Margaret M. Bird

Protein kinase C-delta C2-like domain is a binding site for actin and enables actin redistribution in neutrophils

Neutrophils play a key role in host-defence mechanisms against invading pathogens, using their capacity to migrate, engulf micro-organisms and produce toxic radicals. Protein kinase C (PKC) isotypes are important intracellular regulators of these processes in neutrophils. PKC isotypes themselves are controlled by interactions with lipids, Ca(2+) and proteins. The C2-like domain of PKC-delta (deltaC2) has been identified as a protein-interaction domain in this PKC isotype. In the present paper we have investigated the contribution of protein interactions at this domain to the regulation/function of PKC-delta in neutrophils. Using affinity chromatography we identified actin as a deltaC2 binding partner in these cells. Fluorescein-labelled deltaC2, microinjected into immobilized neutrophils, interacts with filamentous actin (F-actin) inside the cell. PKC-delta co-localizes with F-actin in neutrophils, in lamellipodia at the leading edge of the cell. Stimulation with phorbol ester or IgG-opsonized Staphylococcus aureus results in co-ordinated redistribution of PKC-delta and F-actin, and a PKC-delta inhibitor inhibits these changes. Microinjection of deltaC2 also inhibits F-actin redistribution. Thus PKC-delta binds to F-actin through its C2 domain, and these interactions are important in regulating actin redistribution in neutrophils.

Acetylcholine as a regulator of neurite outgrowth and motility in cultured embryonic mouse spinal cord.

Time-lapse photography was used to examine the effects of acetylcholine on the outgrowth and motility of neurites in cultures of mouse spinal cord. Addition of acetylcholine to the culture medium at concentrations of 100, 10 and 1 microM, caused inhibition of neurite outgrowth. Outgrowth rates were significantly different from those of controls at all concentrations tested. The motility of the growth cones was significantly reduced by treatment with 100 and 10 microM acetylcholine. Blockade of the muscarinic receptor by atropine did not significantly alter the effects of acetylcholine on outgrowth or motility. However when the nicotinic antagonist mecamylamine was added at the same time as acetylcholine the inhibitory effects of the acetylcholine were blocked and no changes in the rate of neurite outgrowth or in growth cone motility were detected. These results suggest that acetylcholine may have a role in the regulation of process outgrowth within the spinal cord with these effects mediated through the nicotinic receptor.

Cellular uptake of the prion protein fragment PrP106-126 in vitro.

The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrPc). This pathological isoform (PrPSc) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrPSc in affected tissue. To investigate the possible uptake mechanisms that may be required for PrPSc-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrPc expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrPSc in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.

The development of synapses in vitro between previously dissociated chick spinal cord neurons.

SummaryThe formation and development of synaptic contacts between dissociated chick spinal cord neurons has been investigated. By the 6th day in vitro “immature” profiles with few vesicles were observed. By 14–18 days “mature” types with numerous vesicles were found, indistinguishable from those of newly hatched chick spinal cord. After this period degeneration occurred, and was especially marked in the post-synaptic element. Such degeneration could be postponed by the addition of small numbers of somatic muscle cells. The Kanaseki and Kadota (1969) technique was applied to the study of coated vesicles at various stages of synaptic development.

Microtubule-synaptic vesicle associations in cultured rat spinal cord neurons

SummaryThis paper describes new ultrastructural features of neural processes and of synapses in cultured CNS tissue treated with albumin before fixation using a modification of the technique recently introduced by Gray (1975). Nerve fibre bundles in explants of foetal spinal cord grown in vitro for 15–18 days were transected microsurgically. After transection the cultures were exposed to 20% albumin in distilled water and then fixed in unbuffered osmium tetroxide followed by unbuffered glutaraldehyde.In this material, but not in controls (injured but not exposed to albumin; exposed to albumin without injury) microtubules were found within many axonal varicosities, often situated close to presynaptic membrane specializations. These microtubules were closely associated with vesicles resembling synaptic vesicles, which were occasionally aligned in rows along the microtubules. Similar vesicle-microtubule associations were also found in non-terminal axons. Microtubules were also observed very close to some postsynaptic densities.The possibility that the microtubule-vesicle associations are involved in vesicle movements (along axons and/or within axon terminals) is discussed. A more direct involvement of microtubules in terminals in the mechanism of transmitter release is also considered.

Patterns of expression of brain-derived neurotrophic factor and tyrosine kinase B mRNAs and distribution and ultrastructural localization of their proteins in the visual pathway of the adult rat.

We have examined the cellular and subcellular distribution and the patterns of expression of brain-derived neurotrophic factor (BDNF), and of its high affinity receptor, tyrosine kinase B (TrkB), in retinorecipient regions of the brain, including the superior colliculus, the lateral geniculate nucleus and the olivary pretectal nucleus. In the retinorecipient layers of the superior colliculus, BDNF protein and mRNA were present in the cell bodies of a subpopulation of neurons, and BDNF protein was present in the neuropil as punctate or fiber-like structures. In the lateral geniculate nucleus, however, BDNF mRNA was not detected, and BDNF protein was restricted to punctate and fiber-like structures in the neuropil, especially in the most superficial part of the dorsal lateral geniculate nucleus, just below the optic tract. At the ultrastructural level, BDNF protein was localized predominantly to axon terminals containing round synaptic vesicles and pale mitochondria with irregular cristae, which made asymmetric (Gray type I) synaptic specializations (R-boutons). Enucleation of one eye was followed by loss of BDNF immunoreactivity and disappearance of BDNF-positive R-boutons in the contralateral visual centers, confirming the retinal origin of at least most of these terminals. TrkB was present in postsynaptic densities apposed to immunoreactive R-boutons in the superior colliculus and lateral geniculate nucleus, and was also associated with axonal and dendritic microtubules. These findings suggest that BDNF is synthesized by a subpopulation of retinal ganglion cells and axonally transported to visual centers where this neurotrophin is assumed to play important roles in visual system maintenance and/or in modulating the excitatory retinal input to neurons in these centers.

Role of glutamate in the regulation of the outgrowth and motility of neurites from mouse spinal cord neurons in culture.

The excitatory amino acid glutamate has been shown to be toxic to a number of neuronal cell types both in vitro and in vivo. It has also been shown to be capable of controlling the development of neurons grown in vitro. Using time‐lapse video microscopy techniques the effects of glutamate on the rate of neurite outgrowth and growth cone motility were examined on cultured mouse spinal cord neurons. Concentrations in the range of 1 to 100 µM caused a significant inhibition of neurite outgrowth and concentrations of 10 and 100 µM significantly inhibited growth cone activity. In addition it was shown that the kainate/AMPA receptor antagonist (±)3‐(2‐carbvoxypiperazin‐4‐yl)‐propyl‐l‐phosphonic acid, but not the NMDA receptor antagonist 6,7‐dinitroquinoxaline‐2,3‐dione, was capable of blocking the inhibitory actions of glutamate on both outgrowth and motility. These results show that, at least in the culture system employed, glutamate might have a role in regulating neuronal development and function.

The culture of previously dissociated embryonic chick spinal cord cells on feeder layers of liver and kidney, and the development of paraformaldehyde induced fluorescence upon the former

SummarySpinal cord cells from embryonic chicks were cultured upon liver and kidney feeder layers of similar species origin. Successful cultures were obtained with inocula of cord cells containing as few as 15 000 cells ml−1, whereas without feeder layers at least 200 000 ml−1 are ordinarily required. Upon liver, many neurons and processes became intensely fluorescent, a property seldom shared by those grown upon kidney. Many processes upon liver contained large numbers of dense-cored vesicles, significantly larger and more numerous than in those grown upon kidney. We conclude that association with liver feeder layers has the consequence of producing in cord cells fluorescence and ultrastructural characteristics appropriate to catecholamine content, through a mechanism as yet unknown.

Neurite outgrowth‐regulating properties of GABA and the effect of serum on mouse spinal cord neurons in culture

Time‐lapse photography was used to examine the effects of γ‐aminobutyric acid (GABA) on the outgrowth and motility of neurites in cultures from mouse spinal cord. GABA at concentrations of 100, 10 and 1 μm caused significant inhibition of neurite outgrowth and the motility of growth cones was significantly reduced by treatment with 100 and 10 μm GABA. This effect was mimicked by the GABAB receptor agonist baclofen, whereas the GABAA receptor agonist muscimol had no effect. The effect of GABA on outgrowth and motility seems to be dependent on the type of serum employed. The results reported here were obtained only when heat‐inactivated serum was used and not when non heat‐inactivated serum was added to the culture medium. They suggest that GABA has a role in the regulation of process outgrowth within the embryonic mouse spinal cord.

The development and ultrastructure of previously dissociated foetal human cerebral cortical cells in vitro.

SummaryCells from foetal human cerebral cortex were mechanically dissociated and subsequently maintained in vitro for periods ranging between three and twenty-eight days.The ultrastructure of these cells at different stages of their development in culture was extensively examined. Nuclear and cytoplasmic features were extremely variable and a wide range of cell types was evidently represented. Of the three principal cell types found i.e. neurons, neuroglia and mesenchymal cells, only a minority of cells was classified with confidence, particularly during the first two weeks in culture.Extensive intercellular junctions of the adhaerens variety, common after 14 days in vitro were present at an earlier stage of development than synaptic profiles. First indications of synapse formation were observed after 21 days in vitro and after 24 days presynaptic sites filled with synaptic vesicles and with well defined presynaptic and postsynaptic thickenings were found. The significance of some of the features observed are both considered and discussed.