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treated with combinations of opioids, anticonvulsants and antidepressants. Over the years, many animal models have been designed to explore neuropathic pain. One of the more common involves selectively damaging sensory fibers of a tributary of the sciatic nerve with a ligature, leading to mechanical allodynia and thermal hyperalgesia in the affected paw. Alternatively, investigators have used subcutaneous inflammatory agents, such as complete Freunds adjuvant (CFA) or formalin, to produce a peripheral inflammation with secondary changes in the plasticity of the CNS. Each method has some clinical predictability as well disadvantagesand some investigators examine both models, as in the current study. Earlier, this group showed that several genes were regulated in the dorsal horn of the spinal cord in rats following nerve injury5. Upregulated genes included that encoding GTP cyclohydrolase, the ratelimiting enzyme responsible for synthesizing BH4. Other genes, such as that encoding the serotonin 5HT3A receptor, were downregulated. In the current study, the authors confirmed the upregulation of GTP cyclohydrolase in primary sensory neurons of the dorsal root ganglion (DRG) following nerve injury, as well as more modest increases in sepiapterin reductase and quinoid dihydropteridine reductase, two other enzymes involved in the generation of BH4. They also observed increases in the levels of neuronal NO synthase and tryptophan hydroxylase, which produces serotonin. They then confirmed the role of BH4 in neuropathic and inflammatory pain directly by inhibiting GTP cyclohydrolase or administering BH4. Enzymatic cofactors are often considered mundane, lacking the excitement of neurotransmitters themselves. Therefore, a role of GTP cyclohydrolase and BH4 in pain modulation is an unexpected, but interesting, observation. Treating neuropathic pain often requires polypharmacy because of the limited efficacy of single agents3, consistent with the known involvement of many transmitters in this process4,6. Thus, GTP cyclohydrolase modulation of BH4 seems to be pulling together many of the various areas of investigation and providing a unifying link. The authors did not specifically address the role of monoamines in neuropathic pain even though BH4 modulated their synthesis in the current studybut they did address the role of NO. NO has long been implicated in pain modulation and opioid action7, but its actions can be complex. Although in vivo inhibitors of neuronal NO synthase reverse opioid tolerance and facilitate opioid analgesia, there are neuronal NO splice variants that have opposing actions8. In the current study, NO facilitated the hypersensitivity due to nerve injury and BH4 was important in regulating this activity. Lowering BH4 levels with a GTP cyclohydrolase inhibitor also reduced NO levels seen in the DRG following the injury, consistent with the known regulatory actions of BH4 on the enzyme. Inhibiting NO synthase also reversed hypersensitivity, providing further confirmation of the role of NO in neuropathic pain. The most intriguing aspect of the study is the correlation between the animal models and clinical pain. The authors identified a painprotective haplotype of the GCH1 gene with an allelic frequency of approximately 15%. This haplotype was associated with decreased pain sensitivity in low back pain patients following herniated disc surgery and in normal volunteers in an experimental pain model. Many highly reproducible and robust observations on pain mechanisms in animal models are difficult to replicate in clinical populations. Thus, it is surprising that the clinical associations in this paper are so clear. It will be important to confirm them in larger populations. Pain has often been thought to be multifactorial owing to the wide range of transmitter systems involved. The involvement of GCH1 and BH4 in several transmitter systems associated with neuropathic and inflammatory pain may help explain this unexpected robust correlation between the GCH1 polymorphisms and clinical pain sensitivity. This robust association should not be perceived as a problem but rather as a pleasant surprise. Clearly, more needs to be done. The experimental focus was limited almost entirely to the sensory neurons of the DRG, whereas pain is modulated throughout the neuroaxis. Also, as the authors point out, the specific molecular mechanisms by which the GCH1 haplotype exerts its pain protective effects need to be determined. Finally, the extension of this type of study to other types of pain is eagerly anticipated.
1. Tegeder, I. et al. Nat. Med. 12, 12691277 (2006). 2. Woolf, C.J. & Max, M.B. Anesthesiology 95, 241 249 (2001). 3. Inturrisi, C.E. Clin. J. Pain 18, S313 (2002). 4. Fields, H.L., Heinricher, M.M. & Mason, P. Annu. Rev. Neurosci. 14, 219245 (1991). 5. Costigan, M. et al. BMC Neurosci. 3, 16 (2002). 6. Dickenson, A.H. Br. J. Anaesth. 75, 193200 (1995). 7. Kitto, K.F., Haley, J.E. & Wilcox, G.L. Neurosci. Lett. 148, 15 (1992). 8. Kolesnikov, Y.A. et al. Proc. Natl. Acad. Sci. USA 94, 82208225 (1997).

2006 Nature Publishing Group http://www.nature.com/naturemedicine

Feeding signals and drugs meet in the midbrain

Richard Simerly
Three studies show that overeating may share neurobiological mechanisms with the addictive properties of drugs of abuse.
Motivation is a complex behavior that is influenced by both internal cues (autonomic sensory information and hormones) and environmental stimuli (aroma of savory foods). Both drug addiction and obesity are caused by patterns of behavior that are reinforced by repetitive rewards, in spite of adverse consequences. Animals seek food, and the rewarding properties of food strongly influence this behavior. Similarly, drug addiction relies on the action of compounds like cocaine to stimulate brain regions that convey pleasure, which provides motivation and promotes drug-seeking behavior. Two studies, in Neuron1,2 and the Journal of Clinical Investigation3, report that hormones known to regulate feeding also regulate neuronal activity in a key site in the brain that plays a central role in mediating drug addiction. Little is known about the neural mechanisms underlying motivational behavior associated with eating. But during drug-seeking behavior, certain compounds activate regions

Richard Simerly is at the Saban Research Institute, Childrens Hospital Los Angeles, University of Southern California, 4650 Sunset Boulevard, Los Angeles, California 90027, USA. E-mail: rsimerly@usc.edu

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Decision/learning

PFC Amygdala

Hippocampus

Behavior/feedback

Reward integration

Behavior

Nucleus accumbens

Ventral pallidum

Lateral hypothalamus

VTA
Dopamine

Leptin

ARH
Energy sensing

Ghrelin

PVH

DMH
Hypothalamic integration

Autonomic regulation

Figure 1 Major neural pathways impacted by leptin and ghrelin that mediate reward and energy balance. Both leptin and ghrelin regulate the activity of neurons in the ventral tegmental area (VTA) of the midbrain and arcuate nucleus of the hypothalamus (ARH). There is a convergence of opposing hormonal signals onto divergent neural pathways affecting motivated behavior and control of autonomic outflow from the hypothalamus. For additional detail on the organization and function of these pathways see refs. 8 and 9. DMH, dorsomedial nucleus of the hypothalamus; PFC, prefrontal cortex; PVH, paraventricular nucleus of the hypothalamus.

of the central nervous system that reinforce natural stimuli. A key neurobiological substrate involved in incentive motivation is the mesolimbic dopaminergic pathway. This pathway extends from dopamine neurons in the ventral tegmental area (VTA) of the midbrain to the nucleus accumbens (ACB), prefrontal cortex, hippocampus and amygdala of the forebrain (Fig. 1 and ref. 4). The VTA contains a discrete population of dopaminergic neurons that are activated by a variety of addictive substances including cocaine, amphetamine and alcohol. The behavioral sensitization that occurs with repeated exposure to drugs like amphetamine is associated with a hypersensitivity of mesolimbic dopaminergic neurotransmission in regions such as the ACB (ref. 4). This sensitization leads to a craving of substances in the absence of need or proportional benefit and pleasure5. Although it has been known for some time that energy balance is maintained through the regulation of body fat, our appreciation

of how peripheral signals such as leptin and ghrelin act on the brain to regulate ingestive behavior is relatively recent. Leptin is secreted by adipocytes into the blood in proportion to total body fat and acts on the hypothalamus to reduce food intake, increase energy expenditure and promote weight loss6. Ghrelin, in contrast, is secreted by endocrine cells in the stomach and is thought to function as an anticipatory signal that promotes feeding by regulating many of the same hypothalamic neuronal populations as leptin7. Animals increase food intake in response to negative energy balance, but will also eat independent of caloric valuedriven by the rewarding or hedonistic aspects of food8. Thus, both food and drugs powerfully reinforce behavior. Abundant evidence supports an incentive role for dopamine in the regulation of ingestive behavior as well as in the reinforcing properties of addictive drugs4,9. Dopaminedeficient mice die of starvation, and the ability of leptin to control food intake is dependent on dopamine10. Overlapping sites of action

are also suggested by the observation that leptin modulates incentive motivation11, drug-relapse behavior12 and amphetamineinduced locomotion13. The expression of leptin receptors by neurons in the VTA and the prominence of mesolimbic dopamine as a substrate for incentive motivation recently led two groups to study the potential role of leptin on dopaminergic neurotransmission in the VTA. The results not only identify the VTA as an important site for the regulation of feeding by leptin1, but also suggest that leptin is generally involved in the modulation of motivated behaviors2. To establish a direct mode of action on VTA dopamine neurons, Hommel et al.1 used duallabel histochemistry to show that dopamine neurons expressed leptin receptors. Moreover, either systemic or local injection of leptin activated janus kinasesignal transducer and activator of transcription (JAK-STAT) signaling in VTA neuronsas evidenced by the phosphorylation of STAT3, the signature of downstream leptin receptor signaling in the central nervous system. In addition, electrophysiological recordings showed that leptin reduced the firing rate of dopamine neurons in vitro and in vivo, suggesting that leptin exerts a direct inhibitory influence on dopamine neurons in the VTA (ref. 1). Injection of leptin directly into the VTA also reduced food intake, independent of locomotor activity, and virally mediated suppression of leptin receptor expression increased food intake. Knockdown of leptin receptors in the VTA also caused hyperphagia on a high-fat diet and increased consumption of a 0.2% sucrose solution, consistent with the notion that leptin increases incentive for highly palatable foods. In the same issue of Neuron, Fulton et al.2 show that dopamine and GABA neurons of the VTA both express leptin receptors. Moreover, the authors used retrograde labeling together with immunohistochemistry to show that leptin induces STAT3 phosphorylation, specifically within a subpopulation of VTA neurons that project to the ACB. In addition, they used a genetic approach to study leptins role in the regulation of mesolimbic dopamine. Dopamine levels were lower in the ACB of obese, leptin-deficient (ob/ob) mice, and leptin treatment increased the synthesis and activity of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in the VTA-to-ACB pathway of ob/ob mice. Furthermore, the authors used electrochemical methods to show that evoked dopamine release was decreased in ob/ob mice compared with wild-type controls, in the absence of differences in dopamine reuptake. Although this

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finding may seem difficult to reconcile with the leptin-induced reduction in VTA neuronal activity reported by Hommel et al.1, the differences may be due to alterations in dopamine receptor dynamics that may occur during lifelong leptin deficiency in ob/ob mice. Both studies, however, argue strongly that leptin regulates mesolimbic neuronal activity. Importantly, Fulton et al. show that leptin can enhance sensitization to amphetamine2, a phenomenon mediated by mesolimbic dopamine14. Leptin-deficient mice failed to show the locomotor sensitization normally seen after repeated amphetamine treatment, but behavioral sensitization was completely restored by chronic treatment of ob/ob mice with leptin2. Just as leptin and ghrelin appear to have opposing actions in the hypothalamus, the same relationship appears to hold in the mesolimbic dopamine pathway. In the Journal of Clinical Investigation, Abizaid et al.3 report that ghrelin triggers increased activity of dopaminergic neurons and enhances dopamine turnover in the VTA. As opposed to the inhibitory effects of leptin described in Neuron, Abizaid et al. show that local injection of ghrelin into the VTA stimulates food intake, in agreement with another recently published study15. Moreover, the enhanced feeding normally seen following systemic ghrelin treatment was blocked by infusion of a specific antagonist to the putative receptor for ghrelin, the growth hormone secetagogue receptor GHS-R1a (ref. 3). Furthermore, the stimulatory action of ghrelin on dopamine neurons was blocked by glutamate receptor antagonists but was unaffected by blockade of GABAergic neurotransmission, suggesting that the enhanced excitability caused by ghrelin depends on glutamatergic inputs. Moreover, morphological studies showed that ghrelin treatment increased overall numbers of synapses on dopamine neurons, with notable increases in what were presumably excitatory inputs (judged on morphological grounds), whereas those that appeared structurally to be GABAergic were reduced. Abizaid et al. found that neither the morphological changes nor the stimulatory effects of ghrelin on the activity of dopamine neurons in the VTA were apparent in GHSR1a knockout mice. Studies of how leptin and ghrelin regulate energy balance have focused primarily on the hypothalamus. The findings described in this trio of studies considerably expand the scope of functional neural systems affected by these important regulatory hormones. Because leptin receptors are expressed in several targets of VTA neurons and leptin has been implicated in hippocampal synaptic plasticity as well as in the regulation of mood16, leptin and ghrelin may modulate neural circuitry in a variety of sites that modulate motivation. Moreover, the convergent actions of leptin and ghrelin on the ability of mesolimbic dopaminergic neurons to regulate motivation suggest common cellular mechanisms for obesity and drug addictionand imaging studies in humans have identified common signaling events between the central actions of drugs of abuse and food-related stimuli4. Future investigations of how metabolic signals affect motivational behavior will be informed by the vast literature on mesolimbic dopamine and the neurobiology of addiction, which may result in common targets for therapeutic intervention.
1. Hommel, J.D. et al. Neuron 51, 801810 (2006). 2. Fulton, S. et al. Neuron 51, 811822 (2006). 3. Abizaid, A. et al. J. Clin. Invest. published online 20 October 2006 (doi: 10.1172/JCI29867). 4. Volkow, N.D. & Wise, R.A. Nat. Neurosci. 8, 555560 (2005). 5. Robinson, T.E. & Berridge, K.C. Brain Res. Brain Res. Rev. 18, 247291 (1993). 6. Benoit, S.C., Clegg, D.J., Seeley, R.J. & Woods, S.C. Recent Prog. Horm. Res. 59, 267285 (2004). 7. Zigman, J.M. & Elmquist, J.K. Endocrinology 144, 37493756 (2003). 8. Saper, C.B., Chou, T.C. & Elmquist, J.K. Neuron 36, 199211 (2002). 9. Kelley, A.E. Neurosci. Biobehav. Rev. 27, 765776 (2004). 10. Szczypka, M.S., Rainey, M.A. & Palmiter, R.D. Nat. Genet. 25, 102104 (2000). 11. Fulton, S., Richard, D., Woodside, B. & Shizgal, P. Behav. Brain Res. 155, 319329 (2004). 12. Figlewicz, D.P. et al. Behav. Neurosci. 118, 479487 (2004). 13. Hao, J., Cabeza de Vaca, S. & Carr, K.D. Physiol. Behav. 83, 377381 (2004). 14. Vezina, P. Neurosci. Biobehav. Rev. 27, 827839 (2004). 15. Naleid, A.M., Grace, M.K., Cummings, D.E. & Levine, A.S. Peptides 26, 22742279 (2005). 16. Lu, X.Y., Kim, C.S., Frazer, A. & Zhang, W. Proc. Natl. Acad. Sci. USA 103, 15931598 (2006).

2006 Nature Publishing Group http://www.nature.com/naturemedicine

Virus scores a perfect 10

Paul Klenerman & Burkhard Ludewig
Blockade of a single cytokineinterleukin-10can re-energize T cells exhausted by persistent virus infection. The findings have implications for controlling HIV and other persistent viruses (pages 13011309).
Forming a long-term relationship with a virus involves a tricky decision for the host. A strong immune response might lead to better control of the pathogen, but, at the same time, such responses can potentially cause substantial tissue damage, so sometimes a little
Burkhard Ludewig is in the Research Department, Kantonal Hospital St. Gallen, CH-9007 St. Gallen, Switzerland. Paul Klenerman is in the Nuffield Department of Medicine, Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Road, Oxford OX1 3SY, UK. E-mail: paul.klenerman@ndm.ox.ac.uk or Burkhard.Ludewig@kssg.ch

tolerance is required. A pair of recent studies have defined one key molecular switch in this important area of co-evolution between the host and virus. Brooks et al.1 and Ejrnaes et al.2 studied the immune responses to lymphocytic choriomeningitis virus (LCMV), a natural pathogen of mice and a versatile model for immunologists. LCMVs exist as diverse strains, and different combinations of virus and mouse strain lead to different infectious outcomes. In most cases, viral replication is rapidly controlled, largely through CD8+ T cells with the capacity to kill infected cells and secrete antiviral cytokines. However, certain LCMV strains, if given

in sufficient doses, can set up long-term persistence with viremia. In doing so, these virus strains overcome the CD8+ T-cell response, and it now seems that one negatively regulating cytokineinterleukin-10 (IL-10)plays a critical role in this process. These findings suggest that it might be possible to modulate the immune system in persistent virus infection in ways that improve the outcome for the host. Over a decade ago, Rolf Zinkernagels group observed that the development of long-term viremia after LCMV infection was associated with a decline in the number and function of antiviral CD8+ T cells3. This phenomenon

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