Opposing effects of 5,7-DHT infusions into the orbitofrontal cortex and amygdala on flexible responding.

Central serotonin is implicated in a variety of emotional and behavioral control processes. Serotonin depletion can lead to exaggerated aversive processing and deficient response inhibition, effects that have been linked to serotonin's actions in the amygdala and orbitofrontal cortex (OFC), respectively. However, a direct comparison of serotonin manipulations within the OFC and amygdala in the same experimental context has not been undertaken. This study compared the effects of infusing the serotonin neurotoxin, 5,7-dihydroxytryptamine into the OFC and amygdala of marmosets performing an appetitive test of response inhibition. Marmosets had to learn to inhibit a prepotent response tendency to choose a box containing high-incentive food and instead choose a box containing low-incentive food, to obtain reward. OFC infusions caused long-lasting reductions in serotonin tissue levels, as revealed at postmortem, and exaggerated prepotent responses. In contrast, the significantly reduced prepotent responses following amygdala infusions occurred at a time when serotonin tissue levels had undergone considerable recovery, but there remained residual reductions in extracellular serotonin, in vivo. These opposing behavioral effects of serotonin manipulations in the same experimental context may be understood in terms of the top-down regulatory control of the amygdala by the OFC.

The role of the orbitofrontal cortex and medial striatum in the regulation of prepotent responses to food rewards.

An impairment in learning to inhibit prepotent responses to positive stimuli is associated with damage to the orbitofrontal cortex (OFC) in rats, monkeys, and humans performing discrimination reversal, extinction, and detour reaching tasks. In contrast, a recent study showed that OFC-lesioned rhesus monkeys could learn to select the smaller of 2 quantities of food reward in order to receive the larger reward, at an equivalent rate to controls, despite the requirement to inhibit a prepotent response. Given this result, the aim of the present study was to further specify the contexts under which the OFC regulates responding and to identify additional components of limbic circuitry that contribute to such regulation. Marmosets with lesions of the OFC and medial striatum (MS), but not the amygdala, made more prepotent responses to a clear Perspex box containing high incentive food before learning to choose the box containing low incentive food, to obtain reward. However, having learned the incongruent incentive discrimination OFC- and MS-lesioned monkeys were impaired upon reversal of the reward contingencies, repeatedly selecting the previously rewarded low incentive object. These findings identify the critical contribution of the OFC and MS in the regulation of responding by affective cues.

Uncoupling of behavioral and autonomic responses after lesions of the primate orbitofrontal cortex.

Successful adaptation to changes in an animal's emotional and motivational environment depends on behavioral flexibility accompanied by changes in bodily responses, e.g., autonomic and endocrine, which support the change in behavior. Here, we identify the orbitofrontal cortex (OFC) as pivotal in the flexible regulation and coordination of behavioral and autonomic responses during adaptation. Using an appetitive Pavlovian task, we demonstrate that OFC lesions in the marmoset (i) impair an animal's ability to rapidly suppress its appetitive cardiovascular arousal upon termination of a conditioned stimulus and (ii) cause an uncoupling of the behavioral and autonomic components of the adaptive response after reversal of the reward contingencies. These findings highlight the role of the OFC in emotional regulation and are highly relevant to our understanding of disorders such as schizophrenia and autism in which uncoupling of emotional responses may contribute to the experiential distress and disadvantageous behavior associated with these disorders.

Mood, neuropsychological function and cognitions in premenstrual dysphoric disorder.

BACKGROUND: Neuropsychological function and cognitive correlates of depression have not previously been examined in a rigorously defined population of patients suffering from premenstrual dysphoric disorder (PMDD). METHOD: MOOD, neuropsychological function and cognition were measured in 10 PMDD patients and 10 age-matched controls in both phases of the menstrual cycle in a random order, counter-balanced design. RESULTS: The BDI was significantly elevated in the luteal phase of PMDD patients only while other cognitive measures showed no significant differences. Working memory was impaired in the luteal phase of the menstrual cycle with no significant differences between PMDD and control subjects. CONCLUSION: Despite the small sample size, these results show that the BDI is sensitive to the mood fluctuations of PMDD patients. An impairment in working memory was also found although this is a general menstrual cycle effect.

Effects of adrenalectomy on 8-OH-DPAT induced hypothermia in mice.

Complex interactions exist between the hypothalamic-pituitary-adrenal (HPA) axis and the serotonergic system, and it has been suggested that these interactions may be fundamental to the pathophysiology and treatment of depressive illnesses. It has previously been found that chronic administration of corticosterone leads to adrenal suppression and an attenuation of somatodendritic 5-HT1A receptor function. Adrenalectomy (ADX) has been shown to cause an increase in postsynaptic 5-HT1A receptor numbers and possibly function. However, other reports have suggested that ADX does not alter somatodendritic 5-HT1A receptor mRNA or binding, though little is known of the effect of ADX on the function of somatodendritic 5-HT1A receptors. This study investigated the effect of markedly reducing corticosterone levels by ADX on 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT)-induced hypothermia in mice, an in vivo model of somatodendritic 5-HT1A receptor function. The degree of 8-OH-DPAT-induced hypothermia did not differ between control, sham, and ADX animals 14 days post operatively. Although repeated administration of corticosterone attenuates somatodendritic 5-HT1A receptor function, these data demonstrate that lowering of corticosteroid levels by ADX have no effect. This suggests that the effects of repeated corticosterone administration is not mediated by a secondary adrenal suppression. The difference in the effects of ADX on somatodendritic as opposed to postsynaptic 5-HT1A receptors may reflect the differential expression of corticosteroid receptor subtypes at postsynaptic and somatodendritic sites.

Ha-ras induction of the invasive phenotype results in up-regulation of epidermal growth factor receptors and altered responsiveness to epidermal growth factor in human papillary transitional cell carcinoma cells.

Recent studies have shown that orthotopic (transurethral) transplantation of human bladder cancer cell lines into nude mice permits tumor growth that more accurately reflects their clinical malignant status in the original host. We have previously demonstrated that transfection and overexpression of normal or mutated c-Ha-ras genes into a noninvasive human papillary transitional cell carcinoma cell line confer upon these cells an invasive phenotype in vivo with behavior remarkably similar to the clinical behavior of high grade bladder carcinomas. Since elevated expression levels of the epidermal growth factor receptor (EGF-R), in addition to that of c-Ha-ras, have been correlated with transitional cell carcinoma progression, we sought to determine whether up-regulation of the EGF-R had occurred in the invasive high ras expressors and if so, what functional significance this might have. Our results show that invasive cell lines which overexpress the c-Ha-ras gene also have increased epidermal EGF-R expression. This was found to occur at both the protein and mRNA levels, and analysis of the EGF-R promoter/enhancer sequences has revealed a putative AP-1 site which may possibly enhancer sequences has revealed a putative AP-1 site which may possibly serve as a ras response element. In addition, we found that the cells overexpressing the EGF-R had acquired a positive sensitivity to the stimulatory mitogenic effects of EGF. Hence, the results obtained suggest a role for either a normal or a mutated overexpressing Ha-ras in up-regulating the surface EGF-R, possibly through an AP-1 site during human bladder carcinoma progression; they also highlight the potential that EGF may have in cooperating with this EGF-R up-regulation to help mediate enhanced tumor growth.

Cloning and overexpression of TGF-beta 1 cDNA in a mammary adenocarcinoma: in vitro and in vivo effects.

It has been suggested that transforming growth factor beta (TGF-beta) may be a potential negative autocrine growth regulator of carcinomas including mammary carcinomas. To directly test this hypothesis we have cloned and expressed human TGF-beta 1 cDNA in a murine mammary adenocarcinoma which is normally growth-inhibited by addition of exogenous TGF-beta in vitro. A number of transfectants over-expressing the foreign TGF-beta 1 mRNA were selected and compared to transfectants which did not overexpress the exogenous TGF-beta 1 cDNAS. Cell lines overexpressing the transfected TGF-beta 1 mRNA were found to produce total levels of TGF-beta 7 to 10 fold greater than the parental cells or control transfected clones. However, when levels of active fractions of TGF-beta were compared in cell lines overexpressing TGF-beta 1 to those which did not, no differences were found. This suggests that the activation mechanism is not necessarily induced or altered by increasing levels of latent TGF-beta 1 production in a given tumor cell line. The basal in vitro doubling time of TGF-beta 1 overexpressing clones was identical to the control populations. Similarly, in vivo tumor growth rates after s.c. injection were similar to that of the parental line. Thus the precise role of TGF-beta in mediating either the in vitro or in vivo growth control of a sensitive mammary adenocarcinoma cell line remains unclear. It may be that cellular over-secretion of latent TGF-beta must be coupled with enhanced cellular TGF-beta activation prior to any observed effect on growth rate in vitro or in vivo; this latter event may constitute the "rate-limiting" step of TGF-beta activity on tumor behavior.

Alteration of the tumorigenic and metastatic properties of neoplastic cells is associated with the process of calcium phosphate-mediated DNA transfection.

During the course of studies designed to assess the effect of human Ha-ras gene expression on the malignant behavior of transfected mouse tumor cells we noted that the process of Ca3(PO4)2-mediated DNA transfection was itself associated with profound alterations in tumorigenic or metastatic behavior. The cell line used as a recipient for these studies was a tumorigenic nonmetastatic CBA/J mouse mammary adenocarcinoma line called SP1. When cotransfected with plasmids containing the neo gene (pSV2neo) and the activated Ha-ras gene (pT24-c3), cells from the pooled (5-10 colonies) G418-resistant colonies gave rise to spontaneous lung metastases in 85% of mice after subcutaneous inoculation. However, we noted that 17% of control mice inoculated with G418-resistant pSV2neo-transfected SP1 cells also had lung metastases and that this number approached 100% as the inoculum comprised a greater pool size (50-100 colonies). When cell lines established from isolated pSV2neo-transfected colonies were examined, 3/16 were found to be metastatic. We also found that 3/16 clones grew slowly, or not at all, in CBA/J mice, whereas they grew readily in athymic (nude) mice. The increase in immunogenicity of two out of three of these latter clones was accompanied by expression of the class I H-2Dk major histocompatibility complex antigen that was not detectable in the parental SP1 cells. At least some of these results would appear to be due to exposure to Ca3(PO4)2 alone, as we found that it resulted in 5/20 (25%) clones manifesting metastatic properties. Our results suggest that heritable changes in malignant behavior of transfected tumor cells can be observed at high frequency subsequent to the process of Ca3(PO4)2-mediated DNA transfection, and these changes may be brought about in part by inherited disturbances in expression of recipient cell genes.

Adoptive immune therapy in mice bearing poorly immunogenic metastases, using T lymphocytes stimulated in vitro against highly immunogenic mutant sublines.

MDW3, a highly immunogenic and non-tumorigenic (tum-) mutant of the poorly immunogenic metastatic murine tumor called MDAY-D2, has been employed in an immune therapy scheme for the treatment of widespread established visceral MDAY-D2 metastases in syngeneic mice. MDW3 was selected from a mutagenized population of MDAY-D2 cells for the ability to grow in the presence of toxic concentrations of wheat-germ agglutinin (WGA) in vitro. The mutant expresses a common tumor-associated antigen (TAA) present on MDAY-D2 as well as a new antigen whose presence enhances the anti-TAA cell-mediated immune response in vivo and in mixed lymphocyte tumor cultures (MLTC) in vitro. For immune therapy, spleen cells from DBA/2 mice which had rejected an inoculum of MDW3 cells were restimulated in MLTC and injected i.v. into MDAY-D2 tumor-bearing mice. Two protocols were used. In the first, mice were given an i.v. injection of 10(3) MDAY-D2 cells ("artificial metastasis") and subsequently treated with 400 R whole-body irradiation and MDW3-stimulated T cells. Such mice had a 75% long-term survival rate, whereas 400 R alone, or no treatment, resulted in 25% and 0% long-term survivors, respectively. In the second protocol, treatment of mice bearing a 12-day-old subcutaneous MDAY-D2 tumor by surgical removal of the solid tumor, 400 R whole-body irradiation, and systemic administration of MDW3-stimulated spleen cells, resulted in a 75-100% survival rate, whereas omitting any part of the treatment resulted in 0-50% survival rates. The treatment increased splenic anti-TAA CTL activity, and the mice acquired immunity against the new antigen on MDW3, suggesting that the injected lymphocytes were proliferating in the host. The optimal combination of resection, whole-body irradiation and passive infusion of MDW3-stimulated spleen cells was ineffective when used on mice bearing a tumor-antigen-loss variant of MDAY-D2, suggesting that success of our immune therapy protocol required specific recognition of the tumor-associated antigen of MDAY-D2.

A model of human cancer metastasis: extensive spontaneous and artificial metastasis of a human pigmented melanoma and derived variant sublines in nude mice.

The extensive metastatic capacities in nude mice of variants of a human melanoma line, MeWo, were studied. BALB/c nude mice received subcutaneous implants of lung cubes impregnated in vitro with small numbers (less than 1,000) of MeWo cells as a result of previous in vitro coincubation. Such implants always resulted in lethal tumors, despite the fact that injection of dispersed 3 X 10(5) MeWo cells was normally required to obtain a tumor take. A BALB/c nude mouse that had received a MeWo lung cube implant was found to have numerous, large lung nodules 6 months after implantation. Transfer of the metastatic lung nodules to new recipients also led to the appearance of lung metastases. Cell lines established from such metastases, or from primary tumors that arose in nude mice implanted with MeWo-infiltrated cubes, showed a remarkable ability to colonize the lungs after iv injection, in contrast to the parent MeWo cells: Lungs were found to be engorged with hundreds of nodules, many, but not all, being melanotic. Widespread extrapulmonary metastases were also observed, but only after iv injection of MeWo sublines established from metastases. Karyotype analysis revealed that lung colonies established by iv cell injection had a near diploid (i.e., wild type) number of human chromosomes (mode: 44), whereas cell lines from spontaneous metastases possessed a near tetraploid number, a possible consequence of either selection or tumor progression in vivo. The results indicate that human tumor variants with unusually aggressive metastatic capabilities can be obtained in adult nude mice, similar in nature to some highly metastatic variants derived from mouse tumors (e.g., the B16 melanoma). The availability of such variants should be a valuable aid to study aspects of tumor cell heterogeneity, progression, and metastasis from a human cancer cell perspective.

Single-step selection of unique human melanoma variants displaying unusually aggressive metastatic behavior in nude athymic mice.

Using two different approaches, variants from a pigmented human melanoma cell line called MeWo were selected in a single step which displayed an unusually aggressive ability to metastasize in adult athymic nude mice. The first set of variants was obtained by recovery, and establishment in culture, of 'spontaneous' lung metastases obtained 5-6 months after subcutaneous inoculation of the parent MeWo line. That these metastases arose by a nonrandom process, and were authentic variants, was shown by the fact that they were always highly aneuploid, having predominantly near-triploid or near-tetraploid chromosome numbers. In contrast, the parent MeWo cells had a predominant hypodiploid chromosome mode with a minor population (less than 10%) of near-tetraploid cells. A second set of variants was obtained through in vitro selection of cloned lectin-resistant (Lecr) aneuploid 'membrane mutants' from MeWo, using wheat germ agglutinin (WGA) as the selective agent. Some of these mutants manifested an extraordinary ability to disseminate widely and extensively to many extrapulmonary sites after intravenous inoculation of the cells; furthermore, the metastases, even those less than 0.5 mm in diameter ('pinhead' sized), were easily visible because of the remarkably intense pigmented nature of the mutant cells. These results provide a promising direction to take for the derivation of heterogenous sublines of human tumors which not only metastasize aggressively in nude mice, but which do so in a manner not unlike what is actually observed in their natural host.

Carcinogenicity of tumor cell populations: origin of a putative H-2 isoantigenic loss variant tumor.

The properties of an unusual mouse tumor capable of extremely rapid and widespread spontaneous metastatic growth were recently described; this tumor, called MDAY-D2, at first appeared to be an H-2Kk loss variant of an (A X DBA/2)F1 (H-2KkDd) sarcoma called MDAY and was obtained by serial ip passage of MDAY in DBA/2 (KdDd) mice. The studies described here were concerned with the analysis of the origin of MDAY-D2; i.e., was it a true variant or a newly induced DBA/2 tumor? Several approaches were used, most of which exploited defined cell surface alloantigenic systems as natural genetic markers. The results indicated that MDAY-D2 was indeed a newly induced DBA/2 tumor and, furthermore, that MDAY was a homozygous A-strain tumor, probably a T-cell lymphoma. Thus a) MDAY was found to be Ly-1.2+, Ly-2.2+, and Thy-1.2+, but Ly-6.2-, whereas the opposite pattern was observed with MDAY-D2; b) MDAY possessed the private and public H-2 specificities associated with H-2k and H-2Dd, but not H-2Dk [i.e., it typed as an A-strain (H-2a) tumor, not as (A X DBA/2)F1]; c) MDAY-D2 possessed private and public specificities associated with H-2Kd and H-2Dd and was found to be H-2Kk-negative [i.e., it typed as a DBA/2 (H-2d) tumor]; d) serial injection of clonally derived ouabain-resistant H-2Kk-positive MDAY cells into DBA/2 hosts led to the rapid development of an MDAY-D2 (H-2d-positive) tumor that was fully ouabain-sensitive. Several findings did not support a contaminant theory to explain induction of MDAY-D2. The rapid induction of a tumor after injection of allogeneic tumor cells may have importance in relation to oncogenesis, tumor variant formation, and tumor progression. The results showed that tumor cells themselves can be potent carcinogens.