Testing the Challenge Hypothesis in Stumptail Macaque Males: The Role of Testosterone and Glucocorticoid Metabolites in Aggressive and Mating Behavior.

The "challenge hypothesis" predicts higher male-male aggressive behavior along with increases in testosterone levels during times of reproductive challenges and social instability. In addition, in some primate species, higher glucocorticoid levels can be observed as well, but this is usually modulated by dominance rank. We studied rank-related aggressive behavior, mating activity, and fecal testosterone and glucocorticoid metabolites (fTm and fGCm) in male stumptail macaques (Macaca arctoides) in order to test some predictions of the "challenge hypothesis". Over a 20-month period, we collected data on aggressive behavior and copulation, as well as fecal samples (n = 700) to quantify fTm and fGCm in seven adult stumptail males living in captivity. During periods of mating activity, male-to-male aggression increased in higher- and middle-ranking males. Neither fTm nor fGCm levels predicted male-to-male aggression. fGCm levels (but not fTm) were positively associated with male-to-female aggression; however, this association was pronounced during periods of mating activity. fGCm levels differed according to social rank, with middle-ranking males having the highest levels. Both hormones were higher during periods of mating activity, but only in higher- and middle-ranking males. Taken together, our findings partially support the challenge hypothesis in a non-seasonal primate and shed some light on the unique social and mating system of the stumptail macaque.

Effects of Intermittent Fasting on Hypothalamus-Pituitary-Thyroid Axis, Palatable Food Intake, and Body Weight in Stressed Rats.

Dietary regimens that are focused on diminishing total caloric intake and restricting palatable food ingestion are the most common strategies for weight control. However, restrictive diet therapies have low adherence rates in obese patients, particularly in stressed individuals. Moreover, food restriction downregulates the hypothalamic-pituitary-thyroid axis (HPT) function, hindering weight loss. Intermittent fasting (IF) has emerged as an option to treat obesity. We compared the effects of IF to an all-day feeding schedule on palatable diet (PD)-stress (S)-induced hyperphagia, HPT axis function, accumbal thyrotropin-releasing hormone (TRH), and dopamine D2 receptor expression in association with adipocyte size and PPARƔ coactivator 1α (PGC1α) and uncoupling protein 1 (UCP1) expression in stressed vs. non-stressed rats. After 5 weeks, S-PD rats showed an increased energy intake and adipocyte size, fewer beige cells, and HPT axis deceleration-associated low PGC1α and UCP1 expression, as well as decreased accumbal TRH and D2 expression. Interestingly, IF reversed those parameters to control values and increased the number of beige adipocytes, UCP1, and PGC1α mRNAs, which may favor a greater energy expenditure and a reduced body weight, even in stressed rats. Our results showed that IF modulated the limbic dopaminergic and TRHergic systems that regulate feeding and HPT axis function, which controls the metabolic rate, supporting this regimen as a suitable non-pharmacologic strategy to treat obesity, even in stressed individuals.

Intrauterine Zn Deficiency Favors Thyrotropin-Releasing Hormone-Increasing Effects on Thyrotropin Serum Levels and Induces Subclinical Hypothyroidism in Weaned Rats.

Individuals who consume a diet deficient in zinc (Zn-deficient) develop alterations in hypothalamic-pituitary-thyroid axis function, i.e., a low metabolic rate and cold insensitivity. Although those disturbances are related to primary hypothyroidism, intrauterine or postnatal Zn-deficient adults have an increased thyrotropin (TSH) concentration, but unchanged thyroid hormone (TH) levels and decreased body weight. This does not support the view that the hypothyroidism develops due to a low Zn intake. In addition, intrauterine or postnatal Zn-deficiency in weaned and adult rats reduces the activity of pyroglutamyl aminopeptidase II (PPII) in the medial-basal hypothalamus (MBH). PPII is an enzyme that degrades thyrotropin-releasing hormone (TRH). This hypothalamic peptide stimulates its receptor in adenohypophysis, thereby increasing TSH release. We analyzed whether earlier low TH is responsible for the high TSH levels reported in adults, or if TRH release is enhanced by Zn deficiency at weaning. Dams were fed a 2 ppm Zn-deficient diet in the period from one week prior to gestation and up to three weeks after delivery. We found a high release of hypothalamic TRH, which along with reduced MBH PPII activity, increased TSH levels in Zn-deficient pups independently of changes in TH concentration. We found that primary hypothyroidism did not develop in intrauterine Zn-deficient weaned rats and we confirmed that metal deficiency enhances TSH levels since early-life, favoring subclinical hypothyroidism development which remains into adulthood.

Mct8 and trh co-expression throughout the hypothalamic paraventricular nucleus is modified by dehydration-induced anorexia in rats.

Thyrotropin-releasing hormone (TRH) is a neuropeptide with endocrine and neuromodulatory effects. TRH from the paraventricular hypothalamic nucleus (PVN) participates in the control of energy homeostasis; as a neuromodulator TRH has anorexigenic effects. Negative energy balance decreases PVN TRH expression and TSH concentration; in contrast, a particular model of anorexia (dehydration) induces in rats a paradoxical increase in TRH expression in hypophysiotropic cells from caudal PVN and high TSH serum levels, despite their apparent hypothalamic hyperthyroidism and low body weight. We compared here the mRNA co-expression pattern of one of the brain thyroid hormones' transporters, the monocarboxylate transporter-8 (MCT8) with that of TRH in PVN subdivisions of dehydration-induced anorexic (DIA) and control rats. Our aim was to identify whether a low MCT8 expression in anorexic rats could contribute to their high TRH mRNA content.We registered daily food intake and body weight of 7-day DIA and control rats and analyzed TRH and MCT8 mRNA co-expression throughout the PVN by double in situ hybridization assays. We found that DIA rats showed increased number of TRHergic cells in caudal PVN, as well as a decreased percentage of TRH-expressing neurons that co-expressed MCT8 mRNA signal. Results suggest that the reduced proportion of double TRH/MCT8 expressing cells may be limiting the entry of hypothalamic triiodothyronine to the greater number of TRH-expressing neurons from caudal PVN and be in part responsible for the high TRH expression in anorexia rats and for the lack of adaptation of their hypothalamic-pituitary-thyroid axis to their low food intake.

Mediobasal hypothalamic and adenohypophyseal TRH-degrading enzyme (PPII) is down-regulated by zinc deficiency.

Thyrotropin-releasing hormone (TRH) synthesized in hypothalamic paraventricular nucleus directs hypothalamus-pituitary-thyroid (HPT) axis function, regulating thyrotropin release from adenohypophysis and thyroid hormones serum concentration. Pyroglutamyl aminopeptidase II (PPII), a Zn-dependent metallopeptidase located in adenohypophysis and medial-basal-hypothalamus degrades TRH released from the median eminence and participates in HPT axis function by regulating TRH-induced thyrotropin release from adenohypophysis. It is unknown whether dietary Zn deficiency down-regulates PPII. Our aim was to compare adenohypohyseal and medial-basal-hypothalamic PPII activity and expression of adult rats fed a Zn-deficient diet (2ppm) throughout their lifespan (DD), prenatally (DC) or after weaning (CD) vs. that of animals fed a control diet (20ppm:CC). Female rats consumed a Zn-deficient or control diet from two weeks before gestation and up to the end of lactation. We analyzed adenohypophyseal and medial-basal-hypothalamic PPII activity of dams and male offspring when adults; its relation to median eminence TRH, serum thyrotropin, leptin and thyroid hormones concentration. Offspring ate the same diet as their dams (CC, DD) or were switched from dietary regime after weaning (CD, DC) and until 2.5 months of age. DD males showed decreased adenohypophyseal and medial-basal-hypothalamic PPII activity, along with high thyrotropin serum concentration. Post-weaning Zn-deficiency (CD) decreased PPII activity only in adenohypophysis and increased thyrotropin circulating levels. Zn-replenishment (DC) normalized PPII activity in both regions and serum thyrotropin concentration. Adenohypophyseal PPII activity decreased and prolactin levels increased in Zn-deficient dams. We concluded that long-term changes in dietary Zn down-regulate PPII activity independently of T3, increasing thyrotropin serum concentration, overall resembling sub-clinical hypothyroidism.

La melatonina induce la síntesis y liberación de las encefalinas en el cerebro de la rata / Melatonin induces enkephalins synthesis and release in the rat brain

Since enkephalins discovery in 1975, several opioid peptides have been included in neuroscience research. Enkephalins have been involved in the homeostasis maintenance of the organism, mostly with cellular and molecular mechanisms implicated in antinociception and narcotic responses. Moreover, enkephalins have been shown to be involved in the control of stress, regulation of cardiovascular functions, modulating primary immune responses, in addition to cellular differentiation processes. As opioid peptides appear to modulate several bioactivities and physiological responses in organisms, this posits that several modifications should occur during their synthesis, cell release, and receptor binding in target cells. At present, it has been demonstrated that the endogenous opioid system (EOS), displays a circadian rhythm, in which its tissue content, presynaptic release, and receptor's number reaches its maximal concentration during the dark phase (24:00h) and the minimal during the early morning (05:00 h). Recently, our group reported that functional pinealectomy disrupts the enkephalin circadian rhythm and significantly reduces the tissue content of opioid peptides in the rat brain. However, the effect was shown to be specific to the hour along the 24h daytime. There were no significant changes during the light period, only during the dark period (01:00h), when the enkephalin tissue content decreased in the experimental group. The effect was reverted when pinealectomized rats were injected with single doses of melatonin (MEL) (150µg/kg i.p.). If the lack of melatonin in the rat brain significantly reduced the enkephalin tissue content, and its exogenous administration re-established the enkephalin tissue levels, it is possible that the hormone is involved in the enkephalin synthesis. In this paper we provide further evidence that supports the relation between melatonin and opioid peptides synthesis and release. In addition, we studied the effect of darkness and melatonin administration in enkephalin tissue levels. Finally, we analyzed the luzindole effect as a melatonin receptor antagonist in the pinealectomized rat brain. Material and methods Subjects: Male Wistar rats were housed in a light and temperature controlled room. Water and pellet food were available ad libitum. This group was subdivided in: 1. Functional pinealectomy group (FP). Rats were housed individually during 15 days in a room with continuous light (<50lux). 2. FP rats were housed in a dark room during four or six hours. 3. FP rats were injected with melatonin (150, 300, 600µg/kg s.c.). 4. FP rats were injected with Luzindole (187.5, 375, 750µg/kg i.p.). After 30 min, the animals were injected with melatonin (150µg/kg). 5. FP rats were injected with melatonin (800µg/ kg) and subjected to the in vitro release processes. The rats were sacrificed by decapitation and the blood collected for melatonin serum determination. The brains were removed and processed for an analytical preparative procedure for the enkephalin determination by radioimmunoassay technique. The in vitro release methodology was performed as follows: tissue samples were homogenized by applying 8 strokes with a Thomas grinder system. The homogenates were centrifuged at 4,000rpm, 4°C during 10 min. Supernatants were recovered and centrifuged at 12,000rpm at 4°C for 20 min. Supernatants were discarded and pellets were resuspended in the homogeneization buffer (1:9 w/v). Samples were placed on top of a Percoll gradient density (23%, 15%, and 10%) and centrifuged at 20,000 rpm at 4°C for 25min. The synaptosomal enriched fraction (15-23%) was obtained and diluted in 1mL of Krebs buffer (mM: NaCl 119, KCl 4.6, CaCl2·2H2O 1.25, KH2PO4 0.85 MgSO4 0.84, NaHCO3 24.8, sucrose 10). Buffer was gasified with a mixture of C0(2) 95% and O2 5%, pH 7.4. 800µL aliquots were placed into plastic chambers. After 20 min of stabilization with Krebs buffer, three different superfusates were collected: 1. basal, 2. potassium [50mM], and 3. post-stimulus (Krebs buffer without potassium). Samples were collected into HCl 0.1N, boiled and subsequently loaded into Amberlite XAD-2 columns (8 × 0.7cm) for solid-phase peptide extraction. The flow rate was held constant at 0.5 mL min-1 and elution of the whole peptide fraction was carried out using a continuous gradient with absolute methanol. Eluted samples were lyophilized and resuspended in 2mL of distilled water and finally stored at -20°C for further quantification of IR-Enkephalin using standard radioimmunoassay procedures. The results showed that functional pinealectomy reduced the opioid tissue content in the different brain structures assayed. The lack of melatonin significantly decreased the enkephalin tissue content when compared to the control group. However, tissue levels of enkephalin material were completely restored after four and six hours of administration of different doses of exogenous melatonin administration to the rats. As continuous light decreases the melatonin content in the brain, darkness should be able to counteract the aforementioned effect. Our results showed that tissue levels of enkephalin material were increased over 200% and 300%, after exposing animals to a four or six-hour period of darkness, when compared to animals exposed to continuous light. Luzindole was used to abolish any melatonin activity via activation of its membrane brain receptors. Our experiments showed that different doses of the antagonist were not able to obliterate the increased content of opioid peptides induced with melatonin administration in the tested brain tissues.

En la actualidad se reconoce que el Sistema Endógeno Opioide (SEO) participa en la regulación y control de la homeostasis; por lo tanto se requiere que los procesos bioquímicos que dan lugar a su síntesis, liberación y unión a receptores se encuentren reguladas de manera endocrina. Diversas líneas de investigación han demostrado que la concentración y liberación presináptica de las encefalinas no permanece constante durante un ciclo de 24 horas. Por el contrario, su síntesis y liberación alcanzan su máxima concentración durante la fase de oscuridad y la mínima durante las primeras horas de la mañana. Recientemente, nuestro grupo de trabajo ha demostrado que la ausencia de melatonina (MEL), por efecto de la pinealectomía funcional, rompe el ritmo circádico y reduce significativamente la concentración de las encefalinas durante la fase de oscuridad. Si la ausencia de la hormona abate la concentración de opioides, es factible que la MEL se encuentre involucrada en la síntesis de las encefalinas. En el presente trabajo se muestran los resultados del efecto de la pinealectomía funcional sobre la concentración tisular y la liberación de los opioides, así como el efecto de la administración exógena de MEL y de su antagonista el luzindol (LZ). Diseño experimental Control Naive. Se utilizaron ratas macho de la cepa Wistar (200-250g), que permanecieron en un cuarto con un ciclo de luz y oscuridad controlada. La fase de luz duró 12 horas y comenzó a las 06:00h. A su vez, este grupo fue subdividido en: 1. Luz Continua (LC): Para abatir la concentración sérica de la hormona, se usó el modelo experimental conocido como pinealectomía funcional. 2. Luz Continua + Oscuridad: La luz abate la concentración de MEL y la exposición a la oscuridad revierte dicho efecto. Un grupo de ratas sometido a la luz continua se colocó en un cuarto con oscuridad durante cuatro y seis horas. 3. Luz continua+melatonina: Para analizar el efecto de la MEL sobre el contenido tisular de opioides, se administró una dosis de MEL (150, 300 y 600µg/Kg s.c.). 4. Luz continua+luzindol (LZ)+MEL: Si la melatonina ejerce su mecanismo de acción al unirse a sus receptores presentes en la membrana plasmática, entonces su efecto podría ser revertido por la presencia del LZ. Por lo tanto, a tres grupos de ratas sometidas a la pinealectomía funcional se les inyectó con LZ a una dosis de 187.5, 375, 750 µg/kg. s.c. Después de transcurridos 30min., los animales fueron inyectados con MEL (150µg/kg. s.c.). 5. Liberación: Se utilizaron tres grupos de ratas: a) control, b) luz continua y c) luz continua+MEL (800µg/kg. s.c.). Post mortem la amígdala fue sometida al proceso preparativo para la obtención de los sinaptosomas. Resultados Los resultados obtenidos en el presente trabajo nos muestran que la pinealectomía funcional reduce significativamente la concentración de encefalinas en todas las estructuras cerebrales analizadas. Sin embargo, el efecto es revertido tanto por la administración exógena de MEL, como por la exposición a la oscuridad. Las diferentes dosis administradas del LZ bloquearon parcialmente el efecto estimulante de la MEL sobre la síntesis de los opioides. Por último, la pinealectomía funcional redujo significativamente la liberación presináptica de encefalinas, misma que se pudo reestablecer con la administración exógena de la hormona. Discusión Las evidencias experimentales del presente trabajo sugieren que la MEL se encuentra relacionada con la síntesis y la liberación de las encefalinas. Por un lado, la pinealectomía funcional redujo significativamente el contenido tisular de encefalinas, pero el efecto fue revertido tanto con la administración exógena de la hormona como con la exposición a la oscuridad. La administración del LZ sólo fue capaz de bloquear parcialmente el efecto estimulante de la MEL sobre la síntesis de encefalinas. El efecto de la MEL sobre la liberación de opioides pone de manifiesto la relación funcional entre ambos sistemas. A menor cantidad de opioides por efecto de la pinealectomía, menor liberación presináptica y por el contrario, una vez administrada la hormona los valores de la síntesis-liberación se restablecen. Conclusión La MEL puede estar involucrada en la síntesis y la liberación de los péptidos opioides.

Variación estacional de péptidos opioides en el cerebro de la rata albina / Stational variation of opioid peptides in the albino rat brain

Desde hace más de 20 años, se ha estudiado intensamente el significado fisiológico de los péptidos opioides en el organismo. Su extensa distribución en el sistema nerviosos central y periférico, así como la de sus receptores estereoespecíficos, han permitido sugerir que los opioides contribuyen a mantener la homeostasis celular. El precursor de los péptidos opioides, es una proteína de 248 aminoácidos denominada proencefalina A, la cual se almacena en los gránulos de secreción producidos por el aparato de Golgi y sufre cambios postraduccionales en su viaje hacia las terminales sinápticas. De la molécula del precursor se originan 4 copias de Metencefalina, una copia de Leu-encefalina, una copia del heptapéptido (Met-encefalina-Arg6-Phe7), y una copia de octapéptido (ME-Arg6-Gly7-Leu8), los cuales se almacenan en las vesículas sinápticas para ser liberados y ejercer su función. Desafortunadamente hasta el momento son desconocidos los cambios que la estacionalidad puede ocasionar sobre la bioquímica del sistema endógeno opioide (SEO). En el presente trabajo, se estudiaron los posibles cambios en la concentración de opioides en el cuerpo estriado y en la amígdala del lóbulo temporal de la rata albina a lo largo de un ciclo estacional. Un grupo de 10 ratas macho de la cepa Wistar (250-300 g), fueron sacrificadas el día 10 de cada mes. Su cerebro fue removido, el cuerpo estriado y la amígdala fueron disecados con rapidez. Ambas estructuras fueron homogeneizadas, centrifugadas y purificadas. El contenido cerebral de 4 péptidos opioides, la Met-encefalina, la Leu-encefalina, la hepta y el octapéptido fue determinado con la técnica de radioinmunoensayo. Los resultados indican que la esticionalidad modifica los niveles de opioides a los largo del año, de manera específica para cada péptido y estructura analizada. Con excepción de la Met-encefalina, los otros 3 péptidos analizados presentaron su menor concentración en los meses de noviembre, diciembre y enero, comparados con los 9 meses restantes. La disminución significativa de los opioides en invierno, puede indicar que la liberación presináptica de los mismos sea mayor en este periodo. Es durante este periodo que los roedores silvestres como Microtus mexicanus, Reithrodontomys megallotis y Peromyscuss maniculatus, tienen la menor disponibilidad de alimento durante el año, lo que ocasiona cambios en las estrategias de la reproducción y el alumbramiento, proceso en el cual, los opioides estimulan la secreción...