Parallel processing of social signals by the mammalian main and accessory olfactory systems.

The mammalian olfactory system has evolved complex mechanisms to detect a vast range of molecular cues. In rodents, the olfactory system comprises several distinct subsystems. Current interest has focused on the exact role that each of these subsystems plays in detecting molecular information and regulating chemosensory-dependent behaviors. Here, we summarize recent results showing that the mouse main and accessory olfactory systems detect, at least in part, overlapping sets of social chemosignals. These findings give rise to a model that involves parallel processing of the same molecular cues in both systems. Together with previous work, this model will lead to a better understanding of the general organization of chemical communication in mammals and give a new direction for future experiments.

Sexually dimorphic activation of galanin neurones in the ferret's dorsomedial preoptic area/anterior hypothalamus after mating.

Male ferrets in breeding condition possess three times as many galanin-immunoreactive (IR) neurones as oestrous females in the sexually dimorphic dorsomedial preoptic area/anterior hypothalamus (dmPOA/AH). Using Fos-IR as a marker of activation, we investigated whether mating with intromission differentially activates this sexually dimorphic group of galanin-IR neurones in male and female ferrets. Male ferrets that intromitted had a significantly greater percentage of galanin-IR neurones in the dmPOA/AH that were colabelled with nuclear Fos-IR than oestrous females that received an intromission. Intromissive stimulation augmented Fos-IR in an equal percentage of galanin-IR neurones in both sexes in the medial amygdala (MA) and bed nucleus of the stria terminalis (BNST). Peripheral anosmia induced by bilateral occlusion of males' nares did not reduce the mating-induced activation of galanin-IR neurones in the dmPOA/AH, and there was a significant correlation among individual males between intromission duration and the percentage of dmPOA/AH galanin-IR neurones colabelled with Fos-IR. Exposure of castrated, testosterone propionate-treated male ferrets to either soiled bedding or to volatile odours from oestrous females failed to induce nuclear Fos-IR in galanin-IR neurones located in the dmPOA/AH, BNST or MA, suggesting that the mating-induced activation of galanin-IR forebrain neurones in male ferrets depends more on genital-somatosensory than on olfactory inputs. The observed sex dimorphism in the mating-induced activation of galanin-IR neurones in the dmPOA/AH raises the possibility that these neurones perform a mating-dependent function that occurs only in males.

The ferret's vomeronasal organ and accessory olfactory bulb: effect of hormone manipulation in adult males and females.

The male ferret, a carnivore, was recently shown to possess a vomeronasal organ (VNO). We compared the morphology of the VNO and its associated accessory olfactory bulb (AOB) in male and female ferrets that were killed in adulthood. The volume and surface area of the VNO neuroepithelium were similar in adult gonadectomized male and female ferrets regardless of whether they were treated with testosterone propionate (TP) or oil vehicle. An AOB was localized bilaterally in the medial caudal part of the olfactory bulbs of adult ferrets using soybean agglutin binding and immunostaining for luteinizing hormone-releasing hormone and tyrosine hydroxylase as well as Nissl staining of coronal, horizontal, and sagittal brain sections. There was no effect of sex or TP treatment on AOB cell layer volume in adult gonadectomized animals. We found the ferret's AOB to be more medially located and much smaller than previously reported in this species, thus highlighting the importance of using several histochemical markers to characterize this structure in any previously unexamined species. Adult male and female ferrets both have a VNO and an associated AOB. More research is needed to determine what role, if any, this accessory olfactory system plays in mediating behavioral and neuroendocrine responses to pheromones in ferrets of either sex.

Nares occlusion eliminates heterosexual partner selection without disrupting coitus in ferrets of both sexes.

Using an airtight Y maze and a new method to induce peripheral anosmia in ferrets, we assessed the contribution of conspecific odors, either alone or in combination with visual and auditory signals, to heterosexual partner preference. Sexually naive ferrets were gonadectomized and treated with sex steroids, after which their nares were either bilaterally occluded using dental impression material or were sham-occluded. Behavioral and histological evidence suggested that nares occlusion blocked access of odors to the main olfactory epithelium for the duration of the study. Sham-occluded females and males preferred to approach odor only or odor plus visual plus auditory cues from opposite-sex conspecifics, whereas nares-occluded ferrets approached opposite- and same-sex cues equally. All ferrets subsequently mated successfully in tests conducted in a small chamber. When retested in the Y maze, sham-occluded females and males again preferred to approach odor-only or odor plus visual plus auditory cues from opposite-sex ferrets, whereas nares-occluded subjects showed no such preference even in tests when a brief physical interaction with tethered stimulus ferrets was allowed after each trial. Our results show that in the ferret, a carnivore, the detection and processing of volatile odors from conspecifics by the main olfactory system is required for heterosexual mate choice.

Maternal odours induce Fos in the main but not the accessory olfactory bulbs of neonatal male and female ferrets.

Previous research demonstrated that exposing gonadectomized adult ferrets to odours in oestrous female bedding induced nuclear Fos-immunoreactivity (Fos-IR; a marker of neuronal activity) in the main as opposed to the accessory olfactory system in a sexually dimorphic fashion, which was further augmented in both sexes by treatment with testosterone propionate. Ferrets are born in an altricial state and presumably use maternal odour cues to locate the nipples until the eyes open after postnatal (P) day 23. We investigated whether maternal odours augment neuronal Fos preferentially in the main versus accessory olfactory system of neonatal male and female ferret kits. Circulating testosterone levels peak in male ferrets on postnatal day P15, and mothers provide maximal anogenital stimulation (AGS) to males at this same age. Therefore, we assessed the ability of maternal odours to augment Fos-IR in the accessory olfactory bulb (AOB), the main olfactory bulb (MOB) and other forebrain regions of male and female ferret kits on P15 and investigated whether artificial AGS (provided with a paintbrush) would further enhance any effects of maternal odours. After separation from their mothers for 4 h, groups of male and female kits that were placed for 1.5 h with their anaesthetized mother had significantly more Fos-IR cells in the MOB granule cell layer and in the anterior-cortical amygdala, but not in the AOB cell layer, compared to control kits that were left on the heating pad. Artificial AGS failed to amplify these effects of maternal odours. Maternal odours (with or without concurrent AGS) failed to augment neuronal Fos-IR in medial amygdaloid and hypothalamic regions that are activated in adult ferrets by social odours. In neonatal ferrets of both sexes, as in adults, socially relevant odours are detected by the main olfactory epithelium and initially processed by the MOB and the anterior-cortical amygdala. In neonates, unlike adults, medial amygdaloid and hypothalamic neurones either do not respond to these inputs or respond in a manner that fails to induce Fos expression.

Mating induces gonadotropin-releasing hormone neuronal activation in anosmic female ferrets.

In females of both spontaneously and induced ovulating species, pheromones from male conspecifics can directly stimulate GnRH neuronal activity, thereby inducing pituitary LH secretion and stimulating the onset of estrus. However, whether pheromones contribute to the steroid- or mating-induced preovulatory activation of GnRH neurons is less clear. Previous studies in the ferret, an induced ovulator, raised the possibility that olfactory cues contribute to the ability of genital-somatosensory stimulation to activate GnRH neurons in the mediobasal hypothalamus (MBH). In the present study the percentage of GnRH neurons colabeled with Fos-immunoreactivity (IR), used as a marker for neuronal activation, was investigated in the MBH of mated gonadectomized, estradiol-treated female ferrets in which both nares were occluded. In addition, the percentage of GnRH neurons colabeled with Fos-IR was examined in the MBH of gonadectomized, estradiol-treated female ferrets exposed to male bedding. Bilateral nares occlusion successfully blocked mating or odor-induced increments in Fos-IR in central olfactory regions, including the cortical and medial amygdala. By contrast, the percentage of GnRH neurons expressing Fos-IR did not differ between mated nares- and sham-occluded females. Exposure to male bedding alone failed to induce Fos-IR in MBH GnRH neurons. Thus, the mating-induced preovulatory activation of GnRH neurons in the female ferret's MBH appears to rely solely on genital-somatosensory as opposed to olfactory inputs.

Inducible mutagenesis in TEPC 2372, a mouse plasmacytoma cell line that harbors the transgenic shuttle vector lambdaLIZ.

The plasmacytoma cell line, TEPC 2372, was derived from a malignant plasma cell tumor that developed in the peritoneal cavity of a BALB/c mouse that harbored the transgenic shuttle vector for the assessment of mutagenesis in vivo, lambdaLIZ. TEPC 2372 was found to display the typical features of a BALB/c plasmacytoma. It consisted of pleomorphic plasma cells that secreted a monoclonal immunoglobulin (IgG2b/lambda), was initially dependent on the presence of IL-6 to grow in cell culture, contained a hyperdiploid chromosome complement with a tendency to undergo tetraploidization, and harbored a constitutively active c-myc gene by virtue of a T(6;15) chromosomal translocation. TEPC 2372 was further characterized by the ability to respond to in vitro exposure with 4-NQO (4-nitroquinoline-1-oxide), an oxidative model mutagen, with a vigorous dose-dependent increase in mutagenesis that peaked at a 7.85-fold elevation of mutant rates in lambdaLIZ when compared to background mutant rates in untreated controls. Cotreatment with 4-NQO and BSO (buthionine sulfoximine), a glutathione-depleting compound that causes endogenous oxidative stress, resulted in a 9.03-fold increase in the mutant frequency in lambdaLIZ. These results demonstrated that TEPC 2372, the malignant plasma cell counterpart of the lambdaLIZ-based in vivo mutagenesis assay, may be useful as an in vitro reference point for the further elucidation of oxidative mutagenesis in lymphoid tissues.

Intraoperative PAR corneal topography system (CTS): comparison of its keratometric readings to manual keratometer, auto-keratometer, EyeSys Corneal Analysis system, and slit lamp PAR CTS in healthy eyes.

PURPOSE: To compare the keratometric readings obtained from Intraoperative PAR Corneal Topography System (IOPAR) to those produced by manual keratometer (Mnl-Km), autokeratometer (Auto-Km), EyeSys CAS (EyeSys Corneal Analysis System) and slit lamp PAR CTS in healthy eyes. METHODS: All instruments were calibrated prior to use and only data from the best image obtained was used for statistical analysis. Simulated keratometry readings obtained from the central 3-mm zone of the corneas by IOPAR, including flat (K1) and steep (K2) keratometry readings, average keratometric power (AK), astigmatism (As) (difference between steep and flat keratometry readings) and the axis of the steep meridian (Ax) were compared to those from four other units. The latter units were also compared among themselves. Statistical analysis was done for right and left eyes separately. For each variable, average differences between the measurements taken from pairs of instruments were estimated, with corresponding 95% confidence intervals. The degree of agreement between pairs of instruments on individual measurements was additionally assessed, via the use of "Bland-Altman"-type plots, and estimates of the proportion of cases achieving satisfactory agreement. Additionally, for every variable, the average of the measurements taken from the different instruments were compared. RESULTS: Forty-five (22 right and 23 left) normal corneas of 26 volunteers were examined. On the average, IOPAR tended to measure K1 higher than slit lamp PAR CTS system. Because K2 measurements taken by the IOPAR were higher than that of all other instruments, the As measurements, on the average, were also higher than that of others, with the exception of the PAR CTS. For the same reasons, the IOPAR produced average AK readings that were higher than those taken by EyeSys CAS and PAR CTS. When the individual measurements taken by the IOPAR were compared with each of the other units, according to the arbitrary designation of satisfactory agreement within +/-0.5 D (for K1, K2, AK, and As) and +/-20 degrees (for Ax), for almost all parameters, proportion of differences that were within the agreement range varied from 0.33 to 0.82, with wide confidence intervals (confidence interval lower limits ranging from 0.20 to 0.61 and upper limits ranging from 0.62 to 0.94). CONCLUSIONS: IOPAR is a clinically useful topographic system, producing qualitative and quantitative data in the operating environment that, in normal corneas, on the average, matches those produced by the other units in the clinic. When individually analyzed, its keratometric measurements may show greater variations with respect to other units. Further studies with multiple examiners, in corneas with high or irregular astigmatism are required to establish its reproducibility and efficacy.

Steroidal modulation of scent investigation and marking behaviors in male and female ferrets (Mustela putorius furo).

Gonadectomized male and female ferrets (Mustela putorius furo) given either testosterone propionate (TP) or oil vehicle preferred to investigate the side of a test cage previously soiled by a breeding male or female as opposed to a clean side. Male and female ferrets receiving TP showed more urogenital wiping than oil-treated animals in either side of the test cage. In a 2nd experiment, ferrets treated sequentially with TP, oil, and estradiol benzoate (EB) were given simultaneous access to sides of a test cage previously soiled by either a breeding female or male. Either EB or TP treatment of females and TP treatment of males facilitated the investigation of odors of opposite-sex ferrets. Females given TP and males given either TP or EB showed increased urogenital wiping in both sides of the test cage. Sex steroids modulate scent investigation and marking in adult ferrets in a sexually differentiated fashion.

Elevated mutant frequencies in lymphoid tissues persist throughout plasmacytoma development in BALB/c.lambdaLIZ mice.

Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, the mean mutant frequencies in the target gene, lacI, were found to be significantly increased in lymphoid tissues of congenic BALB/c.lambdaLIZ N5 mice in the terminal stage of a plasmacytoma induction experiment, 213-280 days after the first i.p. injection of the plasmacytomagenic agent pristane (2,6,10,14-tetramethylpentadecane). In plasmacytoma-bearing mice (n = 7), mutant frequencies in the spleens and mesenteric lymph nodes were elevated 2.46-fold and 5.35-fold, respectively, when compared with age-matched controls. In plasmacytoma-negative mice (n = 11), mutant frequencies were increased 2.30-fold (spleens) and 3.48-fold (mesenteric nodes). These results, interpreted in conjunction with our previous findings (K. Felix et al., Cancer Res., 58: 1616-1619, 1998) of approximately 3-fold elevations in pristane-induced splenic mutagenesis on day 42 postpristane, indicate that increased mutant levels in lymphoid tissues persist throughout plasmacytomagenesis in genetically susceptible BALB/c mice.

Transgenic shuttle vector assays for assessing oxidative B-cell mutagenesis in vivo.

The recent development of transgenic mutagenicity assays provides new opportunities for evaluating mutagenic processes in vivo. To asses mutant frequencies in tissue B cells, we decided to take advantage of two such assays that utilize the transgenic shuttle vectors, lambda LIZ and pUR288. Our main interest in this research is to test two basic premises of inflammation-induced plasmacytoma development in genetically susceptible BALB/c mice; i.e., the possibility that plasmacytoma precursor cells may become targets of phagocyte-mediated oxidative mutagenesis in situ and the prospect that plasmacytoma susceptibility/resistance genes may contribute to these phenotypes by enhancing/reducing oxidative mutagenesis in B cells. Based on our preliminary experience with the lambda LIZ and pUR288 transgenic in vivo mutagenicity tests, we propose to employ these assays as broadly applicable tools for assessing overall mutagenesis during normal and aberrant (malignant) B-cell development. Furthermore, transgenic shuttle vector assays appear to lend themselves as ideal methods to associate general B-cell mutagenesis with the peculiar, B cell-typical somatic hypermutation processes that target the V(D)J gene segment, the proto-oncogene bcl-6 and perhaps other, still unknown loci.

Neuronal Fos activation in olfactory bulb and forebrain of male rats having erections in the presence of inaccessible estrous females.

Volatile odors from estrous female rats are necessary and sufficient to induce non-contact penile erections in male rats. It is not known whether these pheromones are detected by the accessory as opposed to the main olfactory system or whether they are processed by forebrain regions that receive olfactory inputs. Using nuclear Fos immunoreactivity as a marker of neuronal activation, we asked how the detection and processing of distal cues from inaccessible estrous females, which elicited non-contact penile erections, compared with the processing of sensory cues from soiled estrous bedding which did not elicit non-contact penile erections. In Experiment 1, groups of sexually experienced males were given one of five treatments. A control group was placed on clean bedding. A second group displayed non-contact penile erections when exposed to the smell, sight and sound of an estrous female restrained behind a permeable barrier. A third group was exposed to the same stimuli as the second (an estrous female) but failed to exhibit non-contact penile erections during the first hour of testing. A fourth group was placed on soiled estrous bedding, and a fifth group was allowed two ejaculations with an estrous female. All males were perfused with 4% paraformaldehyde 2 h after the onset of these respective treatments, and their brains were later processed for Fos immunoreactivity. Non-contact penile erections were observed in males that were exposed to distal cues from an estrous female but not in males exposed to soiled estrous bedding. Males that displayed non-contact penile erections or that were exposed to estrous bedding showed significantly more neuronal Fos immunoreactivity than clean-bedding controls in the nucleus accumbens core and shell, anterior and posterior medial amygdala, bed nucleus of the stria terminalis and the medial preoptic nucleus. Even greater neuronal Fos responses occurred in these regions in mated males. In Experiment 2 these same treatments were given to another cohort of sexually experienced males. Increased neuronal Fos immunoreactivity was observed in the granule and mitral cell layers of the accessory olfactory bulb of males that were either mated or exposed to estrous bedding, but not in males that displayed non-contact penile erections in response to distal cues from an estrous female. The volatile odors which presumably caused non-contact penile erections failed to stimulate significant neuronal Fos immunoreactivity in five main olfactory bulb sites examined. Even so, it seems likely that these pheromones are detected via the main olfactory system and are subsequently processed by the same projection circuit that responds to other pheromones present in estrous bedding that are incapable of eliciting non-contact penile erections.

Sex difference and testosterone modulation of pheromone-induced NeuronalFos in the Ferret's main olfactory bulb and hypothalamus.

A carnivore, the ferret possesses a vomeronasal organ--accessory olfactory bulb (VNO-AOB) projection to the hypothalamus; however, little is known about its function. Pheromones in soiled bedding from estrous female ferrets or an artificial peppermint odor significantly augmented nuclear Fos protein immunoreactivity (Fos-IR), a marker of neural activation, in several main olfactory bulb (MOB) sites but not in the AOB of gonadectomized male and females. Testosterone propionate (TP) significantly augmented the MOB's neuronal Fos responses to estrous females' pheromones, but not to peppermint. Estrous odors, but not peppermint, also augmented neuronal Fos-IR in the medial preoptic area (mPOA) of female, but not male, subjects. Pheromones in soiled bedding from breeding male ferrets significantly augmented neuronal Fos-IR in the MOB and in the medial amygdala of gonadectomized, TP-treated male and female subjects. Again, male pheromones failed to influence neuronal Fos-IR in the AOB of either sex, and only females showed significant increases in neuronal Fos-IR in the lateral aspect of the ventromedial nucleus and mPOA. These results point to an essential role among higher mammals of the main olfactory epithelium-MOB projection to the hypothalamus in detecting and processing pheromones. Gonadectomized ferrets showed significant increases in sniffing behavior when placed on either female or male bedding. This occurred regardless of whether they had received TP or oil vehicle, suggesting that testosterone's facilitation of neuronal Fos responses to estrous females' odors in the MOB of both sexes cannot be attributed to increased scent gathering. Androgen receptor-IR was present in the MOB granule cell layer of male and female ferrets, raising the possibility that testosterone acts directly on these cells to augment their responsiveness to pheromones.

Association of elevated mutagenesis in the spleen with genetic susceptibility to induced plasmacytoma development in mice.

Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, mean mutant rates were determined in the spleen of mice exposed to sustained oxidative stress and were found to be increased approximately 3-fold in plasmacytoma-susceptible BALB/c and C.D2-Idh1-Pep3 mice, but not in plasmacytoma-resistant DBA/2N mice. This finding suggests a correlation between the genetic susceptibility to inflammation-induced peritoneal plasmacytomagenesis and the phenotype of increased mutagenesis in lymphoid tissues, raising the possibility that plasmacytoma resistance genes may inhibit tumor development by minimizing oxidative mutagenesis in B cells.

Indications and results of combined anterior-posterior approaches for spine tumor surgery.

Spinal instrumentation currently allows gross-total resection and reconstruction in cases of malignancies at all levels of the spine. The authors analyzed the results in 110 patients who underwent surgery for primary and metastatic spinal tumors over a 5-year period (1989-1993) at a single institution. Major primary sites of tumor included breast (14 cases), chordoma (14 cases), lung (12 cases), kidney (11 cases), sarcoma (13 cases), plasmacytoma (10 cases), and others (36 cases). Prior to surgery, 55 patients (50%) had received prior treatment. Forty-eight patients (44%) were nonambulatory, and severe paraparesis was present in 20 patients. Fifty-three patients (48%) underwent combined anterior-posterior resection and instrumentation. 33 (30%) underwent anterior resection with instrumentation, 18 (16%) underwent anterior or posterior resection alone, and the remaining six patients (5%) underwent posterior resection and instrumentation. Major indications for anterior-posterior resection included three-column involvement, high-grade instability, involvement of contiguous vertebral bodies, and solitary metastases. Postoperatively, 90 patients improved neurologically. The overall median survival was 16 months, with 46% of patients surviving 2 years. Fifty-three patients (48%) suffered postoperative complications. Despite the high incidence of complications, the majority of patients reported improvement in their quality of life at follow-up review. Our findings suggest that half of all patients with spinal malignancies require combined anterior-posterior surgery for adequate tumor removal and stabilization.

Growth factor expression in normal peritoneum of patients with gynecologic carcinoma.

Both epidermal growth factor receptor (EGFR) and HER-2/neu (neu) have been found to be of prognostic importance in epithelial ovarian and endometrial carcinoma, but alterations in proto-oncogene expression of normal tissues of patients with gynecologic malignancies are unknown. Patients (118) undergoing laparotomy for gynecologic indications (78 ovarian cancer, 11 endometrial cancer, 19 benign gynecologic disease, 10 other cancers) had biopsies of normal peritoneum for quantitative assessment of neu and EGFR concentrations. Patients undergoing exploration for gynecologic malignancy were found to have significantly higher median neu expression in the peritoneal biopsies than patients with benign gynecologic disease (P = 0.002). Most patients in this study were found to have ovarian cancer, and median peritoneal neu expression was found to be significantly higher in patients with ovarian cancer versus benign ovarian masses (P = 0.0008) or any benign gynecologic disease (P = 0.004). No significant alteration of unbound EGFR was found in peritoneal biopsies of any of the groups of patients. No associations were found for a history of breast cancer, presence of ascites, or menopausal status with alteration of neu or EGFR expression in normal peritoneum. These findings of altered expression of neu in normal tissues of patients with ovarian cancer are suggestive of the presence of proto-oncogene alterations in loco-regional tissues of the peritoneum, such as might be seen if a paracrine influence existed between tumor and peritoneal cells. Alternatively, the alterations may represent subtle alterations of proto-oncogene expression of germ-line tissues.

Mitochondrial genetics of mammalian cells: a mouse antimycin-resistant mutant with a probable alteration of cytochrome b.

Mouse LA9 antimycin-resistant mutants (ANT-R) were isolated and characterized. Genetic analyses established that this phenotype is encoded within the mtDNA: (1) the ANT-R phenotype showed frequent mitotic segregation and reassortment in hybrid clonal lines; (2) it was transmitted directly in cybrid crosses; and (3) it was cotransmitted in cybrid crosses with the mitochondrial CAP-R marker. Furthermore, the genetic studies suggested that the LA9 CAP-R ANT-R cells were heteroplasmic and contained at least two mtDNA genotypes, cap-r ant-s and cap-s ant-r. Cellular respiration of the ANT-R mutant was markedly more resistant to inhibition by antimycin than that of the parental ANT-S cells. The increased resistance of cellular respiration was entirely accounted for by an increase in the resistance of mitochondrial succinate-cytochrome c oxidoreductase to antimycin inhibition. There was no detectable change in the specific activity of the oxidoreductase in mitochondria of resistant ANT-R cells nor in the sensitivity of the complex to three other specific inhibitors of the complex: TTFA, myxothiazol, and HQNO. Taken together, these studies indicate that the ANT-R phenotype is most likely encoded within the mitochondrial cytochrome b gene and, more specifically, within an antimycin binding domain.