Genetic predisposition

A number of years ago, Ron Racine questioned the possible importance of genetic underpinnings to kindling. Subsequently he embarked on a selective breeding program, which began with a parent mixture of two outbred strains, that showed strong genetic control in the rate of amygdala kindling. Within six generations, there was no overlap in the distribution rates of the Fast versus Slow amygdala kindlers.Shortly after this time, the strains were established at my laboratory, where they continue to display their unique kindling rates despite the relaxation of further selection procedures (Racine et al., 1999).

Over the intervening years, these strains have provided us with a plethora offindings concerning all aspects of kindling and many behavioral attributes that are under genetic control. Moreover, we continue to collaborate with many other researchers to determine the mechanisms underlying their differential seizure predispositions.After considerable accumulation of base data, we have now begun to publish many of the findings derived from these two strains. For example, although LTP and LTD in the perforant pathway are similar in the two strains (Racine et al., 1999), the Slow rats are much "smarter" than Fast rats in all behavioral tasks (including both appetite and fear motivated, as well as several spatial learning variants, e.g., Mohapel & McIntyre, 1998, Anisman et al., 2000). Part of the reason for this learning difference is because the Fast rats show two interesting and important behavioral comorbidities - often reported in epileptic children: impulsivity and an attention deficit disorder (McIntyre & Anisman, 2000). Of course, as there are no genetically-based animal models for these two common human conditions, we continue to sculpt the models with those venues in mind.

Determining the molecular mechanisms that differentiate these two strains currently is our biggest challenge. We have eliminated several potential differences between the strains in several transmitter systems, including catecholamine, cholinergic and glutaminergic systems. However, one large difference is evident, which relates to GABAergic mechanisms. We have shown that the Fast rats maintain high expression of the embryonic GABAA subunits (including the a2, a3, a5) and low expression of the predominant adult subtype (a1) in the amygdala and adjacent piriform and perirhinal cortices, while the Slow rats exhibit the opposite profile (underexpression of the embryonic forms and overexpression of the adult form)(Poulter et al., 1999).

Most recently, we have determined that these different receptor phenotypes are associated with unique behavior at a physiological level (McIntyre et al., submitted). The spontaneous miniature inhibitory postsynaptic currents (mIPSCs) are much smaller and deactivate more slowly in the Fast compared to the Slow rats; in addition, the smallest and slowest mIPSCs in the Fast rats are uniquely associated with inhibitory interneurons. This is very important, since it is generally believed that the interneurons determine the timing and oscillatory patterns of the principle neurons. By contrast, all neuronal phenotypes in the Slow rats receive only large and rapidly deactivating mIPSCs. These very different GABA response profiles in the two strains are likely integral components in their differential seizure predispositions and behavioral attributes. These phenotypes are also highly correlated to their differential sensitivity to positive versus negative GABAergic modulators, like pentobarbital and diazepam versus picrotoxin and bicuculline (Racine and McIntyre, in preparation).

We are continuing studies on all avenues provided by these rats. In the learning and memory studies, we are examining the strains in many tasks to further clarify their differences, e.g., how do the two strains attend to and use various cues; and, with special pre-training, how can we behaviorally turn a Fast rat into to a Slow rat ('remedial programs'). We have discovered recently several ways to achieve this outcome. This is similar to therapeutic approaches that define 'special accomodations' to assist and normalize needy individuals.

In the experiments on impulsivity (which is often defined behaviorally as an inability to withhold responses), we are studying this disposition in 'go/no go', DRL and other paradigms, but the most potent example of impulsivity is the absolute inability of the Fast males to suppress the immediate mounting of a conspecific female - independent of her estrous state! Needless to say, such interactions with non-estrous females precipitate a great deal of aggression. This socially inappropriate behavior is never seen in Slow rats. Included in these behavioral experiments, we are concurrently determining the GABA, glutamate and aspartate overflow in the amygdalae and other structures via bilateral microdialysis with laser-induced capillary electrophoresis. This procedure is nicely defining lateralized differences in the two amygdalae associated with various natural behaviors, including aggression. The latter, of course, is not uncommon in TLE with an amygdala origin.

These kinds of experiments will continue into the rats' advanced years, since our anecdotal data indicate that most naïve Fast rats (who have been maintained as pets by students) eventually become spontaneously epileptic. In addition to increased seizure proness later in life, the Fast rats are more sensitive to a febrile challenge early in life (21 days). Although their febrile seizure does not precipitate obvious brain damage (silver and Fluoro-Jade), it does result in a substantial and abnormal accumulation of IgGs in limbic neurons. We will continue to study this important genetically-based phenomenon and its significance for seizure sensitive and behavioral changes later in life.

Continuing molecular experiments in Fast and Slow rats will address the changes in GABAA receptor subtypes associated with kindling. In studies with Michael Poulter, we are now examining the differences in GABA mechanisms between the strains before, during and long after kindling. We are seeing different locations (synaptic versus extrasynaptic) for the various GABAA receptor subtypes, which should speak to the manner in which GABA modulates network activity in Fast and Slow rats before and after kindling. In addition, the physiology associated with the different GABAA receptor subtypes, described above, predicts different oscillatory behavior in the network. We are now testing this in whole animals by determining the coherence between various recording sites in the amygdala, parahippcampal and hippocampal structures during several behaviors (quiet immobility, EEG spindling, walking, exploration and learning) before, during and after amygdala versus hippocampal kindling. Coupled with this whole animal electrophysiology, we are again doing bilateral amygdala microdialysis (and in the hippocampus in future experiments) during those same events. In other planned in vivo and in vitro experiments, we will determine GABA manufacture, release and reuptake in several limbic structures of our strains before, during and after kindling. And, finally, weaving through many of these studies with Fast and Slow rats are collaborative projects with other laboratories examining the expression and modulation of gap junction proteins and communication during various stages of epileptogenesis as well as status epilepticus. In this regard, we have observed clear differences between the strains in the expression of connexin-32, which is a gap junction protein associated with oligodendrocytes, where the Fast rats have much reduced expression in several forebrain structures compared to the Slow rats. Interestingly, this result involving the brain's 'myelinators' is also highly correlated with our recent findings, using both differential display and microarrays, of very reduced expression of several developmentally-regulated myelin associated proteins in the Fast rats compared to the Slow rats e.g., one such protein is the proteolipid protein. These two techniques, when applied to our strains, are continuing to provide us with many new possibilities for differentiating those individuals who are naturally seizure-prone compared to those that are seizure-resistant, with respect to kindling development and, possible, TLE.