Michael Weick

Spectral and Temporal Sensitivity of Cone-Mediated Responses in Mouse Retinal Ganglion Cells

The retina uses two photoreceptor types to encode the wide range of light intensities in the natural environment. Rods mediate vision in dim light, whereas cones mediate vision in bright light. Mouse photoreceptors include only 3% cones, and the majority of these coexpress two opsins (short- and middle-wavelength sensitive, S and M), with peak sensitivity to either ultraviolet (360 nm) or green light (508 nm). The M/S-opsin ratio varies across the retina but has not been characterized functionally, preventing quantitative study of cone-mediated vision. Furthermore, physiological and behavioral measurements suggested that mouse retina supports relatively slow temporal processing (peak sensitivity, ∼2–5 Hz) compared to primates; however, past studies used visible wavelengths that are inefficient at stimulating mouse S-opsin. Here, we measured the M/S-opsin expression ratio across the mouse retina, as reflected by ganglion cell responses in vitro, and probed cone-mediated ganglion cell temporal properties using ultraviolet light stimulation and linear systems analysis. From recordings in mice lacking rod function (Gnat1−/−, Rho−/−), we estimate ∼70% M-opsin expression in far dorsal retina, dropping to <5% M-opsin expression throughout ventral retina. In mice lacking cone function (Gnat2cpfl3), light-adapted rod-mediated responses peaked at ∼5–7 Hz. In wild-type mice, cone-mediated responses peaked at ∼10 Hz, with substantial responsiveness up to ∼30 Hz. Therefore, despite the small percentage of cones, cone-mediated responses in mouse ganglion cells are fast and robust, similar to those in primates. These measurements enable quantitative analysis of cone-mediated responses at all levels of the visual system.

Neurite Branch Retraction Is Caused by a Threshold-Dependent Mechanical Impact

Recent results indicate that, in addition to chemical cues, mechanical stimuli may also impact neuronal growth. For instance, unlike most other cell types, neurons prefer soft substrates. However, the mechanisms responsible for the neuronal affinity for soft substrates have not yet been identified. In this study, we show that, in vitro, neurons continuously probe their mechanical environment. Growth cones visibly deform substrates with a compliance commensurate with their own. To understand the sensing of stiff substrates by growth cones, we investigated their precise temporal response to well-defined mechanical stress. When the applied stress exceeded a threshold of 274 +/- 41 pN/microm(2), neurons retracted and re-extended their processes, thereby enabling exploration of alternative directions. A calcium influx through stretch-activated ion channels and the detachment of adhesion sites were prerequisites for this retraction. Our data illustrate how growing neurons may detect and avoid stiff substrates--as a mechanism involved in axonal branch pruning--and provide what we believe is novel support of the idea that mechanics may act as guidance cue for neuronal growth.

Glutamate-evoked alterations of glial and neuronal cell morphology in the guinea pig retina

Neuronal activity is accompanied by transmembranous ion fluxes that cause cell volume changes. In whole mounts of the guinea pig retina, application of glutamate resulted in fast swelling of neuronal cell bodies in the ganglion cell layer (GCL) and the inner nuclear layer (INL) (by ∼40%) and a concomitant decrease of the thickness of glial cell processes in the inner plexiform layer (IPL) (by ∼40%) that was accompanied by an elongation of the glial cells, by a thickening of the whole retinal tissue, and by a shrinkage of the extracellular space (by ∼18%). The half-maximal effect of glutamate was observed at ∼250 μm, after ∼4 min. The swelling was caused predominantly by AMPA-kainate receptor-mediated influx of Na+ into retinal neurons. Similar but transient morphological alterations were induced by high K+ and dopamine, which caused release of endogenous glutamate and subsequent activation of AMPA-kainate receptors. Apparently, retinal glutamatergic transmission is accompanied by neuronal cell swelling that causes compensatory morphological alterations of glial cells. The effect of dopamine was elicitable only during light adaptation but not in the dark, and glutamate and high K+ induced strong ereffects in the dark than in the light. This suggests that not only the endogenous release of dopamine but also the responsiveness of glutamatergic neurons to dopamine is regulated by light-dark adaptation. Similar morphological alterations (neuronal swelling and decreased glial process thickness) were observed in whole mounts isolated immediately after experimental retinal ischemia, suggesting an involvement of AMPA-kainate receptor activation in putative neurotoxic cell swelling in the postischemic retina.

Activation of P2Y receptors stimulates potassium and cation currents in acutely isolated human Müller (glial) cells

The ability of various neurotransmitters/neuroactive substances to induce fast, transient rises of Ca2+‐activated K+ currents (IBK) caused by release of Ca2+ from intracellular stores was investigated in Müller glial cells of the human retina. Müller cells were enzymatically isolated from retinas of healthy donors or of patients with proliferative vitreoretinopathy, and the transmembrane ionic currents were recorded using the whole‐cell and the cell‐attached patch‐clamp techniques. The results of the screening experiments indicate that human Müller cells express, in addition to GABAA and perhaps glutamatergic and cholinergic receptors, predominantly P2 receptors. ATP and other nucleotides exerted two effects on membrane currents: repetitive transient increases of the IBK amplitude and, in a subpopulation of cells investigated, the appearance of a transient cation conductance at negative potentials. ATP and UTP increased dose‐dependently the IBK amplitude with half‐maximal effects at 0.33 and 0.50 μM, respectively. Since several different P2 receptor agonists increased the IBK, it is assumed that human Müller cells express a mixture of different types of P2Y receptors. In cell‐attached patches, extracellular application of ATP or UTP transiently increased the open probability of single putative BK channels. The increase of IBK and the appearance of the cation conductance in whole‐cell records were abolished when intracellular Ca2+ was buffered by a high‐EGTA pipette solution or when IP3 was included in the pipette solution. The expression of agonist‐evoked transient cation currents was found to be stronger in cells from patients as compared to cells from healthy donors. It is concluded that human Müller glial cells express P2Y receptors that, via IP3 formation, cause intracellular Ca2+ release. The increased intracellular Ca2+ concentration stimulates the activity of BK channels and may induce opening of cation channels. Both the ATP‐induced activity of BK channels and the increased expression of Ca2+‐gated cation channels may be important in respect to proliferative Müller cell gliosis. GLIA 37:139–152, 2002.

Glutamate transport by retinal Müller cells in glutamate/aspartate transporter-knockout mice

Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal‐to‐noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1–5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Müller cells in GLAST−/− mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the Km value for glutamate uptake was similar in wild‐type and GLAST−/− mouse retinas, but the Vmax was ∼50% lower in the mutant. In Na+‐free medium, the Vmax was further reduced by 40%. In patch‐clamp recordings of dissociated Müller cells from GLAST−/− mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Müller cells. [3H]D‐Aspartate uptake autoradiography, however, showed that Na+‐dependent, high‐affinity transporters account for most of the glutamate uptake by Müller cells, and that Na+‐independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Müller cells in the GLAST−/− mouse retina is not due to known glutamate transporters—cystine‐glutamate exchanger, ASCT‐1, AGT‐1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Müller cells, new Na+‐dependent, high‐affinity glutamate transporters remain to be identified.

Glial cell-mediated spread of retinal degeneration during detachment: A hypothesis based upon studies in rabbits

In human subjects with peripheral retinal detachments, visual deficits are not restricted to the detached retina but are also present in the non-detached tissue. Based upon studies on a rabbit model of rhegmatogenous retinal detachment, we propose a glial cell-mediated mechanism of spread of retinal degeneration into non-detached retinal areas which may also have importance for the understanding of alterations in the human retina. Both detached and attached portions of the rabbit retina display photoreceptor cell degeneration and cystic degeneration of the innermost layers. An inverse mode of photoreceptor cell degeneration in the attached tissue suggests a disturbed support of the photoreceptor cells by Müller cells which show various indications of gliosis (increased expression of intermediate filaments, cell hypertrophy, decreased plasma membrane K(+) conductance, increased Ca(2+) responsiveness to purinergic stimulation) in both detached and attached tissues. We propose that gliotic alterations of Müller cells contribute to the degeneration of the attached retina, via disturbance of glial homeostasis mechanisms. A down-regulation of the K(+) conductance of Müller cells may prevent effective retinal K(+) and water clearance, and may favor photoreceptor cell degeneration and edema development.

NMDA receptor contributions to visual contrast coding.

In the retina, it is not well understood how visual processing depends on AMPA- and NMDA-type glutamate receptors. Here we investigated how these receptors contribute to contrast coding in identified guinea pig ganglion cell types in vitro. NMDA-mediated responses were negligible in ON alpha cells but substantial in OFF alpha and delta cells. OFF delta cell NMDA receptors were composed of GluN2B subunits. Using a novel deconvolution method, we determined the individual contributions of AMPA, NMDA, and inhibitory currents to light responses of each cell type. OFF alpha and delta cells used NMDA receptors for encoding either the full contrast range (alpha), including near-threshold responses, or only a high range (delta). However, contrast sensitivity depended substantially on NMDA receptors only in OFF alpha cells. NMDA receptors contribute to visual contrast coding in a cell-type-specific manner. Certain cell types generate excitatory responses using primarily AMPA receptors or disinhibition.

Electrophysiological alterations and upregulation of ATP receptors in retinal glial Müller cells from rats infected with the Borna disease virus.

Infection with the neurotropic Borna disease virus (BDV) causes an immune‐mediated neurological disease in a broad range of species. In addition to encephalitis, BDV‐infected Lewis rats develop a retinitis histologically characterized by the loss of most retinal neurons. By contrast, the dominating retinal macroglia, the Müller cells, do not degenerate. It is known from several models of neurodegeneration that glial cells may survive but undergo significant alterations of their physiological parameters. This prompted us to study the electrophysiology and ATP‐induced changes of intracellular Ca2+‐concentration ([Ca2+]i) in Müller cells from BDV‐infected rat retinae. Freshly isolated cells were used for whole‐cell patch‐clamp recordings. Whereas neither zero current potentials nor membrane resistances showed significant alterations, the membrane capacitance increased in cells from BDV‐infected rats during survival times of up to 8 months. This process was accompanied by a decrease in K+ current densities. Müller cells from BDV‐infected rats were characterized by expression of a prominent fast‐inactivating A‐type K+ current which was rarely found in control cells. Moreover, the number of cells displaying Na+ currents was slightly increased after BDV‐infection. ATP evoked increases in [Ca2+]i in Müller cells within retinal wholemounts of both control and BDV‐infected animals. However, the number of ATP‐responding isolated cells increased from 24% (age‐matched controls) to 78% (cells from animals ≥18 weeks after infection). We conclude that in BDV‐induced retinopathy, reactive rat Müller cells change their physiological parameters but these changes are different from those in Müller cells during proliferative vitreoretinopathy in man and rabbit. GLIA 35:213–223, 2001.

Pathological effects of glyoxalase I inhibition in SH-SY5Y neuroblastoma cells

In Alzheimers disease (AD), in aging, and under conditions of oxidative stress, the levels of reactive carbonyl compounds continuously increase. Accumulating carbonyl levels might be caused by an impaired enzymatic detoxification system. The major dicarbonyl detoxifying system is the glyoxalase system, which removes methylglyoxal in order to minimize cellular impairment. Although a reduced activity of glyoxalase I was evident in aging brains, it is not known how raising the intracellular methylglyoxal level influences neuronal function and the phosphorylation pattern of tau protein, which is known to be abnormally hyperphosphorylated in AD. To simulate a reduced glyoxalase I activity, we applied an inhibitor of glyoxalase I, p‐bromobenzylglutathione cyclopentyl diester (pBrBzGSCp2), to SH‐SY5Y neuroblastoma cells to induce chronically elevated methylglyoxal concentrations. We have shown that 10 μM pBrBzGSCp2 leads to a fourfold elevation of the methylglyoxal level after 24 hr. In addition, glyoxalase I inhibition leads to reduced cell viability, strongly retracted neuritis, increase in [Ca2+]i, and activation of caspase‐3. However, pBrBzGSCp2 did not lead to tau “hyper”‐phosphorylation despite activation of p38 mitogen‐activated protein kinase and c‐Jun NH2‐terminal kinase but rather activated protein phosphatases 2 and induced tau dephosphorylation at the Ser202/Thr205 and Ser396/Ser404 epitopes. Preincubation with the carbonyl scavenger aminoguanidine prevented tau dephosphorylation, indicating the specific effect of methylglyoxal. Also, pretreatment with the inhibitor okadaic acid prevented tau dephosphorylation, indicating that methylglyoxal activates PP‐2A. In summary, our data suggest that a reduced glyoxalase I activity mimics some changes associated with neurodegeneration, such as neurite retraction and apoptotic cell death.

Transmission of phase-coupling accuracy from the auditory nerve to spherical bushy cells in the Mongolian gerbil

The phase of low-frequency sinusoids is encoded in phase-coupled discharges of spherical bushy cells (SBCs) of the anteroventral cochlear nucleus and transmitted to the medial superior olive, where binaural input-coincidence is used for processing of sound source localization. SBCs are innervated by auditory nerve fibers through large, excitatory synapses (endbulbs of Held) and by inhibitory inputs, which effectively reduce SBC discharge rates. Here we monitor presynaptic potentials of endbulb-terminals and postsynaptic spikes of SBCs in extracellular single unit recordings in vivo. We compare postsynaptic phase-coupling of SBCs and their presynaptic immediate auditory nerve input. In all but one SBC discharge rates at the characteristic frequency were reduced pre-to-postsynaptically and phase-coupling accuracy was increased in one-third of them. We investigated the contribution of systemic inhibition on spike timing in SBCs by iontophoretic application of glycine- and GABA-receptor antagonists (strychnine, bicuculline). Discharge rate increased in one-third of the units during antagonist application, which was accompanied by a deterioration of phase-coupling accuracy in half of those units. These results suggest that the phase-coupling accuracy is improved in a subpopulation of SBCs during transmission from the auditory nerve to the SBCs by reduction of spike rates.