Katsuko Morigiwa

Differential expression of ionotropic glutamate receptor subunits in the outer retina

Ionotropic glutamate receptors (iGluRs) are extremely diverse in their subunit compositions. To understand the functional consequences of this diversity, it is necessary to know the subunits that are expressed by known cell types. By using immunocytochemistry with light and electron microscopy, we localized several subunits (GluR2/3, GluR4, and GluR6/7) in cat retinal neurons, postsynaptic to photoreceptors. Type A horizontal cells express all three subunits strongly, whereas type B horizontal cells express GluR2/3 strongly, GluR6/7 weakly, and do not express GluR4. When they are present, the subunits are expressed strongly throughout the cytoplasm of the somata and primary dendrites; however, in the terminals, they are concentrated at the postsynaptic region, just opposite the presumed site of photoreceptor glutamate release. Surprisingly, all bipolar cell classes (OFF cone bipolar cells, ON cone bipolar cells, and rod bipolar cells) express at least one iGluR subunit at their dendritic tips. Cone bipolar cells forming basal contacts with the cones (presumably OFF cells) express all three subunits in association with the electron‐dense postsynaptic membrane. Invaginating dendrites of cone bipolar cells (presumably ON cells) express GluR2/3 and GluR4. Rod bipolar cells (ON cells) express GluR2/3 in their invaginating dendrites. The function of iGluRs in horizontal cells and OFF bipolar cells clearly is to mediate their light responses. GluR6/7 subunit in the receptor of these cells may be responsible for the dopamine‐mediated enhancement of glutamate responses that have been observed previously in these cells. The function of iGluRs in ON bipolar cells remains an enigma. J. Comp. Neurol. 405:173–184, 1999.

Morphological comparisons between outer and inner ramifying alpha cells of the albino rat retina

SummaryThe somato-dendritic morphologies of large ganglion cells were studied by intracellular injections of Lucifer yellow in perfusedin vitro preparations of the albino rat retina. The ganglion cells were prelabeled with retrogradely transported granular blue or labeled with acridine orange dropped into the perfusate ofin vitro preparations. After the dye injection, somato-dendritic morphologies were successfully studied for 210 cells, the majority of which had a large soma more than 20 µm in diameter and were identified as alpha cells. According to the level of dendritic extensions within the inner plexiform layer (IPL) these alpha cells were further classified into inner ramifying (inner) and outer ramifying (outer) cells. Both qualitative and quantitative observations led us to conclude the following:1) The outer cells have a spherical soma with relatively few primary dendrites, while inner cells have a large polygonal soma with more primary dendrites.2) The dendritic field of inner cells was always larger than that of outer cells at every retinal location. The dendritic field diameter tended to increase as a function of retinal eccentricity from the optic disk, the tendency being more clear among inner cells.3) The dendrites of outer cells branch more frequently in the proximal part of the dendritic field while those of inner cells branch more distally.4) Total dendritic length of outer cells increases linearly with eccentricity whereas that of the inner cells does not change much irrespective of retinal location.

P2 purinoceptor expression and functional changes of hypoxia-activated cultured rat retinal microglia

P(2) purinoceptors appear to modulate microglia function, but their role in hypoxic microglia has not been investigated. We examined in postnatal rat retinal microglia cultured under hypoxic (1% oxygen) condition, their P2 expression, proliferation and cytokine release in the presence or absence of the P2 receptor agonists and antagonists. Fura-2 fluorescence measurements of intracellular Ca(2+) rises to P2 receptor agonists and antagonists indicated that both P(2U) and P(2Z) were expressed in hypoxic microglia. Hypoxia induced BrdU incorporation and release of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as well. The P(2U) agonist, UTP, maintained the BrdU incorporation, whereas the P(2Z) agonist, BzATP, suppressed it, but significantly enhanced IL-1beta and TNF-alpha release, suggesting that the P(2U) response may underlie the mitotic activity, and that of P(2Z), the IL-1beta and TNF-alpha release of hypoxia-activated microglia.

A Novel Connection between Rods and ON Cone Bipolar Cells Revealed by Ectopic Metabotropic Glutamate Receptor 7 (mGluR7) in mGluR6-Deficient Mouse Retinas

Since the discovery of direct chemical synapses between rod photoreceptor and OFF cone bipolar cells in mouse retinas, whether the ON cone bipolar cell also receive direct chemical input from rod has been a pending question. In finding that metabotropic glutamate receptor 7 (mGluR7) was uniquely expressed in dendrites of ON cone bipolar cells in the mGluR6-deficient mouse retina, we used this ectopic mGluR7 immunoreactivity as a specific marker for the ON cone bipolar to search for its rod connection. Here, we show that a certain type of ON cone bipolar cell forms ribbon-associated synapses not only with cones, but also rods. This finding was verified in the wild-type mouse retina by three-dimensional reconstruction of bipolar cells from serial electron micrographs. These ON cone bipolars were further identified as corresponding to type 7 of mouse bipolar cell described by Ghosh et al. (2004) and also to the green fluorescent protein (GFP)-labeled type 7 bipolars in the α-gustducin-GFP transgenic mouse. Our findings suggest that, in mice, rod signals bifurcate into a third ON and OFF pathway in addition to the two known routes to cone bipolar cells: (1) via rod chemical synapse → rod bipolar → AII amacrine → ON and OFF cone bipolar cells; (2) via rod–cone gap junction → cone chemical synapse → ON and OFF cone bipolar cells; and (3) via rod chemical synapse → ON and OFF cone bipolar cells. This third novel pathway is thought to transmit fast and moderately light-sensitive rod signals, functioning to smooth out the intensity changes at the scotopic–mesopic interface.

Effects of EEG synchronization on visual responses of the cat's geniculate relay cells: a comparison among Y,X and W cells

Single unit activities of Y, X and W cells were recorded in the cat lateral geniculate nucleus to study how their responses to stationary light spots change with the cortical EEG. The shift from desynchronized to synchronized EEG drastically suppressed W cell activities, depressed both transient and sustained components of X cell responses, but did not affect the transient component of Y cell responses. This was ascribed to different inhibitory circuitries in the three parallel pathways and their differential modulation by the ascending brainstem activities.

Intraretinal axons of ganglion cells in the Japanese monkey (Macaca fuscata): conduction velocity and diameter distribution

In anesthetized and immobilized Japanese monkeys (Macaca fuscata), intraretinal conduction velocities of the ganglion cell axons were measured. The field potentials elicited by optic chiasm shocks consisted of fast and slow components with estimated conduction velocities of 1.19 and 0.72 m/s in recordings from the optic nerve fiber layer, and 1.65 and 1.00 m/s in recordings from the ganglion cell layer. Single cell recordings verified that the time course of the fast component corresponded to the antidromic spike latencies of Y-like cells, whereas that of the slow component covered the latency range of both X-like and W-like cells. In an electron microscopic study of the cross-sections of the intraretinal optic nerve fiber bundles, the axon diameter histograms of large samples (n = 3000-6000) all showed a unimodal distribution with a sharp peak at 0.3-0.6 micron and a long tail extending to 2-3 micron. The mean diameter was largest in the ventral and nasal bundles, smallest in the papillomacular bundle and intermediate in the dorsal, upper arcuate and lower arcuate bundles. However, diameter histograms of a small number of regional axons (n = 255-300) showed a broad tail distinct from the peak at 0.3-0.6 micron, enabling us to segregate a group of larger axons from the medium-sized to small axons. From such regional axon diameter histograms we estimated the mean relative occurrences of the larger axons (7.1-11.3%) and their mean diameters (0.9-1.3 micron). We further applied this relative frequency to the unimodal distribution of the histograms with larger samples in the upper and lower arcuate bundles and estimated the mean axon diameter of the large axons (1.1 micron) and that of the medium-sized to small axons (slightly below 0.5 micron). Finally, in studying the relation between axon diameter and conduction velocity in the two arcuate fiber bundles, we found it to be somewhat different from that previously reported for the cat retina.

Functional and Morphological Restoration of Intracranial Brachial Lesion of the Retinocollicular Pathway by Peripheral Nerve Autografts in Adult Hamsters

Axons of adult mammals can regenerate through peripheral nerve grafts and restore the retinocollicular pathway if lesioned proximal to the retinal ganglion cell somata. Whether the grafting and subsequent reinnervation of the superior colliculus (SC) is possible in distal axotomy in the brain is a question of clinical relevance. We have deafferented the SC of adult hamsters at its brachium thus axotomizing the retinal ganglion cell axons rostral to its synaptic contact with the SC neurons. After unilateral brachium transection, a short segment of the autologous sciatic nerve was grafted to bridge the lesioned site to the SC (n = 28). As controls the brachium was transected and left ungrafted (n = 12). Functional restoration was examined 3 to 75 weeks later in grafted (n = 16) and control (n = 5) animals by recording visual evoked responses from the collicular cells. Prior to recording the grafts were visually evaluated and categorized into successfully (n = 8) and unsuccessfully (n = 8) grafted groups. To diffuse flash stimuli applied to the contralateral eye, visual evoked field potentials were recorded from all successfully grafted, but not in unsuccessfully grafted (with the exception of one animal) nor control animals. Unitary spike responses to diffuse flash stimuli were recorded exclusively from three successfully grafted animals. Morphological reinnervation was examined in the remaining grafted (n = 12) and control (n = 7) animals by anterogradely labeling the regenerating retinal axons with WGA-HRP. Axons in the grafts and their terminals in the superficial layers of the SC were clearly labeled in 8 of the grafted and none of the controls. From these results we conclude that the brachium of the SC is conducive to axonal regeneration and the peripheral nerve graft is indeed effective in restoring distally axotomized visual pathway in adult mammals.

Optic nerve regeneration by peripheral nerve transplant

We studied the morphology of regenerated retinal ganglion cells and their axons in adult rodents after axotomy and autologous transplantation of the sciatic nerve. Regenerated ganglion cells, backlabeled with rhodamine dextran, were of similar size to or larger than those of intact cells in control animals. Dendrites and occasionally axons as well showed abnormal morphologies in most cells, though some cells appeared quite normal. Cross-sections of the regenerated axons, observed by electron microscopy, were always attached to either the Schwann cell cytoplasm or the basal lamina. The immunoreactive structures to anti-laminin antibody were quite irregular in the cross-sectioned graft and, compared with those of the intact sciatic nerve, they were generally smaller. Their appearance closely resembled that of the basal lamina in the graft observed by electron microscopy. These observations, taken together, suggest that the laminin-rich basal laminae of Schwann cells are essentially important for the regeneration of retinal axons in adult rodents.

Retinal inputs and laminar distributions of the dorsal lateral geniculate nucleus relay cells in the eastern chipmunk (Tamias sibiricus asiaticus)

SummaryRetinal inputs and their laminar distributions in the dorsal lateral geniculate nucleus (LGNd) of the eastern chipmunk (Tamias sibiricus asiaticus) were studied using histological and microelectrode recording techniques. A previous anatomical study (Fukuda et al. 1986a) indicated that the chipmunk LGNd had five laminae: contralaterally (contra) innervated lamina 1 and ipsilaterally (ipsi) innervated lamina 2 in its ventromedial part; laminae 3a (contra), 3b (ipsi) and 3c (contra) in its dorsolateral part. We have confirmed this finding in our present anatomical study and have also noted another ipsilaterally innervated thin lamina 0, medial to lamina 1. In our electrophysiological study, however, we were unable to record units from lamina 0 and to investigate it functionally. We recorded 232 units from laminae 1, 2 and the 3 complex, of which 95 were identified as Y-like, 46 as W-like, 15 as X-like, and 8 as mixed Y/W-like cells; the rest were either unclassified or visually unresponsive. In laminae 1 and 2, only Y-like and X-like cells were recorded, whereas in the laminae 3 complex W-like cells were recorded as well. The results suggest that the chipmunk laminae 1,2 and 3 complex correspond relatively well to the cat laminae A, A1 and C complex, respectively. In the chipmunk LGNd, however, there were more Y-like cells in laminae 1 and 2, and a few X-like cells of which some were color sensitive. Also, lamina 3a had a concentration of mixed-type cells with Y-like receptive field properties and W-like OX latencies. As for retinotopy, the dorsoventral transition of the contralateral visual field (laminae 1, 3a, 3c) is represented along the dorsoventral dimension of the chipmunk LGNd, whereas the temporonasal transition is represented in the rostrocaudal direction. Receptive field positions of the ipsilaterally innervated relay cells are limited to the central overlapping field of the contralateral visual fields of both eyes. Relay cells with visual fields having elevations of below -20° had relatively fast latency range and Ylike properties.