Spatiotemporal testing and modeling of catfish retinal neurons.

The responses of retinal neurons depend on the interaction of both temporal and spatial aspects of a light stimulus. We developed a linear spatiotemporal model of receptor and horizontal cell layers in the catfish retina based on reciprocal interactions between both layers and coupling within each. Horizontal cell transfer properties were measured experimentally using white-noise intensity modulated light spots of different diameters and were compared with analytical predictions based on the model. Good agreement was obtained with a reasonable choice of model space-constants and feedback parameters. Furthermore, the same set of parameter values determined from spot experiments enabled accurate prediction of experimental horizontal cell responses to traveling gratings. The proposed feedback connections from horizontal cells to receptors quicken the time-course of responses in both layers and sharpen receptive fields.     

Models for the cross-correlation between retinal ganglion cells

Neighboring ganglion cells in the vertebrate retina not only respond to the same stimuli but also display cross-correlated activity on a millisecond time scale. Recent studies of this cross-correlation have indicated that simple linear addition of common variability to each ganglion cell signal does not account for the observations (Levine 1997). In this report, Monte Carlo simulations of various linear and nonlinear models are presented that confirm the earlier speculations. Models in which common variability alters the leakages of a pair of leaky integrate-and-fire neurons account for the data and predict the cross-correlogram lag without invoking temporal delay lines. Received: 23 August 1996 / Received after review process: 18 June 1998 / Accepted in revised form: 13 July 1998     

Adrenergic retinal neurons

Summary Adrenergic retinal neurons have been studied in cynomolgus monkeys, cats, rabbits, guinea-pigs, rats, and mice with the fluorescence technique of Falck and Hillarp. With some species variations, three adrenergic fibre layers have been observed: an outer adrenergic fibre layer (all species) at the border between the inner nuclear and inner plexiform layers, a middle adrenergic fibre layer (rabbits, guinea-pigs, rats, and mice) in the middle of the inner plexiform layer, and an inner adrenergic fibre layer (rabbits) at the border between the inner plexiform layer and the ganglion cell layer. Similarly, three kinds of adrenergic nerve cells have been found: a somewhat heterogenous group of outer adrenergic cells (all species) situated in the innermost cell rows of the inner nuclear layer, eremite cells (rabbits, guinea-pigs, rats, and mice) within the inner plexiform layer and alloganglionic cells (all species) with a position and appearance resembling some of the ordinary non-adrenergic cells of the ganglion cell layer. All the adrenergic cells are star-shaped with slender branching processes running to the different adrenergic layers.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under grant AF EOAR 66-14 through the European Office of Aerospace Research (OAR), United States Air Force, by the United States Public Health Service (grant no. NB 05236-02), by the Swedish Medical Research Council (grant no. B 66-320), and by the Faculty of Medicine, University of Lund, Sweden.     

Spatio-temporal convergence (STC) in otolith neurons

It has been recently demonstrated that some primary otolith afferents and most otolith-related vestibular nuclei neurons encode two spatial dimensions that can be described by two vectors in temporal and spatial quadrature. These cells are called broadly-tuned neurons. They are characterized by a non-zero tuning ratio which is defined as the ratio of the minimum over the maximum sensitivity of the neuron. Broadly-tuned neurons exhibit response gains that do not vary according to the cosine of the angle between the stimulus direction and the cell's maximum sensitivity vector and response phase values that depend on stimulus orientation. These responses were observed during stimulation with pure linear acceleration and can be explained by spatio-temporal convergence (STC) of primary otolith afferents and/or otolith hair cells. Simulations of STC of the inputs to primary otolith afferents and vestibular nuclei neurons have revealed interesting characteristics: First, in the case of two narrowly-tuned input signals, the largest tuning ratio is achieved when the input signals are of equal gain. The smaller the phase difference between the input vectors, the larger the orientation differences that are required to produce a certain tuning ratio. Orientation and temporal phase differences of 30–40° create tuning ratios of approximately 0.10–0.15 in target neurons. Second, in the case of multiple input signals, the larger the number of converging inputs, the smaller the tuning ratio of the target neuron. The tuning ratio depends on the number of input units, as long as there are not more than about 10. For more than 10–20 input vectors, the tuning ratio becomes almost independent of the number of inputs. Further, if the inputs comprise two populations (with different gain and phase values at a given stimulus frequency), the largest tuning ratio is obtained when the larger population has a smaller gain. These findings are discussed in the context of known anatomical and physiological characteristics of innervation patterns of primary otolith afferents and their possible convergence onto vestibular nuclei neurons.     

Spontaneous activity of dopaminergic retinal neurons

Dopaminergic local circuit neurons in the retina (DA cells) show robust, spontaneous, tetrodotoxin-sensitive pacemaking. To investigate the mechanism underlying this behavior, we characterized the sodium current and a subset of the potassium currents in the cells in voltage-clamp experiments. We found that there is a persistent component of the sodium current in DA cells which activates at more depolarized potentials than the transient component of the current. The transient component was completely inactivated at -50 mV, but DA cells remained able to fire spontaneous action potentials when potassium channels were partially blocked and the membrane potential remained above -40 mV. Based on these electrophysiological data, we developed a reduced computer model that reproduced the major features of DA cells. In simulations at the physiological resting potential, the persistent component of the sodium current was both necessary and sufficient to account for spontaneous activity, and the major contribution of the transient component of the sodium current was to initiate the depolarization of the model cell during the interspike interval. When tonic inhibition was simulated by lowering the input impedance of the model cell, the transient component played a larger role.     

Contour of auto- and cross-correlation histograms of the spike flows of monosynaptically connected neurons

By methods of neuronal interaction modelling--biomathematical (computer controlled experiment on molluscs neurones) and mathematical--in wide physiological ranges of parameters values, characterizing properties and conditions of neurones and synapses functioning, the forms were studied of auto- and cross-correlation histograms of impulse flows of neurones at forward and backward monosynaptic connections between them. Specific form is established of cross-correlation histogram of impulse flows of interconnected neurones in conditions typical of CNS of mammals, when the neurones are subjected to intensive random afferent synaptic bombardment and do not reveal any pace-maker properties. It is also shown that random afferent synaptic bombardment prevents the appearance of excitation reverberation in closed neuronal circuits.     

Two-photon cross-correlation analysis of intracellular reactions with variable stoichiometry

We successfully demonstrate the effectiveness of two-photon fluorescence cross-correlation spectroscopy (TPCCS) to study the complex binding stoichiometry of calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaMKII). Practical considerations are made for developing an intracellular cross-correlation assay, including characterization of the fluorescent molecules involved, calibration procedures of the setup, and optimal measurement conditions. Potential pitfalls and artifacts are discussed, and the complex stoichiometry of the molecular system is accounted for by a new experimental and theoretical framework for TPCCS. Our tailored model accommodates up to 12 red-labeled CaMs binding to a single green-labeled dodecameric CaMKII holoenzyme and accounts for the probability distributions of bound ligand as well as the respective changes in fluorescence emission upon binding. The model was experimentally demonstrated both in solution and in living cells by analyzing the binding of Alexa 633(C2)CaM to eGFP-CaMKII under different biochemical conditions known to induce the basal, activated, and autophosphorylated forms of the enzyme. Key binding parameters, such as binding degree, concentrations of reactants, and binding affinities, were determined under varying conditions with certain assumptions. TPCCS thus offers the unique ability to test our biochemical understanding of protein dynamics in the intracellular milieu.     

Adrenergic retinal neurons of some new world monkeys

Summary The retina of Aotes monkeys, Cebus monkeys, squirrel monkeys, and marmosets were investigated. Adrenergic perikarya were found in the innermost cell rows of the inner nuclear layer of all the investigated species. In addition, the Cebus monkey was found to have a special type of adrenergic neurons in the inner nuclear layer. This cell type was called the adrenergic pleomorph cell. Its processes ramify in the inner nuclear and inner plexiform layers. Adrenergic terminals occur in three more or less well developed sublayers of the inner plexiform layer, the layers being best developed in the Cebus monkey. Adrenergic terminals were also found around the cells of the inner nuclear layer and at the horizontal cells, where a scant sublayer is formed. More than one adrenergic sublayer of the inner plexiform layer has not been observed in primates previously, nor have the adrenergic terminals in the inner nuclear layer been observed previously in any species. The adrenergic pleomorph cells of the Cebus monkey also seem to be unique. The marked differences even between animals as closely related as some platyrhine monkeys call for caution when comparing the detailed function of the retina in different animals.This study was supported by grants from the Swedish Medical Research Council (B69-14X-2321-02) and the Faculty of Medicine, University of Lund, and was carried out within a research group sponsored by the Swedish Medical Research Council (projects No. B69-14X-56-05C and B69-14X-712-04C).     

Differentiation of embryonic stem cells into retinal neurons

Mouse embryonic stem (ES) cells are continuous cell lines derived from the inner mass of blastocysts. Neural progenitors derived from these cells serve as an excellent model for controlled neural differentiation and as such have tremendous potential to understand and treat neurodegenerative diseases. Here, we demonstrate that ES cell-derived neural progenitors express regulatory factors needed for retinal differentiation and that in response to epigenetic cues a subset of them differentiate along photoreceptor lineage. During the differentiation, they activate photoreceptor regulatory genes, suggesting that ES cell-derived neural progenitors recruit mechanisms normally used for photoreceptor differentiation in vivo. These observations suggest that ES cells can serve as an excellent model for understanding mechanisms that regulate specification of retinal neurons and as an unlimited source of neural progenitors for treating degenerative diseases of the retina by cell replacement.     

Possibilities of detecting the plasticity of monosynaptic excitable connections during the analysis of impulse activity of neurons using cross-correlation histograms and Cox's method

By the method of mathematical modelling, stationary irregular exogenous impulse activity of two monosynaptically connected neurones (N1 and N2; excitatory connection) was reproduced. The degree of impulse flows N1 and N2 dependence was estimated by the value P12, proportional to the height of crosscorrelation histogram peak, and by Cox's coefficient beta 12. The dynamics was studied of P12, beta 12 and of values P1* and P2*, proportional to mean interpulse intervals of N1 and N2, at changes of connection efficiency, neurones excitability and of influence of afferent synaptic bombardment on them. The following signs of modification of existing between N1 and N2 monosynaptic excitatory connection were established: 1) change of P12 in direction opposite to that of P1* and P2* change; 2) differently directed changes of beta 12 and P2* of postsynaptic N2.     

Determining protease activity in vivo by fluorescence cross-correlation analysis

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions.     

The functional characteristics of electroreceptive central neurons of the sea catfish Plotosus anguillaris

With the use of microelectrode techniques (extracellular recordings) and the method of post-stimulus histograms, the functional characteristics of medulla oblongata neurons of sea catfish Plotosus were investigated under stimulation of electroreceptors by a homogeneous electric field of different duration, intensity, and direction. Two types of the cells possessing, accordingly, tonic or phase activity were registered among 66 neurons investigated. The mode of responses (inhibition or acceleration) of tonic neurons to the direction of the applied electric current is typical for central neurons of fresh-water catfish connected with ampullae's electroreceptors. Neurons showing a substantial response to fields of an intensity less than 1 microV/cm were registered. The reactions were most pronounced with the duration of electric stimuli in the range of 20-200 ms; however, particularly sensitive neurons showed distinct responses to stimuli of duration of 5 and even 2 ms. Thus, for the first time a high sensitivity of ampullae's electroreceptors to high-frequency stimulus was discovered, which allows one to expand the range of studying electric signals used by weakly electric fish for electrolocation and communication.     

Indoleamine accumulation by retinal neurons exposed to blood

Exposing rabbit retinas for one minute to an incubation medium containing 10 microliters of blood diluted in 20 ml of medium was sufficient to produce serotonin-like immunoreactivity in some of the retinal indoleamine-accumulating neurons. Retinas from rabbits that had been perfused before the eyes were removed had no detectable immunoreactivity. Our results support the conjecture that the serotonin sometimes detected in the retina originates in the blood. Why the cells have a carrier for a molecule that they do not normally contain remains unclear.     

Mammalian retinal horizontal cells are unconventional GABAergic neurons

Lateral interactions at the first retinal synapse have been initially proposed to involve GABA by transporter-mediated release from horizontal cells, onto GABA(A) receptors expressed on cone photoreceptor terminals and/or bipolar cell dendrites. However, in the mammalian retina, horizontal cells do not seem to contain GABA systematically or to express membrane GABA transporters. We here report that mouse retinal horizontal cells express GAD65 and/or GAD67 mRNA, and were weakly but consistently immunostained for GAD65/67. While GABA was readily detected after intracardiac perfusion, it was lost during classical preparation for histology or electrophysiology. It could not be restored by incubation in a GABA-containing medium, confirming the absence of membrane GABA transporters in these cells. However, GABA was synthesized de novo from glutamate or glutamine, upon addition of pyridoxal 5'-phosphate, a cofactor of GAD65/67. Mouse horizontal cells are thus atypical GABAergic neurons, with no functional GABA uptake, but a glutamate and/or glutamine transport system allowing GABA synthesis, probably depending physiologically from glutamate released by photoreceptors. Our results suggest that the role of GABA in lateral inhibition may have been underestimated, at least in mammals, and that tissue pre-incubation with glutamine and pyridoxal 5'-phosphate should yield a more precise estimate of outer retinal processing.