Effects of Various Doses of Methylene Blue on Radial Maze Acquisition in Rats

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Effects of Various Doses of Methylene Blue on
Radial Maze Acquisition in Rats

• Fiorucci, J., Castrillon, N., Rodriguez, I.,
  Khan, S., Rodriguez, M., Antonakopoulos, A.,
  Villegas, F.
• Queensborough Community College, Department of
  Biology, Research Initiative for Minority
  Students Program, Bayside, NY 11364 York
  College of the City University of New York,
  Department of Psychology, Behavioral Neuroscience
  Laboratory, Jamaica, NY 11435

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Abstract

• Methylene Blue (MB) has been shown to enhance
  memory of inhibitory avoidance (Martinez et al.,
  1978) and improves reference memory and reversal
  in a Hole Board Maze test in rats (Callaway et
  al., 2001, 2004). MB enhances brain energy
  metabolism when administered in low doses. The
  aim of this study is to evaluate the effects of
  various dosages of MB on spatial memory test.
  Methods Thirty-two male Long-Evans rats were
  used in this study. Prior to habituation training
  (HT) procedures rats were brought down to 85 of
  their body weight. After five days of HT animals
  were randomly assigned to one of eight
  experimental groups (with four animals per
  group) saline, 0.5, 1.0, 3.0, 4.0, 5.0, 6.0 and
  8.0 mg/kg. Following the HT the animals were
  tested using a delayed non-matching-to- sample
  (DNMTS) procedure for 20 consecutive days. During
  the pre-delay session, four of the eight radial
  arms were blocked with removable doors and the
  remaining four were baited with food reward
  pellets. After entering all four open arms the
  rat was returned to its home cage for a
  five-minute delay. During the post-delay
  session, the rats were returned to the maze and
  all eight arms were open. However, only those
  arms that were blocked during the pre-delay
  session were baited with food pellets. After the
  completion of every trial (Pre-delay and
  Post-delay), rats were injected intraperitoneally
  with their corresponding dose. Results and
  Conclusion The results of our study show that
  all dosage groups had increases in total number
  of mean errors, retroactive and proactive, in a
  dose dependent manner when compared to saline.

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Introduction

• Nootropic Drugs a class of drugs that act as
  cognitive enhancers.
• MB is used to treat Methemoglobinemia (condition
  when the blood cannot deliver oxygen where it is
  needed in the body) (Spahr et al., 1980).
• Methylene Blue (MB) enhances energy metabolism
  in the brain when administered in low
  doses(Visarius et al., 1997).
• MB has been shown to enhance memory in inhibitory
  avoidance in mice and rats (Martinez et al.,
  1978).
• MB has been shown to improve reference memory and
  reversal in the Hole Board Maze test in rats
  (Callaway et al., 2001 2004).
• The aim of this study was to evaluate the effects
  of various dosages of MB on a spatial 8-arm
  radial maze memory test.

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Research Methods

• Subjects
• Thirty- two male Long-Evans Rats, weighing
  approximately 250mg at the start
• of the experiment, are housed two to a cage.
• Animals were maintained on a light-dark cycle
  (12hr on and 12 hr off). Animals
• are tested during the hours of 8am to 12pm in the
  light cycle.
• Prior to the habituation training (HT) procedures
  the animals are food deprived
• and maintained at 85 of their body weight.
  One-week prior to HT
• procedures, the rats are handled daily for five
  days.
• After the HT procedures, animals are randomly
  assigned to one of eight
• experimental groups (saline (n4), 0.5mg/kg
  (n4), 1.0mg/kg, (n4),
• 3.0mg/kg (n4), 4.0mg/kg (n4), 5.0mg/kg (n4),
  6.0mg/kg (n4), and
• 8.0mg/kg (n4).

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• Apparatus
• 8-Arm Radial Maze a maze with an octagonal
  center platform (62cm
• diameter) with eight arms (70cm long and 10cm
  wide) radiating from the
• platform and elevated 110cm above the floor (Fig.
  1).
• Each arm was framed by clear Plexiglas side rails
  and had a submerged cup
• containing a 94mg chocolate-flavored food pellet
  as a reward.
• Removable doors (10cm x 15cm) made of clear
  Plexiglas are used to block
• entrance of arms during the pre-delay sessions of
  the delayed non-matching-
• to-sample (DNMTS) procedures.
• The radial maze was located in a room equipped
  with overhead fluorescent
• lighting and with distinctive visual cues through
  out the room.

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• SMART Video Tracking System
• SMART is a video tracking system that allows the
  experimenter to visually track
• the rats traversing on the 8-arm radial maze.
  This is done via a video camera,
• located in the ceiling above the maze, and the
  information is then stored onto a
• computer hard drive.
• SMART allows the experimenter to define zones
  (arms and center platform).
• SMART provides the experimenter with speed of
  animal, distance traveled,
• number of entrances to zones, length of time
  spent in each zone, and time of
• trial run. SMART allows the experimenter to
  access recordings of the animals
• path on the maze.
• Data Collection
• Data were collected by using the SMART video
  tracking system along with data
• sheets for manual tracking. The data sheets had
  templates of the 8-arm radial
• maze. In order to manually track the rat the
  experimenter viewed the rat
• traversing the maze using the SMART system and a
  computer monitor.

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• Preliminary Habituation Training Procedures
• The animals are handled for 5 consecutive days
  prior to the habituation training
• (HT) procedures. During HT procedures rats were
  allowed to explore the radial
• maze for a 5-min period for 5 days. Following HT
  procedures, the rats were
• tested using a standard DNMTS procedure on the
  Spatial Memory Acquisition
• Task for 20 consecutive days.
• Using shaping techniques, food pellets were first
  placed on the center platform
• near door openings and on the arms leading to the
  food cups. As daily
• sessions proceeded the food pellets were removed
  from the center platform
• and placed closer to the food cups.

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• MB Treatment Spatial Memory Task Acquisition
• Before the daily DNMTS trails rats were weighed.
• The eight radial arms were baited with food
  pellets. The pre-delay session
• consisted of four of the arms being randomly
  blocked with removable doors
• and rats were placed in the center platform to
  enter the open arms (Fig. 2).
• After entering all 4 open arms to retrieve the
  food pellet rewards the rats were
• returned to their home cage for a 5-min delay.
• During the post-delay session, rats were returned
  to the maze where all 8
• arms were opened. However, only those arms that
  were blocked during the
• pre-delay session allowed rats to obtain food
  reward pellets.
• Following the daily DNMTS trials rats were
  injected with Methylene Blue IP.
• Task acquisition performance involved the
  subjects retaining information about
• the pre-delay configuration of open and closed
  arms in reference to external

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• Dependent Measure and Analysis
• During the pre-delay and post-delay session of
  the DNMTS procedure number
• of entries, re-entries, and latency times were
  recorded.
• Post-delay errors (dependent measure) were scored
  as follows
• 1-Retroactive errors (RE) occur when the rat
  searched an arm from which food
• had been obtained in the pre-delay session.
• AND
• 2-Proactive errors (PE) occur when the rat
  searched an arm that had already
• been visited during the post-delay session.
• The data were analyzed using a one-way analysis
  of variance (ANOVA).

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Results

• Descriptive statistics are presented in Table 1
  for each of the dependent
• measures (number of proactive errors, number of
  retroactive errors, and
• number of total errors) and the eight dosage
  groups.
• Table 2 consists of the computed one-way ANOVAs
  for the dependent
• measures and the dosage groups. Significant
  differences were found between
• groups for the following number of proactive
  errors (F(7,575) 2.38, p lt .021),
• number of retroactive errors (F(7,575) 4.33, p
  lt .00), and for total number of
• errors (F(7,575) 4.47, p lt .00).
• Graph 1 illustrates the total number of errors
  (proactive errors retroactive
• errors) over the course of twenty days for the
  groups.
• Tukeys HSD (Honestly Significant Difference)
  post hoc test was used to determine
• the nature of the differences between the dose
  groups. When comparing
• proactive errors, Tukeys revealed no significant
  differences. Tukeys did find
• differences for retroactive errors and for total
  number of errors between the
• groups.

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Figure 1. 8-arm Radial Maze and Visual Cues
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Table 1 Descriptive statistics are presented for
Number of Proactive Errors, Number of Retroactive
Errors, and Total Number of Errors for saline and
seven doses of methylene blue.
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Table 2 Computed one-way ANOVAs are presented for
the dependent measures saline and seven dosage
groups.
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• Graph 1 Total number of errors is presented for
  saline and seven dosage groups
• during the course of the experiment.

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Discussion

• Our studys aim was to evaluate the effects of
  various dosages of MB on a
• spatial memory test. The results indicate that
  no differences were found
• between dosage groups in proactive errors.
  However, significant differences
• were found in retroactive errors made by rats
  receiving the two highest doses
• of MB when compared to saline and 0.5 mg/kg MB.
  Also rats receiving the 8.0
• mg/kg MB versus 1.0mg/kg MB made significant
  increases of retroactive errors.
• Our results showed that all dose groups of MB had
  no memory enhancing
• effect when compared to saline, but significant
  memory deficits were found in
• animals receiving the highest doses of MB (6.0
  and 8.0mg/kg) when compared
• to all other groups.
• These effects may be explained by the study of
  Peter et al., 2000, who found
• that a progressive dose of 10 mg/kg accumulates
  in the brain and reaches a
• concentration of over 10x greater in the brain
  than in the blood. Doses as
• large as 50 mg/kg were found to be detrimental
  and lead to methemoglobin
• formation when tested in mice (Martinez et al.,
  1978).
• In future studies we would like to test lower
  dosages of Methylene Blue such as
• 0.12 and 0.25 mg/kg in order to evaluate memory
  enhancing effects when
• compared to saline.

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