Exploring Models of Exposure to Primary Care Careers in Training: a Narrative Review.

Multiple models of clinical exposure to primary care exist within undergraduate medical education (UME) and graduate medical education (GME). In this narrative review, we explore the evidence behind these different models of exposure, their alignment with positive promoters of primary care careers, and the pros and cons of each. Without positive exposure to primary care during training, sustaining the future primary care work force becomes increasingly challenging. Here, we explore multiple models of clinical exposure in UME, including longitudinal integrated clerkships, primary care tracks, and primary care clerkships. Within GME, we will review the impact of primary care tracks, Area Health Education Centers, block scheduling models, and continuity clinic scheduling models. The goal of this narrative review is to allow educators to think broadly and intentionally about the array of models to develop positive primary care experiences and perceptions in training, ultimately sustaining the primary care workforce.

Exploring Communication about Fall Risk and Prevention between Internal Medicine Residents and Geriatric Patients: A Needs Assessment.

OBJECTIVES: In the United States, falls are the leading cause of fatal and nonfatal injuries for geriatric patients. With a growing aging population, medical trainees must gain experience with geriatric assessments, including fall risk and prevention. To the authors' knowledge, no prior studies have explored who most often initiates fall discussions between Internal Medicine (IM) residents and geriatric (age 65 years and older) patients. Our objective was to determine who most often initiates fall discussions between IM residents and geriatric patients and the barriers to having these discussions. METHODS: This 2023 quantitative needs assessment used surveys distributed to ambulatory geriatric patients, IM residents, and attending physicians within an urban IM resident continuity clinic. We used the Stopping Elderly Accidents, Death & Injuries assessment from the Centers for Disease Control and Prevention to determine patient fall risk. RESULTS: Response rates were 46%, 51%, and 67% for patients, residents, and attendings, respectively. Of the 39 patients who were assessed, 51% were at risk of falling. Eighty-seven percent of patients have had a fall discussion with their residents, and 59% reported these were resident initiated; however, 75% of 28 residents reported initiating fall conversations rarely, and all 4 attendings said that they started these discussions most of the time while staffing patients with residents. Modifiable resident-identified barriers to discussing falls included forgetfulness and lack of knowledge regarding completing a fall risk assessment. CONCLUSIONS: Most patients have conversations about falling with their physicians, but discrepancies exist regarding who initiates them. Data from this study suggest that attendings may be instrumental in starting these conversations. Reminder systems and fall risk didactic curricula may increase resident-initiated fall discussions.

Importance of Low- and Moderate-Grade Adverse Events in Patients' Treatment Experience and Treatment Discontinuation: An Analysis of the E1912 Trial.

PURPOSE: Despite defined grades of 1 to 5 for adverse events (AEs) on the basis of Common Terminology Criteria for Adverse Events criteria, mild (G1) and moderate (G2) AEs are often not reported in phase III trials. This under-reporting may inhibit our ability to understand patient toxicity burden. We analyze the relationship between the grades of AEs experienced with patient side-effect bother and treatment discontinuation. METHODS: We analyzed a phase III Eastern Cooperative Oncology Group-American College of Radiology Imaging Network trial with comprehensive AE data. The Likert response Functional Assessment of Cancer Therapy-GP5 item, "I am bothered by side effects of treatment" was used to define side-effect bother. Bayesian mixed models were used to assess the impact of G1 and G2 AE counts on patient side-effect bother and treatment discontinuation. AEs were further analyzed on the basis of symptomatology (symptomatic or asymptomatic). The results are given as odds ratios (ORs) and 95% credible interval (CrI). RESULTS: Each additional G1 and G2 AEs experienced during a treatment cycle increased the odds of increased self-reported patient side-effect bother by 13% (95% CrI, 1.06 to 1.21) and 35% (95% CrI, 1.19 to 1.54), respectively. Furthermore, only AEs defined as symptomatic were associated with increased side-effect bother, with asymptomatic AEs showing no association regardless of grade. Count of G2 AEs increased the odds of treatment discontinuation by 59% (95% CrI, 1.32 to 1.95), with symptomatic G2 AEs showing a stronger association (OR, 1.75; 95% CrI, 1.28 to 2.39) relative to asymptomatic G2 AEs (OR, 1.45; 95% CrI, 1.12 to 1.89). CONCLUSION: Low- and moderate-grade AEs are related to increased odds of increased patient side-effect bother and treatment discontinuation, with symptomatic AEs demonstrating greater magnitude of association than asymptomatic. Our findings suggest that limiting AE capture to grade 3+ misses important contributors to treatment side-effect bother and discontinuation.

Treatment of myelodysplastic syndrome patients with erythropoietin with or without granulocyte colony-stimulating factor: results of a prospective randomized phase 3 trial by the Eastern Cooperative Oncology Group (E1996).

This phase 3 prospective randomized trial evaluated the efficacy and long-term safety of erythropoietin (EPO) with or without granulocyte colony-stimulating factor plus supportive care (SC; n = 53) versus SC alone (n = 57) for the treatment of anemic patients with lower-risk myelodysplastic syndromes. The response rates in the EPO versus SC alone arms were 36% versus 9.6%, respectively, at the initial treatment step, 47% in the EPO arm, including subsequent steps. Responding patients had significantly lower serum EPO levels (45% vs 5% responses for levels < 200 mU/mL vs > or = 200 mU/mL) and improvement in multiple quality-of-life domains. With prolonged follow-up (median, 5.8 years), no differences were found in overall survival of patients in the EPO versus SC arms (median, 3.1 vs 2.6 years) or in the incidence of transformation to acute myeloid leukemia (7.5% and 10.5% patients, respectively). Increased survival was demonstrated for erythroid responders versus nonresponders (median, 5.5 vs 2.3 years). Flow cytometric analysis showed that the percentage of P-glycoprotein(+) CD34(+) marrow blasts was positively correlated with longer overall survival. In comparison with SC alone, patients receiving EPO with or without granulocyte colony-stimulating factor plus SC had improved erythroid responses, similar survival, and incidence of acute myeloid leukemia transformation.

Estradiol-sertraline synergy in ovariectomized rats.

This study investigated estradiol (E(2)) modulation of the antidepressant effects of a selective serotonin (5-HT) reuptake inhibitor (SSRI; sertraline) and a tricyclic antidepressant (imipramine) as measured by the forced swim test (FST) followed by assessment of gene and protein expression for the 5-HT transporter (SERT) and multiple 5-HT receptors. Female Sprague-Dawley rats were ovariectomized (OVX) and two-thirds of the rats received E(2) implants (OVE). 4 weeks later, implants were withdrawn in half of the OVE rats (OVW) to capture a time point when E(2) levels were rapidly declining. Rats in each hormone group were treated with vehicle, sertraline (10 mg/kg) or imipramine (10 mg/kg), 24, 5 and 1h before the FST. Immediately after the FST, midbrain, hippocampus and prefrontal cortex tissue was removed and frozen for analysis of gene expression via quantitative real-time PCR (midbrain tissue) and protein expression via Western blot (prefrontal cortex and hippocampal tissue). In the FST, sertraline decreased immobility and increased swimming in OVE rats, as well as increased swimming in OVW rats. In contrast, no sertraline effect was observed in OVX rats. Rats treated with imipramine showed increased climbing but no changes in immobility or swimming. No changes in protein expression were detected in any treatment group. However, in vehicle-treated rats, E(2) increased midbrain SERT mRNA expression, with no effect on midbrain mRNA for the 5-HT receptors. In sertraline-treated rats, E(2) decreased 5-HT(2A) receptor mRNA, and E(2)-withdrawal increased 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptor mRNA. In imipramine-treated rats, E(2) (and E(2)-withdrawal) did not affect mRNA expression for any of the target genes. Thus, E(2) synergized behaviorally and neurochemically with an SSRI but not a tricyclic antidepressant.

Use of surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) to study protein expression in a rat model of cocaine withdrawal.

Surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) is an analytical technology for proteomic analysis that combines chromatography and mass spectrometry. At present, this technology is most commonly being exploited for the simultaneous measurement of numerous proteins in serum, but has also been utilized in organ tissue, although rarely in the brain. We applied SELDI-TOF MS technology to study protein expression in the brain of rats withdrawn from repeated cocaine exposure. Our goals were to optimize sample preparation and ProteinChip Array protocols for brain tissue, to verify the reproducibility of SELDI-TOF mass spectra and to determine whether SELDI-TOF MS detects differentially expressed proteins in cocaine- versus saline-treated rats. Consequently, we have developed an optimal protocol and generated a reproducible spectral pattern with six dominant peaks in all test samples. We have detected two smaller peaks (m/z: 5179, 5030) that were significantly increased (p < 0.05) in cocaine-treated rats compared to saline-treated rats. In summary, the application of SELDI-TOF MS to the study of protein expression in a rat model of cocaine withdrawal is feasible and has the potential to generate new hypotheses.

Chronic treatment with a serotonin(2) receptor (5-HT(2)R) agonist modulates the behavioral and cellular response to (+)-3,4-methylenedioxymethamphetamine [(+)-MDMA].

3,4-Methylenedioxymethamphetamine [MDMA; ecstasy] evokes a multifaceted subjective experience in human users which includes stimulation, feelings of well-being, mood elevation, empathy towards others as well as distortions in time, sensation and perception. Aspects of this unique psychopharmacology of MDMA are thought to be related to its potent actions to release serotonin (5-HT) and indirectly stimulate the 5-HT(2A) receptor (5-HT(2A)R). In the present studies, we examined the interrelationship between down-regulation of 5-HT(2A)R expression and the behaviorally stimulatory effects generated by acute administration of (+)-MDMA, the most potent enantiomer of (+/-)-MDMA. Male Sprague-Dawley rats were chronically treated with the preferential 5-HT(2A)R agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) which has been shown to down-regulate expression of the 5-HT(2A)R, but not the closely related 5-HT(2C)R. While chronic DOI treatment did not alter the functional sensitivity of either the 5-HT(2A)R or 5-HT(2C)R, this regimen enhanced (+)-MDMA-evoked hyperactivity. Subsequent analysis of c-Fos and 5-HT(2A)R immunoreactivity in brain sections demonstrated that DOI treatment decreased the number of (+)-MDMA-induced c-Fos immunopositive nuclei and 5-HT(2A)R immunostaining in select cortical and striatal areas. These results indicate that chronic DOI exposure results in an enhanced behavioral response to (+)-MDMA and in a pattern of neuronal activation which resembles that seen in psychostimulant sensitization. These data also suggest that expression of the 5-HT(2A)R in the NAc and PFC may play a role in the sensitivity to the locomotor-stimulating effects of (+)-MDMA and in the processes of neural regulation upon repeated psychostimulant administration.

Estrous cycle influence on individual differences in the response to novelty and cocaine in female rats.

In rats, individual differences in vulnerability to self-administration of drugs of abuse can be predicted by individual locomotor responses to a novel environment. This phenomenon has been well described for male rats, however very little information is available with regard to female rats and the added complication of estrous cycle hormone changes influencing activity levels. This study was designed to explore the relationship between individual responsiveness to exposure to a novel environment, the estrous cycle, and the response to cocaine in intact, cycling female rats. Locomotor activity of naive female rats was measured upon exposure to a novel environment followed by determination of estrous cycle stage and level of circulating estradiol. Rats were identified as high-responder (HR; 15% most active) or low-responder (LR; 15% least active) rats based on the locomotor response. Hyperactivity in response to cocaine was greater in HR than in LR rats. These data in combination with evaluation of the stage of estrous suggest that the estrous cycle interacts with individual phenotypic characteristics to modify the sensitivity to cocaine in female animals.

Validation of a selective serotonin 5-HT(2C) receptor antibody for utilization in fluorescence immunohistochemistry studies.

Although radioligand binding studies have shown that the serotonin 5-HT(2C) receptor (5-HT(2C)R) is widely expressed throughout the brain, more detailed knowledge of 5-HT(2C)R distribution within different neuronal populations will aid in understanding the mechanisms through which this receptor acts. Double-label immunohistochemical procedures can be utilized to examine the localization of receptors within specific neuronal populations. In order to conduct such studies, however, it was first necessary to examine the utility and specificity of two commercially available anti-5-HT(2C)R antibodies [from Santa Cruz (SC) and BD PharMingen (PH)]. In male Sprague-Dawley rats, both antibodies produced widespread immunoreactivity (IR) throughout the brain area chosen for study, the ventral tegmental area, which is the origin of the dopamine mesocorticoaccumbens "reward" pathway. Co-labeling with the SC and PH 5-HT(2C)R antibodies demonstrated that IR for the two antibodies largely overlapped. However, SC 5-HT(2C)R IR was more concentrated within IR cell bodies and was more consistent among assays than the PH 5-HT(2C)R IR. Thus, the SC 5-HT(2C)R antibody was chosen for subsequent studies. When examined in 5-HT(2C)R knockout vs. wild-type mice, the SC 5-HT(2C)R antibody produced widespread IR in wild-type, but not 5-HT(2C)R knockout, mice. In addition, 5-HT(2C)R-IR was not present in either native CHO cells, known to be devoid of 5-HT(2A)R or 5-HT(2C)R, or in CHO cells transfected with the 5-HT(2A)R. Thus, these studies suggest that the SC 5-HT(2C)R antibody produces reliable staining selective for 5-HT(2C)R vs. 5-HT(2A)R in rodent brains and is therefore suitable for use in future immunofluorescence 5-HT(2C)R localization studies.

Intracellular signaling involved in estrogen regulation of serotonin reuptake.

17beta-estradiol (E2) regulates neuronal activity via genomic and rapid, non-genomic mechanisms. The rat serotonergic neuronal cell line (RN46A) was used to investigate the rapid effects of E2 on serotonin (5-HT) reuptake and on potential intracellular signaling pathways. RN46A cells express the serotonin transporter (SERT) and estrogen receptor (ER)beta, but not ERalpha. Fifteen minute E2 treatment (10(-9)M) decreased 5-HT uptake. Intracellular cAMP levels were not increased by 15 min E2 treatment; however, E2 caused an increase in intracellular Ca2+ levels, with a maximum response within the first minute. The response was E2 specific, since other steroids (17alpha-estradiol, testosterone, and progesterone) had no effect. The ER antagonist ICI 182,780 blocked the rapid E2 effects on intracellular Ca2+ levels as did the selective ER modulator tamoxifen. In summary, changes in intracellular Ca2+ levels caused by E2 and mediated through ERbeta may be responsible for observed rapid effects of E2 on SERT activity.

Estrogen regulation of gene expression in the brain: a possible mechanism altering the response to psychostimulants in female rats.

Acute behavioral responses to cocaine are more pronounced in female than in male rats. We have shown that 3 weeks of treatment with 17beta-estradiol (E(2)) implants significantly enhanced the hyperactivity induced by cocaine in ovariectomized (OVX) rats. The ligand-bound estrogen receptor (ER) functions as a transcription factor to regulate the expression of E-responsive genes. Thus, we hypothesized that estrogen (E) modulates the behavioral response to cocaine via regulation of expression of components of dopamine (DA) and serotonin (5-HT) systems in mesolimbic nuclei important in the response to cocaine as well as the hypothalamus, a brain area known to be E-responsive. Adult female Sprague-Dawley rats were OVX; half of them then received E(2) implant (OVX+E) (n=8/group, two groups). Twenty-seven days later, brain tissue was collected to study E(2) effects on mRNA expression for DA D(1)-like (D(1)) and D(2)-like (D(2S), D(2L), D(3)) receptors, DA transporter (DAT), 5-HT(1A), 5-HT(1B), 5-HT(2A), 5-HT(2C) receptors, and 5-HT transporter (SERT) as well as ERalpha and ERbeta in amygdala, hypothalamus, nucleus accumbens, midbrain, and ventral tegmental area (VTA). We found that E(2) implants in OVX rats increased mRNA levels for D(1) receptor in hypothalamus, D(2L) receptor in midbrain, and D(3) receptor in VTA, and decreased D(3) receptor mRNA levels in midbrain relative to OVX controls. E(2) also increased 5-HT(2C) receptor mRNA levels in midbrain and hypothalamus. In addition, E(2) decreased mRNA levels for ERalpha in amygdala and hypothalamus and ERbeta in amygdala. The present study demonstrates that E can regulate mRNA expression for specific DA and 5-HT receptors in a region-specific manner and suggests that such modifications may contribute to the behavioral response to cocaine.

Estrogen effects on the hyperactivity induced by (+)-MDMA and cocaine in female rats.

This study compared the effects of estrogen (E) on the hyperactivity induced by (+)-3,4-methylenedioxymethamphetamine (MDMA) with E effects on cocaine-evoked hyperactivity in female rats. Sprague-Dawley rats were ovariectomized (OVX); half of them received a 17beta-estradiol (E2) implant (OVX + E). Three weeks later, rats received saline, (+)-MDMA (1, 2, or 4 mg/kg) or cocaine (5, 10, or 20 mg/kg), and locomotor activity was monitored. OVX + E rats exhibited greater locomotor hyperactivity in response to both psychostimulants than did OVX rats. The enhanced response to cocaine appeared within 5 min following drug injection whereas the enhanced response to (+)-MDMA was delayed for approximately 30 min. The differential effects of E on hyperactivity may be due to the unique profiles of DA and 5-HT in response to (+)-MDMA and cocaine.

Influence of estrous cycle and estradiol on behavioral sensitization to cocaine in female rats.

The hypotheses that the estrous cycle and estradiol modulate behavioral sensitization to cocaine in female rats were assessed. In an analysis of sensitization across the estrous cycle, female rats were administered saline or cocaine (15 mg/kg) twice daily for 5 days. Sensitization developed in the intact female rats as measured by the significant increase in stimulant behaviors seen between day 1 and day 5 of treatment. Rats were challenged with cocaine (5 mg/kg) at 3 days following discontinuation of drug treatment. The expression of sensitization as measured between cocaine and saline-treated rats was evident only in female rats in diestus at the time of the challenge test with cocaine. To explore the role of estradiol in sensitization, female rats were ovariectomized or ovariectomized and implanted with estradiol for two weeks prior to treatment with cocaine (15 mg/kg) twice daily for 5 days. Sensitization developed in both ovariectomized and ovariectomized+estradiol rats treated with cocaine as measured by the significant increase in stimulant-like behaviors seen between day 1 and day 5 of treatment. Rats were challenged with 5 mg/kg of cocaine at 3, 13 and 34 days following discontinuation of drug treatment. While neither hormone treatment group exposed to the cocaine regimen expressed sensitization at 3 days of withdrawal, both groups exhibited sensitization at 13 and 34 days following discontinuation of cocaine treatment. The estradiol-treated groups exhibited higher levels of activity relative to their untreated cohorts in both saline or cocaine treatment groups. These results suggest that detection of sensitization in female rats is not only influenced by injection regimen and length of abstinence but also by the presence of estrogens which effectively enhance the response to an acute cocaine challenge in the presence or absence of prior cocaine exposure.

Elevated Expression of Serotonin 5-HT(2A) Receptors in the Rat Ventral Tegmental Area Enhances Vulnerability to the Behavioral Effects of Cocaine.

The dopamine mesocorticoaccumbens pathway which originates in the ventral tegmental area (VTA) and projects to the nucleus accumbens and prefrontal cortex is a circuit important in mediating the actions of psychostimulants. The function of this circuit is modulated by the actions of serotonin (5-HT) at 5-HT(2A) receptors (5-HT(2A)R) localized to the VTA. In the present study, we tested the hypothesis that virally mediated overexpression of 5-HT(2A)R in the VTA would increase cocaine-evoked locomotor activity in the absence of alterations in basal locomotor activity. A plasmid containing the gene for the 5-HT(2A)R linked to a synthetic marker peptide (Flag) was created and the construct was packaged in an adeno-associated virus vector (rAAV-5-HT(2A)R-Flag). This viral vector (2 µl; 10(9-10) transducing units/ml) was unilaterally infused into the VTA of male rats, while control animals received an intra-VTA infusion of Ringer's solution. Virus-pretreated rats exhibited normal spontaneous locomotor activity measured in a modified open-field apparatus at 7, 14, and 21 days following infusion. After an injection of cocaine (15 mg/kg, ip), both horizontal hyperactivity and rearing were significantly enhanced in virus-treated rats (p < 0.05). Immunohistochemical analysis confirmed expression of Flag and overexpression of the 5-HT(2A)R protein. These data indicate that the vulnerability of adult male rats to hyperactivity induced by cocaine is enhanced following increased levels of expression of the 5-HT(2A)R in the VTA and suggest that the 5-HT(2A)R receptor in the VTA plays a role in regulation of responsiveness to cocaine.

Nurse coaching to explore and modify patient attitudinal barriers interfering with effective cancer pain management.

PURPOSE/OBJECTIVES: To describe a complex coaching intervention to help patients with cancer pain explore beliefs and attitudinal barriers interfering with pain management. Patients were coached to explore beliefs about pain, communications about pain management, and the use of analgesics and nonpharmacologic interventions. DATA SOURCES: Published journal articles, abstracts, and psychology textbooks. DATA SYNTHESIS: Personal beliefs, related attitudinal barriers, and associated behaviors impede patient adherence to and success with pain management treatments. Interventions targeting beliefs help patients overcome attitudinal barriers, improve treatment adherence, and obtain better pain relief. CONCLUSIONS: Coaching patients to explore beliefs reduces ineffective behaviors and improves pain treatment adherence. IMPLICATIONS FOR NURSING: A coaching intervention incorporating assessment of patient beliefs promotes self-management, self-efficacy, and adherence to pain management treatment plans. Advanced practice nurses should consider incorporating this intervention into their communications with patients experiencing cancer pain.

Serotonin regulation of serotonin uptake in RN46A cells.

AIM: The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT(1A), 5-HT(1B), 5-HT(2A), and 5-HT(2C)) and as cAMP and intracellular Ca(2+) are modulated by different 5-HT receptors, we studied both pathways. METHODS: 5-HT uptake was determined as imipramine-inhibitable uptake of [(3)H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca(2+) changes were determined using the ratiometric method of intracellular Ca(2+) imaging. RESULTS: For uptake experiments, cells were kept for 30 min either with or without 1 microM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [(3)H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca(2+) levels either; however, adding 1 microM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca(2+) levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca(2+) levels. CONCLUSIONS: We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca(2+) levels. This suggests that 5-HT may utilize intracellular Ca(2+) to regulate 5-HT uptake.

Estradiol effects on the dopamine transporter - protein levels, subcellular location, and function.

BACKGROUND: The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause. RESULTS: The activity of the DA transporter (DAT) was measured by the specific uptake of 3H-DA. We found that low concentrations (10(-14) to 10(-8) M) of 17beta-estradiol (E2) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E2 exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-alpha (ERalpha) were elevated in cell extracts by a 20 ng/ml 2 day NGFbeta treatment, while ERbeta was not. DAT, ERalpha and ERbeta were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E2. At cell densities above 15,000 cells per well, the E2-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E2 treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20-30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5-60 min E2 treatment, though immunoblot analyses demonstrated no total cellular loss of protein. CONCLUSION: Our results suggest that physiological levels of E2 may act to sequester DAT in intracellular compartments where the transporter's second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types.

Selective estrogen receptor modulator effects in the rat brain.

The effects in the brain of selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene have not yet been fully elucidated. Based upon the hypothesis that serotonin (5-HT)-steroid hormone interactions are important in mood regulation, we have compared six SERMs (tamoxifen, raloxifene, levormeloxifene, NNC 45-0781, NNC 45-0320, NNC 45-1506) with 17beta-estradiol (E(2)) in terms of their ability to regulate mRNA levels of estrogen receptor (ER)alpha, ER beta, 5-HT(1A) receptor, and 5-HT reuptake transporter (SERT) in the midbrain, amygdala, and hypothalamus of ovariectomized (OVX) rats. Female rats (n = 6/group, 8 groups total) were OVX and allowed to recover for 2 weeks. During the third post-OVX week, rats were injected subcutaneously with E(2) (0.1 mg/kg) or one of the SERMs (5 mg/kg) once per day for 7 days. Twenty-four hours after the last injection, tissue was collected for the determination of mRNA levels by ribonuclease protection assay (RPA). E(2) treatment significantly decreased mRNA levels for ER alpha, ER beta, and SERT in midbrain and ER alpha in hypothalamus. Tamoxifen increased ER beta mRNA levels in hypothalamus, while raloxifene increased ER beta mRNA levels in amygdala. NNC 45-0320 decreased ER alpha mRNA in hypothalamus and decreased ER beta mRNA in amygdala. These results suggest that while SERMs are not full estrogen receptor agonists in the brain, the agonist/antagonist profiles for individual SERMs may differ among brain areas. This raises the possibility of developing new SERMs for selective functions in specific brain areas.