PATHWAYS OF CLINICAL FUNCTION AND DISABILITY 168 forebrains have been characterized (Zhuo, Gebhart 1990a, 1990b, 1991, 1992, 1997; Calejesan, Kim, Zhuo 2000). Biphasic modulation of spinal nociceptive trans- mission from the RVM, perhaps re ecting the differ- ent types of neurons identi ed in this area, offer  ne regulation of spinal sensory thresholds and responses. While descending inhibition is primarily involved in regulating suprathreshold responses to noxious stimuli, descending facilitation reduces the neuronal thresh- old to nociceptive stimulation (Zhuo, Gebhart 1990a, 1990b, 1991, 1992, 1997). Descending facilitation has a general impact on spinal sensory transmission, induc- ing sensory inputs from cutaneous and visceral organs (Zhuo, Sengupta, Gebhart 2002; Zhuo, Gebhart 2002; Zhuo 2007) (Fig. 6.13). Descending facilitation can be activated under physiological conditions, and one physiological function of descending facilitation is to enhance the ability of animals to detect potential dan- gerous signals in the environment. Indeed, neurons in the RVM not only respond to noxious stimuli, but also show “learning”-type changes during repetitive noxious stimuli. More importantly, RVM neurons can undergo plastic changes during and after tissue injury and in ammation. ACC-Induced Facilitation It is well documented that the descending endogenous analgesia system, including the PAG and RVM, plays an important role in modulation of nociceptive trans- mission and morphine- and cannabinoid-produced analgesia. Neurons in the PAG receive inputs from different nuclei of higher structures, including the cingulated ACC. Electrical stimulation of ACC at high intensities (up to 500 µA) of electrical stimulation did not produce any antinociceptive effect. Instead, at most sites within the ACC, electrical stimulation produced signi cant facilitation of the TF re ex (i.e. decreases in TF latency). Activation of mGluRs within the ACC also produced facilitatory effects in both anesthetized rats or freely moving mice (Calejesan, Kim, Zhuo 2000; Tang et al. 2006). Descending facil- itation from the ACC apparently relays at the RVM (Calejesan, Kim, Zhuo 2000) (see Fig. 6.14). Descending Facilitation Maintains Chronic Pain Descending facilitation is likely activated after the injury, contributing to secondary hyperalge- sia (Calejesan, Ch’ang, Zhuo 1998; Robinson et al. 2002b). Blocking descending facilitation by lesion of the RVM or spinal blockade of serotonin receptors is antinociceptive (Urban, Gebhart 1999; Porreca, Ossipov, Gebhart 2002; Robinson et al. 2004). The descending facilitatory system therefore serves as a 30 mmHg 30 mmHg Control 10 Hz 10 s Electrical stimulation A B C Glutamate 600 Total no. imps/20 s Response to distention Glutamate (5 nmoles) 400 200 0 c stim 25 µA P 10.30 NGC NRM LC Sp5 NPGCL 1 mm Pyr NGCα 01 4 Time (min) 710 Figure 6.13 Descending facilitation of spinal visceral pain transmission. Example of facilitation of spinal visceral transmission produced by electrical stimulation and glutamate in the nucleus raphe magnus (NRM). (A) Peristimulus time histograms (1-second binwidth) and cor- responding ocillographic records in the absence (top histograms) and presence (bottom histograms) of electrical stimulation (25 µA) and glutamate (5 nmoles) given in the same site in NRM. The intensity and duration of colorectal distension is illustrated below; the period of electrical stimulation (25 seconds) is indicated by the arrows. (B) Summary of the data illustrated in (A) and time course of effect of gluta- mate given in NRM. The point above c represents the response to 30-mmHg colorectal distension; the point above stimulation represents the response to the same intensity of distension during stimulation in NRM. (C) Site of stimulation and injection of glutamate. Chapter 6: Neurobiology of Chronic Pain 169 7 AB C 6 5 4 3 –15 30 20 10 –10 –20 Spinal cord +: 5-HT RVM ACC Descending facilitation 0 –10 –5 0 5 10 Time, min Pre CNQX * Recovery TF latency, s Facilitation (%) CNQX (0.5 µL) 15 20 25 30 35 40 45 50 Without stimulation Stimulation at 50 µA Figure 6.14 ACC controls RVM-generated descending facilitation. (A) A model shows supraspinal control of RVM-generated descending facilitation of spinal nociception by ACC) neurons. (B) An example illustrates that CNQX microinjection into the RVM reversibly blocks facilitation of the TF refl ex produced by electrical stimulation at a site within the ACC; TF response latencies measured without stimula- tion were represented by open squares. TF latencies measured with stimulation were represented by fi lled squares; (C) Summary data showing mean facilitation (% of control) before CNQX injection into the RVM (Pre); after (within 10 minutes); and 30 minutes after (30 min post). double-edged blade in the central nervous system. On one hand, it allows neurons in different parts of the brain to communicate with each other and enhances sensitivity to potentially dangerous signals; on the other hand, prolonged facilitation of spinal nocicep- tive transmission after injury speeds up central plastic changes related to chronic pain (Table 6.3). CONCLUSIONS AND FUTURE DIRECTIONS Finally, I would like to review and propose three key cellular models for future investigations of chronic pain. I would like to emphasize that integrative experimental approaches are essential for future studies to avoid the misleading discoveries; work at different sensory synapses are equally critical such as spinal cord synapses, cortical synapse, and brainstem synapses that dictate descending facilita- tory and inhibitory modulations. Table 6.4 summa- rizes likely key mechanisms for chronic pain. They include Plasticity of sensory synaptic transmission: excitatory 1. (glutamate) and inhibitory (GABA, Gly) transmission Anatomic structural changes: synaptic reorganiza-2. tion (e.g. changes in spines), cortical reorganization, neuronal phenotype switch, cortical gray matter loss Long-term alteration in descending modulation: 3. enhanced descending facilitation or loss of tonic descending inhibitory in uences In summary, progress made in basic neurobiology investigations has signi cantly helped us understand the fundamental mechanism for pain or physiologi- cal pain processes, both at the peripheral spinal cord level and at the cortical level. Studies of central plas- ticity, including LTP/LTD in sensory synapses, start to provide useful cellular models for our understanding of chronic pain. Novel mechanisms revealed at molec- ular and cellular levels will signi cantly affect our future approaches to search and design novel drugs for treating chronic pain in patients. A I thank funding supports from the EJLB-CIHR Michael Smith Chair in Neurosciences PATHWAYS OF CLINICAL FUNCTION AND DISABILITY 170 Basbaum AI, Fields HL. 1984. Endogenous pain control system: brainstem spinal pathways and endorphin cir- cuitry. Annu Rev Neurosci. 7:309–338. Birbaumer N, Lutzenberger W, Montoya P et al. 1997. Effects of regional anesthesia on phantom limb pain are mirrored in changes in cortical reorganization. J Neurosci. 17:5503 –5508. Bredt DS, Nicoll RA. 2003. AMPA receptor traf cking at excitatory synapses. Neuron. 40:361–379. Calejesan AA, Ch’ang MH-C, Zhuo M. 1998. Spinal sero- tonergic receptors mediate facilitation of a nociecep- tive re ex by subcutaneous formalin injection into the hindpaw in rats. Brain Res. 798:46–54. and Mental Health in Canada, CIHR operating grants, Canada Research Chair, and NeuroCanada Brain repair program. REFERENCES Apkarian AV, Sosa Y, Sonty S et al. 2004. Chronic back pain is associated with decreased prefrontal and thalamic gray matter density. J Neurosci. 24:10410–10415. Bardoni R, Magherini PC, MacDermott AB. 1998. NMDA EPSCs at glutamatergic synapses in the spinal cord dor- sal horn of the postnatal rat. J Neurosci. 18:6558–6567. Table 6.4 Proposed Key Neurobiological Mechanisms for Chronic Pain Proposed Model Synaptic Consequences Key References Plasticity of synaptic transmission Silent synapse LTP Loss of LTD Microglia disinhibition Recruit AMPA responses into NS speci c cells Enhanced existing AMPA responses GluR1 mediated LTP Enhanced glutamate release Fail to depotentiate enhanced responses Switching GABA currents Li, Zhuo 1998 Ikeda et al. 2003 Zhao et al. 2005 Zhao et al. 2006 Wei et al. 1999 Coull et al. 2003 Structural reorganization Phenotype switch Structural sprouting Cortical reorganization Neuronal cell death Neurons making new transmitters such as SP Sprouting  bers Growth of new cortical connections Loss of neurons due to cell death Woolf et al. 1992 Neumann et al. 1996 Flor et al. 1995 Apkarian et al. 2004 Altered descending modulation Loss of descending inhibition Enhanced descending facilitation Activity in the PAG, RVM neuron failed to produce analgesic effects in the spinal cord Enhanced facilitatory in uences from the ACC and RVM Wei et al. 1999 Robinson et al. 2002 Urban et al. 1999 Calejesan et al. 2000 Zhuo, Gebhart 1997 Table 6.3 Comparison of Endogenous Facilitation and Analgesia Systems Descending Facilitation Descending Analgesia Central origin ACC; RVM PAG; RVM Neurotransmitter Glutamate; neurotension Glutamate; opioids Stimulation intensity 5–25 µA 50–100 µA Stimulation–response Function (SRF) Reduced threshold Reduced peak response without affecting threshold Response latency 200 ms 90 ms Laterality Bilateral Bilateral Spinal pathways Ventrolateral funiculi (VLF)/ventral funiculi (VF) Dorsolateral funiculi (DLF) Spinal neurotransmitter 5-HT Ach; NE; 5-HT Synaptic mechanism AMPA receptor traf cking Enhanced AMPA receptor–mediated EPSCs Inhibit presynaptic transmitter release; reduced AMPA receptor–mediated EPSCs Sensory modality Non-nociceptive Nociceptive Mechanical Thermal Non-nociceptive Nociceptive Mechanical Thermal Origin of sensory inputs Somatosensory Visceral Somatosensory Visceral Chapter 6: Neurobiology of Chronic Pain 171 dorsal horn neurones of the rat spinal cord. J Physiol. 492:867–876. Hutchison WD, Davis KD, Lozano AM, Tasker RR, Dostrovsky JO. 1999. Pain-related neurons in the human cingulate cortex. Nat Neurosci. 2:403–405. Ikeda H, Heinke B, Ruscheweyh R, Sandkuhler J. 2003. Synaptic plasticity in spinal lamina I projection neurons that mediate hyperalgesia. Science. 299:1237–1240. Jasmin L, Rabkin S, Granato A, Boudah A, Ohara P. 2003. Analgesia and hyperalgesia from GABA-mediated modulation of the cerebral cortex. Nature. 424(6946): 316–320 Ji RR, Kohno T, Moore KA, Woolf CJ. 2003. Central sensi- tization and LTP: do pain and memory share similar mechanisms? Trends Neurosci. 26:696–705. Johansen JP, Fields HL, Manning BH. 2002. The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulated cortex. Proc Natl Acad Sci U S A. 98:8077–8082. Jones EG, Pons TP. 1998. Thalamic and brainstem contribu- tions to large-scale plasticity of primate somatosensory cortex. Science. 282:1121–1125. Kaas JH. 1998. Phantoms of the brain. Nature. 391:331–333 Kaas JH, Florence SL, Jain N. 1999. Subcortical contributions to massive cortical reorganizations. Neuron. 22:657–660. Kempermann G, Kuhn HG, Gage FH. 1997. More hippo- campal neurons in adult mice living in an enriched environment. Nature. 386:493–495 Kerchner GA, Wei F, Wang G-D et al. 2001. ‘smart’ mice feel more pain, or are they just better learners? Nat Neurosci. 4:453–454. Kerchner GA, Wang GD, Qiu C-S, Huettner JE, Zhuo M. 2001a. Direct presynaptic regulation of GABA/Glycine release by kainate receptors in the dorsal horn: an ionotropic mechanism. Neuron. 32:477–488. Kerchner GA, Wilding TJ, Li P, Zhuo M, Huettner JE. 2001b. Presynaptic kainate receptors regulate spinal sensory transmission. J Neuroscience. 21:59–66. Kerchner GA, Wilding TJ, Huettner JE, Zhuo M. 2002. Kainate receptor subunits underlying presynaptic regulation of transmission release in the dorsal horn. J Neuroscience. 22:8010–8017. Kohno T, Kumamoto E, Higashi H, Shimoji K, Yoshimura M. 1999. Actions of opioids on excitatory and inhibitory transmission in substantia gelatinosa of adult at spinal cord. J Physiol. 518:803–813. Lee DE, Kim SJ, Zhuo M. 1999. Comparison of behavioral responses to noxious cold and heat in mice. Brain Res. 845:117–121. Li P, Calejesan AA, Zhuo M. 1998. ATP P 2X receptors and sensory transmission between primary afferent  bers and spinal dorsal horn neurons in rats. J Neurophysiol. 80:3356–3360. Li P, Zhuo M. 1998. Silent glutamatergic synapses and noci- ception in mammalian spinal cord. Nature. 393:695–698. Li P, Wilding TJ, Kim SJ, Calejesan AA, Huettner JE, Zhuo M. 1999a. Kainate-receptor-mediated sensory synaptic transmission in mammalian spinal cord. Nature. 397:161–164. Li P, Kerchner GA, Sala C et al. 1999b. AMPA receptor- PDZ protein interaction mediate synaptic plasticity in spinal cord. Nat Neurosci. 2:972–977. Calejesan AA, Kim SJ, Zhuo M. 2000. Descending facilita- tory modulation of a behavioral nociceptive response by stimulation in the adult rat anterior cingulate cor- tex. Eur J Pain. 4:83–96. Carroll RC, Beattie EC, von Zastrow M, Malenka RC. 2001. Role of AMPA receptor endocytosis in synaptic plastic- ity. Nat Rev Neurosci. 2(5):315–324. Casey KL. 1999. Forebrain mechanisms of nociception and pain: analysis through imaging. Proc Natl Acad Sci U S A. 96:7668–7674. Caterina MJ, Julius D. 2001. The vanilloid receptor: a molec- ular gateway to the pain pathway. Annu Rev Neurosci. 24:487–517. Colburn RW, Lubin ML, Stone DJ Jr et al. 2007. Attenuated cold sensitivity in TRPM8 null mice. Neuron. 54:379–386. Coull JA, Boudreau D, Bachand K et al. 2003. Trans- synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature. 424:938–942. Dahlqvist P, Zhao L, Johansson IM et al. 1999. Environmen- tal enrichment alters nerve growth factor-induced gene A and glucocorticoid receptor messenger RNA expression after middle cerebral artery occlusion in rats. Neuroscience. 93:527–535. Dhaka A, Murray AN, Mathur J, Earley TJ, Petrus MJ, Patapoutian A. 2007. TRPM8 is required for cold sensation in mice. Neuron. 54:371–378. Dong H, O’Brien RJ, Fung ET, Lanahan AA, Worley PF, Huganir RL. 1997. GRIP: a synaptic PDZ domain- containing protein that interacts with AMPA recep- tors. Nature. 386:279–284. Dong H, Zhang P, Song I, Petralia RS, Liao D, Huganir RL. 1999. Characterization of the glutamate receptor- interacting proteins GRIP1 and GRIP2. J Neurosci. 19:6930–6941. Duffy SN, Craddock KJ, Abel T, Nguyen PV. 2001. Environ- mental enrichment modi es the PKA-dependence of hippocampal LTP and improves hippocampus- dependent memory. Learn Mem. 8:26–34 Flor H, Elbert T, Knetcht S et al. 1995. Phantom-limb pain as a perceptual correlate of cortical reorganization following arm amputation. Nature. 375:482–484. Flor H, Nikolajsen L, Jensen TS. 2006. Phantom limb pain: a case of maladaptive CNS plasticity? Nat Rev Neurosci. 7(11):873–81. Florence SL, Taub HB, Kaas JH. 1998. Large-scale sprout- ing of cortical connections after peripheral injury in adult macaque monkeys. Science. 282:1117–1121 Gebhart GF. 1986. Modulatory effects of descending sys- tems on spinal dorsal horn neurons, In TL. Yaksh, ed. Spinal Afferent Processing. New York: Plenum Press, 391–426. Gebhart GF, Randich A. 1990. Brainstem modulation of nociception, In WR. Klemm and RP. Vettes, eds. Brainstem Mechanisms of Behavior. Wiley and Sons: New York, 315–352. Grudt TJ, Williams JT, Travagli RA. 1995. Inhibition by 5-hydroxytryptamine and noradrenaline in substan- tia gelatinosa of guinea-pig spinal trigeminal nucleus. J Physiol. 485:113–120. Hori Y, Endo K, Takahashi T. 1996. Long-lasting synap- tic facilitation induced by serotonin in super cial PATHWAYS OF CLINICAL FUNCTION AND DISABILITY 172 Ramachandran VS, Rogers-Ramachandran DC, Stewart M. 1992. Perceptual correlates of massive cortical reorga- nization. Science. 258:1159–1160. Ramachandran VS, Rogers-Ramachandran DC, Cobb S. 1995. Touching the phantom limb. Nature. 377:489–490 Rampon C, Tang YP, Goodhouse J, Shimizu E, Kyin M, Tsien JZ. 2000. Enrichment induces structural changes and recovery from nonspatial memory de cits in CA1 NMDAR1-knockout mice. Nat Neurosci. 3:238–244 Ribeiro-da-Silva A, Coimbra A. 1982. Two types of synaptic glomeruli and their distribution in laminae I–III of the rat spinal cord. J Comp Neurol. 209:176–186. Robinson DA, Wei F, Wang GD et al. 2002a. Oxytocin mediates stress-induced analgesia. J Physiol. (Lond). 540:593–606. Robinson D, Calejesan AA, Zhuo M. 2002b. Long-lasting changes in rostral ventral medulla neuronal activity following in ammation. J Pain. 3:292–300. Robinson DA, Calejesan AA, Wei F, Gebhart GF, Zhuo M. 2004. Endogenous facilitation: from molecular mecha- nisms to persistent pain. Curr Neurovasc Res. 1:11–20. Shumyatsky GP, Tsvetkov E, Malleret G et al. 2002. Identi cation of a signaling network in lateral nucleus of amygdala important for inhibiting memory speci - cally related to learned fear. Cell. 111:905–918 Sikes RW, Vogt BA. 1992. Nociceptive neurons in area 24 of rabbit cingulate cortex. J Neurophysiol. 68:1720–1732. Song I, Huganir RL. 2002. Regulation of AMPA receptors during synaptic plasticity. Trends Neurosci. 25:578–588. Sun YG, Chen ZF. 2007. A gastrin-releasing peptide recep- tor mediates the itch sensation in the spinal cord. Nature. 448:700–703. Tachibana M, Wenthold RJ, Morioka H, Petralia RS. 1994. Light and electron microscopic immunocy- tochemical localization of AMPA-selective gluta- mate receptors in the rat spinal cord. J Comp Neurol. 344:431–454. Talbot JD, Marrett S, Evans AC, Meyer E, Bushnell MC, Duncan GH. 1991. Multiple representations of pain in human cerebral cortex. Science. 251:1355–1358. Tang YP, Xhimizu E, Dube GR et al. 1999. Genetic enhancement of learning and memory in mice. Nature. 401:63–69. Tang J, Ko S, Ding HK, Qiu CS, Calejesan AA, and Zhuo, M. 2005. Pavlovian fear memory induced by activation in the anterior cingulate cortex. Mol Pain. 1, 6. Tao YX, Rumbaugh G, Wang GD et al. 2003. Impaired NMDA receptor-mediated postsynaptic function and blunted NMDA receptor-dependent persistent pain in mice lacking postsynaptic density-93 protein. J Neurosci. 23:6703–6712. Todd AJ. 1996. GABA and glycine in synaptic glomeruli of the rat spinal dorsal horn. Eur J Neurosci. 8:2492–2498. Toyoda H, Wu LJ, Zhao MG, Xu H, Zhuo M. 2007a. Time- dependent postsynaptic AMPA GluR1 receptor recruit- ment in the cingulate synaptic potentiation. Dev Neurobiol. 67:498–509. Toyoda H, Wu LJ, Zhao MG, Xu H, Jia Z, Zhuo M. 2007b. Long-term depression requires postsynaptic AMPA GluR2 receptor in adult mouse cingulate cortex. J Cell Physiol. 211:336–343. Li P, Zhuo M. 2001. Substance P and neurokinin A mediate sensory synaptic transmission in young rat dorsal horn neurons. Brain Res Bull. 55:521–531. Liauw J, Wang GD, Zhuo M. 2003. NMDA receptors con- tribute to synaptic transmission in anterior cingulate cortex of adult mice. Sheng Li Xue Bao. 55:373–380. Liauw J, Wu LJ, Zhuo M. 2005. Calcium-stimulated adeny- lyl cyclases required for long-term potentiation in the anterior cingulate cortex. J Neurophysiol. 94:878–882. Lorenz J, Kohlhoff H, Hansen HC, Kunze K, Bromm B. 1998. Abeta- ber mediated activation of cingulate cor- tex as correlate of central post-stroke pain. Neuroreport. 9:659–663. Lumpkin EA, Caterina MJ. 2007. Mechanisms of sensory transduction in the skin. Nature. 445:858–865. Malcangio M, Bowery NG. 1996. GABA and its receptors in the spinal cord. Trends Pharmacol Sci. 17, 457–462. Malinow R, Malenka RC. 2002. AMPA receptor traf cking and synaptic plasticity. Annu Rev Neurosci. 25:103–126. McKemy DD. 2005. How cold is it? TRPM8 and TRPA1 in the molecular logic of cold sensation. Mol Pain. 1:16. Merzenich MM, Nelson RJ, Stryker MP, Cynader MS, Schoppmann A, Zook JM. 1984. Somatosensory corti- cal map changes following digit amputation in adult monkeys. J Comp Neurol. 224:591–605. Merzenich M. 1998. Long-term change of mind. Science. 282:1062–1063. Nakatsuka T, Gu JG. 2001. ATP P2X receptor-mediated enhancement of glutamate release and evoked EPSCs in dorsal horn neurons of the rat spinal cord. J Neurosci. 21:6522–6531. Nakatsuka T, Furue H, Yoshimura M, Gu JG. 2002. Activation of central terminal vanilloid receptor-1 receptors and alpha beta-methylene-ATP-sensitive P2X receptors rev eals a converged synaptic activity onto the deep dorsal horn neurons of the spinal cord. J Neurosci. 22:1228–1237. Neumann S, Doubell TP, Leslie T, Woolf CJ. 1996. In ammatory pain hypersensitivity mediated by phe- notypic switch in myelinated primary sensory neurons. Nature. 384:360–364. Passafaro M, Piech V, Sheng M. 2001. Subunit-speci c tem- poral and spatial patterns of AMPA receptor exocytosis in hippocampal neurons. Nat Neurosci. 4:917–926. Pons TP, Garraghty PE, Ommaya AK, Kaas JH, Taub E, Mishkin M. 1991. Massive cortical reorganization after sensory deafferentation in adult macaques. Science. 252:1857–1860. Popratiloff A, Weinberg RJ, Rustioni A. 1996. AMPA recep- tor subunits underlying terminals of  ne-caliber pri- mary afferent  bers. J Neurosci. 16:3363–3372. Porreca F, Ossipov MH, Gebhart GF. 2002. Chronic pain and medullary descending facilitation. Trends Neurosci. 25:319–325. Rainville P, Duncan GH, Price DD, Carrier B, Bushnell MC. 1997. Pain affect encoded in human anterior cingulate but not somatosensory cortex. Science. 277:968–971. Rainville P, Bushnell MC, Duncan GH. 2001. Representation of acute and persistent pain in the human CNS: poten- tial implications for chemical intolerance. Ann N Y Acad Sci. 933:130–141. Chapter 6: Neurobiology of Chronic Pain 173 Yoshimura M, North RA. 1983. Substantia gelatinosa neu- rones hyperpolarized in vitro by enkephalin. Nature. 305:529–530. Zeng H, Gragerov A, Hohmann JG et al. 2006. Neuromedin U receptor 2-de cient mice display differential responses in sensory perception, stress, and feeding. Mol Cell Biol. 26:9352–9363. Zhao MG, Toyoda H, Lee YS et al. 2005. Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory. Neuron. 47:859–872. Zhao MG, Ko SW, Wu LJ et al. 2006. Enhanced presynaptic neurotransmitter release in the anterior cingulate cor- tex of mice with chronic pain. J Neurosci. 26:8923–8930. Zhuo M. 2000. Silent glutamatergic synapses and long-term facilitation in spinal dorsal horn neurons. Prog. Brain Res. 129:101–113. Zhuo M. 2002. Glutamate receptors and persistent pain: targeting forebrain NR2B subunits. Drug Discov Today. 7:259–267. Zhuo M. 2003. Synaptic and molecular mechanisms for pain and memory. Acta Physiologica Sinica. 55:1–8. Zhuo M. 2007. “Descending Facilitation,” Encyclopedia of Pain. CD-ROM, Springer Reference: Springer. Zhuo M, Gebhart GF. 1990a. Characterization of descend- ing inhibition and facilitation from the nuclei reticu- laris gigantocellularis and gigantocellularis pars alpha in the rat. Pain. 42:337–350. Zhuo M, Gebhart GF. 1990b. Spinal cholinergic and mono- aminergic receptors mediate descending inhibition from the nuclei reticularis gigantocellularis and gigan- tocellularis pars alpha in the rat. Brain Res. 535:67–78. Zhuo M, Gebhart GF. 1991. Spinal serotonin receptors mediate descending inhibition and facilitation from the nuclei reticularis gigantocellularis and gigantocel- lularis pars alpha in the rat. Brain Res. 550:35–48. Zhuo M, Gebhart GF. 1992. Characterization of descend- ing facilitation and inhibition of spinal nociceptive transmission from the nuclei reticularis gigantocel- lularis and gigantocellularis pars alpha in the rat. J Neurophysiol. 67:1599–1614. Zhuo M, Gebhart GF. 1997. Biphasic modulation of spinal nociceptive transmission from the medullary raphe nuclei in the rat. J Neurophysiol. 78:746–758. Zhuo M, Small SA, Kandel ER, Hawkins RD. 1993. Nitric oxide and carbon monoxide produce activity-dependent long- term synaptic enhancement in hippocampus. Science. 260:1946–1950. Zhuo M, Hu Y, Schultz C, Kandel ER, Hawkins RD. 1994. Role of guanylyl cyclase and cGMP-dependent pro- tein kinase in long-term potentiation in hippocampus. Nature. 368:635–639. Zhuo M, Gebhart GF. 1997. Biphasic modulation of spinal nociceptive transmission from the medullary raphe nuclei in the rat. J Neurophysiol. 78:746–758. Zhuo M, Sengupta, JN, Gebhart GF. 2002. Biphasic mod- ulation of spinal visceral nociceptive transmission from the rostroventral medial medulla in the rat. J Neurophysiol. 87:2225–2236. Zhuo M, Gebhart GF. 2002. Modulation of noxious and non- noxious spinal mechanical transmission from the rostral medial medulla in the rat. J Neurophysiol. 88:2928–2941. Travagli RA, Williams JT. 1996. Endogenous monoamines inhibit glutamate transmission in the spinal trigemi- nal nucleus of the guinea-pig. J Physiol. 491:177–185. Urban MO, Gebhart GF. 1999. Supraspinal contribu- tions to hyperalgesia. Proc Natl Acad Sci U S A. 96:7687–7692. van Praag H, Christie BR, Sejnowski TJ, Gage FH. 1999. Running enhances neurogenesis, learning, and long- term potentiation in mice. Proc Natl Acad Sci U S A. 96:13427–13431. Wei F, Li P, Zhuo M. 1999. Loss of synaptic depression in mammalian anterior cingulate cortex after amputa- tion. J Neurosci. 19:9346–9354. Wei F, Dubner R, Ren K. 1999. Dorsolateral funiculus- lesions unmask inhibitory or disfacilitatory mec- hanisms which modulate the effects of innocuous mechanical stimulation on spinal Fos expression after in ammation. Brain Res. 820:112–116. Wei F, Wang GD, Kerchner GA et al. 2001. Genetic enhance- ment of in ammatory pain by forebrain NR2B over- expression. Nat Neurosci. 4:164–169. Wei F, Zhuo M. 2001. Potentiation of synaptic responses in the anterior cingulate cortex following digital amputa- tion in rat. J Physiol. (Lond.). 532:823–833. Wang GD, Zhuo M. 2002. Synergistic enhancement of glutamate-mediated responses by serotonin and for- skolin in adult mouse spinal dorsal horn neurons. J Neurophysiol. 87:732–739. Wei F, Qiu CS, Kim SJ et al. 2002a. Genetic elimination of behavioral sensitization in mice lacking calmodulin- stimulated adenylyl cyclases. Neuron. 36:713–726. Wei F, Qiu CS, Liauw J et al. 2002b. Calcium calmodulin- dependent protein kinase IV is required for fear memory. Nat Neurosci. 6:573–579. Wei F, Vadakkan KI, Toyoda H et al. 2006. Calcium calmodulin-stimulated adenylyl cyclases contribute to activation of extracellular signal-regulated kinase in spinal dorsal horn neurons in adult rats and mice. J Neurosci. 26:851–861. Williams BM, Luo Y, Ward C et al. 2001. Environ - mental enrichment: effects on spatial memory and hippocampal CREB immunoreactivity. Physiol Behav. 73:649–658. Willis WD Jr. 1988. Anatomy and physiology of descending control of nociceptive responses of dorsal horn neu- rons: comprehensive review. Prog Brain Res. 77:1–29. Willis WD. 2002. Long-term potentiation in spinothalamic neurons. Brain Res Brain Res Rev. 40:202–214. Woolf CJ, Shortland P, Coggeshall RE. 1992. Peripheral nerve injury triggers central sprouting of myelinated afferents. Nature. 355:75–78. Woolf CJ, Salter MW. 2000. Neuronal plasticity: increasing the gain in pain. Science. 288:1765–1769. Wu LJ, Toyoda H, Zhao MG et al. 2005. Upregulation of forebrain NMDA NR2B receptors contributes to behavioral sensitization after in ammation. J Neurosci. 25:11107–11116. Xu H, Wu LJ, Zhao MG et al. 2006. Presynaptic regulation of the inhibitory transmission by GluR5-containing kainate receptors in spinal substantia gelatinosa. Mol Pain. 2:29. 174 Chapter 7 PHYSIOLOGICAL EFFECTS AND DISEASE MANIFESTATIONS OF PERFORMANCE-ENHANCING ANDROGENIC–ANABOLIC STEROIDS, GROWTH HORMONE, AND INSULIN Michael R. Graham, Julien S. Baker, Peter Evans, and Bruce Davies ABSTRACT Anabolic–androgenic steroids (AASs) were the fi rst identifi ed doping agents and can be used to increase muscle mass and strength in adult males. Despite successful detection and convictions by sporting antidoping agencies, they are still being used to increase physical performance and improve appear- ance. Their use does not appear to be diminishing. The adverse side effects and potential dangers of AAS use have been well documented. Recent epide- miological research has identifi ed that the designer drugs, growth hormone (GH) and insulin, are also being used because of the belief that they improve sporting performance. GH and insulin are currently undetectable by urinalysis. The objective of this chap- ter is to summarize the classifi cation of these drugs, their prevalence, and patterns of use. The physiology of GH and its pathophysiology in the disease states of defi ciency and excess and in catabolic states has been discussed and a distinction made on the differ- ent effects between therapeutic use in replacement and abuse in a sporting context. The history, physi- ology, and pathophysiology of insulin in therapeutic replacement and its abuse in a sporting context have also been identifi ed. A suggestion has been made on potential mechanisms of the effects of the designer drugs GH and insulin. Keywords: abuse, drugs, GH, insulin, steroids. Chapter 7: Androgenic–Anabolic Steroids, Growth Hormone, and Insulin 175 WHAT ARE ANABOLIC–ANDROGENIC STEROIDS? A nabolic–androgenic steroids (AASs) are a group of synthetic compounds similar in chemical structure to the natural anabolic steroid testosterone (T) (Fig. 7.1) (Haupt, Rovere 1984). T, the predominant circulating testic- ular androgen, is both an active hormone and a pro- hormone for the formation of a more active androgen, the 5α-reduced steroid dihydrotestosterone (DHT). Physiological studies of steroid hormone metabo- lism in the postnatal state demonstrated that DHT is formed in target tissues from circulating T and is a more potent androgen than T in several bioassay systems (Wilson, Leihy, Shaw et al. 2002). Genetic evidence indicates that these two andro- gens work via a common intracellular receptor. The androgen receptor (AR) is an intracellular ligand- dependent protein that modulates the expression of genes and mediates biological actions of physiological androgens (T and 5α-DHT) in a cell-speci c manner (Janne, Palvimo, Kallio et al. 1993). During embryonic life, androgens cause the for- mation of the male urogenital tract and hence are responsible for development of the tissues that serve as the major sites of androgen action in postnatal life. It has been generally assumed that androgens virilize the male fetus by the same mechanisms as in the adult, namely, by the conversion of circulating T to DHT in target tissues. A role for steroid 5α-reduction in androgen action became apparent with the  ndings in 1968 that DHT, the 5α-reduced derivative of T, is formed in many androgen target tissues where it binds to the AR (Bruchovsky, Wilson 1968). DHT binds to the AR more tightly than T, primarily as a result of stabilization of the AR complex and at low concentrations is as effective as T is at high concentra- tions in enhancing the transcription of one response element (Deslypere, Young, Wilson et al. 1992). This  nding clearly indicated that some effects of DHT are the result of ampli cation of the T signal. Loss of function mutations of the steroid 5 α - reductase 2 gene impairs virilization of the urogenital sinus and external genitalia in males (Wilson, Grif n, Russell et al. 1993). In summary, DHT formation both acts as a gen- eral ampli er of androgen action and conveys speci c function to the androgen–AR complex. The mech- anism by which the speci c function is mediated is unknown. The enzyme aromatase controls the androgen/ estrogen ratio by catalyzing the conversion of T into estradiol (E2). Therefore, the regulation of E2 syn- thesis by aromatase is thought to be critical in sexual development and differentiation (Kroon, Munday, Westcott et al. 2005). Synthetic T was  rst synthesized from cholesterol in 1935 (Ruckzika, Wettstein, Kaegi 1935). T is syn- thesized by the interstitial Leydig cells of the testes, which are primarily under the control of the gonado- trophins secreted by the pituitary gland. Approximately 95% of circulating T originates directly from testicular secretion (Ruckzika, Wettstein, Kaegi 1935). Following secretion, T is then trans- ported via the blood to target organs and speci c receptor sites. The bodily functions which are under Figure 7.1 The structure of testosterone. The structural modifi cations to the A- and B-rings of this steroid increase the anabolic activity; substitution at carbon atom position 17 (C-17) confers oral activity. I.M., intramuscular. Reproduced with kind permission from Annals of Clinical Biochemistry 2003; 40:321–356. Attachment of 7α-methyl group B A O 4 3 2 1 7 CD OH 17 Attachment of 17α-alkyl group confers oral activity Esterification confers depot activity for I.M. administration Removal of the angular methyl group Introduction of double bond A ttachment of various groups at C-2 A ttachment of pyrazole ring to the A-ring Attachment of chlorine or hydroxyl group A ttachment of methyl group PATHWAYS OF CLINICAL FUNCTION AND DISABILITY 176 In 1993, a report investigating abuse of AASs in 21 gymnasia in England, Scotland, and Wales found that 119 (9.1%) of the 1310 male respondents to the questionnaire and 8 (2.3%) of the 349 female respon- dents had taken AASs. The youngest abuser was aged 16. The prevalence of abuse of AASs in the gymnasia ranged from 0% (in three gymnasia) to 46% (28 of 61 respondents). The response rate to the questionnaire was 59% (1677/2834) (Korkia, Stimson 1993). In 1997, 100 AAS-using athletes were surveyed and high rates of polypharmacy (80%) with a wide array of drug abuse were reported among this sample group (Evans 1997). Another study in 1996 examined AAS abuse among 176 abusers (171 men and 5 women) and highlighted that 37% of respondents indicated a need for more knowledge of drug effects among drug workers and a less prejudiced attitude against drug dependency from general practitioners (Pates, Barry 1996). In 2001, 69% of 107 respondents of hardcore weight lifters were identi ed as abusing AASs, high- lighting that AAS abuse was certainly not on the decline (Grace, Baker, Davies 2001). Recent surveys conducted by Baker et al. (2006) and Parkinson and Evans (2006) have estimated that AASs are being abused by more than 1 million UK citizens and more than 3 million Americans. PREVALENCE AND PATTERNS OF GROWTH HORMONE AND INSULIN ABUSE GH appeared in the underground doping literature in 1981 (Duchaine 1983). Insulin-dependent diabet- ics are selling insulin pen- lls on the black market to bodybuilders. Unused aliquots are being resold, with the added risk of needle sharing and potential HIV and hepatitis C infection. Extensive literature research identi es very few cases of rhGH or insulin abuse by athletes. The few cases of rhGH abuse that have been published are case histories of individuals who have been arrested in possession at international tournaments. The pos- session of rhGH by the Chinese swimmers bound for the 1998 World Swimming Championships and simi- lar problems at the Tour de France cycling event in 1998 suggested abuse at an elite level (Wallace, Cuneo, Baxter et al. 1999). Approximately 1500 vials were stolen from an Australian wholesale chemist 6 months before the Sydney Olympics in 2000 (Sonksen 2001). The few cases of insulin abuse that have been high- lighted are those that have been admitted to hospi- tal following accidental overdose (Konrad, Schupfer, Wietlisbach et al. 1998; Evans, Lynch 2003). Dawson (2001) reports that 10% of 450 patients attending his direct control of T that have relevance to the athlete can be divided into two broad classi cations: Androgenic functions—male hormonal effects 1. (male-characteristic determining) Anabolic functions—constructive or muscle 2. building The clinical advantages of a pure anabolic agent were recognized many years ago and work was under- taken by a number of drug companies to modify the T molecule with a view to maximizing the ana- bolic effect and minimizing the androgenic activ- ity (Hershberger, Shipley, Meyer 1953). Some of the structural modi cations to testosterone to dissociate the anabolic from the androgenic effects are shown in Figure 7.1. The extent of the dissociation differs depending on the modi cation but there is no AAS that has an anabolic effect in an athlete without an androgenic effect (Di Pasquale 1990). DOPING IN SPORT AASs were the  rst identi ed doping agents to be banned in sport by the International Olympic Committee (IOC) Medical Commission in Athens in 1961. Evidence suggests that they increase muscle mass and strength and are abused to increase physical performance and improve appearance (Bhasin et al. 1996). The adverse side effects and potential dan- gers of AAS abuse are well documented (Ferenchick, Hirokawa, Mammen et al. 1995). The prevalence of AAS use has risen dramatically over the last two decades and has  ltered into all aspects of society. Subsequent published work indicated the concomitant abuse of recombinant human growth hor- mone (rhGH) and insulin (Grace, Baker, Davies 2001). Sportspersons are taking rhGH and insulin, separately or in combination, as doping agents to increase skele- tal muscle mass and improve performance (Ehrnborg, Bengtsson, Rosen 2000; Jenkins 2001; Sonksen 2001). Contemporary research has assessed the effects of taking supraphysiological levels of rhGH, but has not assessed the effects of taking rhGH and insulin in combination in a sporting context. Recent research suggests that rhGH administration in AAS abstinence may indeed improve sporting performance (Graham, Davies, Hullin et al. 2007b; Graham, Baker, Evans et al. 2008). THE PREVALENCE OF ANABOLIC– ANDROGENIC STEROID ABUSE A questionnaire study conducted by Perry and Littlepage (1992) found that 39% of 160 respondents were regular AAS abusers. Chapter 7: Androgenic–Anabolic Steroids, Growth Hormone, and Insulin 177 Cleavage of the GH receptor also yields a circu- lating GH-binding protein (GHBP), which prolongs the half-life and mediates the cellular transport of GH. GH activates the GH receptor, to which the intracellular Janus kinase 2 (JAK2) tyrosine kinase binds. Both the receptor and JAK2 protein are phos- phorylated, and signal transducers and activators of transcription (STAT) proteins bind to this complex. STAT proteins are then phosphorylated and trans- located to the nucleus, which initiates transcription of GH target proteins (Argetsinger, Campbell, Yang et al. 1993). Intracellular GH signaling is suppressed by several proteins, especially the suppressors of cytokine signal- ing (SOCS). GH induces the synthesis of peripheral insulin-like growth factor 1 (IGF-1) (Le Roith, Scavo, Butler 2001) and both circulating (endocrine) and local (autocrine and paracrine) IGF-1 induce cell proliferation and inhibit apoptosis (O’Reilly, Rojo, She et al. 2006). IGF-binding proteins (IGFBP) and their proteases regulate the access of ligands to the IGF-1 receptor, either enhancing or attenuating the action of IGF-1. Levels of IGF-1 are at the highest during late adoles- cence and decline throughout adulthood; these levels are determined by sex and genetic factors (Milani, Carmichael, Welkowitz et al. 2004). The production of IGF-1 is suppressed in malnourished patients as well as in patients with liver disease, hypothyroidism, or poorly controlled diabetes. IGF-1 levels usually re ect the secretory activity of GH and IGF-1 is one of a number of potential markers for identi cation of rhGH administration in sport (Powrie, Bassett, Rosen et al. 2007). In conjunction with GH, IGF-1 has varying differ- ential effects on protein, glucose, lipid, and calcium metabolism (Mauras, Attie, Reiter et al. 2000), and therefore, on body composition. Direct effects result from the interaction of GH with its speci c receptors on target cells. In the adipocyte, GH stimulates the cell to break down triglyceride (TG) and suppresses its ability to uptake and accumulate circulating lipids. Indirect effects are mediated primarily by IGF-1. Many of the growth-promoting effects of GH are due to the action of IGF-1 on its target cells. In most tissues, IGF-1 has local autocrine and paracrine actions, but the liver actively secretes IGF-1 and its binding proteins into the circulation (Mauras, Attie, Reiter et al. 2000). Little is known about the expression of skeletal muscle– speci c isoforms of IGF-1 gene in response to exercise in humans or about the in uence of age and physi- cal training status. Greig et al. (2006) reported that a single bout of isometric exercise stimulated the expres- sion of mRNA for the IGF-1 splice variants IGF-1Ea and IGF-1Ec (mechano growth factor [MGF]) within 2.5 hours, which lasts for at least 2 days after exercise. needle-exchange programme self-prescribe insulin for nontherapeutic purposes. The covert nature of its abuse precludes exact  gures. A recent questionnaire survey by Baker et al. (2006) has shown an increase in the abuse of insulin from 8% to 14% and an increase in the abuse of growth hormone (GH) from 6% to 24% in comparison to a survey conducted by Grace et al. (2001). HISTORY OF GROWTH HORMONE Physiological Aspects A cascade of interacting transcription factors and genetic elements normally determines the ability of the somatotroph cells in the anterior pituitary to syn- thesize and secrete the polypeptide human growth hormone (hGH). The development and proliferation of somatotrophs are largely determined by a gene called the Prophet of Pit-1 (PROP1), which controls the embryonic development of cells of the Pit-1 (POU1F1) transcription factor lineage. Pit-1 binds to the GH promoter within the cell nucleus, a step that leads to the development and proliferation of somatotrophs and GH transcription. Once translated, GH is secre- ted as a 191–amino acid, 4-helix bundle protein (70% to 80%) and a less abundant 176–amino acid form (20% to 30%), (Baumann 1991; Wu, Bidlingmaier, Dall et al. 1999) entering the circulation in a pulsatile manner under dual hypothalamic control through hypothalamic-releasing and hypothalamic-inhibiting hormones that traverse the hypophysial portal sys- tem and act directly on speci c somatotroph surface receptors (Melmed 2006). Growth hormone–releasing hormone (GHRH) induces the synthesis and secretion of GH, and soma- tostatin suppresses the secretion of GH. GH is also regulated by ghrelin, a GH secretagogue–receptor ligand (Kojima, Hosoda, Date et al. 1999) that is syn- thesized mainly in the gastrointestinal tract (GIT). In healthy persons, the GH level is usually unde- tectable (<0.2 μg/L) throughout most of the day. There are approximately 10 intermittent pulses of GH per 24 hours, most often at night, when the level can be as high as 30 μg/L (Melmed 2006). Fasting increases the secretion of GH, whereas aging and obesity are associated with suppressed secre- tory bursts of the hormone (Iranmanesh, Lizarralde, Velduis et al. 1991). The action of GH is mediated by a GH receptor, which is expressed mainly in the liver and in carti- lage and is composed of preformed dimers that undergo conformational change when occupied by a GH ligand, promoting signaling (Brown, Adams, Pelekanos et al. 2005). [...]... production and utilisation Br Med J 1:1239–1 242 Brown RJ, Adams JJ, Pelekanos RA et al 2005 Model for growth hormone receptor activation based on subunit rotation within a receptor dimer Nat Struct Mol Biol 12:8 14 821 Bruchovsky N, Wilson JD 1968 The conversion of testosterone to 5-alpha-androstan-17-beta-ol-3-one by rat prostate in vivo and in vitro J Biol Chem 243 :2012–2021 Burdet L, de Muralt B, Schutz... both PCV and body iron stores are components of the IR syndrome The ability of insulin and of IGF-1, whose effective activity is increased in the context of IR to boost activity of the transcription factor hypoxia-inducible factor- 1- (HIF- 1- ), may be at least partially responsible for this association HIF- 1- , which functions physiologically as a detector of both hypoxia and iron deficiency, promotes... GH in healthy male adults Portes et al (2000) demonstrated that long-term rhGH replacement therapy in A-OGHD significantly decreases serum free T4 and rT3 levels and increases serum T3 levels These changes are independent of TSH and result from increased peripheral conversion of T4 to T3 A-OGHD does not induce hypothyroidism but simply reveals previously unrecognized cases whose serum free T4 values... in cases was similar to that in healthy controls (Markussis, Beshyah, Fisher et al 1992; Valcavi, Gaddi O, Zini et al 1995) In younger GHD adults, the systolic BP (SBP) has been found to be lower (Thuesen, Jørgensen, Müller et al 19 94) , but was increased by GH replacement (Theusen, Jørgensen, Müller et al 19 94) Short-term, placebo-controlled GH-replacement trials for 4 to 12 months in GHD have demonstrated... upregulatory impact of hypoxia on intestinal iron absorption Insulin/IGF-1 may also influence erythropoiesis more directly as they are growth factors for developing reticulocytes Conversely, the activation of HIF- 1- associated with iron deficiency may be responsible for the increased glucose tolerance noted in iron-deficient animals; HIF- 1- promotes efficient glucose uptake and glycolysis, a sensible adaptation... 260:36 42 Baker JS, Graham MR, Davies B 2006 “Steroid” and prescription medicine abuse in the health and fitness community: a regional study Eur J Intern Med 17 :47 9 48 4 Barbieri M, Ragno E, Benvenuti E et al 2001 New aspects of the insulin resistance syndrome: impact on haematological parameters Diabetologia 44 :1232–1237 Barbour LA, Mizanoor Rahman S, Gurevich I et al 2005 Increased P85alpha is a potent... increase the metabolism of thyroxin (tetra-iodothyronine [T4]), enhancing the conversion of T4 to triiodothyronine (T3) (Sato, Suzukui, Taketani et al 1977) The lowering of serum free T4 supported the work of Grunfeld et al (1988) where T4 was significantly lowered by 8%, T3 was significantly increased by 21%, and TSH was significantly decreased by 54% after 4 days of low-dose rhGH administration (0.125 mg/day)... Effect of long-term growth hormone administration on pituitary-thyroid function in idiopathic hypopituitarism Acta Pediatr Scand 68 :40 5 40 9 Caidahl K, Eden S, Bengtsson BA 19 94 Cardiovascular and renal effects of growth hormone Clin Endocrinol (Oxf) 40 :393 40 0 Cameron JD, Bulpitt CJ, Pinto ES, Rajkumar C 2003 The ageing of elastic and muscular arteries: a comparison of diabetic and non-diabetic subjects... 91:5 14 521 Bondanelli M, Ambrosio MR, degli Uberti EC 2001 Pathogenesis and prevalence of hypertension in acromegaly Pituitary 4: 239– 249 Boroujerdi MA, Umpleby AM, Jones RH, Sonksen PH 1995 A simulation model for glucose kinetics and estimates of glucose utilization rate in type I diabetic patients Am J Physiol 268:766–7 74 Borson-Chazot F, Serusclat A, Kalfallah Y et al 1999 Decrease in carotid intima-media... Thomas, Kyd et al 19 94) Transiliac bone biopsies of patients with A-OGHD after 6 to 12 months of rhGH treatment showed an increase in cortical thickness, increased bone formation, and decreased bone resorption Trabecular bone volume remained unchanged (Bravenboer, Holzmann, de Boer et al 1997) RhGH treatment in A-OGHD for 10 years induced a sustained increase in total, lumbar (L2-L4), and femur neck . Light and electron microscopic immunocy- tochemical localization of AMPA-selective gluta- mate receptors in the rat spinal cord. J Comp Neurol. 344 :43 1 45 4. Talbot JD, Marrett S, Evans AC, Meyer. Jørgensen, Müller et al. 19 94) , but was increased by GH replacement (Theusen, Jørgensen, Müller et al. 19 94) . Short-term, placebo-controlled GH-replacement trials for 4 to 12 months in GHD have. Neurosci. 8: 249 2– 249 8. Toyoda H, Wu LJ, Zhao MG, Xu H, Zhuo M. 2007a. Time- dependent postsynaptic AMPA GluR1 receptor recruit- ment in the cingulate synaptic potentiation. Dev Neurobiol. 67 :49 8–509. Toyoda