Alterations of local cerebral glucose utilization in lean and obese fa/fa rats after acute adrenalectomy

An animal model often used to investigate the aetiology of obesity is the genetically obese fa/fa rat. It has many abnormalities, including hyperphagia, hyper-insulinemia, insulin resistance, low cerebral glucose utilization and an overactive hypothalamo-pituitary adrenal (HPA) axis with resulting hypercorticism. Due to the latter consideration, the aim of this work was to study the impact of acute adrenalectomy (ADX) on the local cerebral glucose utilization (LCGU) of lean and obese fa/fa rats. ADX resulted in discrete increases in LCGU of regions common to both lean and obese rats. These common regions were found to belong or be related to the limbic system. Within this system, the LCGU of the brain of obese rats was either normalized to lean sham operated values or increased by ADX to a similar degree in both groups on a percentage basis. It was concluded that the LCGU of both lean and obese animals appears to be negatively regulated, albeit to different extents, by glucocorticoids. Such negative regulation is particularly salient within the limbic system of the lean rat and even more so in the fa/fa rat. It is suggested that the long-term hypercorticism of obese fa/fa rats due to abnormal regulation of the HPA axis may result in a decreased LCGU in limbic and related regions of the brain of fa/fa rats and contribute to the expression of the obese phenotype.     

NMDA and AMPA glutamate receptor subtypes in the thoracic spinal cord in lean and obese–diabetic ob/ob mice

Quantitative autoradiography was used to characterise the binding of selective radiolabelled antagonists for the N-methyl- -aspartate (NMDA) receptor and the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor in the dorsal, intermediate and ventral subregions of the grey matter of the upper thoracic spinal cord in male and female lean and obese–diabetic (ob/ob) mice. The density of binding sites for both receptor subtypes was greater in diabetic mice, in all three subregions of the grey matter, than the corresponding subregions in the lean mice. The affinity of the binding site for the NMDA antagonist was significantly higher in obese mice than lean mice, consistent with the presence of two subpopulations of NMDA receptors with different ligand binding affinities in obese mice. The increase in expression of the glutamate receptor subtypes, and altered ligand affinity for the NMDA receptor subtype in the obese mice may be causally involved in the peripheral neuropathies which can accompany diabetes mellitus.     

Altered α1-adrenoceptor binding in intact and adrenalectomized obese Zucker rats (fa/fa)

While many autonomic and metabolic defects associated with genetic obesity in the Zucker rat are corrected by adrenalectomy (Adx), brain adrenoceptor function has not been examined in this context. Here, 3 weeks after Adx or sham surgery, brains of 11 weeks old lean (Fa/Fa) and obese (fa/fa) male Zucker rats were assayed for α₁-([³H]prazosin; [³H]PRZ) and α₂-adrenoceptor ([³H]paraminoclonidine; [³H]PAC) binding by autoradiography. By genotype, obese rats had 19–256% higher [³H]PRZ binding than lean rats in the amygdala (central [ACN], basolateral [ABL], basomedial [ABM] and medial [MAN] nuclei [n.]), hypothalamus (dorsomedial n. [DMN] and lateral [LH]) and somatosensory cortex. In the ABL and ACN, increased maximal binding (B_max) in obese rats was associated with decreased affinity (increased K_d). Three weeks after surgery, sham-operated obese rats gained 27% more weight than lean rats but lean and obese Adx rats gained the same amount of weight. Adx reduced [³H]PRZ binding in both lean and obese rats by 37–70% in the amygdala (ABM, ACN, MAN) compared to sham-operated rats. But, Adx selectively reduced [³H]PRZ binding only in lean rats in the ABL, DMN, ventromedial hypothalamic n. (VMN) and ventroposteromedial thalamic n. In most areas, decreases in maximal binding (B_max) associated with Adx were accompanied by decreases in K_d. Unlike [³H]PRZ binding, there was no consistent genotype difference in [³H]PAC binding although Adx was followed by increased binding in obese and decreased binding in lean rats in the ABL. In only the VMN, obese rats had a 21% higher α₂-toα₁-adrenoceptor ratio than lean White widespread differences in brain α₁-adrenoceptor binding between lean and obese rats may be important, the selectively higher VMN α₂-/α₁-ratio may be critical to the pathogenesis of obesity.     

Altered hypothalamic c-Fos-like immunoreactivity in diet-induced obese mice

High-fat diet can induce obesity. However, it is not known if the neural activity of the hypothalamus is altered under high-fat diet. The aim of the present study is to search for the altered hypothalamic neuronal activity in C57BI/6J mice fed a high-fat diet for 15 weeks. Hypothalamic c-Fos-like immunoreactivity (FLI) and serum leptin were measured after mice were fed a high-fat diet for 15 weeks. Our results demonstrate that increased body weight and serum leptin are accompanied by an elevated neuronal c-Fos-like immunoreactivity in the lateral hypothalamus, the lateral part of the dorsomedial hypothalamic and perifornical nuclei of diet-induced obese mice. Fasting increases FLI neurons in the arcuate hypothalamic nucleus and decreases FLI neurons in the lateral hypothalamic area and dorsomedial hypothalamic nucleus of both diet-induced obese and lean mice. The current data suggest that constantly activated status of these neurons in the hypothalamus may be responsible for differences in body weight and serum leptin between obese and lean mice.     

c-fos proto-oncogene change in relation to REM sleep duration

Auditory stimulation has been shown to increase REM sleep periods in cats and humans. This effect has been attributed to an elevation of the level of excitability in a variety of brain stem neuronal groups. Fos-like immunostaining (FLI) has been useful in constructing maps of post-synaptic neuronal activity with single cell resolution, and has been suggested to be tightly correlated with ongoing neuronal activity. This study used FLI to quantify neurons from structures expressing c-fos in brain stem areas in animals with normal REMs and compared them with those showing extended REM periods. The results basically indicated that brain stem areas which in other studies have been described as having REM-ON cells, showed an increase in FLI, while no FLI changes occurred in areas described as having REM-OFF cells. These results are discussed in terms of the possibility that REM maintenance is related to a widespread increase in brain stem excitability.     

Meal-induced changes in extracellular 5-HT in medial hypothalamus of lean (Fa/Fa) and obese (fa/fa) Zucker rats

Hypothalamic serotonin (5-HT) is involved in appetite regulation and sympathetic stimulation of thermogenesis. This study tested the hypothesis that the enhanced energetic efficiency of obese Zucker rats involves blunted serotonergic release within the medial hypothalamus (MH). We used microdialysis and HPLC–EC to measure dynamic changes in extracellular 5-HT levels in the MH of 10–13-week-old male lean (Fa/Fa) and obese (fa/fa) Zucker rats before and after a meal. No differences were noted in basal levels of 5-HT between lean and obese rats. Consistent with the suggestion that hypothalamic 5-HT plays a physiological role in feeding, extracellular 5-HT levels increased significantly in both lean and obese rats given a meal. This increase was observed in the 20 min interval in which they ate the 8.1 kcal meal and remained for an additional 60 min. The net release of 5-HT during the meal interval was comparable in the lean (1.46±0.38 fmol/μl) and obese (1.21±0.82 fmol/μl) rats. However, the 5-HT levels of the leans (1.80±0.29 fmol/μl) plateaued in the next 20 min interval, whereas they continued rising (2.74±0.53 fmol/μl) in obese rats and were significantly higher than those in the leans during the 40 and 60 min intervals after the meal was presented. This resulted in a total net release during the meal plus the next three 20 min intervals that was significantly higher in obese (9.83±1.16 fmol/μl) than in lean (5.59±0.85 fmol/μl) rats. Thus, the enhanced energetic efficiency of the obese Zucker rats may not be associated with attenuated serotonin release in response to a meal. Rather their enhanced release of 5-HT in the MH may reflect compensatory mechanisms for the elevated orexigen NPY, the reduction in meal-induced CCK release, and/or a functional resistance to 5-HT.     

Altered neuroanatomical organization in the central nervous system of the genetically obese (ob/ob) mouse

Genetically obese (C57BL/6J ob/ob) mice have significantly reduced brain weights (-14.6%) and cortical brain volumes (-7.9%) compared to lean control mice (C57BL/6J +/+). Morphometric analyses of soma cross-sectional areas of individual neurons in select brain region also reveals significant alterations in the ob/ob mouse. Neurons from 8 out of 9 brain regions, including the ventromedial hypothalamic nucleus, show significantly decreased soma cross-sectional areas in ob/ob mice compared to controls. Only lateral hypothalamic area neurons have equivalent soma cross-sectional areas for these two mouse strains. The decreased brain weight and volume coupled with the observed morphometric changes in individual neuronal soma size suggest that the ob/ob mouse brain differs considerably from that of controls. These differences may underlie some of the endocrine abnormalities seen in this genetic obesity syndrome.     

Increases in α-adrenergic receptors in the hypothalamus of the genetically obese mouse (ob/ob)

Despite significantly elevated hypothalamic norepinephrine levels, genetically obese mice (ob/ob) had more hypothalamic α-adrenergic receptors than their lean littermates. This receptor increase appeared to be specific to the α-receptors in the hypothalamus since no change was found in the number of α-receptors in the cortex or in the dopamine and muscarinic receptors in the cortex and striatum.     

Increased brain microvascular MMP-9 and incidence of haemorrhagic transformation in obese mice after experimental stroke

Obesity is an independent risk factor for stroke and is associated with poorer outcome after stroke. We investigated whether this poorer outcome is related to brain microvascular disruption. Focal cerebral ischaemia was induced in lean or obese (ob/ob) mice by transient middle cerebral artery occlusion. The incidence of haemorrhagic transformation and the volume of ischaemic brain damage were significantly greater in obese mice. Blood–brain barrier permeability and brain microvascular MMP-9 expression were also markedly increased in obese mice. These effects were independent of leptin or glycaemic status, suggesting that obesity potentiates brain microvascular disruption after experimental stroke.     

Seizures and Proto-Oncogene Expression offosin the Brain of Adult Genetically Epilepsy-Prone Rats

The mechanisms and brain circuitry that render genetically epilepsy-prone rats (GEPRs) susceptible to acoustically induced seizures are not completely known. The present study explores the neuroanatomy of acoustically induced seizures by immunohistochemical analysis of the proto-oncoproteinfosafter intense acoustic stimulation (AS) with and without seizures. Acoustic stimulation induced tonic convulsions in GEPR-9s, but not in control rats. Locations of brain nuclei showingfos-like immunoreactive (FLI) neurons following AS with and without seizures were mapped. Semiquantitative methods were used to compare FLI neuron numerical densities in AS control rats and GEPRs. Many brain areas exhibited profound FLI in AS control rats and GEPRs. Unexpectedly, the cochlear nuclei and the central nucleus of the inferior colliculi (ICc), both of which are requisite for AGS initiation, exhibited a diminishedfosexpression in animals having seizures compared to AS controls. In contrast, GEPRs displayed a significant increase in FLI neurons within the dorsal cortex of the IC (ICd) compared to AS controls. This finding may suggest a seizure-related amplification of the auditory signal between the ICc and the ICd. Other nuclei, known to be involved in auditory transmission (i.e., superior olivary complex; trapezoid nucleus; dorsal nucleus of the lateral lemniscus, DNLL), did not show differential FLI densities between seizure and AS control animals. In contrast, seizure-induced FLI was observed in many nonauditory brain nuclei. Of particular interest was the identification of an intensely labeled nucleus in the GEPR. This nucleus resides in the most posterior and dorsal–lateral part of the pedunculopontine tegmental nucleus–pars compacta (PPTn-pc) immediately adjacent to the DNLL and extends posteriorly into the superior lateral subnucleus of the lateral parabrachial area (SLPBn). Therefore, we have tentatively termed this nucleus the PPSLPBn. The PPSLPBn lies in a region previously described as a mesencephalic locomotor region and a suspected functional involvement of this nucleus in display of seizure activity is under investigation. Other brain stem nuclei showing differentialfosexpression between GEPRs and AS control rats are also described.     

Conditioned taste aversion and c-Fos expression in cholestatic rats

Rats in which a ligation of the bile duct (BDL) was paired with a saccharin taste developed a persistent conditioned taste aversion in both preference and taste reactivity tests. All BDL animals regardless of pairing had increased c-Fos-like immunoreactivity (FLI) in the area postrema and the nucleus of the solitary tract. This FLI may reflect the illness associated with BDL, but there was no evidence of conditioned FLI.     

Increased beta-endorphin and dynorphin concentrations in discrete hypothalamic regions of genetically obese (ob/ob) mice.

Disturbances in hypothalamic beta-endorphin and dynorphin levels were investigated in non-fasted genetically obese (ob/ob) and homozygous lean mice at 14-15 weeks of age. Eight brain regions were microdissected from fresh, unfixed brain slices, and opioid peptide concentrations were determined in tissue micropunches by radioimmunoassay. A two-fold and five-fold increase in beta-endorphin levels in ob/ob versus lean mice were found in the ventromedial and dorsomedial hypothalamic nuclei respectively. Dynorphin levels were comparable between ob/ob and lean mice in the anterior, lateral, ventromedial and paraventricular hypothalamic areas, but a 5-fold increase in dynorphin concentrations was detected in the dorsomedial hypothalamic nucleus of the ob/ob mouse. These results demonstrate that increased concentrations of beta-endorphin and dynorphin occur in discrete hypothalamic nuclei, which are known to influence food intake and glucose homeostasis. This could signify an important central defect contributing to hyperphagia and glucoregulatory dysfunction in obese mice.     

Induction of c-fos gene expression by spinal cord transection in Sus scrofa

Functional responses of primary sensory afferents and spinal cord were monitored in swine subjected to a high cervical (C1) spinal transection. Two and a half hours after transection, dorsal root ganglia and cervical and thoracolumbar spinal segments were processed immunocytochemically for the c-fos gene product, Fos and related antigens. In spinal-transected animals, Fos-like immunoreactivity (FLI) was induced in spinal laminae I, V, VII and X and the intermediolateral cell column but not in sensory ganglia as compared to controls: spinal-intact age-matched littermates. Spinal laminae expressing FLI harbor sympathetic and somatic interneurons and may aid in maintaining sympathetic outflow.     

Hypothalamic control of brown adipose tissue in Zucker lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres

Electrophysiological stimulation of the hypothalamic ventromedial nucleus (VMN) resulted in an increase in interscapular brown adipose tissue (BAT) temperature in both lean and obese (fa/fa) rats. Graded stimulations resulted in progressively larger temperature increases in both lean and obese (fa/fa) groups. Both intraperitoneal injection of propranolol and surgical denervation (but not sham denervation) abolished the increase in BAT temperature following VMN stimulation, in both lean and obese (fa/fa) groups. Electrical stimulation of the supraoptic region, and certain anterior hypothalamic regions also resulted in increases in BAT temperature of lean and obese (fa/fa) rats, but stimulation of the dorsomedial nucleus and regions of the lateral hypothalamus did not affect BAT temperature. All hypothalamic regions capable of activating BAT gave a similar maximum rise in temperature for a given stimulus in lean and obese (fa/fa) rats. These results suggest that the efferent sympathetic pathway from the VMN and other hypothalamic regions of BAT is normal in the obese (fa/fa) rat.     

Decreased striatal D2 dopamine receptors in obese Zucker rats: changes during aging

The specific binding of [³H]YM-09151-2 was used to investigate the possible differences in age-associated changes in striatal D₂ dopamine (DA) receptor properties in genetically obese (fa/fa) Zucker rats and their lean3(Fa/?) littermates. The maximal binding sites (B_max) of D₂ DA receptors was found to decline with age in both obese and lean rats: the rate of decline in receptor B_max was slightly higher in lean than obese rats. However. the B_max of D₂ DA receptor in 6-, 12- and 18-month-old obese rats was significantly lower compared to the age-matched lean rats. These data indicate that obesity decreases the number of striatal D₂ DA receptors without affecting the rate at which receptor number decreases with age.     

Nucleus Accumbens D1 Receptor–Expressing Spiny Projection Neurons Control Food Motivation and Obesity

BackgroundObesity is a chronic relapsing disorder that is caused by an excess of caloric intake relative to energy expenditure. There is growing recognition that food motivation is altered in people with obesity. However, it remains unclear how brain circuits that control food motivation are altered in obese animals.MethodsUsing a novel behavioral assay that quantifies work during food seeking, in vivo and ex vivo cell-specific recordings, and a synaptic blocking technique, we tested the hypothesis that activity of circuits promoting appetitive behavior in the core of the nucleus accumbens (NAc) is enhanced in the obese state, particularly during food seeking.ResultsWe first confirmed that mice made obese with ad libitum exposure to a high fat diet work harder than lean mice to obtain food, consistent with an increase in food motivation in obese mice. We observed greater activation of D₁ receptor–expressing NAc spiny projection neurons (NAc D1^SPNs) during food seeking in obese mice relative to lean mice. This enhanced activity was not observed in D₂ receptor–expressing neurons (D2^SPNs). Consistent with these in vivo findings, both intrinsic excitability and excitatory drive onto D1^SPNs were enhanced in obese mice relative to lean mice, and these measures were selective for D1^SPNs. Finally, blocking synaptic transmission from D1^SPNs, but not D2^SPNs, in the NAc core decreased physical work during food seeking and, critically, attenuated high fat diet–induced weight gain.ConclusionsThese experiments demonstrate the necessity of NAc core D1^SPNs in food motivation and the development of diet-induced obesity, establishing these neurons as a potential therapeutic target for preventing obesity.     

Fluorescent histochemical demonstration of catecholamines in brown adipose tissue from obese (ob/ob) and lean mice acclimated at different temperatures

Fluorescent histochemistry was used to visualize catecholamines in brown adipose tissue (BAT) of lean and genetically obese mice after they had been acclimated at different temperatures. At all temperatures, strong catecholamine-dependent fluorescence, attributable to the sympathetic innervation, was seen around the blood vessels of BAT from both lean and obese animals. Additionally, catecholamine-dependent fluorescent varicosities, in direct contact with the adipocytes were seen in abundance in lean mice acclimated at 23 degrees, 13 degrees or 4 degrees C and in obese mice acclimated at 13 degrees C. This latter compartment was greatly reduced in lean mice acclimated at 33 degrees C and in obese mice acclimated at 23 degrees and 33 degrees C. Three acute treatments (pretreatment with a monoamine oxidase inhibitor; 24 h food deprivation; and short-term cold exposure followed by short-term warm exposure) all increased the varicose fluorescence associated with adipocytes in obese mice housed at 23 degrees C, which suggests that the low resting level in these animals is attributable, at least in part, to subthreshold concentrations of catecholamines in existing varicosities rather than the absence of sympathetic varicosities per se. These results are in accordance with the results from noradrenaline turnover studies which suggest that the difference in sympathetic nervous system (SNS) activity in BAT from lean and obese (ob/ob) mice is best demonstrated at normal environmental temperatures. The reduced SNS activity in BAT of obese mice (which our studies show to be at the 'cellular' level) is likely to be a major factor in their reduced non-shivering thermogenesis and resultant high efficiency of energy storage as previously suggested by other workers.     

Effect of opiates on the release of cholecystokinin from in vitro hypothalamus and frontal cortex of Zucker lean (Fa/−) and obese (fa/fa) rats

Opiates, morphine and [d-Ala²-d-Leu⁵]-enkephalin (DADLE), inhibited the K⁺-stimulated release of cholecystokinin (CCK) from the hypothalamus of both Zucker obese (fa/fa) and lean (Fa/−) rats, in vitro. Morphine and DADLE did not inhibit the K⁺-stimulated release of CCK from frontal cortex from either strain. The opiates did not affect basal efflux of CCK and their effects were all blocked by equimolar concentrations of naloxone. These studies indicate a regional specificity for the effect of opiates on CCK release, and may provide evidence for a cellular mechanism by which endogenous opiates modulate feeding behavior.     

The effect of stimulus duration on noxious-stimulus induced c-fos expression in the rodent spinal cord

C-fos is a proto-oncogene that is expressed within some neurons following depolarization. The protein product,fos, has been proposed as an anatomical marker for neuronal activity following noxious peripheral stimulation. However, the literature on noxious-stimulus induced fos expression contains several puzzling observations on the time course and laminar distribution of neuronal labeling within the spinal cord. This study has analyzed the effect of stimulus duration on the expression of fos-like immunoreactivity (FLI) within the spinal cord of anesthetized rats. In order to examine the time course of fos expression following brief periods of stimulation, we required a type of stimulus that was intense enough to activate nociceptors but that did not produce tissue damage. We have therefore employed pulsed, high intensity electrical stimulation, with stimulus durations ranging from 3 s to 24 h. The results indicate that stimulus duration has a profound effect upon the number of labeled cells, the intensity of neuronal labeling, the laminar pattern of FLI, and the time course of fos expression. Brief stimulation periods induce relatively few and relatively lightly labeled neurons, located predominantly within the most superficial laminae of the dorsal horn. Maximal immunoreactivity appears approximately 2 h after stimulation has ceased, and disappears within hours. Continuous stimulation produces many more labeled cells, darker labeling, and FLI within both dorsal and ventral laminar regions. Maximal FLI is seen after approximately 4.5 h of continuous stimulation, with reduction in the number of labeled cells thereafter. These data indicate that the results of any study employing c-fos as a marker for neuronal activity may be affected by the duration of the exciting stimulus.     

Obese male mice (ob/ob) are normally sensitive to the satiating effect of CCK-8

The ob/ob mouse has a defect in short-term satiety mechanisms because ob/ob mice eat larger meals than leans and have abnormal postprandial behaviors. We suggested that this defect involved a failure to release CCK normally in response to ingested nutrients and/or decreased receptor sensitivity to CCK. McLaughlin and Baile reported that female obese mice were less sensitive to the satiating effect of CCK-8, a result consistent with the hypothesis of decreased receptor sensitivity. To investigate this possibility further, we determined the sensitivity to exogenous CCK-8 of obese and lean male mice. Adult male C57Bl/6J ob/ob and male +/+ controls were injected with CCK-8 (1, 2, 4, and 8 micrograms/kg, IP) 15 min prior to the presentation of solid food (Noyes pellets) after 4.5 hr food deprivation in the dark. Food intake (FI) was measured at 30 min and 150 min. CCK-8 decreased FI during the first 30 min in both obese and lean mice (p less than 0.01). The threshold dose for inhibition of FI was 2 micrograms/kg in obese and 4 micrograms/kg in lean. Since obese mice weighed approximately twice as much as lean mice, their total dose of CCK-8 was equal to that of lean mice. Thus, obese male mice were at least as sensitive to the satiating effect of CCK-8 as lean male mice. These results do not confirm McLaughlin and Baile's result in female mice eating in the light and they suggest that the defect in satiety in obese male mice is not the result of decreased sensitivity of CCK receptors.