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Endocrinology 142(10):4560 – 4567 Copyright © 2001 by The Endocrine Society
Enhanced Spontaneous Locomotor Activity in Bovine GH Transgenic Mice Involves Peripheral Mechanisms MOHAMMAD BOHLOOLY-Y, BOB OLSSON, AMEL GRITLI-LINDE, OLA BRUSEHED, ¨ DERPALM, AND JAN TO ¨ RNELL OLLE G. P. ISAKSSON, CLAES OHLSSON, BO SO Departments of Physiology (M.B.-Y., B.O., O.B., J.T.), Oral Biochemistry (A.G.-L.), and Pharmacology (B.S.), Go¨teborg University, Go¨teborg, Sweden S-40530; Research Centre for Endocrinology and Metabolism (M.B.-Y., B.O., O.B., O.G.P.I., C.O., J.T.), Sahlgrenska Hospital, Go¨teborg, Sweden S-41345; and AstraZeneca Transgenic Centre (J.T.), AstraZeneca Research and Development, Mo¨lndal, Sweden S-43183 Clinical and experimental studies indicate a role for GH in mechanisms related to anhedonia/hedonia, psychic energy, and reward. Recently we showed that transgenic mice with general overexpression of bovine GH display increased spontaneous locomotor activity. In the present study, we investigated whether this behavioral change is owing to a direct action of GH in the central nervous system or to peripheral GH actions. A transgenic construct, containing the glial fibrillary acidic protein promoter directing specific expression of bovine GH to the central nervous system, was designed. The central nervous system–specific expression of bovine GH in the glial fibrillary acidic protein-bovine GH transgenic mice was confirmed, but no effect on spontaneous locomotor activity was observed. Serum bovine GH levels were increased
P
ATIENTS SUFFERING FROM GH deficiency have a decreased quality of life (1, 2). The psychic symptoms observed are tiredness and lack of energy, impaired cognitive functions (memory and concentration), and irritability. Dramatic positive effects have been observed when the normal levels of GH are reconstituted by substitution (3). Transgenic mice, generally overexpressing bovine GH (bGH) under control of the metallothionein promoter (MtbGH), display an increased spontaneous activity (4), supporting data from substituted GH-deficient patients. The Mt-bGH transgenic animals also show an increased stimulatory response to amphetamine, suggesting sensitization of the mesocorticolimbic dopamine system, a part of the brain reward system that has been linked to anhedonia/hedonia and psychic drive. In addition, the Mt-bGH transgenic mice show signs of altered dopamine metabolism in mesencephalon, diencephalon, and brain stem, which could be linked to the behavioral effects observed. Alterations of brain serotonin (5-HT) neurochemistry were also noted, and this neurotransmitter has been implicated in the regulation of mood (5) and locomotor activity (6) in humans and mice, respectively. These data indicate that the enhanced locomotor activity of Mt-bGH transgenic mice could provide a useful model to study the mechanisms behind the positive psychic effects of GH. Abbreviations: BBB, Blood brain barrier; bGH, bovine GH; CNS, central nervous system; GFAP, glial fibrillary acidic protein; GHR, GH receptor; 5-HT, brain serotonin; Mt-bGH, overexpression of bGH under control of the metallothionein promoter.
in glial fibrillary acidic protein– bovine GH transgenic mice but clearly lower than in transgenic mice with general overexpression of bovine GH. In contrast to the transgenic mice with general overexpression of bovine GH, glial fibrillary acidic protein-bovine GH mice did not display any difference in serum IGF-I levels. The levels of free T3 and the conversion of the free T4 to free T3 were only increased in transgenic mice with general overexpression of bovine GH, but serum corticosterone levels were similarly increased in both transgenic models. These results suggest that free T3 and/or IGF-I, affecting dopamine and serotonin systems in the central nervous system, may mediate the enhanced locomotor activity observed in transgenic mice with general overexpression of bovine GH. (Endocrinology 142: 4560 – 4567, 2001)
The psychic effects of GH may be exerted by direct actions of GH in the central nervous system (CNS). Some data suggest that GH passes the blood brain barrier (BBB) from the peripheral circulation (7), and in humans GH receptors (GHRs) are present in the choroid plexus, hippocampus, hypothalamus, and pituitary gland (8). In the rat, GHRs are found in the same areas but also in capsula interna, parietal cortex, tegmentum, mamillary bodies, and the temporal cortex (9). Some of these brain regions have been implicated in emotional responses and psychic drive. Another possibility is that the psychic effects of GH are mediated via some substance(s) produced in peripheral tissues that secondarily influences CNS. For example, serum levels of corticosteroids are elevated in GH transgenic mice (10), and corticosteroids may influence brain dopamine systems (11, 12) as well as their sensitivity to drugs of abuse (13). It is also well known that corticosteroids can increase the general well-being in humans and even produce hypomania, mania, and psychosis (14). IGF-I is another peripheral hormone with increased serum levels in the Mt-bGH transgenic mice, and this molecule may also pass the BBB. Thus, endothelial cells lining brain microvessels contain IGF receptors that may internalize IGFs, and infused 125I-IGF-I has been reported to rapidly cross the BBB and enter the brain parenchyma (15). Also interestingly, IGF-I may influence the function of brain dopamine neurons (16). Finally, placebocontrolled trials have demonstrated that GH administration stimulates the peripheral conversion of T4 to the biologically more active T3 in both normal and obese adults as well as in
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GH-deficient adults (17, 18). It is well established that thyroid hormones may influence psychic drive and mood (19). To discriminate between direct effects of GH in the CNS and its peripheral effects, we designed a transgenic construct to limit the expression of GH to the CNS. The effect of CNS-specific expression of the bGH transgene on spontaneous locomotor activity and serum levels of the above mentioned hormones was studied and compared with that of normal controls and mice with a general overexpression of bGH. Materials and Methods Transgene construction and generation of transgenic mice The bGH gene was ligated into the C-3123 plasmid (kindly provided by Dr. Lennart Mucke, Department of Neurology, University of California, San Francisco, CA) containing the glial fibrillary acidic protein (GFAP) promoter, an SV40 intron and an SV40 polyA signal (GFAPbGH; Fig. 1). The GFAP-bGH DNA fragment was excised by restriction enzyme cleavage using SfiI, separated by gel electrophoresis through a 1% agarose gel, cut out, isolated using isotachophoresis (20) and precipitated by ethanol. Transgenic mice were generated by standard microinjection procedure (21) of fertilized C57BL/6JxCBA zygotes. To identify transgenic animals, DNA was extracted from 0.5-cm tail biopsies from 2-wk-old mice. The tails were digested in lysis buffer (1% SDS, 2 mg/ml proteinase K (Merck KgaA, Darmstadt, Germany), 50 mm Tris, 100 mm EDTA, 100 mm NaCl; final pH 8.0) at 56 C overnight. The DNA from the digested tails was extracted using phenol/chloroform and precipitated by ethanol. DNA was digested with BamHI (Promega Corp., Madison, WI), separated by electrophoresis, transferred to an N⫹ Hybond (Amersham, Little Chalfont, Buckinghamshire, UK) nylon membrane by Southern blot (22) and hybridized with a 1.2-kb PvuII fragment digested from the bGH gene. The environment of the animal rooms was controlled with a 12-h light-dark cycle (0730 h-1730 h, with a 1-h dawn/sunset function), a relative humidity between 45–55% and a temperature of 20 C. The mice had free access to tap water and standard pellet chow (R-34, Lactamin, Vadstena, Sweden). The study was performed after prior approval from the local ethical committee for animal experimentation at the Go¨teborg University, Go¨teborg, Sweden. The mice were anesthetized with ketamine hydrochloride (77 mg/kg; Ketalar, Parke-Davis, Detroit, MI) and
FIG. 1. The GFAP-bGH plasmid. Digestion by SfiI generated the injection fragment containing GFAP promoter, SV40 splice, bGH gene, and SV40 polyA.
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xylazine (9 mg/kg; Rompun, Bayer Corp., Lever-Kusen, Germany) and killed by heart puncture. The organs used for RNA and protein preparations were excised, immediately frozen in liquid nitrogen, and stored at ⫺135 C.
RNA analysis Total RNA was isolated from frozen tissues as described by Chomczynski and Sacchi (23). First-strand cDNA synthesis was performed at 42 C for 15 min with 1 g RNA as template in the presence of 10 mm Tris-HCl; pH 9.0, 50 mm KCl, 0.1% Triton X-100, 5 mm MgCl2, 0.5 g/l oligo(dT)15 primer (Promega Corp.), 15 U/g AMV-RT (Promega Corp.), 1 U/l RNAsin (Promega Corp.), and 1 mm of each dNTPs (Promega Corp.). The reverse transcription reaction was terminated by heat inactivation at 95 C for 5 min and then incubated at 4 C for 5 min. The second-strand PCR was performed at 94 C for 30 sec and 30 cycles of sequential incubations at 94 C for 30 sec, 55 C for 30 sec, and 72 C for 2 min using a reaction mixture containing 10 l of the first-strand reaction from all tissues except brain, which contained 2 l, 10 mm Tris-HCl (pH 8.3), 1.5 mm MgCl2, 50 mm KCl, 0.2 mm of each dNTP, 0.5 U Taq DNA polymerase, and 0.4 m each of primers ex2 b-GHs (5⬘TCCCTGCTCCTGGCTTTCGC-3⬘) and ex4 b-GHa (5⬘-GCAGTGAGATGCGAAGCAGC). The PCR product was analyzed by electrophoresis.
Protein analysis by Western blotting Tissues were prepared by homogenization in PE buffer (10 mm potassium phosphate buffer; pH 6.8, and 1 mm EDTA) containing 10 mm 3-([3-cholamidopropyl] dimethyl-ammonio)1-propanesulphonate (Roche Molecular Biochemicals, Mannheim, Germany), aprotinin (1 mg/ml; Roche Molecular Biochemicals), leupeptin (1 mg/ml; Roche Molecular Biochemicals), pepstatin (1 mg/ml; Roche Molecular Biochemicals), and pefablock (1 mg/ml; Roche Molecular Biochemicals). The homogenate was sonicated and centrifuged (10,000 g for 10 min at 4 C) and protein concentrations measured using a protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Thirty-five micrograms total protein from each sample was mixed with sample buffer (62.5 mm Tris-HCl, pH 6.8, 2.3% SDS, 1% glycerol, 5% mercaptoethanol, and 0.001% bromphenol blue) and loaded on 10% NuPAGE Bis-Tris (Novex, San Diego, CA) gels. The proteins were transferred to a polyvinyldifluoride membrane (Amersham Pharmacia Biotech) using a Novex blotting system. The membranes were incubated with bGH-specific antisera raised in monkey (dilution 1:1000; kindly provided by Dr. A. F. Parlow, Pituitary Hormones and Antisera Center, Torrance, CA). Prestained standards (SeeBlue,
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Novex) were used as size markers. Immunoreactive protein was visualized by chemiluminescence using alkaline phosphatase conjugated secondary goat antimonkey antibodies (dilution 1:30,000) (Sigma Chemicals Co., St. Louis, MO) and CDP-star (Tropix, Bedford, MA) as substrate. The membrane was exposed to ECL film (Amersham Pharmacia Biotech) at room temperature for 1 min.
Immunohistochemistry and in situ hybridization Brains from adult GFAP-bGH mice and control littermates were fixed at 4 C overnight in 4% paraformaldehyde in PBS and processed for paraffin embedding. The sections (6 m thick) were rehydrated and nonspecific staining was preblocked with 5% normal rabbit serum in TBS (20 mm Tris-HCl, pH 7.6, and 150 mm NaCl) containing 0.1% Triton X-100. Sections were then incubated overnight at 4 C with bGH antisera raised in monkey (diluted 1:250 in TBS containing 0.2% BSA). For detection of immunoreactivity, alkaline phosphatase-conjugated rabbit antimonkey antibodies were used followed by incubation with a chromogenic substrate for alkaline phosphatase, nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate in NTMT buffer (100 mm NaCl, 100 mm Tris-HCl, pH 9.5, 50 mm MgCl2, 0.1% Tween-20). After color development, brain sections were postfixed in 4% paraformaldehyde in PBS and mounted. Sections adjacent to the ones processed for immunohistochemistry were used for in situ hybridization to localize bGH mRNA expression. A digoxigenin-labeled bGH antisense riboprobe was generated from a Hind-III-linearized 1.2-kb fragment of a GFAP-bGH subclone construct and in vitro transcribed with T3 RNA polymerase. Hybridization and posthybridization washes were essentially the same as described for whole-mount in situ hybridization (24). The transcripts were visualized with a horseradish peroxidase-conjugated mouse antidigoxigenin antibody (Roche Molecular Biochemicals) together with the Tyramide signal amplification kit (NEN Life Science Products Inc., Boston, MA) according to the manufacturer’s instructions.
Analyses of GH, IGF-I, corticosterone, and thyroid hormone levels The concentration of circulating bGH was determined in two lines by RIA (antisera kindly provided by Dr. A. F. Parlow, Pituitary Hormones and Antisera Center, Torrance, CA). The assay was carried out in 200 l PBS (pH 7.4) containing 0,5% BSA (RIA grade, Sigma), 1.25 l mouse serum, anti-bGH antiserum (1:400,000), and 125I-labeled bGH. The bGH standard (0.1–50 ng/tube) contained 1.25 l normal mouse serum. After overnight incubation (4 C), the bound hormone was precipitated by adding 1 ml of a mixture of polyethylene glycol (16%, wt/vol, final concentration), bovine ␥-globulin (2 mg/ml; Cohn fraction II and III; Sigma), and Triton X-100 (0.02% in 0.05 m Tris-HCL, pH 8,5). The samples were further incubated for 30 min in 4 C, centrifuged, and supernatants were aspirated. The pellets were counted for ␥-radioactivity. The IGF-I concentration in serum was determined by RIA (Nicols Institute Diagnostics, San Juan Capistrano, CA) after acid-ethanol extraction, according to the manufacturer’s protocol in a double assay. Serum corticosterone levels were determined by RIA (ICN Biomedicals, Inc., Diagnostics Division, Costa Mesa, CA) according to the manufacturer’s protocol. All samples were collected between 1020 h and 1145 h. FT3 and FT4 were measured in serum using the Amerlex-MAB kits as described (25).
Locomotor activity studies Locomotor activity was measured using activity meters (Digiscan animal activity monitor, model AZYCCM Tao, Omnitech Electronics, Columbus, OH) that were placed in eight identical sound- and lightattenuating boxes containing a weak light and fan. The activity meter was equipped with three rows of infrared photosensors, each row consisting of 16 sensors placed 2.5 cm apart. Two rows were placed in a 90-degree angle along the front and side of the floor of the cage, and the third row was placed 10 cm above the floor to measure vertical activity. The activity meters were connected to an analyzer system (Omnitech Electronics), and the data were collected using LabVIEW (National Instruments, Austin, TX) computer software.
Bohlooly-Y et al. • Locomotor Activity in bGH
Six-month-old GFAP-bGH, Mt-bGH transgenic mice and control littermates were placed in transparent plastic boxes and put into the activity meters. Locomotor activity was recorded for 60 min. All experiments were performed between 0900 h and 1600 h in a randomized (regarding the individual mouse) but balanced order, with respect to groups, boxes, and time of day. Three types of experiments were performed: one with Mt-bGH mice and their littermate controls, a second with one line of the GFAP-bGH mice and their littermate controls, and a third with another line of the GFAP-bGH mice and littermate controls. The activity of littermate controls, in absolute terms, differed slightly among the three experiments, whereas the overall pattern of activity (i.e. an initial high activity followed by a gradual decline) was identical in all experiments. To facilitate comparisons among the three experiments, the cumulated locomotor activity at each time point is expressed in percent of the locomotor activity displayed by the controls during the first 5 min. The pattern of activity was judged on the basis of the habituation of the animals to the new environment. The pattern in control animals is that an initial high activity if followed by a decrease.
Statistics The locomotor activity data were statistically evaluated using a twofactor ANOVA for repeated measures; all other comparisons among groups were made by unpaired t test. Values presented as mean ⫾ sem P ⬍ 0.05 was considered significant.
Results Animals
To generate GFAP-bGH transgenic animals on the C57 BL/ 6JxCBA genetic background, 110 injected C57 BL/6JxCBA embryos were implanted into five C57 BL/6JxCBA foster mothers, resulting in 22 newborn mice. Five mice that carried the GFAPbGH transgene (founder animals) were identified using Southern blot analysis. Two founder mice were infertile. Offspring from the three remaining founders were analyzed and showed a similar phenotype. mRNA analysis by RT-PCR
RNA was isolated from 10 different organs and RT-PCR was used to examine expression of bGH. Specific mRNA for bGH was strongly expressed in the brain and showed weak expression in lung and kidney in two lines (data not shown). The third line expressed bGH only in the brain (Fig. 2). Protein analysis by Western blotting
Protein was isolated from the same organs as for RT-PCR (see above), and the expression of bGH was examined by Western blotting. Brains from all three lines showed high and specific expression of bGH, whereas no expression was found in any other tissue examined (Fig. 3). Bovine GH expression in brain detected by immunohistochemistry and in situ hybridization
Immunohistochemistry for bGH of brain sections from GFAP-bGH mice showed immunostaining of astrocytes in both cerebrum and cerebellum (Fig. 4, A, C, and E). Sections from littermate control brains showed only staining at background levels (Fig. 4, B, D, and F). In brains from GFAP-bGH transgenic mice, bGH-specific mRNA exhibited similar expression pattern to that found by immunohistochemistry (Fig. 4, G and I). No signal above background was detected in brain sections from littermate control mice (Fig. 4, H and J). Astrocyte expression of the
Bohlooly-Y et al. • Locomotor Activity in bGH
FIG. 2. Expression analysis by RT-PCR of bGH mRNA in brains from GFAP-bGH transgenic mice and littermates, using a sense primer in exon 2 of the bGH gene and an antisense primer located in exon 4 of the bGH gene. Templates used are as follows: lane 1, brain cDNA from a littermate control; lane 2– 4, brain cDNA from GFAP-bGH line 1–3; lane 5, DNA ladder (1 kb⫹; Life Technologies, Inc.).
bGH protein and mRNA was found in the gray (protoplasmic astrocytes) as well as in the white matter (fibrous astrocytes). Furthermore, although bGH mRNA in astrocytes was confined to the perinuclear region (Fig. 4, I and K), immunostaining for bGH protein showed a starlike shape owing to immunoreactivity of the long, highly branched processes (Fig. 4, L and M). Hormone levels
Serum bGH levels were significantly increased in GFAPbGH transgenic mice from the two analyzed lines. The serum levels of bGH in line 3 were 334 ⫾ 53 ng/ml (n ⫽ 8) and line 2 were 207 ⫾ 21 ng/ml (n ⫽ 8) (P ⬍ 0.05 vs. littermate controls). This was considerably lower than previously reported for the Mt-bGH transgenic animals (26). No difference in serum IGF-I levels was observed in GFAP-bGH transgenic mice, compared with the littermate controls, whereas Mt-bGH transgenic mice displayed significantly elevated levels, compared with the littermate controls (Table 1). Serum corticosterone levels were significantly increased in both the GFAP-bGH and the Mt-bGH transgenic mice, compared with their littermate controls (Table 1). In GFAP-bGH transgenic mice, the serum levels of T3 were significantly decreased (28%), but the serum levels of T4 did not differ from the littermate controls. In the Mt-bGH, the levels of T3 were significantly increased (25%), and the T4 levels were significantly decreased (47%). The conversion of T3 to T4 in the Mt-bGH transgenic mice were significantly increased (49%) (Table 1).
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FIG. 3. Western blotting analysis of transgenic mice from different lines and a littermate control in 10 different organs using a bGH antibody. Lane 1 in panel A–D, Untreated serum from Mt-bGH transgenic mice used as a positive control (2 l in C, 4 l in D, 8 l in A, and 16 l in B). Lanes 2–11 in panel A–D, 35 g of total protein from different organs. Panel A, Protein samples from a littermate control. Panels B, C, and D, Protein samples from the GFAP-bGH transgenic mice from lines 1, 2, and 3.
Locomotor activity of Mt-bGH and GFAP-bGH transgenic mice
The spontaneous locomotor activity of Mt-bGH transgenic mice during 60 min was significantly increased, compared with littermate controls (Fig. 5; two-factor repeated-measure ANOVA, group effect). F(1,22) ⫽ 5.6; P ⫽ 0.0273). There was a decay of locomotor activity over time in the whole material (time effect: F(11,242) ⫽ 14.5; P ⬍ 0.0001), but the pattern of locomotor activity was different in the two groups, as indicated by a significant interaction term (F(11,242) ⫽ 2.5; P ⫽ 0.0053). Thus, whereas there was a gradual decline over time in the littermate controls, Mt-bGH transgenic mice maintained approximately the same degree of locomotor activity during the first 40 min, after which they gradually became less active but, however, never reached the same low activity level by 60 min as the littermate controls. Line 2 of GFAPbGH mice displayed patterns and degrees of locomotor activity that were almost identical to those of their littermate controls (Fig. 5; group effect; F(1,14) ⫽ 0.0005; P ⫽ 0.9830; time effect; F(11,154) ⫽ 19.3; P ⫽ 0.0001; interaction term; F(11,154) ⫽ 0.8; P ⫽ 0.6649). Line 3 of GFAP-bGH mice did not significantly differ from their controls as regards the amount of locomotor activity displayed during 60 min (group effect; F(1,14) ⫽ 0.5; P ⫽ 0.4951; time effect; F(11,154) ⫽ 12.4; P ⬍ 0.0001), whereas they differed as regards their locomotor activity pattern (interaction term;
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Bohlooly-Y et al. • Locomotor Activity in bGH
FIG. 4. Immunohistochemistry and in situ hybridization of bGH in the CNS of GFAP-bGH transgenic mice and littermate controls. In brains from GFAP-bGH transgenic mice, bGH immunoreactivity is confined to astrocytes in the cerebrum, cerebellum, and spinal cord (A and C). High magnification of the cerebellum section in C, showing the presence of bGH immunoreactivity in astrocytes in the granular and molecular layers (E). Sections from wild-type brains exhibit immunostaining at background levels (B, D, and F). In situ hybridization showing the expression of bGH mRNA in sections from GFAP-bGH transgenic mice (G, I, and K), which appears as black dots, representing mRNA confined to the perinuclear region of astrocytes. This is well visualized at higher magnification in the cerebellum (I) and in the brain stem (K). No signal above background was detected on wild-type sections (H and J). In contrast to the perinuclear cytoplasmic localization of bGH mRNA in astrocytes, immunostaining for bGH protein was also found in the numerous processes of these star-shaped cells (L and M).
F(11,154) ⫽ 2.7; P ⫽ 0.0034). These mice displayed an almost constant intermediate-low activity level over time. Altogether, both the amount and patterns of locomotor activity of both lines of GFAP-GH mice differed clearly from those of Mt-bGH transgenic mice. Discussion
In line with our previous study, Mt-bGH transgenic mice displayed an increased spontaneous locomotor activity and signs of a disturbed habituation process (4). These behavioral alterations could, however, not be demonstrated in the GFAP-bGH transgenic mice, with an expression of bGH restricted to CNS as demonstrated by RT-PCR, Western blot,
and immunohistochemistry. The CNS-produced bGH is likely to be biologically active because we see an effect on food intake and expression of genes related to appetite control in hypothalamus in the GFAP-bGH transgenic animals (Bohlooly-y, manuscript in preparation). This suggests that the increased locomotor activity observed in the transgenic mice, generally overexpressing bGH controlled by the metallothionein promoter, is not owing to signaling through GHR in the CNS but rather involves other, presumably peripheral mechanisms. The mice differ in size that could potentially result in altered locomotor activity. However, arguing for an effect unrelated to size is the locomotor activity pattern of the
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TABLE 1. Effects of CNS-directed overexpression (GFAP-bGH) and general overexpression (Mt-bGH) of bGH on serum levels of IGF-I, corticosterone, T3, T4, and conversion of T4 to T3
Wild-type GFAP-bGH-transgenic Mt-bGH-transgenic
IGF-I (g/liter)
Corticosterone (g/liter)
T3 (pmol/liter)
T4 (pmol/liter)
T3/T4
208 ⫾ 17 229 ⫾ 9 378 ⫾ 24a
199 ⫾ 24 763 ⫾ 64a 745 ⫾ 135a
5.4 ⫾ 0.4 4.2 ⫾ 0.2a 7.2 ⫾ 0.2a
13.7 ⫾ 0.6 13.7 ⫾ 0.6 9.3 ⫾ 1.1a
0.41 ⫾ 0.039 0.31 ⫾ 0.009 0.81 ⫾ 0.069a
The performed analyses are described in Materials and Methods. Values are presented as mean ⫾ SEM. a P ⬍ 0.05 vs. littermate controls (unpaired t test). IGF-I (n ⫽ 5–7). Corticosterone, wild-type, GFAP-bGH transgenic line 3 (n ⫽ 6 – 8), and Mt-bGH transgenic (n ⫽ 3). T3, T4, T3/T4, wild-type (n ⫽ 12), GFAP-bGH line 3 (n ⫽ 7), and Mt-bGH transgenic mice (n ⫽ 5). Only male mice were used in this study.
FIG. 5. Locomotor activity in Mt-bGH and GFAP-bGH transgenic mice and respective littermates. A, Male Mt-bGH transgenic mice and littermate controls (P ⫽ 0.027); B, male GFAP-bGH transgenic mice from line 3 and littermate controls (P ⫽ 0.495); and C, male GFAPbGH transgenic mice from line 2 and littermate controls (P ⫽ 0.983). Statistics were determined by two-factor repeated-measure ANOVA (see Results) where P ⬍ 0.05 was considered significant and n ⫽ 12 in all groups.
Mt-bGH transgenic mice. If the difference in locomotor activity had been an effect of size, one would have expected a higher initial degree of activity followed by a decline in parallel with that of controls. Neither of these was observed. Furthermore, no correlation between size and locomotor activity score has previously been observed with this method among control mice (weight range 25– 45 g) (27). Three tentative peripheral factors involved in mediating the increased locomotor activity of Mt-bGH transgenic mice were examined in the present study: corticosterone, IGF-I, and thyroid hormones. Studies have indicated that corticosterone may influence both dopamine release (12) and dopamine receptor sensitivity (11). Brain dopaminergic systems are heavily implicated in regulation of locomotor activity (13). Indeed, supporting previous observations (10), serum corticosterone levels were markedly increased in generally overexpressing Mt-bGH transgenic mice. However, a similar increase was observed in GFAP-bGH transgenic mice expressing bGH limited to the CNS, in which no difference in locomotor activity, compared with the littermate controls, was observed. This suggests that the locomotor stimulatory effects of GH are not mediated by corticosteroids but via some other peripheral factor. Furthermore, these findings indicate that the enhanced corticosterone secretion observed in Mt-bGH transgenic mice may be owing to a CNS action of GH, suggesting that central GHR may be involved in the regulation of the hypothalamic-pituitary-adrenal axis. IGF-I levels were increased in Mt-bGH transgenic mice, but no difference was observed in GFAP-bGH transgenic mice, compared with littermate controls. It has been demonstrated that IGF-I crosses the BBB and supports dopaminergic neurons (16). Moreover, in vivo studies have shown that IGF-I promotes the survival of dopamine and noradrenaline neurons when exposed to neurotoxins (28). It has also been demonstrated that a local infusion of IGF-I may stimulate axonal regeneration, enhance functional recovery, and increase nerve sprouting in association with peripheral nerve injury (29 –33), whereas no effects on composite motor function or in the learning ability of uninjured animals has been observed (34). It is therefore unclear whether IGF-I produces any trophic effects in the uninjured situation, as is the case in the present study. However, IGF-I could alter the locomotor activity through its documented induction of catecholamine synthesizing enzymes (35). IGF-I could be produced in the brain (36) and potentially also as response to the local GH production in our transgenic model. However, we could not detect any statistically significant changes in IGF-I mRNA levels among the brains from GFAP-bGH, Mt-bGH
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transgenic mice, and littermate controls (data not shown). Furthermore, the weight of the brains of GFAP-bGH transgenic mice were not increased in relation to body weight (data not shown) in contrast to transgenic mice overexpressing IGF-I (37). Supporting previous findings that GH increases the conversion of T4 into T3 (38), a clear increase in the T3/T4 conversion ratio was observed in the Mt-bGH transgenic animals. In contrast, in GFAP-bGH transgenic mice that did not display locomotor hyperactivity, no alteration in the conversion of T4 to T3 was observed. Thus, the increased locomotor activity seen in the Mt-bGH transgenic mice may, at least in part, be related to an increased conversion of T4 into the more biologically active T3 and an increased signaling through the thyroid hormone system. When T3 is given to mice, the spontaneous activity is increased (39), further supporting the hypothesis that the elevated T3 levels may be important for the increased locomotor activity in the Mt-bGH transgenic mice. Indeed, both T3 and T4 cross the BBB when peripherally injected, and both acute and chronic T3 administration increases the concentration of serotonin in the rat frontal cortex but not in the hippocampus (40 – 42). This T3-induced increase of 5-HT levels is produced either by modifying 5-HT reuptake and turnover rate or by acting on the neurotransmitter metabolism (43, 44). The effects of thyroid hormones on the levels of serotonin metabolites may be species specific because, in the hyperthyroid rat, 5-HIAA levels are decreased (45) but an increase is seen in the hyperthyroid mouse (46). Interestingly, serotonergic agonists markedly enhance behavioral activity in hyperthyroid rats (47), and serotonin has also been suggested to stimulate locomotor activity in mice (6). In the Mt-bGH transgenic mice, enhanced levels of 5-HT and 5-HIAA were previously observed in the striatum, mesencephalon, and cortex. Novelty- and amphetamine-induced hyperactivity were also observed in Mt-bGH transgenic mice (4). In this context it should be recalled that amphetamine releases not only dopamine but also serotonin, and both transmitters appear to contribute to the behavioral activation produced in mice by pharmacological manipulations that raise their synaptic levels (48). Also, more recent studies suggest that amphetamine-induced locomotor stimulation involves activation of postsynaptic 5-HT2A receptors (49, 50). One hypothesis could therefore be that in the MtbGH transgenic mice, high circulating levels of GH increase the conversion of T4 to T3 that secondarily enhances the function of the brain serotonin system. This in turn promotes locomotor activity in response to challenges that increase 5-HT release, such as exposure to amphetamine or, possibly, novelty stress. Also, the thyroid status in humans is of great importance for psychic well-being. Thus, hyperthyroidism and hypothyroidism may be associated with anxiety and depression, respectively (51), and these disorders are related to brain monoamine function (5, 52). T3 supplementation has been shown to have a beneficial effect on depression in several studies. For instance, among patients refractory to tricyclic antidepressant drug therapy, those treated with T3 augmentation were twice as likely to respond as controls (53). Taken together with the present results, it may be suggested that
Bohlooly-Y et al. • Locomotor Activity in bGH
Mt-bGH mice could be a useful model for studying mechanisms underlying the interaction between thyroid hormones and monoaminergic systems. This could be of value not only for understanding the positive psychic effects of GH but also for optimization of treatment of affective disorders. In conclusion, the present results indicate that the enhanced spontaneous locomotor activity in Mt-bGH mice is not primarily owing to activation of GHR in the CNS but involves some peripheral factor(s). Furthermore, the data suggest that especially T3 but also IGF-I are two major candidates in this context. However, a firm determination of whether either or both of these are involved requires further experimentation. It should also be pointed out that the present data do not rule out an involvement of direct GHRmediated effects, other than on spontaneous locomotor activity, in the CNS. Acknowledgments We would like to thank Maud Pettersson for excellent technical assistance and Professor Anders Linde for the excellent assistance with the art work. Received March 28, 2001. Accepted June 27, 2001. Address all correspondence and requests for reprints to: Jan To¨ rnell, AstraZeneca Transgenic Centre, AstraZeneca R&D, S-43183 Mo¨ lndal, Sweden. E-mail:
[email protected]. This work was supported by the Swedish Medical Research Council (Grant 4250, 2789, 14100), the Swedish Cancer Foundation, the Swedish Foundation for Strategic Research, the Lundberg Foundation, the Swedish Medical Society, AstraZeneca R&D, Pharmacia-Upjohn, Novo Nordisk Foundation, the Sahlgrenska University Foundation, and the Swedish Association Against Rheumatic Disease.
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