Four types of glial cells could be distinguished in the grey matter of rat spinal cord slices at postnatal days 1-19 (P1-P19), based on their pattern of membrane currents as revealed by the whole cell patch clamp technique, and by their morphological and immunocytochemical features. The recorded cells were labelled with Lucifer Yellow, which allowed the subsequent identification of cells using cell-type-specific markers. Astrocytes were identified by positive staining for glial fibrillary acidic protein (GFAP). These were morphologically characterized by multiple, very fine and short processes and electrophysiologically by symmetrical, non-decaying K+ selective currents. Oligodendrocytes were identified by a typical oligodendrocyte-like morphology, lack of GFAP staining and positive labelling with a combination of O1 and O4 antibodies (markers of the oligodendrocyte lineage), and their membrane was dominated by symmetrical, passive, decaying K+ currents. The third population of glial cells was also characterized by positive staining for O1/O4 or only for O4 antigens, lack of GFAP staining and, in some cells, oligodendrocyte-like morphology. However, these cells could be distinguished by the presence of inwardly rectifying (KIR), delayed outwardly rectifying (KDR) and A-type K+ currents (KA), representing the most likely glial precursor cells of the oligodendrocyte lineage. The fourth population of glial cells had small somata and a widespread network of long processes with no apparent orientation preference. In one case, processes were positively labelled with GFAP, while 30% were characterized by faint, diffuse staining. These cells expressed a complex pattern of voltage-gated channels, namely Na+, KDR, KA and KIR channels. In contrast to neurons, the amplitude of Na+ currents was at least one order of magnitude smaller than the K+ currents, and none of these cells showed the ability to generate action potentials in the current clamp mode. Since none of these cells could be labelled by oligodendrocyte markers we assume that they were either astrocytes or glial precursor cells of the astrocyte lineage. The four cell types were found in all regions of the grey matter. When randomly accessing the glial cells, the probability of recording from the oligodendrocyte precursor cells and the glial cells with Na+ currents decreased during development. At P1-P3, 50% of the cells revealed the Na+ current, while at P13-P15 only 18% did. Concomitantly, the number of glial cells with astrocyte- and oligodendrocyte-like membrane currents increased from 19 and 12% to 41 and 35.5% respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

In the neonatal rat spinal cord, four types of glial cells, namely astrocytes, oligodendrocytes and two types of precursor cells, can be distinguished based on their membrane current patterns and distinct morphological features. In the present study, we demonstrate that these cells respond to the inhibitory neurotransmitters glycine and GABA, as revealed with the whole-cell recording configuration of the patch-clamp technique. All astrocytes and glial precursor cells and a subpopulation of oligodendrocytes responded to glycine. The involvement of glycine receptors was inferred from the observation that the response was blocked by strychnine and that the induced current reversed close to the Cl- equilibrium potential. GABA induced large membrane currents in astrocytes and precursor cells while oligodendrocytes showed only small responses. The GABA-activated current was due to the activation of GABAA receptors since muscimol mimicked and bicuculline blocked the response; moreover, the reversal potential was close to the Cl- equilibrium potential. Besides the increase in a Cl- conductance, GABAA receptor activation also induced a block of the resting K+ conductance, as observed previously in Bergmann glial cells. Our experiments show that while glial GABAA receptors are found in many brain regions and the spinal cord, glial glycine receptors have so far been detected only in the spinal cord. The restricted coexpression of glial and neuronal glycine receptors in a defined central nervous system grey matter area implies that such glial receptors may be involved in synaptic transmission.

Summary:The authors investigated the time course of leuko cyte infiltration compared with microglial activation in adult rat brain slices after permanent middle cerebral artery occlusion (MCAO). To distinguish peripheral leukocytes from microglia, the blood cells were prelabeled in vivo with Rhodamine 6G (Rhod6G) IV before induction of ischemia. At specific times after infarct, invading leukocytes, microglia, and endothelial cells were labeled in situ with isolectin (IL)B4-FITC (ILB4). Six hours after MCAO only a few of the ILB4+ cells were colabeled by Rhod6G. These cells expressed the voltage-gated inwardly and outwardly rectifying K+ currents characteristic of macrophages. The majority of the ILB4+ cells were Rhod6G and expressed a lack of voltage-gated channels, recently de scribed for ramified microglial cells in brain slices, or exhibited It has been known for over 75 years that focal cerebral ischemia induces an inflammatory response in the brain (Spielmeyer, 1922). Only recently, however, has it been recognized that brain inflammation may have a major impact on the extent of tissue damage and the subsequent recovery. Inflammation occurs with immediate and de layed cellular responses after ischemia. Because early recruitment of patients has been a major obstacle in stroke therapy, inflammation is a promising target for intervention that extends the time window for treatment considerably (Dirnagl et aI., 1999). Understanding isch emia-induced inflammation at the cellular and molecular level is a prerequisite for the development of new anti inflammatory strategies in stroke. Inflammation involves Supported by the DFG (SFB 507), BMBF, the Deutsche Schlagan fallstiftung, and the Hermann and Lilly Schilling Foundation.Address correspondence and reprint requests to Prof. Dr. Helmut Kettenmann, Max-Delbruck-Center for Mol Med. Cellular Neurosci ences, Robert-Rossle-Str.IO, 13092 Berlin-Buch, Germany.only an inward rectifier current, a unique marker for cultured (but unstimulated) microglia. Forty-eight hours after MCAO, all blood-borne and the majority of Rhod6G-cells expressed outward and inward currents indicating that the intrinsic mi croglial population exhibited physiologic features of stimu lated, cultured microglia. The ILB4+/Rhod6G-intrinsic mi croglial population was more abundant in the border zone of the infarct and their morphology changed from radial to ame boid. Within this zone, the authors observed rapidly migrating cells and recorded this movement by time-lapse microscopy. The current findings indicate that microglial cells acquire physiologic features of leukocytes at a later time point after MCAO.

Oligodendrocytes express two gap junction proteins, connexin32 (Cx32) and Cx45. To test for functional coupling between oligodendrocytes, cells were filled with the (Cx32‐permeable) dyes Lucifer Yellow (LY) and Neurobiotin®. Cells in slices from rat spinal cord were dialyzed via the patch pipette containing the dye while recording with the patch‐clamp technique. The dye‐labeled cells were identified as oligodendrocytes by their characteristic pattern of membrane currents and by morphology. In gray matter, 18% of the injected cells (N = 94) were coupled to more than three adjacent cells (slices from postnatal day 1 to 19). In contrast, in white matter, the dye was restricted to the injected cell (N = 63 for Lucifer Yellow injection only; N = 11 for LY and Neurobiotin®) indicating a lack of functional coupling. Immunolabeling of Cx32 in mature oligodendrocytes of white matter revealed that the gap junction protein is localized on the cell bodies and abaxonal processes which occupy non‐overlapping territories. In immature white matter and gray matter, Cx32 is mostly concentrated in the somatic region of the cells. In addition to Cx32, we have obtained immunocytochemical data that oligodendrocytes can express Cx45 with a labeling pattern different from the Cx32 expression. Two alternative interpretations of the coupling data are discussed: 1) that the presence of Cx32 in mature white matter oligodendrocytes does not serve for communication between cells, but rather for communication within oligodendrocytes in the sense of autocellular coupling, or 2) that the glial syncytium is furnished with a high degree of functional rectification at the oligodendrocytic side. GLIA 24:108–120, 1998. © 1998 Wiley‐Liss, Inc.

We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in gliat cells from rat spinal cord. In the present study, the patch-clamp technique was combined with the reverse transcription-mediated PCR to analyze the glycine receptor-subunit expression in individual glial cells of rats age 3-18 days. Using the patch-clamp technique in the whole-cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit-specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all glial cell types investigated, transcripts of the al subunit, but not of a2 or a3 subunits, were detected. In addition, about one-half the glial cells analyzed contained p3-subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown responses in the glial cells.

Basic fibroblast growth factor (bFGF; FGF‐2) has potent trophic effects on developing and toxically impaired midbrain dopaminergic (DAergic) neurons which are crucially affected in Parkinson's disease. The trophic effects of FGF‐2 are largely indirect, both in vitro and in vivo, and possibly involve intermediate actions of astrocytes and other glial cells. To further investigate the cellular and molecular mechanisms underlying the restorative actions of FGF‐2, and to analyse in more detail the changes within astroglial cells in the MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine)‐lesioned striatum, we have studied striatal expression and regulation of connexin‐43 (cx43), the principal gap junction protein of astroglial cells, along with the expression of glial fibrillary acidic protein (GFAP), FGF‐2, and functional coupling. Our results show an immediate, yet transient increase in cx43 mRNA, and a sustained increase in FGF‐2 mRNA, GFAP‐positive cells, and cx43‐immunoreactive punctata following the MPTP lesion, without any induction of functional coupling between astrocytes and other glial cells as revealed by dye coupling of patched cells. Unilateral administration of FGF‐2 in a piece of gelfoam caused a further increase in cx43‐positive punctata immediately adjacent to the implant, which was more pronounced than after application of a gelfoam containing the non‐trophic control protein in cytochrome C. These changes were parallelled by a small increase in cx43 protein determined by Western blot, but not by alterations in the coupling state of cells in the vicinity of the gelfoam implant. Although our data indicate that MPTP and exogenous FGF‐2 may alter expression and protein levels of cx43, they do not support the notion that increases in cellular coupling may underly the trophic and widespread actions of FGF‐2 in the MPTP‐model of Parkinson's disease. © 1996 Wiley‐Liss, Inc.

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El objetivo fue caracterizar la disponibilidad y promoción de alcohol asociados a los locales de venta y consumo de alcohol en Madrid, así como explorar las diferencias en su distribución en función de la tipología del local y las características socioeconómicas del área. Se utilizó el instrumento OHCITIES para caracterizar locales situados en 42 secciones censales de Madrid durante 2016. Se registró la densidad de locales y el número de locales con amplios horarios de apertura (12 o más horas). Se registró cualquier tipo de promoción asociada al local visible desde el exterior. Se compararon los porcentajes de características de disponibilidad y promoción asociada a los locales de consumo y venta de alcohol utilizando el test de chi cuadrado y la prueba exacta de Fisher. Se estimó la densidad de disponibilidad y promoción por sección censal y se exploró su distribución en función de las características socioeconómicas del área mediante el test de Kruskal-Wallis. Se registraron 324 locales, 241 de consumo y 83 de venta. La mayoría tenía un horario amplio de apertura (73,77%) y algún elemento promocional (89,51%). Los locales de consumo tenían horarios más amplios de apertura y más elementos promocionales que los de venta (p < 0,001). Se encontraron mayor densidad de locales, amplitud de horarios y elementos promocionales en áreas de nivel socioeconómico alto (todos p < 0,001). La disponibilidad y promoción estuvieron asociadas con los locales de venta y consumo de alcohol en Madrid. Futuras políticas cuyo objetivo sea la prevención del consumo de alcohol deben tener en cuenta la influencia de los tipos de locales y las características socioeconómicas del área en la distribución de la disponibilidad y promoción de alcohol. Palabras clave: Disponibilidad de alcohol; Locales de venta de alcohol; Promoción de alcohol; Nivel socioeconómico; Desigualdades. We aimed to characterize the availability and promotion of alcohol at alcohol outlets in Madrid and to compare them according to type of outlet and area-level socioeconomic status. We used the OHCITIES instrument to characterise the alcohol outlets in 42 census tracts of Madrid in 2016. We specified alcohol availability as the density of alcohol outlets and the number of alcohol outlets with extended opening hours (12 or more). We registered any type of promotion associated to alcohol outlets that could be perceived from outside the outlet. We calculated and compared proportions of availability and promotion by alcohol outlet (on-and off-premise) using chi-squared and Fisher Exact tests. We estimated the availability and promotion of alcohol densities per census tract according to area-level socioeconomic status. To assess statistical significance, we used Kruskal-Wallis tests. We recorded 324 alcohol outlets, 241 on-premise and 83 off-premise. Most of the outlets had extended opening hours (73.77%) and at least one sign promoting alcohol (89.51%). More on-premise outlets had extended opening hours and higher presence of alcohol promotion than off-premise (p < 0.001)...