Long, intrinsic horizontal axons radiating through and beyond rat barrel cortex have spatial distributions similar to horizontal spreads of activity evoked by whisker stimulation.

Stimulation of a single whisker evokes a peak of activity that is centered over the associated barrel in rat primary somatosensory cortex, and yet the evoked local field potential and the intrinsic signal optical imaging response spread symmetrically away from this barrel for over 3.5 mm to cross cytoarchitectonic borders into other "unimodal" sensory cortical areas. To determine whether long horizontal axons have the spatial distribution necessary to underlie this activity spread, we injected adeno-associated viral vectors into barrel cortex and characterized labeled axons extending from the injection site in transverse sections of flattened cortex. Combined qualitative and quantitative analyses revealed labeled axons radiating diffusely in all directions for over 3.5 mm from supragranular injection sites, with density declining over distance. The projection pattern was similar at four different cortical depths, including infragranular laminae. Infragranular vector injections produced patterns similar to the supragranular injections. Long horizontal axons were detected both using a vector with a permissive cytomegalovirus promoter to label all neuronal subtypes and using a calcium/calmodulin-dependent protein kinase II α vector to restrict labeling to excitatory cortical pyramidal neurons. Individual axons were successfully reconstructed from series of supragranular sections, indicating that they traversed gray matter only. Reconstructed axons extended from the injection site, left the barrel field, branched, and sometimes crossed into other sensory cortices identified by cytochrome oxidase staining. Thus, radiations of long horizontal axons indeed have the spatial characteristics necessary to explain horizontal activity spreads. These axons may contribute to multimodal cortical responses and various forms of cortical neural plasticity.

Comparing the functional representations of central and border whiskers in rat primary somatosensory cortex.

The anatomical representations of the large facial whiskers, termed barrels, are topographically organized and highly segregated in the posteromedial barrel subfield (PMBSF) of rat layer IV primary somatosensory cortex. Although the functional representations of single whiskers are aligned with their appropriate barrels, their areal extents are rather large, spreading outward from the appropriate barrel along the tangential plane and thereby spanning multiple neighboring and non-neighboring barrels and septal regions. To date, single-whisker functional representations have been characterized primarily for whiskers whose corresponding barrels are located centrally within the PMBSF (central whiskers). Using intrinsic signal imaging verified with post-imaging single-unit recording, we demonstrate that border whiskers, whose barrels are located at the borders of the PMBSF, also evoke large activity areas that are similar in size to those of central whiskers but spread beyond the PMBSF and sometimes beyond primary somatosensory cortex into the neighboring dysgranular zones. This study indicates that the large functional representation of a single whisker is a basic functional feature of the rat whisker-to-barrel system and, combined with results from other studies, suggest that a large functional representation of a small, point-like area on the sensory epithelium may be a functional feature of primary sensory cortex in general.

Malformation of the functional organization of somatosensory cortex in adult ephrin-A5 knock-out mice revealed by in vivo functional imaging.

The molecular mechanisms that coordinate the functional organization of the mammalian neocortex are largely unknown. We tested the involvement of a putative guidance label, ephrin-A5, in the functional organization of the somatosensory cortex by quantifying the functional representations of individual whiskers in vivo in adult ephrin-A5 knock-out mice, using intrinsic signal optical imaging. In wild-type mice ephrin-A5 is expressed in a gradient in the somatosensory cortex during development. In adult ephrin-A5 knock-out mice, we found a spatial gradient of change in the amount of cortical territory shared by individual whisker functional representations across the somatosensory cortex, as well as a gradient of change in the distance between the functional representations. Both gradients of change were in correspondence with the developmental expression gradient of ephrin-A5 in wild-type mice. These changes involved malformations of the cortical spacing of the thalamocortical components, without concurrent malformations of the intracortical components of individual whisker functional representations. Overall, these results suggest that a developmental guidance label, such as ephrin-A5, is involved in establishing certain spatial relationships of the functional organization of the adult neocortex, and they underscore the advantage of investigating gene manipulation using in vivo functional imaging.

Visualizing and quantifying evoked cortical activity assessed with intrinsic signal imaging.

Intrinsic signal imaging (ISI) measures changes in light reflectance from the illuminated cortex (intrinsic signals or IS) attributed to various vascular and metabolic sources that, when using illumination in the 600 nm range, appear to co-localize with neuronal activity. Given the multiple sources contributing to the collected IS, the common practice of averaging across an extended post-stimulus time epoch before dividing by baseline data typically visualizes evoked IS overlying both the cortical tissue and the large surface blood vessels. In rat PMBSF, the contribution from these vessels are problematic as they do not co-localize with known PMBSF function. Determining a means for quantifying the evoked IS area poses an additional challenge. Here, we describe how exploiting IS collected shortly after stimulus onset (within 1.5 s), which coincides with fast oxygen consumption of active neurons, visualizes evoked IS overlying the cortical tissue without the large surface vessels. We also describe how the use of absolute thresholds combined with a baseline determined from data collected immediately prior to stimulus onset (within 1 s) targets most precisely a specific evoked IS amplitude, a method that should be especially useful when evoked areas are expected to occupy a substantial portion of the total imaged area and/or when peak activity is expected to differ between subjects.

A mapping label required for normal scale of body representation in the cortex.

The neocortical primary somatosensory area (S1) consists of a map of the body surface. The cortical area devoted to different regions, such as parts of the face or hands, reflects their functional importance. Here we investigated the role of genetically determined positional labels in neocortical mapping. Ephrin-A5 was expressed in a medial > lateral gradient across S1, whereas its receptor EphA4 was in a matching gradient across the thalamic ventrobasal (VB) complex, which provides S1 input. Ephrin-A5 had topographically specific effects on VB axon guidance in vitro. Ephrin-A5 gene disruption caused graded, topographically specific distortion in the S1 body map, with medial regions contracted and lateral regions expanded, changing relative areas up to 50% in developing and adult mice. These results provide evidence for within-area thalamocortical mapping labels and show that a genetic difference can cause a lasting change in relative scale of different regions within a topographic map.

Two directions of plasticity in the sensory-deprived adult cortex.

Damage or deprivation of a localized region of the skin surface has been shown to induce a selective expansion of adjacent skin surface representations in the adult somatosensory cortex. Here, we use repeated optical imaging in conjunction with single unit recordings to assess the plasticity of a single whisker's functional representation in the adult rat. We observed a large-scale expansion of a single whisker's functional representation following innocuous removal of all neighboring whiskers. Surprisingly, the same manipulation can also induce a large-scale contraction of the representation if the animal is removed from its home cage and given a brief opportunity to use its whiskers for active exploration of a different environment. Both the expansion and contraction reverse upon regrowth of the deprived whiskers. Thus, allowing the animal to use its deprived receptor organ in active exploration can determine the direction of plasticity in the adult cortex.

Varying the degree of single-whisker stimulation differentially affects phases of intrinsic signals in rat barrel cortex.

Using intrinsic signal optical imaging (ISI), we have shown previously that the point spread of evoked activity in the rat barrel cortex in response to single-whisker stimulation encompasses a surprisingly large area. Given that our typical stimulation consists of five deflections at 5 Hz, the large area of evoked activity might have resulted from repetitive stimulation. Thus in the present study, we use ISI through the thinned skull to determine whether decreasing the degree of single-whisker stimulation decreases the area of the cortical point spread. We additionally outline a protocol to quantify stimulus-related differences in the temporal characteristics of intrinsic signals at a fine spatial scale. In 10 adult rats, whisker C2 was stimulated randomly with either one or five deflections delivered in a rostral-to-caudal fashion. Each deflection consisted of a 0.5-mm displacement of the whisker as measured at the point of contact, 15 mm from the snout. The number of whisker deflections did not affect the area or peak magnitude of the cortical point spread based on the intrinsic signal activity occurring from 0.5 up to 1.5 s poststimulus onset. In contrast, the magnitude and time course of intrinsic signal activity collected after 1.5-s poststimulus onset did reflect the difference in the degree of stimulation. Thus decreasing the degree of stimulation differentially affected the early and late phases of the evoked intrinsic signal response. The implications of the present results are discussed in respect to probable differences in the signal source underlying the early versus later phases of evoked intrinsic signals.

Areal extent quantification of functional representations using intrinsic signal optical imaging.

An important parameter often investigated in the characterization of cortical functional organization is the areal extent of functional modules. Because it allows the visualization of functional modules with high spatial resolution in a noninvasive way to the cortex, intrinsic signal optical imaging (ISI) can be employed for the quantification of these areal extents. The present paper describes the use of the normalized threshold analysis of areal extent quantification for the objective assessment of single-whisker functional representations in the primary somatosensory cortex of adult rats. As the success of areal extent quantification depends on the ability of ISI to allow visualization of cortical representations with minimal stimulus-dependent blood vessel representations, which are commonly encountered by ISI, the present paper also describes the further development of the intratrial analysis of visualization for minimizing these vessel representations. Both analyses are discussed with respect to their advantages as well as their inherent limitations.

Variability and interhemispheric asymmetry of single-whisker functional representations in rat barrel cortex.

1. The rat whisker-to-barrel system was used to investigate the variability and interhemispheric asymmetry in the functional organization of primary somatosensory cortex as assessed with intrinsic signal optical imaging. The areal extent of whisker D1 functional representation was determined for both the left and right barrel cortex of each of 10 adult male rats. The average size of whisker D1 functional representation and the amount of variability away from this average across animals were determined. In addition, interhemispheric asymmetry was addressed at both the population level and the individual level. The degree of side preference for thigmotactic scanning (typical whisker-related rodent behavior) was determined for each rat in an attempt to find a behavioral correlate for the degree of interhemispheric asymmetry in the size of whisker D1 functional representation. 2. The average areal extent of whisker D1 functional representation (defined as area at half-height) was large (1.95 +/- 0.14 mm2, mean +/- SE, N = 10 rats), suggesting that stimulation of a single whisker evokes activity over a large cortical area that includes other whisker representations. 3. The average size of whisker D1 functional representation was not significantly different between the left (1.86 +/- 0.21 mm2) and right (2.04 +/- 0.15 mm2) hemispheric side, suggesting that interhemispheric functional asymmetry of barrel cortex is not systematic toward a specific hemispheric side at the population level. 4. The degree of variability in the size of whisker D1 functional representation from the left hemisphere ranged between 54.6% smaller than to 50.6% larger than the left average areal extent. A large degree of variability was also observed for the right D1 representation, 37.6% smaller than to 34.9% larger than the right average areal extent. Thus it appears that a large variability in the size of unmanipulated single-whisker functional representations exists across animals from the same species and is not exclusive to a particular hemispheric side. 5. In 5 of 10 rats, the size of whisker D1 functional representation between the two hemispheres differed by > or = 25% within an individual animal. Of these five rats, four had a larger representation in their right hemisphere. The degree and direction of behavioral asymmetry was not linearly correlated with the interhemispheric asymmetry in the size of D1 functional representation (r = 0.494). 6. The large size of a single-whisker functional representation as defined with intrinsic signal optical imaging is discussed with respect to previous anatomic and 2-deoxyglucose autoradiography studies, whereas the large variability in this size across animals is discussed with respect to the individuality of each animal. In addition, the results of the present study have implications for projects that plan to investigate relative changes in the size of single-whisker functional representations.

RAPID and opposite effects of BDNF and NGF on the functional organization of the adult cortex in vivo.

The adult cortex is thought to undergo plastic changes that are closely dependent on neuronal activity (reviewed in ref. 1), although it is not yet known what molecules are involved. Neurotrophins and their receptors have been implicated in several aspects of developmental plasticity, and their expression in the adult cortex suggests additional roles in adult plasticity. To examine these potential roles in vivo, we used intrinsic-signal optical imaging to quantify the effects of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on the functional representation of a stimulated whisker in the 'barrel' subdivision of the rat somatosensory cortex. Topical application of BDNF resulted in a rapid and long-lasting decrease in the size of a whisker representation, and a decrease in the amplitude of the activity-dependent intrinsic signal. In contrast, NGF application resulted in a rapid but transient increase in the size of a representation, and an increase in the amplitude of the activity-dependent intrinsic signal. These results demonstrate that neurotrophins can rapidly modulate stimulus-dependent activity in adult cortex, and suggest a role for neurotrophins in regulating adult cortical plasticity.

Quantitative long-term imaging of the functional representation of a whisker in rat barrel cortex.

In this study, we implement chronic optical imaging of intrinsic signals in rat barrel cortex and repeatedly quantify the functional representation of a single whisker over time. The success of chronic imaging for more than 1 month enabled an evaluation of the normal dynamic range of this sensory representation. In individual animals for a period of several weeks, we found that: (i) the average spatial extent of the quantified functional representation of whisker C2 is surprisingly large--1.71 mm2 (area at half-height); (ii) the location of the functional representation is consistent; and (iii) there are ongoing but nonsystematic changes in spatiotemporal characteristics such as the size, shape, and response amplitude of the functional representation. These results support a modified description of the functional organization of barrel cortex, where although a precisely located module corresponds to a specific whisker, this module is dynamic, large, and overlaps considerably with the modules of many other whiskers.

Suprathreshold auditory cortex activation visualized by intrinsic signal optical imaging.

The suprathreshold tonotopic organization of rat and guinea pig auditory cortex was investigated using intrinsic signal optical imaging through a thinned skull. Optical imaging revealed that suprathreshold pure sine wave tone stimulation (25-80 dB) evoked activity over large cortical areas that were tonotopically organized. Three-dimensional surface plots of the activated areas revealed "patchy' auditory-evoked activity consisting of numerous local peaks and valleys building to a maximum. Subsequent detailed electrophysiological mapping in the same subjects confirmed the localization of auditory-evoked activity based on optical imaging, including responses to a test frequency at cortical loci more than 2 octaves away from the threshold-defined isofrequency contour. The success of this technique in visualizing auditory cortex functional organization at suprathreshold stimulus levels will allow for future investigations of auditory cortex frequency representation, including representational plasticity induced by a variety of experimental manipulations.

Wavelet analysis for brain-function imaging.

The authors present a new algorithmic procedure for the analysis of brain images. This procedure is specifically designed to image the activity and functional organization of the brain. The authors' results are tested on data collected and previously analyzed with the technique known as in vivo optical imaging of intrinsic signals. The authors' procedure enhances the applicability of this technique and facilitates the extension of the underlying ideas to other imaging problems (e.g., functional MRI). The authors' thrust is two fold. First, they give a systematic method to control the blood vessel artifacts which typically reduce the dynamic range of the image. They propose a mathematical model for the vibrations in time of the veins and arteries and they design a new method for cleaning the images of the vessels with the highest time variations. This procedure is based on the analysis of the singularities of the images. The use of wavelet transform is of crucial importance in characterizing the singularities and reconstructing appropriate versions of the original images. The second important component of the authors' work is the analysis of the time evolution of the fine structure of the images. They show that, once the images have been cleaned of the blood vessel vibrations/variations, the principal component of the time evolutions of the signals is due to the functional activity following the stimuli. The part of the brain where this function takes place can be localized and delineated with precision.

Cortical point-spread function and long-range lateral interactions revealed by real-time optical imaging of macaque monkey primary visual cortex.

Processing of retinal images is carried out in the myriad dendritic arborizations of cortical neurons. Such processing involves complex dendritic integration of numerous inputs, and the subsequent output is transmitted to multiple targets by extensive axonal arbors. Thus far, details of this intricate processing remained unexaminable. This report describes the usefulness of real-time optical imaging in the study of population activity and the exploration of cortical dendritic processing. In contrast to single-unit recordings, optical signals primarily measure the changes in transmembrane potential of a population of neuronal elements, including the often elusive subthreshold synaptic potentials that impinge on the extensive arborization of cortical cells. By using small visual stimuli with sharp borders and real-time imaging of cortical responses, we found that shortly after its onset, cortical activity spreads from its retinotopic site of initiation, covering an area at least 10 times larger, in upper cortical layers. The activity spreads at velocities from 100 to 250 microns/msec. Near the V1/V2 border the direct activation is anisotropic and we detected also anisotropic spread; the "space constant" for the spread was approximately 2.7 mm parallel to the border and approximately 1.5 mm along the perpendicular axis. In addition, we found cortical interactions between cortical activities evoked by a small "center stimulus" and by large "surround stimuli" positioned outside the classical receptive field. All of the surround stimuli used suppressed the cortical response to the center stimulus. Under some stimulus conditions iso-orientation suppression was more pronounced than orthogonal-orientation suppression. The orientation dependence of the suppression and its dependency on the size of some specific stimuli indicate that at least part of the center surround inhibitory interaction was of cortical origin. This findings reported here raise the possibility that distributed processing over a very large cortical area plays a major role in the processing of visual information by the primary visual cortex of the primate.

Characterization of functional organization within rat barrel cortex using intrinsic signal optical imaging through a thinned skull.

We used optical imaging of intrinsic signals to characterize the functional representations of mystacial vibrissae (whiskers) in rat somatosensory cortex. Stimulation of individual whiskers for 2 s at 5 Hz resulted in a discrete area of functional activity in the cortex. Images of whisker representations were collected both through the dura and through a thinned skull. We characterized the functional representation of a whisker both spatially and temporally with two-dimensional images and three-dimensional surface plots of intrinsic signal development in the cortex in response to whisker stimulation. Single unit recordings verified that the representation of the whisker obtained with optical imaging corresponded with the electrophysiological response area of that whisker in the cortex. Lesions in the center of the functional activity were found to be in the center of the dense cytochrome oxidase patch for the corresponding whisker. In addition, a 3 x 3 matrix of whiskers was stimulated and the distances between the centers of the imaged representations and the distances between the centers of the layer IV cytochrome oxidase staining of the nine whiskers were found to be highly correlated (r = 0.98). This study shows a striking correspondence among imaging, physiology, and anatomy in the rat somatosensory cortex. Furthermore, the ability to use optical imaging through a thinned skull should allow investigations into the long-term changes in a sensory representation within a single animal.

High-resolution optical imaging of functional brain architecture in the awake monkey.

Optical imaging of the functional architecture of cortex, based on intrinsic signals, is a useful tool for the study of the development, organization, and function of the living mammalian brain. This relatively noninvasive technique is based on small activity-dependent changes of the optical properties of cortex. Thus far, functional imaging has been performed only on anesthetized animals. Here we establish that this technique is also suitable for exploring the brain of awake behaving primates. We designed a chronic sealed chamber and mounted it on the skull of a cynomolgus monkey (Macaca fascicularis) over the primary visual cortex to permit imaging through a transparent glass window. Restriction of head position alone was sufficient to eliminate movement noise in awake monkey imaging experiments. High-resolution imaging of the ocular dominance columns and the cytochrome oxidase blobs was achieved simply by taking pictures of the exposed cortex when the awake monkey was viewing video movies alternatively with each eye. Furthermore, the functional maps could be obtained without synchronization of the data acquisition to the animal's respiration and the electrocardiogram. The wavelength dependency and time course of the intrinsic signal were similar in anesthetized and awake monkeys, indicating that the signal sources were the same. We therefore conclude that optical imaging is well suited for exploring functional organization related to higher cognitive brain functions of the primate as well as providing a diagnostic tool for delineating functional cortical borders and assessing proper functions of human patients during neurosurgery.

Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high-resolution optical imaging of intrinsic signals.

We have shown previously the existence of small, activity-dependent changes in intrinsic optical properties of cortex that are useful for optical imaging of cortical functional architecture. In this study we introduce a higher resolution optical imaging system that offers spatial and temporal resolution exceeding that achieved by most alternative imaging techniques for imaging cortical functional architecture or for monitoring local changes in cerebral blood volume or oxygen saturation. In addition, we investigated the mechanisms responsible for the activity-dependent intrinsic signals evoked by sensory stimuli, and studied their origins and wavelength dependence. These studies enabled high-resolution visualization of cortical functional architecture at wavelengths ranging from 480 to 940 nm. With the use of near-infrared illumination it was possible to image cortical functional architecture through the intact dura or even through a thinned skull. In addition, the same imaging technique proved useful for imaging and discriminating sensory-evoked, activity-dependent changes in local blood volume and oxygen saturation (oxygen delivery). Illumination at 570 nm allowed imaging of activity-dependent blood volume increases, whereas at 600-630 nm, the predominant signal probably originated from activity-dependent oxygen delivery from capillaries. The onset of oxygen delivery started prior to the blood volume increase. Thus, optical imaging based on intrinsic signals is a minimally invasive procedure for monitoring short- and long-term changes in cerebral activity.

Functional organization of primate visual cortex revealed by high resolution optical imaging.

A high spatial resolution optical imaging system was developed to visualize cerebral cortical activity in vivo. This method is based on activity-dependent intrinsic signals and does not use voltage-sensitive dyes. Images of the living monkey striate (VI) and extrastriate (V2) visual cortex, taken during visual stimulation, were analyzed to yield maps of the distribution of cells with various functional properties. The cytochrome oxidase--rich blobs of V1 and the stripes of V2 were imaged in the living brain. In V2, no ocular dominance organization was seen, while regions of poor orientation tuning colocalized to every other cytochrome oxidase stripe. The orientation tuning of other regions of V2 appeared organized as modules that are larger and more uniform than those in V1.

Recurring discharge patterns in multiple spike trains. I. Detection.

We present a procedure to detect recurring discharge patterns in multiple spike trains. Such recurring patterns can include many spikes and involve from three to many spike trains. The pattern detection procedure is based on calculating the exact probability of randomly obtaining each individually recurring pattern. The statistical evaluation is based on the use of 2 x 2 contingency tables and the application of Fisher's exact test. Several simulations are applied to evaluate the method. Findings based on applying the procedure to simultaneously recorded spike and event trains are described in a companion paper (Frostig et al. 1990).

Recurring discharge patterns in multiple spike trains. II. Application in forebrain areas related to cardiac and respiratory control during different sleep-waking states.

Simultaneously recorded spike trains were obtained using microwire bundles from unrestrained, drug-free cats during different sleep-waking states in forebrain areas associated with cardiac and respiratory activity. Cardiac and respiratory activity was simultaneously recorded with the spike trains. We applied the recurring discharge patterns detection procedure described in a companion paper (Frostig et al. 1990) to the spike and cardiorespiratory trains. The pattern detection procedure was applied to detect only precise (in time and structure) recurring patterns. Recurring discharge patterns were detected in all simultaneously recorded groups. Recurring discharge patterns were composed of up to ten spikes per pattern and involved up to four simultaneously recorded spike trains. Fourty-two percent of the recurring patterns contained cardiac and/or respiratory events in addition to neuronal spikes. When patterns were compared over different sleep-waking states it was found the the same units produced different patterns in different states, that patterns were significantly more compact in time during quiet sleep, and that changes in the discharge rates accompanying changes in sleep-waking states were not correlated with changes in pattern rate.