Essentially pure (>95%) cultures of microglia were established from neopallia of newborn rats and used for whole-cell patch-clamp recording of electrophysiological properties and for proliferation studies. Two types of cultures were examined: 1) "Primary" cultures were grown in culture medium with serum and used within 3 weeks of isolation; 2) and "Colony-stimulating factor (CSF)-1-stimulated" cultures were derived from 3-week-old "primary" cultures by passaging and culturing them for several weeks longer in the presence of conditioned medium enriched in CSF-1. Microglia in the "primary" cultures expressed: 1) an inwardly rectifying K+ current (Kir) that was inhibited by Ba2+; 2) an outwardly rectifying K+ current (Kv) with many similarities to the cloned Kv1.3 channel of lymphocytes, including block by nanomolar concentrations of charybdotoxin (ChTX) and margatoxin (MgTX); and 3) an outwardly rectifying anion current with time- and voltage-independent gating. The anion current is activated reversibly under cell swelling conditions, i.e., after exposure to a hypo-osmotic bathing medium. The anion channels are highly permeable to Cl-, measurably permeable to gluconate (P(gluconate)/ PCl = 0.34), and blocked by flufenamic acid, 4-nitro-2-(3-phenylpropylamino)- benzoic acid (NPPB), and 6, 7-dichloro-2-cyclopentyl-2, 3-dihydro-2-methyl-1-oxo-1H-inden-5-yl (oxy) acetic acid (IAA-94). Microglia in the "CSF-1-stimulated" cultures expressed Kir and Cl- current, but not Kv current. Proliferation in the latter type of cultures could be slowed by omission of the CSF-1 enriched supernatant for 2 days and stimulated by adding back the conditioned medium. This "CSF-1-stimulated" proliferation was inhibited by Ba2+ (Kir blocker), and the Cl(-)-channel blockers flufenamic acid, NPPB, and IAA-94, whereas the Kv blockers ChTX and MgTX had no effect. Thus, Kir and Cl- channels appear to be necessary for "CSF-1-stimulated" proliferation of rat microglia, and there is no evidence that even a transient activation of Kv is necessary.

Properties of K+ and Cl- channels and their involvement in proliferation of rat microglial cells

We examined the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the morphology, cell motility, cytoskeletal organization, and phagocytic activity of microglia in tissue cultures initiated from neopallia of newborn C3H/OuJ mice. Normally, the microglia in our cultures are non-migratory and Mac-1 positive, have ameboid cell morphology, no polarity, many short processes that extend into lamellipodia in opposing directions, and undulating cell membrane projections. When 1-5 micrograms/ml LPS is added to such cultures, some cells acquire polarity by forming a large lamellipodium and begin to migrate. Two hours later migration ceases; the membrane undulations stop; and the cells become non-polar, assume a large, round, flat shape, and gradually develop many microspikes all over the cell body. Those cells that do not transform into large, round, flat cells enlarge and extend numerous lamellipodia in opposing directions. We found that the cytoskeleton of microglia is composed of actin, vimentin-containing intermediate filaments (IF) and microtubules (MT). Vimentin-containing IF and MT form dense networks that radiate into the cell periphery, whereas F-actin is diffusely arranged throughout the cytoplasm. The LPS-treated cells show changes in the organization of the main components of the cytoskeleton. F-actin is reorganized by the formation of bundles underneath and parallel to the cell membrane and other bundles projecting into the cores of the microspikes. The vimentin-containing IF dense network reorganizes into two condensed rings, with fine strands of IF extended between the two rings and the MT networks become less dense and extend throughout the cytoplasm. The LPS treatment potentiates the phagocytic activity of the microglia. However, approximately 30% of microglia lose the expression of MHC class II antigens.

Effect of bacterial wall lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures

The hematopoietic cytokine, colony-stimulating factor 1, is also a growth factor in the CNS: Congenital absence of CSF-1 in mice results in abnormal microglial response and increased neuron vulnerability to injury ☆

A voltage-gated K+ conductance resembling that of the human ether-a-go-go -related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance–voltage relation was half maximal at −59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (−14 vs. −39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol . 111:795–805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with K i = ∼40 μM, and the HERG-selective blocker, E-4031, blocked with K i = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.

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HERG-like K+ Channels in Microglia

We report that neurons in the central nervous system express colony stimulating factor-1 receptor (CSF-1R) mRNA and protein and that the expression has regional specificity. The presence of CSF-1R in neurons was demonstrated by the use of four different types of antibodies to CSF-1R and the presence of CSF-1R mRNA by in situ hybridization using oligonucleotide probe. In the steady state in most areas of the brain, CSF-1R is weakly expressed in only a few neurons. In the cerebellum, brainstem, and spinal cord, however, CSF-1R is expressed constitutively in greater numbers of neurons. After cerebral cortex ischemic injury, neurons in the area next to the ischemic lesion markedly upregulate CSF-1R. It is also upregulated in the contralateral cortex and in many other areas of the brain and spinal cord. We demonstrated that in cultures the ligand CSF-1 binds to its receptor (CSF-1R) in neurons and that reduction of the number of apoptotic neurons and potentiation of neuron survival is CSF-1 dose dependent. We propose that CSF-1/CSF-1R signaling is an important regulatory pathway between neurons, microglia, and astrocytes.

Expression of colony stimulating factor‐1 receptor (CSF‐1R) by CNS neurons in mice

Microglia have been known as a cellular entity of the CNS for about 73 years (del Rio Hortega, 1919 a,b,c,d). Their morphology, based on silver carbonate stains (del Rio Hortega, 1932) and more recently, on immunocytochemistry (Perry and Gordon, 1991), has been described; their distribution has been determined by immunocytochemistry (Lawson et al., 1990); and their response to pathological changes has been examined by classical histological methods, electron microscopy and by immunocytochemistry (Dickson et al., 1991; Polak et al., 1982, Streit and Kreutzberg, 1988). Their function, however, other than that denoted by the term “brain macrophages”, is only now being investigated. Microglia are components of the neuron-glia and the neuroimmune networks (Benveniste, 1988; Goetzl and Spector, 1980). They communicate by means of a number of signaling molecules. Because of the complexity of the networks and multiple signalling, study of microglia function in situ is difficult. Recently, in several laboratories, microglia and brain macrophages have been isolated and grown in tissue culture (reviewed by Hao et al., 1991), and under these conditions the secretion of trophic factors and response to various ligands can be evaluated and cell interactions postulated.

Paracrine and Autocrine Signalling in Regulation of Microglia Survival

The spermatheca of Melanoplus sanguinipes consists of a preapical and an apical diverticulum, and a long, thin ductus seminalis. Histologically, the three components are identical. The wall of the spermatheca includes a basement membrane, secretory and epithelial cells, and a cuticular intima. Small, discrete bundles of muscle occur outside the basement membrane. In each secretory cell is a large central cavity which connects with a cuticular channel (efferent ductule) running through the epithelial cell to the spermathecal lumen. During sexual maturation, light- and dark-staining vesicles accumulate in the secretory cells and discharge their contents into the central cavity. Simultaneously, glycogen accumulates in the epithelial cells. Allatectomy of newly emerged females renders the secretory cells unable to produce material, an effect which can be reversed by topical application of synthetic juvenile hormone. The secretion contains protein and acidic mucopolysaccharide. After insemination the quantitie...

The spermatheca of Melanoplus sanguinipes (Fabr.). I. Morphology, histology, and histochemistry

The spermathecal wall of Melanoplus sanguinipes includes a thick intima, secretory units, a basal lamella, and small bundles of muscle. Each secretory unit consists of a secretory cell and an epithelial cell, the latter surrounding the former on all sides except that adjacent to the basal lamella. The secretory cell shows the ultrastructure typical of cells engaged in the synthesis of protein for export. Distally the cell is invaginated to form a central cavity lined with microvilli. Secretion is released into the central cavity and travels via the end apparatus and efferent ductule to the spermathecal lumen. Basally, the secretory cell's plasma membrane is much folded and large numbers of mitochondria are concentrated in this region, along with small glycogen deposits, features perhaps related to the uptake of raw materials for manufacture of secretion. The epithelial cell flanges contain bundles of longitudinally arranged microtubules and large deposits of glycogen. Next to the basal lamella, the plasma...

The spermatheca of Melanoplus sanguinipes (Fabr.). II. Ultrastructure

The ejaculatory duct of the migratory grasshopper ( Melanoplus sanguinipes [Fabr.]) (Orthoptera : Acrididae) is divisible into 3 regions: upper ejaculatory duct (UED) into whose anterior end the accessory glands and vasa deferentia empty; the funnel characterized by its slit-like lumen; and the lower ejaculatory duct (LED). Anteriorly, the UED has a keyhole-shaped lumen surrounded by a thin intima and highly columnar epithelial cells whose most conspicuous feature is massive aggregations of microtubules. More posteriorly, the UED lumen differentiates into dorsal and ventral chambers, the former having a thick cuticular lining armed with spines. In the hindmost part of the UED, the ventral chamber expands to obliterate the dorsal chamber; its cuticular lining thickens, and conspicuous lateral evaginations develop. The thick cuticle includes 3 distinct layers and on its surface carries numerous spatulate processes. In this region, the epithelial cells develop numerous short microvilli beneath which are many mitochondria. As the funnel is reached, the intima becomes extremely thick, and the epithelial cells lack microvilli and most microtubules. Within the funnel, a new, very distinct form of cuticle appears, which is in “units”, each associated with an epithelial cell and having a rounded epicuticular cap. The new cuticle arises ventrally but rapidly spreads to encircle the entire lumen, at which point the LED is considered to begin. Beneath this new cuticle, the epithelial cells are columnar, have long microvilli, numerous mitochondria in the apical cytoplasm, and rough endoplasmic reticulum basally. Apically, adjacent cells are tightly apposed; however, prominent intercellular channels develop more basally. The ejaculatory duct's features are briefly discussed in terms of its role in spermatophore formation.

Ijaz Ahmed

Papers

Paracrine and Autocrine Signalling in Regulation of Microglia Survival

The spermatheca of Melanoplus sanguinipes (Fabr.). I. Morphology, histology, and histochemistry

The spermatheca of Melanoplus sanguinipes (Fabr.). II. Ultrastructure

Ejaculatory duct of the migratory grasshopper, Melanoplus sanguinipes (fabr.) (Orthoptera : Acrididae): A histological and ultrastructural analysis