Laser-deinsulated, printed-circuit electrodes integrated into the floor of culture chambers have been used to monitor the spontaneous activity of mouse spinal monolayer cell cultures. This technique has allowed a multisite analysis of activity over long periods of time in closed chambers. In 63 cultures investigated 3-5 weeks after seeding, 89% included single- or multiunit bursting. Based on a subset of 40 cultures in which all electrodes were sequentially scanned, bursting was found on 41% of the active electrodes (approximately 38% of all units monitored). A total of 35% of the electrodes monitoring spontaneous bursting activity revealed rhythmic sequences that were usually coupled among multiple electrodes. Although most of this coupling was in-phase, three out of 40 cultures exhibited antiphasic bursting. In all cases where coupling was observed, each electrode monitored different burst compositions, demonstrating that the activity was generated by different units. Some rhythmic patterns persisted for over 12 hr and were observed in 400 mm2 monolayer cultures, as well as in much smaller 3 mm2 adhesion islands. The addition of 10 mM MgCl2 consistently blocked both random and patterned (i.e., bursting) spontaneous activity at all recording sites. Strychnine (10(-6) M) typically increased firing frequencies and either disrupted pretest bursting or generated rhythmic activity from random phasic patterns. In certain cases, strychnine also blocked activity on specific electrodes, indicating that glycine is not the only inhibitory transmitter involved. The spontaneous appearance of rhythmic activity in low-density, monolayer cell cultures established from dissociated and randomly seeded spinal tissue can be explained by one or a combination of two hypotheses: an inherent specificity of some interconnections in developing mammalian cultures and the generation of organized activity by random circuits at certain stages of complexity.
The current paper presents our initial efforts to establish an in vitro spinal preparation for investigating locomotor pattern generation in mice. We have characterized the step cycle timing from EMG activity in the gastrocnemius (G) and tibialis anterior (TA) muscles of freely moving intact adult as well as neonatal mice and then compared those data with rhythmic EMG activity in an isolated spinal cord-hindlimb preparation. The motor output during the first four days of life was evaluated in an effort to identify the optimal post-partum period for in vitro locomotor studies. The in vitro pattern generating capabilities of the lumbosacral region were tested in both nonhemisected and hemisected preparations. Spontaneous as well as NMDA evoked in vitro activity in the antagonist set of hindlimb muscles included sequences of: 1) synchronous bursting; 2) mixed synchrony and alternation; and/or 3) irregular alternations. The alternating bursting observed in vitro was more often an alternation of sequences rather than a cycle-to-cycle phasing between G and TA muscles. In summary, while there was evidence of reciprocal inhibition in neonates, the circuitry for cycle-to-cycle alternation between antagonists was found to be labile.
Rhythmic motoneuronal activity was recorded from decerebrated, paralyzed stingrays and compared with electromyograms recorded from the same animals. Before and after paralysis, a rostral-to-caudal sequence of alternation occurred between dorsal (elevator) and ventral (depressor) efferents. The swimming pattern was thus observed in the absence of phasic afferent input, and this constitutes fictive locomotion. After paralysis, both the intersegmental delay (time between activation at progressively caudal recording sites) and the burst duration remained linearly related to the swim cycle period. In many instances, neither the slope nor the intercept was significantly altered by immobilization. The intercepts all fell near the origin, indicating that the fictive rhythm remains constant phase coupled. Although the swimming rhythm was obtained after paralysis, some differences occurred. These included fewer and shorter spontaneous sequences, a greater range of cycle periods, and longer burst durations. During fictive swimming, the burst duration:cycle period ratio usually increased to 0.53 from 0.39 observed before paralysis. Therefore, the silent periods seen between burst discharges in antagonist efferents during movement were often absent after paralysis. Mechanical stimulation of the tail reduced both cycle periods and burst durations; however, the burst:cycle ratio remained greater than or equal to 0.50. The linear relation between burst duration and cycle period found for spontaneous sequences was not changed by stimulation of the tail. During fictive swimming the inter- and intrasegmental coupling that characterizes stingray swimming becomes labile. Abnormal coupling appears more often during sequences with long swim cycles. Intrasegmental coupling is tighter than intersegmental coupling at any cycle period. Rhythmic activity at one segmental level can be independent of activity at other levels. This suggests that multiple oscillator circuits exist that are not dependent on propriospinal circuits interconnecting different segments. Rhythmicity in elevator and depressor motoneurons is not dependent on reciprocal connections between the circuitry driving the motor nuclei. Therefore, separate oscillators for elevators and depressors appear to be present within one spinal segment.
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