Sensitization Procedure Research Articles

Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis

Considering the importance of CBB staining in visualizing proteins in 2-DE gels, any improvement in the existing protocols with high sensitivity and good MS compatibility is of significant importance. In this study, we systematically evaluated the effects of different staining parameters on CBB methods by 1-DE and 2-DE, and demonstrated that G-250 was more suitable for visualizing low-abundant proteins as well as generating more spots than R-250, whereas R-250 had a superior capability for quick staining of high-abundant proteins. The staining produced by mixing G-250 and R-250 in different ratios showed similar sensitivity. Compared with acetic acid, phosphoric acid produced more protein spots. Ammonium-based stain demonstrated a superior sensitivity than the aluminum-based one. Based on these findings, a new protocol using CBB G-250, ammonium sulfate and phosphoric acid (GAP) was developed by incorporating the fixation, sensitization and staining procedures together. The comparison of GAP with other methods revealed that GAP generated more protein spots and had wider applications. The identification of 11 proteins demonstrated that GAP was not only compatible with MS but also obviously reduced in vitro protein modification, and thus could be a preferable protocol in the future proteomic analysis.

Reversing Bacterial Resistance to Antibiotics by Phage-Mediated Delivery of Dominant Sensitive Genes

Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that resensitizes pathogens to approved antibiotics therefore holds key advantages. We present a proof of principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, the genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages carrying the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene's sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage's ability to kill pathogens in the infected host, but instead, on its ability to deliver genetic constructs into the bacteria and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phage will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

Amylin is a novel neuropeptide with potential maternal functions in the rat

Amylin, a 37-aa pancreatic peptide, was found to be expressed in the preoptic area of mother rats in our recent microarray study. Here, we report a marked increase in amylin expression around parturition and show that amylin mRNA level remains elevated as long as the pups are not removed from the dams. Amylin expression is also induced in maternally behaving (sensitized) nonlactating but not in nonsensitized nulliparous females or in females that did not become maternal despite the sensitization procedure. Immunohistochemistry verified the increased amylin peptide expression in maternally behaving rats and demonstrated the same expression pattern of amylin as in situ hybridization histochemistry. Ovariectomy had no effect on the activation of amylin neurons, suggesting sexual steroid-independent mechanisms. In subsequent functional experiments, mothers were separated from their pups for 22 h. On return of the pups, neuronal activation was found in the mother's preoptic area, with a distribution pattern similar to amylin-expressing neurons. Subsequent double labeling revealed that 86-93% of amylin neurons were activated by pup exposure. The results implicate amylin in the control of maternal adaptations, possibly exerting its actions on maternal behaviors via amylin receptors present in brain regions to which preoptic neurons project.

Infliximab alleviates inflammation and ex vivo airway hyperreactivity in asthmatic E3 rats

Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of asthma, and neutralization of TNF-α is an effective therapy for inflammatory diseases. The present study tested the idea that a TNF-α antibody, infliximab, may be useful in the management of asthma. E3 rats were immunized with ovalbumin (OVA)/alum and received infliximab intra-peritoneally. Two weeks later, OVA-PBS was instilled intranasally daily for 7 days. Bronchoalveolar lavage fluids (BALFs), serum and lung homogenates were collected for analysis of cells and inflammatory mediators. Contractile responses of lobar-bronchus segments to agonists were functionally tested. Pulmonary tissues were investigated using histological examination. The results showed that the sensitized 'model E3 rats' exhibited an increase in the total amount of inflammatory cells, primarily eosinophils, in BALF and pulmonary tissue, as well as epithelial damage. Serum levels of IgE increased and so did the levels of nitric oxide, inducible nitric oxide synthase, TNF-α and IL-4, IL-5 and IL-13 in lung homogenate and serum. Furthermore, the contractile responses in bronchi induced by endothelin-1, sarafotoxin 6c and bradykinin increased and isoprenaline-induced relaxations decreased. All these changes induced by the sensitization procedure were reduced by the infliximab treatment. The results suggest that infliximab prevents the development of local airway inflammation and antagonizes changes of the bronchial smooth muscle receptor phenotype, thereby blocking the development of airway smooth muscle hyperreactivity of asthmatic rats.

Developing a passive sensitization procedure for basophils to diagnose IgE-mediated drug hypersensitivity (151.19)

Abstract Introduction: The basophil activation test is quite well established for protein allergens. It is normally done with the patients’ basophils and requires fresh cells and rapid processing. It would be a particularly interesting test for drug allergy, as only very few drug specific in vitro IgE assays exist and it seems to be a highly relevant read out system. Objective: To set up a passive sensitization protocol to test IgE-mediated drug hypersensitivity. Methods: PMBCs of basophil donors are incubated with lactic acid (pH=3.9) to remove surface bound IgE. Afterwards, re-sensitization with patient serum and subsequent stimulation with serial dilutions of drugs. Degranulation can be measured by flow cytometry (anti-CCR3-APC/anti-CD63-PE). Results: We were able to establish a well functional procedure for protein allergens and for some drug allergens (cephalosporines, NMBAs). Passive sensitization assays using serum of patients with well-documented IgE-mediated amoxicillin allergy were reproducibly false negative. Conclusion: Using defined donor basophils for passive sensitizations with serum would have several advantages over direct assays, as it would facilitate diagnosis, as patient’s sera is easy to store and as it would provide a direct proof of an IgE-mediated hypersensitivity reaction. However, a better understanding of how small drugs are actually able to cross-link cell surface bound IgE seems to be a prerequisite to improve this assay and to widen its application.

Effect Sizes in Experimental Pain Produced by Gender, Genetic Variants and Sensitization Procedures

BackgroundVarious effects on pain have been reported with respect to their statistical significance, but a standardized measure of effect size has been rarely added. Such a measure would ease comparison of the magnitude of the effects across studies, for example the effect of gender on heat pain with the effect of a genetic variant on pressure pain.Methodology/Principal FindingsEffect sizes on pain thresholds to stimuli consisting of heat, cold, blunt pressure, punctuate pressure and electrical current, administered to 125 subjects, were analyzed for 29 common variants in eight human genes reportedly modulating pain, gender and sensitization procedures using capsaicin or menthol. The genotype explained 0–5.9% of the total interindividual variance in pain thresholds to various stimuli and produced mainly small effects (Cohen's d 0–1.8). The largest effect had the TRPA1 rs13255063T/rs11988795G haplotype explaining >5% of the variance in electrical pain thresholds and conferring lower pain sensitivity to homozygous carriers. Gender produced larger effect sizes than most variant alleles (1–14.8% explained variance, Cohen's d 0.2–0.8), with higher pain sensitivity in women than in men. Sensitization by capsaicin or menthol explained up to 63% of the total variance (4.7–62.8%) and produced largest effects according to Cohen's d (0.4–2.6), especially heat sensitization by capsaicin (Cohen's d = 2.6).ConclusionsSensitization, gender and genetic variants produce effects on pain in the mentioned order of effect sizes. The present report may provide a basis for comparative discussions of factors influencing pain.

Ethanol-induced locomotor sensitization in DBA/2J mice is associated with alterations in GABA A subunit gene expression and behavioral sensitivity to GABA A acting drugs

Repeated exposure to ethanol may produce increased sensitivity to its acute locomotor stimulant actions, a process referred to as locomotor sensitization. Neuroadaptation within certain brain circuits, including those possessing GABA A receptors, may underlie locomotor sensitization to ethanol. Indeed, GABA A receptors are documented mediators of ethanol's cellular and behavioral actions. Moreover, because subunit composition of this receptor is predictive of its pharmacology, it is possible that alterations in subunit composition contribute to the expression of locomotor sensitization to ethanol. The goal of the present study was to determine if alterations in GABA A subunit composition are associated with the expression of locomotor sensitization in DBA/2J mice, a strain known to be particularly susceptible to the development of this behavioral phenomenon. Following a modified 14 day sensitization procedure (Phillips et al., 1994) relative changes in GABA A subunit gene expression were assessed in discrete mesolimbic brain regions. To determine if the observed changes in gene expression produced functional changes in the locomotor responses to drugs known to either preferentially or generally activate GABA A receptors normally possessing the significantly altered subunits, separate cohorts of animals were challenged with one of several low doses of zolpidem (α1-selective), etomidate (β2/3-selective), or flurazepam (γ2-directed) and assessed for locomotor alterations. Sensitized animals displayed increased expression of the α1, β2, and γ2 (v1) subunits in the Nucleus Accumbens (NAc) but not Ventral Tegmental Area (VTA). Additionally, sensitized animals displayed altered sensitivity to the locomotor actions of etomidate and flurazepam. These results support the hypothesis that neuroadaptive changes in GABA A subunit composition participate in the expression of locomotor sensitization.

Induction of eosinophil-infiltrating drug photoallergy in mice

Drug photoallergy is one of the highly incident adverse effects. Several different histological patterns have been recognized. To establish a murine model of the eosinophil-infiltrating type of drug photoallergy by using afloqualone (AQ), a representative photosensitive drug. AKR/J mice were sensitized by intraperitoneal injection of afloqualone solution (2mg/kg/mouse) and irradiation of shaved abdomen with ultraviolet A light (UVA) (12J/cm(2)). This sensitization procedure was repeated 2-12 times, and 3 days after the last immunization, mice were challenged by a subcutaneous injection of AQ solution and irradiation of the same site with UVA. The draining lymph node cells (LNCs) were used for transfer and cytokine production studies, and the challenged skin was analyzed for chemokine expression. More than 10 times of sensitization induced a massive infiltrate of eosinophils and lymphocytes at the challenged site. AKR/J mice were a high responder strain. The sensitivity was transferred with 5-8 x 10(7) immune lymph node and spleen cells into naïve mice. CD4(+) T cells were mainly responsible for this sensitivity, since 1 x 10(7) CD4(+) cells alone induced a high level of sensitivity, but CD8(+) T cells evoked the sensitivity to a lesser degree. Culture supernatants from AQ-photoimmuned lymph node cells contained a higher level of IL-4 and lower interferon-gamma than those from mice immunized with dinitrofluorobenzene. Finally, the skin of AQ-photochallenged site exhibited high expression of CCL24/eotaxin-2, a chemokine for eosinophils. It is suggested that eosinophilic drug photoallergy is mediated by sensitized Th2 cells and locally produced eosinophil-attracting chemokines.

Does context influence the duration of locomotor sensitization to ethanol in female DBA/2J mice?

Repeated exposure to ethanol produces a progressive increase in locomotor sensitivity, referred to as locomotor sensitization. Locomotor sensitization may persist for some time after termination of repeated drug exposure, and context appears to facilitate expression of the behavioral phenomenon. However, many unanswered questions remain concerning the persistence of and degree to which context influences locomotor sensitization to alcohol (ethanol). The goal of the present work was to determine the duration of locomotor sensitization to ethanol and the degree to which context dependence positively influences the induction, expression, and persistence of the behavioral phenomenon in female DBA/2J mice. Sensitized (with or without ethanol-paired exposure to the testing chamber) and non-sensitized saline control mice were left undisturbed in their home cages until subsequent ethanol challenge and testing in the locomotor activity testing chambers 7, 14, 21, 28, 42, 56, and/or 70 days after cessation of the ethanol sensitization procedure. Retro-orbital sinus bloods were sampled to determine whether the sensitization procedure had altered blood ethanol clearance rates. Locomotor sensitization persisted through post-sensitization day 14, and repeated paring of the drug and testing context prolonged the expression of this phenomenon through at least post-sensitization day 28. Blood ethanol concentrations did not differ. Locomotor sensitization to ethanol persists for some time after cessation of repeated ethanol exposure, and the association of contextual cues with the ethanol experience lengthens this persistence. The present data lay the groundwork for investigations into the neuroadaptive changes that underlie locomotor sensitization to ethanol in mice.

THE fickle MUTATION OF A CYTOPLASMIC TYROSINE KINASE EFFECTS SENSITIZATION BUT NOT DISHABITUATION IN DROSOPHILA MELANOGASTER

fickle is a P-element mutation identified from a screen for defects in courtship behavior and disrupts the fly homolog of Bruton's tyrosine kinase (Btk) gene (Baba et al., ). Here, we show that habituation of the olfactory jump reflex also is defective in fickle. Unlike, the prototypical memory mutants, rutabaga and dunce, which habituate more slowly than normal, fickle flies habituate faster than normal. fickle's faster-than-normal response decrement did not appear to be due to sensorimotor fatigue, and dishabituation of the jump response was normal. Based on a long-standing “two opponent process” theory of habituation, these data suggested that behavioral sensitization might be defective in fickle. To test this hypothesis, we designed a olfactory sensitization procedure, using the same stimuli to habituate (odor) and dishabituate (vortexing) flies. Mutant flies failed to show any sensitization with this procedure. Our study reveals a “genetic dissection” of sensitization and dishabituation and, for the first time, provides a biological confirmation of the two opponent process theory of habituation.

A model of allergen-driven human airway contraction: β 2 pathway dysfunction without cytokine involvement

In our previous in vitro model, allergen incubation of passively sensitized human airways reduced the response to salbutamol. However, whether cytokines play a role in this model is still unknown. To investigate interleukin 1beta and tumor necrosis factor a expression in allergen-challenged human airways. Nonasthmatic airways (n = 13) were passively sensitized by overnight atopic serum incubation and then challenged with allergen for 1 hour (n = 9). After repeated washouts, airways were immersed in physiologic salt solution for 6 hours and finally in formaldehyde for immunohistochemical studies. The effect of co-incubation in anti-interleukin 1beta and anti-tumor necrosis factor a specific neutralizing antibodies on salbutamol response was also studied (n = 4). No differences were found among control, sensitized, and challenged rings in the number of inflammatory cells. The percentage of basement membrane covered by epithelium was similar in the different conditions. There was a higher percentage of degranulating to total mast cells in allergen-challenged rings than in sensitized rings (P < .001). A significant correlation was observed between allergen-induced contraction and mast cell degranulation (r = 0.88; P < .001). The sensitization procedure was validated by paired allergen-induced contractions. No expression of the 2 cytokines was detectable up to 6 hours after allergen challenge, and specific neutralizing antibodies did not attenuate the impaired response to salbutamol in allergen-challenged rings. These data suggest that in our in vitro model of allergic inflammation, beta2 pathway dysfunction can occur without cytokine involvement, thus supporting previous results that suggest a role for leukotrienes.

Characterization of a highly photorefractive RF-sputtered SiO2-GeO2 waveguide

We present the characterization of highly photorefractive Er3+/Yb3+-doped 75SiO2-25GeO2 planar waveguides, single mode at 1550 nm, deposited by radio-frequency-magnetron-sputtering (RFMS) technique. Details of the deposition process are reported. The material presents an intense absorption band (alpha approximately 10;3/10;4 cm;-1) in the UV region. Irradiations by a KrF excimer laser source at lambda = 248 nm have produced large positive (up to 310-3) refractive index changes, without the need of particular sensitization procedures. Direct measurements of UV photo-induced volume densification demonstrates that glass compaction accounts for large part of the refractive index change. Highly efficient photo-induced phase gratings have thus been fabricated in the waveguide.

Dielectric properties of titanate based ceramic capacitors using electroless Ni as contact electrodes

The electroless plating Ni was uniformly coated on the ceramic capacitors based on BaTiO 3, SrTiO 3, CaTiO 3, PbTiO 3, Bi 2/3TiO 3, etc. as the contact electrodes of these ceramic capacitors. Their structural and electrical properties were characterized using XRD, SEM, and impedance analyser, respectively. Results indicated that the Ni electroded capacitors had larger dielectric capacitances, smaller loss tangents, and higher breakdown voltages than Ag electroded counterparts. Through introducing complex agents in the electroless solution and cleaning process after sensitization and activation procedures, the negative effect of Bi 3+ ions on the surface resistance of the Ni coating was effectively eliminated, and good Ni coatings were obtained.

A model of chronic IgE-mediated food allergy in ovalbumin-sensitized mice.

Food allergy is most frequently the result of IgE-mediated hypersensitivity reactions. Here, we describe a chronic model in which some of the intestinal and systemic consequences of continuous egg white solution ingestion by ovalbumin-sensitized eight-week-old BALB/c mice, 6 animals per group, of both sexes, were investigated. There was a 20% loss of body weight that began one week after antigen exposure and persisted throughout the experiment (3 weeks). The sensitization procedure induced the production of anti-ovalbumin IgG1 and IgE, which were enhanced by oral antigen exposure (129% for IgG1 and 164% for IgE, compared to sensitization values). Intestinal changes were determined by jejunum edema at 6 h (45% Evans blue extravasation) and by a significant eosinophil infiltration with a peak at 48 h. By day 21 of continuous antigen exposure, histological findings were mild, with mast cell hyperplasia (100%) and increased mucus production (483%). Altogether, our data clearly demonstrate that, although immune stimulation was persistently occurring in response to continuous oral antigen exposure, regulatory mechanisms were occurring in the intestinal mucosa, preventing overt pathology. The experimental model described here reproduces the clinical and pathological changes of mild chronic food allergy and may be useful for mechanistic studies of this common clinical condition.

I DDT: Fundamentals and test generation

It is the time to explore the fundamentals of IDDT testing when extensive work has been done for IDDT testing since it was proposed. This paper precisely defines the concept of average transient current (IDDT) of CMOS digital ICs, and experimentally analyzes the feasibility of IDDT test generation at gate level. Based on the SPICE simulation results, the paper suggests a formula to calculate IDDT by means of counting only logical up-transitions, which enables IDDT test generation at logic level. The Bayesian optimization algorithm is utilized for IDDT test generation. Experimental results show that about 25% stuck-open faults are with IDDT testability larger than 2.5, and likely to be IDDT testable. It is also found that most IDDT testable faults are located near the primary inputs of a circuit under test. IDDT test generation does not require fault sensitization procedure compared with stuck-at fault test generation. Furthermore, some redundant stuck-at faults can be detected by using IDDT testing.

Role of halogens in the mechanism of sensitization of uncooled PbSe infrared photodetectors

Lead selenide layers were obtained by thermal evaporation in vacuum on thermally oxidized silicon. As-deposited layers present no response to infrared radiation and an activation procedure is mandatory. The sensitization process described in this work consists of a thermal treatment developed in two different stages: The first step in an iodine and oxygen rich atmosphere at 220 °C, and the second one at a higher temperature (450 °C) on air. After this treatment, the developed photodetectors show high sensitivity to medium wavelength infrared radiation at room temperature. We have analyzed the structural, compositional, electrical, and morphological changes of the films occurred during the activation process. After processing many films, we conclude that iodine plays a key role in the PbSe sensitization. This halogen behaves as a transport agent during the PbSe recrystallization process, and promotes a fast growth of PbSe microcrystals. Oxygen is trapped into the PbSe lattice during the recrystallization process, as it happens in chemically deposited PbSe films. The introduction of halogens in the PbSe sensitization procedure is a highly efficient technique for the incorporation of oxygen to the semiconductor lattice in electrically active positions.

Gabapentin suppresses cutaneous hyperalgesia following heat-capsaicin sensitization.

The anticonvulsant gabapentin, proven effective for neuropathic pain in two large, placebo-controlled clinical trials, is widely used for treatment of chronic pain. Preclinical studies have demonstrated analgesic and antiallodynic effects in models involving neuronal sensitization and nerve injury, without affecting acute pain transmission. The aim of the present study was to link data from animal models and clinical trials for chronic pain by investigating the effect of gabapentin on acute nociception and experimentally induced cutaneous hyperalgesia in healthy volunteers. The human experimental hyperalgesia model, the heat-capsaicin sensitization model, was induced in 25 healthy male volunteers. Subjects received oral gabapentin (1,200 mg) or placebo after heat-capsaicin sensitization was established on the forearm. The primary outcome measures were the sizes of the areas of secondary hyperalgesia to von Frey hair and brush stimulation on the forearm. Secondary outcome measures were as follows: (1) size of secondary hyperalgesia area in response to brief thermal sensitization procedure on the thigh; (2) heat pain detection thresholds in normal and sensitized skin; and (3) painfulness of 1 min of 45 degrees C stimulation in normal skin. Oral gabapentin profoundly suppressed established cutaneous sensitization on the forearm and prevented development of cutaneous sensitization on the thigh. Thermal nociception in normal skin was unchanged. Side effects were modest. The results link preclinical findings with results from clinical trials of neuropathic pain. The results further suggest that gabapentin may prove effective in acute pain disorders involving neuronal sensitization, such as postoperative pain and acute herpetic pain, and could prove effective in prevention of chronic pain.

Repeated exposure to low levels of sulfur dioxide (SO 2) enhances the development of ovalbumin-induced asthmatic reactions in guinea pigs

Sulfur dioxide (SO2) is one of the major air pollutants. It is known to aggravate asthma symptoms in human beings, but few studies have focused on the effects of SO2 upon the development of bronchial asthma in animal models. This study was undertaken to evaluate the role of SO2 upon the development of ovalbumin (OA)-induced asthmatic reactions in guinea pigs. Guinea pigs were divided into four groups: (1) OA- and SO2-exposed group (n = 12), (2) SO2-exposed group (n = 12), (3) OA-exposed group (n = 11), and (4) saline-exposed group (n = 7). Guinea pigs of the first and second groups were exposed to 0.1 ppm SO2 for 5 hours a day on 5 consecutive days. Guinea pigs in the first and third groups inhaled 0.1% OA aerosols for 45 minutes a day on days 3, 4, and 5. One week after the sensitization procedure, all the guinea pigs underwent bronchial challenge with 1.0% OA aerosols, using unrestricted whole-body plethysmography. Bronchoalveolar lavage and histopathologic examination were performed 24 hours after the bronchial challenge. Increases in enhanced pause (Penh), as an index of airway obstruction, after the bronchial challenge was significantly higher in OA- and SO2-exposed group (group 1) than the other groups (P < .05, respectively). Eosinophil counts in bronchoalveolar lavage fluids were also significantly higher in group 1 than in the other groups (P < .05, respectively). Histopathologic findings of bronchial and lung tissue in the group 1 showed an infiltration of inflammatory cells, bronchiolar epithelial damage, and mucus and cell plug in the lumen, but no significant abnormalities were observed in the other groups. These results indicate that repeated exposure to low levels of sulfur dioxide may enhance the development of ovalbumin-induced asthmatic reactions in guinea pigs.

Experimental induction of contact allergy in dogs by means of certain sensitization procedures

Three contact allergens; dinitrochlorobenzene (DNCB), nickel and formalin, were determined allergenicities. Contact allergies were induced in 24 dogs and 15 guinea pigs (used as a comparative animal species) by means of Draize test, Maximization test, Open Epicutaneous test and Buehler test, and evaluated by means of closed patch testing. Results showed that, in dogs, nickel produced allergic responses of the skin in the Open Epicutaneous test, but formalin did not produced it. In the Maximization test, however, both nickel and formalin failed to induce a contact allergy, although DNCB showed a high allergenicity. The appropriate concentration of test solutions for the closed patch test in dogs were 1.0% for DNCB, 0.1% for nickel and 1:10 dilution for formalin, respectively.

The differential effect of the anxiolytic agent 8-OH-DPAT during lactation is independent of pup withdrawal and maternal behavior.

Injection of the serotonergic agonist, 8-hydroxy-2-(di-n-propylamino-tetralin (8-OH-DPAT) (0.5 mg/kg ip) produced a clear anxiolytic-like effect (as measured in the burying behavior test), after parturition, which remains until day 6 of lactation. Thereafter 8-OH-DPAT completely lacked action. In order to analyze whether lactation prevented the action of 8-OH-DPAT, dams were separated from their pups for five consecutive days. The blockade of the anxiolytic effect of 8-OH-DPAT does not disappear by isolation of the mothers from their offspring or from neighboring pups. Finally, to investigate the possible role of maternal behavior in the blockade of the anxiolytic effect of 8-OH-DPAT a third experiment was made in which ovariectomized females were rendered maternal by the sensitization procedure. These females respond normally to the antianxiety actions of 8-OH-DPAT. Results suggest that a long-term clue triggered by lactation, possibly related to prolactin secretion, interferes with the anxiolytic effect of 8-OH-DPAT.