JY Lee, JY Kang, SY Lee, SY Cho, DK Jin

Abstract

Objectives: The aim of this study was to investigate the effects of recombinant human GH (rhGH, henceforth designated GH) on the gene expression of GABA_B receptor subunits and GH/insulin-like growth factor (IGF) axis genes IGF-1, IGF-1R, IGF-2 and IGF-2R in the brain regions of Prader-Willi syndrome (Snord116del) mice, a dwarf strain exhibiting cognitive impairment. Methods: Snord116del mice were treated with GH (1.0 mg/kg) or saline for seven days before decapitation and tissue dissection. The collected brain tissues were analysed for mRNA content using quantitative PCR (qPCR) in the cerebellum, hippocampus and cerebral cortex. Results: In the cerebellum, GH restored the mRNA expression level of the GABA_B1 receptor subunit (GABA_BR1) and IGF-1R. Furthermore, a significant positive correlation was found between the level of GABA_BR1 mRNA and the expression of the IGF-1R transcript. GH also induced an increase in the mRNA expression of IGF-2 and IGF-2R. Conclusions: These data suggest a modulatory effect of GH on the expression of GABA_BR1 and GH/IGF-1 axis genes in cerebellum may provide a mechanism for the GH-induced brain function in PWS patients.

Introduction

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by the lack of paternally expressed imprinted genes on human chromosome 15q11-q13.¹ PWS is characterised by hypothalamic dysfunction including growth hormone (GH) deficiency (GHD) with short stature, hyperphagia, obesity, neurobehavioural abnormalities, and cognitive impairment.² Clinical studies have shown that GH replacement therapy improves cognitive development in infants and adults with PWS,^3-5 prevents cognitive deterioration and improves cognitive skills in children with PWS.⁶ Early GH therapy has been reported to increase the rate of language and neurodevelopment in infants with PWS.⁷ However, the physiological and molecular mechanisms underlying the improvement in cognitive function after GH treatment have not been investigated in PWS.

GH/insulin-like growth factor (IGF)-1 axis is involved in the growth, development and function of the central nervous system (CNS).⁸ Individuals with GHD show cognitive impairment, which can be ameliorated by GH treatment.⁹ GH administration attenuates cognitive deficits and improves memory in hypophysectomised rodents.¹⁰ Another mediator of GH effects, IGF-2, has been proposed as a novel cognitive enhancer.¹¹ The presence of binding sites for GH and IGF-1 in the brain suggested that GH crosses the blood-brain barrier,^12,13 although the mechanisms behind the actions of GH on brain function remain unclear.

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the CNS, acting via GABA_A and GABA_B receptors. GABA_B receptors are heterodimer and composed of GABA_B receptor subunit 1 (GABA_BR1) and GABA_BR2,¹⁴ which are responsible for the neuromodulatory effect of GABA.^15,16 Recent studies have reported that exogenous GH increases the abundance of the GABA_B receptor in the area of the rat brain associated with cognition¹⁷ and GABA_BR1 gene expression in hypophysectomised rat.¹⁸ These findings indicate the possible correlation between GH-induced cognitive function and the GABA_B receptor.

The Snord116 deletion (Snord116del) mouse, a genetic model of PWS, is a dwarf strain caused by the deletion of the Snord116 C/D box snoRNA cluster. This is characterised by a subset of PWS symptoms such as growth retardation,^19,20 elevated anxiety and deficiency in motor learning ability.²⁰ Although not obese,^19,20 Snord116del mice are hyperghrelinemic and moderately hyperphagic.²⁰ This mice have early-onset postnatal growth deficiency and exhibit the decrease in liver Igf-1 mRNA and serum IGF-1,²⁰ suggesting that GH/IGF axis undergoes decreased GH concentrations in the brain and this decline of GH levels could be associated with changes of certain CNS functions such as impairment of cognitive function.⁸

Since GH is an important regulator of developmental and cognitive functions in the CNS, the aim of this study was to investigate the effects of GH on the expressions of GABA_B receptor subunits as well as the GH/IGF axis gene in specific brain regions known to be affected by GH treatment^21,22 in Snord116del mice.

Materials and Methods

Animals and Drug Treatment

All animal experiments were carried out in accordance with a protocol approved by the Institutional Animal Care and Use Committee, Laboratory Animal Research Center, Samsung Biomedical Research Institute (Seoul, Korea). Mice were housed under standard vivarium conditions and provided food and water ad libitum. Snord116del mice (B6(Cg)-Snord116tm1.1Uta/J) were obtained from The Jackson Laboratory (Bar Harbor, 352 Maine, USA). Male Snord116del mice and their wild-type littermates aged six-twelve months with C57BL/6J background were used and genotyped as described.²⁰ At the start of the experiment, the mice were three weeks old (n=6-8 for each group), corresponding to adolescent age. Male Snord116del mice were injected subcutaneously with 1.0 mg/kg GH (Growtropin, provided from Dong-A Pharmaceutical Co., Yongin-si, Korea) or saline, once daily for seven days. Male C57BL/6J littermates were used as controls and given saline injections. All animals were weighed every day to monitor their biological response in weight gain. On day 8 of the experiment, the mice were sacrificed and the cerebellum, hippocampus and cerebral cortex were dissected using a brain matrix. The collected tissues were immediately placed in RNALater solution (Applied Biosystems, Foster City, CA, USA) and stored at 4°C prior to total RNA extraction.

RNA Extraction and cDNA Synthesis

Brain tissues were prepared for RNA extraction using the RNeasy Lipid Tissue Mini Kit (QIAGEN, MD, USA), according to the protocol provided by the manufacturer. Briefly, the tissue samples were quickly homogenised in 1000 ml Qiazol tissue lyzer (Qiagen, Sollentuna, Sweden), and 200 ml chloroform was then added to each sample. The samples were centrifuged at 4°C (12,000 g, 15 minutes) and 70% ethanol was added to the supernatant. Mini Spin columns were then used to elute the samples. The quantification of total RNA was later assessed in a NanoDrop^® ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, USA) to provide total RNA concentrations and a preliminary quality control. For quality control, RNA purity and integrity were evaluated by denaturing gel electrophoresis, OD 260/280 ratio and analysed on Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, USA). The conversion of total RNA to cDNA was performed using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) in a final reaction volume of 20 ml. In total, 2 mg RNA was used for the synthesis. The cycling parameters were as follows: 37°C for 60 minutes and 95°C for 5 minutes.

Quantitative Polymerase Chain Reaction

The expression of six genes (Gabbr1, Gabbr2, Igf-1, Igf-1r, Igf-2 and Igf-2r) was quantified using a TaqMan^® Gene Expression Assay (Applied Biosystems), which included a TaqMan^® real-time quantitative polymerase chain reaction (qPCR) in the cerebellum, hippocampus and cerebral cortex. qPCR analysis was performed using a PRISM 7900HT Sequence Detection System (Applied Biosystems) with SDS 2.3 software (Applied Biosystems) in 384-well plates containing cDNA template (10 ng), primers, probes and TaqMan^® Universal PCR Master Mix to a final volume of 10 ml/well. Each assay included individual samples for a specific gene in triplicate, with corresponding negative controls. Cycling parameters were as follows: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minutes. Predesigned gene-specific primers and probes were used to detect each gene (Applied Biosystems), presented in Table 1. For all primers, more details can be obtained from http://www.appliedbiosystems.com. The amount of each transcript was normalised to the amount of GAPDH expressed in the same sample.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism 5.0b (GraphPad Software, Inc., La Jolla, USA). The weight measurements were analysed using two-way repeated ANOVA. The results from the qPCR were analysed using one-way ANOVA with a post hoc Student-Newman-Keuls test for the statistical analysis of the differences between the groups. The correlation was tested by simple regression analysis. Values are presented as mean ± SEM and p-value less than 0.05 were considered significant.

Results

Compared to the WT mice, the Snord116del mice with GHD exhibited reduced body weight, and GH treatment significantly increased gains in body weight (Figure 1). This indicates that the administered GH was physiologically active and had an expected systemic effect on body growth.

Figure 1 Effects of GH treatment on body weight. Mice received daily subcutaneous injections of GH (1.0 mg/kg) or saline injection for 7 days. Body weights were measured over 8 days. Data represent mean ± S.E.M. n=7-8 for each group. *p<0.05, **p<0.01, ***p<0.001. Abbreviations: GH, growth hormone; Del, Snord116del mice injected with saline; Del + GH, Snord116del mice injected with recombinant growth hormone; WT, wild-type mice injected with saline.

The expression of six genes (Gabbr1, Gabbr2, Igf-1, Igf-1r, Igf-2 and Igf-2r) in the cerebellum, hippocampus and cerebral cortex was analysed in Snord116del mice treated with GH (Del + GH) or saline (Del) and wild-type (WT) mice with saline. The results from the gene expression analysis of Gabbr1 and Gabbr2 in the cerebellum, hippocampus and cerebral cortex are displayed in Figure 2. In the cerebellum, there were significant differences between the treatment groups regarding the mRNA expression of Gabbr1 (p<0.05) where both the Del + GH and WT groups showed increased Gabbr1 mRNA expression compared with the Del group, but no effect on the Gabbr2 expression was observed. However, the administration of GH did not alter the expression of Gabbr1 or Gabbr2 in the hippocampus and cerebral cortex.

Figure 2 Effects of GH treatment on mRNA expression of GABABR1 and GABABR2 in the cerebellum, hippocampus and cerebral cortex. Values are expressed as mean ± SEM, n=7-8/group. *p<0.05, **p<0.01. Abbreviations: GH, growth hormone; Gabbr1, GABAB1 receptor subunit; Gabbr2, GABAB2 receptor subunit; Del, Snord116del mice injected with saline; Del + GH, Snord116del mice injected with recombinant growth hormone; WT, wild-type mice injected with saline.

The results from the gene expression analysis of Igf-1, Igf-1r, Igf-2 and Igf-2r in the cerebellum, hippocampus and cerebral cortex are shown in Figures 3 and 4. There was a significant difference between the treatment groups regarding the Igf-1r, Igf-2 and Igf-2r expression in the cerebellum. A significant decrease of Igf-1r mRNA expression was found in the Del group compared to the WT group and GH administration induced an increase of Igf-1r expression (p<0.05). In addition, alterations of Igf-2 and Igf-2r mRNA expression were found; the Del + GH group had increased the Igf-2 and Igf-2r expression (p<0.05). However, GH administration did not alter the expression of Igf-1, Igf-1r, Igf-2 and Igf-2r in the hippocampus and cerebral cortex (Figures 3 & 4). By comparing the expression of the gene transcripts for IGF-1R, IGF-2 and IGF-2R with those of the GABAB receptor subunits, a significant positive correlation was observed in cerebellum, between the level of IGF-1R mRNA and the level of the transcript for the Gabbr1 (r²=0.62, p<0.05) (Figure 5). However, no significant correlation could be seen between the expression level of mRNA for IGF-2, IGF-2R and any other GABAB receptor subunits in any brain regions.

Figure 3 Effects of GH treatment on mRNA expression of IGF-1 and IGF-1R in the cerebellum, hippocampus and cerebral cortex. Values are expressed as mean ± SEM, n=7-8/group. *p<0.05. Abbreviations: GH, growth hormone; Igf-1, insulin-like growth factor 1; Igf-1r, insulin-like growth factor 1 receptor; Del, Snord116del mice injected with saline; Del + GH, Snord116del mice injected with recombinant growth hormone; WT, wild-type mice injected with saline.

Figure 4 Effects of GH treatment on mRNA expression of IGF-2 and IGF-2R in the cerebellum, hippocampus and cerebral cortex. Values are expressed as mean ± SEM, n=7-8/group. *p<0.05. Abbreviations: GH, growth hormone; Igf-2, insulin-like growth factor 2; Igf-2r, insulin-like growth factor 2 receptor; Del, Snord116del mice injected with saline; Del + GH, Snord116del mice injected with recombinant growth hormone; WT, wild-type mice injected with saline.

Figure 5 Simple-regression analysis of the correlation between the GABABR1 and IGF-1R mRNA in the cerebellum of Snord116del mice (n=7) treated with GH (1.0 mg/kg).

Discussion

This is the first study, to our knowledge, to examine the effects of GH administration on the expression of GABA_B receptor subunits and GH/IGF-1 axis genes in specific regions of the PWS (Snord116del) mice brain.

The present study demonstrated that in comparison to wild-type mice, both the expression of GABA_BR1 and IGF-1R transcripts are markedly decreased in the cerebellum of Snord116del mice and GH increases the expression of GABA_BR1 and IGF-1R transcripts. GABA_B receptor has been shown to be important for neuronal excitability and plasticity and is suggested to be involved in the regulation of long-term potentiation, which is the cellular mechanism for learning and memory.^16,23 GH treatment has been reported to affect the functionality and density of GABA_B receptors in the area of the brain associated with cognition.¹⁷ Other studies revealing that GH administration up-regulated the expression of GABA_BR1 transcript in rat brain further have validated the connection between GH and GABA_B system.^18,24

We also detected a significant positive correlation between the mRNA level of IGF-1R and GABA_BR1 in the cerebellum. This finding, indicative of an IGF-1R-mediated effect on the function of the GABA_B receptor, is in agreement with a recent observation that the activation of the GABA_B receptor induces IGF-1R transactivation leading to survival signaling in the cerebellum.²⁵ Thus, several studies have suggested that the GABA_B receptor protects the brain from ischaemic damage and improves memory,^26-28 providing evidence that stimulation of the GABA_B receptor may be involved in a mechanism by which GH regulates brain function, including a cognitive and neuroprotective effect.

Of particular interest from the present study is the GH-induced increase in the gene expression for IGF-2 and IGF-2R in the cerebellum. IGF-2, another mediator of GH action, is known to be important for brain development and to have neurotrophic or neuroprotective properties.^29,30 IGF-2 signaling has been implicated in cognitive function and it is suggested that the effect of IGF-2 as a memory enhancer is selectively mediated by IGF-2R. It was shown that IGF-2 promoted IGF-2R-dependent, persistent long-term potentiation, demonstrated by memory improvement.¹¹ While the precise mechanisms by which IGF-2 and IGF-2R are regulated remain to be investigated, our data suggest the possibility that IGF-2/IGF-2R signaling could have an important role in GH-induced cognitive function in Snord116del mice.

On the contrary to the effects seen in the cerebellum, the expression of GABA_BR1, GABA_BR2, IGF-1, IGF-1R, IGF-2 and IGF-2R in the hippocampus and cerebral cortex was unaffected by GH administration. The GH activity may be different regionally, because the brain is highly heterogeneously functional. Several potential mechanisms, such as differences in blood-brain barrier permeability and the distribution of GH receptor (GHR), may account for differences in the effects of GH on GABA_B receptor subunits and GH/IGF axis expression in specific brain regions. When analysing the results in the present study we did not find any significant difference in cerebral GHR mRNA expression between the treatment groups (data not shown). Additionally, the amount of GH binding protein may affect the response to GH by modulating the bound fraction of GH in Snord116del mice.

The clinical phenotype of individuals with PWS, including mental retardation, hypotonia, motor delay, and poor fine motor skills, support the idea that cerebellar development may be abnormal in these individuals. Several autopsy studies of individuals with PWS have shown abnormalities in the white matter of the cerebellum and one found partial hypoplasia of the right cerebellar hemisphere.^31,32 Additionally, quantitative structural magnetic resonance imaging (MRI) studies have been performed in patients with PWS that reported abnormalities in the cerebellum.^33-36 Many genetic disorders are associated with compromised cerebellar development.³⁷ In addition to being important for motor control, the cerebellum has connections to areas in the cerebrum which are relevant to cognition and behaviour.³⁸ A large structural imaging study found that there was a significant relationship between general cognitive ability (IQ) and the volume of the cerebellum.³⁹ Importantly, individuals with PWS have decreased cerebellar volumes and lower general cognitive ability (GIA) compared to controls.³⁶ Moreover, the neurobehavioural test of Snord116del mice has reported impaired motor learning in the rotarod test²⁰ that is a suitable test for evaluation of cerebellar deficits in rodents.^40,41 The rotarod task measures motor coordination and can also measure motor learning.⁴² The cerebellum is highly implicated in the functioning of this task, and it is a well documented site of action for other learning paradigms.⁴³

The local expression of GH and the presence of its receptor GHR in the cerebellum indicate that cerebellum is an autocrine and/or paracrine site of GH action.⁴⁴ As it is known that GH and IGF-I increase brain growth, myelination, and has neuroprotective properties ^45-47 we could speculate that if the GH treatment had any effect on the brain, it would have a positive effect in terms of brain normalisation.

In conclusion, this study demonstrates that GH restores the gene expression of GABA_BR1 and IGF-1R and increases IGF-2 and IGF-2R in the cerebellum of Snord116del mice. The alterations of GABA_BR1 and IGF-1R observed in Snord116del mice could, at least partly, account for cognitive impairment. Because GHD during early life could impair proper brain development, thereby leading to cognitive deficits, it is suggested from the present study that a modulatory effect of GH on the expression of GABA_BR1 and GH/IGF-1 axis genes in brain may provide a mechanism for the GH-induced brain function in Snord116del mice, genetic models of PWS.

Acknowledgments

This paper was supported by Sungkyun Research Fund, Sungkyunkwan University, 2014 (# 2013-0716-000).

Declaration of Interest

The authors declare that there is no conflict of interest.

References

1. Cassidy SB. Prader-Willi syndrome. J Med Genet. 1997;34:917-23.

2. Swaab DF, Purba JS, Hofman MA. Alterations in the hypothalamic paraventricular nucleus and its oxytocin neurons (putative satiety cells) in Prader-Willi syndrome: a study of five cases. J Clin Endocrinol Metab 1995;80:573-9.

3. Festen DA, Wevers M, Lindgren AC, et al. Mental and motor development before and during growth hormone treatment in infants and toddlers with Prader-Willi syndrome. Clin Endocrinol (Oxf) 2008;68:919-25.

4. Myers SE, Whitman BY, Carrel AL, Moerchen V, Bekx MT, Allen DB. Two years of growth hormone therapy in young children with Prader-Willi syndrome: physical and neurodevelopmental benefits. Am J Med Genet A 2007;143A:443-8.

5. Hoybye C, Thoren M, Bohm B. Cognitive, emotional, physical and social effects of growth hormone treatment in adults with Prader-Willi syndrome. J Intellect Disabil Res 2005;49:245-52.

6. Siemensma EP, Tummers-de Lind van Wijngaarden RF, Festen DA, et al. Beneficial effects of growth hormone treatment on cognition in children with Prader-Willi syndrome: a randomized controlled trial and longitudinal study. J Clin Endocrinol Metab 2012;97:2307-14.

7. Cho SY, Jin DK. Issues in Infants with Prader-Will Syndrome: Special Review on Early Dietary Intervention and Early Use of Growth Hormone. Annals of Pediatric Endocrinology & Metabolism 2012;17:145.

8. Sonntag WE, Ramsey M, Carter CS. Growth hormone and insulin-like growth factor-1 (IGF-1) and their influence on cognitive aging. Ageing Res Rev 2005;4:195-212.

9. Nyberg F, Hallberg M. Growth hormone and cognitive function. Nat Rev Endocrinol 2013;9:357-65.

10. Le Greves M, Zhou Q, Berg M, et al. Growth hormone replacement in hypophysectomized rats affects spatial performance and hippocampal levels of NMDA receptor subunit and PSD-95 gene transcript levels. Exp Brain Res 2006;173:267-73.

11. Chen DY, Stern SA, Garcia-Osta A, et al. A critical role for IGF-II in memory consolidation and enhancement. Nature 2011;469:491-7.

12. Nyberg F, Burman P. Growth hormone and its receptors in the central nervous system-location and functional significance. Horm Res 1996;45:18-22.

13. Pan W, Yu Y, Cain CM, Nyberg F, Couraud PO, Kastin AJ. Permeation of growth hormone across the blood-brain barrier. Endocrinology 2005;146:4898-904.

14. Jones KA, Borowsky B, Tamm JA, et al. GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2. Nature 1998;396:674-9.

15. Goudet C, Magnaghi V, Landry M, Nagy F, Gereau RWt, Pin JP. Metabotropic receptors for glutamate and GABA in pain. Brain Res Rev 2009;60:43-56.

16. Benarroch EE. GABAB receptors: structure, functions, and clinical implications. Neurology 2012;78:578-84.

17. Gronbladh A, Johansson J, Nyberg F, Hallberg M. Recombinant human growth hormone affects the density and functionality of GABAB receptors in the male rat brain. Neuroendocrinology 2013;97:203-11.

18. Walser M, Hansen A, Svensson PA, et al. Peripheral administration of bovine GH regulates the expression of cerebrocortical beta-globin, GABAB receptor 1, and the Lissencephaly-1 protein (LIS-1) in adult hypophysectomized rats. Growth Horm IGF Res 2011;21:16-24.

19. Skryabin BV, Gubar LV, Seeger B, et al. Deletion of the MBII-85 snoRNA gene cluster in mice results in postnatal growth retardation. PLoS Genet 2007;3:e235.

20. Ding F, Li HH, Zhang S, et al. SnoRNA Snord116 (Pwcr1/MBII-85) deletion causes growth deficiency and hyperphagia in mice. PLoS One 2008;3:e1709.

21. Aberg ND, Carlsson B, Rosengren L, et al. Growth hormone increases connexin-43 expression in the cerebral cortex and hypothalamus. Endocrinology 2000;141:3879-86.

22. Aramburo C, Alba-Betancourt C, Luna M, Harvey S. Expression and function of growth hormone in the nervous system: a brief review. Gen Comp Endocrinol 2014;203:35-42.

23. Davies CH, Starkey SJ, Pozza MF, Collingridge GL. GABA autoreceptors regulate the induction of LTP. Nature 1991;349:609-11.

24. Gronbladh A, Johansson J, Nyberg F, Hallberg M. Administration of growth hormone and nandrolone decanoate alters mRNA expression of the GABA receptor subunits as well as of the GH receptor, IGF-1, and IGF-2 in rat brain. Growth Horm IGF Res 2014;24:60-6.

25. Tu H, Xu C, Zhang W, et al. GABAB receptor activation protects neurons from apoptosis via IGF-1 receptor transactivation. J Neurosci 2010;30:749-59.

26. Zhang F, Li C, Wang R, et al. Activation of GABA receptors attenuates neuronal apoptosis through inhibiting the tyrosine phosphorylation of NR2A by Src after cerebral ischemia and reperfusion. Neuroscience 2007;150:938-49.

27. Xu J, Li C, Yin XH, Zhang GY. Additive neuroprotection of GABA A and GABA B receptor agonists in cerebral ischemic injury via PI-3K/Akt pathway inhibiting the ASK1-JNK cascade. Neuropharmacology 2008;54:1029-40.

28. Li CJ, Lu Y, Zhou M, et al. Activation of GABAB receptors ameliorates cognitive impairment via restoring the balance of HCN1/HCN2 surface expression in the hippocampal CA1 area in rats with chronic cerebral hypoperfusion. Mol Neurobiol 2014;50:704-20.

29. Russo VC, Gluckman PD, Feldman EL, Werther GA. The insulin-like growth factor system and its pleiotropic functions in brain. Endocr Rev 2005;26:916-43.

30. Rotwein P, Burgess SK, Milbrandt JD, Krause JE. Differential expression of insulin-like growth factor genes in rat central nervous system. Proc Natl Acad Sci U S A 1988;85:265-9.

31. Miller JL, Couch JA, Schmalfuss I, He G, Liu Y, Driscoll DJ. Intracranial abnormalities detected by three-dimensional magnetic resonance imaging in Prader-Willi syndrome. Am J Med Genet A 2007;143A:476-83.

32. Hayashi M, Itoh M, Kabasawa Y, Hayashi H, Satoh J, Morimatsu Y. A neuropathological study of a case of the Prader-Willi syndrome with an interstitial deletion of the proximal long arm of chromosome 15. Brain Dev 1992;14:58-62.

33. Lukoshe A, White T, Schmidt MN, van der Lugt A, Hokken-Koelega AC. Divergent structural brain abnormalities between different genetic subtypes of children with Prader-Willi syndrome. J Neurodev Disord 2013;5:31.

34. Honea RA, Holsen LM, Lepping RJ, et al. The neuroanatomy of genetic subtype differences in Prader-Willi syndrome. Am J Med Genet B Neuropsychiatr Genet 2012;159B:243-53.

35. Ogura K, Fujii T, Abe N, et al. Small gray matter volume in orbitofrontal cortex in Prader-Willi syndrome: a voxel-based MRI study. Hum Brain Mapp 2011;32:1059-66.

36. Miller JL, Couch J, Schwenk K, et al. Early childhood obesity is associated with compromised cerebellar development. Dev Neuropsychol 2009;34:272-83.

37. Steinlin M. The cerebellum in cognitive processes: supporting studies in children. Cerebellum 2007;6:237-41.

38. Rapoport M, van Reekum R, Mayberg H. The role of the cerebellum in cognition and behavior: a selective review. J Neuropsychiatry Clin Neurosci 2000;12:193-8.

39. Pangelinan MM, Zhang G, VanMeter JW, Clark JE, Hatfield BD, Haufler AJ. Beyond age and gender: relationships between cortical and subcortical brain volume and cognitive-motor abilities in school-age children. Neuroimage 2011;54:3093-100.

40. Lalonde R, Bensoula AN, Filali M. Rotorod sensorimotor learning in cerebellar mutant mice. Neurosci Res 1995;22:423-6.

41. Caston J, Jones N, Stelz T. Role of preoperative and postoperative sensorimotor training on restoration of the equilibrium behavior in adult mice following cerebellectomy. Neurobiol Learn Mem 1995;64:195-202.

42. Crawley JN. Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Res 1999;835:18-26.

43. Liu SJ, Lachamp P, Liu Y, Savtchouk I, Sun L. Long-term synaptic plasticity in cerebellar stellate cells. Cerebellum 2008;7:559-62.

44. Alba-Betancourt C, Aramburo C, Avila-Mendoza J, et al. Expression, cellular distribution, and heterogeneity of growth hormone in the chicken cerebellum during development. Gen Comp Endocrinol 2011;170:528-40.

45. Carson MJ, Behringer RR, Brinster RL, McMorris FA. Insulin-like growth factor I increases brain growth and central nervous system myelination in transgenic mice. Neuron 1993;10:729-40.

46. Alba-Betancourt C, Luna-Acosta JL, Ramirez-Martinez CE, et al. Neuro-protective effects of growth hormone (GH) after hypoxia-ischemia injury in embryonic chicken cerebellum. Gen Comp Endocrinol 2013;183:17-31.

47. Noguchi T, Sugiasaki T, Tsukada Y. Microcephalic cerebrum with hypomyelination in the growth hormone-deficient mouse (lit). Neurochem Res 1985;10:1097-106.