Changing pattern of expression of parvalbumin immunoreactivity during human fetal spinal cord development.

Expression of the calcium binding protein parvalbumin (PV) by different classes of spinal neuron has been shown to be developmentally regulated in both rat and monkey. From postmortem studies of eight human cervical spinal cords ranging in age from 11 to 35 weeks postconceptional age, we report that parvalbumin immunoreactivity is similarly plastic in human lower cervical spinal cord development, with many changes occurring prenatally. At 11-14 weeks postconceptional age, there was prominent immunostaining of primary sensory afferents that could be seen coursing through the dorsal horn and extensively innervating the motoneuron pools. Motoneurons were also found to be clearly immunoreactive for choline acetyltransferase by this age. A few ventral horn neurons that were not motoneurons were also parvalbumin immunoreactive. By 24-27 weeks postconceptional age, sensory afferents were still immunoreactive, as were many other axons throughout the white matter. In addition, many ventral horn neurons were now immunoreactive as well as a few dorsal horn neurons. By 31-35 weeks postconceptional age, there was extensive immunostaining of neurons throughout the spinal cord, including a few moderately immunoreactive motoneurons. There were many immunopositive axons in all the white matter tracts except the corticospinal tracts; however, staining of sensory axons traversing the grey matter was less prominent by this age. In the rat, expression of PV by primary sensory neurons coincides with the onset of fetal limb movement. The onset of expression of PV in ventral horn neurons coincides with later developmental events after the arrival of corticospinal inputs, whereas widespread PV immunoreactivity in dorsal horn neurons marks the attainment of a mature pattern of PV expression. The extent to which expression of PV immunoreactivity can be taken to indicate landmarks in human development will be discussed.

The effect of a peripheral nerve lesion on calbindin D28k immunoreactivity in the cervical ventral horn of developing and adult rats.

Expression of calbindin D28k (CB) immunoreactivity by putative Renshaw cells is substantially downregulated by sciatic motoneuron axotomy in the adult rat. The present study investigated the effect of median and ulnar nerve lesion at different ages on ventral horn CB immunoreactivity 7 days after the injury to see whether similar results were obtained in the cervical cord and during development. Two major differences were observed. First, axotomy induced CB immunoreactivity in some motoneurons, confirmed by retrograde labeling of the injured neurons with fast blue (FB). Observation of fluorescent phagocytic microglia revealed that some motoneuron death occurred following lesions at postnatal day 2 (P2) and P7, but not at P21 or P63. A significantly higher proportion of remaining FB labeled motoneurons expressed CB following lesion at P2 (mean 33% +/- 7.6 SD) and P7 (30.6% +/- 5.2) than at P28 (14.0% +/- 1.9). Second, CB expression by putative Renshaw cells was not significantly downregulated ipsilateral to the lesion. CB immunofluorescent putative Renshaw cells were counted in sections containing FB labeled motoneurons. No consistent differences in the numbers of Renshaw cells ipsilateral and contralateral to the lesion were found at any age. To confirm that these neurons really were Renshaw cells, the mediators of recurrent inhibition to cholinergic motoneurons, we employed double-immunofluorescence labeling with confocal microscopy. The group of CB immunopositive neurons located among the converging ventral roots in the cervical cord were closely apposed by many axon terminals immunoreactive for (i) vesicular acetylcholine transporter and (ii) cholera toxin B localized to motor axon collaterals by injection of this tracer into a distal forelimb muscle. We conclude that motoneuron axotomy need not always downregulate CB expression in associated Renshaw cells. In addition, some brachial motoneurons respond to axotomy by expressing CB.

Gephyrin-like immunoreactivity is a marker for growing axons in the central nervous system of the immature rat.

The membrane-associated protein gephyrin can form part of the glycine receptor complex at inhibitory synapses. This study provides evidence for gephyrin localisation in the developing axons of the rat brain and spinal cord, with particular reference to the corticospinal tract. Using a well-characterised monoclonal antibody (MAb 7a) gephyrin-like immunoreactivity was found expressed by growing axons, disappearing as these axons became myelinated. Immunoelectron microscopy localised the antigen to the interior of small, unmyelinated axons in the dorsal funiculus of young rats. Western blot analysis of cervical spinal cord from different post-natal ages only detected one immunoreactive band at ages when immunohistochemistry revealed gephyrin-like immunoreactivity in both the grey matter and corticospinal tract. Furthermore, the molecular weight of this band corresponded to that of gephyrin, suggesting it is present both at synapses and in axons. The known tubulin binding ability of the gephyrin may have a role in stabilisation of microtubules in growing axons.

Developmental expression of parvalbumin by rat lower cervical spinal cord neurones and the effect of early lesions to the motor cortex.

Expression of calcium binding proteins (CaBPs), increasing neuronal activity and phases of synapse elimination are widely believed to be linked during development. We have employed immunocytochemistry to study the expression of the CaBP parvalbumin (PV) during the postnatal development of the lower cervical spinal cord and investigated how early lesions to the motor cortex, at the onset of corticospinal synaptogenesis, perturb the normal pattern of PV expression. This study confirms previous observations that in normal rats PV-like immunoreactivity is confined to large sensory afferents for at least 10 days postnatally (P10) and that the adult pattern of expression emerges from about P18 and involves mainly dorsal horn neurones. However, the study has also demonstrated a transient wave of expression in ventral horn neurones which reaches a maximum between P14-18 and declines thereafter. Unilateral lesions made at P7 to the forelimb motor cortex, which sends an almost completely crossed projection to the spinal cord, resulted in reduced neuronal expression of PV in the lower cervical spinal cord contralaterally at a range of ages (P14-31). The median ratio of PV positive neurones contralateral/ipsilateral to the lesion in spinal cord segments C7 and C8 was significantly lower (p < 0.01) at 56.0% (34.5-76.8 95% confidence limits, n = 14) than in sham operated controls (99.7%, range 93.7-113.6, n = 5). The lesion affected the transient wave of expression seen in ventral horn neurones during the third postnatal week as well as dorsal horn expression at older ages. We conclude that there is considerable plasticity in PV immunoreactivity during spinal cord development. PV is transiently expressed by ventral horn neurones at an age when movement control is functionally maturing. Early cortical lesions disrupt this transient phase of expression but also alter mature patterns of PV localisation. This suggests a critical role for corticospinal pathways in guiding maturation of segmental spinal cord circuitry.

A new small particle packing for faster analysis with high resolution.

The need for fast and efficient separations of complex samples such as pharmaceuticals and biologicals led to the development of fast, efficient, and reproducible 3.5 microns columns. Separations using 3.5 microns column are 30-50% faster at equal plate-count compared to 5 microns columns. The results show that the 3.5 microns columns (100 mm length) give the same efficiency and resolution of drug impurities as the 5 microns columns (150 mm length). For many analytical methods, switching to 3.5 microns columns saves time and reduces costs. Separation methodologies using 5 microns columns are easily modified to accommodate 3.5 microns columns of the same chemistry because efficiency, resolution and sensitivity remain the same. It is shown that 3.5 microns columns have lifetimes comparable to 5 microns columns of the same brand.