Spinal Kittens Research Articles

Transplants Enhance Locomotion in Neonatal Kittens Whose Spinal Cords Are Transected: A Behavioral and Anatomical Study

We have studied the locomotor development of kittens that received complete low thoracic spinal cord transections and embryonic spinal cord transplants as newborns. Embryonic spinal cord (E21-E26) transplanted into the site of a transection integrated well with the host spinal cord and promoted the development of overground locomotion. Spinalized kittens with transplants were first distinguished from spinalized kittens during the 2nd and 3rd postnatal weeks when kittens with transplants positioned their hindlimbs underneath their bodies which promoted support of the hindquarters. By postnatal Week 6, kittens with transplants exhibited overground locomotion characterized by full weight support and moderate balance control. By 20 weeks of age, as many as 96% of the step cycles showed full weight support and as few as 2% of the step cycles were interrupted by a fall. Most kittens also showed coordination between the forelimbs and the hindlimbs. They differed from normal in the precocious onset of reflex stepping and in the less precise interlimb coordination and more precarious balance during overground locomotion. The overground locomotor performance of kittens with transplant greatly exceeded that of spinal kittens without transplants since few spinalized kittens showed any full-weight-supported step cycles and none showed coordination between the forelimbs and the hindlimbs. In the absence of a transplant, no fibers could grow across the lesion site. In the presence of a transplant, fibers grew across the lesion site and established anatomical connectivity with the host. Host segmental systems identified by the presence of calcitonin gene-related peptide- and substance P-immunoreactive fibers were found throughout the transplants. Descending host systems of supraspinal origin were identified by serotonin- and dopamine β-hydroxylase-immunoreactive fibers throughout the transplants. The growth of supraspinal axons into the transplant, and in one case into the caudal host spinal cord, provided a possible anatomical basis for the development of coordinated overground locomotion.

Development of Locomotor Behavior in the Spinal Kitten

This study was undertaken to determine the locomotor capability of kittens whose spinal cords were transected at birth. The postnatal development of reflex and goal-directed locomotion was examined during the first 5 postnatal months in kittens that received low thoracic spinal cord transections as newborns. Some spinal kittens developed aberrant quadrupedal forms of locomotion. The onset of quadrupedal locomotion, however, was delayed by 2-3 months compared to the normal kitten (42) and deteriorated by 5 months of age. Qualitative and quantitative analyses demonstrated that the quadrupedal locomotion was abnormal. Although some step cycles were characterized by full weight support, the typical hindlimb step cycle of the best performing cat showed inadequate weight support and balance. No spinal cat was able to coordinate the hindlimbs with the forelimbs during overground locomotion on a runaway or during quadrupedal locomotion on a treadmill. Neuroanatomical tracing with WGA-HRP and immunocytochemical techniques showed no axonal regeneration or growth into or across the lesion sites. The aberrant form of quadrupedal locomotion developed without descending input to the caudal spinal cord. The variability in performance among animals suggested that compensatory strategies were important factors in the spinal kitten's achievement of quadrupedal locomotion. Hindlimb weight-supported stepping during quadrupedal locomotion in some animals underscored the capacity of the isolated caudal spinal cord to generate both rhythmical stepping movements and weight support. The maintenance of developmentally immature, but functional, hindlimb postures suggested that the development of the isolated caudal spinal cord was arrested in the absence of descending input.

Neuromuscular patterns of stereotypic hindlimb behaviors in the first two postnatal months. II. Stepping in spinal kittens

From birth to postnatal day 60, neuromuscular patterns for airstepping and treadmill stepping were assessed in kittens spinalized (T12) at birth (Day-1) or at the end of the second postnatal week (Day-14). Within 72 h after spinalization, all kittens displayed stepping motions, but exteroceptive facilitation (e.g. tail pinch) was required to initiate and sustain both behaviors. In Day-14 spinal kittens, the hindlimbs spontaneously and alternately airstepped, but in Day-1 spinal kittens exteroceptive stimulation was usually necessary to evoke airstepping, and the hindlimbs stepped synchronously. Kittens in both groups developed atypical neuromuscular patterns; flexor bursts were nearly twice as long in duration as extensor bursts. Development of bipedal treadmill stepping was similar for Day-1 and Day-14 spinal kittens, but differed from that for normal kittens9. Tested at the same belt speeds, stepping was more easily elicited in spinal kittens, bouts of repetitive stepping were longer, and cycle periods were shorter than in normal kittens until postnatal week 6. Spinal kittens, however, seldom exhibited adequate weight support during hindlimbs stepping, and the neuromuscular patterns associated with bipedal stepping were atypical. For spinal kittens, the relationship between the extensor burst duration and the cycle period was reduced substantially, and flexor activity was initiated earlier in the step cycle and was longer in duration than that for normal kittens. These atypical intralimb synergies may have been the consequence of altered lumbosacral circuits produced by the spinal transection. It is also possible that these spinal circuits, lacking rostral input, were particularly susceptible to abnormal motion-dependent feedback resulting from reduced hindlimb weight support.

Neuromuscular patterns of stereotypic hindlimb behaviors in the first two postnatal months. III. Scratching and the paw-shake response in kittens

Neuromuscular patterns of scratching and the paw-shake response were studied in normal kittens from birth to postnatal day 60. Onset of both behaviors coincided with the development of secure weight-bearing posture and occurred on postnatal day 21 for scratching and postnatal day 26 for paw shaking. At onset, cycle periods for scratching (5–6 Hz) and paw shaking (8–10 Hz) were similar to that for adult cats, and EMG patterns were adult-like. The scratch cycle consisted of reciprocal flexor and extensor bursts of equal duration, while the shake cycle consisted of coactive knee extensor and ankle flexor bursts alternately active with ankle extensor bursts. The lack of scratching and paw shaking during the first 3 postnatal weeks and the adult-like EMG patterns at onset are consistent with the hypothesis that pattern-generating circuits within lumbosacral segments are available early in development but inhibited by the rostral neuraxis until postural control is sufficient to accommodate the response. To eliminate rostral inputs, including descending input critical for postural control, kittens were spinalized at the T12 level, and onset of paw shaking was accelerated. In kittens spinalized at birth, paw-shake onset occurred on postnatal day 14, while in kittens spinalized on postnatal day 14, onset occurred 48 h after spinalization. In all spinal kittens, however, knee extensor activity was disrupted and not normal by postnatal day 60. Mature neuromuscular patterns for scratching and paw shaking are available at onset of the behavior during normal development. Spinalization hastens the onset of paw shaking but the normal neuromuscular synergy is disrupted as well as the temporal structure of the multicycle response. Disruptions following spinalization may be due to altered development of spinal pattern generators or abberant feedback from atypical hindlimb motions due to a retardation of hindlimb growth and an alteration of muscle contractile properties in spinal kittens.

Interfering with inhibition may improve motor function

Motor behavior not seen in newborn cats can be revealed by spinal transection and is therefore normally suppressed. Motor performance of these spinal kittens after reaching adulthood surpasses that of chronic adult-operated spinal cats but the latter display a significant improvement when GABAergic inhibition is blocked pharmacologically. Thus, inhibitory processes influence normal motor development as well as recovery of function after neurological damage.

Early onset of hindlimb paw-shake responses in spinal kittens: new perspective in motor development

Normal kittens and kittens spinalized (T12) at 14 days of age were tested for onset of paw-shake responses (PSR) in fore and hindlimbs. In normals, onset followed a cephalocaudal pattern that was coincident with the development of stable posture, 21–28 days of age. In spinals, onset of hindlimb PSRs preceded that of the forelimb and occurred soon after cordotomy, independent of posture development. These findings suggest that in the neonate, spinal networks responsible for coordinating PSRs are normally inhibited until the response can be supported by stable posture.

Absence of hind limb tactile placing in spinal cats and kittens

Tactile placing and associated responses of the fore and hind paws to a light tactile stimulus were studied in normal young-adult cats and kittens and their spinal littermates. All spinal transections were performed at T12 on the 14th postnatal day. In the first study, responses of normal and spinal young-adult cats were compared at 9 to 10 months of age. Frequency of forelimb tactile placing (FL-TP) was similar for both groups, but hind limb tactile placing (HL-TP), seldom elicited in the normal cats, was never elicited in the spinal cats. Due to the absence of HL-TP in the spinal young-adult cats, a second study was conducted. Kittens, 1 to 62 days of age, were tested to determine if tactile placing was present prior to supraspinal maturation. The initial withdrawal ifsponse to the light tactile stimulus was equally developed in forelimbs and hind limbs at birth. In normal kittens, development of FL-TP preceded that of HL-TP by 2 weeks. During the first week after transection, forelimb responses of spinal kittens were more frequent than those of their normal littermates, suggesting an enhancement of motor responses proximal to the lesion, the difference decreasing thereafter. Frequency of hind limb withdrawal was not immediately altered after transection, and airstepping was easily triggered within 48 h after cordotomy. During tactile-triggered airstepping, the hind paw occasionally contacted the placing surface, but HL-TP was not observed in spinal kittens during the testing period. The absence of HL-TP in all spinal cats tested suggests that tactile placing is not a spinal reflex.

Organization of the sacral parasympathetic reflex pathways to the urinary bladder and large intestine.

Electrophysiological and horseradish peroxidase (HRP) techniques have provided new insights into the organization of the sacral parasympathetic reflex pathways to the large intestine and urinary bladder. The innervation of the two organs arises from separate groups of sacral preganglionic cells: (1) a dorsal band of cells in laminae V and VI providing an input to the intestine; and (2) a lateral band of cells in lamina VII providing an input to the bladder. These two groups of cells were separated by an interband region containing tract cells and interneurons. Neurons in the interband region received a visceral afferent input and exhibited firing correlated with the activity of intestine and urinary bladder. It seems reasonable therefore to consider the interband region as a third component of the sacral parasympathetic nucleus. Anterograde transport of HRP revealed that visceral afferents from the intestine and bladder projected into the parasympathetic nucleus. Most of the projections were collaterals from afferent axons in Lissauer's tract that passed in lamina I laterally and medially around the dorsal horn. These afferent collaterals were located in close proximity to preganglionic perikarya and dendrites in laminae I, V and VI. The proximity of visceral afferents and efferents in the sacral cord probably reflects the existence of polysynaptic rather than monosynaptic connections since electrophysiological studies revealed that both the defecation and micturition reflexes occurred with very long central delays (45-70 msec). The reflex pathways mediating defecation and micturition in cats with an intact neuraxis were markedly different. Defecation was dependent upon a spinal reflex with unmyelinated (C-fiber) peripheral afferent and efferent limbs. On the other hand, micturition was mediated by a spinobulbospinal pathway with myelinated peripheral afferent (A-fiber) and efferent axons (B-fiber). Transection of the spinal cord at T12-L2 blocked the micturition reflex but only transiently depressed the defecation reflex. In chronic spinal cats the micturition reflex recovered 1-2 weeks after spinalization; however, in these animals bladder-to-bladder micturition reflexes were elicited by C-fiber rather than A-fiber afferents. The C-fiber afferent-evoked reflex was weak or undetectable in animals with an intact neuraxis. Transection of the spinal cord also changed the micturition reflex in neonatal kittens (age 5-28 days). In neonates with an intact neuraxis bladder-to-bladder reflexes occurred via a long latency spinobulbospinal pathway (325-430 msec). The long latency is attributable to the slow conduction velocity in immature unmyelinated peripheral and central axons. In chronic spinal kittens (3-7 days after spinalization) the long latency reflex was abolished and a shorter latency (90-150 msec) bladder reflex was unmasked. The emergence of this spinal pathway may reflect axonal sprouting and the formation of new reflex connections within the sacral parasympathetic nucleus.

The locomotion of the low spinal cat. II. Interlimb coordination.

The interaction of the two hindlimbs were investigated by an analysis of the muscular activity and the movements in 14 chronic spinal kittens during treadmill locomotion (i.e. in kittens subjected to a transection of the spinal cord (Th10--12)) one or two weeks after birth). At low speed the limbs were alternating (walk or trot). At higher they were activated more simultaneous, as during gallop. The two limbs could walk at different velocities, as during walking in a circle, when the two belts of the treadmill were driven at different speeds. The duration of the support phases was mainly influenced by the speed of the belt on which the limb was walking. The limbs could still maintain a common rhythm up to a two or three fold speed difference, as the flexion or the first extension phase of the limb walking on the "fast" belt was prolonged and the flexion phase of "slow limb" was shortened. At extreme speed differences the limb on the "fast belt" performed 2, 3 and even 4 steps during one stepcycle of the "slow limb". The placement of the feet was found to maintain the most stable relationship during alternating gaits at different speed differences. It is concluded that all phases of the step cycle are modifiable and that there are several mechanisms coordinating the limbs within the spinal cord.

Neuromuscular responses to gait perturbations in freely moving cats.

If an obstacle impedes the forward swing of a cat's foot, the animal responds by rapidly lifting the foot over the obstacle. In freely moving cats, the electrical activity of hindlimb flexors and extensors was recorded during such reactions elicited both mechanically and electrically. The sequencing of muscle activity was more complex and longer in duration in the mechanically elicited reactions. Anaesthesia of the foot dorsum abolished responses in ankle extensors and knee flexors, and converted the responses of ankle flexors to simple stretch reflexes. Although our findings closely resemble those reported for chronic spinal kittens, there are interesting points of difference, which should be taken into account if the notion of a purely spinal mediation of the placing reaction during stepping is to be accepted.

Tactile placing reactions in chronic spinal kittens.

AbstractTransection of the spinal cord at the level of Th 12 to L 1 was performed in 6–14 days old kittens. The kittens were kept for several months after the operation. During this period tactile stimuli applied to the dorsum of the hindpaw could evoke a flexion followed by an extension and a placing of the limb in a more rostral position. These reactions have been studied by recording the EMG activity and filming the movement. It is concluded that it must be regarded as tactile placing, which contrary to what has hitherto been assumed is not a reflex relayed in higher nervous structures, but rather a complex form of spinal reflex.

After-effects of repetitive stimulation in spinal neurons

Spontaneous activity of interneurons before and after repetitive stimulation at 0.1–0.5/sec was recorded in acute experiments on spinal cats and kittens. Using the dynamic selective correlation method a search was made for areas of spontaneous activity with the same distribution of action potentials in time as in the averaged evoked response to a single stimulus. In the case of some neurons portions of the background which correlate reliably in structure with the evoked response repeated at an interval equal to or a multiple of the interval of stimulation. Reproduction of the rhythm of stimulation in the spontaneous activity is intensified with an increase in the total duration of preceding stimulation with the same input and shows positive correlation with the degree of posttetanic potentiation. The facts obtained are evidence of prolonged after-processes in spinal neurons.

Spinal kittens: Effect of clamping of the thoracic aorta on long-term increases in electromyograms due to a conditioning routine

Conditioning and pseudoconditioning routines have different effects on EMG responses recorded from the semitendinosus muscle of spinal kittens pinched on tail (CS) and toe (US) 50 times daily for 5 days. The response to tail pinches habituates during each day. However, conditioning routines cause increases in responses between days 5 and 1. Pseudoconditioning routines in which CS and US are separated by 10 sec have no comparable effect. Clamping thoracic aortas for 30 min severely reduces conditioning routines' effectiveness in producing increased responses. Because thoracic clamping selectively destroys the spinal cord interneurons, we conclude that interneurons are essential to the production by conditioning routines of long-term increases in responsiveness of isolated lumbosacral spinal cord.

Spinal kittens: Long-term increases in electromyograms due to a conditioning routine☆

Abstract The effects of conditioning and pseudo-conditioning routines on the habituation of electromyographic responses of spinal kittens to the pinches of the tail (CS) and toe (US) were compared for 5 days. Habituating animals in a conditioning routine caused daily increases in their responses to tail pinches. Habituating them in a pseudo-conditioning routine had no comparable effect. Hence, a conditioning routine appears to cause long-term increases in the responsiveness of the isolated lumbo-sacral spinal cord.