Open Journal of Social Sciences, 2014, 2, 86-89
Published Online May 2014 in SciRes. http://www.scirp.org/journal/jss
How to cite this paper: Xu, X. and Zmolek, K. (2014) Nitric Oxide Scavenger Carboxy-PTIO Impaired Memory of Avoidance
Conditioning in Goldfish. Open Journal of Social Sciences, 2, 86-89. http://dx.doi.org/10.4236/jss.2014.25017
Nitric Oxide Scavenger Carboxy-PTIO
Impaired Memory of Avoidance
Conditioning in Goldfish
X. Xu, K. Zmolek
Department of Psychology, Grand Valley State University, Allendale, MI 49401, USA
Received January 2014
The long-term potentiation (LTP), an activity-induced increase in the efficacy of neurotransmis-
sion, has long been conceived to be a physiological correlate of learning and memory. Investiga-
tions of synaptic transmission suggest that the postsynaptic N-methyl-D-aspartate (NMDA) recep-
tor is the upstream mediator of LTP, while nitric oxide (NO), a retrograde messenger from post-
synaptic neurons to presynaptic neuron, is the downstream mediator of LTP. Our previous studies
showed that microinjections of NMDA receptor antagonist D-AP5 to the goldfish telencephalon
prior to trainings impaired learning of avoidance conditioning in goldfish. However, microinjec-
tions of D-AP5 to the goldfish telencephalon immediately following trainings did not impair mem-
ory of avoidance conditioning. C arboxy -PTIO is a NO scavenger that prevents NO from reaching the
presynaptic neurons. The present study investigated the effects of microinjections of carboxy-
PTIO to the goldfish telencephalon immediately following trainings on avoidance conditioning.
The results showed that microinjections of carboxy-PTIO to the goldfish telencephalon imme-
diately following trainings impaired memory of avoidance conditioning.
Avoidance Conditioning, Memory Consolidation, Nitric oxide, Goldfi sh
Several neurochemical steps of long-term potentiation (LTP), an activity-induced increase in the efficacy of
neurotransmission, have been identified. Investigations of synaptic transmission show that the neurochemical
steps involved in the LTP initially require activation of postsynaptic receptors known as N-methy l -D-aspartate
(NMDA) receptors . Activation of NMDA receptors leads to the influx of Ca2+ into postsynaptic neurons .
The influx of Ca2+ into postsynaptic neurons activates the nitric oxide synthase (NOS) enzyme that leads to the
synthesis of nitric oxide (NO), a retrograde messenger carrying signals backward from postsynaptic to presy-
naptic neurons . NO then triggers further biochemical reactions that lead to LTP . Thus, the NMDA recep-
tor is the upstream mediator of LTP, while NO is the downstream mediator of LTP .
X. Xu, K. Zmolek
LTP has long been conceived to be a physiological correlate of learning and memory . Our previous stu-
dies showed that NMDA receptor antagonists impaired learning when given before training, but did not impair
memory when given after training  . Furthermore, microinjections of NMDA receptor antagonist D-AP5 to
the goldfish telencephalon prior to trainings impaired learning of active avoidance conditioning through its inte-
raction with telencephalic NMDA receptors in goldfish . However, microinjections of D-AP5 to the goldfish
telencephalon immediately following trainings did not impair memory of avoidance conditioning  Carboxy-
PTIO is a NO scavenger that prevents NO from reaching the presynaptic neurons. The present study investigated
the effects of microinjections of carboxy-PTIO to the goldfish telencephalon immediately following trainings on
active avoidance conditioning.
2.1. Subjects and Experimental Drug
Goldfish (Carassius auratus L.), which were 8 - 10 cm in length and obtained from local pet stores, were kept in
large tanks for several weeks prior to experiments. During experiments, fish were kept in individual compart-
ments of partitioned tanks at 20˚C ± 1˚C with a 12 h light-dark cycle (0700-1900 light). Experiments were con-
ducted during the light cycle. 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt
(carboxy-PTIO) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in saline.
The goldfish telencephalon is located at the anterior end of the head and right beneath the skull. The skull of the
goldfish has characteristic sutures that are visible without removing any tissue. Because the skull of the goldfish
used in the study was easy to penetrate with a needle without harming the goldfish, a 26-gauge needle with a
rubber stopper making the length of needle 2 mm was used to make needle holes in the skull relative to the su-
tures. The punctures were made on line with the posterior margins of the iris of the eyes and 0.5 mm lateral to
the midline bilaterally. A 10 μl Hamilton syringe with a 7 mm length needle was used to make injections
through the punctures at 7 mm ventral to the top of the skull. The injection was 0.5 μl per side and 1 μl total per
fish. Repeated injections were made through the same punctures and at the same depth, therefore to the same
location in the brain. The accuracy of the placement of the microinjection was determined by injecting methy-
lene blue dye through the same punctures and at the same depth at the end of testing. Fish were sacrificed with
an overdose of tricaine and the skull of fish was then removed to verify the location of microinjections. Fish in
which both sides of telencephalon were dyed blue were retained in the study.
Fish were trained and tested individually in three identical fish shuttle-boxes connected to a Smart Control (Med
Associates Inc., Albans, Vermont). The fish shuttle-box consisted of a water-filled tank (41 cm in length × 20.5
cm in width × 10.5 cm in height) separated by an opaque barrier (20.5 cm in width × 10.5 cm in height) into two
equal compartments. A rectangular opening (8 cm in width × 2.5 cm in height) in the barrier allowed fish to
swim freely from one side of the tank to the other. The crossing movement of the fish was monitored by four
infrared light beams and their corresponding detectors located on the long sides of the tank. There was a light
(75W, 125V, DC) at each end of the tank and there were two stainless steel electrode plates (18 cm in length ×
19.5 cm in width) at the top and bottom of each compartment. The water in fish shuttle-boxes was completely
changed between fish.
2.4. Active Avoidance Paradigm
Fish were placed in the shuttle-boxes for 5 min, and then a trial began with the onset of the light on the side of
the fish’s location. After the light was on for 20 seconds, a repetitive mild electrical shock (1 V/cm DC, pulsed
200 ms on and 800 ms off) was administered, along with the light, for 20 seconds through the water by means of
electrodes. At the end of 40 seconds or upon a crossing response by fish during the 40 seconds, both the light
and electrical shock was switched off and the trial ended. After an intertrial interval (ITI) ranging from 25 - 55
seconds, another trial began.
X. Xu, K. Zmolek
Fish initially swam through the opening only after receiving several shocks. The crossing response following
the onset of both light signal and electrical shock to escape the electrical bodyshock is defined as an escape re-
sponse. During the training sessions, fish gradually learned to swim from the lighted end to the dark end to avoid
an electric bodyshock. The crossing response following the onset of the light signal but before the onset of elec-
trical shock to avoid the electrical bodyshock is defined as an avoidance response. Fish were trained semiweekly
on Experimental Days 1, 4, and 8. On Experimental Day 11, fish were tested for retention of learning. A training
session consisted of 20 trials, and the testing session consisted of 10 trials. The measurements were the number
of avoidances, escapes and crossings during ITI; and the measurements were then converted into percentages.
To reduce the rate of crossing during ITI, crossing during ITI was immediately followed by a single bodyshock.
All experiments were fully automated through the Smart Control and a single 486 computer that programmed
stimuli, monitored and recorded behavior of fish. Fish that showed ITI crossing 30% or less during training and
testing sessions were retained in the study. Percentage of avoidance responses during the test session was used
as an indicator of retention.
Fish were randomly divided into groups, and a hole was made in their skull bilaterally. Groups of fish received
no injection, microinjection of saline, or microinjection of 10 μg/μl of carboxy-PTIO to their telencephalon im-
mediately following training sessions on Experimental Days 1, 4, and 8. On Experimental Day 11, fish were
tested without injection.
Because there were no differences in avoidance responses during testing between no-injection and saline-in-
jected fish, those fish were combined into one control group. A t test on the percentages of avoidance responses
during testing indicated a significant difference between the control fish and fish that fish received carboxy-
PTIO immediately following trainings. Fish that received carboxy-PTIO displayed significantly fewer avoidance
responses during testing compared to the control fish. The fish that received carboxy-PTIO showed a mean of
12.5% avoidance responses, while the control fish showed a mean of 60% avoidance responses (Figure 1).
4. Discussions & Conclusions
The control group was trained and tested using the same methods as the carboxy-PTIO fish, but received either
no injections or injections with saline. There was no significant difference between fish that received no injec-
Figure 1. Post-training injection of carboxy-PTIO.
X. Xu, K. Zmolek
tions and fish that received saline injections immediately following training. Therefore, neither the microinjec-
tion procedure itself nor the microinjection of saline to the goldfish telencephalon impaired active avoidance
conditioning. Microinjections of carboxy-PTIO immediately following training impaired avoidance conditioning,
suggesting that extracellular NO may be necessary for forming memory of avoidance conditioning in goldfish.
The results of the current study confirm the finding that telencephalic NO is involved in memory consolidation
. Thus, our previous results with D-AP5  and current results with carboxy-PTIO together suggest that the
NMDA receptors are involved in learning or the process that is completed during training, whereas the NO is
involved in memory consolidation or the process that is normally completed sometime following the learning
This work was supported in part by a Grand Valley State University grant-in-aid.
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