Non-telecentric two-photon microscopy for 3D random access mesoscale imaging.

Diffraction-limited two-photon microscopy permits minimally invasive optical monitoring of neuronal activity. However, most conventional two-photon microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing a non-telecentric optical design, we present a low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view. Moreover, rapid laser-focus control via an electrically tunable lens allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in zebrafish, mice and fruit flies.

OFF bipolar cells express distinct types of dendritic glutamate receptors in the mouse retina.

Parallel representations of the visual world are already established at the very first synapse of the visual system. Cone photoreceptors, which hyperpolarize in response to light, forward the visual signal onto distinct types of ON and OFF cone bipolar cells (BCs). In the case of OFF BCs, the glutamatergic cone input is integrated by ionotropic glutamate receptors, giving rise to a sign-preserving mode of synaptic transmission. The combination of glutamate receptor (GluR) subunits, i.e. AMPA or kainate subunits, importantly contributes to shaping the OFF bipolar cells' distinct response properties. The mouse is one of the few mammals in which the (most likely) complete set of (five) retinal OFF BC types is identified. However, it is not clear which GluR subtypes are expressed by the different mouse OFF BC types. We addressed this question by combining immunolabeling, electrical whole-cell recordings and pharmacology, and present evidence that the different types of OFF BCs express distinct types of glutamate receptors: Type 1 BCs exclusively expressed AMPA receptors, whereas type 2 and type 3a BCs expressed kainate receptors of different subunit compositions. Additionally, we found that two OFF BC types (3b and 4) very likely express both AMPA and kainate receptors but, interestingly, the two receptor subunits were not co-localized at the same dendritic site. The complex, BC type-specific expression pattern of GluRs we describe here supports their essential role in establishing parallel pathways at the first synapse of the mouse visual system.

Dendritic processing.

Dendritic processing multiplies the computational power of a single neuron by enabling the processing of inputs in a spatio-temporally differentiated manner. Recently, the development of new and refined optical, electrophysiological and molecular-biological techniques has led to new insights into dendritic function and revealed an astonishing plethora of computational mechanisms.

Light-evoked responses of bipolar cells in a mammalian retina.

We recorded light-evoked responses from rod and cone bipolar cells using patch-clamp techniques in a slice preparation of the rat retina. Rod bipolar cells responded to light with a sustained depolarization (ON response) followed at light offset by a slight hyperpolarization. ON and OFF cone bipolar cells were encountered, both with diverse temporal properties. The responses of rod bipolar cells were composed primarily of two components, a nonspecific cation current and a chloride current. The chloride current was reduced greatly in axotomized cells and could be suppressed by coapplication of the GABA(A) antagonist bicuculline and the GABA(C) antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid. This suggests that it largely reflects feedback from GABAergic amacrine cells. The response latency of intact rod bipolar cells was shorter than that of the axotomized cells, and the sensitivity curve covered more than twice the dynamic range. Application of the GABA receptor antagonists partially mimicked the effects of axotomy. These findings suggest that functional properties of the axon terminal system-notably synaptic feedback from amacrine cells-play an important role in defining the response properties of mammalian bipolar cells.

Spatial order within but not between types of retinal neurons.

We studied the mosaics of six types of retinal neurons, asking how the position of a cell relates to the positions of other cells of that same type and also to cells of different types. Every neuron studied was found to be nonrandomly positioned: Cells of a particular type were evenly spaced. However, all cells were positioned randomly with respect to members of the other cell classes. This was true even when the cells were known to be synaptically connected. It is consistent with a concept of developmental pattern formation in which (i) the number of cells of a particular type and their laminar distribution are specified, and (ii) the final spatial position of each cell is controlled exclusively by a rule that prevents cells of the same type from being positioned close to each other. This sequence would imply that a cell's final position is independent of the cell's position at the time of its specification, and we suggest a reason why, in laminar structures containing many cell types, it might be desirable for this to be so.

Different contributions of GABAA and GABAC receptors to rod and cone bipolar cells in a rat retinal slice preparation.

Whole cell currents were recorded from rod and cone bipolar cells in a slice preparation of the rat retina. Use of the gramicidin D perforated-patch technique prevented loss of intracellular compounds. The recorded cells were identified morphologically by injection with Lucifer yellow. During the recordings, the cells were isolated synaptically by extracellular cobalt. To distinguish the gamma-aminobutyric acid (GABA) receptors pharmacologically, the GABAA receptor antagonist, bicuculline, and the GABAC receptor antagonist, 3-aminopropyl(methyl)phosphinic acid, were used. In all bipolar cells tested, application of GABA induced postsynaptic chloride currents that hyperpolarized the cells from their resting potential of about -40 mV. GABA was applied at different concentrations to allow for the different affinity of GABA at GABAA and GABAC receptors. At a GABA concentration of 25 microM, in the case of rod bipolar cells, approximately 70% of the current was found to be mediated by GABAC receptors. In the case of cone bipolar cells, only approximately 20% of the current was mediated by GABAC receptors. Furthermore, this GABAC-mediated fraction varied among the different morphological types of cone bipolar cells, supporting the hypothesis of distinct functional roles for the different types of cone bipolar cells. There is evidence that the efficacy of GABAC receptors is modulated by glutamate through metabotropic glutamate receptors. We tested this hypothesis by applying agonists of metabotropic glutamate receptors (mGluR)1/5 to rod bipolar cells. The specific agonist (+/-)-trans-azetidine-2, 4-dicarboxylic acid and the potent mGluR agonist quisqualic acid reduced the amplitude of the GABAC responses by 10-30%. This suggests a functional role for the modulation of GABAC receptors by the metabotropic glutamate receptors mGluR1/5.

Glutamate responses of bipolar cells in a slice preparation of the rat retina.

Whole-cell currents from >70 voltage-clamped bipolar cells were recorded in a slice preparation of the rat retina. The recorded cells were identified and classified by intracellular staining with Lucifer yellow. Glutamate, the specific agonists (+/-)-2-amino-4-phosphonobutyric acid (AP-4) and kainate (KA), and the antagonist 6-cyanoquinoxaline-2,3-dione (CNQX) were applied. The cells could be isolated from presynaptic influences by the co-application of bicuculline, strychnine, and cobalt ions. Responses to AP-4 were elicited only from bipolar cells with axons stratifying in the inner part of the inner plexiform layer (IPL). AP-4 caused an outward current in these cells attributable to the closure of nonspecific cation channels. Responses to kainate representing a direct action of the drug on the recorded cells were observed only in bipolar cells with axons stratifying in the outer part of the IPL. KA caused a CNQX-sensitive inward current in these cells, associated with openings of nonspecific cation channels. The results predict that cone bipolar (CB) cells with axons terminating in the outer IPL are OFF-bipolars, whereas those with axons terminating in the inner IPL are ON-bipolars. Most of the cells expressed GABA-gated Cl- conductances. In rod bipolar and in some CB cells, only part of the GABA-induced currents could be blocked by the application of bicuculline, suggesting the presence of GABAc receptors in addition to GABAA receptors.

Immunocytochemical identification of cone bipolar cells in the rat retina.

We studied the morphology of bipolar cells in fixed vertical tissue sections of the rat retina by injecting the cells with Lucifer Yellow and neurobiotin. In addition to the rod bipolar cell, nine different putative cone bipolar cell types were distinguished according to the position of their somata in the inner nuclear layer and the branching pattern and stratification level of their axon terminals in the inner plexiform layer. Some of these bipolar cell populations were labeled immunocytochemically in vertical and horizontal sections using antibodies against the calcium-binding protein recoverin, the glutamate transporter GLT-1, the alpha isoform of the protein kinase C, and the Purkinje cell marker L7. These immunocytochemically labeled cell types were characterized in terms of cell density and distribution. We found that rod bipolar cells and GLT-1-positive cone bipolar cells occur at higher densities in a small region located in the upper central retina. This area probably corresponds to the central area, which is the region of highest ganglion cell density. A second peak of rod bipolar cell density in the lower temporal periphery matches the retinal area of binocular overlap. The population densities of the immunocytochemically characterized bipolar cells indicate that at least 50% of all bipolar cells are cone bipolar cells. The variety and total number of cone bipolar cells is surprising because the retina of the rat contains 99% rods. Our findings suggest that cone bipolar cells may play a more important role in the visual system of the rat than previously thought.

Co-stratification of GABAA receptors with the directionally selective circuitry of the rat retina.

Direction-selective (DS) ganglion cells of the mammalian retina have their dendrites in the inner plexiform layer (IPL) confined to two narrow strata. The same strata are also occupied by the dendrites of cholinergic amacrine cells which are probably presynaptic to the DS ganglion cells. GABA is known to play a crucial role in creating DS responses. We examined the types of GABAA receptors expressed by the cholinergic amacrine cells and also those expressed by their presynaptic and postsynaptic neurons, by applying immunocytochemical markers to vertical sections of rat retinas. Double-labelling experiments with antibodies against choline acetyltransferase (ChAT) and specific antibodies against different GABAA receptor subunits were performed. Cholinergic amacrine cells seem to express an unusual combination of GABAA receptor subunits consisting of alpha 2-, beta 1-, beta 2/3-, gamma 2-, and delta-subunits. Bipolar cells, which could provide synaptic input to the DS circuitry, were stained with antibodies against the glutamate transporter GLT-1. The axon terminals of these bipolar cells are narrowly stratified in close proximity to the dendritic plexus of displaced cholinergic amacrine cells. The retinal distribution of synaptoporin, a synaptic vesicle associated protein, was studied. Strong reduction of immunolabelling was observed in the two cholinergic strata. The anatomical findings are discussed in the context of models of the DS circuitry of the mammalian retina.