Research Article
A comparison of immunocytochemical markers to identify bipolar cell types in human and monkey retina
- SILKE HAVERKAMP, FRANCOISE HAESELEER, ANITA HENDRICKSON
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- Published online by Cambridge University Press:
- 30 March 2004, pp. 589-600
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As more human retinas affected with genetic or immune-based diseases become available for morphological analysis, it is important to identify immunocytochemical markers for specific subtypes of retinal neurons. In this study, we have focused on bipolar cell markers in central retina. We have done single and double labeling using several antisera previously utilized in macaque monkey or human retinal studies and two new antisera (1) to correlate combinations of antisera labeling with morphological types of bipolar cells in human retina, and (2) to compare human labeling patterns with those in monkey retina. Human bipolar cells showed a wide range of labeling patterns with at least ten different bipolar cell types identified from their anatomy and marker content. Many bipolar cell bodies in the outer part of the inner nuclear layer contained combinations of protein kinase C alpha (PKCα), Islet-1, glycine, and Goα. Bipolar cells labeled with these markers had axons terminating in the inner half of the inner plexiform layer (IPL), consistent with ON bipolar cells. Bipolar cell bodies adjacent to the amacrine cells and with axons in the outer half of the IPL contained combinations of recoverin, glutamate transporter-1, and PKCβ, or CD15 and calbindin. Bipolar cells labeled with these markers were presumed OFF bipolar cells. Calcium-binding protein 5 (CaB5) labeled both putative ON and OFF bipolar cells. Using this cell labeling as a criteria, most cell bodies close to the horizontal cells were ON bipolar cells and almost all bipolar cells adjacent to the amacrine cells were OFF with a band in the middle 2–3 cell bodies thick containing intermixed ON and OFF bipolar cells. Differences were found between human and monkey bipolar cell types labeled by calbindin, CaB5, and CD15. Two new types were identified. One was morphologically similar to the DB3, but labeled for CD15 and CaB5. The other had a calbindin-labeled cell body adjacent to the horizontal cell bodies, but did not contain any accepted ON markers. These results support the use of macaque monkey retina as a model for human, but caution against the assumption that all labeling patterns are identical in the two primates.
Melanopsin and non-melanopsin expressing retinal ganglion cells innervate the hypothalamic suprachiasmatic nucleus
- PATRICIA J. SOLLARS, CYNTHIA A. SMERASKI, JESSICA D. KAUFMAN, MALCOLM D. OGILVIE, IGNACIO PROVENCIO, GARY E. PICKARD
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- Published online by Cambridge University Press:
- 30 March 2004, pp. 601-610
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Retinal input to the hypothalamic suprachiasmatic nucleus (SCN) synchronizes the SCN circadian oscillator to the external day/night cycle. Retinal ganglion cells that innervate the SCN via the retinohypothalamic tract are intrinsically light sensitive and express melanopsin. In this study, we provide data indicating that not all SCN-projecting retinal ganglion cells express melanopsin. To determine the proportion of ganglion cells afferent to the SCN that express melanopsin, ganglion cells were labeled following transsynaptic retrograde transport of a recombinant of the Bartha strain of pseudorabies virus (PRV152) constructed to express the enhanced green fluorescent protein (EGFP). PRV152 injected into the anterior chamber of the eye retrogradely infects four retinorecipient nuclei in the brain via autonomic circuits to the eye, resulting in transneuronally labeled ganglion cells in the contralateral retina 96 h after intraocular infection. In animals with large bilateral lesions of the lateral geniculate body/optic tract, ganglion cells labeled with PRV152 are retrogradely infected from only the SCN. In these animals, most PRV152-infected ganglion cells were immunoreactive for melanopsin. However, a significant percentage (10–20%) of EGFP-labeled ganglion cells did not express melanopsin. These data suggest that in addition to the intrinsically light-sensitive melanopsin-expressing ganglion cells, conventional ganglion cells also innervate the SCN. Thus, it appears that the rod/cone system of photoreceptors may provide signals to the SCN circadian system independent of intrinsically light-sensitive melanopsin ganglion cells.
Activation of protein kinase C reduces GLAST in the plasma membrane of rat Müller cells in primary culture
- ZHIQING WANG, WEI LI, CHERYL K. MITCHELL, LOUVENIA CARTER-DAWSON
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- 30 March 2004, pp. 611-619
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In this study, a Müller cell culture preparation from young rats was used to investigate the regulation of GLAST transport activity in native cells. Immunohistochemical analysis confirmed GLAST to be the predominant glutamate transporter expressed by the cells through five passages. [3H]-glutamate uptake assays showed the typical Na+-dependent glutamate transport which was blocked by L-(-)-threo-3-hydroxyaspartate (L-THA), a competitive inhibitor. Glutamate transport was decreased significantly in Müller cells exposed to phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator. A similar effect on [3H]-D-aspartate (nonmetabolizable glutamate analog) uptake ruled out the possibility that the decrease was a consequence of altered metabolism. However, PMA did not affect Na+-dependent [3H]-glycine transport, indicating the absence of a nonspecific change in the electrochemical gradients. The PMA effect on glutamate uptake was evidenced by partial blocking with a specific PKC inhibitor, bisindolymaleimide II (Bis II). Activation of PKC did not change the Km, but the Vmax was significantly reduced. Image analysis of Müller cells with biotinylated cell membranes immunolabeled with GLAST shows a reduction of GLAST in the plasma membrane. In conclusion, these data show that rat Müller cells in primary cultures express GLAST and that PKC activation affects GLAST transport activity by decreasing cell surface expression.
The unitary event amplitude of mouse retinal on-cone bipolar cells
- AMY BERNTSON, W. ROWLAND TAYLOR
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- 30 March 2004, pp. 621-626
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Light-evoked synaptic currents were recorded from on-cone bipolar cells in the mouse retina. Fluctuations in the synaptic current observed during maintained light steps were analyzed in order to estimate the amplitude of the underlying unitary event. The maximal synaptic current variance was 5-fold larger than the maximum expected from fluctuations in the number of active postsynaptic channels. Due to uncertainty in the contribution from channel variance, we calculated a range of values for the unitary event amplitude. The observed variance could be accounted for if 30–39 synaptic sites randomly generated unitary events with a waveform identical to the flash-response, and an amplitude of −3.1 to −2.4 pA. The amplitude is consistent with gating about five mGluR6 channels. The shape of the variance–mean relation suggests that in bright light transmitter release approaches zero, while in darkness transmitter release saturates the postsynaptic response. Thus the on-cone bipolar cell synapse is operating over its entire possible range. If it is assumed that the postsynaptic response saturates when one unitary event occurs per integration time, then a lower bound for the unitary event rate is 18 events/s/synaptic site. If the unitary event is generated by a single synaptic vesicle, the results suggest the total vesicle cycling rate available for encoding the on-cone bipolar cell signal is about 540–700 s−1.
GABAA and GABAC receptor antagonists increase retinal cyclic GMP levels through nitric oxide synthase
- DOU YU, WILLIAM D. ELDRED
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- 30 March 2004, pp. 627-637
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The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal transduction pathway plays a role in every retinal cell type. Previous studies have shown that excitatory glutamatergic synaptic pathways can increase cGMP-like immunoreactivity (cGMP-LI) in retina through stimulation of NO production, but little is known about the role of synaptic inhibition in the modulation of cGMP-LI. Gamma-amino-n-butyric acid (GABA) plays critical roles in modulating excitatory synaptic pathways in the retina. Therefore, we used GABA receptor antagonists to explore the role of GABAergic inhibitory synaptic pathways on the modulation of the NO/cGMP signal-transduction system. Cyclic GMP immunocytochemistry was used to investigate the effects of the GABA receptor antagonists bicuculline, picrotoxin, and (1,2,5,6-tetrahyropyridin-4-yl) methylphosphinic acid (TPMPA) on levels of cGMP-LI. Cyclic GMP-LI was strongly increased in response to the GABAA receptor antagonist bicuculline, while the GABAC receptor antagonist TPMPA had little effect on cGMP-LI. The GABAA/GABAC receptor antagonist, picrotoxin, caused a moderate increase in cGMP-LI, which was mimicked by the combination of bicuculline and TPMPA. The nitric oxide synthase inhibitor, S-methyl-L-thiocitrulline (SMTC), blocked the increased cGMP-LI in response to stimulation with either bicuculline or picrotoxin. Treatments with either of the glutamate receptor antagonists (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) partially blocked the increases in cGMP-LI seen in response to bicuculline, but a combination of MK-801 and CNQX completely eliminated these increases. These results suggest that inhibitory synaptic pathways involving both types of GABA receptors work through excitatory glutamatergic receptors to regulate the NO/cGMP signal-transduction pathway in retina.
Morphology of the turtle accessory optic system
- JOHN MARTIN, NAOKI KOGO, TIAN XING FAN, MICHAEL ARIEL
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- 30 March 2004, pp. 639-649
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Neural signals of the moving visual world are detected by a subclass of retinal ganglion cells that project to the accessory optic system in the vertebrate brainstem. We studied the dendritic morphologies and direction tuning of these brainstem neurons in turtle (Pseudemys scripta elegans) to understand their role in visual processing. Full-field checkerboard patterns were drifted on the contralateral retina while whole-cell recordings were made in the basal optic nucleus in an intact brainstem preparation in vitro. Neurobiotin diffused into the neurons during the recording and was subsequently localized in brain sections. Neuronal morphologies were traced using appropriate computer software to analyze their position in the brainstem. Most labeled neurons were fusiform in shape and had numerous varicosities along their processes. The majority of dendritic trees spread out in a transverse plane perpendicular to the rostrocaudal axis of the nucleus. Neurons near the brainstem surface were often oriented tangential to that surface, whereas more cells at the dorsal side of the nucleus were oriented radial to the brainstem surface. Further analysis of Nissl-stained neurons revealed the largest neurons are located in the rostral and medial portions of the nucleus although neurons are most densely packed in the middle of the nucleus. The preferred directions of the visual responses of the neurons in this sample did not correlate with their morphology and position in the nucleus. Therefore, the morphology of the cells in the turtle accessory optic system appears dependent on its position within the nucleus while its visual responses may depend on the synaptic inputs that contact each cell.
Rabbit retinal ganglion cell responses to nicotine can be mediated by β2-containing nicotinic acetylcholine receptors
- CHRISTIANNE E. STRANG, FRANKLIN R. AMTHOR, KENT T. KEYSER
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- Published online by Cambridge University Press:
- 30 March 2004, pp. 651-662
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Acetylcholine (ACh) affects the response properties of many retinal ganglion cells (GCs) through the activation of nicotinic acetylcholine receptors (nAChRs). To date there have been few studies directly correlating the expression of specific nAChR subtypes with the physiological and morphological characteristics of specific retinal GCs. This study was designed to correlate responses to nicotine application with immunohistochemical evidence of nAChR expression in physiologically and morphologically identified ganglion cells. Extracellular recordings were used to physiologically identify rabbit retinal GCs, based on responses to light stimulation. Cells were then tested for responses to nicotine application and/or for expression of nAChRs, as judged by immunoreactivity to mAb210, an nAChR antibody. The morphologies of many physiologically identified cells were also determined by dye injection. More than three-fourths of ganglion cells tested responded to nicotine application under cobalt-induced synaptic blockade. The nicotine sensitivity was consistent with nAChR immunoreactivity and was also correlated with specific morphological subgroups of GCs. Overall, approximately two-thirds of all physiologically identified GCs that were processed for immunohistochemistry displayed immunoreactivity. In total, 18 of 22 physiologically identified cells demonstrated both sensitivity to nicotine application under synaptic blockade and mAb210 immunoreactivity (mAb210-IR). Thus, mAb210-IR is likely to represent functional nAChRs that can modulate retinal information processing and visual functioning via direct excitation of a number of GC classes.
Complete flatmounting of the macaque cerebral cortex
- LAWRENCE C. SINCICH, DANIEL L. ADAMS, JONATHAN C. HORTON
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- 30 March 2004, pp. 663-686
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The elaborate folding of the brain surface has posed a practical impediment to investigators engaged in mapping the areas of the cerebral cortex. This obstacle has been overcome partially by the development of methods to erase the sulci and gyri by physically flattening the cortex prior to sectioning. In this study, we have prepared a step-by-step atlas of the flatmounting process for the entire cerebral cortex in the macaque monkey. The cortex was dissected from the white matter, unfolded, and flattened in a single piece of tissue by making three relieving cuts. The flatmount was sectioned at 60–75 μm and processed for cytochrome oxidase (CO) or myelin. From animal to animal there was nearly a twofold variation in the surface area of individual cortical regions, and of the whole cortex. In each specimen, a close correlation was found between V1 surface area (mean = 1343 mm2), V2 surface area (mean = 1012 mm2), hippocampal area (mean = 181 mm2), and total cerebral cortex area (mean = 10,430 mm2). The complete pattern of CO stripes in area V2 was labeled clearly in several cases; the number of cycles of thick-pale-thin-pale stripes ranged from 26 to 34. Characteristic patterns of strong CO activity were encountered in areas V3, MT, auditory and somatosensory cortex. In some animals we made injections of a retrograde tracer, gold-conjugated cholera toxin B subunit, into area V2 to identify all sources of cortical input. In addition to previously described inputs, we identified three new regions in the occipitotemporal region that project to V2. Flatmounting the cerebral cortex is a simple, efficient method that can be used routinely for mapping areas and connections in the macaque brain, the most widely used primate model of the human brain.
Visual detection deficits following inactivation of the superior colliculus in the cat
- MARNIE C. FITZMAURICE, VIVIAN M. CIARAMITARO, LARRY A. PALMER, ALAN C. ROSENQUIST
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- 30 March 2004, pp. 687-701
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Lesion or inactivation of the superior colliculus (SC) of the cat results in an animal that fails to orient toward peripheral visual stimuli which normally evoke a brisk, reflexive orienting response. A failure to orient toward a visual stimulus could be the result of a sensory impairment (a failure to detect the visual stimulus) or a motor impairment (an inability to generate the orienting response). Either mechanism could explain the deficit observed during SC inactivation since neurons in the SC can carry visual sensory signals as well as motor commands involved in the generation of head and eye movements. We investigated the effects of SC inactivation in the cat in two ways. First, we tested cats in a visual detection task that required the animals to press a central, stationary foot pedal to indicate detection of a peripheral visual stimulus. Such a motor response does not involve any components of the orienting response and is unlikely to depend on SC motor commands. A deficit in this task would indicate that the SC plays an important role in the detection of visual targets even in a task that does not require visual orienting. Second, to further investigate the visual orienting deficit observed during SC inactivation and to make direct comparisons between detection and orienting performance, we tested cats in a standard perimetry paradigm. Performance in both tasks was tested following focal inactivation of the SC with microinjections of muscimol at various depths and rostral/caudal locations throughout the SC. Our results reveal a dramatic deficit in both the visual detection task and the visual orienting task following inactivation of the SC with muscimol.
ERRATUM
Structure of glutamate analogs that activate the ON bipolar cell metabotropic glutamate receptor in vertebrate retina
- NING TIAN, MALCOLM M. SLAUGHTER
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- Published online by Cambridge University Press:
- 30 March 2004, p. 703
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There are errors in the chemical structures for Figure 1 as it was originally printed. There should be a carbon atom between the cyclopropyl ring and the phosphonate in both the trans cyclopropyl AP4 and the cis cyclopropyl AP4. Figure 1 is printed below as it should be.
(article appeared in Visual Neuroscience (2003), 20, 231–240)