ABSTRACT:
Propranolol is a non-selective ß-blocker that has recently proven useful in the
treatment of social anxiety and Post Traumatic Stress Disorder. Recollection of a memory
requires that the memory pathway be strengthened by continual re-visitation. This re¬
visitation requires the activation of B-adrenergic receptors by catecholamines such as
epinephrine and norepinephrine, two important signalers in the fight-or-flight stress
response. Propranolol blocks this activation and thus prevents the strengthening and
maintenance of memory pathways. Most previous studies have utilized either human or
rodent study subjects. Zebra fish provide a unique opportunity to examine an organism
with a different scale of neuro-complexity and a more accessible genome, which could
eventually lead to greater understanding of the coupling between B-receptors and
memory. The fish (Danio rerio) were trained to associate tapping on the glass of their
aquarium with a positive (Tap+Food), negative (Tap-Net), or neutral experience (Tap).
Control fish (No Tap) were not exposed to tapping during the three week training
process. After re-activation of the memory, propranolol was dissolved in the water at a
concentration of 27uM. The anxiety levels and memory retention of the fish were then
tested after 1, 2, 3 and 18 hours. Preliminary data suggests that propranolol has a major
impact on the anxiety levels of the negative, neutral and control fish and may have some
effect on fish receiving the positive reinforcement regime. Additionally, propranolol
decreases the strength and accuracy of spatial memory in fish trained using positive
reinforcement. Propranolol also differently affects paired associative learning and non¬
paired habituation learning, suggesting that these pathways are differently dependent on
B-receptors. This study demonstrates that ß-receptors and memory recall are tightly
coupled throughout vertebrate evolution, despite differences in neuro-complexity. Future
studies should utilize a larger number of replicates to yield statistical significance and a
slightly lower concentration of propranolol as 27uM seems to be at the threshold of
toxicity in these fish.
INTRODUCTION:
Memory is a dynamic process that undergoes constant reorganization and
restructuring. Memories are initially labile and sensitive to disruption and are then
consolidated and stored as long-term memories (Nader 2003). When such a memory is
recalled, or reactivated, it must be reconsolidated via a separate but similar process and
stored in the context of the new situation (Lee et al 2004). Reconsolidation strengthens
memories and must follow reactivation or the memory will be forgotten (Misanin et al
1968, Przybyslawski & Sara 1997
Recent studies have shown that B-adrenergic receptors are an important part of
Long-Term Potentiation, a major pathway implicated in learning and memory (Gelinas &
Nguyen 2005). Propranolol, a lipophilic non-selective ß-blocker, blocks the stimulation
of B-adrenergic receptors by catecholamines such as epinephrine and norepinephrine
(Sigma-Aldrich Co.). These catecholamines also normally function in the fight-or-flight
response caused by a stressful physical or environmental stimulus.
In rats, treatment with propranolol following reactivation of an emotional or non¬
emotional memory caused memory impairment (Przybyslawski et al 1999). This post¬
reactivation treatment was a more effective memory dampener than propranolol treatment
immediately after initial training, suggesting that ß-receptors are more important in
reconsolidation than consolidation in rats. Similar studies have shown that giving humans
propranolol immediately after experiencing or recalling a traumatic experience has
memory dampening effects (Pitman et al 2002, Brunet et al 2008). Additionally,
propranolol is more successful at dampening or blocking emotionally charged memories
(Cahill et al 1994). These characteristics of propranolol make it potentially useful as a
treatment for Post Traumatic Stress Disorder.
Most previous studies have utilized either rodents or humans as model organisms.
In this study, zebra fish (Danio rerio) provide a unique opportunity to examine an
organism with far less complex neurological organization and a more accessible genome.
which could eventually lead to a greater understanding of how the coupling of memory
reconsolidation and ß-receptors functions.
MATERIALS AND METHODS:
Conditioning
Four 2-gallon glass aquaria were set up with five small to medium sized zebra fish
(Danio rerio) in each. One tank was assigned to each of the four conditioning treatments:
Neutral/Tap, Negative/Tap-Net, Positive/Tap-Food or Control. The Tap treatment
consisted of one full minute of tapping on the front of the aquarium during each session.
In the Tap-Net tank, 3 seconds of tapping was immediately followed by a 20 second long
chase with a small net. In the Tap-Food tank, 3 seconds of tapping occurred right before
the fish received a portion of their daily diet. The control tank was not exposed to any
tapping and had no conditioning sessions.
Over the course of 20 days, the fish received up to 7 sessions a day, with at least
30 minutes between each. Due to a high initial mortality rate, new fish were added to
each tank on the 6“ day, bringing the total number of fish in each tank up to seven. Notes
were taken regarding the number of sessions it took for fish to learn their behavior. A
learned behavior was defined as correctly targeting to the front of the tank three sessions
in a row.
Water chemistry and testing of memory and anxiety
Each tank was buffered with a 2mM solution of HEPES sodium salt (minimum
99.5%). On the morning of the 21“ day, the side of each tank received five seconds of
tapping to reactivate the memory. Observations were made regarding the amount of time
it took the fish to return to normal behavior, a proxy for anxiety as well as the percent of
fish that went to the trained location following tapping in the positive and negative tanks.
Then each of the four tanks got 0.05 g of (*)-Propranolol hydrochloride
(minimum 99%) added to the water for a concentration of approximately 27 uM. This
concentration was determined based on previous tests of Propranolol concentration and a
literature search. Then the fish were again tested on their behaviors after 1, 2, 3, and 18
hours using the process described above.
RESULTS:
The effects of propranolol on memory
In the positive treatment, propranolol decreased not only the ability of the fish to
go to their trained location but the accuracy with which they did it (Fig 1). Before the
administration of the drug, 100% of the fish swam to the top left corner of the tank
following tapping. This quickly dropped off following exposure to propranolol and
between 1 and 3 hours only 70-85% of the fish went to the front of the tank. Although the
graph shows that 100% of the fish swam to the correct location after 18 hours, they did so
with less spatial accuracy than they had before the administration of the drug. They did
not all swim to the top left corner of the tank but were instead spread out in the front half
of the tank.
The negative treatment did not produce a clear pattern following treatment with
propranolol. For the first two hours after propranolol administration, fewer fish were
entering the front half of the tank. Then after three hours, the fish returned to their pre-
exposure levels (Fig 1). It took the negatively reinforced fish significantly longer to
initially learn their behavior (p20.03, Fig 2) and the highest level of accuracy achieved
during the experiment was only 67%, which is 3% below the lowest levels achieved by
the fish with the positive experience.
Additionally, the fish in the negative treatment tank (as well as in the neutral/Tap
tank and the control/No Tap tank) were too disoriented at 18 hours to be tested again,
possibly indicating an overdose. Interestingly, these fish showed no signs of propranolol
overdose during the first 3 hours. After 68 hours, they were nearly fully recovered but
had seemingly forgotten how they are normally fed. Instead of rushing up to the surface
when food was dropped in (as they had previously done and as the positive treatment fish
did), they allowed the food to drop to the bottom before consuming it.
The effects of propranolol on anxiety
The amount of time it took the fish to return to their normal behavior following
tapping on their tanks was used as a proxy for anxiety levels. The control tank shows an
initial increase in anxiety following tapping and propranolol exposure (Fig 3A). After 3
hours, this anxiety decreases dramatically. The neutral fish initially had very low anxiety
levels and almost completely ignored the tapping. This lasted for the first two hours but at
three hours the anxiety level increased dramatically (Fig 3A).
The positive fish showed only a slight increase in anxiety following exposure to
propranolol (Fig 3B). The negative fish had the highest anxiety levels before propranolol
exposure, which is to be expected based on their treatment. Following drug
administration, there was an immediate and lasting drop in anxiety (Fig 3B)
DISCUSSION:
Propranolol and memory
Post-reactivation administration of propranolol decreased the intensity and
accuracy of the memory of the learned behavior in the positive fish, thus showing that
propranolol can have an effect on the retention of non-emotional memories. It is difficult
to determine from these data if the effect is stronger or weaker than an emotional memory
such as that of the negative fish due to their low levels of learning.
As previously mentioned, propranolol has the ability to block both emotional and
non-emotional memories in rats (Przybyslawski et al 1999). Because propranolol also
seems to dampen non-emotional memories in zebra fish, it is likely that the coupling of
ß-adrenergic receptors and the reconsolidation of emotional memories has been
conserved throughout vertebrate evolution. Further experimentation is required to
examine the effect of propranolol on an emotional memory in zebra fish.
Propranolol and anxiety
The negative fish show changing anxiety levels most consistent with literature on
propranolol and its potential use as a medication for Post Traumatic Stress Disorder.
Following administration of propranolol, the stress response was greatly reduced,
possibly indicating a disruption of reconsolidation. A similar disruption of