Faculty Bibliography

A quantitative analysis of two rat syndromes of myoclonus are presented, modeling myoclonic epilepsy and postanoxic myoclonus. Like the human conditions, both of the models benefit therapeutically from drugs that act on the serotonin system. The rat model of myoclonic epilepsy is associated with a profound loss of serotonin throughout the brain (except in the striatum) and is generated by an oscillator that is synchronized around the midline. The rat model of posthypoxic myoclonus does not demonstrate a significant reduction in serotonin in any location of its brain and is generated by a non-oscillating circuit in the medulla. Although some forms of myoclonic epilepsy may benefit from serotonin drugs because they are caused by a decrease in brain serotonin, our data indicate that posthypoxic myoclonus is not caused by a decrease in the serotonergic innervation of any region of the brain. That the raphe nuclei do not degenerate after global brain ischemia was noted by C. David Marsden in a discussion of the histologic findings of three of his human cases of posthypoxic myoclonus (page 117 of reference 10) and led him to question the hypothesis that posthypoxic myoclonus was due to a loss of serotonin neurons. Our data confirm his observation in the rat, but also indicate that density of serotonin fibers and terminals throughout the brain is not reduced by the brain ischemia that produces posthypoxic myoclonus. It remains to be determined whether the physiologic responsiveness of serotonin neurons is altered by global brain ischemia and whether changes in serotonin release or serotonin receptor properties are associated with posthypoxic myoclonus. The stability of the serotonin system in posthypoxic myoclonic rats is remarkable when one considers the wide range of disorders that is produced by the prolonged brain ischemia. The inability of the most severely posthypoxic myoclonic rats to perform 7-Hz tongue protrusions indicates substantial physiologic disruption of brainstem motor function. Moreover, the posthypoxic myoclonic rat suffers from ataxia, seizures, retrograde amnesia, and impaired ability to learn. The wide spectrum of these deficits is sharply constrasted by its apparently intact serotonin system. We have identified the inferior olive as a locus that may generate the rhythmic components of tremor and myoclonus in syndromes that are truly associated with a dramatic loss of brainstem serotonin. Serotonin acts within the inferior olive to constrain its rhythmic firing. Without intraolivary serotonin, olivary neurons are predisposed to oscillate continuously, providing a substrate upon which sustained rhythmic spiking may be superimposed. It is clear that such unconstrained rhythmicity produces synchronized whole-body tremor at 10 Hz (33, 41-43). The effects of serotonin to suppress olivocerebellar rhythmicity are mediated by postsynaptic 5-HT2 receptors that reduce the magnitude of the low-threshold calcium conductance, IT. It is notable that dysregulation of this conductance has been associated with hyper-rhythmic states in the thalamus underlying cognitive disorders ranging from depression to tinnitus (49), indicating a common mechanism underlying a variety of neurologic conditions. The identification of a specific brainstem locus (inferior olive), serotonin receptor 5-HT2, and ionic current IT involved in a form of rhythmic myoclonus may provide multiple clues toward which future pharmacotherapies can be directed

1. The effects of N-methyl-D-aspartate (NMDA) receptor activation and blockade on subthreshold membrane potential oscillations of inferior olivary neurones were studied in brainstem slices from 12- to 21-day-old rats. 2. Dizocilpine (MK-801), a non-competitive NMDA antagonist, at 1-45 &mgr;M abolished spontaneous subthreshold oscillations, without affecting membrane potential, input resistance, or the low-threshold calcium current, I(T). Ketamine (100 &mgr;M), a non-competitive NMDA antagonist, and L-689,560 (20 &mgr;M), an antagonist at the glycine site of the NMDA receptor, also abolished the oscillations, while the competitive non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20-50 &mgr;M) had no effect. 3. NMDA (100 &mgr;M) induced 4.1 Hz subthreshold oscillations and reversibly depolarized olivary neurones by 13.7 mV. In contrast, 10 &mgr;M alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and 20 &mgr;M kainic acid depolarized the membrane equivalently but did not induce oscillations. 4. Both NMDA-induced and spontaneous subthreshold oscillations were unaffected by 1 &mgr;M tetrodotoxin and were prevented by substituting extracellular calcium with cobalt. 5. Removing magnesium from the perfusate did not affect spontaneous subthreshold oscillations but did prevent NMDA-induced oscillations. 6. NMDA-induced oscillations were resistant to 50 &mgr;M mibefradil, an I(T) blocker, in contrast to spontaneous oscillations. Both oscillations were inhibited by 20 &mgr;M nifedipine, an L-type calcium channel antagonist, and 200 nM omega-agatoxin IVA, a P-type calcium channel blocker. Bay K 8644 (10 &mgr;M), an L-type Ca(2+) agonist, significantly enhanced the amplitude of both spontaneous and NMDA-induced oscillations. 7. The data indicate that NMDA receptor activation induces olivary neurones to manifest high amplitude membrane potential oscillations in part mediated by L- and P- but not T-type calcium currents. Moreover, the data demonstrate that NMDA receptor currents are necessary for generation of spontaneous subthreshold oscillations in the inferior olive

The effect of serotonin on membrane potential oscillations of inferior olivary neurones was studied in brainstem slices from 10- to 19-day-old rats. Serotonin at 50 and 5 microM induced a mean depolarization of 9.4 and 7.7 mV, respectively, that was preceded by a reversible suppression of subthreshold membrane potential oscillations. These effects were not changed by 1 microM tetrodotoxin and the suppression of subthreshold oscillations persisted after current-mediated restoration of resting potential. In spontaneously active neurones, serotonin abolished the rhythmicity of action potential firing without affecting spike frequency. Serotonin reduced the slope of the calcium-mediated rebound spike and both the duration and amplitude of the subsequent afterhyperpolarization. Serotonin also shifted the voltage dependence of the rebound spike to more negative values. Hyperpolarizing current pulses (200 ms) revealed that serotonin increased the pre-rectification and steady-state components of membrane resistance by 37 and 38 %, respectively, in 66 % of neurones, but decreased these parameters by 14 and 20% in the remaining cells. The serotonin effects were antagonized by 5 microM methysergide or 1-5 microM ketanserin and were mimicked by 10-20 microM dimethoxy-4-iodoamphetamine but not 10 microM 8-hydroxy-2-(di-N-propylamino)-tetralin. The data indicate that serotonin suppresses the rhythmic activity of olivary neurones via 5-HT2 receptors by inhibition of the T-type calcium current in combination with membrane depolarization due to activation of a cation current (Ih) and block of a resting K+ current (fast IK(ir)). This modulatory action of serotonin may account for the differential propensity of olivary neurones to fire rhythmically during different behavioural states in vivo