Transcription of a new zinc finger gene, rKr1, is localized to subtypes of neurons in the adult rat CNS.

Proteins which share zinc finger DNA binding motifs comprise one of the main families of transcription factors. We have previously described rKr1, a new rat Cys2/Hys2 zinc finger gene of the Krüppel gene family. This gene is predominantly expressed in the nervous system, with highest abundance in neurons and with lower abundance in developing oligodendrocytes of the CNS. Here, we have undertaken a detailed anatomical analysis of rKr1 expression in the adult brain of the rat using in situ hybridization. Our results show that rKr1 is expressed in a specific manner in defined subpopulations of neurons in many regions of the adult brain. Moderate levels of rKr1 mRNA were detectable in some structures of the telencephalon (e.g. cerebral cortex and hippocampus) and a few nuclei of the thalamus. The highest degree of labelling was seen in both upper and lower motor neurons of the mesencephalon and rhombencephalon (e.g. red nucleus, gigantocellular reticular nuclei, motor nuclei of the cranial nerves). High levels of rKr1 expression were also present in spinal motoneurons and dorsal root ganglion cells. In order to determine if rKr1 gene expression can be regulated, we have examined the expression pattern of rKr1 in the facial nucleus in response to facial nerve lesion. The expression of rKr1 in the facial nucleus showed a differential downregulation, reaching lowest levels 1 week after transection of the facial nerve. By 3 weeks after lesion, expression of rKr1 on the operated side of the brain reached normal levels and was identical to that of the unoperated side. These data suggest that rKr1 could be involved in the maintenance of the phenotypic differentiation of specific neuronal subtypes including motoneurons.

Axonal regeneration contributes to repair of injured brainstem-spinal neurons in embryonic chick.

Recent results have demonstrated complete anatomical and functional repair of descending brainstem-spinal projections in chicken embryos that underwent thoracic spinal cord transection prior to embryonic day 13 (E13) of the 21 d developmental period. To determine to what extent axonal regeneration was contributing to this repair process, we conducted experiments using a double retrograde tract-tracing protocol. On E8-E13, the upper lumbar spinal cord was injected with the first fluorescent tracing dye to label those brainstem-spinal neurons projecting to the lumbar cord at that time. One to two days later (on E10-E15), the upper to mid-thoracic spinal cord was completely transected. After an additional 7-8 d, a different second fluorescent tracing dye was injected into the lumbar cord at least 5 mm caudal to the site of transection. Finally, 2 d later on E19 to postnatal day 4, the CNS was fixed and sectioned. Brainstem and spinal cord tissue sections were then viewed with epifluorescence microscopy. In comparison to nontrasected control animals, our findings indicated that there were relatively normal numbers of double-labeled brainstem-spinal neurons after a transection prior to E13, whereas the number of double-labeled and second-labeled brainstem-spinal neurons decreases after an E13-E15 transection. In addition, at each subsequent stage of development from E10 to E12, there was a greater number of double-labeled brainstem-spinal neurons (indicating regeneration of previously severed axons) than cell bodies labeled with the second fluorescent tracer alone (indicating subsequent development of late brainstem-spinal projections). Assessment of voluntary open-field locomotion (hatchling chicks) and/or brainstem-evoked locomotion (embryonic or hatchling) indicated that functional recovery of animals transected prior to E13 was indistinguishable from that observed in control chicks (sham operated or unoperated). Taken together, these data suggest that regeneration of previously axotomized fibers contributes to the observed anatomical and functional recovery after an embryonic spinal cord transection.

Suppression of the onset of myelination extends the permissive period for the functional repair of embryonic spinal cord.

In an embryonic chicken, transection of the thoracic spinal cord prior to embryonic day (E) 13 (of the 21-day developmental period) results in complete neuroanatomical repair and functional locomotor recovery. Conversely, repair rapidly diminishes following a transection on E13-E14 and is nonexistent after an E15 transection. The myelination of fiber tracts within the spinal cord also begins on E13, coincident with the transition from permissive to restrictive repair periods. The onset of myelination can be delayed (dysmyelination) until later in development by the direct injection into the thoracic cord on E9-E12 of a monoclonal antibody to galactocerebroside, plus homologous complement. In such a dysmyelinated embryo, a subsequent transection of the thoracic cord as late as E15 resulted in complete neuroanatomical repair and functional recovery (i.e., extended the permissive period for repair).

Functional repair of transected spinal cord in embryonic chick.

The purpose of this study was to determine the developmental stage of the chick embryo when descending spinal tracts lose the capacity for anatomical and functional repair after complete transection of the thoracic spinal cord. Previous studies have demonstrated that the first reticulospinal projections descend to the lumbar cord by embryonic day (E) 5. A comparison of the distribution and density of retrogradely labelled brainstem-spinal neurons in embryos versus hatchling chicks suggests that the descent of all brainstem-spinal projections is essentially complete to lumbar levels between E10 and El2. Transections and control sham operations were performed on different embryos from E3 through E14 of development. After a recovery period of 5-18 days, the extent of anatomical repair was assessed by injecting a small volume of a retrograde tract-tracing chemical into the upper lumbar spinal cord, caudal to the transection site. The brainstem nuclei were then examined for the number and distribution of retrogradely labelled brainstem-spinal neurons. In comparison to control animals, anatomical recovery appeared to be complete for embryos transected as late as E12, whereas thoracic cord transections conducted on E13-E14 resulted in reduced labelling of most brainstem-spinal nuclei. In addition, a number of E3-E6 transected embryos were allowed to hatch and with some assistance a few E7-E14 transected embryos also hatched. Functional recovery was assessed by behavioral observations and by focal electrical stimulation of brainstem locomotor regions (known to have direct projections to the lumbar spinal cord). Brainstem stimulation experiments were undertaken on transected and control embryos, either in ovo on E18-E20 or after hatching. Leg and wing muscle electromyographic recordings were used to monitor any brainstem evoked motor activity. Voluntary open-field locomotion (hatchling chicks) or brainstem evoked locomotion (embryonic or hatchling) in animals transected on or before E12 was indistinguishable from that observed in control (i.e. sham-operated or unoperated) chicks, indicating that complete functional recovery had occurred. In contrast, chicks transected on or after El3 showed reduced functional recovery. Since a previous study has shown that neurogenesis in chick brainstem-spinal neurons is complete prior to E5, the possible intrinsic neuronal mechanisms underlying the repair of descending supraspinal pathways are: (1) subsequent projections from later developing (undamaged) neurons, or (2) regrowth of previously axotomized projections (regeneration). For the E5-E12 chick embryos examined in this study, significant descending supraspinal fibers are present within the thoracic cord at the time of transection. Even if the transection is made at E12, when descending projections have completed their development to the lumbar cord, there is still a similar number and distribution of brainstem-spinal neurons labelled afterward (when compared to controls). This suggests that regeneration of previously axotomized projections may account for some of the observed anatomical and functional repair of brainstem-spinal pathways.

Stimulation of the brainstem reticular formation evokes locomotor activity in embryonic chicken (in ovo).

This study was designed to examine the period of embryonic chick development during which descending brainstem-spinal projections, originating from defined avian brainstem locomotor regions, become functionally active. Locomotor activity was examined using a new in ovo preparation for the focal electrical stimulation of embryonic brainstem locomotor regions. Embryos or hatchlings were anesthetized and mounted in a stereotaxic apparatus. Leg and wing muscle electromyographic (EMG) recordings were used to monitor any brainstem-stimulated motor activity. At present, we have been successful in demonstrating coordinated brainstem-evoked locomotion in embryos as early as embryonic day 15. The patterns of evoked locomotor activity were similar to locomotion evoked in hatchling chicks and were of 4 types: (1) alternating hindlimb movements ('stepping'), (2) synchronous (in-phase) hindlimb movements ('hatching'), (3) synchronous wing movements ('flapping'), and (4) simultaneous 'stepping' and 'flapping'. The cycle durations of evoked embryonic hindlimb movements are shorter than those observed for hatchling chicks. The present results are the first direct demonstration of functional connections between descending supraspinal neurons and spinal locomotor circuits at such an early stage of embryonic development. With modifications in technique, it may be possible to demonstrate functional connections at even earlier stages of embryonic development.

Direction-selective adaptation in simple and complex cells in cat striate cortex.

1. The selectivity of adaptation to unidirectional motion was examined in neurons of the cat striate cortex. Following prolonged stimulation with a unidirectional high-contrast grating, the responsivity of cortical neurons was reduced. In many units this decrease was restricted to the direction of prior stimulation. This selective adaptation produced changes in the degree of direction selectivity of the cortical units (as measured by the ratio of the response to motion in the preferred direction to that in the nonpreferred direction). 2. The initial strength of the directional preference of a given cortical unit did not determine the degree of direction-selective adaptation. Indeed, even non-direction-selective units could exhibit pronounced direction-selective adaptation. The degree of direction-selective adaptation was also independent of the overall decrease in responsivity during adaptation. 3. There was no difference between simple and complex cells in the total amount of adaptation observed. The selectivity of the adaptation, however, did differ between these two cell types. As a group, simple cells showed significant direction-selective adaptation, whereas complex cells did not. The directional preference of most simple cells decreased following preferred direction adaptation and many highly direction selective simple cells became non-direction selective. In addition, simple cells became significantly more direction selective following nonpreferred direction adaptation. 4. Some complex cells also demonstrated direction-selective adaptation. There was, however, much more variability among complex cells than simple cells. Some complex cells actually increased direction selectivity following preferred direction adaptation. These differences between simple and complex cells suggest that changes in direction selectivity following unidirectional adaptation are not due to simple neuronal fatigue of the unit being recorded, but depend on selective adaptation of afferent inputs to the unit. 5. The spontaneous activity of many cortical neurons decreased following preferred direction adaptation but increased following adaptation in the nonpreferred direction. The response to a stationary grating also decreased following preferred direction adaptation. However, there was very little change in the response to a stationary grating following adaptation in the nonpreferred direction.