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Background: Attention-deficit/hyperactivity disorder (ADHD) is a developmental disorder characterised by symptoms of inattention, impulsivity and hyperactivity. The spontaneously hypertensive rat (SHR) is a well-characterised model of this disorder and has been shown to exhibit dopamine dysregulation, one of the hypothesised causes of ADHD. Since stress experienced in the early stages of life can have long-lasting effects on behaviour, it was considered that early life stress may alter development of the dopaminergic system and thereby contribute to the behavioural characteristics of SHR. It was hypothesized that maternal separation would alter dopamine regulation by the transporter (DAT) in ways that distinguish SHR from control rat strains.Methods: SHR and control Wistar-Kyoto (WKY) rats were subjected to maternal separation for 3 hours per day from postnatal day 2 to 14. Rats were tested for separation-induced anxiety-like behaviour followed by in vivo chronoamperometry to determine whether changes had occurred in striatal clearance of dopamine by DAT. The rate of disappearance of ejected dopamine was used as a measure of DAT function.Results: Consistent with a model for ADHD, SHR were more active than WKY in the open field. SHR entered the inner zone more frequently and covered a significantly greater distance than WKY. Maternal separation increased the time that WKY spent in the closed arms and latency to enter the open arms of the elevated plus maze, consistent with other rat strains. Of note is that, maternal separation failed to produce anxiety-like behaviour in SHR. Analysis of the chronoamperometric data revealed that there was no difference in DAT function in the striatum of non-separated SHR and WKY. Maternal separation decreased the rate of dopamine clearance (k -1) in SHR striatum. Consistent with this observation, the dopamine clearance time (T100) was increased in SHR. These results suggest that the chronic mild stress of maternal separation impaired the function of striatal DAT in SHR.Conclusions: The present findings suggest that maternal separation failed to alter the behaviour of SHR in the open field and elevated plus maze. However, maternal separation altered the dopaminergic system by decreasing surface expression of DAT and/or the affinity of DAT for dopamine, increasing the time to clear dopamine from the extracellular fluid in the striatum of SHR. © 2011 Womersley et al; licensee BioMed Central Ltd.
Ascorbic acid, dopamine-HCl, urethane and Nafion® were purchased from Sigma Chemical Company (St. Louis, MO, U.S.A). All other chemicals were of the highest standard and were purchased from Merck Chemicals (Germany). Carbon fibre electrodes were purchased from Quanteon LLC (Lexington, KY, U.S.A). Teflon-coated silver wire was purchased from A-M Systems (Carlsborg, WA, U.S.A). Three month old male and female inbred WKY (Harlan, UK) and SHR rats (Charles River Laboratories, USA) were obtained from the University of Cape Town Animal Unit and paired in cages for breeding. The date of birth of the litter was designated postnatal day 0 (P0). On P2, litters were culled to 8 pups to ensure equal nourishment during the early postnatal period. Male rats were preferentially selected to avoid the confounding effects of fluctuating hormones during the female oestrus cycle on brain function and behaviour in later experiments. This selection produced an average of 4.3 males and 3.7 females in WKY NMS litters, 4.6 males and 3 females in WKY MS litters, 6.3 males and 1.2 females in SHR NMS litters, and 5.2 males and 1.7 females in SHR MS litters. On P2, litters were designated as either maternally separated experimental rats or non-maternally separated control rats to give a total of 21 non-separated WKY; 22 maternally separated WKY; 20 non-separated SHR and 29 separated SHR. Rats were housed in the University of Cape Town satellite animal facility in cages with woodchip bedding and a 12 hour light/dark cycle (lights on at 06h00). Rats had ad libitum access to food and water. This study was conducted in accordance with international guidelines (South African National Standard: The care and use of animals for scientific purposes. 1st edition, 2008) and approved by the University of Cape Town Faculty of Health Sciences Animal Ethics Committee. The maternal separation protocol involved removal of the dam from the pups for 3 hours per day between 09h00 and 13h00 from P2 to P14 [40]. Pups were transferred in the home cage to a different room to prohibit communication with the dam by means of ultrasound vocalisation [40]. The temperature within the home cage was maintained at 31-33°C by infrared heating lamps so as to avoid the risk of possible hypothermia. After 3 hours the pups were returned to the animal facility and the dams returned to their home cages. Rats designated as controls were maintained in the home cage at all times until weaning. The cleaning routine was standard across both separated and non-separated rats with approximately half of the soiled wood-chip bedding removed every third day in the first week and every second day in the second and third weeks following birth. Thus handling of pups was consistent across both maternally separated and non-separated experimental groups. On P21 rats were weaned and male rats separated from their female littermates (the ratio of male to female pups in maternally separated and non-separated litters did not differ significantly within strains but SHR litters tended to have a higher ratio of male to female pups than WKY). The male rats were housed in groups of two to four rats per cage for the remainder of the project. On P28, rats were taken to the behavioural facility and allowed to acclimatise to the behavioural testing room for a minimum of 1 hour prior to testing in the open field and elevated plus maze. The open field test measures total distance covered in a 100 cm × 100 cm (floor) × 50 cm (walls) black box, as well as time spent and number of entries into the inner zone (70 cm × 70 cm) of the box. The tests were conducted in an isolated noise-free room. The lighting in the room was 50 lux. More time spent in the inner zone indicated increased exploration, and was defined as reduced anxiety [41,42]. The distance covered provided an indication of the rat’s locomotor activity [41,42]. Each rat was tested in a single trial of 5 minutes. All behavioural tests were recorded with a Sony Handicam DCR-SX 83E for later analysis with Noldus Ethovision XT 7.0 (Noldus Information Technology, Wageningen, The Netherlands). After the open field test, the rat was returned to the home cage and a minimum of 1 hour was allowed to pass before the rat was tested in the elevated the plus maze which has open and closed arms, elevated 50 cm above the ground. The animal was placed in the centre of the maze, facing an open arm and allowed to explore the maze for 5 minutes. Time spent in the open arms suggested decreased anxiety-like behaviour while time spent in the closed arms suggested increased anxiety-like behaviour [43]. Gloves were used throughout the behavioural experiments. The maze was cleaned after every test with 70% alcohol to ensure that the rat’s behaviour was not affected by the scent of another rat. High-speed in vivo chronoamperometric measurement of extracellular dopamine was carried out using the FAST-16 system (Quanteon LLC, Lexington, KY, U.S.A) with Nafion-coated carbon fibre microelectrodes (electrode tips, 30 μm outer diameter and 150 μm length) [44,45]. This technique has the benefit of high spatial resolution and second-by-second temporal resolution, which make it superior to in vivo microdialysis coupled with high-performance liquid chromatography techniques [46]. The Nafion® coating (5% solution in alcohol at 200°C) was used to enhance the sensitivity and selectivity of the electrodes for dopamine over other electroactive molecules [45,47]. Prior to use in vivo, electrodes were calibrated in 0.05 M phosphate-buffered saline solution (10 mM NaH2PO4, 40 mM Na2HPO4, 100 mM NaCl) to which a known amount of ascorbic acid and incremental amounts of dopamine were added to determine selectivity of the electrodes for dopamine and to generate a calibration curve of current versus dopamine concentration. A square wave potential (0 to +0.55 V vs. a glass RE-5 Ag/AgCl reference electrode) was applied to the carbon fibre microelectrode for 100 ms to create a 5 Hz waveform repeated at 1 Hz intervals to cause the oxidation and subsequent reduction of dopamine at the microelectrode surface. Dopamine has a specific red/ox ratio (~0.7-0.9) which was used as a ‘chemical fingerprint’ to confirm specific dopamine recording [47]. Changes in extracellular concentrations of dopamine in vivo were expressed as changes from a stable baseline response of the microelectrode. Only carbon fibre microelectrodes that gave a selectivity for dopamine over ascorbic acid greater than 250:1, a limit of detection less than 0.1 μM dopamine, and a correlation coefficient greater than 0.997, were used (Table (Table1).1). A glass micropipette (1 mm od, 0.58 mm id) was pulled and bumped to produce a micropipette tip with an inner diameter of approximately 10 μm. The microelectrode and micropipette were aligned in parallel and joined by sticky wax (Kerr Corporation, Orange, CA, USA) such that their tips were 180 – 220 μm apart. A miniature Ag/AgCl reference electrode was prepared from Teflon-coated silver wire by removing ~3 mm of the Teflon sheath at one end, exposing the tip and anodizing it at +10 volts versus platinum wire in a solution of 1 M HCl saturated with NaCl for 10-15 minutes. The anodized Ag/AgCl reference electrodes were stored in 3 M NaCl prior to use in vivo [48]. Carbon fibre electrode calibration parameters Calibration parameters are displayed as median, 25% and 75% quartiles. Between P49 and P54 rats were deeply anesthetized with 25% urethane at a dose of 1.25 g/kg to produce non-recoverable terminal anaesthesia. Urethane was chosen because it affected the activity of neurotransmitter systems less than other anaesthetic agents [49]. Rats were placed in a stereotactic frame, on a heated cushion maintained at 37°C by circulating water. A midline incision was made, the scalp was reflected and a burr hole drilled to provide access to the right striatum. A burr hole was similarly drilled above the left posterior cortex for the placement of a miniature Ag/AgCl reference electrode. The microelectrode assembly was positioned at +1 mm anterior to Bregma, 2.5 mm lateral to the sagittal suture and lowered 3 mm into the rat brain under stereotactic guidance using the flat skull coordinates of Paxinos and Watson [50]. High-speed chronoamperometric electrochemical measurements were continuously made with the carbon fibre microelectrode using a FAST16 mkI recording system (Quanteon, LLC, Lexington, Kentucky, USA). After a baseline period of 1 hour, a 200 μM dopamine solution (0.2 mM dopamine plus 100 μM ascorbic acid, in normal saline adjusted to pH 7.2-7.4) was ejected into the striatum using a Picospritzer II (Parker Instrumentation, Parker Hannifin Corporation). The volume of ejected dopamine was varied by finely adjusting the pressure and time controls on the picospritzer to achieve a peak amplitude between 0.75 and 1.5 μM dopamine. The microelectrode assembly was lowered by 0.5 mm increments from 3.5 to 5.0 mm ventral to the cortical surface and striatal clearance of a pressure ejected pulse of dopamine was recorded. The amplitude, defined as the difference between baseline and peak dopamine concentration, first-order rate constant (k-1), and clearance time (T100) were measured (Figure (Figure1).1). The first-order rate constant k-1 is calculated from the decay of dopamine concentration versus time. It is indicative of dopamine uptake efficiency and as such, is indirectly proportional to the clearance time [51,52]. T100 represents the time taken for the dopamine concentration to return from peak amplitude to the baseline value prior to the ejection of dopamine. Independent recordings of dopamine clearance were made at 0.5 mm intervals (between 3.5 and 5 mm ventral to the cortical surface) in the striatum. A total of 38 recordings were obtained from 10 non-separated WKY (WKY NMS), 24 recordings from 7 maternally separated WKY (WKY MS), 25 recordings from 7 non-separated SHR (SHR NMS) and 31 recordings from 8 maternally separated SHR (SHR MS). Representative graph showing dopamine clearance in rat striatum. The amplitude of the peak was measured as the maximum change in dopamine concentration from baseline. T100 represents the time taken for the dopamine concentration to return from maximum amplitude to baseline. The k-1 is the first order rate constant. It provides a measure of the rate of decay of dopamine concentration over time, to provide a measure of DAT efficiency. After completion of the experiment, the rats were cervically dislocated and decapitated. The brain was removed from the skull and placement of the electrodes confirmed to be in the rat striatum and thus all data resulting from these recordings were included in the analysis. All data were analysed using Statistica 10. The Shapiro-Wilk’s test was used to test for normality and all data were found to be non-normally distributed. Therefore non-parametric statistics in the form of the Kruskal-Wallis and Mann-Whitney U tests were used for between group comparisons. Significance was defined as p < 0.05. Due to the data being non-normally distributed, results are displayed as median and interquartile range. Graphs were prepared using Graph Pad Prism 5.
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