Anxiolytic effect of serotonin depletion in the novelty-induced hypophagia test


Rationale Relatively little is known about the neural mechanisms underlying anxiety in the novelty-induced hypophagia test, the only known anxiety test that is responsive to chronic but not acute or subchronic antide- pressant treatment.

Objectives The goal of the present experiment was to characterize the role of serotonin in the ability of novelty to suppress feeding.
Materials and methods Pair-housed male Sprague–Dawley rats were trained to eat graham cracker crumbs individually in their home cage (15 min/day). After stable daily intakes were obtained, the animals were depleted of serotonin using 4-chloro-DL-phenylalanine (150 mg kg−1 day−1×2 days). Forty-eight hours later, central serotonin was restored by the administration of the peripheral L-aromatic amino acid decarboxylase inhibitor, benserazide (10 mg/kg), followed 15 min later with the immediate precursor of serotonin, 5- hydroxy-L-tryptophan (30 mg/kg). Thirty minutes later, the animals were given access to graham cracker crumbs in a novel environment.

Results The animals demonstrated increased latencies to approach the food and reduced food intake in the novel environment. This effect was attenuated by serotonin depletion. Repletion of central serotonin restored the inhibitory response to novelty. The analysis of serotonin content in different brain regions confirmed that serotonin was depleted by greater than 90%, whereas the repletion treatment resulted in serotonin levels similar to nondepleted animals.

Conclusions Acute depletion of serotonin acts to reduce anxiety behavior as measured by an inhibitory anxiety response during exposure to novel stimuli. These findings are in agreement with the proposed general role for serotonin in behavioral inhibition and that reductions of serotonin facilitate the adoption of more active coping responses to stress.

Keywords : Novelty . Hypophagia . Serotonin . 5-HT. PCPA . Depletion . Anxiety


The novelty-induced hypophagia (NIH) test measures the suppression of food intake by exposure to a novel environment (Dulawa and Hen 2005; Dulawa et al. 2004). The NIH test is a recent modification of an older test, the novelty-induced suppression of feeding (NSF; Bodnoff et al. 1988) test, that minimizes secondary motivational and feeding effects by omitting food deprivation and including home cage assessments (Dulawa and Hen 2005; Dulawa et al. 2004; Merali et al. 2003, 2004). The NIH test is one of the few animal tests of anxiety that are responsive to chronic antidepressant treatment (for review see Borsini et al. 2002). Because the delayed response to antidepressants in this test is similar to the delay required to reduce anxiety in psychiatric patients, the NIH test has promise for the understanding of what allows antidepressants to become effective after chronic treatment.

For many years, it has been known that serotonin plays an important role in anxiety and its treatment; however, the exact nature of this relationship is not entirely clear (Lucki 1996). Serotonin has traditionally been thought to be responsible for the behavioral inhibition that accompanies anxiety, at least in part, because the release of serotonin is increased by stress and anxiety (Chaouloff 2000; Kirby et al. 1997; Yoshioka et al. 1995). Likewise, the ability of benzodiazepines to decrease serotonin release while de- creasing anxiety has contributed to the notion that reduc- tions of serotonin may be anxiolytic (Rex et al. 1993; Stein et al. 1975; Tye et al. 1979; Yoshioka et al. 1995). Buspirone and a series of related compounds have been shown to produce anxiolytic behavioral effects in animals (Fernandez-Guasti and Lopez-Rubalcava 1990; Gammans et al. 1992; Oshima et al. 2001), in part, by stimulating 5- hydroxytryptamine 1A autoreceptors in the dorsal raphe that inhibits the release of serotonin in forebrain regions (Higgins et al. 1992). On the other hand, clinical findings demonstrate that selective serotonin reuptake inhibitors (SSRIs) that would increase extracellular serotonin levels are effective to treat anxiety disorders when given repeat- edly (Nutt 2001). The required lag time between the start of treatment with SSRIs and the onset of clinical benefit combined with the aforementioned effects of depletion leave the direct role of serotonin in anxiety and its treatment unclear.

The present study examined the role of direct manipu- lations of serotonin transmission in the NIH test. The NIH test in rats and mice has developed recent prominence due to its responsiveness to chronic rather than acute or subchronic treatment with antidepressants including the SSRIs (Dulawa and Hen 2005). However, little is known about the neural mechanisms that mediate anxiety behavior and antidepressant efficacy in the NIH test because this test has not been extensively studied. The purpose of this study was to examine the role of serotonin in performance in the NIH test. The effects of serotonin depletion were studied following the inhibition of synthesis using the tryptophan hydroxylase inhibitor 4-chloro-DL-phenylalanine (PCPA; Weissman and Koe 1965). Secondly, the effects of increasing serotonin content by administration of its immediate precursor 5-hydroxy-L-tryptophan (5-HTP) in combination with the peripheral decarboxylase inhibitor benserazide were studied in normal and previously depleted animals.

Materials and methods


Sixty male Sprague–Dawley rats were obtained from Charles River Laboratories (Wilmington, MA), weighing 225–250 g on arrival, and housed two per polycarbonate cage. Rats were maintained in a temperature-controlled environment (22±1°C) under a 12-h light–dark cycle, with lights turned on at 0700 hours, and with food and water freely available. Animals were not food deprived during any part of the experiment. Home cages were lined with Bed-o’Cobs® (The Andersons, Maulmee, OH) bedding material. Procedures were conducted with the approval of the University of Pennsylvania Institutional Animal Care and Use Committee and within the guidelines of the National Research Council’s (1996) Guide for the Care and Use of Laboratory Animals.

Novelty-induced hypophagia

The methods used here were modeled after the methods used by Merali et al. (2004) with some modifications including changes to accommodate pair-housed animals. Ninety minutes before each daily feeding session, an opaque Plexiglas divider was inserted into the home cage, separating the two animals so that individual intakes could be measured. Each individual rat was presented with 8– 10 g of graham cracker crumbs for 15 min in their home cage. The animals were trained to consume the palatable snack in the home cage for 8 days. This was the time required to reach stable intakes and latencies to eat. After the 8-day training period, half of the animals were injected with PCPA (n =30; 150 mg kg−1 day−1) once daily for 2 days to induce serotonin depletion, whereas the remaining animals were injected with 0.9% saline (n=30). Graham crackers were not presented on days when PCPA was administered. The animals were then left undisturbed in the colony for 1 day before a final home cage test. The next day, novelty-induced suppression of feeding was assessed by giving the animals access to the familiar palatable food (graham cracker crumbs) in a novel environment. The novel environment consisted of a polycarbonate cage of the same dimensions as the home cage (48×26×20 cm). Unlike the home cage, the novel cage was brightly lit and had a wire, rather than bedding lined, floor. Bright lighting was achieved by placing a 60-W, 120-V light fixture, 8 in. above the cage lid, directly above the food bowl. Forty-five minutes before the novel cage test, animals from each pretreatment group (n =15/group) were injected with the peripheral inhibitor of L-aromatic amino acid decarboxyl- ase; benserazide HCl (10 mg/kg) followed 15 min later (30 min before the test) by injection with the serotonin precursor, 5-HTP (30 mg/kg; n =15/group). Control animals were injected with saline (0.9%) in place of benserazide and phosphate buffered saline (pH=6.0) in place of 5-HTP. Test sessions were videotaped so that the latency to begin eating (s) in either the home or novel cage could be measured by a blind observer. Total food consumed (g) during the 15-min block was measured by weighing the food before and after it was presented to the animal. During the early training sessions, the food was sometimes buried. In this case a value of 0 was recorded. However, this was never observed in later sessions. During the novel cage test, spilling was prevented by attaching the food bowl to the novel cage with Velcro. Immediately after the novel cage test, the rats were killed, the brains were removed, and the amygdala, frontal cortex, and hippocampus were removed by gross dissection. Tissue samples were weighed, flash frozen, and stored at −80° until high-performance liquid chromatography (HPLC) analysis of serotonin tissue content.

Locomotor assessment

Digital videos of the home and novel cage sessions were analyzed by an IBM-compatible computer-running SMART II Video Tracking System software (San Diego Instru- ments). Distance calibration of this software made it possible to determine the distance traveled (cm) during each of the 15-min test sessions.


All drugs were obtained from Sigma-Aldrich (St. Louis, MO). The doses administered were expressed as the freebase weight. The tryptophan hydroxylase inhibitor PCPA methyl ester hydrochloride (150 mg/2 ml) and the peripheral L-aromatic amino acid decarboxylase inhibitor benserazide HCl (10 mg/ml) were dissolved in sterile deionized water. The immediate precursor of serotonin, 5- HTP (30 mg/2 ml), was dissolved in an acidic (pH=2.0) phosphate-buffered saline solution, and then the pH of the solution was adjusted to 6.0. These doses were chosen based on previous studies demonstrating that they were sufficient to induce substantial decreases in serotonin and to restore serotonin to near-basal levels (Harvey et al. 1975; Minabe et al. 1996).

Analysis of tissue contents

The tissue samples were homogenized (Tekmar, Cincinnati, OH) in a 100-μM ethylenediamine tetraacetic acid solution dissolved in 0.1-N perchloric acid (15 μl/mg of tissue). After centrifugation for 15 min at 15,000 rpm (2–8°C), the supernatant was filtered using 0.45-μM nylon Costar Spin- X centrifuge filters. The filtered supernatant was assayed for serotonin content by HPLC coupled with electrochem- ical detection (EC).

The HPLC–EC system (Bioanalytical Systems, West Lafayette, IN) consisted of a PM-80 pump, a Sample Sentinel autosampler, and a LC-4C electrochemical detec- tor. The samples (12 μl) were injected and pumped through a reverse phase microbore column (ODS 3 μm, 1×100 mm, Bioanalytical Systems) at a flow rate of 0.6 ml/min with electrodetection at +0.6 V. Separation for serotonin was accomplished by using an HPLC mobile phase consisting of 90-mM sodium acetate, 35-mM citric acid, 0.34 mM EDTA, 1.2-mM sodium octyl sulfate, and 9% methanol (v/v; Mayorga et al. 2001) with the addition of 2% dimethylacetamide (v/v). The pH of the mobile phase was adjusted to 4.2 with 12 N HCl.

Statistical procedures

To determine the effect of serotonin depletion on baseline food intake, a two-way mixed analysis of variance (ANOVA) was conducted with the within-subjects factor session (pre-, post-depletion) and the between-subjects factor depletion (vehicle, PCPA). The analyses conducted for the effects of novelty on behavior were three-way mixed ANOVAs. The independent variables for these analyses were the within-subjects factor of environment (home, novel) and between-subjects factors of depletion (vehicle, PCPA) and repletion (vehicle, benserazide/5-HTP). The dependent variables were latency to begin eating (s) and food intake (g). Follow-up comparisons were conducted using Fisher’s PLSD test. To follow-up the complex interactions of Environment by Depletion and Repletion, difference scores were calculated between the home- and novel-cage-dependent variables (intake difference score, novel cage intake−home cage intake; latency difference score, novel cage latency−home cage latency) and one-way ANOVAs followed by Fisher’s PLSD tests were conducted. The effect of depletion on locomotor activity during the home cage test was analyzed by a one-way between- subjects ANOVA with the factor depletion (vehicle, PCPA). Locomotor effects in the novel cage were analyzed by a two-way between-subjects ANOVA, with the between- subjects factors of depletion (vehicle, PCPA) and repletion (vehicle, benserazide/5-HTP). Similarly, tissue serotonin content for each brain area analyzed by two-way between- subjects ANOVA with the between-subjects factors of depletion and repletion (vehicle, benserazide/5-HTP). Fol- low-up comparisons were conducted using Fisher’s PLSD test.


Baseline food intake

A two-way mixed ANOVA comparing total food intake during the final home cage training session and the home cage test after serotonin depletion revealed significant main effects of PCPA treatment [F(1.58) =5.57, p <0.05] and home cage session [F(1.58) =7.08, p <0.05], and a signif- icant interaction [F(1.58)=9.39, p<0.005]. Follow-up comparisons revealed that the main effects were the result of the interaction such that intakes between the PCPA (4.75± 0.26) and saline- (5.01±0.29) treated groups did not differ before depletion (p=0.50); however, PCPA treatment (3.76±0.24) significantly reduced food consumed in the home cage relative to saline treatment (5.08±0.28; p< 0.001). Novelty effects on food consumed The effects of novelty on food consumption are illustrated in Fig. 1. The exposure to the novel environment resulted in a significant suppression of food intake that was attenuated by serotonin depletion and restored by serotonin repletion. This conclusion was supported by a three-way ANOVA (environment×depletion×repletion) yielding significant main effects of environment [F(1.56) =247.35, p <0.0001], depletion [F(1.56) =6.26, p <0.01] and repletion [F(1.56) = 4.08, p <0.05], as well as significant environment×depletion [F(1.56) = 13.01, p < 0.001], environment×repletion [F(1.56) = 24.06, p < 0.0001] and environment×deple- tion× repletion [F(1.56) =5.07, p<0.05] interactions. Fol- low-up Fisher’s PLSD tests comparing intake across environments within each treatment demonstrated that food intake was reduced in the novel environment compared to the home environment in all treatment groups (saline–vehicle p<0.0001, saline–5-HTP p<0.0001, PCPA-vehicle p <0.05, PCPA–5-HTP p <0.0001) and that the PCPA–5-HTP and saline–5-HTP animals ate less than both saline–vehicle- (p <0.0001 and p <0.001) and PCPA–vehicle- (p <0.001 and p <0.05) treated animals. Because all of the groups showed a suppression of intake in the novel environment relative to the home cage, the complex interaction was examined by conducting a two-way ANOVA on difference scores calculated between home and novel cage. This analysis revealed significant main effects of depletion [F(1.56) = 13.01, p <0.001] and repletion [F(1.56) =24.06, p<0.0001] and a significant depletion×repletion interaction [F(1.56) = 9.28, p <0.05]. The follow-up Fisher’s PLSD comparisons demonstrated that PCPA–vehicle-treated animals showed less novelty-induced suppression of feeding compared to all other groups (ps<0.0001). No other pairwise compar- isons were significant. However, there was a trend toward saline–5-HTP animals having greater novelty-induced suppression of feeding relative to saline–vehicle-treated animals (p =0.065). Fig. 1 a Mean (±SEM) graham cracker intakes (g) during the 15-min home (open bars) or novel (dark bars) cage tests.Asterisks, significantly reduced novel cage intake compared to home cage intake. Ampersand, significantly different from sa- line–vehicle-treated animals within the same test cage group. Dagger, significantly different from PCPA–vehicle-treated ani- mals within the same test cage group. b Mean (±SEM) differ- ence score for graham cracker intakes (novel cage intake− home cage intake). One sharp, significantly less difference in intake between the home and novel cage compared to all other groups. n=15 per group. One symbol, p<0.05; two symbols, p<0.001; three symbols, p<0.0001. Novelty effects on latency to begin eating Figure 2 shows the effects of novelty on latency to begin eating. The exposure to novelty had similar effects on latency to begin eating as food intake. However, these data were more variable, and effects of repletion were revealed that may suggest a floor effect for food intake. This conclusion was supported by a three-way ANOVA (envi- ronment×depletion×repletion), yielding significant main effects of environment [F(1.56) =96.95, p <0.0001] and repletion [F(1.56) =17.73, p <0.0001], as well as significant environment×repletion [F(1.56) = 20.09, p < 0.0001] and environment×depletion×repletion [F(1.56) = 5.28, p < 0.05] interactions. Follow-up Fisher’s PLSD tests compar- ing latency to begin eating across environments within each treatment demonstrated that latency to begin eating was increased in the novel environment compared to the home environment in all treatment groups (saline–vehicle p <0.001, saline–5-HTP p<0.001, PCPA–vehicle p <0.001, PCPA–5-HTP p < 0.0001). Because all of the groups showed a greater latency to begin eating in the novel environment relative to the home cage, the complex interaction was examined by conducting a two-way ANOVA on difference scores calculated between home and novel cage. This analysis revealed a significant main effect of repletion [F(1.56) =20.10, p<0.0001] and a significant depletion×repletion interaction [F(1.56) =5.28, p <0.05]. Follow-up Fisher’s PLSD comparisons demonstrated that PCPA–5-HTP-treated animals showed greater novelty- induced suppression of feeding by novelty compared to all other groups (0.0001< p <0.01). In addition, PCPA– vehicle-treated animals had shorter latencies compared to saline–5-HTP-treated animals (p <0.05). Fig. 2 a Mean (±SEM) latency to begin eating (s) during the 15-min home (open bars) or novel (dark bars) cage tests. Asterisks, significantly increased novel cage latency to begin eating compared to home cage latency. Ampersand, significant- ly different from PCPA–vehicle- treated animals within the same test cage group. Dagger, signif- icantly different from PCPA–5- HTP-treated animals within the same test cage group. One sym- bol, p<0.05; two symbols, p< 0.001; three symbolsp <0.0001. b Mean (±SEM) difference score for latency to begin eating (novel cage latency−home cage latency). Three sharps, signifi- cantly greater difference in la- tency between the home and novel cage compared to all other groups (saline–vehicle, p< 0.0001; saline–5-HTP, p <0.01; PCPA–vehicle, p<0.0001); one sharp, p<0.05, n=15 per group. Locomotor assessment Locomotor activity during the home cage test was not altered by serotonin depletion. Comparison of activity levels during the home cage test after serotonin depletion indicated that distance traveled (cm) did not differ between depleted (1,192.03 ± 83.47) and nondepleted animals (1,171.03 ± 47.77) according to one-way ANOVA. In contrast, depleted animals (1,200.19± 72.68) were less active than nondepleted animals (1,591.63± 59.36) during the novel cage test regardless of their repletion treatment as evidenced by a significant main effect of depletion [F(1.56) = 34.47, p < 0.0001] but no effect of repletion or of the interaction. Tissue serotonin content Serotonin tissue contents in the amygdala, frontal cortex, and hippocampus are presented in Table 1. The PCPA treatment resulted in serotonin levels that were less than 10% of saline- treated animals. Repletion of serotonin using benserazide plus 5-HTP resulted in serotonin levels that were similar to and, in some cases, higher than nondepleted animals. In addition, benserazide plus 5-HTP treatment in nondepleted animals increased serotonin levels to approximately 170% of vehicle-treated animals. These general conclusions were supported by two-way ANOVAs demonstrating significant effects of depletion [F(1.56)=105.99, p<0.0001]; [F(1.56)= 301.00, p<0.0001]; [F(1.56)=70.21, p<0.0001], repletion [F(1.56)=163.51, p<0.0001]; [F(1.56)=656.52, p<0.0001]; [F(1,56)=204.21, p<0.0001], and their interaction [F(1.56)= 7.80, p <0.01]; [F(1.56) = 53.36, p < 0.0001]; [F(1.56) = 20.48, p<0.0001] in the amygdala, frontal cortex, and hippocampus, respectively. Regression analyses To better understand the relationship between serotonin content and the behavioral outcomes, regression analyses were conducted between the behavioral-dependent vari- ables (intake difference score, novel cage intake, latency difference score, novel cage latency) and serotonin content Discussion The NIH test is one of the few animal behavior tests with predictive validity for antidepressant efficacy in the treatment of anxiety. Antidepressants are used to treat a number of clinical anxiety disorders including generalized anxiety disorder, panic disorder, social anxiety disorder, and obses- sive–compulsive disorder. Antidepressants are also effective at reducing anxiety that may be a co-morbid manifestation of depression. The ability of exposure to a novel environment to inhibit performance of a well-trained behavioral response in the NIH test may resemble fear in new or uncontrolled circumstances in anxious patients that could produce serious deleterious consequences for their social functioning. As a test sensitive to the anxiolytic effects of chronic antidepres- sant treatments, the NIH is responsive to both acute treatment with benzodiazepines and to chronic, but not acute or subchronic, treatment with antidepressants (Dulawa and Hen 2005; Dulawa et al. 2004; Merali et al. 2003, 2004). Apart from NIH, isolation-induced calling in the guinea pig pups is the only anxiety test that is sensitive to all classes of antidepressants (Borsini et al. 2002), although reduction in isolation-induced calling requires only acute antidepressant treatment. Such animal tests of chronic antidepressant treatment are important biological models for understanding the neural mechanisms associated with the anti-anxiety effects of antidepressant treatments. The development of more effective and rapidly acting treatments for anxiety and depression may depend on understanding the mechanisms causing the substantial latency between the initiation of clinical treatment and the appearance of symptomatic relief with currently used antidepressant drugs. The depletion of serotonin resulted in a pattern of behaviors associated with a reduction in anxiety in the NIH test, shown for the first time. In particular, novel cage food intake increased after serotonin depletion to a level that was similar to home cage intake although not significantly greater than saline treatment. In contrast, repletion of serotonin restored the intake-suppressing effects of novelty to levels that were similar to those in animals that were not depleted of serotonin. Increased serotonin content by treatment with the immediate precur- sor to serotonin 5-HTP returned the depleted animals to control performance. However, the same treatment did not result in a clear anxiogenic effect in the animals that had not been depleted of serotonin. A trend (p =0.06) was observed for increased serotonin content to induce a greater novelty-induced suppression of intake compared to the control treatment. A previous study examining a dose- response function of 5-HTP in a modified light–dark exploration test using mice suggested that increased anxiety was only observed when serotonin levels were very high. In that study, the most effective anxiogenic dose of 5-HTP resulted in cortical serotonin levels that were more than 220% of baseline levels (Artaiz et al. 1998). In the present study, the dose of 5-HTP was chosen to bring depleted animals back to baseline levels. It is possible that the resultant serotonin levels in nondepleted animals were not high enough to evoke anxiety levels that were statistically different from control values. Further studies examining larger increases in serotonin are required. The findings presented here are in agreement with previous findings suggesting that acute antidepressant treatment enhanced the effects of novelty in the NSF test (Bodnoff et al. 1989) and extend a broader literature showing that depletion of serotonin produces anxiolytic effects in animal tests of anxiety including shock-probe burying (Treit et al. 1993), elevated plus maze (Kshama et al. 1990; Treit et al. 1993), Vogel conflict (Soderpalm and Engel 1989), light/dark exploration (Artaiz et al. 1998; Kshama et al. 1990) and crossing (Koprowska et al. 1999), and social interaction (File and Hyde 1977). Overall, these findings suggest that serotonin is important in the adoption of a passive strategy in response to novel stimuli and provide insight as to how antidepressants might work to modify this unique test that is sensitive to the anxiolytic effects of chronic antidepressant treatment. The examination of the latency to begin eating provided less clear-cut results. In our hands, latency scores tended to be more variable and may have been influenced by floor effects in the home cage. Similar findings have been reported previously (Dulawa and Hen 2005). Home cage feeding was established through many training sessions that resulted in very short approach latencies that may not have been correlated with changes in home cage intake due to overtraining (R =−0.17). Consequently, changes in the relationship between home and novel cage latencies could not be used to interpret the pattern of results. Although serotonin depletion seemed to reduce anxiety as evidenced by intakes that were similar between the home and novel cage, a significant reduction in latency in the novel cage was not observed. Similarly, nondepleted animals given 5- HTP to increase serotonin demonstrated a tendency toward increased anxiety compared to control animals in terms of decreased intake but showed no difference in latency. Interestingly, animals treated with 5-HTP showed longer novel cage latencies than depleted animals treated with vehicle, although the control animals did not differ from depleted animals. Finally, animals that had been previously depleted of serotonin and then given 5-HTP demonstrated the longest novel cage latencies of any group, suggesting a possible sensitization to the anxiogenic effects of serotonin, although they showed decreased food intake in the novel cage relative only to depleted animals. Taken together, although latency to begin eating and total intake were correlated measures, these data suggest that latency to begin SSRIs to become effective in the treatment of anxiety. One is that chronic administration of antidepressants downregulate the 5-HT2C receptor (Hollander et al. 1991; Kennett et al.eating and intake may be influenced by floor and ceiling effects, respectively. Regression analyses clearly demon- strated that serotonin levels in all three areas examined were most highly correlated with the difference in intake between the home and novel cage. However, the lack of complete agreement between intake and latency outcomes in this test may suggest that these two measures are mediated by different neural mechanisms. Fig. 3 Scatter plots for the correlation between intake difference scores and serotonin tissue content in the a amygdala, b hippocampus, and c frontal cortex. Correlations were all significant, ps<0.0001. It is possible that the depletion of serotonin resulted in intakes that were similar in the home and novel cages because of serotonin’s effects on satiety. It has been known for some time that serotonin initiates satiety to arrest ongoing consumption and prevent further bouts of eating (Halford et al. 2005). A lack of such an input might result in sustained eating. However, if this were a likely explanation for the present findings, the relationship between serotonin and intakes would also be reflected in the home cage by comparing intakes between depleted and intact animals. Home cage intakes were actually decreased after serotonin depletion, suggesting that feeding in the novel cage is not explained by the apparent necessity of serotonin to stop a feeding bout. It has been known for some time that PCPA-induced serotonin depletion in rats can result in hyperactivity (Borbely et al. 1973; Marsden and Curzon 1976, 1977) that could account for the observed differential intakes between depleted and nondepleted animals. That is, hyperactivity could act as a competing behavior to decrease intakes in the home cage and serve to overcome novelty- induced suppression of locomotion in the novel cage. However, the present findings regarding locomotion in the home and novel cage do not favor this interpretation. Specifically, serotonin depletion did not yield significant differences in home cage locomotion despite differences in graham cracker intakes. Further, serotonin depletion resulted in decreased locomotion in the novel cage regardless of repletion treatment. Taken together, these data provide dissociation between locomotion and intake and suggest that nonspecific effects of PCPA on locomotion do not likely explain the observed effects.Although reductions of serotonin transmission produce anxiolytic-like behavioral effects in animals, it is chronic administration with a number of antidepressant drugs that produce prolonged increases in extracellular 5-HT levels that has become the most preferred method to treat a number of anxiety disorders (Vaswani et al. 2003). Several theories have been suggested to resolve this apparent paradox. It is presumed that alterations in receptors or signaling mecha- nisms occur over prolonged treatment that might allow 1994; Quested et al. 1997), a receptor associated with the production of panic and anxiety symptoms when activated in human volunteers (Germine et al. 1994). Another idea is that chronic administration of antidepressants may produce increases of neurosteroids that have anxiolytic effects by activating gamma aminobutyric acid mechanisms known to be associated with anxiety reduction (Czlonkowska et al. 2003). Yet another set of studies suggests that chronic antidepressant treatment stimulates different forms of neuro- plasticity that counteracts the effects of chronic stress and is required for antidepressant efficacy (Duman 2004; Santarelli et al. 2003). Despite these possible explanations for the apparent disconnect between the acute and chronic effects of SSRIs, the lack of preclinical tests that are sensitive to chronic, but not acute, antidepressant treatments have made advances in this field illusive. In summary, the production of anxiolytic effects in the NIH by the depletion of serotonin was demonstrated for the first time. These findings contribute to the greater under- standing of this important test that has high predictive validity for the efficacy of antidepressant treatment of anxiety disorders, closely models the delayed time course of antidepressant efficacy in humans, and may provide insight into the etiology of some anxiety symptoms. Continued investigations into the mechanisms underlying the anxiety provoked by novelty and the ability of antidepressants to attenuate it will undoubtedly lead to the development of new more rapidly acting antidepressants and may provide insight about the etiology of anxiety disorders.