Michael Wilkinson

Leptin Immunoreactivity Is Localized to Neurons in Rat Brain

Leptin is secreted from adipocytes and is thought to enter the brain to regulate and coordinate metabolism, feeding behaviour, energy balance and reproduction. It is now clear that there are many additional sites of leptin production, including human placenta, ovary, stomach, skeletal muscle, mammary gland, pituitary gland and brain. In the present work, we employed double-label immunofluorescent histochemistry to establish the neuronal localization of leptin immunoreactivity (IR). To accomplish this, we used the neuron-specific marker NeuN to label cells in the arcuate nucleus (ARC), piriform cortex and hippocampus. In the supraoptic nucleus (SON) and paraventricular nucleus (PVN), we used antisera to oxytocin and vasopressin as neuronal markers. Double labelling revealed leptin IR in neurons of the ARC and piriform cortex. Leptin IR was confined to the nucleus and to distinct perinuclear sites. In contrast, neurons in the CA 2/CA 3 region of the hippocampus showed little nuclear staining. Leptin IR was clustered around the nucleus in these cells. Neurons of the dentate gyrus exhibited both nuclear and perinuclear localization of leptin IR. In the SON/PVN, most oxytocin- and vasopressin-IR neurons also contained leptin IR, often in perinuclear sites. In conclusion, the neuronal, perinuclear localization of leptin IR in rat brain corresponds closely to that of leptin receptor (OB-R) IR, which has also been detected intracellularly. Our observation of leptin IR associated with cell nuclei suggests the existence of an OB-R distinct from the well-described membrane forms.

Adipokine Gene Expression in Brain and Pituitary Gland

The brain has been recognized as a prominent site of peptide biosynthesis for more than 30 years, and many neuropeptides are now known to be common to gut and brain. With these precedents in mind it is remarkable that adipose-derived peptides like leptin have attracted minimal attention as brain-derived putative neuromodulators of energy balance. This review outlines the evidence that several adipose-specific genes are also expressed in the central nervous system and pituitary gland. We, and others, confirmed that the genes for leptin, resistin, adiponectin, FIAF (fasting-induced adipose factor) and adiponutrin are expressed and regulated in these tissues. For example, leptin mRNA was readily detectable in human, rat, sheep and pig brain, but not in the mouse. Leptin expression in rat brain and pituitary was regulated through development, by food restriction, and following traumatic brain injury. In contrast, hypothalamic resistin mRNA was unaffected by age or by fasting, but was significantly depleted by food restriction in mouse pituitary gland. Similar results were seen in the ob/ob mouse, and we noted a marked reduction in resistin-positive hypothalamic nerve fibres. Resistin and fiaf mRNA were also upregulated in hypoxic/ischaemic mouse brain. Our studies on the regulation of neuronal adipokines were greatly aided by the availability of clonal hypothalamic neuronal cell lines. One of these, N-1, expresses both rstn and fiaf together with several other neuropeptides and receptors involved in energy homeostasis. Selective silencing of rstn revealed an autocrine/paracrine regulatory system, mediated through socs-3 expression that may influence the feedback effects of insulin and leptin in vivo. A similar convergence of signals in the pituitary gland could also influence anterior pituitary hormone secretion. In conclusion, the evidence is suggestive that brain and pituitary-derived adipokines represent a local regulatory circuit that may fine tune the feedback effects of adipose hormones in the control of energy balance.

KiSS-1 mRNA in adipose tissue is regulated by sex hormones and food intake

Hypothalamic KiSS-1 gene expression is critical for the maintenance of reproductive function, and levels are attenuated by sex hormones and by food restriction, providing a link between fat mass and fertility. We hypothesized that adipose tissue (FAT) would express KiSS-1. KiSS-1 mRNA was quantified in FAT, hypothalamus (HYP) and pituitary gland (PIT) using realtime RT-PCR. FAT KiSS-1 expression was sensitive to sex steroids and to nutritional status. Gonadectomized rats given estradiol (E; females) or testosterone (T; males) revealed striking increases in KiSS-1 mRNA in FAT (E: 8-fold, p<0.01; T: 5-fold, p<0.01). In contrast, HYP KiSS-1 expression was reduced by E/T, whereas PIT expression was reduced by gonadectomy only in females, reversed by E. Food restriction (18 h) increased FAT KiSS-1 mRNA in both sexes (2.5-4.0-fold, p<0.01), but decreased levels in male PIT and female HYP. Conversely, FAT expression was reduced in rats fed a high fat diet (HFD), as well as in obese Zucker rats, whereas PIT expression was increased in Zucker rats (p<0.05) but not by HFD. In contrast HYP KiSS-1 mRNA was elevated by HFD. Experiments in which the arcuate nucleus was damaged by an excitotoxic lesion revealed that hypothalamic KiSS-1 mRNA was significantly reduced, whereas FAT levels were unaffected, suggesting that regulation of KiSS-1 in FAT is independent of the hypothalamus. In conclusion, KiSS-1 expression is differentially regulated by sex hormones, food intake and obesity in FAT, HYP and PIT. Kisspeptins of adipose tissue origin may act as adipokines or as local regulators of adipocyte function.

Resistin expression and regulation in mouse pituitary

Resistin, a new adipocytokine, is expressed in human, rat and mouse adipose tissue. Its putative role as a mediator of insulin resistance is controversial. We hypothesized that resistin, in common with leptin, has multiple roles in non‐adipose tissues. Using reverse transcription polymerase chain reaction (RT‐PCR) we show that the resistin gene (Retn) is expressed in mouse brain (hypothalamus and cortex) and pituitary gland. Immunohistochemistry revealed resistin protein in the arcuate nucleus and pituitary gland. Semi‐quantitative RT‐PCR analysis indicated that Retn mRNA is developmentally regulated in the pituitary. Expression was lowest at birth, increased abruptly between postnatal days 14 and 25 (four‐fold; P<0.001), and declined thereafter. This peak in pituitary Retn mRNA was unaffected by early weaning but was abolished by neonatal treatment with monosodium glutamate, suggesting that the basal hypothalamus regulates pituitary Retn. Although the role(s) of endogenous resistin in mouse brain and pituitary remains to be determined, it may be distinct from its controversial involvement in insulin resistance. Our data suggest that local resistin expression could have functional implications during prepubertal maturation of the hypothalamic–pituitary system.

The heart reinnervates after transplantation

BACKGROUND Whether cardiac reinnervation occurs after transplantation remains controversial. If reinnervation does occur, how sympathetic and parasympathetic efferent neurons do this remains unknown. METHODS Power spectral analysis of heart rate variability was assessed for 1 year after cardiac autotransplantation in 9 dogs. After induction of anesthesia 13 months after transplantation, cardiac and intrinsic cardiac neuronal responses elicited by both electrical stimulation of parasympathetic or sympathetic efferent neurons and systemic or local coronary artery administration of nicotine (5 microg/kg), angiotensin II (0.75 microg/kg), and tyramine (1.2 microg/kg) were studied. The transmembrane electrical properties of intrinsic cardiac neurons were studied in vitro. Ventricular tissue catecholamine content, alpha-tubulin expression, and beta-adrenergic receptor density and affinity were studied. The presence of axons crossing suture lines was sought histologically. RESULTS Nerves were identified crossing suture lines. Electrical or chemical (ie, nicotine or angiotensin II) activation of sympathetic efferent neurons enhanced cardiodynamics, as did tyramine. Stimulating vagal efferent preganglionic axons induced bradycardia in half of the dogs. Functional reinnervation did not correlate with specific power spectra derived from rate variability in the conscious state. Responding to nicotine and angiotensin II in situ, transplanted intrinsic cardiac neurons generated spontaneous activity. These neurons displayed nicotine-dependent synaptic inputs in vitro. Ventricular tissue had normal beta-adrenergic receptor affinity and density but reduced catecholamine and alpha-tubulin contents. CONCLUSIONS The intrinsic cardiac nervous system receives reduced input from extracardiac sympathetic efferent neurons after transplantation and inconsistent input from parasympathetic efferent preganglionic neurons. These heterogeneous neuronal inputs are not reflected in heart rate variability or ventricular beta-adrenergic receptor function. Transplanted angiotensin II-sensitive intrinsic cardiac neurons exert greater cardiac control than do nicotine-sensitive ones. The intrinsic cardiac nervous system remodels itself after cardiac transplantation, and this indicates that direct assessment of extracardiac and intrinsic cardiac neuronal behavior is required to fully understand cardiac control after transplantation.

Methylphenidate regulates Activity regulated cytoskeletal associated but not Brain-derived neurotrophic factor gene expression in the developing rat striatum

Methylphenidate (MPH) is a psychostimulant drug used to treat attention deficit hyperactivity disorder in children. To explore the central effects of chronic MPH, we investigated the expression of an effector immediate early gene, activity regulated cytoskeletal associated (arc), and the neurotrophin, brain-derived neurotrophic factor (bdnf) in the brain of immature and adult rats following repeated MPH. Prepubertal (postnatal day (PD) 25-38) and adult (PD 53-66) male rats were injected once daily for: a) 14 days with saline or MPH (2 or 10 mg/kg; s.c.) or b) 13 days with saline followed by a single dose of MPH (2 or 10 mg/kg; s.c.). To determine possible long-term effects of MPH, prepubertal rats were allowed a drug-free period of 4 weeks following the 14 days of treatment, and then were given a challenge dose of MPH. We demonstrated, for the first time, that an acute injection of MPH increased levels of activity-regulated cytoskeletal protein (ARC) and arc mRNA in the prepubertal rat striatum and cingulate/frontal cortex. This response was significantly attenuated by chronic MPH. The desensitization in arc expression observed in prepubertal rats persisted in the adult striatum following a later MPH challenge. In contrast to these data we observed little effect of MPH on bdnf expression. We also developed an effective, non-stressful technique to treat freely moving immature rats with oral MPH. Consistent with the results described above, we observed that oral MPH (7.5 and 10 mg/kg) also increased arc expression in the prepubertal rat striatum. However, unlike the effects of injected MPH, repeated oral MPH (7.5 mg/kg) did not alter the normal arc response. This result raises the important possibility that oral doses of MPH that reproduce clinically relevant blood levels of MPH may not down-regulate gene expression, at least in the short term (14 days). We confirmed, using mass spectrometry, that the oral doses of MPH used in our experiments yielded blood levels within the clinical range observed in children. The novel oral administration paradigm that we describe thus provides a clinically relevant animal model to further explore the effects of chronic drug exposure on central gene expression in the developing rat brain.

Odor-induced sexual maturation and expression of c-fos in the olfactory system of juvenile female mice

Exposure to the urine or soiled bedding odors of novel adult males is known to accelerate puberty in juvenile female mice. To determine what part of the olfactory system is activated by these odors, the expression of c-fos in the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB) of juvenile female mice was examined after their exposure to male-soiled bedding, peppermint odor or their own bedding. Fos-like immunoreactivity was found throughout the AOB of the juvenile female mice exposed to the bedding odors of adult males for 3 h. In contrast, dense staining was found in the granular cell layer of the MOB of mice exposed to peppermint odors for 3 h, whereas mice exposed to their own bedding failed to show immunostaining in the AOB and only slight or no staining in the MOB. These results indicate that social odors stimulate the expression of c-fos in the AOB while non-social odors activate the MOB. This method allows the identification of individual cells activated by the different odors and will be useful in locating other areas of the brain involved in the neuroendocrine changes underlying odor-induced precocious puberty.

Developmental regulation of leptin gene expression in rat brain and pituitary

We have previously reported the expression of leptin mRNA and protein in adult rat brain and pituitary gland. We report here the presence of leptin and leptin receptor mRNA in neonatal female rat brain and pituitary using RT-PCR as well as leptin and leptin receptor immunoreactivity in neonatal rat brain. In addition, we describe age-related changes in leptin mRNA expression in female rat brain and pituitary from postnatal day 2 to 28, evaluated using semi-quantitative RT-PCR analysis. Age-related differences in leptin (ob) mRNA levels were tissue-dependent. The most striking developmental changes were noted in the pituitary and cerebral cortex. In the pituitary, ob mRNA levels were maximal during postnatal days 7-14 and fell sharply by postnatal day 22. In cortex, ob mRNA levels were low in neonatal pups (day 2-7) but increased significantly between postnatal days 14 and 28. Leptin mRNA was detectable at postnatal day 2 in hypothalamus and subcutaneous fat. No significant differences in the level of expression were observed between postnatal day 2 and 28. Serum leptin levels were highest at day 7-14 and decreased significantly by day 21-28, coincident with the fall in pituitary leptin expression. The high levels of leptin expression in the neonatal pituitary suggest that this gland may contribute to the circulating leptin levels during early postnatal development, when adipose deposits are minimal. These data indicate that regulation of leptin gene expression in the postnatal period is tissue-dependent, a finding, which suggests that local leptin expression may have important functional significance in the development of the brain-pituitary system.

Leptin receptors are developmentally regulated in rat pituitary and hypothalamus

We have previously reported that leptin is expressed in adult rat brain and pituitary gland, though the role of leptin in these sites has not been determined. Leptin mRNA is developmentally regulated in the brain and pituitary of male and female rats during early postnatal development, suggesting a role in the maturation of the brain-pituitary system. Here, we sought to extend our previous studies by evaluating (1) the ontogeny of leptin receptor mRNA levels in rat brain and pituitary and (2) pituitary leptin protein levels in neonatal and pre-pubertal rats. Pituitary leptin concentration was highest shortly after birth (postnatal day (PD) 4, 25 ng/mg protein) and fell significantly throughout postnatal development and into adulthood (PD 60, 3.5 ng/mg protein; P<0.005) coincident with a decline in pituitary leptin mRNA levels. Significant age-related effects on leptin receptor mRNA levels were also observed in the pituitary and the hypothalamus of male and female rats using semi-quantitative RT-PCR analysis. In the pituitary, the short form (OBRa) mRNA levels were highest in neonatal rats (PD 4) but declined throughout postnatal development (PD 4-22) paralleling the fall in pituitary leptin mRNA and protein levels. The long form (OBRb) mRNA levels were unaffected by age between PD 4 and 22. In contrast, hypothalamic, levels of OBRb mRNA were very low to undetectable shortly after birth (PD 4) and rose significantly between PD 4 and 14/22 while levels of OBRa mRNA were not significantly different between PD 4 and 22. Immunohistochemical detection of leptin receptor immunoreactivity (all forms) revealed the presence of OBR-like protein in pituitary and hypothalamus as early as PD 4. Cortical leptin receptor mRNA levels were similar throughout early postnatal development. No gender-related differences in leptin receptor mRNA levels were noted in brain or pituitary. In conclusion, these data, together with our previous work, indicate that the neonatal pituitary gland expresses leptin and leptin receptors at levels far in excess of those observed in mature rats. The pituitary is thus quite different from adipose tissue, hypothalamus and cerebral cortex, in which neonatal leptin expression is lowest at birth. Since neonatal pituitary leptin receptor expression is also elevated, it is possible that pituitary-derived leptin plays some role in the development of the hypothalamic-pituitary system.

Peripubertal Treatment with N-Methyl-D-Aspartic Acid or Neonatally with Monosodium Glutamate Accelerates Sexual Maturation in Female Rats, an Effect Reversed by MK-801

Recent reports from several laboratories have implicated the excitatory neurotransmitter glutamate as a component in the neural regulation of sexual maturation. In the rat we have previously proposed that a hypothalamic opioid restraint mechanism may ultimately be overridden by maturation of an excitatory drive, culminating in first ovulation. We have now investigated whether glutamate may be the excitatory factor. Treatment of immature female rats with single, daily injections of two N-methyl-D-aspartate (NMDA) antagonists--dextrorphan (18 mg/kg) and MK-801 (0.1 mg/kg)--beginning on the 27th postnatal day, significantly delayed the timing of vaginal opening (VO). Interestingly, treated rats reached VO in spite of continued antagonist treatment. The antagonist effect was reversed by preinjection of NMDA, suggesting that endogenous glutamate exerts its effect via an NMDA-subtype glutamate receptor. Injection of NMDA alone (15 mg/kg; once daily) produced a striking synchronization of VO such that all treated rats showed VO over a 24-hour period compared to a normal distribution of several days for control rats. In a model of first ovulation, i.e., rats induced to ovulate by pregnant mare serum, MK-801 (1 mg/kg) arrested treated rats at proestrus. This was readily reversible after discontinuing injections. A lower dose of MK-801 (0.1 mg/kg/day) was ineffective in delaying ovulation. In a second series of experiments we studied the consequences of a neonatal hypothalamic lesion which destroys glutamate-sensitive neurons.(ABSTRACT TRUNCATED AT 250 WORDS)