Yuanzhong Xu

An Obligate Role of Oxytocin Neurons in Diet Induced Energy Expenditure

Oxytocin neurons represent one of the major subsets of neurons in the paraventricular hypothalamus (PVH), a critical brain region for energy homeostasis. Despite substantial evidence supporting a role of oxytocin in body weight regulation, it remains controversial whether oxytocin neurons directly regulate body weight homeostasis, feeding or energy expenditure. Pharmacologic doses of oxytocin suppress feeding through a proposed melanocortin responsive projection from the PVH to the hindbrain. In contrast, deficiency in oxytocin or its receptor leads to reduced energy expenditure without feeding abnormalities. To test the physiological function of oxytocin neurons, we specifically ablated oxytocin neurons in adult mice. Our results show that oxytocin neuron ablation in adult animals has no effect on body weight, food intake or energy expenditure on a regular diet. Interestingly, male mice lacking oxytocin neurons are more sensitive to high fat diet-induced obesity due solely to reduced energy expenditure. In addition, despite a normal food intake, these mice exhibit a blunted food intake response to leptin administration. Thus, our study suggests that oxytocin neurons are required to resist the obesity associated with a high fat diet; but their role in feeding is permissive and can be compensated for by redundant pathways.

Calcitonin gene-related peptide dynamics in rat dorsal root ganglia and spinal cord following different sciatic nerve injuries.

We examined calcitonin gene-related peptide (CGRP) expression dynamics in the dorsal root ganglia (DRGs) and spinal cords of adult rats subjected to one of the following three types of unilateral sciatic nerve injury: crush (SNC), ligation (SNL), or transection combined with subsequent neurorrhaphy (SNT). Following SNC, CGRP immunoreactivity (IR) was increased in ipsilateral primary sensory neurons of L4-L5 DRGs, laminae I-II and spinal motoneurons; an area of CGRP-labeled fibers in ipsilateral laminae III-V was also increased in size following SNC. CGRP up-regulation exhibited a distinct temporospatial pattern and expression levels had returned to baseline levels by the end of the 28-day test period. Similar to SNC, SNT also resulted in an increase of CGRP-IR in these areas, though to a slightly lesser degree in the three latter areas. By contrast SNL, which is associated with complete blockade of axonal transport, induced a sustained decrease in CGRP-IR in primary sensory neurons of L4-L5 DRGs and superficial laminae (I-IIo), as well as in an ipsilateral area occupied by CGRP-labeled fibers. Interestingly, SNL did not affect CGRP-IR in spinal motoneurons, but did result in an accumulation of nerve growth factor (NGF) distal to ligature that was apparent as early as 1 day post-injury and persisted throughout the experimental period. These findings indicate that the nature of peripheral nerve injury has an impact on CGRP expression dynamics and that the response involves target tissues in vivo. Our results have important implications for elucidating the mechanisms of nerve regeneration.

Brain Expression of Cre Recombinase Driven by Pancreas-Specific Promoters

Cre‐loxP technology enables specific examination of the function and development of individual nuclei in the complex brain network. However, for most brain regions, the utilization of this technique has been hindered by the lack of mouse lines with Cre expression restricted to these regions. Here, we identified brain expressions of three transgenic Cre lines previously thought to be pancreas‐specific. Cre expression driven by the rat‐insulin promoter (Rip‐Cre) was found mainly in the arcuate nucleus, and to a lesser degree in other hypothalamic regions. Cre expression driven by the neurogenin 3 promoter (Ngn3‐Cre mice) was found in the ventromedial hypothalamus. Cre expression driven by the pancreas‐duodenum homeobox 1 promoter (Pdx1‐Cre) was found in several hypothalamic nuclei, the dorsal raphe and inferior olivary nuclei. Interestingly, Pdx1‐Cre mediated deletion of vesicular GABA transporter led to postnatal growth retardation while Ngn3‐Cre mediated deletion had no effects, suggesting a role for Pdx1‐Cre neurons, but not pancreas, in the regulation of postnatal growth. These results demonstrate the potential for these Cre lines to study the function and development of brain neurons. genesis 48:628–634, 2010.

Glutamate Mediates the Function of Melanocortin Receptor 4 on Sim1 Neurons in Body Weight Regulation

The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing brain regions, which include the paraventricular nucleus of hypothalamus (PVH), represent key brain sites that mediate melanocortin action. We conditionally restored MC4R expression in Sim1 neurons in the background of Mc4r-null mice. The restoration dramatically reduced obesity in Mc4r-null mice. The anti-obesity effect was completely reversed by selective disruption of glutamate release from those same Sim1 neurons. The reversal was caused by lower energy expenditure and hyperphagia. Corroboratively, selective disruption of glutamate release from adult PVH neurons led to rapid obesity development via reduced energy expenditure and hyperphagia. Thus, this study establishes glutamate as the primary neurotransmitter that mediates MC4Rs on Sim1 neurons in body weight regulation.

Cytokine-induced activation of glial cells in the mouse brain is enhanced at an advanced age.

Numerous neurological diseases which include neuroinflammatory components exhibit an age-related prevalence. The aging process is characterized by an increase of inflammatory mediators both systemically and in the brain, which may prime glial cells. However, little information is available on age-related changes in the glial response of the healthy aging brain to an inflammatory challenge. This problem was here examined using a mixture of the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha, which was injected intracerebroventricularly in young (2-3.5 months), middle-aged (10-11 months) and aged (18-21 months) mice. Vehicle (phosphate-buffered saline) was used as control. After a survival of 1 or 2 days (all age groups) or 4 days (young and middle-aged animals), immunohistochemically labeled astrocytes and microglia were investigated both qualitatively and quantitatively. In all age groups, astrocytes were markedly activated in periventricular as well as in deeper brain regions 2 days following cytokine treatment, whereas microglia activation was already evident at 24 h. Interestingly, cytokine-induced activation of both astrocytes and microglia was significantly more marked in the brain of aged animals, in which it included numerous ameboid microglia, than of younger age groups. Moderate astrocytic activation was also seen in the hippocampal CA1 field of vehicle-treated aged mice. FluoroJade B histochemistry and the terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling technique, performed at 2 days after cytokine administration, did not reveal ongoing cell death phenomena in young or aged animals. This indicated that glial cell changes were not secondary to neuronal death. Altogether, the findings demonstrate for the first time enhanced activation of glial cells in the old brain, compared with young and middle-aged subjects, in response to cytokine exposure. Interestingly, the results also suggest that such enhancement does not develop gradually since youth, but appears characterized by relatively late onset.

Role of GABA Release From Leptin Receptor-Expressing Neurons in Body Weight Regulation

It is well established that leptin regulates energy balance largely through isoform B leptin receptor-expressing neurons (LepR neurons) in the brain and that leptin activates one subset of LepR neurons (leptin-excited neurons) while inhibiting the other (leptin-inhibited neurons). However, the neurotransmitters released from LepR neurons that mediate leptin action in the brain are not well understood. Previous results demonstrate that leptin mainly acts on γ-aminobutyric acid (GABA)ergic neurons to reduce body weight, and that leptin activates proopiomelanocortin neuron activity by reducing GABA release onto these neurons, suggesting a body weight-promoting role for GABA released from leptin-inhibited neurons. To directly examine the role of GABA release from LepR neurons in body weight regulation, mice with disruption of GABA release specifically from LepR neurons were generated by deletion of vesicular GABA transporter in LepR neurons. Interestingly, these mice developed mild obesity on chow diet and were sensitive to diet-induced obesity, which were associated with higher food intake and lower energy expenditure. Moreover, these mice showed blunted responses in both food intake and body weight to acute leptin administration. These results demonstrate that GABA plays an important role in mediating leptin action. In combination with the previous studies that leptin reduces GABA release onto proopiomelanocortin neurons through leptin-inhibited neurons and that disruption of GABA release from agouti gene-related protein neurons, one subset of LepR-inhibited neurons, leads to a lean phenotype, our results suggest that, under our experimental conditions, GABA release from leptin-excited neuron dominates over leptin-inhibited ones.

Dynamic changes in Robo2 and Slit1 expression in adult rat dorsal root ganglion and sciatic nerve after peripheral and central axonal injury

Robos are transmembrane receptors that mediate Slit signaling to repel growth cone outgrowth and neural migration in the developing central nervous system. Their distribution and function in the peripheral nervous system remains unclear. In the present study, we examined expression of Slit1 and Robo2 in adult rat dorsal root ganglion (DRG), spinal cord and sciatic nerve after peripheral nerve injury (axotomy). In control rats, Slit1 and Robo2 mRNA and protein were expressed at basic levels in the L5 and L6 DRGs. Sciatic transection resulted in a significant up-regulation of both Robo2 and Slit1 mRNA and protein (p<0.05 versus control). The peak of Slit1 and Robo2 expression occurred at days 7 and 14, respectively, and returned to control levels at days 28 and 21 post-axotomy, respectively. By contrast, injury to the central axons of the DRG by dorsal rhizotomy did not up-regulate Slit1 and Robo2 expression. Robo2 staining was stronger in small diameter neurons than in large diameter neurons in control DRG. Interestingly, post-axotomy, Robo2 immunostaining increased in the large diameter neurons and the number of Robo2 positive large diameter neurons increased significantly relative to controls. Non-neuronal cells surrounding the primary sensory neurons, including the satellite cells, were Slit1-positive, and Slit1 protein was expressed in the myelin sheath and non-neural cells in both intact and degenerating sciatic nerve axons. Sciatic nerve transection also led to an accumulation of Slit1 protein in peripheral region of the traumatic neuroma. In conclusion, we report an altered expression and redistribution of Robo2 and Slit1 in the DRG and sciatic nerve trunk after peripheral axotomy. Our results indicate that Slit1 and Robo2 likely play an important role in regeneration after peripheral nerve injury.

Glutamate release mediates leptin action on energy expenditure

Restricting energy expenditure is an adaptive response to food shortage. Despite being insulated with massive amount of fat tissues, leptin-deficient mice lose the ability to maintain their body temperature and develop deep hypothermia, which can be suppressed by exogenous leptin, suggesting an important role for leptin in energy expenditure regulation. However, the mechanism underlying the leptin action is not clear. We generated mice with disruption of glutamate release from leptin receptor-expressing neurons by deleting vesicular glutamate transporter 2 in these neurons, and found that these mice developed mild obesity purely due to reduced energy expenditure, exhibited bouts of rapidly reduced energy expenditure, body temperature and locomotion. In addition, these mice exhibited lower energy expenditure and body temperature in response to fasting and were defective in leptin-mediated thermogenic action in brown adipose tissues. Taken together, our results identify a role for glutamate release in mediating leptin action on energy expenditure.

Glial transcripts and immune-challenged glia in the suprachiasmatic nucleus of young and aged mice.

Biological rhythms are frequently disturbed with advancing age, and aging-related changes of glia in the hypothalamic suprachiasmatic nucleus (SCN), the master circadian pacemaker, require special attention. In particular, astrocytes contribute to SCN function, and aging is associated with increased inflammatory activity in the brain, in which microglia could be especially implicated. On this basis, we investigated in the SCN of young and old mice glial transcripts and cell features, and the glial cell response to a central inflammatory challenge. Quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR) was used to analyze the expression of mRNAs encoding the astrocytic glial fibrillary acidic protein and the microglial antigen CD11b. Both these transcripts, here investigated in the SCN for the first time, were significantly increased in the old SCN. Glial cell phenotyping with immunohistochemistry revealed hypertrophic and intensely stained astrocytes and microglia in the aged SCN. In both age groups, microglia were scattered throughout the SCN and astrocytes were prominent in the ventral portion, where retinal fibers are densest; in the aged SCN, astrocytes were also numerous in the dorsal portion. After intracerebroventricular injections of a mixture of interferon-γ and tumor necrosis factor-α, or phosphate-buffered saline as control, immunolabeling was evaluated with stereological cell counts and confocal microscopy. Phenotypic features of astrocyte and microglia activation in response to cytokine injections were markedly enhanced in the aged SCN. Subregional variations in glial cell density were also documented in the aged compared to the young SCN. Altogether, the findings show increases in the expression of glial transcripts and hypertrophy of astrocytes and microglia in the aged SCN, as well as age-dependent variation in the responses of immune-challenged SCN glia. The data thus point out an involvement of glia in aging-related changes of the biological clock. (Author correspondence: marina.bentivoglio@univr.it)

Regulation of cytokine signaling and T-cell recruitment in the aging mouse brain in response to central inflammatory challenge

Aging is often accompanied by increased levels of inflammatory molecules in the organism, but age-related changes in the brain response to inflammatory challenges still require clarification. We here investigated in mice whether cytokine signaling and T-cell neuroinvasion undergo age-related changes. We first analyzed the expression of molecules involved in T-cell infiltration and cytokine signaling regulation in the septum and hippocampus of 2-3 months and 20- to 24-month-old mice at 4h after intracerebroventricular injections of tumor necrosis factor (TNF)-alpha or interferon-gammaversus saline injections. Transcripts of the chemokine CXCL9, intercellular adhesion molecule (ICAM)-1 and suppressor of cytokine signaling molecules (SOCS) 1 and 3 were increased in both age groups after cytokine injection; microglia-derived matrix metalloproteinase (MMP) 12 mRNA was induced in old mice also after control saline injections. Age-related changes in ICAM-1 protein expression and T-cell infiltration were then analyzed in mice of 3-4, 8-9 and 15-16 months at 48h after TNF-alpha injections. ICAM-1 immunoreactivity, and Western blotting in striatum, septum, hippocampus and hypothalamus showed progressive age-related enhancement of TNF-alpha-elicited ICAM-1 upregulation. Double immunofluorescence revealed ICAM-1 expression in microglia and astrocytic processes. CD3(+), CD4(+) and CD8(+) T-cells exhibited progressive age-related increases in brain parenchyma and choroid plexus after cytokine exposure. The findings indicate that the brain responses to inflammatory challenges are not only preserved with advancing age, but also include gradual amplification of ICAM-1 expression and T-cell recruitment. The data highlight molecular and cellular correlates of age-related increase of brain sensitivity to inflammatory stimuli, which could be involved in altered brain vulnerability during aging.