Elsevier

Neurotoxicology and Teratology

Volume 25, Issue 4, July–August 2003, Pages 419-426
Neurotoxicology and Teratology

Learning impairment caused by a toxin produced by Pfiesteria piscicida infused into the hippocampus of rats

https://doi.org/10.1016/S0892-0362(03)00011-4Get rights and content

Abstract

Pfiesteria piscicida, an estuarine dinoflagellate, which has been shown to kill fish, has also been associated with neurocognitive deficits in humans. With a rat model, we have demonstrated the cause-and-effect relationship between Pfiesteria exposure and learning impairment. In several studies, we have replicated the finding in Sprague–Dawley rats that exposure to fixed acute doses of Pfiesteria cells or filtrates caused radial-arm maze learning impairment. Recently, this finding of Pfiesteria-induced learning impairment in rats has been independently replicated in another laboratory as well. We have demonstrated significant Pfiesteria-induced learning impairment in both the win-shift and repeated-acquisition tasks in the radial-arm maze and in reversal learning in a visual operant signal detection task. These learning impairments have been seen as long as 10 weeks after a single acute exposure to Pfiesteria. In the current study, we used a hydrophilic toxin isolated from clonal P. piscicida cultures (PfTx) and tested its effect when applied locally to the ventral hippocampus on repeated acquisition of rats in the radial-arm maze. Toxin exposure impaired choice accuracy in the radial-arm maze repeated acquisition procedure. The PfTx-induced impairment was seen at the beginning of the session and the early learning deficit was persistent across 6 weeks of testing after a single administration of the toxin. Eventually, with enough practice, in each session, the PfTx-exposed rats did learn that session's problem as did control rats. This model has demonstrated the cause-and-effect relationship between exposure to a hydrophilic toxin produced by P. piscicida and learning impairment, and specifically that the ventral hippocampus was critically involved.

Introduction

Pfiesteria piscicida Steidinger and Burkholder, a toxic dinoflagellate, has been associated with mass fish kills in estuaries of the East Coast of North America [3], [4], [12], [26]. Adverse health effects were found in humans after accidental exposure to P. piscicida cultures in laboratory facilities, with a hallmark of cognitive disturbance [13], [32]. Cognitive deficits were also seen in a cohort of people exposed to Pfiesteria in field conditions in Maryland [15]. The syndrome of adverse health effects has been termed possible estuary-associated syndrome (PEAS) by the Centers for Disease Control and Prevention [5], [6]. PEAS includes a variety of impairments including cognitive deficits, a deficit in visual contrast sensitivity, pulmonary impairment, gastrointestinal disruptions, and immunologic dysfunction [16], [17], [32], [33], [35]. In a series of controlled laboratory experimental studies, we demonstrated, using a rat model, a cause-and-effect relationship between Pfiesteria exposure and persisting learning impairment (for a review, see Ref. [22]). We have used this model to help identify a critical toxin and its action in a specific brain region underlying Pfiesteria-induced learning impairment.

We have conducted a series of studies to characterize P. piscicida exposure effects on cognitive performance in the radial-arm maze [21], [23], [25], in which learning impairments resulted from acute exposure to Pfiesteria by injection before the onset of radial-arm maze training. Subcutaneous exposure to P. piscicida cells or sterile filtrates in which cells had been removed caused significant impairments in learning behavior. These studies were the foundation for the rat model of Pfiesteria-induced cognitive impairment [22]. The Pfiesteria-treated rats were significantly delayed but were not incapable of learning. The Pfiesteria-exposed rats improved during the additional phase of training such that they overcame the significant deficits seen earlier. The delayed acquisition was not a demonstration of recovery because additional training was required. With true recovery, training on a new task would proceed at the same rate as controls. Recently, Duncan et al. [8] found a very similar radial-arm maze learning impairment in rats after exposure to P. piscicida. They extended this finding with a study showing long-lasting learning impairments caused by exposure to the similar species, P. shumaywae. In the current study, we used the repeated acquisition procedure to examine the persistence of the learning impairment with new learning being repeatedly tested over 6 weeks after acute exposure to Pfiesteria extracts.

In this rat model of Pfiesteria-induced cognitive impairment, the deficits were relatively selective to learning function. Subtle effects were seen with tests of exploratory behavior and locomotion [22]; however, memory and attention did not seem to be significantly affected by acute Pfiesteria exposure in this model. When Pfiesteria was administered after rats were pretrained on the radial-arm maze, no deficit in memory was observed. To verify the effect on learning, Pfiesteria-treated rats were subsequently given a new task to learn, repeated acquisition, where they showed a significant deficit. The learning impairment was evident at least 10 weeks after a single exposure. Because the same sensorimotor, motivational, and working memory functions were required in the memory task, which was not significantly affected, this result supported the existence of a relatively selective Pfiesteria effect on learning. In a recent study, we found that Pfiesteria exposure did not significantly affect attentional performance [30]. Rats were trained before Pfiesteria exposure on a demanding signal detection task measuring sustained attention. No effect of Pfiesteria exposure was seen on maintenance of choice accuracy in this task. However, when the response contingencies were switched (i.e., the correct response lever when the visual signal was presented was switched from the left to the right, or visa versa), there was a significant Pfiesteria-induced impairment. All the rats had to relearn the task and the Pfiesteria-exposed rats had significantly more difficulty than controls in this relearning. Again, acute Pfiesteria exposure in rats was found to significantly impair learning without significant effects on another cognitive function, in this case attention.

It is important to keep in mind that, to date, this model has only tested the consequences of acute Pfiesteria exposure. More extensive chronic Pfiesteria exposure may result in more extensive neurobehavioral impairment, as has been seen in people exposed in accidental laboratory and field exposures [13], [15], [32], [34], [35]. Other physiological systems, in addition to the nervous system and immune and respiratory functions, have been shown to be affected in humans. Additional future development of the rodent model would be needed to encompass these systems. Currently, we are delving further into the neurocognitive aspects of Pfiesteria intoxication to help determine the mechanisms of its effects.

With the current series of studies, we have developed a reliable model of the neurocognitive deficits caused by Pfiesteria exposure. The work presented here breaks new ground by testing a hydrophilic toxin [9], [19], [27] isolated from clonal P. piscicida cells cultured in the absence of fish (PfTx). The specific neural systems involved in the learning impairment in rats caused by Pfiesteria administration in previous studies were examined in the current study by direct local bilateral infusion of PfTx into the ventral hippocampus.

Section snippets

Toxin preparation

The toxin used for the current study was isolated and partially purified from clean, clonal cultures of toxic flagellated stages of P. piscicida, in the absence of fish or other microbes except for benign axenic algal (cryptomonad) prey. The P. piscicida cultures were provided by the Center for Applied Aquatic Ecology, North Carolina State University (laboratory of Drs. JoAnn Burkholder and Howard Glasgow). The cultures had been cloned following flow cytometric techniques to characterize the

Repeated acquisition

Bilateral infusion of a hydrophilic toxin produced by P. piscicida (PfTx) into the ventral hippocampus of rats caused persisting learning impairment. Overall, there was a highly significant main effect of Trial [F(4,48)=7.82, P<.0001] reflecting the learning that took place over the course of each session. Both control and PfTx-treated groups showed learning; however, the toxin-treated group learned more slowly. There was a significant Pfiesteria×Trial interaction [F(4,48)=3.78, P<.01]. The

Discussion

Acute infusion into the ventral hippocampus of a hydrophilic toxin (PfTx) produced by P. piscicida caused significant persisting impairment in the learning of rats in the repeated acquisition procedure on the radial-arm maze. PfTx selectively impaired the early phase of learning as shown by the significant toxin-induced accuracy impairment on the first two trials of the five-trial session. PfTx-treated rats caught up with controls during the later part of the session to eventually learn the

Acknowledgements

The authors thank Jennifer Blackwelder (Duke University) for her help with radial-arm maze testing and Stacie Dover (NOAA) for calibration of the hydrophilic toxin from P. piscicida. This research was supported by the National Oceanic and Atmospheric Administration, Duke University, an anonymous foundation, the North Carolina General Assembly, and the Department of Botany at North Carolina State University.

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    This work was funded by the National Oceanic and Atmospheric Administration National Ocean Service (NOAA-NOS). The NOS does not approve, recommend, or endorse any proprietary product or material mentioned in this publication. No reference shall be made to NOS, or to this publication furnished by NOS, in any advertising or sales promotion which would indicate or imply that NOS approves, recommends, or endorses any proprietary product or proprietary material mentioned herein, or which has as its purpose any intent to cause directly or indirectly the advertised product to be used or purchased because of NOS publication.

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