Innovations In Clinical Neuroscience

JAN-FEB 2017

A peer-reviewed, evidence-based journal for clinicians in the field of neuroscience

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[ V O L U M E 1 4 , N U M B E R 1 – 2 , J A N U A R Y – F E B R U A R Y 2 0 1 7 ] Innovations in CLINICAL NEUROSCIENCE 45 camera. After removal of each animal, t he maze was cleaned with a wet cloth and wiped dry. The behaviors of the mice recorded on videotapes were later scored by a trained observer blinded to the treatments. The number of entries a nd the amount of time spent in each zone was recorded. Behavioral measures included percent time spent in open arms (% open time) expressed as a percentage of total time on both open and enclosed arms. Arm entry was defined as all four paws having crossed the dividing line between an arm and the central area. This test was repeated on 12th day after MDMA gavage for recording its effects during recovery period. 52,53 Open field. Locomotor activity was assessed by the open field test (OFT). The recording area was limited by gray Plexiglas (40cm×40cm×40cm). A painted grid divided the Plexiglas floor into 16 identical squares each measuring 10cm×10cm. The Plexiglas floor was located on a table 50cm above the floor, and was illuminated by three white 40- watt fluorescent indirect and homogenous lamps. Black curtains surrounded the recording area, and behavior was monitored using an overhead video camera. Following gavage of MDMA, the mice were immediately placed in the open field and behaviors were monitored for 60 minutes by the experimenter. 54 Animals were re-tested on ninth day post-gavage to assess effects on locomotor activity during recovery period. 55 Mice were monitored for frequency of line-crossing and rearing behaviors. Frequency of these behaviors was assessed in the peripheral zone (defined as the 12 squares directly adjacent to the walls) and central zone (all remaining squares). 2 Tissue fixation and Nissl staining. After the second behavioral assessment, the mice were anesthetized with ketamine 100mg/kg and xylesin 10mg/kg i.p and perfused transcardially with 0.9% saline, followed by 10% buffered formalin. Brains were immediately removed and post-fixed in the same fixative overnight at 40ºC. The brains were then embedded in paraffin. They were coronally sectioned 30 m thick and stained with Nissl stain (Cresyl fast violet). The sections containing the hippocampus from the anterior to posterior parts were taken according to the mouse brain atlas of Paxinos. 56 Cell count of the hippocampal CA1 neurons was then undertaken using stereology. Stereological analysis. The numerical density of the hippocampal CA1 neurons was estimated using the optical dissector technique. 57,58 The optical dissector setting included an Eclipse microscope (E200, Nikon, Tokyo, Japan) with a high-numerical- aperture (NA=1.25)×100 oil-immersion objective with a video camera that was connected to a monitor and an electronic microcator with digital readout (MT12, Heidnehain, Traunreut, Germany) for measuring the movements in the Z-direction with 0.5- m precision. A computer-generated counting frame was superimposed on the screen using a stereology software system (Stereolite, SUMS, Shiraz, Iran). The neuronal density was defined with following formula: NV=ΣQ/[ΣP×a(f)×h], where ΣQ is the number of neurons counted within the sampling volume, ΣP is number of dissector, a(f)=0.0033mm 2 is the area of the sampling frame, and h=0.01mm is the height of the dissector. 5 9 Neuronal nuclei were counted in approximately 600 (±1) of the sections evaluated during the entire experiment, counting all the groups and 20 (± 5) optical dissectors per hippocampus in each animal, providing the mean the coefficient of error (CE) on the estimates of total number of eight percent. Cells were identified as neurons if they had a nucleolus, dendritic processes, euchromatin material within the nucleus, and nuclei surrounded by cytoplasm. 60,61 This study was based on a systematic, uniform, random sampling that allowed unbiased and efficient estimates of all parameters under study to be obtained. Cell counts were performed in the CA1 region of the hippocampus on both sides. Chemicals. All the chemicals were purchased from Sigma Chemical Company unless otherwise mentioned. Statistical analysis. Data are expressed as the mean ± standard error of the mean (SEM). Statistical significance was determined by pairwise comparison with one-way ANOVA FIGURE 8. Numerical density (neuron/mm 3 ) of hippocampal CA1 neurons of AL-AL, AL-IF, IF-IF and IF-AL subgroups. Ad libitum (AL), Intermittent feeding (IF), Control (C).(Analysis was done by one-way ANOVA; significance was determined by post hoc Tukey test). #Significant difference between the AL-AL and AL-IF subgroups (20 and 60 mg/kg).(### p < 0.001) ★Significant difference between the AL C and AL-AL subgroups (20 and 60 mg/kg).(p< 0.05)

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