Dietary supplementation with a specific combination of high protein, leucine, and fish oil improves muscle function and daily activity in tumour-bearing cachectic mice.

Cancer cachexia is characterised by metabolic alterations leading to loss of adipose tissue and lean body mass and directly compromises physical performance and the quality of life of cancer patients. In a murine cancer cachectic model, the effects of dietary supplementation with a specific combination of high protein, leucine and fish oil on weight loss, muscle function and physical activity were investigated. Male CD2F1 mice, 6-7 weeks old, were divided into body weight-matched groups: (1) control, (2) tumour-bearing, and (3) tumour-bearing receiving experimental diets. Tumours were induced by s.c. inoculation with murine colon adenocarcinoma (C26) cells. Food intake, body mass, tumour size and 24 h-activity were monitored. Then, 20 days after tumour/vehicle inoculation, the animals were killed and muscle function was tested ex vivo. Tumour-bearing mice showed reduced carcass, muscle and fat mass compared with controls. EDL muscle performance and total daily activity were impaired in the tumour-bearing mice. Addition of single nutrients resulted in no or modest effects. However, supplementation of the diet with the all-in combination of high protein, leucine and fish oil significantly reduced loss of carcass, muscle and fat mass (loss in mass 45, 52 and 65% of TB-con, respectively (P<0.02)) and improved muscle performance (loss of max force reduced to 55-64% of TB-con (P<0.05)). Moreover, total daily activity normalised after intervention with the specific nutritional combination (50% of the reduction in activity of TB-con (P<0.05)). In conclusion, a nutritional combination of high protein, leucine and fish oil reduced cachectic symptoms and improved functional performance in cancer cachectic mice. Comparison of the nutritional combination with its individual modules revealed additive effects of the single components provided.

Direct effects of doxorubicin on skeletal muscle contribute to fatigue.

Chemotherapy-induced fatigue is a multidimensional symptom. Oxidative stress has been proposed as a working mechanism for anthracycline-induced cardiotoxicity. In this study, doxorubicin (DOX) was tested on skeletal muscle function. Doxorubicin induced impaired ex vivo skeletal muscle relaxation followed in time by contraction impediment, which could be explained by DOX-induced changes in Ca(2+) responses of myotubes in vitro. The Ca(2+) responses in skeletal muscle, however, could not be explained by oxidative stress.

The stimulatory effect of vasopressin on the luteinizing hormone surge in ovariectomized, estradiol-treated rats is time-dependent.

The luteinizing hormone surge in the female rat is not only induced by the positive feedback of estradiol, but also by circadian signals originating in the suprachiasmatic nucleus (SCN). In a previous study we showed that administration of vasopressin, an SCN transmitter present in preoptic projections, induced an LH surge in animals bearing complete lesions of the SCN. This strongly suggests vasopressin as a stimulatory circadian signal for the timing of the LH surge. In the present study we investigated during which time window vasopressin may act in the medial preoptic area to stimulate LH secretion in SCN-intact female rats. Vasopressin or a specific V1a receptor antagonist was administered into the MPO by a reverse microdialysis technique during different time windows, and plasma LH concentrations were measured. Vasopressin stimulated the LH surge in 30% of the animals, when administered during the second half of the light period, but during the first half of the light period no effects were observed. Administration of the V1a receptor antagonist, however, did not affect the LH surge. These data confirm our previous results that vasopressin is a stimulatory factor for the LH surge also in SCN-intact animals, and indicate that a certain time window is available for such stimulation. We hypothesize that vasopressin in the SCN-intact animal may act as a circadian signal during a specific time window to induce the LH surge. The time window is the result of other SCN regulatory systems that are involved in the preparation of the LH surge.

Control of the estradiol-induced prolactin surge by the suprachiasmatic nucleus.

In the present study we investigated how the suprachiasmatic nucleus (SCN) controls the E(2)-induced PRL surge in female rats. First, the role of vasopressin (VP), a SCN transmitter present in medial preoptic area (MPO) projections and rhythmically released by SCN neurons, as a circadian signal for the E(2)-induced PRL surge was investigated. Using a reverse microdialysis technique, VP was administered in the MPO during the PRL surge, resulting in a suppression of the surge. VP administration before the surge did not affect PRL secretion. Also, administration of a V1a receptor antagonist before the surge was ineffective. Second, lesions of the SCN were made that resulted in constant basal PRL levels, suggesting that with removal of the SCN a stimulatory factor for PRL secretion disappeared. Indeed, the PRL secretory response to blockade of pituitary dopamine receptors was significantly reduced in SCN-lesioned animals. These data suggest that the afternoon decrease of VP release in the MPO by SCN terminals enables the PRL surge to occur, and may thus be a circadian signal for the PRL surge. Simultaneously the SCN is involved in the regulation of the secretory capacity of the pituitary, possibly via specific PRL-releasing factors.

Changes in estrogen-alpha receptor immunoreactivity during the estrous cycle in lactating dairy cattle.

Estradiol is one of the most important hormones in the regulation of estrous behavior, which is at a very low level of expression in the modern dairy cow. In the present study the neuroanatomical distribution of estrogen receptors of the alpha-subtype (ER-alpha) in the bovine hypothalamic area is determined with immunocytochemical methods, at various stages of the estrous cycle. During the luteal phase of the cycle, ER-alpha immunoreactive cells were found in most of the nuclei that are known to express ER-alpha immunoreactivity in other species, like the Bed nucleus of the Stria terminalis, Medial preoptic area, Ventromedial hypothalamus and Arcuate nucleus. During estrus and metestrus, however, no ER-alpha immunoreactive cells could be detected in those areas, except for a few in the caudal Arcuate nucleus. The results from the present study indicate that there is a coherent regulation and timing of physiological and behavioral events around ovulation, in which estradiol and its receptor play a key role.

Vasopressin induces a luteinizing hormone surge in ovariectomized, estradiol-treated rats with lesions of the suprachiasmatic nucleus.

The luteinizing hormone surge in the female rat is the result of the integration of multiple signals within the medial preoptic area. The medial preoptic area contains gonadotropin-releasing hormone neurons that are responsible for the release of luteinizing hormone, neurons containing estrogen receptors and terminals originating from the suprachiasmatic nucleus with, for example, vasopressin as neurotransmitter. Both the medial preoptic area and suprachiasmatic nucleus are crucial for the occurrence of luteinizing hormone surges, since lesioning of either nucleus prevents pre-ovulatory and steroid-induced luteinizing hormone surges. In this study, we investigated whether vasopressin in the medial preoptic area could be the daily neuronal signal from the suprachiasmatic nucleus responsible for the timing of the luteinizing hormone surge. Vasopressin (50 ng/microl) or Ringer solution was administered by reverse microdialysis from Zeitgeber times 7.5 to 12.5 into the medial preoptic area of ovariectomized, estradiol-treated rats. The suprachiasmatic nucleus was lesioned to remove all cyclic luteinizing hormone secretion. This was evaluated by monitoring behavioral activity; animals that were arrhythmic were included in the experiments. Hourly blood samples were taken to measure plasma luteinizing hormone levels. Preoptic vasopressin administration induced a surge-like luteinizing hormone pattern in suprachiasmatic nucleus-lesioned animals, whereas constant, basal luteinizing hormone levels were found in the control animals. These data show that vasopressin, by itself, is able to trigger the luteinizing hormone surge in suprachiasmatic nucleus-lesioned rats. We propose that vasopressin is a timing signal from the suprachiasmatic nucleus responsible for the activation of the hypothalamo-pituitary-gonadal axis in the female rat.

GABAergic regulation of light-induced c-Fos immunoreactivity within the suprachiasmatic nucleus.

Analysis of the photic induction of c-Fos immunoreactivity (-ir) within the suprachiasmatic nucleus (SCN) has proven to be a powerful tool with which to study the neurochemical mechanisms involved in phase shifting the circadian clock. Some systemically administered GABAergic drugs inhibit light-induced phase shifts and c-Fos-ir, whereas others inhibit light-induced phase shifts without affecting c-Fos-ir. More recently, we have found that injection of GABAergic drugs directly into the SCN region can have dramatically different effects on light-induced phase shifts than following their systemic administration. The present study investigated the effects of GABA(A) and GABA(B) agonists and antagonists injected into the SCN region on c-Fos-ir within the SCN. Microinjection of either a GABA(A) agonist, muscimol, or a GABA(B) agonist, baclofen, into the SCN region significantly reduced light-induced c-Fos-ir within the SCN when administered before light exposure at circadian time (CT) 13.5 or CT 19. In contrast, microinjection of a GABA(A) antagonist, bicuculline, but not a GABA(B) antagonist, CGP-35348, into the SCN region increased light-induced c-Fos-ir within the SCN when administered before light exposure at CT 13.5 or CT 19. These data indicate that GABAergic agonists and antagonists injected directly into the SCN region alter light-induced Fos-ir in a manner similar to their effects on light-induced phase shifts. Comparison of these data with previous studies examining the effects of systemically administered GABAergic drugs suggests that GABA(B)-active drugs have similar effects whether given systemically or within the SCN, but that GABA(A)-active drugs have more complex effects on c-fos induction and have multiple sites of action.

Fluctuating estrogen and progesterone receptor expression in brainstem norepinephrine neurons through the rat estrous cycle.

Norepinephrine (NE) neurons within the nucleus tractus solitarii (NTS; A2 neurons) and ventrolateral medulla (A1 neurons) represent gonadal steroid-dependent components of several neural networks regulating reproduction. Previous studies have shown that both A1 and A2 neurons express estrogen receptors (ERs). Using double labeling immunocytochemistry we report here that substantial numbers of NE neurons located within the NTS express progesterone receptor (PR) immunoreactivity, whereas few PRs are found in ventrolateral medulla. The evaluation of ERa and PR immunoreactivity in NE neurons through the estrous cycle revealed a fluctuating pattern of expression for both receptors within the NTS. The percentage of A2 neurons expressing PR immunoreactivity was low on metestrus and diestrus (3-7%), but increased significantly to approximately 24% on proestrous morning and remained at intermediate levels until estrus. The pattern of ERalpha immunoreactivity in A2 neurons was more variable, but a similar increment from 11% to 40% of NE neurons expressing ERa was found from diestrus to proestrus. Experiments in ovariectomized, estrogen-treated and estrogen-plus progesterone-treated rats revealed that PR immunoreactivity in A2 neurons was induced strongly by estrogen treatment, whereas progesterone had no significant effect. The numbers of ERalpha-positive NE neurons were not influenced by steroid treatment. These observations provide direct evidence for PRs in NE neurons of the brainstem and show that cyclical patterns of gonadal steroid receptor expression exist in A2, but not A1, neurons through the rat estrous cycle. The expression of PR in A2 neurons appears to be driven principally by circulating estrogen concentrations. The fluctuating levels of ERalpha and PR expression in these brainstem NE neurons may help generate cyclical patterns of biosynthetic and electrical activity within reproductive neural networks.

Central administration of antiserum to vasoactive intestinal peptide delays and reduces luteinizing hormone and prolactin surges in ovariectomized, estrogen-treated rats.

The present study investigated the role of hypothalamic VIP in the regulation of the LH and PRL surge using immunoneutralization of endogenous VIP in mature ovariectomized (OVX), estradiol benzoate (EB)-treated female Wistar rats. We compared the effect of intracerebroventricular (i.c.v.) injections of a VIP antiserum (VIP-Ab) with that of saline (Ctr) on LH and PRL profiles in two separate groups of rats following two subcutaneous EB injections on days 8 and 9 after OVX. VIP-Ab or Ctr injections were given during the second half of the dark period, i.e. at 22:00 h (day 9), and, in addition, the following morning, i.e. at 08:00 h (day 10), just before the expected onset of the LH surge. Hourly blood samples were collected between 09:00 and 18:00 h on day 10. In addition, we studied the reproducibility of EB-induced LH and PRL surges and compared the effect of Ctr and VIP-Ab treatment on sequential surges in individual OVX females, i.e. 10 and 23 days after OVX, using each animal as its own control. Although we observeda large variation in the height and timing of LH and PRL peak levels between EB-treated females, the characteristics of successive surges of individual rats were highly reproducible. This reproducibility suggests that differences in functioning of the suprachiasmatic nucleus as well as in the response of the hypothalamus to steroid feedback largely explain the normal variation in hormone responses between rats. The VIP-Ab treatment resulted in a significant delay in the time course and a strong reduction of the magnitude of the afternoon LH and PRL surge. When analyzed within individual females, the effect of VIP-Ab treatment was even more pronounced due to a reduction in variability when each animal was used as its own control. These results suggest that hypothalamic VIP is an important regulator of both the timing and the magnitude of the EB-induced LH and PRL surge in the OVX rat, and suggest that its role may be stimulatory in this respect.

Gender-specific apposition between vasoactive intestinal peptide-containing axons and gonadotrophin-releasing hormone-producing neurons in the rat.

Light microscopic double labeling immunocytochemistry for vasoactive intestinal peptide (VIP) and gonadotrophin-releasing hormone (GnRH) was carried out on the hypothalami of male and female rats. It was found that the number of VIP boutons that terminate on GnRH neurons and the percentage of GnRH neurons contacted by VIP axons were higher in females than in males. This sexual dimorphic interaction of VIP fibers and GnRH neurons may indicate the involvement of VIP in the gender-specific regulation of gonadotrophin release in rats.

Evidence for a direct neuronal pathway from the suprachiasmatic nucleus to the gonadotropin-releasing hormone system: combined tracing and light and electron microscopic immunocytochemical studies.

The timing and occurrence of the preovulatory luteinizing hormone (LH) surge in the female rodent are critically dependent on the integrity of the suprachiasmatic nucleus (SCN). Destruction of the SCN leads to a cessation of the ovarian cycle, whereas implantation of estrogen in ovariectomized rats results in daily LH surges. The anatomical substrate for these effects is not known. Previous studies involving lesions of the SCN have suggested the presence of a direct vasoactive intestinal polypeptide (VIP)-containing pathway to gonadotropin-releasing hormone (GnRH) neurons. To further investigate the direct connection between the SCN and the GnRH system, we have used tract-tracing with the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PhaL) in combination with an immunocytochemical staining for GnRH in light and electron microscopic studies. Small, unilateral PhaL deposits, especially when they were placed in the rostral ventrolateral portion of the SCN, revealed a bilateral projection to the preoptic area, where PhaL-immunoreactive fibers were regularly found in close apposition to GnRH neurons. Ultrastructural studies showed synaptic interaction of PhaL-containing fibers with GnRH-immunoreactive (IR) cell bodies, thus demonstrating a direct SCN-GnRH connection. Taken together, these data provide evidence for the existence of a monosynaptic pathway from the SCN to the GnRH system in the hypothalamus of the female rat. We suggest that this pathway may contain at least VIP as a putative transmitter and may play a role in the circadian regulation of the estrous cycle in the female rat.

Synaptic contacts between gonadotropin-releasing hormone-containing fibers and neurons in the suprachiasmatic nucleus and perichiasmatic area: an anatomical substrate for feedback regulation?

The suprachiasmatic nucleus (SCN) is critically involved in the generation and entrainment of circadian rhythms in mammalian species. Both the occurrence and the timing of the luteinizing hormone surge on the afternoon of proestrus in the female rodent are critically dependent on the integrity of the SCN. Recently, we demonstrated the presence of a monosynaptic pathway from the SCN to the gonadotropin releasing hormone (GnRH) neurons in the preoptic area. In addition, we found that interaction between the SCN and the GnRH system may be found close to the SCN, since we observed apposition of SCN efferents and GnRH fibers at the ultrastructural level in that region. The aim of the present study was to investigate the presence of synaptic contacts between GnRH fibers and structures in the SCN and surrounding perichiasmatic area (periSCN). At the light microscopical level, the immunoreactivity for GnRH showed a considerable overlap with the immunoreactivity for vasopressin and vasoactive intestinal peptide, two neuropeptides synthesized by SCN neurons. At the ultrastructural level, we demonstrated synaptic input of GnRH-containing axons on immunocytochemically unidentified structures in the SCN/peri-SCN region. The present results clearly demonstrate that the SCN and periSCN are postsynaptic targets of GnRH fibers. It is hypothesized that the GnRH input in the SCN region represents an anatomical substrate for feedback-control between these systems.

Preferential induction of c-fos immunoreactivity in vasoactive intestinal polypeptide-innervated gonadotropin-releasing hormone neurons during a steroid-induced luteinizing hormone surge in the female rat.

In small rodents, reproduction is critically dependent on the integrity of the circadian oscillator of the brain, the suprachiasmatic nucleus (SCN). Lesions of the SCN induce persistent estrus (anovulation) in intact female rats, whereas estrogen implantation in ovariectomized rats results in daily LH surges, which disappear after SCN lesions. Vasoactive intestinal polypeptide (VIP), a peptide synthesized in cell bodies of the SCN, has been implicated in the regulation of LH release. Recently, we have provided immunocytochemical evidence for a VIP-containing neuronal projection from the SCN to the GnRH system. This suggests that VIP from the SCN may modulate LH release via a direct influence on GnRH neurons. To investigate the involvement of VIP input on GnRH neurons and SCN neurons in the generation of a LH surge, we used immunoreactive c-fos as a marker for cell activation in ovariectomized mature rats and immature rats treated with steroids. VIP-containing fibers were observed in apposition to a substantial portion of the GnRH neurons containing c-fos. Expression of c-fos was more frequently observed in VIP-innervated GnRH neurons than in GnRH neurons in general. This difference in activation was most pronounced during the onset of the LH surge. In SCN neurons, steroid treatment did not induce c-fos immunoreactivity before or during the LH surge. The present results indicate that VIP-containing fibers, possibly originating in the SCN, are involved in the initiation of the LH surge. In view of the reported inhibitory effects of VIP on LH release, it is suggested that the role of VIP input in this respect is permissive.

Immunocytochemical localization of vasoactive intestinal peptide and neuropeptide Y in the bovine ovary.

The distribution of the neuropeptides vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) was studied immunocytochemically in bovine ovaries from 3 mo of gestation up to and including puberty, and from adult cows at three stages of the estrous cycle. The appearance of VIP and NPY immunoreactivity of 4.5-6 mo of gestation coincided with the onset of follicular development. In contrast to NPY, VIP was first found in the cortex. Both VIP and NPY immunoreactivity increased with age. From 9 mo of gestation onwards, VIP and NPY were found around blood vessels and non-vascular smooth muscle cells, in the stroma near preantral follicles, and in the theca externa of antral follicles. In addition, VIP-positive cells were observed exclusively in the granulosa layer of the preovulatory follicle at the time of the LH surge. The distribution of VIP- and NPY-immunoreactive fibers in the ovary may point to an effect of these neuropeptides on various physiological processes, including follicle development and ovarian blood flow. In addition, the presence of VIP-positive cells in the granulosa layer of the preovulatory follicle is indicative of a role for VIP in ovulation.

Lesions of the suprachiasmatic nucleus indicate the presence of a direct vasoactive intestinal polypeptide-containing projection to gonadotrophin-releasing hormone neurons in the female rat.

In non-seasonal breeders like the rat, the influence of the suprachiasmatic nucleus (SCN) on reproduction is most clearly expressed in the female. Complete lesions of the SCN induce persistent oestrus (anovulation) in intact female rats, whereas oestrogen implantation in ovariectomized rats results in daily luteinizing hormone surges. Vasoactive intestinal polypeptide (VIP), a peptide synthesized in cell bodies of the SCN, inhibits the increase in pulsatile luteinizing hormone release observed in ovariectomized female rats. In search of the anatomical basis for these observations, the present study employs an immunocytochemical double staining for VIP and gonadotrophin-releasing hormone (GnRH) at the light microscopical level. It was demonstrated that approximately 45% of the GnRH positive neurons in the diagonal band of Broca, the preoptic and anterior hypothalamic area of female rats are innervated by VIP-containing processes. To investigate whether these VIP-containing fibres represent a direct projection of the SCN to the GnRH system, unilateral thermic SCN lesions were made. Lesions that unilaterally destroyed the majority of the VIP synthesizing cells in the SCN resulted in at least a 50% decrease of the VIP innervation of GnRH cell bodies at the lesioned side compared to the intact side. Lesions not affecting the VIP synthesizing cell population in the SCN did not change the percentage of GnRH neurons innervated by VIP-containing fibres, while partial lesions resulted in intermediate effects. These results indicate that the majority of the light microscopical VIP-containing input on GnRH neurons in the hypothalamus is derived from the SCN.(ABSTRACT TRUNCATED AT 250 WORDS)