.5, P ⬍ 0.001). The effect of corticosterone was brief when 2 ng/ml was administered for 20 min (F ⫽ 7.1, P ⬍ 0.001; Dunnett’s q ⫽ 5.1) or for 120 min (F ⫽ 3.5, P ⬍ 0.03; Dunnett’s q ⫽ 3.3). Similarly, when 20 ng/ml corticosterone was delivered, the 5-HT response began to be reversed before the end of the hormone treatment period (Fig. 2B). Higher doses of corticosterone, either 20 ng for 120 min (F ⫽ 10.7, P ⬍ 0.001; Dunnett’s q ⫽ 6.7) or 200 ng for 20 min (F ⫽ 9.3, P ⬍ 0.001; Dunnett’s q ⫽ 6.7), had greater stimulatory effect on 5-HT overflow than the 2 ng/ml dose. GABA Delivered directly via a microdialysis probe, GABA inhibited (F ⫽ 7.5, P ⬍ 0.04; two-way rm) 5-HT release in lizard hippocampus (Fig. 3). The effect was long-lasting (time effect: F ⫽ 16.6, P ⬍ 0.001). A 20 min exposure produced inhibition that was not reversed by the end of the experimental trial (100 min). Higher doses of GABA had a stronger inhibitory effect on 5-HT release (treatment ⫻ time interaction: F ⫽ 2.4, P ⬍ 0.03). When 1 ng/ml GABA was delivered the inhibition was approximately 40% from baseline levels of 5-HT release (F ⫽ 13.1, P ⬍ 0.001; Dunnett’s q ⫽ 3.6), compared with 20% inhibition caused by 0.1 ng/ml GABA (F ⫽ 5.1, P ⬍ 0.003; Dunnett’s q ⫽ 5.8). Furthermore, preliminary results from experiments using the GABAA antagonist bicuculline (N ⫽ 3) produced a rapid increase in serotonin release, which lasted no more than 40 min (data not presented).

Discussion Serotonergic activity in the hippocampus of the lizard A. carolinensis is rapidly and dynamically influenced by local changes in hormone or transmitter concentrations (Figs. 2 and 3). Local rapid stimulation or inhibition of hippocampal 5-HT release supplies the basis for behavioral adaptations during stressful conditions. Corticosterone and GABA modify hippocampal release of 5-HT in a dose- and time-sensitive manner. Extracellular 5-HT overflow in the hippocampus was markedly decreased by TTx, suggesting that most

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of the 5-HT measured in Anolis hippocampus can be derived from active neuronal release. The remaining 5-HT in the extracellular pool from which the samples are derived may also be neuronal, if basal or background levels do not require action potential for release. Serotonin is delivered broadly to cortical structures from a small number of raphe neurons in the brainstem. Serotonergic activity, however, is highly modifiable in terminal regions by both local interneuron factors, such as GABA, and by peripheral hormonal factors like glucocorticoids (Figs. 2, 3). This suggests that local terminal influences on 5-HT release may periodically enhance, diminish, or overshadow stimuli directed by action potentials from brainstem perikarya. Our data represent localized modification of serotonergic activity, consistent with previous studies that demonstrate region-specific activity associated with specific behavioral responses (Summers et al., 1998; Chaouloff et al., 1999; Summers, 2001), but inconsistent with hypotheses of generalized serotonergic activity associated with arousal or locomotion (Jacobs and Fornal, 1999). There are three reasons the results of our dialysis experiments should be considered regionally specific, not including the fact that hormone or transmitter was delivered by dialysis to a limited region of the brain. The first is that hippocampal GABA is produced by local interneurons, even in lizards (Guirado and Davila, 1999). The second is that glucocorticoid receptors, which are most dense in hippocampus (McEwen et al., 1986; Summers et al., 1994), determine the locality of corticosteroid actions. Finally, neither of the treatments presented here was delivered to the serotonergic cell bodies in the raphe, but rather to terminals in the hippocampus. The effects reported here were most likely generated by stimulation of terminals in hippocampus, rather than by eliciting action potentials from cells in the raphe. Although we believe that much of the regulatory influence on 5-HT release occurs in terminal regions, our data are not inconsistent with recent evidence for identifiable cell groups in the raphe with distinctive output qualities (Lowry et al., 2000). Even though glucocorticoids are secreted systemically, influences on the central nervous system may be exerted locally due to regionally specific receptor expression (McEwen et al., 1986). The serotonergic machinery influenced by glucocorticoids (or absence of, following adrenalectomy) at 5-HT terminals or perikarya of mammals include tryptophan uptake (Hillier et al., 1975), tryptophan hydroxylase activity (Azmitia and McEwen, 1969, 1974), 5-HT synthesis (Millard et al., 1972), and expression of 5-HT1A, 5-HT2, 5-HT7, receptor, and 5-HT transporter mRNAs (Kuroda et al., 1994; Le Corre et al., 1997). Corticosteroids (or lack thereof due to adrenalectomy or metyrapone) have also been demonstrated in mammals to influence cerebral 5-HT concentrations (De Maio, 1959), 5-HT release by synaptosomes from brain tissue in vitro (Sze, 1976), 5-HT1A, 5-HT1B, and 5-HT2 receptor binding (Mendelson and McEwen, 1992; Kuroda et al., 1994), 5-HT1A mediated hyperpolarization (Karten et al., 2001; Joe¨ ls, 2001), 5-HT turn-

over measured by accumulation after pargyline treatment (de Kloet et al., 1982; Korte-Bouws et al., 1996), and the ratio 5-HIAA/5-HT in the hippocampus of A. carolinensis (Summers et al., 2000). Corticosterone directly delivered to the hippocampus of A. carolinensis by microdialysis had a significant stimulatory effect on 5-HT overflow, which was rapid, brief, and dose dependent. The list of corticosteroid influences on hippocampal serotonergic activities is long, but the events measured in our study using in vivo microdialysis probably include only the faster acting mechanisms, and therefore may be mediated by membrane-bound glucocorticoid receptors like those that have been discovered in amphibians and birds (Orchinik et al., 1991; Breuner and Orchinik, 2001). The rapid, short-term, dose-dependent plasticity of the response suggests a temporal framework both sensitive to behavioral mediation (Jacobs and Fornal, 1999) and affecting subsequent behavior (Summers, 2001, 2002). The results of these microdialysis experiments are not only consistent with, but match the time frame of increased serotonergic activity stimulated by aggressive social interaction (Summers, 2001). Perhaps it is not a coincidence that during social stress both plasma corticosterone and hippocampal serotonergic activity are elevated after 10 min of aggressive social interaction (Summers, 2001). However, although the rapid response measured does not preclude conjunctive activation between corticosterone and 5-HT (Jacobs and Fornal, 1999), it does not signify a conjunctive synergism either, as corticosterone stimulates enhanced release of 5-HT. In addition, it is important to note that intrahippocampal administration of corticosterone almost certainly does not thoroughly imitate the effects of peripherally generated corticosterone induced by stress. The flexibility of serotonergic responses in hippocampus suggests an important role for behavior that is increased by the capacity for rapid inhibition by GABA. The time course for GABA inhibition of hippocampal 5-HT release in A. carolinensis is similar to that for glucocorticoid stimulation. However, although the effect is similarly dose dependent, GABA inhibition has a longer duration. Preliminary evidence suggests that the effect may be mediated by GABAA receptors, as the inhibition is reversed by bicuculline and, as with corticosterone, is rapidly achieved and very brief. In the context of stress-related behavioral modifications, these differences may be further modified by the actions of corticosterone, which in mammals affects the composition of GABAA receptors (Orchinik et al., 1995, 2001). Taken together, the actions of glucocorticoids and GABA present behaviorally relevant mechanisms for adaptable modifications to stress-related hippocampal activity in the lizard A. carolinensis. The rapid progression of social stress-induced serotonergic activity (measured as 5-HIAA/ 5-HT) in Anolis hippocampus is diagnostic for social status, and coincident with glucocorticoid secretion (Summers, 2001, 2002). Subordinate males have both delayed and

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prolonged elevated hippocampal serotonergic activity as well as plasma corticosterone secretion (Greenberg et al., 1984; Summers, 2001). This corresponds to the delay observed for eyespot formation among subordinate males (Summers and Greenberg, 1994). Eyespots act as social sign stimuli, inhibiting aggression and influencing social status (Korzan et al., 2002). The 5-HT reuptake inhibitor sertraline delays eyespot formation and reverses social dominance (Larson and Summers, 2001). Artificially creating eyespot darkness with black paint not only establishes dominant social status, but also modifies hippocampal and raphe serotonergic activity (Korzan et al., 2000, 2001, 2002). The evidence suggests that serotonergic activity is pliable, influenced by both behavioral and neuroendocrine machinery. In addition, the reverse is also true. Greater or reduced serotonergic response also influences behavior and neuroendocrine machinery. The hippocampus is a site of flexible and dynamic serotonergic response to both corticosterone and GABA, and is a region pivotal to the functions of memory and stress responsiveness. The relationship between stress and memory is critical for producing adaptive responses to social interaction. Social stress, corticosterone, and 5-HT have significant effects on the structural and neurochemical characteristics of the hippocampus that are important for learning and memory. Presumably, social interaction and the neuroendocrine by-products of aggression and stress that result from it modify social behavior and thereby enhance survival. It follows not only that memory of social and environmental elements that enhance survival is valuable, but that the formation and consolidation of such memory are necessarily part of the evolutionarily conserved neuroendocrine mechanisms that promote continued survival. The state of neuroendocrine machinery that is responsive to social stress and facilitates memories and behavior seems likely to be included among the qualities that distinguish dominant from subordinate individuals. Our results suggest a rapidly acting and responsive system that integrates environmental and internal conditions to produce adaptive behavioral and physiological output.

Acknowledgments This work was supported by NIH Grant P20 RR15567, NSF Grant IBN-9596009, and a grant from the South Dakota Health Research Foundation. We thank David Kappenman for technical support.

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