Multiple circadian oscillators regulate the timing of behavioral and endocrine rhythms in female golden hamsters.

A single daily "surge" in pituitary luteinizing hormone release was observed in ovariectomized-estrogen-treated hamsters expressing an intact circadian rhythm of locomotor activity. In contrast, two luteinizing hormone surges occurred within a single 24-hour period in hamsters whose activity rhythm had dissociated or "split" into two distinct components. These observations indicate that both behavioral and endocrine circadian rhythms are regulated by the same multioscillator system, which seems to be composed of at least two distinct circadian oscillators.

Stereological sex difference during development of the magnocelluar subdivision of the medial preoptic nucleus (MPN mag).

In Syrian hamsters, reproductive behaviors are initiated in the presence of appropriate hormonal and chemosensory cues. These cues are detected and integrated within a highly conserved pathway that converges on a small nuclear group in the lateral aspect of the medial preoptic area, the magnocellular subdivision of the medial preoptic nucleus (MPN mag). The MPN mag plays a critical role in the regulation of male mating behavior--bilateral ablation of the MPN mag eliminates copulation. The MPN mag is sexually differentiated in both neuron number and density, but not in overall volume or volume of individual neurons. The current study used unbiased stereological methods to determine when the MPN mag becomes sexually differentiated. Our data indicate that the MPN mag becomes sexually dimorphic in volume and cell number after the critical period when steroid treatment induces male sexual behavior.

The magnocellular medial preoptic nucleus I. Sources of afferent input.

The magnocellular medial preoptic nucleus plays a crucial role in the regulation of male sexual behavior in Syrian hamsters. Histological and behavioral studies suggest that the magnocellular medial preoptic nucleus regulates male mating behavior by integrating chemosensory and hormonal signals. The present study is the first to systematically identify the afferent connections of the magnocellular medial preoptic nucleus by tracing the uptake of cholera toxin B from deposits in the magnocellular medial preoptic nucleus of adult male Syrian hamsters. Our findings indicate that the magnocellular medial preoptic nucleus receives 1) chemosensory input from areas in the main and accessory olfactory pathways including the posterior medial bed nucleus of the stria terminalis, anterior medial, anterior cortical and posterior cortical nuclei of the amygdala; 2) input from steroid responsive structures such as the posterior medial nucleus of the amygdala, bed nucleus of the stria terminalis, lateral septum, anteroventral periventricular nucleus, medial preoptic nucleus, ventromedial nucleus of the hypothalamus and arcuate nucleus; 3) input from structures in the brainstem such as the subparafascicular thalamic nucleus, peripeduncular nucleus and the premamillary nucleus in the hypothalamus that carry sensory information from the genitalia. The major afferent input to the magnocellular medial preoptic nucleus was confirmed by injecting anterograde tracer biotinylated dextran amine into the anterior medical nucleus of the amygdala, the posterodorsal part of the medial nucleus of the amygdala, the posteromedial part of the bed nucleus of the stria terminalis and the posterointermediate part of the bed nucleus of the stria terminalis. Our results support the hypothesis that the magnocellular medial preoptic nucleus is part of the chemosensory pathway that receives chemosensory and hormonal input to regulate mating behavior and suggest that the magnocellular medial preoptic nucleus may utilize information from the genitalia to regulate male mating behavior.

The bed nucleus of the stria terminalis in the Syrian hamster: subnuclei and connections of the posterior division.

The bed nucleus of the stria terminalis is a key part of a ring of cells extending between the centromedial amygdala and bed nucleus of the stria terminalis referred to as the extended amygdala. The present study describes the architecture of the bed nucleus of the stria terminalis and the connections of subnuclei in posterior bed nucleus of the stria terminalis. The hamster bed nucleus of the stria terminalis is readily allotted to anterior and posterior divisions separated by the fibers of the body of the anterior commissure. The anterior division has four subnuclei: anteromedial, anterointermediate, anterolateral, and anteroventral. Within the posterior division, there are three distinct regions: posteromedial, posterointermediate, and posterolateral. In hamsters, the posterior bed nucleus of the stria terminalis contributes to male sexual behavior, particularly chemoinvestigation. Moreover, the posterior bed nucleus of the stria terminalis is part of a neural circuit essential for mating, including the medial amygdaloid nucleus and medial preoptic area. The connections of bed nucleus of the stria terminalis, posteromedial part, bed nucleus of the stria terminalis, posterointermediate part and bed nucleus of the stria terminalis, posterolateral part were visualized by co-injection of anterograde (Phaseolus vulgaris leucoagglutinin) and retrograde (cholera toxin B) tract tracers. The bed nucleus of the stria terminalis, posterointermediate part and bed nucleus of the stria terminalis, posteromedial part have dense bidirectional connections with medial amygdaloid nucleus and cortical amygdala via the stria terminalis and ventral amygdalofugal pathway. These subnuclei also maintain bidirectional connections with steroid-concentrating areas including lateral septum, medial preoptic area, hypothalamus, and periaqueductal gray. The bed nucleus of the stria terminalis, posterointermediate part and bed nucleus of the stria terminalis, posteromedial part receive projections from the subiculum and send projections to deep mesencephalic nuclei. By contrast, the bed nucleus of the stria terminalis, posterolateral part is connected with the central amygdala, lateral hypothalamus, subthalamic nucleus, nucleus accumbens, substantia innominata, substantia nigra and thalamus. Thus, the bed nucleus of the stria terminalis, posterointermediate part and bed nucleus of the stria terminalis, posteromedial part have similar connections with areas involved in social behaviors. The bed nucleus of the stria terminalis, posterolateral part maintains connections with areas involved in motivational circuits. This supports the concept of distinct circuits within the extended amygdala which differentially link the centromedial amygdala and bed nucleus of the stria terminalis.

Sex differences in the magnocellular subdivision of the medial preoptic nucleus in Syrian hamsters.

Exposure to pheromonal cues initiates male mating behavior. Pheromones are processed within a pathway that converges on the magnocellular subdivision of the medial preoptic nucleus. Lesions of this area eliminate male copulatory behavior, but do not affect anogenital investigation. Exposure to pheromones stimulates cells of the magnocellular subdivision of the medial preoptic nucleus in a sex-specific manner. In this study, we hypothesize that sex differences in cell number may underlie sex differences in pheromone-induced neural stimulation. The current study used unbiased stereological methods to identify sexual dimorphisms in the magnocellular subdivision of the medial preoptic nucleus. Sex differences were found in the number and density of neurons, but not in overall volume or neuron volume. Consequently, the total volume is not sexually differentiated because neurons are more densely packed within the male magnocellular subdivision of the medial preoptic nucleus. These results support the hypothesis that additional neurons in the magnocellular subdivision of the medial preoptic nucleus are critical for the expression of male copulatory behaviors in adulthood. Furthermore, they suggest that sexual differentiation of the magnocellular subdivision of the medial preoptic nucleus is an important process that forms the anatomical basis for sex-specific behavioral responses to pheromonal stimulation.

Forebrain expression of c-fos due to active maternal behaviour in lactating rats.

To reveal brain sites simultaneously active during the expression of maternal behaviour in lactating rats, we used immunocytochemical visualization of the nuclear protein product Fos of the immediate-early gene c-fos as a marker of neuronal activity. After a 48 h separation from their litter, day 7 postpartum dams received a 1 h period of physical interaction with pups either capable or incapable of suckling, inaccessible pups in a wire-mesh box, an empty box, or no stimulation. Physical interaction with pups elicited high levels of pronurturant maternal behaviour (retrieval, licking, mouthing), and suckling elicited nursing behaviour as well. Exposure to the box, with or without pups, elicited high levels of investigatory sniffing, self-grooming, and general activity. Distal stimulation from pups did not differentially activate Fos in any of 20 sites, including olfactory-processing structures such as the piriform cortex and medial amygdala. Physical interaction with pups, with or without suckling, elicited higher levels of Fos-immunoreactive nuclei than that of other conditions in numerous sites, including many previously implicated in maternal behaviour (medial preoptic nucleus, nucleus accumbens, lateral septum, lateral habenula, and the bed nucleus of the stria terminalis). Similar group patterns of Fos expression also occurred in sites not previously implicated in maternal behaviour (somatosensory cortex and paraventricular thalamic nucleus). Interaction with nonsuckling pups elicited the highest levels of Fos in the cortical amygdala, whereas suckling did not activate higher Fos than nonsuckling interaction in any site included in this report, including hypothalamic nuclei involved in lactation (paraventricular, supraoptic, and arcuate). There was little or no Fos in cingulate cortex, olfactory tubercle, medial septum, medial habenula, or ventromedial hypothalamus. These data suggest that trigeminal stimuli received by lactating rats during the performance of pronurturant maternal behaviour promote cellular activity resulting in neuronal expression of c-fos in many forebrain sites including the medial preoptic nucleus, several sites connected with it that are part of the mesotelencephalic dopamine system, and in the somatosensory cortex. In contrast, in these forebrain sites suckling does not elicit greater levels of Fos than that seen in nonsuckled rats and distal stimuli from pups are ineffective in increasing Fos levels compared with non-stimulated controls.

Pheromones induce c-fos in limbic areas regulating male hamster mating behavior.

Hamsters rely on chemosensory cues from females of the same species for the initiation of copulatory behavior. While these cues are detected by both the main and accessory olfactory systems it is the central nuclei in the accessory system that regulate mating behavior. The results of the present study indicate that exposure to vaginal secretions from a female Syrian hamster (FHVS) stimulates c-fos production in the medial nucleus of the amygdala (Me), the bed nucleus of the stria terminalis (BNST) and the medial preoptic area (MPOA). Exposure to vaginal secretions from Djungarian hamsters do not stimulate neurons in these areas. Thus the present results suggest that FHVS stimulates mating behavior by activating neurons within the vomeronasal pathway.

Neuropeptide Y in hamster limbic nuclei: lack of colocalization with substance P.

The medial nucleus of the amygdala (Me), bed nucleus of the stria terminalis (BNST), and medial preoptic area (MPOA) regulate copulation in the male hamster. The present study identified neuropeptide Y-immunoreactive (NPY-IR) neurons in the BNST and Me with the greatest concentration in the posteromedial and posteriordorsal subdivisions of these nuclei, respectively. NPY-IR filters are found in all three nuclei with dense plexi of NPY-IR varicosities in the most medial subdivisions. Substance P neurons are also densely concentrated in the posterior BNST and Me; however, no neurons contained both peptides. Thus, NPY and substance P neurons comprise two distinct populations within the BNST and Me of the hamster.

Gonadal steroids regulate behavioral responses to pheromones by actions on a subdivision of the medial preoptic nucleus.

Mating behavior in male hamsters is initiated by pheromones, detected by two chemosensory systems which converge on the medial nucleus of the amygdala and the bed nucleus of the stria terminalis. Neurons in these areas project to the medial preoptic nucleus. All three of these areas contain androgen receptors. Using Fos as a marker of stimulation we have found that pheromones stimulate neurons in all three areas in intact males but fail to stimulate the magnocellular division of the medial preoptic nucleus of castrates. As this area plays a critical role in the regulation of male mating behavior our results suggest that steroids regulate mating by maintaining the responsiveness of the magnocellular division of the medial preoptic nucleus to pheromones.

Testosterone regulates substance P within neurons of the medial nucleus of the amygdala, the bed nucleus of the stria terminalis and the medial preoptic area of the male golden hamster.

The medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area appear to mediate steroidal regulation of mating behavior in male rodents. The mechanism of action has not been determined. One way testosterone could enhance neuronal function is by increasing neurotransmitter levels, thus altering neuronal transmission. To assess this hypothesis, we examined the effect of castration and testosterone treatment on substance P levels in the neurons of these three brain regions. Brains from male Syrian hamsters that were (1) gonadally intact, (2) castrated for 13 weeks, or (3) castrated for 9 weeks and treated with testosterone for 4 weeks, were processed for substance P, and the numbers of substance P immunoreactive neurons in the medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area were determined. Castration reduced the number of substance P neurons in the bed nucleus of the stria terminalis and medial preoptic area relative to those in intact hamsters; the number of substance P neurons in these regions was restored by testosterone treatment. Castration did not reduce the number of substance P neurons in the medial nucleus of the amygdala; however, testosterone treatment increased the numbers of these neurons when compared to intacts. Thus, testosterone regulates substance P levels in areas that regulate mating behavior. As substance P enhances male copulatory behavior our results suggest that testosterone may regulate copulatory behavior by enhancing substance P levels in medial nucleus of the amygdala, bed nucleus of the stria terminalis and medial preoptic area.

Photoperiodic regulation of substance P immunoreactivity in the mating behavior pathway of the male golden hamster.

Mating behavior in the male golden hamster is regulated by both gonadal steroids and photoperiod. Gonadal steroids may regulate mating behavior by actions on the medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area. Neurons in these areas actively accumulate gonadal steroids and lesions of these nuclei disrupt mating behavior in male hamsters. Photoperiodic regulation of mating behavior is regulated, at least in part, by decreased responsiveness to gonadal steroids. Therefore, we sought to determine if the changes induced by changes in gonadal steroids would mimic those induced by changes in photoperiod. The number of substance P-containing neurons in these areas decrease following castration and are restored with testosterone treatment suggesting that this peptide may mediate steroidal regulation of male mating behavior. To determine the effect of photoperiod on substance P, peptide containing neurons were counted in (1) enucleates (n = 6), (2) enucleated castrates treated with testosterone (n = 6), (3) castrates treated with testosterone (n = 4), and (4) intact controls (n = 6). Bilateral enucleation caused a decrease in the number of substance P neurons in the medial nucleus, bed nucleus of the stria terminalis, and medial preoptic area (P less than 0.05). Testosterone treatment prevented this decrease (P less than 0.05). Thus, a decrease in daylength causes a decrease in substance P in the medial nucleus of the amygdala, the medial bed nucleus of the stria terminalis and the medial preoptic area that is mediated by changes in testosterone levels.

The colocalization of substance P and prodynorphin immunoreactivity in neurons of the medial preoptic area, bed nucleus of the stria terminalis and medial nucleus of the amygdala of the Syrian hamster.

To determine the extent of colocalization of substance P (SP) and prodynorphin peptides within neurons of the medial nucleus of the amygdala (AMe), medial bed nucleus of the stria terminalis (BNSTm) and medial preoptic area (MPOA), we incubated colchicine-treated Syrian hamster brain tissue in an antiserum mixture containing rat anti-SP antibody combined with 1 of 3 rabbit antibodies against prodynorphin peptides: anti-dynorphin A(1-17), anti-dynorphin B(1-13) or anti-C-peptide. This was followed by incubation in a secondary antiserum mixture containing fluorescein-labelled anti-rabbit and rhodamine-labelled anti-rat antibodies. Sections were viewed with an epifluorescence microscope using blue light excitation for fluorescein and green light excitation for rhodamine. Colocalization of SP and prodynorphin labelling was observed in neurons of the caudal parts of AMe, BNSTm and MPOA, areas which are essential for male mating behavior. The colocalization was most extensive in the dorsolateral part of the caudal MPOA, the caudodorsal part of the BNSTm, and in the posterodorsal subdivision of AMe. Although all 3 dynorphin peptides coexisted with SP in these areas, dynorphin B did so less than C-peptide, and dynorphin A less than dynorphin B.

Glucoprivic treatments that induce anestrus, but do not affect food intake, increase FOS-like immunoreactivity in the area postrema and nucleus of the solitary tract in Syrian hamsters.

Animals make a wide variety of physiological and behavioral adjustments in order to maintain caloric homeostasis. For example, most animals increase food intake when the availability of cellular metabolic fuels is low. The area postrema (AP) and adjacent, reciprocally-innervated nucleus of the solitary tract (NTS) are important brain areas for metabolic control of food intake in rats. However, in Syrian hamsters, food intake is not affected by decreases in metabolic fuel availability such as those that occur with food deprivation or with pharmacological inhibitors of metabolic fuels. Hamsters make other adjustments that conserve energy when the availability of metabolic fuels is low. Estrous cycles are inhibited by treatment with a high dose of 2-deoxy-D-glucose (2DG), a drug that inhibits cellular glucose utilization, but not by treatment with methyl palmoxirate (MP) a drug that inhibits fatty acid utilization. Recent data suggest that the AP/NTS is critical for the effects of glucoprivation on estrous cycles. Lesions of the AP/NTS prevent 2DG-induced anestrus. If the AP/NTS is involved in anestrus induced by glucoprivation, it might be predicted that glucoprivic treatments that induce anestrus would change patterns of neural activation, as measured by FOS-like immunoreactivity (FOS-li), in the AP/NTS. We examined FOS-li in females that were either food deprived or fed ad libitum, and in females treated with 2DG, MP or the appropriate vehicle. FOS-li was increased in the AP/NTS only in hamsters food deprived or treated with 2DG, the two treatments that induce anestrus but have no effect on food intake. These results are consistent with the notion that metabolic control of estrous cycles involves detection of decreases in the availability of metabolic fuels in the AP/NTS.

Androgen and estrogen concentrating neurons in chemosensory pathways of the male Syrian hamster brain.

The medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), and medial amygdaloid nucleus (Me) are essential for male sexual behavior in the Syrian hamster. These nuclei received chemosensory stimuli and gonadal steroid signals, both of which are required for mating behavior. The objective of this study was to compare the distribution of androgen- and estrogen-concentrating neurons in MPOA, BNST, and Me in the adult male hamster using steroid autoradiography for estradiol (E2), testosterone (T) and dihydrotestosterone (DHT). Adult males (n = 4 per group) received two i.p. injections of tritiated steroid 4-7 days after castration. Six-microns frozen sections through the brain were mounted onto emulsion-coated slides, and exposed for 11-16 months. In MPOA, BNST, and Me, neurons were more abundant and heavily labelled after [3H]E2 treatment than after either [3H]T or [3H]DHT. Tritiated estradiol- and DHT-labeled cells were found throughout the rostrocaudal extent of Me, with a high concentration in posterodorsal Me. Tritiated testosterone treatment labelled cells largely within posterodorsal Me. In MPOA, the majority of E2-, T-, and DHT-labelled neurons were in the medial preoptic nucleus (MPN) and the preoptic continuation of the posteromedial bed nucleus of the stria terminalis (BNSTpm). Few T-labelled cells were present outside these subdivisions. In the BNST, E2- and DHT-labelled neurons were present in all subdivisions, whereas T labelling was confined to the antero- and posteromedial subdivisions of BNST. These results suggest that the distribution of androgen- and estrogen receptor-containing neurons overlap considerably in nuclei which transmit chemosensory signals in the control of mating behavior.

Expression of c-fos protein in lumbosacral spinal cord in response to vaginocervical stimulation in rats.

The pattern of vaginocervical stimulation-evoked expression of the proto-oncogene c-fos in lumbar 5-sacral 1 segments of the spinal cord of ovariectomized adult rats was mapped using immunocytochemistry. A calibrated force of mechanostimulation was applied to the vaginal cervix of experimental animals and to the perineum of control animals while they were gently restrained. The number of cells expressing c-fos was significantly greater in the experimental than the control animals in laminae I, IV, V-VI and X. The implications of the present findings for elucidating the spinal pathways mediating the various behavioral, neuroendocrine and autonomic effects of vaginocervical stimulation (VS) are discussed.

Connections of the magnocellular medial preoptic nucleus (MPN mag) in male Syrian hamsters. II. The efferents.

The magnocellular medial preoptic nucleus (MPN mag) plays a critical role in the regulation of male copulatory behavior in the Syrian hamster. Our study of the afferents are consistent with the hypothesis that the MPN mag receives input from areas in the chemosensory pathway and nuclear groups that contain receptors for gonadal steroids (Wang and Swann, 2006). The goal of the present study is to identify targets of the MPN mag by describing the location of labeled fibers following an injection of biotinylated dextran amine (BDA) into the MPN mag. Our results indicate that targets of the MPN mag include: (1) brainstem nuclei implicated in regulating male mating behavior in other species, such as the periaqueductal gray, deep mesencephalic nucleus, retrorubral field, ventral tegmental area and lateral paragigantocellular nucleus and (2) steroid-concentrating nuclei in the septum, preoptic area and hypothalamus. The lack of projections from the MPN mag to its chemosensory afferents indicate that the connections of the MPN mag with the posterior medial bed nucleus of the stria terminalis, medial and anterior cortical nuclei of the amygdala are unidirectional, and that chemosensory information flows from the medial amygdala and bed nucleus of the stria terminalis (BST) to the MPN mag. The bidirectional nature of the connections between the MPN mag and steroid-concentrating nuclei suggest that the MPN mag may influence the function of a steroid-concentrating network that regulates behaviors. Together these results support the hypothesis that the MPN mag regulates male mating behavior by integrating chemosensory and hormonal signals and relaying this information to brainstem areas that control motor output.

Testosterone differentially influences sex-specific pheromone-stimulated Fos expression in limbic regions of Syrian hamsters.

The results of the present study indicate that (1) pheromones differentially stimulate neurons in males and females within a pathway that regulates copulatory behavior; and (2) testosterone (T) differentially regulates these sex differences. Exposure to the pheromones in FHVS (female hamster vaginal secretions) induces Fos immunoreactivity (Fos-IR) in the posterior subdivision of the medial nucleus of the amygdala (MeP) and the posteromedial subdivision of the bed nucleus of the stria terminalis (BNSTpm) of both sexes and stimulates the magnocellular subdivision of the medial preoptic nucleus (MPNmag) in males but not in females. Males also show more Fos in the MeP and BNSTpm than females. In the absence of T, gonadectomized males show greater FHVS-stimulated Fos-IR in the BNSTpm and MeP than gonadectomized females. T in females eliminates the sex difference in these regions. Only T-treated males show FHVS-stimulated Fos-IR within the MPNmag, and T has no effect on FHVS-stimulated Fos-IR within MPNmag in females. Thus, T influences FHVS-stimulated Fos-IR in the BNSTpm and MeP of females and the MPNmag of males. T also increases investigation (sniffing and licking) of FHVS in both males and females, but increases copulatory responses only in males. Our results indicate that T in the adult hamster differentially influences neural and behavioral responses to pheromone exposure in males and females. T only partially accounts for observed sex differences, and it is likely that neural organization during development also plays a role in influencing responses to pheromones.

Transfer from long to short days reduces the frequency of pulsatile luteinizing hormone release in intact but not in castrated male golden hamsters.

In the golden hamster, prolonged exposure to short days (i.e. less than 12.5 h of light/day) induces testicular regression that has usually, but not always, been reported to be accompanied by a decrease in mean serum luteinizing hormone (LH) levels. As LH appears to be released in a pulsatile pattern in most mammalian species, we hypothesized that individual variations in serum LH levels over time may obscure significant changes in pituitary LH release in hamsters exposed to short photoperiods. Therefore, we examined the effects of short day exposure on pulsatile LH release in castrated and intact golden hamsters. Intact and castrated male golden hamsters were maintained on long days (light:dark 14:10 h) or transferred to short days (light:dark 6:18 h). Nine to eleven weeks after transfer, animals from all four groups were fitted with intra-atrial cannulas and bled at 10-min intervals for 6 h. Exposure to short days inhibited LH release in the intact male hamsters. All 6 of the males maintained on long days had pulsatile LH release while only 1 of the 4 intact males maintained on short days showed a pulse of LH during the 6-hour bleeding session. All of the castrates on short days had pulsatile patterns of LH release that were similar in amplitude and frequency to those of the castrates on long days. Castrates on long days had significantly greater LH pulse frequency and amplitude than intacts on long days. From these data we conclude that the pulsatile nature of pituitary LH release in the golden hamster mandates frequent sampling over several hours for accurate determination of serum LH levels and for determining the effects of photoperiod on pituitary gonadotropin release.

The effects of the organophosphorous insecticides Dursban and Lorsban on the ciliated epithelium of the frog palate in vitro.

The ciliotoxic potential of the organophosphorous insecticides Dursban and Lorsban, their active ingredient, chlorpyrifos, and their carrier ingredients (Blanks) were assessed. Since chlorpyrifos inhibits acetylcholinesterase, the acetylcholine-innervated ciliated epithelial cultures of frog palate were used as the model. All compounds caused a decrease in frequency of ciliary beat overtime. EC50 values followed the same order as the time to inhibition. The orders were Lorsban > Dursban > chlorpyrifos, and Lorsban > Dursban approximately Lorsban Blank > Dursban Blank. Stimulation of ciliary beating occurred immediately after exposure to all compounds, followed by inhibition. Dursban, Lorsban, and both Blanks elicited stimulatory effects in the presence of atropine. Atropine only blocked the initial stimulatory response with chlorpyrifos. In addition to chlorpyrifos, some component(s) of the inert ingredients were initially stimulatory but ultimately inhibitory to ciliary beating in the frog palate model. All compounds caused mitochondrial damage, including swelling, disruption of cristae, and loss of matrix.

The effects of benomyl and its breakdown products carbendazim and butyl isocyanate on the structure and function of tracheal ciliated cells.

The effects of the fungicide benomyl and its breakdown products, carbendazim and butyl isocyanate, were examined on canine tracheal epithelial tissue in primary culture. Changes in ciliary frequencies were monitored with an optical spectrum analysis system. Serial dilutions of the test compounds were prepared in 100% corn oil and applied to the cell cultures for intervals up to 6 hours and frequencies measured at intervals of 15 minutes to 1 hour. Benomyl and butyl isocyanate caused concentration-dependent decreases in ciliary beat frequency. Benomyl at 300 micrograms/ml (3 mM) caused ciliostasis within 75 minutes of exposure. Butyl isocyanate at a molar concentration three times lower than benomyl (1 mM) caused a similar response, although within 30 minutes. The IBC50 for benomyl was 0.75 mM, while for butyl isocyanate it was 0.52 mM. Carbendazim caused a moderate decrease in frequency over a 6 hour exposure period. Benomyl caused moderate to severe swelling of the mitochondria of ciliated epithelial cells with other cell organelles appearing normal. Butyl isocyanate did not cause any noticeable effect on cell ultrastructure and the apparently low rate of penetration of carbendazim into cells made it impossible to obtain an effect which justified ultrastructural analysis. It appears, at least for benomyl and butyl isocyanate, that while the physiological effect of these two compounds (inhibition of ciliary beat) is the same, the sites of action in the cell may be different.