[Retinal and Cortical Activation by Electrical Stimulation with Retina Implants]. / Retinale und kortikale Aktivierung durch elektrische Stimulation mit Netzhautimplantaten.

In Germany, about 30,000 to 40,000 people suffer from retinitis pigmentosa (RP), which ultimately results in blindness. The only aid to blind RP patients are retinal implants: These have been under development for several years and have now been approved as a medical product. Retinal implants produce visual perceptions in response to electrical stimulation of the degenerated retina and are useful in the everyday life of blind people. However, the currently achievable quality of vision is such that people with a retinal implant are still legally blind. The visual quality that can be achieved with epi- and subretinal implants depends not only on patient-specific factors such as individual history and status of retinal degeneration, but especially on the interface between implant and retina and the quality of the achievable neuronal activation. Biophysical approaches to functional improvements of the implants are founded on the physiology of the retina (cell density, intraretinal interconnections), are based on technical optimisation of the interface (electrode materials, size and density), and exploit the stimulation protocols with which visual information is fed into the degenerated retina (time courses of electrical stimuli, spatiotemporal stimulation pattern). Optimisation of stimulation parameters can be supported by a detailed analysis of cortical responses, with appropriate electrophysiological and optical methods. This article looks at both the physiological and biophysical fundamentals of electrical retinal stimulation, as well as the resulting retinal and cortical activation.

Metaplasticity of horizontal connections in the vicinity of focal laser lesions in rat visual cortex.

Focal cortical injuries are accompanied by a reorganization of the adjacent neuronal networks. An increased synaptic plasticity has been suggested to mediate, at least in part, this functional reorganization. Previous studies showed an increased long-term potentiation (LTP) at synapses formed by ascending fibres projecting onto layers 2/3 pyramidal cells following lesions in rat visual cortex. This could be important to establish new functional connections within a vertical cortical column. Importantly, horizontal intracortical connections constitute an optimal substrate to mediate the functional reorganization across different cortical columns. However, so far little is known about their potential implication in the functional rewiring post-lesion. Here, we investigated possible alterations of synaptic plasticity of horizontal connections in layers 2/3 in an 'ex vivo-in vitro' model of focal laser lesion in rat visual cortex. LTP at these synapses was found to be enhanced post-lesion, whereas long-term depression (LTD) was impaired, revealing a metaplastic shift toward strengthening of these synapses. Furthermore, we disclosed a prolonged decay-time constant of NMDAR-dependent currents, which can contribute to the enhanced LTP. Taken together these data revealed that a laser lesion-induced focal damage of the visual cortex is accompanied by a facilitated potentiation of horizontal synaptic connections in the vicinity of the focal injury. This specific strengthening of synaptic plasticity at horizontal connections in layers 2/3 might be one important cellular mechanism to compensate focal injury-mediated dysfunction in the cerebral cortex.

Functional selectivity of interhemispheric connections in cat visual cortex.

The functional specificity of callosal connections was investigated in visual areas 17 and 18 of adult cats, by combining in vivo optical imaging of intrinsic signals with labeling of callosal axons. Local injections of neuronal tracers were performed in one hemisphere and eight single callosal axons were reconstructed in the opposite hemisphere. The distributions of injection sites and callosal axon terminals were analyzed with respect to functional maps in both hemispheres. Typically, each callosal axon displayed 2 or 3 clusters of synaptic boutons in layer II/III and the upper part of layer IV. These clusters were preferentially distributed in regions representing the same orientation and the same visuotopic location as that at the corresponding injection sites in the opposite hemisphere. The spatial distribution of these clusters was elongated and its main axis correlated well with the preferred orientation at the injection site. These results demonstrate a specific organization of interhemispheric axons that link cortical regions representing the same orientation and the same location of visual stimuli. Visual callosal connections are thus likely involved in the processing of coherent information in terms of shape and position along the midline of the visual field, which may facilitate the fusion of both hemifields into the percept of a single visual scene.

Layout of transcallosal activity in cat visual cortex revealed by optical imaging.

The contribution of interhemispheric connections to functional maps in cat visual cortex was investigated by using optical imaging of intrinsic signals. In order to isolate the functional inputs arriving via the corpus callosum (CC) from other inputs, we used the split-chiasm preparation. The regions activated through the CC in visual areas 17 (A17) and 18 (A18) were localized and characterized by stimulating monocularly split-chiasm cats with moving, high contrast oriented gratings. We found that the CC mediates the activation of orientation selective domains in the transition zone (TZ) between A17 and A18 and occasionally within portions of both of these areas. We observed transcallosally activated orientation domains all along the TZ without any obvious interruption, and these domains were arranged around "pinwheel" centers. Interestingly, the TZ was divided in two parallel regions, which resemble A17 and A18 in their preferred temporal and spatial frequencies. Finally, we demonstrated that orientation maps evoked through the transcallosal and geniculo-cortical pathways were similar within the TZ, indicating a convergence of inputs of matching orientations in this region. These results contribute to a better understanding of the role of the CC in visual perception of orientations and shapes, at the level of the visual cortex.

Changes in intracellular calcium transients and LTP in the surround of visual cortex lesions in rats.

Injury and loss of neurons are observed in the center of a cerebral cortical lesion. Mechanisms of early functional reorganization post-lesion involve changes in the strength of synaptic coupling as measured in long-term potentiation (LTP). Since these changes in LTP may depend on the intraneuronal calcium concentration ([Ca2+]I), the present study analyzed the strength of synaptic LTP combined with measurements of the stimulus-induced peak calcium influx in slices from rat visual cortex in vitro. Slices were analyzed 1-7 days post-lesion by use of electrophysiological and calcium fluorescence imaging techniques. A theta-burst stimulus (TBS) was electrically applied to cortical layer IV, while changes in extracellular field potentials (FPs) and in the corresponding peak calcium influx were recorded in layers II/III. Both the strength of LTP and of the FP mediated peak calcium influx were significantly enhanced 1-6 days post-lesion at a distance of 4 mm from the lesion border. Pharmacological experiments revealed that the expression of LTP was dependent on the activation of NMDA receptors. The area of increased stimulus-evoked peak calcium influx correlated with the enhanced LTP, suggesting that changes in [Ca2+]I mediate the strength of long-term synaptic plasticity following a cortical lesion. This mechanism may support synaptic reorganization in the surround of the deafferented region in rat visual cortex.

Retinotopic map plasticity in adult cat visual cortex is accompanied by changes in Ca2+/calmodulin-dependent protein kinase II alpha autophosphorylation.

In adult cats, the induction of homonymous binocular central retinal lesions causes a dramatic reorganization of the topographic map in the sensory-deprived region of the primary visual cortex. To investigate the possible involvement of the alpha-subunit of the calcium/calmodulin dependent protein kinase type II (alphaCaMKII) in this form of brain plasticity, we performed in situ hybridization and Western blotting experiments to analyze mRNA, protein and autophosphorylation levels of this multifunctional kinase. No differences in the mRNA or protein levels were observed between the central, sensory-deprived and the peripheral, non-deprived regions of area 17 of retinal lesion animals or between corresponding cortical regions of normal control animals. Western blotting with an alphaCaMKII threonine-286 phosphorylation-state specific antiserum consistently showed a small, albeit not significant, increase of alphaCaMKII autophosphorylation in the central versus the peripheral region of cortical area 17, and this both in normal subjects as well as in retinal lesion animals with a 3-day post-lesion survival time. In contrast, a post-lesion survival time of 14 days resulted in a alphaCaMKII autophosphorylation level that was four times higher in visually-deprived area 17 than in the non-deprived cortical region. This increased phosphorylation state is not a direct consequence of the decrease in visual activity in these neurons, because we would have expected to see a similar change at shorter or longer post-lesion survival times or in the visually deprived visual cortex of animals in which the left optic tract and the corpus callosum were surgically cut. No such changes were observed, leading to the conclusion that the phosphorylation changes observed at 14 days are related to a delayed reorganization of the retinotopic map of the striate cortex.

Spatio-temporal plasticity of cortical receptive fields in response to repetitive visual stimulation in the adult cat.

Many psychophysical experiments on perceptual learning in humans show increases of performance that are most probably based on functions of early visual cortical areas. Long-term plasticity of the primary visual cortex has so far been shown in vivo with the use of visual stimuli paired with electrical or pharmacological stimulation at the cellular level. Here, we report that plasticity in the adult visual cortex can be achieved by repetitive visual stimulation. First, spatial receptive field profiles of single units (n=38) in area 17 or 18 of the anesthetized cat were determined with optimally oriented flashing light bars. Then a conditioning protocol was applied to induce associative synaptic plasticity. The receptive field center and an unresponsive region just outside the excitatory receptive field were synchronously stimulated ('costimulation', repetition rate 1 Hz; for 10-75 min). After costimulation the receptive field and its adjacent regions were mapped again. We observed specific increases of the receptive field size, changes of the receptive field subfield structure as well as shifts in response latency. In 37% of the cells the receptive field size increased specifically towards the stimulated side but not towards the non-stimulated opposite side of the receptive field. In addition, changes in the relative strength and size of the on and off subfield regions were observed. These specific alterations were dependent on the level of neuronal activity during costimulation. During recovery, the new responses dropped down to 120% of the preconditioning value on average within 103 min; however, the decay times significantly depended on the response magnitude after costimulation. In the temporal domain, the latency of new responses appeared to be strongly influenced by the latency of the response during costimulation.Twenty-nine percent of the units displayed no receptive field enlargement, most likely because the activity during costimulation was significantly lower than in the cases with enlarged receptive fields. An unspecific receptive field enlargement towards both the stimulated and non-stimulated side was observed in 34% of the tested cells. In contrast to the cells with specifically enlarged receptive fields, the unspecific increase of receptive field size was always accompanied by a strong increase of the general activity level. We conclude that the receptive field changes presumably took place by strengthening of synaptic inputs at the recorded cells in a Hebbian way as previously shown in the visual cortex in vitro and in vivo. The observed receptive field changes may be related to preattentive perceptual learning and could represent a basis of the 'filling in' of cortical scotomas obtained with specific training procedures in human patients suffering from visual cortex lesions.

Increased synaptic plasticity in the surround of visual cortex lesions in rats.

Lesion-induced functional loss is reduced when new synaptic connections are established in the surround of a cortical lesion. For this, long-term synaptic plasticity can play a key role. We studied long-term potentiation (LTP) and long-term depression (LTD) in slices of rat visual cortex with small cortical lesions. Surprisingly, the normal balance between LTP and LTD was significantly altered in the first week following cortical injury. Theta-burst induced LTP was increased, whereas LTD evoked by low frequency stimulation was not affected. The increased potentiation of subcortical inputs after cortical lesions opens a window for facilitated early functional reorganization by repetitive visual training.

Intracellular calcium signals in the surround of rat visual cortex lesions.

Focal lesions of the visual cortex induce deafferentiation, excitotoxic cell death as well as functional reorganization in the surrounding tissue. The intracellular second messenger calcium is involved in a wide range of cellular responses including excitotoxicity and functional reorganization following cortical injuries. We investigated the intracellular calcium concentration [Ca2+]i in neurons of the visual cortex using fluorescence imaging of fura-2 signals in a slice preparation obtained from lesioned and sham-operated cortices. We observed an increase in resting and stimulus evoked [Ca2+]i in the surround of the lesion, which were mediated by NMDA and non-NMDA ionotropic glutamate receptors. This increase in [Ca2+]i might be an important factor for lesion-induced functional reorganization in the rat visual cortex.

Axonal topography of cortical basket cells in relation to orientation, direction, and ocular dominance maps.

The axonal (bouton) distributions of a layer 4 clutch cell (CC), two layer 3 medium-sized basket cells (MBC), and a layer 3 large basket cell (LBC) to orientation, direction, and ocular dominance maps were studied quantitatively. 1) The CC provided exclusively local projections (<380 microm from the soma) and contacted a narrow "niche" of functional representations. 2) The two MBCs emitted local projections (75% and 79% of all boutons), which were engaged with isoorientations (61% and 48%) and isodirections, and long-range projections (25% and 21%, >313 microm and >418 microm), which encountered cross-orientation sites (14% and 12%) and isoorientation sites (7% and 5%). Their direction preferences were mainly perpendicular to or opposite those of local projections. 3) The LBC provided the majority (60%) of its boutons to long-range distances (>437 microm). Locally, LBC boutons showed a rather balanced contribution to isoorientations (19%) and cross-orientations (12%) and preferred isodirections. Remotely, however, cross-orientation sites were preferred (31% vs. 23%) and the directional output was balanced. 4) Monte Carlo simulations revealed that the differences between the orientation specificity of local and long-range projections cannot be explained by a homogeneous lateral distribution of the boutons. 5) There was a similar eye preference in the local and long-range projection fields of the MBCs. The LBC contacted both contra- and ipsilateral eye domains. 6) The basket axons showed little laminar difference in orientation and direction topography. The results suggest that an individual basket cell can mediate a wide range of effects depending on the size and termination pattern of the axonal field.

Topography of orientation centre connections in the primary visual cortex of the cat.

The functional topography of lateral connections to orientation-centre zones was studied by optical imaging of intrinsic signals in combination with tracer injections (fluorescent beads and biocytin) and electrophysiological recordings. Three-dimensional reconstruction of anterogradely labelled axon terminals and retrogradely labelled somata revealed a uniform distribution across all orientations in a non-patchy manner. The overall lateral extent of the labelling was 3-4 mm in layer 3, that is about half of the extent observed for orientation domain connections in the same layer. These bulk injection data are in contrast with the reportedly sharp orientation tuning of neurons of centre zones and suggest that orientation specificity here does not require highly specific connections. Nonetheless, another plausible scenario is that orientation centre connections are orientation specific but their specificity present at the single cell level cannot be revealed by bulk labelling due to their large spatial overlap.

Calculating direction maps from intrinsic signals revealed by optical imaging.

Previous optical imaging studies used the vector-summation (VS) method for calculating direction and orientation preference maps. However, for direction maps it often resulted in direction vectors which showed a steep angle to that of orientation vectors violating the 'aperture rule'. The present report provides a simple procedure for calculating direction preference maps using the 'electro- physiologist's ear' approach. This approach takes into account the strongest directional response component (vector-maximum, VM) in each pixel of the optical image, reminiscent of how electro- physiologists determine direction preference by audio-monitoring of the firing rate of neurons. The major advantage of this method is that the orthogonal relationship between orientation and direction preference vectors is preserved and that for most image pixels direction preference can be faithfully described by a single vector parameter. Here we used the VM method for calculating direction and the VS method for calculating orientation preference maps and quantified their spatial relationship. The results showed that, typically, an iso-orientation domain contained a pair of patches that preferred opposite directions orthogonal to the orientation. Rate-of-change maps for direction revealed that virtually all direction discontinuity lines linked orientation centres. Close to orientation centres, direction discontinuity lines ran chiefly parallel with iso-orientation lines, whereas more remotely they had either parallel or perpendicular courses.

Comparison of orientation maps obtained with different number of stimulus orientations.

The structure of orientation maps computed from a different number of stimulus orientations was studied in visual cortical area 18 of the cat. Single condition maps (SCMs) were obtained to 16 stimulus orientations, of which angle maps were generated using 4, 8, and 16 SCMs corresponding to multiples of 45, 22.5, and 11.25 degrees, respectively. The overall orientation distribution of the three types of maps was compared on a pixel-by-pixel basis. Twenty percent of the pixels of the 4-orientations maps differed by more than +/-17 degrees from those produced by 16 orientations. Maps of 8 orientations differed by 6.4 and 5.8% from those of 4 and 16 orientations, respectively. Structural differences between the maps were mainly found at locations displaying high rate of change in orientation preference, i.e., orientation centers and adjoining short, fracture-like zones. These changes included lateral shifts up to 155 microm (average: 38.7 microm) in the position of orientation centers and appearance/disappearance of orientation centers when compared between different conditions. In general, these changes were three times more frequent between maps of 4/8 and 4/16 orientations than 8/16 orientations. It is concluded that orientation maps should be calculated from activity maps representing 8 or more stimulus orientations.

Changes in response modulation of cat perigeniculate neurons related to EEG state and application of neuromodulators.

Spike activity of single perigeniculate (PGN) neurons was recorded in the anaesthetized (N2O/halothane) and paralysed cat during presentation of moving gratings of optimal spatial frequency. Typically, the ongoing (tonic, spontaneous) activity of PGN cells increased during a rise in EEG delta power accompanied by a reduction and often a total loss of spike rate modulation by the moving grating. The opposite behaviour was found when the EEG delta power vanished. Micro-iontophoretically applied acetylcholine (ACh) had an effect similar to a decrease in EEG delta power, decreasing ongoing activity but increasing the response modulation depth. The opposite effect could be achieved with the excitatory action of serotonin (5-HT), mimicking a strengthened EEG delta power. These data support previous data indicating that PGN neurons contribute to spatio-temporal tuning of subcortical visual activity in a state-dependent way.

Enhanced responsiveness of human extravisual areas to photic stimulation in patients with severely reduced vision.

Lesions in the primary visual cortex induce severe loss of visual perception. Depending on the size of the lesion, the visual field might be affected by small scotomas, hemianopia, or complete loss of vision (cortical blindness). In many cases, the whole visual field of the patient is affected by the lesion, but diffuse light-dark discrimination remains (residual rudimentary vision, RRV). In other cases, a sparing of a few degrees can be found (severely reduced vision, SRV). In a follow-up study, we mapped visually induced cerebral activation of three subjects with SRV using functional magnetic resonance imaging. We were especially interested in the visual areas that would be activated if subjects could perceive the stimulus consciously although information flow from V1 to higher visual areas was strongly reduced or virtually absent. Because subjects were only able to discriminate strong light from darkness, we used goggles flashing intense red light at a frequency of 3 Hz for full visual field stimulation. Besides reduced activation in V1, we found activation in the parietal cortex, the frontal eye fields (FEF), and the supplementary eye fields (SEF). In all patients, FEF activation was pronounced in the right hemisphere. These patterns were never seen in healthy volunteers. In a patient who recovered completely, we observed that extrastriate activation disappeared in parallel with the visual field restitution. This result suggests that damage to the primary visual cortex changes the responsiveness of parietal and extravisual frontal areas in patients with SRV. This unexpected result might be explained by increased stimulus-related activation of attention-related networks.

Cooperative changes in GABA, glutamate and activity levels: the missing link in cortical plasticity.

Different intracortical mechanisms have been reported to contribute to the substantial topographic reorganization of the mammalian primary visual cortex in response to matching lesions in the two retinas: an immediate expansion of receptive fields followed by a gradual shift of excitability into the deprived area and finally axonal sprouting of laterally projecting neurons months after the lesion. To gain insight into the molecular mechanisms of this adult plasticity, we used immunocytochemical and bioanalytical methods to measure the glutamate and GABA neurotransmitter levels in the visual cortex of adult cats with binocular central retinal lesions. Two to four weeks after the lesions, glutamate immunoreactivity was decreased in sensory-deprived cortex as confirmed by HPLC analysis of the glutamate concentration. Within three months normal glutamate immunoreactivity was restored. In addition, the edge of the unresponsive cortex was characterized by markedly increased glutamate immunoreactivity 2-12 weeks postlesion. This glutamate immunoreactivity peak moved into the deprived area over time. These glutamate changes corresponded to decreased spontaneous and visually driven activity in unresponsive cortex and to strikingly increased neuronal activity at the border of this cortical zone. Furthermore, the previously reported decrease in glutamic acid decarboxylase immunoreactivity was found to reflect decreased GABA levels in sensory-deprived cortex. Increased glutamate concentrations and neuronal activity, and decreased GABA concentrations, may be related to changes in synaptic efficiency and could represent a mechanism underlying the retinotopic reorganization that occurs well after the immediate receptive field expansion but long before the late axonal sprouting.

Retrograde signalling with nitric oxide at neocortical synapses.

Long-term changes of synaptic transmission in slices of rat visual cortex were induced by intracellular tetanization: bursts of short depolarizing pulses applied through the intracellular electrode without concomitant presynaptic stimulation. Long-term synaptic changes after this purely postsynaptic induction were associated with alterations of release indices, thus providing a case for retrograde signalling at neocortical synapses. Both long-term potentiation and long-term depression were accompanied by presynaptic changes, indicating that retrograde signalling can achieve both up- and down-regulation of transmitter release. The direction and the magnitude of the amplitude changes induced by a prolonged intracellular tetanization depended on the initial properties of the input. The inputs with initially high paired-pulse facilitation (PPF) ratio, indicative of low release probability, were most often potentiated. The inputs with initially low PPF ratio, indicative of high release probability, were usually depressed or did not change. Thus, prolonged postsynaptic activity can lead to normalization of the weights of nonactivated synapses. The dependence of polarity of synaptic modifications on initial PPF disappeared when plastic changes were induced with a shorter intracellular tetanization, or when the NO signalling pathway was interrupted by inhibition of NO synthase activity or by application of NO scavengers. This indicates that the NO-dependent retrograde signalling system has a relatively high activation threshold. Long-term synaptic modifications, induced by a weak postsynaptic challenge or under blockade of NO signalling, were nevertheless associated with presynaptic changes. This suggests the existence of another retrograde signalling system, additional to the high threshold, NO-dependent system. Therefore, our data provide a clear case for retrograde signalling at neocortical synapses and indicate that multiple retrograde signalling systems, part of which are NO-dependent, are involved.

Effect of partial sensory deprivation on monoaminergic neuromodulators in striate cortex of adult cat.

The role of monoaminergic neuromodulators in the reorganization of cortical topography following limited sensory deprivation in the adult cat was investigated. The total concentrations of dopamine, noradrenaline, serotonin and their major metabolites were measured in the visual cortex of both normal control and experimental animals using microbore high-performance liquid chromatography coupled with electrochemical detection. The experimental animals were subjected to a binocular retinal lesion corresponding to the central 10 degrees of vision and killed two weeks post-lesion. The sensory deprivation was confirmed in area 17 by measuring immediate-early gene zif-268 messenger RNA expression. Following the retinal lesion, the total concentrations of noradrenaline and dopamine were significantly higher in the non-deprived cortex of retinal lesion cats than in the deprived cortex of retinal lesion cats and the cortex of normal animals. This pattern follows the release of the excitatory neurotransmitter glutamate under the same conditions. Serotonin levels were significantly lower in the deprived cortex, and its metabolite 5-hydroxyindole-3-acetic acid was significantly higher in the non-deprived cortex than in deprived cortex and normal cortex. From these results, we suggest that the modulation of noradrenaline, dopamine and serotonin is regulated by visual afferent activity.

Combined physiological-anatomical approaches to study lateral inhibition.

In the visual cortex, large basket cells form the cellular basis of long-range lateral inhibition. The present paper focuses on combinations of methods with which large basket cells can be studied in the context of extensive neuronal representations. In the first approach, the topographic relationship between large basket axons and known functional representations such as orientation, direction, and ocular dominance is analysed. Functional mapping is carried out using extracellular electrode recordings or optical imaging of intrinsic signals followed by 3-dimensional anatomical reconstruction of biocytin stained large basket cells in the same regions. In the second approach, the contribution of lateral inhibition to orientation and direction selectivity is assessed using the GABA inactivation paradigm and direct inhibitory projections from the inactivation to recording sites are demonstrated with biocytin staining and injections of [3H]nipecotic acid, a radioactive marker for GABAergic cells. The limitation of these approaches is that they can only be used in cortical regions which lie on the surface of the brain.