Stepping-like movements in humans with complete spinal cord injury induced by epidural stimulation of the lumbar cord: electromyographic study of compound muscle action potentials.

STUDY DESIGN: It has been previously demonstrated that sustained nonpatterned electric stimulation of the posterior lumbar spinal cord from the epidural space can induce stepping-like movements in subjects with chronic, complete spinal cord injury. In the present paper, we explore physiologically related components of electromyographic (EMG) recordings during the induced stepping-like activity. OBJECTIVES: To examine mechanisms underlying the stepping-like movements activated by electrical epidural stimulation of posterior lumbar cord structures. MATERIALS AND METHODS: The study is based on the assessment of epidural stimulation to control spasticity by simultaneous recordings of the electromyographic activity of quadriceps, hamstrings, tibialis anterior, and triceps surae. We examined induced muscle responses to stimulation frequencies of 2.2-50 Hz in 10 subjects classified as having a motor complete spinal cord injury (ASIA A and B). We evaluated stimulus-triggered time windows 50 ms in length from the original EMG traces. Stimulus-evoked compound muscle action potentials (CMAPs) were analyzed with reference to latency, amplitude, and shape. RESULTS: Epidural stimulation of the posterior lumbosacral cord recruited lower limb muscles in a segmental-selective way, which was characteristic for posterior root stimulation. A 2.2 Hz stimulation elicited stimulus-coupled CMAPs of short latency which were approximately half that of phasic stretch reflex latencies for the respective muscle groups. EMG amplitudes were stimulus-strength dependent. Stimulation at 5-15 and 25-50 Hz elicited sustained tonic and rhythmic activity, respectively, and initiated lower limb extension or stepping-like movements representing different levels of muscle synergies. All EMG responses, even during burst-style phases were composed of separate stimulus-triggered CMAPs with characteristic amplitude modulations. During burst-style phases, a significant increase of CMAP latencies by about 10 ms was observed. CONCLUSION: The muscle activity evoked by epidural lumbar cord stimulation as described in the present study was initiated within the posterior roots. These posterior roots muscle reflex responses (PRMRRs) to 2.2 Hz stimulation were routed through monosynaptic pathways. Sustained stimulation at 5-50 Hz engaged central spinal PRMRR components. We propose that repeated volleys delivered to the lumbar cord via the posterior roots can effectively modify the central state of spinal circuits by temporarily combining them into functional units generating integrated motor behavior of sustained extension and rhythmic flexion/extension movements. This study opens the possibility for developing neuroprostheses for activation of inherent spinal networks involved in generating functional synergistic movements using a single electrode implanted in a localized and stable region.

Initiating extension of the lower limbs in subjects with complete spinal cord injury by epidural lumbar cord stimulation.

We provide evidence that the human spinal cord is able to respond to external afferent input and to generate a sustained extension of the lower extremities when isolated from brain control. The present study demonstrates that sustained, nonpatterned electrical stimulation of the lumbosacral cord--applied at a frequency in the range of 5-15 Hz and a strength above the thresholds for twitches in the thigh and leg muscles--can initiate and retain lower-limb extension in paraplegic subjects with a long history of complete spinal cord injury. We hypothesize that the induced extension is due to tonic input applied by the epidural stimulation to primary sensory afferents. The induced volleys elicit muscle twitches (posterior root muscle-reflex responses) at short and constant latency times and coactivate the configuration of the lumbosacral interneuronal network, presumably via collaterals of the primary sensory neurons and their connectivity with this network. We speculate that the volleys induced externally to the lumbosacral network at a frequency of 5-15 Hz initiate and retain an "extension pattern generator" organization. Once established, this organization would recruit a larger population of motor units in the hip and ankle extensor muscles as compared to the flexors, resulting in an extension movement of the lower limbs. In the electromyograms of the lower-limb muscle groups, such activity is reflected as a characteristic spatiotemporal pattern of compound motor-unit potentials.

Mechanisms of electrical stimulation with neural prostheses.

Individual electric and geometric characteristics of neural substructures can have surprising effects on artificially controlled neural signaling. A rule of thumb approved for the stimulation of long peripheral axons may not hold when the central nervous system is involved. This is demonstrated here with a comparison of results from the electrically stimulated cochlea, retina, and spinal cord. A generalized form of the activating function together with accurate modeling of the neural membrane dynamics are the tools to analyze the excitation mechanisms initiated by neural prostheses. Analysis is sometimes possible with a linear theory, in other cases, simulation of internal calcium concentration or ion channel current fluctuations is needed to see irregularities in spike trains. Spike initiation site can easily change within a single target neuron under constant stimulation conditions of a cochlear implant. Poor myelinization in the soma region of the human cochlear neurons causes firing characteristics different from any animal data. Retinal ganglion cells also generate propagating spikes within the dendritic tree. Bipolar cells in the retina are expected to respond with neurotransmitter release before a spike is generated in the ganglion cell, even when they are far away from the electrode. Epidural stimulation of the lumbar spinal cord predominantly stimulates large sensory axons in the dorsal roots which induce muscle reflex responses. Analysis with the generalized activating function, computer simulations of the nonlinear neural membrane behavior together with experimental and clinical data analysis enlighten our understanding of artificial firing patterns influenced by neural prostheses.

Circadian temperature rhythms in rabbit pups and in their does.

Circadian rhythms of mammals are generated endogenously, the master oscillator system residing in the suprachiasmatic nuclei (SCN). Previous experiments have indicated that the rabbit has a feeding entrainable circadian oscillator (FEO) which is supposed to be of greatest importance during the early infancy of the rabbit. Here we report the course of telemetrically monitored core body temperature of rabbit pups and of their does. Temperature increased from 37.6+/-0.3 degrees C on day 2 to 39.5+/-0.1 degrees C on day 28 of life. The pups showed a 24 h temperature rhythm even during their first days of life. Temperature increased 2 1/2-3 h prior to nursing for 0.4-0.8 degrees C and rose for an additional 0.4-0.6 degrees C immediately after milk ingestion. The anticipatory, but not the postprandial component persisted when nursing was skipped twice. The persistence of a rhythm in the absence of any entraining agent is crucial for its endogenous generation. In the doe, the core body temperature gradually decreased during the last 2/3 of pregnancy. During parturition it steeply rose for 1.5-1.7 degrees C and attained a plateau of 39.7+/-0.2 degrees C during lactation. The circadian rhythm persisted during the whole course of pregnancy and lactation. Thus, in the rabbit an endogenous, feeding entrainable circadian oscillator appears to operate from the first days of life. It is of functional significance in that it alerts the pup in time so that it is able to utilize the singular short presence of the doe for maximal milk intake.

Diversity and development of circadian rhythms in the European rabbit.

Three main concerns underlie this review: 1) The need to draw together the widely dispersed information available on the circadian biology of the rabbit. Although the rabbit is a classic laboratory mammal, this extensive body of information is often overlooked by chronobiologists, and despite several advantages of this species. In terms of its general biology the rabbit is the best studied laboratory mammal in the wild, it demonstrates a wide variety of robust circadian functions, and being a lagomorph, it provides a useful comparison with more commonly studied rodent species. 2) The need to more fully exploit a developmental approach to understanding circadian function, and the particular suitability of the rabbit for this. Female rabbits only visit their altricial young for a few minutes once every 24 h to nurse, and survival of the young depends on the tight circadian-controlled synchronization in behavior and physiology of the two parties. Patterns of circadian rhythmicity in neonatal pups associated with nursing do not form a smooth continuum into weaning and adult life, and may reflect the action of separate mechanisms operating in their own right. 3) Using information from the first two points, to emphasize the diversity and complexity of circadian rhythms underlying behavioral and physiological functions in adult and developing mammals. Information accruing on circadian functions in the rabbit makes it increasingly difficult to account for these in terms of one or two regulatory mechanisms or "oscillators." Thus, it is argued that in addition to the reductionist, molecular approaches currently dominating much of chronobiology, the study of circadian systems as emergent characteristics of whole organisms operating in complex environments merits special attention.

Circadian thermoregulation in suckling rabbit pups.

The rabbit pup is well suited to track the age-dependent development of periodic thermoregulation during the suckling period. Since the litters are regularly nursed once per day for a total of 3 to 4 min, an exogenous, metabolic, nonphotic periodic variable is supposed to have an impact on the 24-h rhythm of body temperature. The authors monitored the course of core body temperature during the suckling period of 20 pups by means of a transmitter implanted intraperitoneally on day 3 postpartum. The 24-h mean rose from an average of 37.8+/-0.3 degrees C on day 4 of life to 39.5+/-0.2 degrees C at weaning on day 27, for 2 out of 20 pups, and day 28, for 18 out of 20 pups. In constant dim illumination, the pups exhibited a 24-h rhythm even on postnatal day 4, which consolidated around days 5 to 7. The rhythm consisted of a significant anticipatory rise of 0.4 to 0.6 degrees C above the respective 24-h mean commencing 2.5 to 3.5 h prior to nursing. Milk intake was followed by a further increase of temperature for an additional 0.3 to 0.6 degrees C. Then the temperature dropped for 1.2 to 1.5 degrees C within 1 to 3 h and returned to average 3 to 5 h later. During a 48-h fast, the rhythm continued to exist, though in a modified shape: the anticipatory component persisted almost unchanged; a further elevation of temperature, however, did not occur. Thus, the anticipatory component apparently is generated endogenously and the second surge represents an exogenous suckling-induced, thermogenic peak. When maternal nursing was advanced for 15 min/day for a total of 5 h, the temperature rhythm of the pups followed the shift of the zeitgeber in parallel. These data confirm the assumption that a circadian rhythm exists during the first postnatal days of the rabbit and that this rhythm is entrained by the 24-h nursing rhythm. The authors suggest that the biological significance of a feeding entrainable oscillator (FEO) in the rabbit might be to activate the pups prior to the periodic nursing visit of the rabbit doe. Thus, the pups are prepared to quantitatively use the one and only short nursing episode per day for maximal milk ingestion.

Ontogeny of the rabbit's circadian rhythms without an external zeitgeber.

We have monitored parturition and different behavioral rhythms of does, the activity of suckling pups and behavioral rhythms of rabbits after weaning until adulthood. All animals were living in continuous light conditions (LL). As a consequence of the free-run of the does parturition, which takes place during the resting period of the animal, occurred at almost any time of the day. The once/day nursing was advanced during the first 7-10 days until it was in synchrony with the time of main activity. The pups significantly anticipated nursing by day 5: the 60 min bin prior to nursing exceeded the 24-h mean by 63 +/- 30%. The anticipatory activity (AA) was entrained by an artificial nursing schedule of T = 24 h, the 60 min bin prior to nursing exceeding the 24-h mean by 123 +/- 50%. Since AA persisted during a 48 h fast, its phase is controlled endogenously. At weaning behavioral rhythms of rabbits free-ran with a main tau < 24 h (average 23:35 +/- 17'), period length extending with increasing age. 7/10 animals had an additional component > 24 h (average: 24:24 +/- 16') which became increasingly dominant, the components < 24 h fading out within 30 days after weaning. Between days 340-350 a stable period length of 24:35 +/- 8' and a consolidation of the circadian rhythms was attained. These results confirm that during the first days of the rabbit's life the circadian system is controlled by a feeding entrainable oscillator, the light entrainable oscillators taking over control later.

Restricted food access and light-dark: impact of conflicting zeitgebers on circadian rhythms of the rabbit.

Free-running circadian rhythms of rabbits were exposed to a 11:55-11:55-h light-dark (LD) schedule. After complete entrainment (63 +/- 22 days), the predominantly nocturnally active rabbits were exposed to an additional zeitgeber, restricted food access (RF), which was imposed during the light period. In five animals RF had the same period (T) as the LD cycle (23:50 h), and in five other animals TRF was 24:10 h. At a period of 23:50 h for both zeitgebers, the rhythms of four animals were stably entrained to RF, while in one animal a component of the rhythm broke away from RF and entrained to the LD zeitgeber. In animals exposed to zeitgebers of different periods most of the activity rhythm also entrained to RF, but 20 +/- 7% of the activity entrained to the LD zeitgeber. The light-entrained activity component merged with the RF component when the zeitgebers crossed, and decomposition occurred when the phase difference exceeded 4-6 h. The results indicate that two circadian oscillator systems exist in the rabbit, one entrained by light-dark cycles and the other by feeding-fasting cycles. Both exert common control over a number of overt behavioral rhythms.

The ontogeny of circadian rhythms in the rabbit.

The nursing rhythms of 14 rabbit does and the activity of 18 litters from birth to days 14-16 were monitored continuously, as were the circadian rhythms of different behavioral functions of 10 young rabbits from weaning until day 380 of life. The does gave birth 2 hr, 26 min +/- 2 hr, 8 min after lights-on in a light-dark cycle (LD 12:12). The first nursing occurred 20 hr, 30 min +/- 2 hr, 21 min after parturition. Does that had continuous access to the next boxes exhibited a regular once-a-day nursing rhythm; the nursing visits to the litters occurred during the dark phase (D). The nursing visits lasted 3 min, 42 sec +/- 25 sec. Nursing advanced during the first 10 days of lactation by an average of 42 +/- 16 min/day, and thus shifted to the first half of D. The pups anticipated nursing by a significant increase of activity, which was established between days 1 and 5 of life. In two does, the nursing rhythm split into two nursing visits per day, one during D and one during the light phase (L). When scheduled to nurse at an "artificial" phase in L with T = 24 hr, the pups' anticipatory activity was entrained by the 24-hr nursing rhythm. During a 48-hr fast, the elevated activity persisted around the phase of prior nursing time, demonstrating that it was controlled by an endogenous oscillator entrained by the zeitgeber of maternal nursing. Of 10 weanlings, 6 first exhibited a trimodal rhythm, which turned bimodal within 3-6 days. Stable entrainment by the external LD zeitgeber was attained by about days 45-80, and full nocturnality was attained by about days 200-250 after weaning. Having attained steady-state conditions, 7 of 10 rabbits exhibited a stable unimodal, nocturnal rhythm in each of the five functions, while the other 3 retained a bimodal rhythm.

A feeding-entrainable circadian oscillator system in the rabbit.

One of the most important properties of biological oscillators is their entrainment by exogenous forces acting cyclically in the environment. Among the environmental cycles, the phenomena recurring with a circadian periodicity are very prominent. The role of circadian zeitgebers is discussed giving a particular importance to the effects of feeding as a cause of masking and entrainment.

Restricted feeding: a nonphotic zeitgeber in the rabbit.

The free-running circadian rhythms of five behavioral functions of the rabbit were masked by unsignalled restricted food access (RF). The rhythms were reorganized immediately, a large part of events being assembled around the end of food availability. In addition to masking a slower process of entrainment was running: a component of anticipatory activity (AA) was established 1-3 h before food access. AA consolidated in continuation of the camouflaged free-running rhythm. The time of AA establishment correlated significantly with the phase angle difference (PAD) between free-running rhythm and RF: it decreased with decreasing positive PAD. With the consolidation of AA, the rhythms had attained a stable phase relation to RF. At the termination of RF, the circadian rhythms started to free-run again, the phase being dependent from that of the preceding RF schedule. The period length of the RF zeitgeber (TRF) had some impact on tau of the circadian rhythm. This aftereffect was most evident after termination of TRF less than 24 h: the free-running rhythm continued for up to 49 days with a tau less than 24 h and turned longer thereafter. The results demonstrate the entrainment of circadian rhythms of the rabbit by RF in addition to masking.

The rabbit: a diurnal or a nocturnal animal?

Although the rabbit is an almost 'classical' laboratory animal, chronobiological research in this species is in its infancy. It appears not even clear, whether the rabbit is a predominantly diurnal, crepuscularly active or nocturnal animal. In an ordinary, non sound-isolated animal room rabbits exhibited elevated locomotor activity, hard faeces excretion, food intake and water intake during elevated external animal-house noise. When the hours of external noise coincided with the 12 h light period 8/15 rabbits displayed a predominantly diurnal pattern, in 3/15 rabbits no preference existed for light or dark time and 4/15 animals had a typically nocturnal pattern. In contrast, in a properly sound-isolated laboratory locomotor activity, hard faeces excretion, food intake, water intake and urine excretion were significantly higher during the dark period of a LD 12:12. In constant light conditions of 7 lux either function free-ran with a circadian period-length greater than 24 h, which, too, is typically in nocturnally active animals. When food access was restricted to 4/12 h of the light period, most events of the five behavioral functions were concentrated around the time of restricted food access (RF), the rabbits now being almost exclusively light active. In the absence of any other zeitgeber RF in fact did entrain the free-running circadian rhythms and, thus, is zeitgeber for the circadian oscillator system of the rabbit. Thus, while the rabbit endogenously is a nocturnally active animal, external noise or scheduled feeding during the light period can turn it to a predominantly diurnal animal.

Circadian rhythms of rabbits during restrictive feeding.

Without a zeitgeber the circadian rhythms of five physiological functions free-ran with a period length greater than 24 h. Restricted feeding time (RF) masked the free-running rhythms. In addition to masking, entrainment with RF occurred. This process was most evident in locomotor activity and visits to the food box. RF thus had zeitgeber properties in these rabbits. However, in most rabbits the RF zeitgeber was not strong enough to entrain the circadian rhythm completely. A small component free-ran during RF. Following return to continuous food access the whole circadian rhythm resumed to free-run again. In some animals its phase was determined by the RF zeitgeber and in others by the small free-running fraction present during RF. The results suggest that in addition to the light-dark-entrainable circadian oscillator system a feeding-entrainable oscillator exists that takes over phase control of the majority of the rhythm during RF.

The internal synchronization of five circadian functions of the rabbit.

Five different physiological functions of the rabbit (hard faeces and urine excretion, food and water intake and locomotor activity) were registered during LD 12:12 and during continuous light conditions (LL). (1) In LD 12:12 a strong synchronization of the five parameters existed. The minima of all functions consistently occurred during the hours of light. The nocturnal percentage of overall 24-hr events was increased significantly in 'hard faeces excretion' (66 +/- 8(S.D.)%), 'water intake' (64 +/- 15(S.D.)%) and 'urine excretion' (58 +/- 10(S.D.)%). The nocturnal percentage of locomotor activity was significantly increased during the dark-hours in 9 out of 14 animals. In the other five individuals prominent peaks were present even during the photoperiod. On the average of all 14 animals 55 +/- 13(S.D.)% of the 24 hr events of locomotor activity occurred during the night. Despite a trough during the cessation of hard faeces excretion the events of food intake were not elevated significantly during the dark hours. (2) During LL the synchronization of the five functions within each animal persisted during the complete 90-day LL period. A free-running circadian rhythm with tau = 24.8 +/- 0.5(S.D.) hr was present in the four rabbits kept in LL conditions within 5-16 days after the withdrawal of the zeitgeber. (3) In addition to the circadian period the power spectrum analysis of data obtained during LD 12:12 revealed significant ultradian periods of an average period length of 11,6 hr (hard faeces and urine excretion), 8 hr (food and water intake, locomotor activity) and 4 hr (food intake, locomotor activity). During the free-run ultradian periods of 8 and 3.2-4.2 hr were significant in almost all parameters. (4) During LL the level of locomotor activity was reduced for 13 +/- 16(S.D.)%, the events of food intake were increased for 17 +/- 12(S.D.)%. (5) The reinserted LD 12:12 zeitgeber re-entrained the circadian rhythms within 25-45 days. (6) These results provided evidence of a predominant nocturnality of the rabbits under investigation.

Monophasic and diphasic patterns of the circadian caecotrophy rhythm of rabbits.

The circadian caecotrophy rhythm was synchronized with the light-dark cycle of 12 : 12 h. During this the rabbits practised caecotrophy regularly during the light period. While most rabbits manifested 1 caecotrophy per 24 h (monophasic caecotrophy), some had an additional caecotrophy during the dark period (diphasic caecotrophy). During continuous light the circadian caecotrophy rhythm ran free monophasically, even in those rabbits which were diphasic under the preceding 12 : 12 regime. The average period length amounted to 24.7 +/- 0.3 h. Following restoration of the 12 : 12 routine animals reestablished their original caecotrophy pattern. In a further test the caecotrophy pattern remained constant during a constant 12 : 12 regime, but changed in 7 of 16 animals when the photoperiod was reduced first to 60 min and then to 2 x 60 min light every 24 h. The reduction of the lit time resulted in an increased occurrence of diphasic animals. Details of synchronization of the caecotrophy rhythm with the different light-dark schedules are given. These results accord with data obtained in nocturnal rodent species.

Rate of movement of marker particles in the digestive tract of the rabbit.

Using small (50-100 micrometer) and large (800-1000 micrometer) polystyrene particles as a marker substance, transit through the gastrointestinal tract of the rabbit was studied during the formation of hard and soft faeces (caecotrophe). Both fractions of the marker were transported at an equal rate during soft faeces formation. During the formation of hard faeces a significantly higher amount of small particles was present in the caecum, whereas in the distal colon and rectum the number of large particles was elevated significantly.