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Evidence that the serotonin transporter does not shift into the cytosol of remaining neurons after neonatal brain injury
Neuroscience Research 73 (2012) 252–256
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Evidence that the serotonin transporter does not shift into the cytosol of remaining neurons after neonatal brain injury Julie A. Wixey, Hanna E. Reinebrant, Kathryn M. Buller ∗ Clinical Neuroscience, Perinatal Research Centre, The University of Queensland, UQ Centre for Clinical Research, Herston, QLD 4029, Australia
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Article history: Received 23 January 2012 Received in revised form 3 April 2012 Accepted 5 April 2012 Available online 15 April 2012 Keywords: Astrocytes Hypoxia-ischemia Microglia Neonate Raphé Serotonin transporter
a b s t r a c t Following neonatal hypoxia-ischemia (HI) serotonin (5-hydroxytryptamine, 5-HT) levels are decreased in the brain. The regulation of brain 5-HT is dependent on the serotonin transporter (SERT) localised at the neuronal pre-synaptic cell membrane. However SERT can also traffic away from the cell membrane into the cytosol and, after injury, may contribute to the cell’s inability to maintain 5-HT levels. Whether this occurs after neonatal HI brain injury is not known. In addition, there is contradictory evidence that glial cells may also contribute to the clearance of 5-HT in the brain. Using a postnatal day 3 (P3) HI rat pup model (right carotid ligation + 30 min 6% O2 ), we found, in both control and P3 HI animals, that SERT is retained on the cell membrane and is not internalised in the cytosol. In addition, SERT was only detected on neurons. We found no evidence of SERT co-localisation on microglia or astrocytes. We conclude that neuronal SERT is the primary regulator of synaptic 5-HT availability in the intact and P3 HI-injured neonatal brain. Furthermore, since concomitant reductions in 5-HT, SERT and serotonergic neurons occur after neonatal HI, it is plausible that the decrease in brain 5-HT is a consequence of SERT being lost as neurons degenerate as opposed to remaining neurons internalising SERT or clearance by glial cells. © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction Serotonin (5-hydroxytryptamine, 5-HT) is critical for brain development (Gaspar et al., 2003). The serotonergic system constitutes a pervasive neural network and serves a diverse array of functions. Thus disruption to this central 5-HT network early in life contributes to a wide range of adverse outcomes including depression, epilepsy, movement disorders, sudden infant death syndrome and autism (Duncan et al., 2010; Gartside et al., 2003; Nemeroff and Owens, 2009; Pardo and Eberhart, 2007). It is pertinent that many of these disorders match those observed in neonates with hypoxic-ischemic (HI) brain injury (Peterson et al., 2003; Anderson and Doyle, 2003; Hack et al., 2004; Ferriero, 2004). Indeed we have demonstrated recently in a neonatal rodent model that exposure to a HI insult significantly disrupts the serotonergic system in the neonatal brain (Reinebrant et al., 2010; Wixey et al., 2011a,b). Damage to this system includes loss of serotonergic raphé neurons, reduced 5-HT levels and reduced expression of the 5-HT transporter (SERT) (Reinebrant et al., 2010; Wixey et al., 2011a,b). It
∗ Corresponding author at: Clinical Neuroscience, Perinatal Research Centre, Building 71/918, The University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, Herston, QLD 4029, Australia. Tel.: +61 7 33466011; fax: +61 7 33465594. E-mail address: [email protected] (K.M. Buller).
is important to understand the mechanisms that contribute to 5HT neuronal damage in order to develop therapeutic interventions that could prevent lifelong functional deficits seen in HI-affected neonates. The SERT consists of 12 trans-membrane domains that span the presynaptic membrane of 5-HT-releasing cells (Blakely et al., 1991). It is the key regulator of extracellular 5-HT levels in the brain. Central SERT terminates 5-HT neurotransmission by the re-uptake of 5-HT from the synapse into the nerve terminals and controls the duration of action and post-synaptic signalling of 5-HT in the brain. Consequently, SERT is a major target for drugs such as selective serotonin reuptake inhibitors (SSRIs) that can increase 5-HT availability in the brain. In the normal adult brain, SERT can traffic from the cellular membrane to sub-cellular or cytosolic compartments. This process of internalisation reduces the capacity of serotonergic neurons to release and re-uptake 5-HT at the cell membrane (Qian et al., 1997; Ramamoorthy et al., 1998). We have found concomitant reductions in SERT and 5-HT in the brain after neonatal HI (Wixey et al., 2011a). These parameters are not abolished and, although serotonergic neurons are also reduced in number after neonatal HI (Wixey et al., 2011a; Reinebrant et al., 2010), the possible internalisation of SERT in remaining neurons may reduce their capacity to release and reuptake 5-HT and therefore contribute to the reduced 5-HT levels one week after injury. This would create the potential for SERT to be recovered back to the cell membrane after brain injury where it
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J.A. Wixey et al. / Neuroscience Research 73 (2012) 252–256
could resume regulating synaptic re-uptake of 5-HT. For example, chronic activation of p38 MAPK can lead to enhanced SERT surface expression (Samuvel et al., 2005; Zhu et al., 2006). Such a mechanism might reveal a new target to manipulate SERT function after brain injury. The efficient re-uptake of 5-HT is primarily dependent on SERT localisation on cell bodies, dendrites and fibres of serotonergic neurons in the central nervous system (Qian et al., 1995). However, although evidence is controversial, it has also been purported that 5-HT uptake can occur by glial cells and thus non-neuronal cells could assist in clearing 5-HT from the synapse. Reports primarily based on in vitro studies, suggest SERT may exist on astrocytes and microglia (Bel et al., 1997; Kubota et al., 2001; Inazu et al., 2001; Horikawa et al., 2010). In direct contrast, others utilising in vivo models have found no evidence of glial SERT in the brain (Sur et al., 1996; Tao-Cheng and Zhou, 1999; Zhou et al., 2000). It is important to examine the localisation of SERT in vivo since microglial cells, for example, can change their morphology and function substantially after brain injury (Kreutzberg, 1996) and after neonatal HI substantial increases in numbers of activated microglia appear in the brain (Ivacko et al., 1996; McRae et al., 1995; Wixey et al., 2009, 2011a). Inhibition of neuroinflammation has also been shown to prevent damage to the 5-HT system following neonatal HI brain injury (Wixey et al., 2011a,b). We hypothesised that neonatal HI brain injury can influence the sub-cellular and cellular phenotypic localisation of SERT in the brain. To test this we used a post-natal day 3 (P3) HI model of neonatal brain injury that incurs significant losses of raphé serotonergic neurons, reduced SERT expression and reduced levels of 5-HT in the brain (Reinebrant et al., 2010; Wixey et al., 2011a,b). We investigated if SERT expression is evident in the cellular membrane and/or cytosol of the non-injured and P3 HI-injured brain. We also examined whether SERT is localised on neurons, microglia and/or astrocytes. 2. Materials and methods 2.1. Animals Sprague–Dawley dams and their pups (10–12 per litter) were used to perform experiments. The animals had access to food and water ad libitum and were kept in rooms maintained at 22 ◦ C on a 12 h light/dark cycle. Surgical procedures and tissue collection were performed between 0900 and 1100 to minimise the possible effects of circadian rhythms. All experiments were performed in accordance with ethical approvals stipulated by the University of Queensland Animal Ethics Committee. Efforts were made to minimise the number of animals used and their suffering. 2.2. Neonatal hypoxic-ischemic insult On post-natal day 3 (P3), pups were assigned randomly to (i) control or (ii) P3 HI groups. The P3 HI insult was produced as described previously (Buller et al., 2008; Carty et al., 2008; Reinebrant et al., 2010). Briefly, pups were anaesthetised using isoflurane (2%; Baxter, IL, USA), the right common carotid artery was isolated and ligated permanently. Pups recovered for 20 min before being exposed to 6% O2 for 30 min at 37 ◦ C in a humidified chamber. Pups were then returned to the dam. The control group underwent the same procedures but the carotid artery was not ligated and the animals were exposed to room-air instead of hypoxia. The mortality rate was < 3.7%. On P10, animals were euthanized by administering a lethal dose of sodium pentobarbitone (80 mg/kg, i.p.; Lethabarb, Virbac, France).
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2.3. Membrane and cytosolic fraction preparation For protein analyses, brains (control n = 6; P3 HI n = 6) were rapidly excised on P10. Frontal cortex (+4.0 to 0.0 mm relative to bregma) and brainstem dorsal raphé (−7.0 to −12.0 mm relative to bregma) regions were collected, immediately frozen and stored at −80 ◦ C (Paxinos and Watson, 1997; Reinebrant et al., 2010). Total protein extracts were separated into two fractions to determine the quantity of SERT in the plasma membrane and cytosolic fraction. Tissue was homogenised in 1 mL homogenisation buffer (0.32 M sucrose, 0.10 mM HEPES pH 7.4, 0.2 mM EDTA and protease inhibitor cocktail) and spun at 1000 × g for 15 min at 4 ◦ C. Supernatant was recovered and spun at 183,000 × g for 45 min at 4 ◦ C. Supernatant was collected for the cytosolic fraction. The resultant pellet was resuspended in HEPES-lysis buffer (0.5 mM HEPES, 0.2 mM EDTA) and constituted the membrane fraction. Total amount of protein in each fraction was determined using a bicinchoninic acid kit (Pierce, Rockford, IL, USA). To determine whether protein preparations were separated successfully, both fractions from a control brain were probed with aspartyl aminopeptidase (AspAP, 1:10,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) because it is predominantly localised in the cytosol (Wilk et al., 1998), early endosome antigen 1 (EEA1, 1:2000; BD Biosciences, San Jose, CA, USA) to detect endosomal proteins in the cytosol (Wang et al., 2005) and sodium-potassium adenosine triphosphatase (Na+ /K+ -ATPase, 1:5000; Chemicon International, CA, USA) as it is predominantly localised in the cellular membrane (Schimmel et al., 1973). 2.4. Western blotting Western blotting was carried out to detect SERT protein expression in the plasma membrane and cytosolic fractions. Samples (16 g per well) were separated by 10% sodium dodecyl sulphate-poly-acrylamide gel electrophoresis using nondenaturing conditions. Separated proteins were transferred to a polyvinylindene difluoride membrane (Bio-Rad, Hercules, CA, USA), blocked with 5% non-fat dried milk in Tris-buffered saline (TBS, pH 7.4) with 0.1% Tween 20 and incubated in the anti-SERT antibody (1:2000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4 ◦ C. SERT was detected using horseradish peroxidise conjugated anti-goat IgG (1:10,000; Sigma, NSW, Australia) and visualised using enhanced chemiluminescence (Amersham, NSW, Australia) on X-ray film (Super RX, FujiFilm Australia, NSW, Australia). Each blot was stripped using Re-blot Plus (Millipore, NSW, Australia) and re-probed with -actin (1:20,000; Imgenex, San Diego, CA, USA). SERT expression levels were determined relative to -actin protein. Chemiluminescence was eliminated for SERT when the antibody was pre-incubated with the control peptide (1:2000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Bands were quantified using commercial software (NIH Image J). Statistical analyses of results were evaluated using Student t-tests and statistical significance was set at p < 0.05. 2.5. Immunohistochemistry On P10, animals (control n = 3; P3 HI n = 3) were perfused via the heart with 1% sodium nitrite solution (in 0.1 M PBS, pH 7.4) followed by 4% formaldehyde (in 0.1 M PBS, pH 7.4). Brains were post-fixed in 4% formaldehyde (in 0.1 M PBS, pH 7.4) then stored in 10% sucrose (in 0.1 M PBS, pH 7.4 at 4 ◦ C). Sections were collected using a cryostat set at 10 m thickness. Forebrain sections were incubated with anti-SERT (1:200) and anti-ionised calcium binding adaptor molecule-1 (Iba-1; 1:1000; WAKO Pure Chemical Industries, Osaka, Japan) or mouse anti-glial fibrillary acidic protein (GFAP; 1:1000; Cell Signalling, Danvers, MA, USA) in 0.1 M PBS
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Fig. 1. Western blot images demonstrating the separation of the cytosolic (S2) and membrane (P2) fraction using the membrane marker Na+ /K+ -ATPase (A), cytosolic marker AspAP (B) and cytosolic endosomal marker EEA1 (C) in the frontal cortex. AspAP, aspartyl aminopeptidase; Na+ /K+ -ATPase, sodium potassium adenosine triphosphatase; EEA1, early endosome antigen 1.
with 0.2% Triton-X for 24 h at room temperature in a humidified chamber. Brainstem sections were incubated in anti-SERT (1:200) and anti-NeuN (1:1000; Chemicon International, CA, USA). The sections were then immersed in an appropriate secondary fluorophore at room temperature for 2 h (Alexafluor 488, Alexafluor 568; 1:200, Molecular Probes, Invitrogen Australia, Victoria, Australia). Sections were mounted in Prolong Gold anti-fade reagent with 4 ,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Invitrogen Australia, Victoria, Australia). Images of brain sections were acquired using an Olympus microscope (BX41) and photographed with a CCD camera (Olympus DP70). 3. Results 3.1. Sub-cellular localisation of SERT Successful separation of the membrane (P2) and cytosolic (S2) sub-cellular fractions was established (Fig. 1). The predominant membrane-localised protein, Na+ /K+ -ATPase, was only detected in the P2 fraction (Fig. 1A) and AspAP was predominantly localised in the S2 fraction (Fig. 1B). In addition, the S2 fraction was enriched with EEA1, a marker for endosomes, but EEA1 was not detectable in the P2 fraction (Fig. 1C). In both the frontal cortex and brainstem of control animals, SERT was only detected in the P2 fraction; not the S2 fraction (Fig. 2A and B). In addition, after P3 HI there was no shift of SERT from the plasma membrane and thus for both the frontal cortex and brainstem tissue, no SERT was detected in the S2 fractions (Fig. 2A and B). However, in P3 HI animals, there was a significant decrease in the expression of SERT in the P2 fractions from the frontal cortex (44%; Fig. 2A and C) and the brainstem (24%; Fig. 2B and D) compared to control animals. These changes are consistent with previous findings using the same P3 HI model and therefore not only confirm there was significant disruption of the serotonergic system but also that the severity of injury was comparable to previous studies (Wixey et al., 2011a; Reinebrant et al., 2010). 3.2. Localisation of SERT in different cellular phenotypes In the frontal cortex of control brains, SERT-positive fibres were in abundance and appeared as fine beaded varicose strands (Fig. 3A and B). In contrast, after P3 HI (Fig. 3C and D), SERT-positive fibres were fewer in number and displayed shorter or damaged processes consistent with previous findings (Wixey et al., 2011a). In addition, a reduced number of SERT-positive neuronal cell bodies was apparent after P3 HI (Fig. 3G and H) compared to control brains (Fig. 3E and F) as we have established earlier (Wixey et al., 2011a; Reinebrant et al., 2010). In the brainstem raphé nuclei, SERTpositive immunolabelling was exclusively found on NeuN-positive
Fig. 2. Western blot images from control and P3 HI animals demonstrating SERT protein is present only in the plasma membrane (P2) fraction, but not the cytosolic (S2) fraction, in both the frontal cortex (A) and brainstem (B). Expression of SERT P2 levels decreased significantly in the frontal cortex and brainstem following P3 HI (C and D). *p < 0.05, **p < 0.01, control versus P3 HI group.
raphé neurons in both the control rat brain and following P3 HI (Fig. 3E–H). Furthermore in control and P3 HI brains, we did not detect co-localisation of SERT-immunolabelling on Iba-1-positive microglia (Fig. 3I–L) or GFAP-positive astrocytes (Fig. 3M–P). 4. Discussion We report novel evidence that neonatal HI brain injury does not alter the cellular distribution of SERT. It is unlikely that SERT shifts away from the neuronal membrane into the cytosol after injury. In addition, we only detected SERT on neurons; there was no indication that SERT is co-localised on Iba-1-positive microglia or GFAP-positive astrocyte cells. Thus our findings show that neuronal membrane SERT is the primary regulator of 5-HT levels in the brain one week after neonatal HI brain injury. Although SERT can traffic between the membrane and the cytosol (Qian et al., 1997; Ramamoorthy et al., 1998) we did not find evidence for this phenomenon in the neonatal rat brain. Our Western blot analyses for AspAP, Na+ /K+ -ATPase and EEA1 confirmed the successful separation of the membrane (P2) and cytosol (S2) fractions however no cytosolic SERT was detected in control or P3 HI tissue. Therefore the reduction in 5-HT after P3 HI is unlikely to reflect a shift of SERT to the cytosol of remaining neurons. These findings are consistent with previous findings whereby disruption to the 5-HT system did not reflect changes in SERT trafficking but rather the toxicity of the insult incurred a loss of SERT (Xie et al., 2006). Furthermore, since we have recently demonstrated that concomitant reductions in 5-HT, SERT and dorsal raphe with graven required serotonergic neurons occur after P3 HI (Reinebrant et al., 2010; Wixey et al., 2011a,b), it seems likely that the decrease in brain 5-HT is a consequence of SERT being lost as 5-HT neurons degenerate. Although we examined only one time point, coincident with increased neuroinflammatory mediators and injury to the serotonergic system after P3 HI (Reinebrant et al., 2010; Wixey et al., 2011a,b), we cannot discount the possibility that SERT internalisation occurs at earlier time points. However, given the present
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Fig. 3. Photomicrographs of coronal sections from control and P3 HI animals through the frontal cortex (A–D, I–P) and dorsal raphé nuclei of the brainstem (E–H) on P10. SERT-positive fibres (red) were clearly visible in the control (A and B, I and J, M and N) and P3 HI (C and D, K and L, O and P) rat forebrain. Co-localisation of SERT-positive neurons was demonstrated on NeuN-positive neurons (green; E–H) in the brainstem. In the frontal cortex, SERT-positive fibres did not co-localise with either Iba-1-positive microglia (green; I–L) or GFAP-positive astrocytes (green; M–P) in control or P3 HI brains. All sections have been stained with DAPI (blue). Scale bars represent 50 m and 25 m for inset photomicrographs.
findings, if earlier internalisation had occurred this was likely to be transient. In both the intact and HI-injured brain, it appears that neuronal SERT, but not glial SERT, is the primary regulator of 5-HT levels in the brain. At least in our model of neonatal brain injury, Iba-1-positive microglial cells and GFAP-positive astrocytes do not appear to possess SERT. We chose two representative regions to examine the localisation of SERT; serotonergic fibre-rich region (frontal cortex) and serotonergic cell body rich region (dorsal raphé nuclei). The cortex elicits substantial increases in numbers of activated microglia using the same neonatal HI model used here (Carty et al., 2008; Wixey et al., 2011a,b) whereas in the dorsal raphé nuclei relatively few activated microglia appear (Wixey et al., 2011a,b). Regardless of these differences, the localisation
and sub-cellular localisation were unequivocal. The predominance of SERT-positive neurons confirms previous reports that SERT is virtually exclusively found on 5-HT neurons in the brainstem dorsal raphé nuclei (Reinebrant et al., 2010; Smith and Porrino, 2008). Although one study suggests that SERT is co-localised on microglia in rodent cultured cell lines as well as primary cultured microglia from a P3 rat pup (Horikawa et al., 2010) no direct functional role for microglial SERT is apparent (Horikawa et al., 2010). The lack of SERT on GFAP-positive astrocytes after neonatal HI concurs with previous in vivo studies in the brain (Sur et al., 1996; Tao-Cheng and Zhou, 1999; Zhou et al., 2000). In contrast, evidence supporting the presence of SERT on astrocytes is based on studies utilising in vitro cell culture models (Bel et al., 1997; Kubota et al., 2001; Inazu et al., 2001) suggesting the discrepancy in reports may
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arise from the methods of detection. Nevertheless we cannot discount the possibility that other astrocyte populations possess SERT because not all astrocytes are GFAP-positive (Pekny and Nilsson, 2005). In conclusion, neuronal membrane SERT is the primary regulator of synaptic 5-HT availability in the intact and P3 HI-injured neonatal brain. Furthermore, since concomitant reductions in 5HT, SERT and serotonergic neurons occur after P3 HI (Reinebrant et al., 2010; Wixey et al., 2011a,b), it is plausible that the decrease in brain 5-HT is a consequence of SERT being lost as neurons degenerate. These are important outcomes that help identify the critical targets to prevent or repair damage to the serotonergic system after neonatal HI. Acknowledgements JAW is supported by an Australian Postgraduate Award. HER is supported by a University of Queensland Research Tuition Award and University of Queensland Research Scholarship. References Anderson, P., Doyle, L.W., 2003. Neurobehavioural outcomes of school-age children born extremely low birth weight or very preterm in the 1990. JAMA 289, 3264–3272. Bel, N., Figueras, G., Vilaro, T., Sunol, C., Artigas, F., 1997. Antidepressant drugs inhibit a Gial 5-hydroxytryptamine transporter in rat brain. Eur. J. Neurosci. 9, 1728–1738. Blakely, R.D., Berson, H.E., Fremeau, R.T., Caron, M.G., Peek, M.M., Prince, H.K., Bradley, C.C., 1991. Cloning and expression of a functional serotonin transporter from rat brain. Nature 354, 66–70. Buller, K.M., Wixey, J.A., Pathipati, P., Carty, M., Colditz, P.B., Williams, C.E., Scheepens, A., 2008. Selective losses of brainstem catecholamine neurons after hypoxia-ischemia in the immature rat pup. Pediatr. Res. 63, 364–369. Carty, M.L., Wixey, J.A., Colditz, P.B., Buller, K.M., 2008. Post-hypoxia-ischemia minocycline treatment attenuates neuroinflammation and white matter injury in the neonatal rat; a comparison of two different dose regimens. Int. J. Dev. Neurosci. 26, 477–485. Duncan, J.R., Paterson, D.S., Hoffman, J.M., Mokler, D.J., Borenstein, N.S., Belliveau, R.A., Krous, H.F., Haas, E.A., Stanley, C., Nattie, E.E., Trachtenberg, F., Kinney, H.C., 2010. Brainstem serotonergic deficiency in sudden infant death syndrome. JAMA 303, 430–437. Ferriero, D.M., 2004. Neonatal brain injury. N. Engl. J. Med. 351, 1985–1995. Gartside, S.E., Johnson, D.A., Leitch, M.M., Troakes, C., Ingram, C.D., 2003. Early life adversity programs changes in central 5-HT neuronal function in adulthood. Eur. J. Neurosci. 17, 2401–2408. Gaspar, P., Cases, O., Maroteaux, L., 2003. The developmental role of serotonin: news from mouse molecular genetics. Nat. Rev. Neurosci. 4, 1002–1012. Hack, M., Youngstrom, E.A., Cartar, L., Schluchter, M., Taylor, H.G., Flannery, D., Klein, N., Borawski, E., 2004. Behavioral outcomes and evidence of psychopathology among very low birth weight infants at age 20 years. Pediatrics 114, 932–940. Horikawa, H., Kato, T.A., Mizoguchi, Y., Monji, A., Seki, Y., Ohkuri, T., Gotoh, L., Yonaha, M., Ueda, T., Hashioka, S., 2010. Inhibitory effects of SSRIs on IFN-␥ induced microglial activation through the regulation of intracellular calcium. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 34, 1306–1316. Inazu, M., Takeda, H., Ikoshi, H., Sugisawa, M., Uchida, Y., Matsumiya, T., 2001. Pharmacological characterization and visualization of the glial serotonin transporter. Neurochem. Int. 39, 39–49. Ivacko, J.A., Sun, R., Silverstein, F.S., 1996. Hypoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats. Pediatr. Res. 39, 39–47. Kreutzberg, G.W., 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318.
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Evidence that the serotonin transporter does not shift into the cytosol of remaining neurons after neonatal brain injury Julie A. Wixey, Hanna E. Reinebrant, Kathryn M. Buller ∗ Clinical Neuroscience, Perinatal Research Centre, The University of Queensland, UQ Centre for Clinical Research, Herston, QLD 4029, Australia
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Article history: Received 23 January 2012 Received in revised form 3 April 2012 Accepted 5 April 2012 Available online 15 April 2012 Keywords: Astrocytes Hypoxia-ischemia Microglia Neonate Raphé Serotonin transporter
a b s t r a c t Following neonatal hypoxia-ischemia (HI) serotonin (5-hydroxytryptamine, 5-HT) levels are decreased in the brain. The regulation of brain 5-HT is dependent on the serotonin transporter (SERT) localised at the neuronal pre-synaptic cell membrane. However SERT can also traffic away from the cell membrane into the cytosol and, after injury, may contribute to the cell’s inability to maintain 5-HT levels. Whether this occurs after neonatal HI brain injury is not known. In addition, there is contradictory evidence that glial cells may also contribute to the clearance of 5-HT in the brain. Using a postnatal day 3 (P3) HI rat pup model (right carotid ligation + 30 min 6% O2 ), we found, in both control and P3 HI animals, that SERT is retained on the cell membrane and is not internalised in the cytosol. In addition, SERT was only detected on neurons. We found no evidence of SERT co-localisation on microglia or astrocytes. We conclude that neuronal SERT is the primary regulator of synaptic 5-HT availability in the intact and P3 HI-injured neonatal brain. Furthermore, since concomitant reductions in 5-HT, SERT and serotonergic neurons occur after neonatal HI, it is plausible that the decrease in brain 5-HT is a consequence of SERT being lost as neurons degenerate as opposed to remaining neurons internalising SERT or clearance by glial cells. © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction Serotonin (5-hydroxytryptamine, 5-HT) is critical for brain development (Gaspar et al., 2003). The serotonergic system constitutes a pervasive neural network and serves a diverse array of functions. Thus disruption to this central 5-HT network early in life contributes to a wide range of adverse outcomes including depression, epilepsy, movement disorders, sudden infant death syndrome and autism (Duncan et al., 2010; Gartside et al., 2003; Nemeroff and Owens, 2009; Pardo and Eberhart, 2007). It is pertinent that many of these disorders match those observed in neonates with hypoxic-ischemic (HI) brain injury (Peterson et al., 2003; Anderson and Doyle, 2003; Hack et al., 2004; Ferriero, 2004). Indeed we have demonstrated recently in a neonatal rodent model that exposure to a HI insult significantly disrupts the serotonergic system in the neonatal brain (Reinebrant et al., 2010; Wixey et al., 2011a,b). Damage to this system includes loss of serotonergic raphé neurons, reduced 5-HT levels and reduced expression of the 5-HT transporter (SERT) (Reinebrant et al., 2010; Wixey et al., 2011a,b). It
∗ Corresponding author at: Clinical Neuroscience, Perinatal Research Centre, Building 71/918, The University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, Herston, QLD 4029, Australia. Tel.: +61 7 33466011; fax: +61 7 33465594. E-mail address: [email protected] (K.M. Buller).
is important to understand the mechanisms that contribute to 5HT neuronal damage in order to develop therapeutic interventions that could prevent lifelong functional deficits seen in HI-affected neonates. The SERT consists of 12 trans-membrane domains that span the presynaptic membrane of 5-HT-releasing cells (Blakely et al., 1991). It is the key regulator of extracellular 5-HT levels in the brain. Central SERT terminates 5-HT neurotransmission by the re-uptake of 5-HT from the synapse into the nerve terminals and controls the duration of action and post-synaptic signalling of 5-HT in the brain. Consequently, SERT is a major target for drugs such as selective serotonin reuptake inhibitors (SSRIs) that can increase 5-HT availability in the brain. In the normal adult brain, SERT can traffic from the cellular membrane to sub-cellular or cytosolic compartments. This process of internalisation reduces the capacity of serotonergic neurons to release and re-uptake 5-HT at the cell membrane (Qian et al., 1997; Ramamoorthy et al., 1998). We have found concomitant reductions in SERT and 5-HT in the brain after neonatal HI (Wixey et al., 2011a). These parameters are not abolished and, although serotonergic neurons are also reduced in number after neonatal HI (Wixey et al., 2011a; Reinebrant et al., 2010), the possible internalisation of SERT in remaining neurons may reduce their capacity to release and reuptake 5-HT and therefore contribute to the reduced 5-HT levels one week after injury. This would create the potential for SERT to be recovered back to the cell membrane after brain injury where it
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could resume regulating synaptic re-uptake of 5-HT. For example, chronic activation of p38 MAPK can lead to enhanced SERT surface expression (Samuvel et al., 2005; Zhu et al., 2006). Such a mechanism might reveal a new target to manipulate SERT function after brain injury. The efficient re-uptake of 5-HT is primarily dependent on SERT localisation on cell bodies, dendrites and fibres of serotonergic neurons in the central nervous system (Qian et al., 1995). However, although evidence is controversial, it has also been purported that 5-HT uptake can occur by glial cells and thus non-neuronal cells could assist in clearing 5-HT from the synapse. Reports primarily based on in vitro studies, suggest SERT may exist on astrocytes and microglia (Bel et al., 1997; Kubota et al., 2001; Inazu et al., 2001; Horikawa et al., 2010). In direct contrast, others utilising in vivo models have found no evidence of glial SERT in the brain (Sur et al., 1996; Tao-Cheng and Zhou, 1999; Zhou et al., 2000). It is important to examine the localisation of SERT in vivo since microglial cells, for example, can change their morphology and function substantially after brain injury (Kreutzberg, 1996) and after neonatal HI substantial increases in numbers of activated microglia appear in the brain (Ivacko et al., 1996; McRae et al., 1995; Wixey et al., 2009, 2011a). Inhibition of neuroinflammation has also been shown to prevent damage to the 5-HT system following neonatal HI brain injury (Wixey et al., 2011a,b). We hypothesised that neonatal HI brain injury can influence the sub-cellular and cellular phenotypic localisation of SERT in the brain. To test this we used a post-natal day 3 (P3) HI model of neonatal brain injury that incurs significant losses of raphé serotonergic neurons, reduced SERT expression and reduced levels of 5-HT in the brain (Reinebrant et al., 2010; Wixey et al., 2011a,b). We investigated if SERT expression is evident in the cellular membrane and/or cytosol of the non-injured and P3 HI-injured brain. We also examined whether SERT is localised on neurons, microglia and/or astrocytes. 2. Materials and methods 2.1. Animals Sprague–Dawley dams and their pups (10–12 per litter) were used to perform experiments. The animals had access to food and water ad libitum and were kept in rooms maintained at 22 ◦ C on a 12 h light/dark cycle. Surgical procedures and tissue collection were performed between 0900 and 1100 to minimise the possible effects of circadian rhythms. All experiments were performed in accordance with ethical approvals stipulated by the University of Queensland Animal Ethics Committee. Efforts were made to minimise the number of animals used and their suffering. 2.2. Neonatal hypoxic-ischemic insult On post-natal day 3 (P3), pups were assigned randomly to (i) control or (ii) P3 HI groups. The P3 HI insult was produced as described previously (Buller et al., 2008; Carty et al., 2008; Reinebrant et al., 2010). Briefly, pups were anaesthetised using isoflurane (2%; Baxter, IL, USA), the right common carotid artery was isolated and ligated permanently. Pups recovered for 20 min before being exposed to 6% O2 for 30 min at 37 ◦ C in a humidified chamber. Pups were then returned to the dam. The control group underwent the same procedures but the carotid artery was not ligated and the animals were exposed to room-air instead of hypoxia. The mortality rate was < 3.7%. On P10, animals were euthanized by administering a lethal dose of sodium pentobarbitone (80 mg/kg, i.p.; Lethabarb, Virbac, France).
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2.3. Membrane and cytosolic fraction preparation For protein analyses, brains (control n = 6; P3 HI n = 6) were rapidly excised on P10. Frontal cortex (+4.0 to 0.0 mm relative to bregma) and brainstem dorsal raphé (−7.0 to −12.0 mm relative to bregma) regions were collected, immediately frozen and stored at −80 ◦ C (Paxinos and Watson, 1997; Reinebrant et al., 2010). Total protein extracts were separated into two fractions to determine the quantity of SERT in the plasma membrane and cytosolic fraction. Tissue was homogenised in 1 mL homogenisation buffer (0.32 M sucrose, 0.10 mM HEPES pH 7.4, 0.2 mM EDTA and protease inhibitor cocktail) and spun at 1000 × g for 15 min at 4 ◦ C. Supernatant was recovered and spun at 183,000 × g for 45 min at 4 ◦ C. Supernatant was collected for the cytosolic fraction. The resultant pellet was resuspended in HEPES-lysis buffer (0.5 mM HEPES, 0.2 mM EDTA) and constituted the membrane fraction. Total amount of protein in each fraction was determined using a bicinchoninic acid kit (Pierce, Rockford, IL, USA). To determine whether protein preparations were separated successfully, both fractions from a control brain were probed with aspartyl aminopeptidase (AspAP, 1:10,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) because it is predominantly localised in the cytosol (Wilk et al., 1998), early endosome antigen 1 (EEA1, 1:2000; BD Biosciences, San Jose, CA, USA) to detect endosomal proteins in the cytosol (Wang et al., 2005) and sodium-potassium adenosine triphosphatase (Na+ /K+ -ATPase, 1:5000; Chemicon International, CA, USA) as it is predominantly localised in the cellular membrane (Schimmel et al., 1973). 2.4. Western blotting Western blotting was carried out to detect SERT protein expression in the plasma membrane and cytosolic fractions. Samples (16 g per well) were separated by 10% sodium dodecyl sulphate-poly-acrylamide gel electrophoresis using nondenaturing conditions. Separated proteins were transferred to a polyvinylindene difluoride membrane (Bio-Rad, Hercules, CA, USA), blocked with 5% non-fat dried milk in Tris-buffered saline (TBS, pH 7.4) with 0.1% Tween 20 and incubated in the anti-SERT antibody (1:2000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4 ◦ C. SERT was detected using horseradish peroxidise conjugated anti-goat IgG (1:10,000; Sigma, NSW, Australia) and visualised using enhanced chemiluminescence (Amersham, NSW, Australia) on X-ray film (Super RX, FujiFilm Australia, NSW, Australia). Each blot was stripped using Re-blot Plus (Millipore, NSW, Australia) and re-probed with -actin (1:20,000; Imgenex, San Diego, CA, USA). SERT expression levels were determined relative to -actin protein. Chemiluminescence was eliminated for SERT when the antibody was pre-incubated with the control peptide (1:2000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Bands were quantified using commercial software (NIH Image J). Statistical analyses of results were evaluated using Student t-tests and statistical significance was set at p < 0.05. 2.5. Immunohistochemistry On P10, animals (control n = 3; P3 HI n = 3) were perfused via the heart with 1% sodium nitrite solution (in 0.1 M PBS, pH 7.4) followed by 4% formaldehyde (in 0.1 M PBS, pH 7.4). Brains were post-fixed in 4% formaldehyde (in 0.1 M PBS, pH 7.4) then stored in 10% sucrose (in 0.1 M PBS, pH 7.4 at 4 ◦ C). Sections were collected using a cryostat set at 10 m thickness. Forebrain sections were incubated with anti-SERT (1:200) and anti-ionised calcium binding adaptor molecule-1 (Iba-1; 1:1000; WAKO Pure Chemical Industries, Osaka, Japan) or mouse anti-glial fibrillary acidic protein (GFAP; 1:1000; Cell Signalling, Danvers, MA, USA) in 0.1 M PBS
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Fig. 1. Western blot images demonstrating the separation of the cytosolic (S2) and membrane (P2) fraction using the membrane marker Na+ /K+ -ATPase (A), cytosolic marker AspAP (B) and cytosolic endosomal marker EEA1 (C) in the frontal cortex. AspAP, aspartyl aminopeptidase; Na+ /K+ -ATPase, sodium potassium adenosine triphosphatase; EEA1, early endosome antigen 1.
with 0.2% Triton-X for 24 h at room temperature in a humidified chamber. Brainstem sections were incubated in anti-SERT (1:200) and anti-NeuN (1:1000; Chemicon International, CA, USA). The sections were then immersed in an appropriate secondary fluorophore at room temperature for 2 h (Alexafluor 488, Alexafluor 568; 1:200, Molecular Probes, Invitrogen Australia, Victoria, Australia). Sections were mounted in Prolong Gold anti-fade reagent with 4 ,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Invitrogen Australia, Victoria, Australia). Images of brain sections were acquired using an Olympus microscope (BX41) and photographed with a CCD camera (Olympus DP70). 3. Results 3.1. Sub-cellular localisation of SERT Successful separation of the membrane (P2) and cytosolic (S2) sub-cellular fractions was established (Fig. 1). The predominant membrane-localised protein, Na+ /K+ -ATPase, was only detected in the P2 fraction (Fig. 1A) and AspAP was predominantly localised in the S2 fraction (Fig. 1B). In addition, the S2 fraction was enriched with EEA1, a marker for endosomes, but EEA1 was not detectable in the P2 fraction (Fig. 1C). In both the frontal cortex and brainstem of control animals, SERT was only detected in the P2 fraction; not the S2 fraction (Fig. 2A and B). In addition, after P3 HI there was no shift of SERT from the plasma membrane and thus for both the frontal cortex and brainstem tissue, no SERT was detected in the S2 fractions (Fig. 2A and B). However, in P3 HI animals, there was a significant decrease in the expression of SERT in the P2 fractions from the frontal cortex (44%; Fig. 2A and C) and the brainstem (24%; Fig. 2B and D) compared to control animals. These changes are consistent with previous findings using the same P3 HI model and therefore not only confirm there was significant disruption of the serotonergic system but also that the severity of injury was comparable to previous studies (Wixey et al., 2011a; Reinebrant et al., 2010). 3.2. Localisation of SERT in different cellular phenotypes In the frontal cortex of control brains, SERT-positive fibres were in abundance and appeared as fine beaded varicose strands (Fig. 3A and B). In contrast, after P3 HI (Fig. 3C and D), SERT-positive fibres were fewer in number and displayed shorter or damaged processes consistent with previous findings (Wixey et al., 2011a). In addition, a reduced number of SERT-positive neuronal cell bodies was apparent after P3 HI (Fig. 3G and H) compared to control brains (Fig. 3E and F) as we have established earlier (Wixey et al., 2011a; Reinebrant et al., 2010). In the brainstem raphé nuclei, SERTpositive immunolabelling was exclusively found on NeuN-positive
Fig. 2. Western blot images from control and P3 HI animals demonstrating SERT protein is present only in the plasma membrane (P2) fraction, but not the cytosolic (S2) fraction, in both the frontal cortex (A) and brainstem (B). Expression of SERT P2 levels decreased significantly in the frontal cortex and brainstem following P3 HI (C and D). *p < 0.05, **p < 0.01, control versus P3 HI group.
raphé neurons in both the control rat brain and following P3 HI (Fig. 3E–H). Furthermore in control and P3 HI brains, we did not detect co-localisation of SERT-immunolabelling on Iba-1-positive microglia (Fig. 3I–L) or GFAP-positive astrocytes (Fig. 3M–P). 4. Discussion We report novel evidence that neonatal HI brain injury does not alter the cellular distribution of SERT. It is unlikely that SERT shifts away from the neuronal membrane into the cytosol after injury. In addition, we only detected SERT on neurons; there was no indication that SERT is co-localised on Iba-1-positive microglia or GFAP-positive astrocyte cells. Thus our findings show that neuronal membrane SERT is the primary regulator of 5-HT levels in the brain one week after neonatal HI brain injury. Although SERT can traffic between the membrane and the cytosol (Qian et al., 1997; Ramamoorthy et al., 1998) we did not find evidence for this phenomenon in the neonatal rat brain. Our Western blot analyses for AspAP, Na+ /K+ -ATPase and EEA1 confirmed the successful separation of the membrane (P2) and cytosol (S2) fractions however no cytosolic SERT was detected in control or P3 HI tissue. Therefore the reduction in 5-HT after P3 HI is unlikely to reflect a shift of SERT to the cytosol of remaining neurons. These findings are consistent with previous findings whereby disruption to the 5-HT system did not reflect changes in SERT trafficking but rather the toxicity of the insult incurred a loss of SERT (Xie et al., 2006). Furthermore, since we have recently demonstrated that concomitant reductions in 5-HT, SERT and dorsal raphe with graven required serotonergic neurons occur after P3 HI (Reinebrant et al., 2010; Wixey et al., 2011a,b), it seems likely that the decrease in brain 5-HT is a consequence of SERT being lost as 5-HT neurons degenerate. Although we examined only one time point, coincident with increased neuroinflammatory mediators and injury to the serotonergic system after P3 HI (Reinebrant et al., 2010; Wixey et al., 2011a,b), we cannot discount the possibility that SERT internalisation occurs at earlier time points. However, given the present
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Fig. 3. Photomicrographs of coronal sections from control and P3 HI animals through the frontal cortex (A–D, I–P) and dorsal raphé nuclei of the brainstem (E–H) on P10. SERT-positive fibres (red) were clearly visible in the control (A and B, I and J, M and N) and P3 HI (C and D, K and L, O and P) rat forebrain. Co-localisation of SERT-positive neurons was demonstrated on NeuN-positive neurons (green; E–H) in the brainstem. In the frontal cortex, SERT-positive fibres did not co-localise with either Iba-1-positive microglia (green; I–L) or GFAP-positive astrocytes (green; M–P) in control or P3 HI brains. All sections have been stained with DAPI (blue). Scale bars represent 50 m and 25 m for inset photomicrographs.
findings, if earlier internalisation had occurred this was likely to be transient. In both the intact and HI-injured brain, it appears that neuronal SERT, but not glial SERT, is the primary regulator of 5-HT levels in the brain. At least in our model of neonatal brain injury, Iba-1-positive microglial cells and GFAP-positive astrocytes do not appear to possess SERT. We chose two representative regions to examine the localisation of SERT; serotonergic fibre-rich region (frontal cortex) and serotonergic cell body rich region (dorsal raphé nuclei). The cortex elicits substantial increases in numbers of activated microglia using the same neonatal HI model used here (Carty et al., 2008; Wixey et al., 2011a,b) whereas in the dorsal raphé nuclei relatively few activated microglia appear (Wixey et al., 2011a,b). Regardless of these differences, the localisation
and sub-cellular localisation were unequivocal. The predominance of SERT-positive neurons confirms previous reports that SERT is virtually exclusively found on 5-HT neurons in the brainstem dorsal raphé nuclei (Reinebrant et al., 2010; Smith and Porrino, 2008). Although one study suggests that SERT is co-localised on microglia in rodent cultured cell lines as well as primary cultured microglia from a P3 rat pup (Horikawa et al., 2010) no direct functional role for microglial SERT is apparent (Horikawa et al., 2010). The lack of SERT on GFAP-positive astrocytes after neonatal HI concurs with previous in vivo studies in the brain (Sur et al., 1996; Tao-Cheng and Zhou, 1999; Zhou et al., 2000). In contrast, evidence supporting the presence of SERT on astrocytes is based on studies utilising in vitro cell culture models (Bel et al., 1997; Kubota et al., 2001; Inazu et al., 2001) suggesting the discrepancy in reports may
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arise from the methods of detection. Nevertheless we cannot discount the possibility that other astrocyte populations possess SERT because not all astrocytes are GFAP-positive (Pekny and Nilsson, 2005). In conclusion, neuronal membrane SERT is the primary regulator of synaptic 5-HT availability in the intact and P3 HI-injured neonatal brain. Furthermore, since concomitant reductions in 5HT, SERT and serotonergic neurons occur after P3 HI (Reinebrant et al., 2010; Wixey et al., 2011a,b), it is plausible that the decrease in brain 5-HT is a consequence of SERT being lost as neurons degenerate. These are important outcomes that help identify the critical targets to prevent or repair damage to the serotonergic system after neonatal HI. Acknowledgements JAW is supported by an Australian Postgraduate Award. HER is supported by a University of Queensland Research Tuition Award and University of Queensland Research Scholarship. References Anderson, P., Doyle, L.W., 2003. Neurobehavioural outcomes of school-age children born extremely low birth weight or very preterm in the 1990. JAMA 289, 3264–3272. Bel, N., Figueras, G., Vilaro, T., Sunol, C., Artigas, F., 1997. Antidepressant drugs inhibit a Gial 5-hydroxytryptamine transporter in rat brain. Eur. J. Neurosci. 9, 1728–1738. Blakely, R.D., Berson, H.E., Fremeau, R.T., Caron, M.G., Peek, M.M., Prince, H.K., Bradley, C.C., 1991. Cloning and expression of a functional serotonin transporter from rat brain. Nature 354, 66–70. Buller, K.M., Wixey, J.A., Pathipati, P., Carty, M., Colditz, P.B., Williams, C.E., Scheepens, A., 2008. Selective losses of brainstem catecholamine neurons after hypoxia-ischemia in the immature rat pup. Pediatr. Res. 63, 364–369. Carty, M.L., Wixey, J.A., Colditz, P.B., Buller, K.M., 2008. Post-hypoxia-ischemia minocycline treatment attenuates neuroinflammation and white matter injury in the neonatal rat; a comparison of two different dose regimens. Int. J. Dev. Neurosci. 26, 477–485. Duncan, J.R., Paterson, D.S., Hoffman, J.M., Mokler, D.J., Borenstein, N.S., Belliveau, R.A., Krous, H.F., Haas, E.A., Stanley, C., Nattie, E.E., Trachtenberg, F., Kinney, H.C., 2010. Brainstem serotonergic deficiency in sudden infant death syndrome. JAMA 303, 430–437. Ferriero, D.M., 2004. Neonatal brain injury. N. Engl. J. Med. 351, 1985–1995. Gartside, S.E., Johnson, D.A., Leitch, M.M., Troakes, C., Ingram, C.D., 2003. Early life adversity programs changes in central 5-HT neuronal function in adulthood. Eur. J. Neurosci. 17, 2401–2408. Gaspar, P., Cases, O., Maroteaux, L., 2003. The developmental role of serotonin: news from mouse molecular genetics. Nat. Rev. Neurosci. 4, 1002–1012. Hack, M., Youngstrom, E.A., Cartar, L., Schluchter, M., Taylor, H.G., Flannery, D., Klein, N., Borawski, E., 2004. Behavioral outcomes and evidence of psychopathology among very low birth weight infants at age 20 years. Pediatrics 114, 932–940. Horikawa, H., Kato, T.A., Mizoguchi, Y., Monji, A., Seki, Y., Ohkuri, T., Gotoh, L., Yonaha, M., Ueda, T., Hashioka, S., 2010. Inhibitory effects of SSRIs on IFN-␥ induced microglial activation through the regulation of intracellular calcium. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 34, 1306–1316. Inazu, M., Takeda, H., Ikoshi, H., Sugisawa, M., Uchida, Y., Matsumiya, T., 2001. Pharmacological characterization and visualization of the glial serotonin transporter. Neurochem. Int. 39, 39–49. Ivacko, J.A., Sun, R., Silverstein, F.S., 1996. Hypoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats. Pediatr. Res. 39, 39–47. Kreutzberg, G.W., 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318.
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