Functional mapping of cortical areas with optical imaging.

Sensory areas in mammalian cortex compute sensory inputs of different modalities in order to perceive the environment. Much is known about the anatomical pattern of inter-laminar connections, which form the basis of the computational process. Nevertheless, less is known about the functional relevance of these wiring patterns. We used intrinsic optical signals (IOSs) in vitro to investigate functional properties of inter-laminar connections in cortical brain slices of rat sensory cortex. By electrical stimulation in layer VI, a columnar-shaped IOS in all cortical areas was found. We detected different laminar patterns of activation in different cortical areas. In primary sensory areas, like primary visual cortex and primary somatosensory cortex, the peak intensity of IOSs occurred in layer IV, which receives the main thalamic input. In secondary sensory areas, like the secondary visual cortex or the secondary somatosensory cortex, the maximum of IOSs amplitude was shifted to layer II/III. In motor areas, IOS peak amplitude is located in layer II/III. In the hind limb area, considered as amalgam between sensory and motor function, a mixture of the activity patterns observed in primary sensory and a motor area occurred with a peak amplitude in layers II and IV. At different stimulation sites within one cortical area, the shape of columnar IOSs remained very similar, reflecting a canonical architecture of functional micro-circuitry. We conclude that both primary and secondary sensory cortical areas display their characteristic functional activation pattern, regardless of their sensory modalities.

Cell swelling and ion redistribution assessed with intrinsic optical signals.

Cell volume changes are associated with alterations of intrinsic optical signals (IOS). In submerged brain slices in vitro, afferent stimulation induces an increase in light transmission. As assessed by measurement of the largely membrane impermeant ion tetramethylammonium (TMA) in the extracellular space, these IOS correlate with the extent and time course of the change of the extracellular space size. They have a high signal to noise ratio and allow measurements of IOS changes in the order of a few percent. Under conditions of reduced net KCl uptake (low Cl solution) a directed spatial buffer mechanism (K syphoning) can be demonstrated in the neocortex with widening of the extracellular space in superficial layers associated with a reduced light transmission and an increase of extracellular K concentration. The nature of the IOS under pathophysiological conditions is less clear. Spreading depressions first cause an increase of light transmission, then a decrease. Such a decrease has also been observed following application of NMDA where it was associated with structural damage. Pharmacological analyses suggest that under physiological conditions changes of extracellular space size are mainly caused by astrocytic volume changes while with strong stimuli and under pathophysiological conditions also neuronal swelling occurs. With reflected light usually signals opposite to those observed with transmitted light are seen. Recording of IOS from interface slices gives very complex signals since under these conditions an increase of light transmission has been reported to be superimposed by a decrease of the signal due to mechanical lensing effects of the slice surface. Depending on the method of measurement and the exact conditions, several mechanisms may contribute to IOS. Under well defined conditions IOS are a useful supplementary tool to monitor changes of extracellular volume both in space and time.

A novel role of vasopressin in the brain: modulation of activity-dependent water flux in the neocortex.

The brain contains an intrinsic vasopressin fiber system the function of which is unknown. It has been demonstrated recently that astrocytes express high levels of a water channel, aquaporin-4 (AQP4). Because vasopressin is known to regulate aquaporin expression and translocation in kidney collecting ducts and thereby control water reabsorption, we hypothesized that vasopressin might serve a similar function in the brain. By recording intrinsic optical signals in an acute cortical slice preparation we showed that evoked neuronal activity generates a radial water flux in the neocortex. The rapid onset and high capacity of this flux suggest that it is mediated through the AQP4-containing astrocytic syncytium that spans the entire thickness of the neocortical mantle. Vasopressin and vasopressin receptor V1a agonists were found to facilitate this flux. V1a antagonists blocked the facilitatory effect of vasopressin and reduced the water flux even in the absence of any exogenous agonist. V2 agonists or antagonists had no effect. These data suggest that vasopressin and V1a receptors play a crucial role in the regulation of brain water and ion homeostasis, most probably by modulating aquaporin-mediated water flux through astrocyte plasma membranes.

From form to function: calcium compartmentalization in dendritic spines.

Dendritic spines compartmentalize calcium, and this could be their main function. We review experimental work on spine calcium dynamics. Calcium influx into spines is mediated by calcium channels and by NMDA and AMPA receptors and is followed by fast diffusional equilibration within the spine head. Calcium decay kinetics are controlled by slower diffusion through the spine neck and by spine calcium pumps. Calcium release occurs in spines, although its role is controversial. Finally, the endogenous calcium buffers in spines remain unknown. Thus, spines are calcium compartments because of their morphologies and local influx and extrusion mechanisms. These studies highlight the richness and heterogeneity of pathways that regulate calcium accumulations in spines and the close relationship between the morphology and function of the spine.

Directed spatial potassium redistribution in rat neocortex.

The functional role of the glial network as a draining system for extracellular potassium (spatial buffer) was investigated in rat neocortical brain slices. After electrical stimulation, extracellular space volume decreased in the middle cortical layers and increased in the upper cortical layers, confirming predictions for a spatial buffer. The widening of extracellular space was associated with an increase in extracellular potassium. The data suggested a delayed redistribution of potassium from middle to superficial cortical layers. Interruption of gap junctions abolished the widening of extracellular space. The data show that a multicellular directed network connected by gap junctions participates in maintaining potassium homeostasis in brain.

Intrinsic optical signals in vitro: a tool to measure alterations in extracellular space with two-dimensional resolution.

In excitable tissues, extensive neuronal activity or pathophysiological conditions, such as spreading depression, ischemic infarct, or epileptic seizure, are accompanied by changes in extracellular space volume. Extracellular space volume, in turn, influences neuronal excitability and extracellular ion concentrations and is, therefore, an important parameter of brain activity. Unfortunately, determination of changes in extracellular space by ion-selective microelectrodes is tedious, restricted to one spot in space at a time and limited in time resolution. In this study we present intrinsic optical signals in vitro as a tool to measure relative changes in extracellular space volume in brain slice preparations with two-dimensional spatial and sufficient time resolution. Evidence is given that the intensity of intrinsic optical signals is linearly correlated to the amplitude of extracellular space volume changes. In contrast, the optical signal is poorly correlated to the concomitant increase in extracellular potassium concentration. We conclude that intrinsic optical signals in vitro are a useful tool to measure the spread of changes in extracellular space volume with high resolution in time and space. In combination with the measurement of the extracellular space at one location using ion-selective microelectrodes, it is possible to calibrate the optical signal to percentile alterations of extracellular space volume.

Intrinsic optical signals in rat neocortical slices measured with near-infrared dark-field microscopy reveal changes in extracellular space.

In the last decade, the measurement of activity-dependent intrinsic optical signals (IOSs) in excitable tissues has become a useful tool for collecting data about spatial patterns of information processing in mammalian brain and spread of excitation. Although the extent of the IOS correlates well with the extent of electrical excitation, its time course is much slower, suggesting that it does not directly monitor the electrical activity. The aim of this study was to investigate the mechanisms responsible for generation of IOSs. Coronal neocortical brain slices of juvenile rats were electrically stimulated at the border of layer VI and the white matter. The induced columnar-shaped IOSs were recorded using dark-field video microscopy. At corresponding locations, alterations in extracellular K+ concentration and extracellular space (ECS) volume were registered using ion-selective microelectrodes. After stimulation, a transient increase of extracellular K+ concentration up to 10 mM and a transient decrease of ECS volume by approximately 4% could be observed. The comparison of the time courses of these parameters yielded considerable differences between extracellular K+ concentration increase and IOS, but obvious similarities between alterations in ECS volume and IOS. To test the hypothesis that changes in IOS reflect changes in ECS, but not extracellular K+ concentration, we recorded under conditions that are known to prevent activity-induced changes in ECS, i.e., in low Cl- solutions and in the presence of furosemide. Both treatments similarly decreased stimulation-induced IOSs and alterations of ECS. However, the effect of these treatments on changes of extracellular K+ was different and did not correspond to the changes of IOS. We conclude that activity-dependent IOSs in rat neocortical slices measured by near-infrared video microscopy reveal changes in ECS. Furthermore, the pharmacological and ion substitutional experiments make it likely that activity-induced IOSs are attributable to cell swelling via a net KCI uptake and a concomitant water influx.

Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation.

Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation were recorded using darkfield infrared-videomicroscopy. Response amplitude was linearly related to stimulation intensity. The intensity of the optical signal reached its maximum 3 s after onset of stimulation and redecayed with a mean time constant of 23 +/- 7.1 s. The optical signal had a columnar shape. The size of the column was independent from stimulation intensity with stimuli of medium amplitudes. The extent of the optical signal corresponded to the extent of the electrical activation. Changes in intrinsic optical properties may be a useful tool for the study of spread of excitation in neuronal tissue in vitro.

A general numerical method evaluating three-dimensional eye rotations by scanning laser ophthalmoscopy.

A general computational method is described to specify completely the rotational state of the eye in three dimensions by scanning laser ophthalmoscopy (SLO). The method uses the simplex algorithm to fit the eye's rotational parameters to data given by n individually selected ocular fundus landmarks before and after the eye rotation. The rotational parameters are expressed as the rotation vector and three spherical Euler angles. The method, which was implemented in the C programming language, can be applied for various eye movement measurements in clinical and laboratory environments, including SLO.