Confocal microscopic study of glial-vascular relationships in the retinas of pigmented rats.

Astroglia are interposed between the cerebral vasculature and neurons, where they may mediate the transfer of substances from the circulation to neurons and couple changes in neuronal activity to changes in cerebral blood flow. The retina is a particularly advantageous model system for studying glial-vascular interactions in situ. Confocal microscopy and three-dimensional image reconstruction were used to study the anatomical relationships between glia and the surface vasculature in retinas acutely isolated from adult pigmented rats. Retinas were immunostained using antibodies directed against the basal lamina surrounding the vasculature as well as antibodies directed against glial fibrillary acidic protein. Surface vessels of all calibers were contacted by the processes of astrocytes. The vitreal surfaces of the large retinal vessels were covered by a meshwork of immunoreactive astrocyte processes of a variety of shapes, whereas the scleral surfaces of the vessels were supported by thick bundles of astrocyte processes. In addition, glial cells were filled intracellularly with the gap junction-permeable tracers Lucifer yellow and Neurobiotin. Intracellular fills clearly demonstrated the presence of astrocytes with somata that were closely apposed to the large retinal vessels. Tracer-filled astrocytes displayed a variety and complexity of shapes that was not apparent in immunostained material. Gap junctional coupling was stronger between astrocytes adjacent to the same artery than between periarterial astrocytes and astrocytes located away from arteries. Significantly fewer Müller cells were labeled when Neurobiotin was injected into astrocytes associated with arteries than when Neurobiotin was injected into astrocytes that were distant from arteries.

Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: phenotypic impact in retina.

The inwardly rectifying potassium channel Kir4.1 has been suggested to underlie the principal K(+) conductance of mammalian Müller cells and to participate in the generation of field potentials and regulation of extracellular K(+) in the retina. To further assess the role of Kir4.1 in the retina, we generated a mouse line with targeted disruption of the Kir4.1 gene (Kir4.1 -/-). Müller cells from Kir4.1 -/- mice were not labeled with an anti-Kir4.1 antibody, although they appeared morphologically normal when stained with an anti-glutamine synthetase antibody. In contrast, in Müller cells from wild-type littermate (Kir4.1 +/+) mice, Kir4.1 was present and localized to the proximal endfeet and perivascular processes. In situ whole-cell patch-clamp recordings showed a 10-fold increase in the input resistance and a large depolarization of Kir4.1 -/- Müller cells compared with Kir4.1 +/+ cells. The slow PIII response of the light-evoked electroretinogram (ERG), which is generated by K(+) fluxes through Müller cells, was totally absent in retinas from Kir4.1 -/- mice. The b-wave of the ERG, in contrast, was spared in the null mice. Overall, these results indicate that Kir4.1 is the principal K(+) channel subunit expressed in mouse Müller glial cells. The highly regulated localization and the functional properties of Kir4.1 in Müller cells suggest the involvement of this channel in the regulation of extracellular K(+) in the mouse retina.

The critical period for ocular dominance plasticity in the Ferret's visual cortex.

Microelectrode recordings and optical imaging of intrinsic signals were used to define the critical period for susceptibility to monocular deprivation (MD) in the primary visual cortex of the ferret. Ferrets were monocularly deprived for 2, 7 or >14 d, beginning between postnatal day 19 (P19) and P110. The responses of visual cortical neurons to stimulation of the two eyes were used to gauge the onset, peak, and decline of the critical period. MDs ending before P32 produced little or no loss of response to the deprived eye. MDs of 7 d or more beginning around P42 produced the greatest effects. A rapid decline in cortical susceptibility to MD was observed after the seventh week of life, such that MDs beginning between P50 and P65 were approximately half as effective as those beginning on P42; MDs beginning after P100 did not reduce the response to the deprived eye below that to the nondeprived eye. At all ages, 2 d deprivations were 55-85% as effective as 7 d of MD. Maps of intrinsic optical responses from the deprived eye were weaker and less well tuned for orientation than those from the nondeprived eye, with the weakest maps seen in the hemisphere ipsilateral to the deprived eye. Analysis of the effects of 7 d and longer deprivations revealed a second period of plasticity in cortical responses in which MD induced an effect like that of strabismus. After P70, MD caused a marked loss of binocular responses with little or no overall loss of response to the deprived eye. The critical period measured here is compared to other features of development in ferret and cat.

Heterotypic coupling between glial cells of the mammalian central nervous system.

Heterotypic coupling, defined as gap-junctional coupling between cells of different classes, may be common among the different types of non-neuronal cells in the central nervous system. Since gap junctions provide a route for the intercellular exchange of signaling molecules, heterotypic coupling may serve to coordinate the activities of many types of "support cells" in the brain. The evidence for heterotypic coupling between astrocytes and oligodendrocytes, astrocytes and retinal Müller glial cells, and astrocytes and ependymal cells is reviewed. The finding that some heterotypic gap junctions are chemically rectifying implies that there is asymmetry between the two sides of these gap junctions, and the connexin composition of heterotypic gap junctions is discussed. Finally, I speculate about the functions of heterotypic gap junctions, including their proposed roles in K+ spatial buffering around axons and in the propagation of intercellular Ca2+ waves between astrocytes and other glial cells.

Modulation of neuronal activity by glial cells in the retina.

Glial-neuronal communication was studied by monitoring the effect of intercellular glial Ca2+ waves on the electrical activity of neighboring neurons in the eyecup preparation of the rat. Calcium waves in astrocytes and Müller cells were initiated with a mechanical stimulus applied to the retinal surface. Changes in the light-evoked spike activity of neurons within the ganglion cell layer occurred when, and only when, these Ca2+ waves reached the neurons. Inhibition of activity was observed in 25 of 53 neurons (mean decrease in spike frequency, 28 +/- 2%). Excitation occurred in another five neurons (mean increase, 27 +/- 5%). Larger amplitude Ca2+ waves were associated with greater modulation of neuronal activity. Thapsigargin, which reduced the amplitude of the glial Ca2+ increases, also reduced the magnitude of neuronal modulation. Bicuculline and strychnine, inhibitory neurotransmitter antagonists, as well as 6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and D(-)-2-amino-7-phosphonoheptanoic acid (D-AP7), glutamate antagonists, reduced the inhibition of neuronal activity associated with glial Ca2+ waves, suggesting that inhibition is mediated by inhibitory interneurons stimulated by glutamate release from glial cells. The results suggest that glial cells are capable of modulating the electrical activity of neurons within the retina and thus, may directly participate in information processing in the CNS.

Asymmetric gap junctional coupling between glial cells in the rat retina.

Gap junctional communication between glial cells is thought to play a role in K+ spatial buffering, in the propagation of inter-astrocytic Ca2+ waves, and in glial-neuronal signaling. In the present study, we characterize dye coupling between astrocytes, and between astrocytes and Müller cells, in the isolated rat retina. Whole-cell patch recordings were obtained from retinal astrocytes and Müller cells and the cells filled with Lucifer Yellow and neurobiotin. Spread of Lucifer Yellow to two to ten neighboring astrocytes occurred in 90% of the astrocyte recordings. After fixation and incubation of the retina with fluorescent conjugated streptavidin, neurobiotin was seen to label clusters of 13-88 astrocytes, as well as > 100 Müller cells. In contrast, when Müller cells were filled with Lucifer Yellow and neurobiotin, both tracers were confined solely to the recorded Müller cell. The uncoupling agents octanol, halothane, and doxyl-stearic acid were tested for their ability to uncouple retinal glia in situ. All three agents eliminated the visible spread of Lucifer Yellow from the injected astrocyte and the spread of neurobiotin into Müller cells. However, only doxyl-stearic acid combined with octanol eliminated the spread of neurobiotin between astrocytes. These results demonstrate that astrocytes in the rat retina are coupled to each other and to Müller cells. The astrocyte-to-Müller cell coupling is asymmetric, allowing transfer of the tracer in the forward direction only. In addition, astrocyte-to-Müller cell coupling is more sensitive to the uncoupling agents tested than is astrocyte-to-astrocyte coupling.

Calcium waves in retinal glial cells.

Calcium signals were recorded from glial cells in acutely isolated rat retina to determine whether Ca2+ waves occur in glial cells of intact central nervous system tissue. Chemical (adenosine triphosphate), electrical, and mechanical stimulation of astrocytes initiated increases in the intracellular concentration of Ca2+ that propagated at approximately 23 micrometers per second through astrocytes and Müller cells as intercellular waves. The Ca2+ waves persisted in the absence of extracellular Ca2+ but were largely abolished by thapsigargin and intracellular heparin, indicating that Ca2+ was released from intracellular stores. The waves did not evoke changes in cell membrane potential but traveled synchronously in astrocytes and Müller cells, suggesting a functional linkage between these two types of glial cells. Such glial Ca2+ waves may constitute an extraneuronal signaling pathway in the central nervous system.

Characterization of "plasma proteins" secreted by cultured rat macroglial cells.

The brain is isolated behind a blood-tissue barrier that restricts the access of circulating proteins to neural cells. There is evidence that some of these proteins are synthesized within the central nervous system. The present study examines the synthesis and secretion of such proteins by cultured macroglial cells. Primary glial cultures were derived from cortical and subcortical regions of neonatal rat brains, and subsequent secondary cultures were enriched in type-1 astrocytes, type-2 astrocytes, or oligodendrocytes. Newly synthesized proteins were immunoprecipitated from the culture media using antisera directed against whole rat serum. All three types of glial cells secreted a range of plasma proteins. In general, type-1 astrocytes secreted more of these proteins than did type-2 astrocytes or oligodendrocytes, although the one-dimensional polyacrylamide gel electrophoresis (PAGE) profiles were specific for each cell type. Anti-sera directed against specific plasma proteins identified three of the most abundant proteins secreted by type-1 astrocytes as transferrin, alpha-2-macroglobulin, and ceruloplasmin. Northern blot analysis of cellular RNA confirmed that type-1 astrocytes contained transferrin mRNA, and that it was more abundant in cultures derived from subcortical regions than from cortical regions. In situ hybridization studies revealed that virtually all type-1 and type-2 astrocytes contained transferrin mRNA. Since the proteins identified in this study have been proposed to have a variety of neurotrophic roles in the central nervous system, these data further extend the range of possible functions that glial cells may serve in the CNS.

Characterization and distribution of angiotensin II binding sites in fetal and neonatal astrocytes from different rat brain regions.

Although angiotensin II (Ang II) binding sites have been extensively investigated in brain, revealing the presence of both AT1 and AT2 subtypes in various areas, the question as to which cells express AT1 and AT2 sites is still open. We report here that primary cultures of astrocytes obtained from various brain regions of fetal (F17) and one-day-old rats express Ang II binding sites belonging only to the AT1 subtype. The binding sites have the same binding profile in all regions tested; however, much less binding was observed in membranes of astrocytes derived from cortical than from subcortical regions and almost none were found in neonatal cortex. In addition, the dispersion method used at the onset of culture affects the number of binding sites present at the end of the culture period.

Relation of cortical cell orientation selectivity to alignment of receptive fields of the geniculocortical afferents that arborize within a single orientation column in ferret visual cortex.

Neurons in the primary visual cortex of higher mammals are arranged in columns, and the neurons in each column respond best to light-dark borders of particular orientations. The basis of cortical cell orientation selectivity is not known. One possible mechanism would be for cortical cells to receive input from several lateral geniculate nucleus (LGN) neurons with receptive fields that are aligned in the visual field (Hubel and Wiesel, 1962). We have investigated the relationship between the arrangement of the receptive fields of geniculocortical afferents and the orientation preferences of cortical cells in the orientation columns to which the afferents provide visual input. Radial microelectrode penetrations were made into primary visual cortex of anesthetized adult sable ferrets. Cortical cells were recorded throughout the depth of the cortex, and their orientation preferences were determined. Cortical cell responses were then eliminated by superfusion of the cortex with either kainic acid (Zahs and Stryker, 1988) or muscimol. After the drug treatment, responses from many single units with distinct receptive fields were recorded. These responses were presumed to be those of geniculocortical afferents, because they had the response properties characteristic of LGN neurons, and because they could be recorded only in cortical layers that receive geniculate input. In 16 of 18 cases, the afferent receptive fields recorded in a single penetration covered an elongated region of visual space. In these penetrations, the best-fit line through the centers of the afferent receptive fields generally paralleled the preferred orientation of cortical cells recorded at the same site in cortex. These results are consistent with the Hubel and Wiesel (1962) model for the construction of oriented visual cortical receptive fields from geniculate inputs with aligned receptive fields.

Production and differential endocrine regulation of atrial natriuretic peptide in neuron-enriched primary cultures.

To identify factors that directly regulate the synthesis and secretion of atrial natriuretic peptide (ANP) in neuronal cells, we have developed a neuron-enriched primary culture system from fetal rat brains. A number of factors proved of importance in maintaining adequate levels of ANP secretion in such cultures: 1) cultures derived from diencephalon produced much more ANP than cultures derived from diencephalon produced with the distribution of ANP-containing cells in the rat brain; 2) brains from rats at gestational day 17 proved a better source of ANP-secreting cells than brains from rats at gestational day 16; 3) the presence of serum was required in the latter stages of the culture period to allow expression of the ANP gene; and 4) the cultures secreted more ANP when maintained at 39 C vs. 37 C. ANP mRNA transcripts in the neuron-enriched primary cultures were analyzed by S1 nuclease protection and shown to have a transcription start site similar to that employed by rat atrium and fetal hypothalamus in vivo. Dexamethasone and T3, in contrast to their stimulatory effect on ANP production in cardiocyte cultures, suppressed both the release of immunoreactive ANP and the levels of ANP mRNA in the neuron-enriched primary cultures. The cultures incorporated [35S]cysteine into immunoprecipitable ANP. HPLC analysis of 35S-labeled products in the medium revealed that, unlike neonatal cardiocyte cultures, the majority of secreted immunoreactive ANP migrated with the processed form(s) of ANP rather than the prohormone.

Organization of primary visual cortex (area 17) in the ferret.

Anatomical and electrophysiological mapping techniques were used to determine topographic organization and arrangement of ocular dominance columns in the primary visual cortex of ferrets. From its border with area 18 on the posterior lateral gyrus, area 17 extends around the caudal pole of the hemisphere and over the splenial gyrus to the caudal bank of the splenial sulcus. The visuotopic map is oriented with the isoazimuth lines approximately parallel to the long axis of the posterior lateral gyrus and the isoelevation lines approximately perpendicular to the isoazimuths. Central azimuths are represented on the posterior lateral gyrus and peripheral azimuths are represented on the splenial gyrus; the inferior visual field maps medially and the superior visual field maps laterally. As in other species, the representation of the central visual field is expanded. The ferret has a considerable degree of binocular vision. Receptive fields driven through the ipsilateral eye extended more than 20 degrees into the contralateral visual field. Within the region of area 17 corresponding to the binocular portion of the visual field, tritiated proline injected into one eye transneuronally labelled an ipsilateral projection as a series of patchy bands roughly complementary to gaps in the labelled contralateral projection. Physiological ocular dominance columns were evident as well in that neurons and groups of neurons recorded in this region showed clustered ocular dominance preferences. Most single neurons studied were binocularly driven.

Segregation of ON and OFF afferents to ferret visual cortex.

1. ON-center and OFF-center cells are found in separate sublaminae of the ferret's lateral geniculate nucleus (LGN). The purpose of these experiments was to determine whether this segregation is maintained in the projection from the LGN to primary visual cortex (area 17). 2. The distribution of the geniculocortical afferents within area 17 was studied by recording in layer IV after cortical neurons were silenced with kainic acid. 3. In 28 radial penetrations made into layer IV of five kainate-treated ferrets, the center types of 289 single units with response characteristics identical to those of geniculate cells were noted. A Monte Carlo analysis of these data demonstrated that the geniculocortical afferents cluster according to center type. 4. There was no tendency for ON and OFF afferents to occupy separate sublayers within layer IV. 5. The organization of the afferents in the plane of layer IV was studied by making closely spaced electrode penetrations across the dorsal exposed surface of the cortex in three kainate-treated ferrets. A Monte Carlo analysis of these results demonstrated that afferents segregate on the basis of center type, as well as on the basis of ocular dominance, into patches in the plane of layer IV. 6. The surface-mapping results and the results of experiments in which electrode penetrations were made tangential to layer IV indicated that center-type patches can extend over several hundred micrometers. A Monte Carlo analysis of the sizes of the ocular dominance patches and center-type patches provided further support for this conclusion.

The projection of the visual field onto the lateral geniculate nucleus of the ferret.

The projection of the visual field onto the dorsal lateral geniculate nucleus (LGN) of the ferret was mapped electrophysiologically. The nucleus contains a single orderly map of the contralateral visual hemifield. The upper visual field is represented dorsally and rostrally in the nucleus; central fields are found in the medial and caudal sections of the LGN; and peripheral fields are represented most laterally. The ipsilateral eye is represented in laminae A1 and C1 up to eccentricities of 20-30 degrees. Lines of projection run perpendicular to the laminar borders. The ferret LGN resembles that of the cat rotated approximately 110 degrees clockwise in the sagittal plane, viewing the right nucleus from its lateral aspect; it differs from the cat in having a larger monocular segment.

On and off sublaminae in the lateral geniculate nucleus of the ferret.

Like the retinal ganglion cells from which they receive their input, most relay neurons in the lateral geniculate nucleus have ON- or OFF-center receptive fields with antagonistic surrounds. In the cat, neurons with these two types of receptive fields are anatomically intermingled, even though the ON and OFF systems are functionally segregated. In the ferret, there is a sublamination of the retinal input to lateral geniculate nucleus laminae A and A1. We have investigated the function of this sublamination by making microelectrode recordings and have found that each sublamina consists of geniculate neurons of a single center type.