Precision of dual-energy X-ray absorptiometry of the knee and heel: methodology and implications for research to reduce bone mineral loss after spinal cord injury.

STUDY DESIGN: Methodological validation of dual-energy x-ray absorptiometry (DXA)-based measures of leg bone mineral density (BMD) based on the guidelines of the International Society for Clinical Densitometry. OBJECTIVES: The primary objective of this study was to determine the precision of BMD estimates at the knee and heel using the manufacturer provided DXA acquisition algorithm. The secondary objective was to determine the smallest change in DXA-based measurement of BMD that should be surpassed (least significant change (LSC)) before suggesting that a biological change has occurred in the distal femur, proximal tibia and calcaneus. SETTING: Academic Research Centre, Canada. METHODS: Ten people with motor-complete SCI of at least 2 years duration and 10 people from the general population volunteered to have four DXA-based measurements taken of their femur, tibia and calcaneus. BMDs for seven regions of interest (RIs) were calculated, as were short-term precision (root-mean-square (RMS) standard deviation (g cm-2), RMS-coefficient of variation (RMS-CV, %)) and LSC. RESULTS: Overall, RMS-CV values were similar between SCI (3.63-10.20%, mean=5.3%) and able-bodied (1.85-5.73%, mean=4%) cohorts, despite lower absolute BMD values at each RIs in those with SCI (35%, heel to 54%, knee; P<0.0001). Precision was highest at the calcaneus and lowest at the femur. Except at the femur, RMS-CV values were under 6%. CONCLUSIONS: For DXA-based estimates of BMD at the distal femur, proximal tibia and calcaneus, these precision values suggest that LSC values >10% are needed to detect differences between treated and untreated groups in studies aimed at reducing bone mineral loss after SCI.

NMDA receptor-mediated oscillatory activity in the neonatal rat spinal cord is serotonin dependent.

The effect of serotonin (5-HT) receptor blockade on rhythmic network activity and on N-methyl--aspartate (NMDA) receptor-induced membrane voltage oscillations was examined using an in vitro neonatal rat spinal cord preparation. Pharmacologically induced rhythmic hindlimb activity, monitored via flexor and extensor electroneurograms or ventral root recordings, was abolished by 5-HT receptor antagonists. Intrinsic motoneuronal voltage oscillations, induced by NMDA in the presence of tetrodotoxin (TTX), either were abolished completely or transformed to long-lasting voltage shifts by 5-HT receptor antagonists. Conversely, 5-HT application facilitated the expression of NMDA-receptor-mediated rhythmic voltage oscillations. The results suggest that an interplay between 5-HT and NMDA receptor actions may be critical for the production of rhythmic motor behavior in the mammalian spinal cord, both at the network and single cell level.

Regional distribution of the locomotor pattern-generating network in the neonatal rat spinal cord.

The regional distribution of spinal cord networks producing locomotor-like, as well as non-locomotor-like, activity was studied with the use of an in vitro neonatal rat preparation. Rhythmic activity was induced by bath application of either serotonin (5-HT), acetylcholine (ACh), N-methyl-D,L-aspartate (NMA), or combined 5-HT/NMA, and was monitored via hindlimb flexor (peroneal) and extensor (tibial) electroneurograms (ENGs) or ventral root recordings. In some experiments, synchronous patterns were produced by the addition of inhibitory amino acid (IAA) receptor antagonists. Selective application of 5-HT to cervical and thoracic cord regions induced rhythmic activity in these segments but failed to evoke hindlimb ENG discharge. Exposure of the isolated lumbar region to 5-HT produced tonic activity only. Application of 5-HT to the whole cord produced locomotor-like activity in hindlimb ENGs that persisted after midsagittal section of the spinal cord from the conus to the thoracolumbar junction. In other experiments, transverse hemisection of the rostral lumbar cord during whole cord exposure to 5-HT abolished rhythmic activity in ipsilateral hindlimb ENGs, suggesting that under these conditions rhythmic activity on one side of the lumbar cord was insufficient to maintain rhythmic activity on the contralateral side. Selective application of NMA or ACh to cervical and/or thoracic cord regions evoked rhythmic activity in these supralumbar segments, as well as rhythmic, but non-locomotor-like, activity in the lumbar region. In contrast to the effect of 5-HT, both NMA and ACh evoked rhythmic activity when applied solely to the lumbar region, and the side-to-side alternation produced by whole cord ACh application was uncoupled by midsagittal lesions of the lumbar region. In the presence of IAA antagonists, the side-to-side coupling of bilaterally synchronous rhythms was maintained despite extensive midsagittal lesions leaving all but one or two segments of either cervical, thoracic, or lumbar cord bilaterally intact, and rhythmic activity could be maintained even in single isolated hemisegments. The effects of 5-HT/NMA were similar to those observed with the use of 5-HT alone, although 5-HT/NMA induced rhythmic activity in hindlimb ENGs when applied selectively to supralumbar regions. The results suggest that 1) a 5-HT-sensitive oscillatory network, capable of producing a locomotor-like pattern of activity, is distributed throughout the supralumbar region of the spinal cord and mediates descending rhythmic drive to lumbar motor centers; 2) NMA- and ACh-sensitive rhythmogenic elements are distributed throughout the spinal cord, including the lumbar region; and 3) the spinal cord contains an extensive propriospinal network of reciprocal inhibitory and excitatory connections characterized by redundantly organized side-to-side projections.

Effects of inhibitory amino acid antagonists on reciprocal inhibitory interactions during rhythmic motor activity in the in vitro neonatal rat spinal cord.

1. The role of inhibitory amino acid transmission in the coordination and generation of rhythmic motor activity was examined with the use of an in vitro neonatal rat spinal cord preparation. Before adding gamma-aminobutyric acid (GABA) or glycine receptor agonists and antagonists, rhythmic motor activity was induced by bath application of acetylcholine (ACh), N-methyl-D,L-aspartate (NMA), or serotonin (5-HT) while monitoring bilateral ankle flexor and extensor electroneurograms (ENGs). The timing of rhythmic flexor and extensor discharge was consistent with that seen during overground locomotion in 27% of 84 bath applications of these substances (n = 65 preparations). 2. Subsequent addition of the GABAA receptor agonist muscimol, the GABAB receptor agonist baclofen, or glycine, abolished rhythmic activity in 95% of the tested applications. 3. GABAB receptor blockade did not disrupt alternating patterns of ENG discharge. However, addition of the GABAA receptor antagonist bicuculline, or the glycine receptor antagonist strychnine, transformed alternating flexor-extensor and left-right activity into patterns characterized by bilaterally synchronous rhythmic activation of all hindlimb ENGs. The onset of individual ENG bursts was more abrupt following bicuculline or strychnine. Strychnine also synchronized high-frequency (4-8 Hz) packets of rhythmic discharge within ENG bursts. 4. Some preparations developed synchronous, but unstable, rhythmic activity in the presence of bicuculline or strychnine alone. However, NMA, 5-HT, or ACh was usually required in addition to these antagonists to promote sustained rhythmic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Some limitations of ventral root recordings for monitoring locomotion in the in vitro neonatal rat spinal cord preparation.

Studies of the in vitro neonatal rat spinal cord have used ventral root recordings, among other methods, to monitor locomotion. However, whether ventral root activity reliably indicates the presence of hindlimb stepping has not been established. In the present study, we use an in vitro spinal cord-hindlimb preparation to analyse lumbar ventral root recordings obtained while simultaneously observing coordinated stepping movements or rhythmic alternation of ankle flexor and extensor nerve activity. During locomotion ventral root patterns included: tonic activity, rhythmic left-right alternation with in-phase activity of ipsilateral roots, and rhythmic activity that was in-phase both bilaterally and ipsilaterally at different segmental levels. Ventral root transection during rhythmic activity demonstrated that ankle flexor and extensor nerve activity depended on motor units coursing through common lumbar roots in 31/39 hindlimbs. These findings suggest that ventral root recordings alone are not a reliable means of monitoring phasic hindlimb flexor and extensor activity during locomotion in the in vitro rat preparation.

A comparison of motor patterns induced by N-methyl-D-aspartate, acetylcholine and serotonin in the in vitro neonatal rat spinal cord.

Using an in vitro preparation from neonatal rat spinal cord, we compared the motor patterns induced by three putative locomotion-inducing substances. N-Methyl-D,L-aspartate (NMA) induced rhythmic hindlimb nerve activity in 17/20 preparations that was characterized by: (a) side-to-side alternation, but co-activated intralimb flexor-extensor pairs in 29%; (b) bilateral co-activation of all flexors and extensors in 24%; and c) rhythmic but poorly coordinated activity in 35%. Acetylcholine induced rhythmic activity in 34/35 preparations, which in 68% of animals was characterized by side-to-side alternation of co-activated intralimb flexor-extensor pairs. Only rarely did NMA (2/20 trials) and acetylcholine (1/35 trials) induce sustained ENG patterns compatible with hindlimb stepping. Serotonin, however, induced rhythmic activity in 22/24 preparations that was consistent with locomotion in intact rats in 13/22 (59%). These findings demonstrate that exogenously applied neurochemicals induce a variety of in vitro motor rhythms although some substances preferentially activate specific patterns. The results also highlight the importance of monitoring flexor and extensor activity from both hindlimbs in order to distinguish locomotor-like patterns from other types of neurochemically-induced rhythms.