The priming effect of rewarding brain stimulation in rats depends on both the cost and strength of reward but survives blockade of D2-like dopamine receptors.

Receipt of an intense reward boosts motivation to work for more of that reward. This phenomenon is called the priming effect of rewards. Using a novel measurement method, we show that the priming effect of rewarding electrical brain stimulation depends on the cost, as well as on the strength, of the anticipated reward. Previous research on the priming effect of electrical brain stimulation utilized a runway paradigm in which running speed serves as the measure of motivation. In the present study, the measure of motivation was the vigour with which rats executed a two-lever response chain, in a standard operant-conditioning chamber, to earn rewarding electrical stimulation of the lateral hypothalamus. In a second experiment, we introduced a modification that entails self-administered priming stimulation and alternating blocks of primed and unprimed trials. Reliable, consistent priming effects of substantial magnitude were obtained in the modified paradigm, which is closely analogous to the runway paradigm. In a third experiment, the modified paradigm served to assess the dependence of the priming effect on dopamine D2-like receptors. The priming effect proved resilient to the effect of eticlopride, a selective D2-like receptor antagonist. These results are discussed within the framework of a new model of brain reward circuitry in which non-dopaminergic medial forebrain bundle fibers and dopamine axons provide parallel inputs to the final common paths for reward and incentive motivation.

Combined effects of the contraceptive hormones, ethinyl estradiol and levonorgestrel, on the use of place and response memory in gonadally-intact female rats.

During maze navigation rats can rely on hippocampus-mediated place memory or striatum-mediated response memory. Ovarian hormones bias whether females use place or response memory to reach a reward. Here, we investigated the impact of the contraceptive hormones, ethinyl estradiol (EE) and levonorgestrel (LNG), on memory bias. A total of 63 gonadally-intact female rats were treated with either 10 µg/kg of EE alone, 20 µg/kg of LNG alone, both 10 µg/kg of EE and 20 µg/kg of LNG together, or a sesame oil injection with 5% ethanol as a vehicle control. Rats in the control condition were tested during the diestrus phase of the estrous cycle in order to control for the low circulating levels of gonadotropin and ovarian hormones that occur with oral contraceptive administration. Rats treated with LNG alone had a bias towards the use of place memory compared to diestrus phase control rats. This bias was not observed if LNG was administered in combination with EE. Rats treated with EE or EE+LNG did not have a statistically significant difference in memory bias compared to rats in the control group. These data show that synthetic hormones contained in oral contraceptives administered to females influence which cognitive strategy is predominantly used during navigation.

The role of progesterone in memory bias during spatial navigation in females.

Rats can use several memory systems to navigate a maze toward a reward. Two of these are place memory and response memory and female rats can be biased to predominantly use one over another. Both progesterone and estrogens have been shown to alter memory bias. Although the effects of estrogens have been well documented, the effects of progesterone remain somewhat unexplored. Mechanisms through which progesterone may be acting to exert its effects are reviewed here. Converging evidence suggests that the actions of progesterone differ depending on the presence of estrogens, frequently acting in opposition to estrogens when administered together. The hippocampus, dorsal striatum, and prefrontal cortex are likely involved, as is the progesterone metabolite, allopregnanolone. There is a need for more research on progesterone and memory bias, especially considering current formulations of hormonal contraceptives include progestins.

Modeling hormonal contraception in female rats: A framework for studies in behavioral neurobiology.

Research on hormonal contraceptives (HC) in animal models is lacking, and as a result, so is our understanding of the impact of HC on the brain and behavior. Here, we provide a review of the pharmacology of HC, as well as the methodology and best practices for designing a model of HC in female rats. We outline specific methodological considerations regarding dosing, route of administration, exposure time/timing, and selecting a control group. We also provide a framework outlining important levels of analysis for thinking about the impact of HC on behavioral and neurobiological outcomes. The purpose of this review is to equip researchers with foundational knowledge, and some basic elements of experimental design for future studies investigating the impact of HC on the brain and behavior of female rats.

Estrogen receptors observed at extranuclear neuronal sites and in glia in the nucleus accumbens core and shell of the female rat: Evidence for localization to catecholaminergic and GABAergic neurons.

Estrogens affect dopamine-dependent diseases/behavior and have rapid effects on dopamine release and receptor availability in the nucleus accumbens (NAc). Low levels of nuclear estrogen receptor (ER) α and ERß are seen in the NAc, which cannot account for the rapid effects of estrogens in this region. G-protein coupled ER 1 (GPER1) is observed at low levels in the NAc shell, which also likely does not account for the array of estrogens' effects in this region. Prior studies demonstrated membrane-associated ERs in the dorsal striatum; these experiments extend those findings to the NAc core and shell. Single- and dual-immunolabeling electron microscopy determined whether ERα, ERß, and GPER1 are at extranuclear sites in the NAc core and shell and whether ERα and GPER1 were localized to catecholaminergic or γ-aminobutyric acid-ergic (GABAergic) neurons. All three ERs are observed, almost exclusively, at extranuclear sites in the NAc, and similarly distributed in the core and shell. ERα, ERß, and GPER1 are primarily in axons and axon terminals suggesting that estrogens affect transmission in the NAc via presynaptic mechanisms. About 10% of these receptors are found on glia. A small proportion of ERα and GPER1 are localized to catecholaminergic terminals, suggesting that binding at these ERs alters release of catecholamines, including dopamine. A larger proportion of ERα and GPER1 are localized to GABAergic dendrites and terminals, suggesting that estrogens alter GABAergic transmission to indirectly affect dopamine transmission in the NAc. Thus, the localization of ERs could account for the rapid effects of estrogen in the NAc.

Progesterone rapidly alters the use of place and response memory during spatial navigation in female rats.

17ß-Estradiol (E2) and progesterone (P) influence place and response memory in female rats in spatial navigation tasks. Use of these memory systems is associated with the hippocampus and the dorsal striatum, respectively. Injections of E2 result in a well-established bias to use place memory, while much less is understood about the role of P. A total of 120 ovariectomized female rats were tested within a dual-solution T-maze task and treated with either low E2 (n = 24), high E2 (10 µg/kg; n = 24), or high E2 in combination with P (500 µg/kg) at three time points before testing: 15 min (n = 24), 1 h (n = 24), and 4 h (n = 24). Given alone, high E2 biases rats to the use of place memory, but this effect is reversed when P is given 1 h or 4 h before testing. This indicates that P may be playing an inhibitory role in the hippocampus during spatial tasks, which is consistent with past findings. Our findings show that P acts rapidly (within an hour) to affect performance during spatial tasks.

Depression, Estrogens, and Neuroinflammation: A Preclinical Review of Ketamine Treatment for Mood Disorders in Women.

Ketamine has been shown to acutely and rapidly ameliorate depression symptoms and suicidality. Given that women suffer from major depression at twice the rate of men, it is important to understand how ketamine works in the female brain. This review explores three themes. First, it examines our current understanding of the etiology of depression in women. Second, it examines preclinical research on ketamine's antidepressant effects at a neurobiological level as well as how ovarian hormones present a unique challenge in interpreting these findings. Lastly, the neuroinflammatory hypothesis of depression is highlighted to help better understand how ovarian hormones might interact with ketamine in the female brain.

Fos expression is increased in oxytocin neurons of female rats with a sexually conditioned mate preference for an individual male rat.

Evidence suggests an important role of Pavlovian learning in sexual partner selection. Female rats that experience paced copulation with a male scented with a neutral odor selectively solicit and receive ejaculations from the scented male relative to an unscented male. Exposure to the conditioned odor alone induces Fos protein in regions of the brain associated with sexual excitation. Here we tested whether female rats can be conditioned to show a sexual preference for an unscented male rat of the same strain. Female Long-Evans rats were given 10 copulatory trials with either a one-hole pacing divider or a four-hole pacing divider in a unilevel chamber with the same conspecific male (n = 16). Females were then given an open-field partner preference test with the paired male versus a novel male. After two reconditioning trials females were exposed to the partner or a novel male to induce Fos expression. Females that paced with the one-hole divider received the first ejaculation and more ejaculations overall from the paired compared to novel male. Fos immunoreactivity within oxytocin neurons in the PVN, mPOA, and VMH was increased in females with a preference that were exposed to the paired male. These data indicate that female rats can form selective sexual preferences for an individual conspecific and that their formation depends on the type of pacing during conditioning. These findings further suggest the involvement of oxytocin in the display of conditioned preferences. Thus, early copulatory experience appears to determine the mating strategy used by female rats.

The priming effect of food persists following blockade of dopamine receptors.

The priming effect of rewards is a boost in the vigor of reward seeking resulting from the previous receipt of a reward. Extensive work has been carried out on the priming effect of electrical brain stimulation, but much less research exists on the priming effect of natural rewards, such as food. While both reinforcement and motivation are linked with dopamine transmission in the brain, the priming effect of rewards does not appear to be dopamine-dependent. In the present study, an operant method was developed to measure the priming effect of food and then applied to investigate whether it is affected by dopamine receptor antagonism. Long-Evans rats were administered saline or one of the three doses (0.01, 0.05, 0.075 mg/kg) of the dopamine D1 receptor family antagonist, SCH23390, or the dopamine D2 receptor family antagonist, eticlopride. Although dopamine receptor antagonism affected pursuit of food, it did not eliminate the priming effect. These data suggest that despite the involvement of dopamine transmission in reinforcement and motivation, the priming effect of food does not depend on dopamine transmission.

Varying the rate of intravenous cocaine infusion influences the temporal dynamics of both drug and dopamine concentrations in the striatum.

The faster drugs of abuse reach the brain, the greater is the risk of addiction. Even small differences in the rate of drug delivery can influence outcome. Infusing cocaine intravenously over 5 vs. 90-100 s promotes sensitization to the psychomotor and incentive motivational effects of the drug and preferentially recruits mesocorticolimbic regions. It remains unclear whether these effects are due to differences in how fast and/or how much drug reaches the brain. Here, we predicted that varying the rate of intravenous cocaine infusion between 5 and 90 s produces different rates of rise of brain drug concentrations, while producing similar peak concentrations. Freely moving male Wistar rats received acute intravenous cocaine infusions (2.0 mg/kg/infusion) over 5, 45 and 90 s. We measured cocaine concentrations in the dorsal striatum using rapid-sampling microdialysis (1 sample/min) and high-performance liquid chromatography-tandem mass spectrometry. We also measured extracellular concentrations of dopamine and other neurochemicals. Regardless of infusion rate, acute cocaine did not change concentrations of non-dopaminergic neurochemicals. Infusion rate did not significantly influence peak concentrations of cocaine or dopamine, but concentrations increased faster following 5-s infusions. We also assessed psychomotor activity as a function of cocaine infusion rate. Infusion rate did not significantly influence total locomotion, but locomotion increased earlier following 5-s infusions. Thus, small differences in the rate of cocaine delivery influence both the rate of rise of drug and dopamine concentrations, and psychomotor activity. A faster rate of rise of drug and dopamine concentrations might be an important issue in making rapidly delivered cocaine more addictive.

17ß-estradiol locally increases phasic dopamine release in the dorsal striatum.

Studies using in vivo microdialysis have shown that 17ß-estradiol (E2) increases dopamine (DA) transmission in the dorsal striatum. Both systemic administration of E2 and local infusion into the dorsal striatum rapidly enhance amphetamine-induced DA release. However, it is not known to what degree these effects reflect tonic and/or phasic DA release. It was hypothesized that E2 acts directly within the DS to rapidly increase phasic DA transmission. In urethane-anesthetized (1.5mL/kg) female rats, we used fast-scan cyclic voltammetry to study the effects of E2 on phasic, electrically-evoked release of DA in the dorsal striatum. Rats were ovariectomized and implanted with a silastic tube containing 5% E2 in cholesterol, previously shown to mimic low physiological serum concentrations of∼20-25pg/mL. DA release was evoked every 1min by delivering biphasic electrical stimulation in the substantia nigra. Local infusions of E2 (244.8pg/µl) into the dorsal striatum increased the amplitude of the electrically evoked DA transients. Behaviorally significant stimuli and events trigger phasic release of DA. The present findings predict that E2 would boost such signaling in behaving subjects.

Interactions between estradiol and haloperidol on perseveration and reversal learning in amphetamine-sensitized female rats.

There are sex differences associated with schizophrenia, as women exhibit later onset of the disorder, less severe symptomatology, and better response to antipsychotic medications. Estrogens are thought to play a role in these sex differences; estrogens facilitate the effects of antipsychotic medications to reduce the positive symptoms of schizophrenia, but it remains unclear whether estrogens protect against the cognitive symptoms of this disorder. Amphetamine sensitization is used to model some symptoms of schizophrenia in rats, including cognitive deficits like excessive perseveration and slower reversal learning. In this experiment female rats were administered a sensitizing regimen of amphetamine to mimic these cognitive symptoms. They were ovariectomized and administered either low or high estradiol replacement as well as chronic administration of the antipsychotic haloperidol, and were assessed in tests of perseveration and reversal learning. Results of these experiments demonstrated that, in amphetamine-sensitized rats, estradiol alone does not affect perseveration or reversal learning. However, low estradiol facilitates a 0.25mg/day dose of haloperidol to reduce perseveration and improve reversal learning. Combined high estradiol and 0.25mg/day haloperidol has no effect on perseveration or reversal learning, but high estradiol facilitates the effects of 0.13mg/day haloperidol to reduce perseveration and improve reversal learning. Thus, in amphetamine-sensitized female rats, 0.25mg/day haloperidol only improved perseveration and reversal learning when estradiol was low, while 0.13mg/day haloperidol only improved these cognitive processes when estradiol was high. These findings suggest that estradiol facilitates the effects of haloperidol to improve perseveration and reversal learning in a dose-dependent manner.

Intra-perirhinal cortex administration of estradiol, but not an ERß agonist, modulates object-recognition memory in ovariectomized rats.

Intra-rhinal cortical infusion of 17-ß estradiol (E2, 244.8pg/µl) enhances performance on the Novel-Object Preference (NOP) test and impairs accuracy on the delayed nonmatching-to-sample (DNMS) task in the same set of ovariectomized rats (Gervais, Jacob, Brake, & Mumby, 2013). These results appear paradoxical, as normal performance on both tests require intact object-recognition memory (ORM) ability. While demonstrating a preference for the novel object requires recognizing the sample object, rodents can recognize the sample object and still fail to demonstrate a preference. Therefore, enhanced NOP test performance is consistent with both improved ORM and increased novel-object exploration independent of memory processes. There is some evidence suggesting that estrogen receptor (ER) ß agonists enhance NOP test performance (Jacome et al., 2010), but no study to date has examined the role of this receptor in DNMS task performance in rodents. The aim of the present study was to determine whether intra-PRh infusion of an ER ß agonist, diarylpropionitrile (DPN, 2µg/µl), has divergent effects on novel-object preference (i.e. novelty preference) and accuracy on the DNMS task. Ovariectomized (OVX) rats (n=7) received chronic low E2 (∼22pg/ml serum) replacement, then intra-PRh infusion of DPN (2µg/µl), E2 (244.8pg/µl), or vehicle before each mixed-delay session (0.5-5min) of the DNMS task. A different set of OVX rats (n=10) received the same infusions before each NOP test trial, and were tested either 4 or 72h later. Consistent with Gervais et al. (2013), intra-PRh E2 reduced accuracy on the DNMS task following a 5-min retention delay and enhanced novelty preference on both tests. Intra-PRh DPN was associated with accuracy that was similar to the vehicle-infusion condition, despite enhancing novelty preference on both tests. The accuracy results suggest that while intra-PRh E2 impairs ORM, ERß does not play a role. However, ERß in the PRh appears to be important for the expression of novelty preference, in a manner that is unaffected by retention delay. These findings suggest that the modulation of novelty preference by intra-PRh E2/ERß may be due to factors unrelated to ORM.

17ß-Estradiol infusions into the dorsal striatum rapidly increase dorsal striatal dopamine release in vivo.

Systemic injections of 17ß-estradiol (E2) in ovariectomized (OVX) female rats rapidly enhance dorsal striatal dopamine (DA) release in response to amphetamine (AMPH). Additionally, a single injection of E2 rapidly (within 30min) enhances amphetamine-induced DA release. In situ studies show that this rapid effect of E2 occurs specifically within the dorsal striatum (DS). The present study investigated the in vivo effects of E2 infused into the DS, medial prefrontal cortex (mPFC) or the substantia nigra (SN) on dorsal striatal DA release. Rats were OVX and implanted with a silastic tube containing 5% E2 in cholesterol, previously shown to mimic low physiological serum concentrations of 18-32pg/ml. Single-probe microdialysis was used to measure extracellular DA levels in the DS. In addition, DA release was measured subsequent to systemic injections of the indirect DA agonist, AMPH (0.5mg/kg SC), administered simultaneously with E2 (0.544µg/100µl) or its vehicle, cyclodextrin (VEH) (0.520µg/100µl). Local infusions of E2 into the DS resulted in a greater amphetamine-induced dorsal striatal DA release in comparison to vehicle. Local infusions of E2 into the mPFC or the SN did not result in an enhancement of amphetamine-induced DA levels in the DS. These studies suggest that increases in dorsal striatal DA release in response to systemic E2 are a consequence of E2 actions within the DS itself.

High estrogen and chronic haloperidol lead to greater amphetamine-induced BOLD activation in awake, amphetamine-sensitized female rats.

The ovarian hormone estrogen has been implicated in schizophrenia symptomatology. Low levels of estrogen are associated with an increase in symptom severity, while exogenous estrogen increases the efficacy of antipsychotic medication, pointing at a possible interaction between estrogen and the dopaminergic system. The aim of this study is to further investigate this interaction in an animal model of some aspects of schizophrenia using awake functional magnetic resonance imaging. Animals receiving 17ß-estradiol and haloperidol were scanned and BOLD activity was assessed in response to amphetamine. High 17ß-estradiol replacement and chronic haloperidol treatment showed increased BOLD activity in regions of interest and neural networks associated with schizophrenia (hippocampal formations, habenula, amygdala, hypothalamus etc.), compared with low, or no 17ß-estradiol. These data show that chronic haloperidol treatment has a sensitizing effect, possibly on the dopaminergic system, and this effect is dependent on hormonal status, with high 17ß-estradiol showing the greatest BOLD increase. Furthermore, these experiments further support the use of imaging techniques in studying schizophrenia, as modeled in the rat, but can be extended to addiction and other disorders.

Modulation of spatial and response strategies by phase of the menstrual cycle in women tested in a virtual navigation task.

Different memory systems are employed to navigate an environment. It has been consistently shown in rodents that estrogen impacts multiple memory system bias such that low estradiol (E2) is associated with increased use of a striatal-mediated response strategy whereas high E2 increases use of a hippocampal-dependent spatial memory. Low E2 also enhances performance on a response-based task whereas high E2 levels improve learning on a spatial task. The purpose of the present cross-sectional study was to investigate navigational strategies in young, healthy, naturally cycling women. Participants were split into either an early follicular (i.e., when E2 levels are low), ovulatory (i.e., when E2 levels are high) or mid/late luteal (i.e., end of the cycle, when E2 levels decrease and progesterone levels rise) phase group, using self-reported date of the menstrual cycle. Serum hormone level measurements (E2, progesterone, testosterone) were used to confirm cycle phase assignment. Participants were administered a verbal memory task as well as a virtual navigation task that can be solved by using either a response or spatial strategy. Women tested in the ovulatory phase, under high E2 conditions, performed better on a verbal memory task than women tested during the other phases of the cycle. Interestingly, women tested in the mid/late luteal phase, when progesterone is high, predominantly used a spatial strategy, whereas the opposite pattern was observed in the early follicular and ovulatory groups. Our data suggest that the specific memory system engaged differs depending on the phase of the menstrual cycle and may be mediated by both E2 and progesterone, rather than E2 alone.

Estrogen receptor α and G-protein coupled estrogen receptor 1 are localized to GABAergic neurons in the dorsal striatum.

Estrogens affect dopamine transmission in the striatum, increasing dopamine availability, maintaining D2 receptor density, and reducing the availability of the dopamine transporter. Some of these effects of estrogens are rapid, suggesting that they are mediated by membrane associated receptors. Recently our group demonstrated that there is extra-nuclear labeling for ERα, ERß, and GPER1 in the striatum, but that ERα and GPER1 are not localized to dopaminergic neurons in this region. GABAergic neurons are the most common type of neuron in the striatum, and changes in GABA transmission affect dopamine transmission. Thus, to determine whether ERα or GPER1 are localized to GABAergic neurons, we double labeled the striatum with antibodies for ERα or GPER1 and GABA and examined them using electron microscopy. Ultrastructural analysis revealed that ERα and GPER1 are localized exclusively to extranuclear sites in the striatum, and ∼35% of the dendrites and axon terminals labeled for these receptors contain GABA immunoreactivity. Binding at membrane-associated ERα and GPER1 could account for rapid estrogen-induced decreases in GABA transmission in the striatum, which, in turn, could affect dopamine transmission in this region.

Ovarian steroids alter dopamine receptor populations in the medial preoptic area of female rats: implications for sexual motivation, desire, and behaviour.

Dopamine (DA) transmission in the medial preoptic area (mPOA) plays a critical role in the control of appetitive sexual behaviour in the female rat. We have shown previously that a DA D1 receptor (D1R)-mediated excitatory state appears to occur in females primed with estradiol benzoate (EB) and progesterone (P), whereas a DA D2 receptor (D2R)-mediated inhibitory state appears to occur in females primed only with EB. The present experiment employed three techniques to better understand what changes occur to DA receptors (DARs) in the mPOA under different hormonal profiles. Ovariectomized females were randomly assigned to one of three steroid treatment groups: EB + P (10 and 500 µg, respectively), EB + Oil, or the control (Oil + Oil), with hormone injections administered at 48 and 4 h prior to euthanizing. First, the number of neurons in the mPOA that contained D1R or D2R was assessed using immunohistochemistry. Second, the mPOA and two control areas (the prelimbic cortex and caudate putamen) were analysed for DAR protein levels using western blot, and DAR functional binding levels using autoradiography. Ovarian steroid hormones affected the two DAR subtypes in opposite ways in the mPOA. All three techniques supported previous behavioural findings that females primed with EB have a lower D1R : D2R ratio, and thus a D2R-mediated system, and females primed with EB + P have a higher D1R : D2R ratio, and thus a D1R-mediated system. This provides strong evidence for a DA-driven pathway of female sexual motivation, desire, and behaviour that is modified by different hormone priming regimens.

Estrogen receptors in the central nervous system and their implication for dopamine-dependent cognition in females.

This article is part of a Special Issue "Estradiol and cognition". Over the past 30 years, research has demonstrated that estrogens not only are important for female reproduction, but also play a role in a diverse array of cognitive functions. Originally, estrogens were thought to have only one receptor, localized exclusively to the cytoplasm and nucleus of cells. However, it is now known that there are at least three estrogen receptors (ERs): ERα, ERß and G-protein coupled ER1 (GPER1). In addition to being localized to nuclei, ERα and ERß are localized to the cell membrane, and GPER1 is also observed at the cell membrane. The mechanism through which ERs are associated with the membrane remains unclear, but palmitoylation of receptors and associations between ERs and caveolin are implicated in membrane association. ERα and ERß are mostly observed in the nucleus using light microscopy unless they are particularly abundant. However, electron microscopy has revealed that ERs are also found at the membrane in complimentary distributions in multiple brain regions, many of which are innervated by dopamine inputs and were previously thought to contain few ERs. In particular, membrane-associated ERs are observed in the prefrontal cortex, dorsal striatum, nucleus accumbens, and hippocampus, all of which are involved in learning and memory. These findings provide a mechanism for the rapid effects of estrogens in these regions. The effects of estrogens on dopamine-dependent cognition likely result from binding at both nuclear and membrane-associated ERs, so elucidating the localization of membrane-associated ERs helps provide a more complete understanding of the cognitive effects of these hormones.

Changes in brain volume in response to estradiol levels, amphetamine sensitization and haloperidol treatment in awake female rats.

Estrogen has been shown to further ameliorate symptoms when administered in conjunction with antipsychotics in patients with schizophrenia. We have previously shown that chronic haloperidol (HAL) treatment reduces amphetamine (AMPH)-induced locomotor activity in AMPH-sensitized rats, but only when paired with high levels of the estrogen, 17-ß estradiol. In addition, we reported estradiol-dependent responses to AMPH in AMPH-sensitized rats as measured by functional magnetic resonance imaging. It is thus clear that estradiol and antipsychotics both affect the rat brain, however the mechanism by which this occurs is unknown. The aim of the current study was to assess this interaction by investigating the effects of estradiol, AMPH and HAL on brain volume changes in awake female rats. Repeated exposure to AMPH resulted in an overall reduction in brain volume, regardless of hormonal status (i.e. no, low or high estradiol). Similarly, chronic HAL treatment further reduced brain volume compared to acute treatment. Hormonal status affected hippocampal volume with rats receiving low estradiol replacement showing larger volume; this difference was no longer significant after repeated exposure to AMPH. Finally, we found changes in volume in response to AMPH throughout hippocampal components (i.e. CA1-CA3 and dentate) as well as components of the mesocortical system. In conclusion, brain volume seems to be influenced by hormonal status, as well as exposure to AMPH and haloperidol treatment. These findings implicate areas where estradiol, amphetamine and antipsychotics may be producing volumetric changes in the brain, pointing the way to where future studies should focus.