Involvement of the Ventral Tegmental Area in a Rodent

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Involvement of the VTA in PTSD,NS Corral Frias et al. systemic injections of a D1 receptor agonist diminishes the Pre Shock Tests. freezing response elicited by conditioned fear Inoue et al. 2000 Pezze and Feldon 2004 Little research has been done. Open field test Rats were placed in the center of a circular. black arena diameter of 152 4 cm no obstacles lined by a. to directly explore the role of dopaminergic systems such. 30 cm high peripheral wall in a dim room The animals were. as the VTA in animal models of PTSD A recent study. allowed to freely move for 5 min before being returned to. however identified the mesolimbic DA system as a key. contributor to the resistance to social defeat in mice their home cage The position of the animals was recorded. automatically using a video tracker 20 30 frames,Krishnan et al 2007 This work showed that as with. per second Animals underwent open field testing before. human PTSD patients vulnerable animals showed deficits. shock and on day 16 after shock The moving time and. in sensitivity to natural rewards sucrose and increases in. distance were calculated This test was used to assess initial. drug seeking behavior cocaine, thigmotaxis Thigmotaxis or the tendency to remain close. Neurophysiologically some studies showed that the mean. to walls has been previously used as an index of anxiety in. firing rate of VTA DA neurons increased during an acute. animals as well as in humans and is indicative of a general. restraint paradigm and that the burst firing of a subset of. bias to safety seeking phobic behavior Kallai et al 2007. DA neurons increased significantly compared with baseline. Treit and Fundytus 1988,Anstrom and Woodward 2005 This increase in burst. firing could persist up to 24 h after a single exposure to. stress Others have shown that stressors increase the Nociception and touch sensitivity tests There is evidence. number of active dopaminergic neurons in VTA when that there are increases in pain sensitivity in PTSD patients. measured within 24 h of the last stress episode Valenti et al Asmundson et al 2002 To assess whether increased. 2011 In contrast other studies demonstrated that long nociception was present in our rat model we tested for. term more than 17 days chronic cold exposure yielded nociception and touch sensitivity using the tail flick test and. fewer spontaneously active VTA and medial substantia the Von Frey test respectively The tests were administered. nigra neurons Moore et al 2001 However in this study the day before shock procedures and again on days 8 and 17. the firing rates in the cells that remained active did not after the shock See Supplementary Methods for a detailed. differ significantly from controls description of these procedures. Taken altogether these studies suggest that dopaminergic. neurons of the VTA are affected by stress and respond. Trauma Inducing Procedure, differently depending on the type of stressor and the delay.
after which their activity is measured Very little research The trauma procedure consisted of a single exposure to an. has been conducted on the long lasting effects of trauma inescapable foot shock day 1 followed by three short. on VTA cell activity Here we study and validate a rodent reexposures to the shock chamber without foot shocks. model of PTSD demonstrate the involvement of the situational reminders SRs on days 3 5 and 7 Louvart. dopaminergic system using systemic injections and demon et al 2005 Pynoos et al 1996 The shock apparatus was. strate a specific role of the VTA by selective bilateral made of plexiglass 54 110 40 cm3 high and was. inactivation We show the presence of trauma related long subdivided into two equal sized compartments separated. term electrophysiological changes in VTA DA cells by a guillotine door a lighter first compartment and a. Preliminary results of this work were presented in abstract darker second compartment Figure 1bA and B On the. form Corral Fr as et al 2010 shock day each rat was placed in the lighter compartment. of the shock box After a 3 min adaptation period the. guillotine door was opened and a bright light located in this. first compartment was turned on for 20 sec The door. MATERIALS AND METHODS remained open until the animal entered the second. compartment After another 3 min adaptation period in. the dark shock compartment the rat received an inescap. We used 63 male Sprague Dawley rats weighing 300 400 g able continuous 2 mA foot shock for 10 sec The sham group. 33 behavioral 16 injected intraperitoneally i p and 14 received the same treatment but the shock was not given. cannula implanted Animals were housed under a 12 h Both sham and shocked animals were reexposed to the. dark light reversed cycle lights off at 0900 hours context of the trauma SRs for 2 min once every 2 days for. Behavioral testing was performed in the dark phase of the 3 days Figure 1bB This was achieved by placing the. cycle Animals had free access to food and water at all times animal in the lighter compartment with the guillotine door. All the protocols used to complete this study were reviewed opened The reexposure to the shock box was kept short to. and approved by the Institutional Animal Care and Use prevent extinction The total time spent in the white. Committee at the University of Arizona compartment and number of crosses into the shock. In order to assess the behavioral effects of stress each rat compartment were measured. was subjected to a battery of tests before and after the To investigate long term behavioral sequelae we tested. trauma inducing procedure so that each rat was its own anxiety behaviors 2 weeks after the shock Animals were. control The anxiety tests given up to 2 weeks after trauma tested in three different apparati the black and white box. were given in a contextual setting different from that of the also referred as light and dark box Costall et al 1989 the. trauma procedure The timing of the experimental proce elevated plus maze Pellow 1985 Walf and Frye 2007 and. dure is shown in Figure 1a and each test is discussed in the open field Ohl 2003 See Supplementary Methods for a. details below or in Supplementary Material detailed description of the anxiety testing methods used. Neuropsychopharmacology,Involvement of the VTA in PTSD. NS Corral Frias et al,Shock Situational No procedures Anxiety. day 1 Reminders 6 days Tests,Fields Reversible Black and White Acute in vivo. inactivation and Box Elevated Plus electrophysiology. SCH23390 Maze Open Field day 18,Pain and Touch Pain and Touch Pain and Touch. sensitivity test day 1 sensitivity test day 8 sensitivity test day 17. Shock Situational Reminder SCH23390 Shock 2 5 Bupiviciane Shock. 3 minute exploration Rats underwent shock, 2 minute exploration Rats underwent shock procedure.
in each compartment procedure immediately after, with the door opened immediately after i p injection. with closed door intra VTA injection,VTA AP 5 2 5 6 AP 5 4. SNr VTA VTA,VTA VTA VTA, Figure 1 Behavioral protocols and histology a General protocol Animals underwent baseline measurements including 4 days of open fields and pain. and touch sensitivity tests They were then exposed to a single inescapable foot shock day 1 followed by reexposures to the nonshock compartment of. the shock cage SRs days 3 5 and 7 To assess long term behavioral sequelae animals were evaluated for anxiety pain and touch sensitivity after 6 days of. rest in their home cage The experiments ended with acute recordings in the VTA In two groups of animals the dopaminergic system was interfered with. immediately before foot shock b Trauma inducing procedure The trauma inducing procedure consisted of a single 2 mA 10 s inescapable foot shock A. followed by three SRs on 3 different days during which the animal was exposed to the safe compartment of the shock box for 2 min B A group of animal. received a single i p injection of D1 receptor antagonist SCH23390 immediately before shock C Another group of animals received 2 5 bupivicaine. hydrochloride or saline delivered in the VTA just before the foot shock D c Histology Recordings were obtained from the peribrachial pigmented. nucleus of the VTA and marked by ejection of Pontamine Sky Blue white arrow A The location of the injection canulae was verified with tyrosine. hydroxylase staining black arrows B, Intraperitoneal Injections of D1 Agonists Rats either received bilateral injections of bupivacaine. hydrochloride 2 5 dissolved in 0 9 saline solution or. A subset of animals was injected with the D1 antagonist. vehicle 0 9 saline solution only once on their shock day. SCH23390 Sigma prior to the shock procedure Rats either immediately before shock Figure 1bD This type of. received 0 1 mg kg of SCH23390 dissolved in 0 9 saline. reversible local anesthetic has been previously used in the. solution or vehicle 0 9 saline solution only once,inactivation of VTA neurons Mahmoodi et al 2011.
immediately before shock Figure 1bC All other phases. Moaddab et al 2009 Seip and Morrell 2009 All injections. of the experiments were identical, were done in awake rats with the internal cannulae. extending 1 mm beyond the tip of the guide cannula and. were performed using a microliter Hamilton syringe. Intracerebral Cannulation and Injections Hamilton Reno NV attached to flexible polyethylene. tubing See Supplementary Methods for a detailed descrip. A group of animals underwent cannulation surgery target tion of spread and mechanisms of action of bupivicaine. ing the peribrachial pigmented nucleus in the VTA Nair. Roberts et al 2008 Rats were anesthetized with isoflurane. Acute In Vivo Electrophysiology, and their body temperature was maintained at 37 1C. using a temperature controlled heating pad Two stainless After all behavioral tests were completed chloral hydrate. steel guide cannulae plastic one were stereotaxically 350 mg kg i p 14 rats was used for the induction. implanted bilaterally 5 4 mm posterior to bregma 0 5 mm and maintenance of anesthesia Cao et al 2010 Supple. lateral to midline and 7 5 mm below the skull Five stainless mental doses of anesthetic 45 mg kg intravenous. steel microscrews were placed into the skull and dental were given when vibrassal movements became evident. cement was used to anchor the cannula guides to the screws Each animal was fitted with a tracheal breathing tube and a. and skull catheter was placed in a lateral tail vein for the intravenous. Neuropsychopharmacology,Involvement of the VTA in PTSD. NS Corral Frias et al, injection of apomorphine Using stereotaxic procedures a days 3 5 and 7 for SRs or days 1 8 and 16 for nociception. small burr hole was drilled in the skull overlying the VTA and touch sensitivity tests as a between factor A signifi. 5 2 5 6 mm posterior to bregma and 0 5 1 mm lateral to cant difference was indicated by po0 05 or po0 01. the midline suture Body temperature was maintained at. 37 38 1C throughout the experiment by a temperature. Localization of Recording Electrodes and Cannulae and. controlled heating pad,Immunohistochemistry, Action potentials were recorded by single barrel glass.
capillary electrodes that were pulled and broken back to a Each rat was perfused transcardially with 0 9 saline and. tip diameter of approximately 1 mm and filled with a fixed with 4 paraformaldehyde in 0 1 M phosphate buffer. solution of 2 Pontamine Sky Blue dye in 0 5 M Na acetate The brain was extracted and cryoprotected overnight in a. The impedance of the electrodes ranged 12 20 MO At the 30 sucrose and 0 02 sodium azide solution Coronal. end of each recording session a DC current 10 mA pulses sections were cut into 50 mm slices on a cryostat The. 10 s on 10 s off for 20 30 min was passed through the electrode tracts were localized and their position in VTA. recording electrode in order to eject the dye which allowed confirmed Figure 1c One out of three slices was stained. for the identification of the location of the recorded cells with Cresyl Violet Another third of the slices was processed. Figure 1c for tyrosine hydroxylase The primary antibody was a rabbit. anti tyrosine hydroxylase from Chemicon 1 10 000 in. Spike Sorting PBS Triton X100 with 3 normal goat serum followed. by a biotinylated secondary antibody diluted 1 1000. Action potentials were isolated using Spike 2 Cambridge biotinylated rabbit anti goat Invitrogen Oregon An. Electronic Design UK Using custom written Matlab code avidin biotin peroxidase enzyme complex was prepared. Mathworks the firing and action potential properties of and applied according to the manufacturer s instructions. VTA cells were determined Putative DA and GABA neurons Vectastain Elite ABC kit Finally sections were incubated. were defined by the mean firing rate as well as the shape of for 5 min in a DAB hydrogen peroxide substrate solution. the extracellular spike waveform Supplementary Material Sigma Aldrich Sections were mounted with cytoseal. Supplementary Figures S2 S4 The mean activity was Stephens Scientific and coverslipped. calculated using 5 min time windows A burst was defined. by a minimum of three successive spikes having an initial. interspike interval ISI p80 ms and ending with an ISI RESULTS. 4160 ms Grace and Bunney 1984 Cells were classified as Validation of The Rat Model of PTSD. putative dopaminergic on the basis of their firing rate and. spike waveforms Supplementary Figures S2 S4 and SRs In order to mimic the condition. Model of Post Traumatic Stress Disorder Nadia S Corral Frias1 Ryan P Lahood2 Kimberly E Edelman Vogelsang3 2007 High comorbidity of PTSD with drug addiction Kofoed et al 1993 Najavits et al 1997 and evidence showing deficits in the brain reward and reinforcement circuits in PTSD patients Elman et al 2009 Hopper et al 2008 also suggest the involvement of dopaminergic systems

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