J Mol Neurosci DOI 10.1007/s12031-013-0071-z
Chronic Exposure to Light Reverses the Effect of Maternal Separation on Proteins in the Prefrontal Cortex J. J. Dimatelis & D. J. Stein & V. A. Russell
Received: 11 May 2013 / Accepted: 8 July 2013 # Springer Science+Business Media New York 2013
Abstract Animals subjected to maternal separation display behavioural and endocrine disturbances, as well as structural and functional changes in the prefrontal cortex and limbic areas. The aim of the present study was to determine the effect of maternal separation and treatment with either chronic constant light exposure or anti-depressant (escitalopram) on proteins in the prefrontal cortex. Four experimental groups of male Sprague–Dawley rats were subjected to (1) normal rearing, (2) maternal separation (3 h per day from postnatal day 2 (P2) to P14), (3) maternal separation followed by chronic light exposure (P42-P63) or (4) maternal separation followed by treatment with the anti-depressant drug, escitalopram (P68-P100). Groups 1–3 were treated with saline as vehicle control for the escitalopram-treated group. At P101, all rats were decapitated, and the prefrontal cortex was collected and stored at −80 °C. Tissue from three rats per group was pooled and proteins determined by isobaric tagging for relative and absolute quantification using matrix-assisted laser desorption/ionisation tandem mass spectrometry. Maternal separation led to disruptions in the prefrontal cortex that included hypometabolism by decreasing energy-related proteins (creatine kinase B, aconitate hydratase), decreased cell signalling (synapsin I, calmodulin, 14-3-3 protein epsilon) and impaired plasticity (spectrin, microtubule-associated protein). Maternal separation also J. J. Dimatelis (*) : V. A. Russell Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Observatory, Anzio Road, Cape Town 7925, South Africa e-mail:
[email protected] V. A. Russell e-mail:
[email protected] D. J. Stein Department of Psychiatry and Mental Health, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town 7925, South Africa e-mail:
[email protected]
increased dihydropyrimidinase-related protein/collapsin response mediator protein (CRMP) and myelin proteolipid protein. Exposure of maternally separated animals to constant light during adolescence reversed the hypometabolic state by increasing energy-related proteins in the prefrontal cortex and increasing cell signalling and cytoskeletal proteins and decreasing the expression of CRMP. Escitalopram had similar effects to light by increasing ATP synthase in maternally separated rats and dissimilar effects by increasing 2',3'-cyclic-nucleotide 3'-phosphodiesterase and myelin proteolipid protein. Constant light exposure during adolescence reversed a range of protein changes in the prefrontal cortex of rats exposed to early maternal separation. The most prominent reversal by light treatment of maternal separationinduced protein increases in the prefrontal cortex was the expression of CRMP which impairs plasticity and neuronal signalling. The effects of light treatment overlapped partially with the effects of escitalopram. Keywords Maternal separation . Depression . Chronic constant light . Escitalopram . Prefrontal cortex
Introduction Maternal separation is an important animal model, which may be relevant to studying a range of psychiatric disorders, including depression (Daniels et al. 2004; Marais et al. 2008; Leussis et al. 2012; Vivinetto et al. 2012; Yoo et al. 2012). It involves the exposure of pups to a mild stressor during early life, a critical developmental period of the animal. Rats subjected to maternal separation are characterised by behavioural changes in later life that are reflective of anxiety- and depressive-like behaviour as measured in the elevated plus maze and forced swim test (Daniels et al. 2004; Aisa et al. 2007; Marais et al. 2008). Maternal separation-induced depressive-like behaviour has been shown to be improved by non-pharmacological interventions such as exercise and
J Mol Neurosci
chronic constant light treatment (Marais et al. 2009a; Dimatelis et al. 2012a). Similarly, reversal of depressivelike behaviour by means of light therapy has also been demonstrated in other animal models of depression (Molina-Hernandez and Tellez-Alcantara 2000; Yilmaz et al. 2004; Schultz et al. 2008; Iyilikci et al. 2009). Clinically, depressed mood as found in seasonal and nonseasonal affective disorder is improved by light exposure (Golden et al. 2005; Terman and Terman 2005; Lam et al. 2006; Even et al. 2008). Depressed mood is associated with decreased metabolic activity in the prefrontal cortex and heightened metabolic activity in limbic areas of the brain (Videbech 2000; Seminowicz et al. 2004; Fitzgerald et al. 2008; Salerian and Altar 2012). The severity of depressed mood has been found to correlate negatively with metabolic activity in the prefrontal cortex and positively with the metabolic activity of the amygdala (Abercrombie et al. 1998; Drevets 1999; Osuch et al. 2000; Drevets et al. 2002). The therapeutic efficacy of antidepressants has been suggested to depend on their ability to restore the metabolic and executive function of the prefrontal cortex (Salerian and Altar 2012). There is some evidence that maternal separation alters energyrelated proteins of the prefrontal cortex as well as other areas (Mu et al. 2007; Marais et al. 2009a; Piubelli et al. 2011; Dimatelis et al. 2012b). These findings raise the question of whether prefrontal cortex metabolic activity is altered during maternal separation and whether light exposure and antidepressant treatment in this model are able to reverse such alterations. In addition to maternal separation affecting the metabolic activity of the brain, it also alters plasticity of brain structures and signalling in these affected areas. In particular, maternal separation alters neurogenesis in the hippocampus, an area vulnerable to stress (Mirescu et al. 2004). Neurotrophic factors that function to maintain structure and synaptic plasticity are affected by maternal separation leading to compromised integrity of brain structures (Duman 2002; Roceri et al. 2002; MacQueen et al. 2003; Marais et al. 2008). Anti-depressants have similarly been shown to restore neurotrophin expression (Castrén and Rantamäki 2010). Thus, the identification of structural and signalling proteins that are affected by depression would be important to establish more effective treatments. The aim of the present study was to assess the effects of maternal separation and treatment with light therapy and an antidepressant (escitalopram) on proteins in the prefrontal cortex.
Material and Methods Ethical Approval The study was authorised by the Faculty of Health Sciences Animal Research Ethics Committee of the University of
Cape Town and affirms that all aspects of the Animals Protection Act (Act 71 of 1962) was adhered to in both the housing of animals and their use in experiments as stipulated in the South African National Standard: The Care and Use of Animals for Scientific Purposes (SANS 10386, 2008). SANS guidelines supports the adherence to the 3 R’s (Replacement, Refinement and Reduction). All efforts were made to minimise animal suffering and to reduce the number of animals used. Animals Male Sprague–Dawley rats were bred in the Satellite Animal Facility at the University of Cape Town. Animals were housed in a temperature-controlled room (23±1 °C) and had ad libitum access to standard rat chow and water. Rats were housed three to four per Plexiglass cages with woodchip bedding. The housing facility was maintained on a 12/12 h light/dark cycle (lights on at 06 h00 and lights off at 18 h00). On postnatal day 2 (P2), rats were sexed and culled to eight males per litter. If the litter had less than eight males, female littermates were added to standardise litter size and to allow for equal suckling from the dam. The experiment consisted of three groups (1) control/non-separated (C), (2) maternally separated (MS) and (3) maternally separated rats exposed to chronic constant light (MS CCL). These groups were further divided into drug-treatment groups and consisted of (a) control non-separated animals that received volume-controlled saline injections (1 ml/kg; intraperitoneal (i.p.)) (CS; n=3), (b) non-separated animals that received escitalopram (5 mg/kg; i.p., CE; n=3), (c) maternally separated animals that received saline (MS S; n=3), (d) maternally separated animals that received escitalopram (MS E; n=3), (e) maternally separated animals exposed to chronic constant light that received saline (MS CCL S; n=3) and (f) maternally separated animals exposed to chronic constant light that received escitalopram (MS CCL E; n=3). On P101 all animals were decapitated and the prefrontal cortices were rapidly dissected, snap frozen in liquid nitrogen and stored at −80 °C until later biochemical analysis. Maternal Separation (MS) Paradigm The date of birth was designated P0, maternal separation commenced 2 days later on P2 and continued until P14 for a 3-h daily period between 09 h00 and 12 h00. This protocol is in accordance with the procedures employed by Ladd et al. (2000). During the separation period, the pups were removed from the dam and kept in a separate cage. To ensure that the pups recognised the odour of the home cage, home cage bedding was transferred with the pups to the separation cage. The dam remained in the home cage. The cage containing the pups was transferred to a room where the pups were kept
J Mol Neurosci
warm under infrared lights (±30–33 °C) during separations. After the 3-h separation period the pups were returned to the housing facility and reunited with the dams in the home cage. Control litters were reared normally without separation from the dams. All rat pups were weaned at P21 and housed according to gender. Chronic Constant Light Treatment (CCL) A group of maternally separated animals was subjected to chronic constant light (MS CCL) for 3 weeks from P42 to P63. The chronic light exposure occurred in a dark room. The rats were placed under lighting of relatively low irradiance (100–120 lux) at the floor of the cage generated by a 40 W clear bulb. After the 3-week period of constant light, animals were returned to their normal 12 h light/dark cycle. Escitalopram Treatment On P68 until P100, rats were either subjected to escitalopram (5 mg/kg pure base, i.p.) injection (Cipla Medpro (Pty) Ltd) (Uys et al. 2006) or volume controlled saline (0.9 % NaCl) treatment (i.p. injection) in the afternoons between 13 h00 and 14 h00 administered in a dedicated room. The dose of 5 mg/kg was based on previous studies which showed that this dose of escitalopram reversed molecular changes induced by trauma and maternal separation (Uys et al. 2006; Marais et al. 2008). Isobaric Tagging for Relative and Absolute Quantitation (iTRAQ) of the Proteome Prefrontal cortex tissue was analyzed. Tissue collected from three rats per group (CS, CE, MS S, MS E, MS CCL S, MS CCL E) was pooled and sonicated in 1 M triethylammonium bicarbonate buffer. After centrifugation at 17,200×g for 30 min at 4 °C, the supernatant was collected and used for isobaric tagging for relative and absolute quantitation (iTRAQ) of proteins using matrix-assisted laser desorption/ionisation tandem mass spectrometry analysis (Liu et al. 2009; Craft et al. 2013) by the Centre for Proteomic and Genomic Research (CPGR) at the University of Cape Town. Each sample was incubated with trypsin and labeled with an 8–plex iTRAQ reagent kit. All eight samples were modified with iTRAQ tags. After mixing the samples, the iTRAQ reagents were removed from the mixture using a strong cation exchange solid-phase extraction device. The sample mixture was submitted to 1 dimensional liquid chromatography using a Pepmap C18 reverse phase column with gradient elution. The chromatogram confirmed the complete digestion and suitable signal intensity to proceed to mass spectrometric analysis. The samples collected from the chromatographic separation were mixed with MALDI matrix through an inline T connector and spotted on
the MALDI source plate. The matrix was spiked with a fivepoint internal calibration mixture. Internal calibration showed that the mass spectrometer was functioning within specifications. Statistics The MS/MS spectra were analyzed with ProteinPilot software (ProteinPilotTM Software 3.0, Applied Biosystems, MDS Analytical Technologies) using the Ratus ratus database. A Paragon Algorithm was used to determine differentially expressed proteins by calculating the average ratio of sample protein to reference protein, for each protein, along with the associated pvalue and error factors. The p value for each protein was derived from a t test, where the sample size (n) was the number of peptides contributing to the identification of a specific protein and calculated as t¼
ðAverage of Log Ratios–Log BiasÞ Standard error of Average of Log Ratios
where Log Ratio=log (ratio of specific protein in test sample to specific protein in reference sample) and Log Bias=log (sample bias). Proteins detected with >95 % confidence and those that differed significantly between the experimental groups (p<0.05) are reported as fold change with respect to the appropriate reference sample. Four comparisons were made which included the following: MS S and CS (Table 1), MS CCL S and MS S (Table 2), CE and CS, as well as MS E and MS S (Table 3). Hence, the Bonferroni correction for multiple comparisons for each protein was applied at p<0.0125.
Results Maternal Separation Leads to Hypometabolic State of the Prefrontal Cortex, Impaired Cytoskeletal Structure and Reduced Signalling Proteins After maternal separation, there was a significant decrease in several groups of proteins in the prefrontal cortex. Energyrelated proteins that decreased were creatine kinase B-type, aconitate hydratase, malate dehydrogenase and glucose-6phosphate isomerase. Neurotransmission and/or signalling proteins that decreased were calmodulin, synapsin-1 and 14-3-3 protein epsilon. Cytoskeletal and associated proteins that decreased were spectrin, microtubule-associated protein and tubulin. In contrast, maternal separation increased proteins that are involved in signalling and structural functions, namely dihydropyrimidinase-related protein or collapsing response-mediated protein (CRMP), myelin proteolipid protein and WD repeat-containing protein 7 (Fig. 1, Table 1, MS S versus CS).
J Mol Neurosci Table 1 Effects of maternal separation on prefrontal cortex proteins Protein Energy metabolism Creatine kinase B-type Aconitate hydratase, mitochondrial Malate dehydrogenase, cytoplasmic Glucose-6-phosphate isomerase Neurotransmission/signalling Calmodulin Dihydropyrimidinase-related protein Synapsin-1 14-3-3 Protein epsilon Cytoskeletal/structural Spectrin alpha chain, brain Microtubule-associated protein Myelin proteolipid protein Tubulin alpha-1A chain Other WD repeat-containing protein 7
Accession no.
%Cov(95)
Peptides
MS S/CS
P value
sp|P07335| sp|Q9ER34| sp|O88989| sp|Q6P6V0|
84.25 39.49 53.59 37.46
57 32 23 34
0.54 0.71 0.61 0.60
0.033 0.038 0.047 0.049
sp|P62161| tr|Q9JMG8| sp|P09951| sp|P62260|
42.95 9.24 56.25 79.22
11 6 39 32
0.54 5.30 0.56 0.70
0.003a 0.007 a 0.009 a 0.030
sp|P16086| tr|Q63724| sp|P60203| sp|P68370|
41.46 23.63 24.91 89.58
100 34 8 138
0.65 0.71 1.67 0.33
0.001 a 0.009 a 0.013 0.026
sp|Q9ERH3|
7.66
9
1.56
0.027
The % Cov (95) refers to the number of amino acids in the identified peptides that match the protein with a confidence of 95 % or higher, divided by the total number of amino acids in the protein and expressed as a percentage. Peptides refer to the number of peptides in the protein that have been identified with a confidence of 95 % or greater MS S maternally separated rats treated with saline, CS non-maternally separated rats treated with saline a
Significant with Bonferroni correction (p<0.0125)
Chronic Constant Light Reverses Maternal Separation Induced Hypometabolism in the Prefrontal Cortex and Increases Cytoskeletal Structural and Signalling Proteins Maternally separated animals subjected to chronic light exposure during adolescence and treated with vehicle (MS CCL S versus MS S) demonstrated increased energy-related proteins (ATP synthase subunit alpha and beta, aconitate hydratase, creatine kinase B-type and nucleoside diphosphate kinase A), signalling proteins (synapsin-1, 14-3-3 protein epsilon, calmodulin and brain acid soluble protein 1) as well as structural proteins (spectrin, microtubule-associated protein and nonerythroid spectrin beta) compared with maternally separated animals treated with vehicle alone (Table 2). Moreover, dihydropyrimidinase-related protein/CRMP and WD repeatcontaining protein 7 were decreased by light exposure in maternally separated animals. Maternally separated rats exposed to constant light for 3 weeks during adolescence had similar levels of these various proteins when compared to controls (Fig. 1, MS CCL S versus CS). Escitalopram Leads to Altered Proteins in the Prefrontal Cortex Escitalopram treatment of maternally separated rats (MS E versus MS S) increased ATP synthase and WD repeat-
containing protein 7, which was similarly changed by chronic exposure to light. WD repeat-containing protein 7 is a multifunctional protein involved in signal transduction, transcription and proteosomal degradation. Escitalopram also increased myelin proteolipid protein and 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNPase) and decreased vinculin, proteins that were not affected by light treatment (Table 3).
Discussion Early life stress decreases energy metabolism and inhibits axonal outgrowth and cell signalling in the prefrontal cortex, effects that are reversed by 3 weeks of constant light exposure during adolescence. Specifically, maternal separation decreased energy-related proteins (creatine kinase B, aconitate hydratase), cell signalling proteins (dihydropyrimidinase-related protein (CRMP), synapsin I, calmodulin, 14-3-3 protein epsilon) and cytoskeletal proteins (spectrin, microtubuleassociated protein, myelin proteolipid protein) in prefrontal cortex. Exposure to light reversed most of these protein changes induced by maternal separation. The effects of light exposure overlapped partially with the effects of escitalopram treatment. The prefrontal cortex and the limbic areas of the brain form the neural substrates of emotional and motivated
J Mol Neurosci Table 2 Effects of chronic constant light (CCL) on prefrontal cortex proteins of maternally separated animals treated with vehicle Protein Energy metabolism ATP synthase subunit alpha, mitochondrial Aconitate hydratase, mitochondrial Creatine kinase B-type ATP synthase subunit beta, mitochondrial Nucleoside diphosphate kinase A Neurotransmission/signalling Synapsin-1 Dihydropyrimidinase-related protein 14-3-3 Protein epsilon Calmodulin Brain acid soluble protein 1 Cytoskeletal/structural Spectrin alpha chain, brain Microtubule-associated protein Non-erythroid spectrin beta Other WD repeat-containing protein 7
Accession no.
%Cov(95)
Peptides
MS CCL S/MS S
Pvalue
sp|P15999| sp|Q9ER34| sp|P07335| sp|P10719| sp|Q05982|
56.96 39.49 84.25 56.71 43.42
38 32 57 37 6
1.85 1.56 1.82 1.43 1.53
0.002a 0.004 a 0.017 0.025 0.022
sp|P09951| tr|Q9JMG8| sp|P62260| sp|P62161| sp|Q05175|
56.25 9.236 79.22 42.95 38.64
39 6 32 11 7
1.67 0.25 1.25 1.50 3.50
0.009 a 0.017 0.018 0.021 0.030
sp|P16086| tr|Q63724| tr|Q6XD99|
41.46 23.63 29.52
100 34 65
1.46 1.34 1.41
0.001 a 0.017 0.019
sp|Q9ERH3|
7.661
9
0.55
0.047
The % Cov (95) refers to the number of amino acids in the identified peptides that match the protein with a confidence of 95 % or higher, divided by the total number of amino acids in the protein and expressed as a percentage. Peptides refer to the number of peptides in the protein that have been identified with a confidence of 95 % or greater MS CCL S maternally separated rats treated with chronic constant light and saline, MS S maternally separated rats treated with saline a
Significant with Bonferroni correction (p<0.0125)
behaviour and have been implicated in various psychiatric disorders, including depression (Drevets 1999). We have previously found that maternally separated rats have increased energy-related proteins in the ventral hippocampus (Marais et al. 2009b). Here we provide evidence that maternal separation is accompanied by decreased energy-related proteins in the prefrontal cortex. These findings are consistent with the clinical literature on depression, which is characterised by hypometabolism in the prefrontal cortex and hypermetabolism in the amygdala, hippocampus and other limbic structures (Drevets 1999; Kennedy et al. 2001; Dunn et al. 2002; Seminowicz et al. 2004; Fitzgerald et al. 2008; Salerian and Altar 2012). Intracellular signalling and effective neurotransmission is well-known to be altered in depression (Nestler et al. 2002). For example, the present study found decreased synapsin-1 levels in response to maternal separation. This presynaptic protein functions by docking vesicles containing neurotransmitters to the actin cytoskeleton in neuronal terminals (Hilfiker et al. 1999) indicating a strong inter-dependence between structural components and signalling. Decreased expression of synapsin-1 is consistent with findings from other animal models of early life stress, the chronic social isolation stress, which similarly results in decreased synapsin-1 levels in the prefrontal cortex (Hermes et al. 2011).
As highlighted earlier, functional communicative networks are particularly dependent on cytoskeletal proteins maintaining active connectivity between brain areas involved in emotion. Stress and thus glucocorticoid exposure affects dendritic length and spine number of neurons which ultimately alters the structure of synaptic connections between neurons (Woolley et al. 1990; Christian et al. 2011). Depression, which is characterised by high basal corticosterone levels, places the brain in a state of dysfunctional connectivity as a result of abnormal cytoskeletal structure (Carroll et al. 1976; Warner-Schmidt and Duman 2006; Tata and Anderson 2010; Wong et al. 2012). We found decreases in cytoskeletal proteins (spectrin, microtubule-associated protein, tubulin and myelin proteolipid protein) in response to maternal separation. This is in line with previous proteomic studies of the hippocampus and nucleus accumbens which found reduced levels of cytoskeletal proteins in response to stress (Piubelli et al. 2011; Dimatelis et al. 2012b). One of the most prominent changes by light treatment of rats subjected to maternal separation is the normalisation of dihydropyrimidinase related protein levels in the prefrontal cortex. Dihydropyrimidinase related protein, also known as CRMP, is an essential regulator of microtubule organisation during axonal growth (Fukata et al. 2002). Myelin-dependent axonal outgrowth is inhibited by CRMP4 interacting with
J Mol Neurosci Table 3 Effects of escitalopram on prefrontal cortex proteins in both maternally separated and non-separated/control animals Protein Neurotransmission/signalling Glutamine synthetase Synaptojanin-1 Cytoskeletal/structural Glycoprotein m6a Other Lactoylglutathione lyase Protein Energy metabolism ATP synthase subunit alpha, mitochondrial Cytoskeletal/structural Myelin proteolipid protein Vinculin 2',3'-Cyclic-nucleotide 3'-phosphodiesterase Other WD repeat-containing protein 7
Accession no.
%Cov(95)
Peptides
CE/CS
Pvalue
sp|P09606| sp|Q62910|
46.38 10.93
21 16
1.77 0.75
0.038 0.042
tr|Q812E9|
15.11
6
1.98
0.041
sp|Q6P7Q4|
10.33
3
0.78
0.049
Accession no.
%Cov(95)
Peptides
MS E/MS S
Pvalue
sp|P15999|
56.96
38
1.92
0.002a
sp|P60203| sp|P85972| sp|P13233|
24.91 4.78 40.95
8 3 17
1.64 0.79 1.42
0.009 a 0.013 0.017
sp|Q9ERH3|
7.66
9
0.70
0.029
The % Cov (95) refers to the number of amino acids in the identified peptides that match the protein with a confidence of 95 % or higher, divided by the total number of amino acids in the protein and expressed as a percentage. Peptides refer to the number of peptides in the protein that have been identified with a confidence of 95 % or greater CE non-maternally separated rats treated with escitalopram, CS non-maternally separated rats treated with saline, MS E maternally separated rats treated with escitalopram, MS S maternally separated rats treated with saline a
Significant with Bonferroni correction (p<0.0125)
RhoA signalling molecules (Alabed et al. 2007). It was found that phosphorylation of GSK 3β by myelin-associated inhibitors leads to dephosphorylation of CRMP4 and the formation of the CRMP/RhoA complex resulting in axonal outgrowth inhibition (Mimura et al. 2006; Alabed et al. 2010). Interestingly, axonal sprouting and regeneration in the adult central nervous system is blocked by the presence of myelin and Fig. 1 Chronic constant light exposure during adolescence (MS+CCL) reversed changes caused by maternal separation (MS) in the prefrontal cortex proteins involved in metabolic activity, signalling and cytoskeletal structure in adult rats
myelin-associated proteins (Alabed et al. 2010). Maternal separation resulted in an overwhelming upregulation of CRMP in the presence of increased myelin proteolipid protein. This is in line with previous findings, where animals subjected to chronic unpredictable stress, another animal model used to induce depression-like behaviour, resulted in increased CRMP and the expression of myelin proteins in the MS S / CS
Creatine kinase B-type
MS CCL S / CS
Aconitate hydratase
Calmodulin Synapsin-1 14-3-3 protein epsilon Dihydropyrimidinase-related protein
Spectrin alpha chain, brain Microtubule-associated protein 0
1
2
3
4
5
6
J Mol Neurosci
prefrontal cortex (Lisowski et al. 2013). The upregulation of myelin proteins in response to chronic unpredictable stress was ascribed to an adaptive stress response mechanism (Lisowski et al. 2013). Similarly, other neurodegenerative disorders like Alzheimer’s disease and Down syndrome display dysregulated expression of CRMP and resultant disrupted neuronal connectivity and wiring of the brain (Lubec et al. 1999; Wietzdoerfer et al. 2001; Castegna et al. 2002). Light treatment is a non-pharmacological intervention that can normalise depressive behaviour and neuropathology. We now show, for the first time, that light treatment reverses most of the protein changes induced by maternal separation in the prefrontal cortex indicating that light treatment targets similar pathways affected by maternal separation and can reverse the effects of maternal separation. Previous work has shown that chronic constant light treatment results in anti-depressive behavioural effects (MolinaHernandez and Tellez-Alcantara 2000; Yilmaz et al. 2004; Schultz et al. 2008; Iyilikci et al. 2009) and has demonstrated that such exposure leads to restoration of decreased muopioid levels in the nucleus accumbens of maternally separated rats (Dimatelis et al. 2012a). Our findings suggest that light therapy works by increasing metabolic proteins in the prefrontal cortex and reversing the changes in signalling and cytoskeletal proteins caused by maternal separation. Chronic constant light treatment of maternally separated animals restored the decrease in signalling proteins (synapsin-1, calmodulin, 14-3-3 protein epsilon and dihydropyrimidinase-related protein (CRMP)) in the prefrontal cortex. We also show that light treatment resulted in increased cytoskeletal proteins (spectrin, microtubule-associated protein, non-erythroid spectrin beta) in maternally separated animals. This is consistent with clinical and animal data indicating that depression affects these functional proteins (Drevets 1999; Ladd et al. 2000; Nestler et al. 2002; Li et al. 2004; Hermes et al. 2011; Wong et al. 2012). The restoration of the expression levels of CRMP in the prefrontal cortex could possibly infer a mechanism of light to restore normal connectivity and signalling in the prefrontal cortex. In the present study, the effects of light exposure overlapped partially with the effects of escitalopram treatment. Light exposure or escitalopram treatment increased mitochondrial ATP synthase protein levels in the prefrontal cortex of maternally separated rats. This is consistent with anti-depressant treatment normalising decreased cerebral blood flow and glucose metabolism in the prefrontal cortex of depressed patients (Buchsbaum et al. 1997; Kennedy et al. 2001). On the other hand, escitalopram increased myelin proteolipid protein and CNPase and decreased vinculin. These findings are consistent with previous work indicating that CNPase is crucial for maintenance of oligodendrocyte integrity and hence neuronal connectivity. CNPase knockout mice have been shown to have low emotionality by displaying decreased anxiety and depressive-
like behaviour after chronic unpredictable stress exposure (Edgar et al. 2011). This study provides new data on the effects of maternal separation, demonstrating that this results in reduced structure and signalling in addition to hypometabolism of the prefrontal cortex. It also provides new data on how light therapy might work, showing for the first time reversal of effects on metabolism, neurotransmission and neuroplasticity-related proteins. Finally, the data here suggest partial overlap between light therapy and a SSRI. Proteomic approaches may ultimately contribute to the development of more targeted approaches for the treatment of depression; our data suggest that interventions that act on reducing CRMP levels may be particularly useful. Acknowledgements The authors would like to acknowledge the contributions of the CPGR, the Institute for the Study of Affective Neuroscience (ISAN, grant no 9276) for financial support, the National Research Foundation (NRF), Harry Crossley Foundation for financial support of the postdoctoral fellow and Cipla Medpro (Pty) Ltd for their kind donation of escitalopram oxalate.
References Abercrombie HC, Schaefer SM, Larson CL et al (1998) Metabolic rate in the right amygdala predicts negative affect in depressed patients. Neuroreport 9:3301–3307 Aisa B, Tordera R, Lasheras B, Del Rio J, Ramirez MJ (2007) Cognitive impairment associated to HPA axis hyperactivity after maternal separation. Psychoneuroendocrinology 32:256–266 Alabed YZ, Pool M, Tone SO, Fournier AE (2007) Identification of CRMP4 as a convergent regulator of axon outgrowth inhibition. J Neurosci 27:1702–1711 Alabed YZ, Pool M, Tone SO, Sutherland C, Fournier AE (2010) GSK3β regulates myelin-dependent axon outgrowth inhibition through CRMP4. J Neurosci 30:5635–5643 Buchsbaum MS, Wu J, Siegel BV et al (1997) Effect of sentraline on regional metabolic rate in patients with affective disorders. Biol Psychiatry 41:15–22 Carroll BJ, Curtis GC, Mendels J (1976) Neuroendocrine regulation of depression. I. Limbic system–adrenocortical dysfunction. Arch Gen Psychiatry 33:1039–1044 Castegna A, Aksenov M, Thongboonkerd V et al (2002) Proteomic identification of oxidatively modified proteins in Alzheimer’s disease brain. Part II: dihydropyrimidinase-related protein 2, αenolase and heat shock cognate 71. J Neurochem 82:1524–1532 Castrén E, Rantamäki T (2010) The role of BDNF and its receptors in depression and antidepressant drug action: reactivation of developmental plasticity. Develop Neuobiol 70:289–297 Christian KM, Miracle AD, Welllman CL, Nakazawa K (2011) Chronic stress-induced hippocampal dendritic retraction requires CA3 NMDA receptors. Neuroscience 174:26–36 Craft GE, Chen A, Nairn AC (2013) Recent advances in quantitative neuroproteomics. Methods 61:186–218 Daniels WM, Pietersen CY, Carstens ME, Stein DJ (2004) Maternal separation in rats leads to anxiety-like behaviour and a blunted ACTH response and altered neurotransmitter levels in response to a subsequent stressor. Metab Brain Dis 19:3–14 Dimatelis JJ, Stein DJ, Russell VA (2012a) Behavioral changes after maternal separation are reversed by chronic constant light treatment. Brain Res 1480:61–71
J Mol Neurosci Dimatelis JJ, Stein DJ, Russell VA, Daniels WM (2012b) Effects of maternal separation and methamphetamine exposure on protein expression in the nucleus accumbens shell and core. Met Brain Dis 27:363–375 Drevets WC (1999) Prefrontal cortical-amygdalar metabolism in major depression. Ann NY Acad Sci 877:614–637 Drevets WC, Price JL, Bardgett ME, Reich T, Todd RD, Raichle ME (2002) Glucose metabolism in the amygdala in depression: relationship to diagnostic subtype and plasma cortisol levels. Pharmacol Biochem Behav 71:431–447 Duman RS (2002) Pathophysiology of depression: the concept of synaptic plasticity. Eur Psychiatry 17:306–310 Dunn RT, Kimbrell TA, Ketter TA et al (2002) Principal components of the beck depression inventory and regional cerebral metabolism in unipolar and bipolar depression. Biol Psychiatry 51:387–399 Edgar NM, Touma C, Palme R, Sibille E (2011) Resilient emotionality and molecular compensation in mice lacking the oligodendrocytespecific gene Cnp1. Transl Psychiatry 1:e42 Even C, Schröder CM, Friedman S, Rouillon F (2008) Efficacy of light therapy in nonseasonal depression: a systematic review. J Affect Disord 108:11–23 Fitzgerald PB, Laird AR, Malle J, Daskalakis ZJ (2008) A metaanalytic study of changes in brain activation in depression. Hum Brain Mapp 29:683–695 Fukata Y, Itoh TJ, Kimura T et al (2002) CRMP-2 binds to tubulin heterodimers to promote microtubule assembly. Nat Cell Biol 4:583–591 Golden RN, Gaynes BN, Ekstrom RD et al (2005) The efficacy of light therapy in the treatment of mood disorders: a review and metaanalysis of the evidence. Am J Psychiatry 162:656–662 Hermes G, Li N, Duman C, Duman R (2011) Post-weaning chronic isolation produces profound behavioural dysregulation with decreases in prefrontal cortex synaptic-associated protein expression in female rats. Physiol Behav 104:354–359 Hilfiker S, Pieribone VA, Czernik AJ, Kao HT, Augustine GJ, Greengard P (1999) Synapsins as regulators of neurotransmitter release. Philos Trans R Soc Lond B Biol Sci 354:269–279 Iyilikci O, Aydin E, Canbeyli R (2009) Blue but not red light stimulation in the dark has antidepressant effect in behavioural despair. Behav Brain Res 203:65–68 Kennedy SH, Evans KR, Krüger S et al (2001) Changes in regional brain glucose metabolism measured with positron emission tomography after paroxetine treatment of major depression. Am J Psychiatry 158:899–905 Ladd CO, Huot RL, Thrivikraman KV, Nemeroff CB, Meaney MJ, Plotsky PM (2000) Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog Brain Res 122:81–103 Lam RW, Levitt AJ, Levitan RD et al (2006) The Can-SAD study: a randomized controlled trial of the effectiveness of light therapy and fluoxetine in patients with winter seasonal affective disorder. Am J Psychiatry 163:805–812 Leussis MP, Freund N, Brenhouse HC, Thompson BS, Anderson SL (2012) Depressive-like behavior in adolescents after maternal separation: sex differences, controllability and GABA. Dev Neurosci 34:210–217 Li X, Nahas Z, Kosel FA, Anderson B, Bohning DE, George MS (2004) Acute left prefrontal transcranial magnetic stimulation in depressed patients is associated with immediately increased activity in prefrontal cortical as well as subcortical regions. Biol Psychiatry 55:882–890 Lisowski P, Wieczorek M, Goscik J et al (2013) Effects of chronic stress on prefrontal cortex transcriptome in mice displaying different genetic backgrounds. J Mol Neurosci 50:33–57 Liu T, Hu J, Li H (2009) iTRAQ-based shotgun neuroproteomics in neuroproteomics. In: Ottens AK, Wang KKW (eds) Methods in Molecular Biology, vol.566. Humana, New York, NY, pp 201–216
Lubec G, Nonaka M, Krapfenbauer K, Gratzer M, Cairns N, Fountoulakis M (1999) Expression of the dihydropyrimidinase related protein 2 (DRP-2) in Down syndrome and Alzheimer’s disease brain is downregulated at the mRNA and dysregulated at the protein level. J Neural Transm Suppl 57:161–177 MacQueen GM, Ramakrishnan K, Ratnasingan R, Chen B, Young LT (2003) Despiramine treatment reduces the long-term behavioural and neurochemical sequelae of early-life maternal separation. Int J Neuropsychopharm 6:391–396 Marais L, Stein DJ, Daniels WMU (2009a) Exercise increases BDNF levels in the striatum and decreases depressive-like behavior in chronically stressed rats. Metab Brain Dis 24:587–597 Marais L, Hattingh SM, Stein DJ, Daniels WMU (2009b) A proteomic analysis of the ventral hippocampus of rats subjected to maternal separation and escitalopram treatment. Metab Brain Dis 24:569–586 Marais L, van Rensburg SJ, van Zyl JM, Stein DJ, Daniels WMU (2008) Maternal separation of rat pups increases the risk of developing depressive-like behaviour after subsequent chronic stress by altering corticosterone and neurotrophin levels in the hippocampus. Neurosci Res 61:106–112 Mimura F, Yamagishi S, Arimura N et al (2006) Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinasedependent mechanism. J Biol Chem 281:15970–15979 Mirescu C, Peters JD, Gould E (2004) Early life experience alters response of adult neurogenesis to stress. Nat Neurosci 7:841–846 Molina-Hernandez M, Tellez-Alcantara P (2000) Long photoperiod regimen may produce antidepressant actions in the male rat. Prog Neuropsychopharmacol Biol Psychiatry 24:105–116 Mu J, Xie P, Yang ZS et al (2007) Neurogenesis and major depression: implications from proteomic analyses of hippocampal proteins in a rat depression model. Neurosci Lett 416:252–256 Nestler EJ, Barrot M, DiLeone RJ, Eisch AJ, Gold SJ, Monteggia LM (2002) Neurobiology of depression. Neuron 34:13–25 Osuch EA, Ketter TA, Kimbrell TA et al (2000) Regional cerebral metabolism associated with anxiety symptoms in affective disorder patients. Biol Psychiatry 48:1020–1023 Piubelli C, Carboni L, Becchi S, Mathé AA, Domenici E (2011) Regulation of cytoskeleton machinery, neurogenesis and energy metabolism pathways in a rat gene-environment model of depression revealed by proteomic analysis. Neuroscience 176:349–380 Roceri M, Hendriks W, Racagni G, Ellenbroek BA, Riva MA (2002) Early maternal deprivation reduces the expression of BDNF and NMDA receptor subunits in rat hippocampus. Mol Psychiatry 7:609–616 Salerian AJ, Altar CA (2012) The prefrontal cortex influence over subcortical and limbic regions governs antidepressant response by N = H/(M + R). Psychiatry Res. doi:10.1016/j.pscychresns.2012.04.019 Seminowicz DA, Mayberg HS, McIntosh AR et al (2004) Limbicfrontal circuitry in major depression: a path modeling metanalysis. NeuroImage 22:409–418 Schultz D, Aksoy A, Canbeyli R (2008) Behavioral despair is differentially affected by the length and timing of photic stimulation in the dark phase of an L/D cycle. Prog Neuropsychopharmacol Biol Psychiatry 32:1257–1262 Tata DA, Anderson BJ (2010) The effects of chronic glucocorticoid exposure on dendritic length, synapse numbers and glial volume in animal models: implications for hippocampal volume reductions in depression. Physiol Behav 99:186–193 Terman M, Terman JS (2005) Light therapy for seasonal and nonseasonal depression: efficacy, protocol, safety, and side effects. CNS Spectrums 10:647–663 Uys JDK, Muller CJF, Marais L, Harvey BH, Stein DJ, Daniels WMU (2006) Early life trauma decreases glucocorticoid receptors in rat dentate gyrus upon adult re-stress: reversal by escitalopram. Neuroscience 137:619–625 Videbech P (2000) PET measures of brain glucose metabolism and blood flow in major depressive disorder: a critical review. Acta Psychiatr Scand 101:11–20
J Mol Neurosci Vivinetto AL, Suárez MM, Rivarola MA (2012) Neurobiological effects of neonatal maternal separation and post-weaning environmental enrichment. Behav Brain Res. doi:10.1016/j.bbr.2012.11.014 Warner-Schmidt JL, Duman RS (2006) Hippocampal neurogenesis: opposing effects of stress and antidepressant treatment. Hippocampus 16:239–249 Wietzdoerfer R, Fountoulakis M, Lubec G (2001) Aberrant expression of dihydropyrimidinase related proteins-2,-3, and −4 in fetal Down syndrome brain. J Neural Transm Suppl 61:95–107 Wong GTH, Chang RCC, Law ACK (2012) A breach in the scaffold: the possible role of cytoskeleton dysfunction in the pathogenesis of major depression. Ageing Res Rev 12:67–75
Woolley CS, Gould E, McEwen BS (1990) Exposure to excess glucocorticoids alters dendritic morphology of adult hippocampal pyramidal neurons. Brain Res 531:225–231 Yilmaz A, Aksoy A, Canbeyli R (2004) A single day of constant light (L/L) provides immunity to behavioural despair in female rats maintained on an L/D cycle. Prog Neuropsychopharmacol Biol Psychiatry 28:1261–1265 Yoo SB, Kim B-T, Kim JY et al (2012) Adolescence fluoxetine increases serotonergic activity in the raphe-hippocampus axis and improves depression-like behaviors in female rats that experienced neonatal maternal separation. Psychoneuroendocrinology. doi:10. 1016/j.psyneuen.2012.08.013