Cellular and subcellular compartmentation of creatine kinase in brain.

Creatine kinase (CK) catalyzes a reversible transphosphorylation reaction that is believed to play a crucial role in the maintenance and channeling of high energy phosphate in tissues with high energy demands. In rat brain development, cytosolic (nonmitochondrial) CK levels increase rapidly during the peak period of myelination and remain high in the adult. The cellular compartmentation of CK was investigated through the use of primary cultures of neurons, oligodendrocytes and astrocytes. The CK activity in cultured oligodendrocytes, which expressed both enzymatic and immunocytochemical markers of myelin, was the highest of the cell types examined and comparable to levels measured in whole adult brain; these observations suggest a role for CK in myelinogenesis. We found that low density, dividing astrocyte cultures also exhibited high B-CK (brain isoenzyme of CK) immunoreactivity, with the nuclear CK staining being especially intense. We studied these cultured astrocytes in more detail with respect to their intense nuclear CK immunoreactivity. Optical sections of astrocyte nuclei taken with a confocal microscope show that the high B-CK present is actually contained within the nucleus, with a nucleoplasmic distribution that does not co-localize with DNA or RNA. To ascertain whether the high nuclear B-CK in proliferating astrocytes correlated with ongoing cell division, we conducted experiments with confluent, nondividing cultures. These results show that both CK enzyme activity and immunoreactivity are high in the nucleus of proliferating astrocytes, and significantly reduced in the nucleus of quiescent, nondividing astrocytes. The high level of CK protein and activity in the nucleus of proliferating astrocytes suggests a role for CK in cell division/nuclear function.(ABSTRACT TRUNCATED AT 250 WORDS)

Denervation does not alter the number of neuronal bungarotoxin binding sites on autonomic neurons in the frog cardiac ganglion.

The binding of neuronal bungarotoxin (n-BuTX; also known as bungarotoxin 3.1, kappa-bungarotoxin, and toxin F) was analyzed in normal and denervated parasympathetic cardiac ganglia of the frog Rana pipiens, n-BuTX blocks both EPSPs and ACh potentials at 5-20 nM, as determined by intracellular recording techniques. Scatchard analysis on homogenates indicates that cardiac ganglia have two classes of binding sites for 125I-n-BuTX: a high-affinity site with an apparent dissociation constant (Kd,app) of 1.7 nM and a Bmax (number of binding sites) of 3.8 fmol/ganglion and a low-affinity site with a Kd,app of 12 microM and a Bmax of 14 pmol/ganglion. alpha-Bungarotoxin does not appear to interfere with the binding of 125I-n-BuTX to either site. The high-affinity binding site is likely to be the functional nicotinic ACh receptor (AChR), given the similarity between its affinity for 125I-n-BuTX and the concentration of n-BuTX required to block AChR function. Light microscopic autoradiographic analysis of 125I-n-BuTX binding to the ganglion cell surface reveals that toxin binding is concentrated at synaptic sites, which were identified using a synaptic vesicle-specific antibody. Scatchard analysis of autoradiographic data reveals that 125I-n-BuTX binding to the neuronal surface is saturable and has a Kd,app similar to that of the high-affinity binding site characterized in homogenates. Surface binding of 125I-n-BuTX is blocked by nicotine, carbachol, and d-tubocurarine (IC50 less than 20 microM), but not by atropine (IC50 greater than 10 mM). Denervation of the heart increases the ACh sensitivity of cardiac ganglion cells but has no effect upon the number of high-affinity binding sites for 125I-n-BuTX in tissue homogenates. Moreover, autoradiographic analysis indicates that denervation does not alter the number of 125I-n-BuTX binding sites on the ganglion cell surface. n-BuTX is as effective in reducing ganglion cell responses to ACh in denervated ganglia as it is in normally innervated ganglia. These results suggest that denervation alters neither the total number of nicotinic AChRs in the cardiac ganglion nor the number found on the surface of ganglion cells. These autonomic neurons thus respond differently to denervation than do skeletal myofibers. The increase in ACh sensitivity displayed by cardiac ganglion cells upon denervation cannot be explained by changes in AChR number.

Creatine kinase activity in postnatal rat brain development and in cultured neurons, astrocytes, and oligodendrocytes.

The development and distribution of cytosolic creatine kinase (CK) activity was studied in rat brain and in cell culture. The activity of CK in whole brain increased almost fivefold during the period from birth to day 40 when adult levels of 18-19 U/mg of protein were attained. The distribution of CK activity was examined in dissected regions of the adult brain and was nonuniform; the cerebellum, the striatum, and the pyramidal tracts contained significantly higher CK activity than did whole brain. The cellular compartmentation of CK was investigated using primary cultures of purified neurons, astrocytes, and oligodendrocytes. The CK activity in neurons increased fourfold greater than that measured at the time of isolation to 4 U/mg of protein. The CK activity in astrocytes cultured for 20 days was 3.5 U/mg of protein and was 1.5-fold greater than that measured at the time of isolation. In contrast, the CK activity in cultured oligodendrocytes (day 20) was three- to fourfold higher than that determined in astrocytes and almost sevenfold higher than the activity measured at the time the cells were isolated. The high levels of CK in cultured oligodendrocytes suggest a role for this enzyme in oligodendrocyte function and/or myelinogenesis.

Deprived somatosensory-motor experience in stumptailed monkey neocortex: dendritic spine density and dendritic branching of layer IIIB pyramidal cells.

Infant macaque monkeys (Macaca arctoides) were individually raised to age 6 months in large clear cubes built into one wall of a control colony that allowed them visual access to it but not tactile contact. The two deprivation conditions (Cond 2 and Cond 3) were equal both in physical size and with respect to partial social isolation. They differed in the degree of somatosensory-motor opportunity available during development in that the Cond 2 chamber was empty, whereas Cond 3 contained ladders, a trapeze, and play objects. Four monkeys from each of these conditions were compared with four colony-reared (Cond CR) monkeys. Neuroanatomical changes were evaluated by using light microscopy in Golgi-Cox-stained neocortex. Dendritic spines on the apical shafts of layer IIIB pyramidal cells were counted in primary motor (MI), somatosensory (SI), and visual (area 17, V1) cortical regions. Layer IIIB pyramidal neurons with somas of medium size were selected from each cortical region and the density of apical dendritic spines determined. The basilar dendritic branches of these same neurons were traced, and the dendritic branching complexity was assessed in order to compare the sensitivity of the dendritic spine and branching measures consequent to deprived rearing. The number of apical dendritic spines was significantly reduced in Cond 2 when compared with either Cond 3 or Cond CR (which did not differ from each other). This occurred in both MI and SI cortex, but not in the visual cortex, the region used as a control for a global brain effect. Branching complexity measured on the same pyramidal neurons was reduced only in MI cortex of Cond 2. These results show spine density, a more direct measure of neuronal connectivity, to be the more sensitive measure of early environmental deprivation. Also, the enriched environment provided by Cond 3 relative to Cond 2 offset the effect of partial social isolation such that both morphometric measures had values comparable to Cond CR monkeys.

Detection of 60-hertz vertical electric fields by rats.

Rats were trained to press levers to indicate the presence or absence of 60-Hz vertical electric fields at intensities from 0 to 27 kV/m (rms). The probability of detecting the field increased as the strength of the field increased. The shape of the detection curve (psychometric function) for most subjects (Ss) was similar whether the discriminative stimulus was the electric field or a tone. Two protocols were used to estimate the minimum field intensity necessary to detect the field (Reiz Limen, RL). The RL was estimated to be 13.3 kV/m (rms) when using one protocol (the staircase method) and 7.9 kV/m (rms) when using another protocol (the method of constant stimuli).