Catherine L. Ryan

Effectiveness of PSD95 Inhibitors in Permanent and Transient Focal Ischemia in the Rat

Background and Purpose— Postsynaptic density-95 inhibitors reduce ischemic brain damage without inhibiting excitatory neurotransmission, circumventing the negative consequences of glutamatergic inhibition. However, their efficacy in permanent ischemia and in providing permanent neuroprotection and neurobehavioral improvement in a practical therapeutic window is unproven. These were tested here under conditions that included fever, which is a common occurrence in clinical stroke. Methods— Six studies were performed in unfasted Sprague-Dawley rats. Two involved permanent pial vessel occlusion in male and female rats. Two involved permanent middle cerebral artery occlusion, which induced severe hyperthermia, and 2 involved transient middle cerebral artery occlusion. Animals were treated with a single intravenous injection of postsynaptic density-95 inhibitors (Tat-NR2B9c[SDV] or Tat-NR2B9c[TDV]) 1 hour or 3 hours after stroke. Infarct volumes and neurobehavior were assessed in a blinded manner at 24 hours (pial vessel occlusion and permanent middle cerebral artery occlusion) or at 62 days (transient middle cerebral artery occlusion). Results— Postsynaptic density-95 inhibitors dramatically reduced infarct size in male and female animals exposed to pial vessel occlusion (>50%), in hyperthermic animals with fever exceeding 39°C exposed to permanent middle cerebral artery occlusion (approximately 50%), and at 62 days poststroke in animals exposed to transient middle cerebral artery occlusion (approximately 80%). Effectiveness of postsynaptic density-95 inhibitors was achieved without the drugs affecting body temperature. In transient middle cerebral artery occlusion, a single dose of postsynaptic density-95 inhibitor given 3 hours after stroke onset permanently maintained reduced infarct size and improved neurobehavior. Conclusions— Postsynaptic density-95 inhibitors administrated 3 hours after stroke onset reduced infarct volumes and improved long-term neurobehavioral functions in a wide therapeutic window. This raises the possibility that they may have future clinical usefulness.

Low doses of domoic acid during postnatal development produce permanent changes in rat behaviour and hippocampal morphology

It is well established that the developing brain is a highly dynamic environment that is susceptible to toxicity produced by a number of pharmacological, chemical and environmental insults. We report herein on permanent behavioural and morphological changes produced by exposing newborn rats to very low (subconvulsive) doses of kainate receptor agonists during a critical window of brain development. Daily treatment of SD rat pups with either 5 or 20 µg/kg of domoic acid (DOM) from postnatal day 8-14 resulted in a permanent and reproducible seizure-like syndrome when animals were exposed to different tests of spatial cognition as adults. Similar results were obtained when animals were treated with equi-efficacious doses of kainic acid (KA; 25 or 100 µg/kg). Treated rats had significant increases in hippocampal mossy fiber staining and reductions in hippocampal cell counts consistent with effects seen in adult rats following acute injections of high doses of kainic acid.In situ hybridization also revealed an elevation in hippocampal brain derived neurotrophic factor (BDNF) mRNA in region CA1 without a corresponding increase in neuropeptide Y (NPY) mRNA. These results provide evidence of long-lasting behavioural and histochemical consequences arising from relatively subtle changes in glutamatergic activity during development, that may be relevant to understanding the aetiology of seizure disorders and other forms of neurological disease.

Comparative behavioural toxicity of domoic acid and kainic acid in neonatal rats

Cumulative behavioural toxicity was measured in groups of male and female rat pups (n=6/sex) at different stages of postnatal development. Dose-response curves (DRCs) for toxicity produced by domoic acid (DOM) were generated using animals on postnatal days (PND) 0, 5, 14, and 22, using a behavioural rating scale. In a subsequent experiment, DRCs for toxicity generated by either DOM or kainic acid were produced in rats at PND 8 and 14 for comparison between the two toxins. DOM was found to be a very potent neurotoxin in newborn rats and the potency of DOM progressively decreased with increasing age (interpolated ED(50)=0.12, 0.15, 0.30, and 1.06 mg/kg at PND 0, 5, 14, and 22, respectively). In addition, the patterns of behavioural expression were found to differ with age. Comparisons between DOM and kainic acid revealed that DOM was approximately six-fold more potent than kainate at both PND 8 and PND 14 and that both toxins were approximately two-fold less potent in PND 14 rats, compared to PND 8. This implies that the mechanism(s) responsible for reduced potency is/are similar between the two compounds. Consistent with previous reports, however, there were both similarities and differences in the observed patterns of behavioural toxicity produced by the two toxins at both ages.

Low doses of non-NMDA glutamate receptor agonists alter neurobehavioural development in the rat

While it is known that glutamate is critical to CNS development and function, less is known about the role of kainate receptors, a subclass of ionotropic glutamate receptors, during ontogeny. This is especially true with respect to the emergence and expression of behaviour. It is also known that the neonatal CNS differs from that of adults with respect to excitatory amino acid (EAA) toxicity. Our aim was to determine the effects of early low-dose stimulation of kainate receptors on physical and behavioural development in the rat. Saline, one of two subtoxic doses of domoic acid (DOM) (5 and 20 microg/kg), or in a parallel study, saline, or one of two pharmacologically equivalent doses of kainic acid (KA) (25 and 100 microg/kg), were systemically administered once daily from postnatal days (PNDs) 8-14. While DOM or KA had no effect on typical measures of toxicity such as weight gain, acoustic startle, ultrasonic vocalizations (UVs), or maternal retrieval, these doses were shown to be physiologically relevant, producing particular group differences in eye opening, conditioned place preference, and activity levels. We conclude that administration of very low doses of selective kainate receptor agonists during the second postnatal week produces changes in neurobehavioural development in the rat.

Persistent changes in learning and memory in rats following neonatal treatment with domoic acid.

The present study examined the effects of neonatal exposure to serial low dose injections of the glutamate agonist, domoic acid (DOM), on learning and memory in two spatial memory tasks in the rat. Neonatal Sprague Dawley rats were given single daily injections of low dose DOM (20 microg/kg) over postnatal days 8-14 and assessed as adolescents and adults in the radial 8-arm maze and the Morris Water Maze, respectively. Our results indicate that the DOM-treated rats showed a complex pattern of lasting alterations in learning and memory performance measures that were task specific. Adolescent DOM-treated animals, regardless of sex, demonstrated superior choice accuracy over seven days of testing in the 8-arm baited version of the radial maze. As adults, these same animals manifested improvements in several performance measures in the water maze. These improvements were also observed in a reversal task. However, when the escape platform was returned to its original position, some regression in search strategies were observed in the DOM-treated animals, especially the females, compared to their saline counterparts. These findings demonstrate that low-dose administration of a selective kainate receptor agonist during critical periods of CNS maturation produces lasting changes in learning and memory in the rat; adding to the ever-expanding body of literature which underscores the importance of optimal glutamatergic signaling to normal neurodevelopmental processes.

Gender-based changes in cognition and emotionality in a new rat model of epilepsy

Summary.Epilepsy research relies heavily on animal models that mimic some, or all, of the clinical symptoms observed. We have previously described a new developmental rat model of epilepsy that demonstrates both behavioural seizures and changes in hippocampal morphology. In the current study we investigated whether these rats also show changes in cognitive performance as measured using the Morris water maze task, and emotionality as measured using the Elevated plus maze task. In the water maze, significant differences between male and female rats were found in several performance variables regardless of treatment. In addition, female but not male rats, treated neonatally with domoic acid had significant impairments in learning new platform locations in the water maze. In the elevated plus maze, a significant proportion of female rats spent more time in the open arm of the maze following prior exposure to the maze whereas this effect was not seen in male rats. We conclude that perinatal treatment with low doses of domoic acid results in significant gender-based changes in cognition and emotionality in adult rats.

Altered responses to novelty and drug reinforcement in adult rats treated neonatally with domoic acid

Activity in the mesocorticolimbic dopamine system is linked to responses to novelty, reward, and drug-seeking behaviours. Glutamate signaling, through kainate receptors, has been shown to modulate dopamine release in this pathway. In the present study, a low, overtly non-convulsive dose of the kainate receptor agonist, domoic acid (DOM), was administered to rat pups over PND 8-14. As juveniles and adolescents, rats were assessed in the open field. During adulthood, rats were tested in an open field, a sucrose consumption task, the playground maze and in a nicotine-induced conditioned place preference paradigm. Domoic acid related effects were found in open field behavior at each time point assessed. Male rats treated neonatally with DOM displayed altered novelty-related behaviour in a novelty trial, as indicated by an increase in time spent exploring familiar objects during the novelty trial of the playground maze. In nicotine-induced conditioned place preference, DOM-treated females developed a conditioned place preference for the nicotine-paired compartment of the test arena, an effect that was maintained for at least a month following the final drug-compartment pairing. The results of this study underscore the importance of the glutamate system in the ontogeny of behaviors that rely on the functional integrity of the midbrain dopamine system.

Altered pre-pulse inhibition in adult rats treated neonatally with domoic acid

Summary.Altered functioning of the glutamate system during critical periods of development is believed to play a role in various neurodevelopmental disorders, such as schizophrenia. Prepulse inhibition (PPI) of the acoustic startle response is deficient in people with schizophrenia. This study investigated the theory that neonatal treatment with domoic acid (DOM), a glutamate agonist, leads to deficient PPI. Results indicate that neonatal treatment with DOM leads to lowered PPI in adult males and an increased startle response in adult females.

NMDA receptor involvement in the effects of low dose domoic acid in neonatal rats.

Summary.We have previously reported that neonatal rats display enhanced sensitivity to domoic acid relative to adults, and that perinatal injections of low doses of domoic acid alter early associational learning in the newborn rat. The current study was designed to further investigate the effects of low dose domoic acid on neonatal odour conditioning and to determine if the observed effects are due in part to an action on NMDA receptors. Groups of rat pups were conditioned to a novel odour on postnatal day (PND) 8, injected with 20 μg/kg domoic acid either alone, or in combination with the NMDA antagonist CPP (or appropriate controls), daily from day 8–14, reexposed to the conditioning odour or a novel odour on day 9, and tested for odour preference on day 13 using a standard 3-choice paradigm. Results indicated that rats treated with domoic acid spent significantly more time over the conditioning odour than did saline-treated rats when tested on PND 13. This effect was antagonized by concomitant injection of CPP, indicating an involvement of NMDA receptors in the actions of DOM in this paradigm. Rats injected with either saline or CPP alone showed the opposite effect, i.e. a preference for the alternate odour. The results indicate that a very low dose of DOM produces a conditioned odour preference in neonatal rats and that this effect is due in part to NMDA receptor involvement, thereby emphasizing a role for both kainate and NMDA glutamate receptors in implicit memory.

Low dose domoic acid in neonatal rats abolishes nicotine induced conditioned place preference during late adolescence

Summary.In this study, neonatal rats were chronically exposed to low, non-convulsive doses of the kainate receptor agonist domoic acid (DOM), or saline. Later, as adolescents, all animals were tested in a nicotine-induced conditioned place preference (CPP) paradigm. As expected, a nicotine-induced CPP was evident in the adolescent control rats, but surprisingly, not in the DOM animals. This study demonstrates the importance of KA receptors in the development of normal adolescent behaviors manifested in response to the rewarding properties of nicotine.