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Single-cell RNA sequencing CRISPR (perturb-seq) screens enable high-throughput investigation of the genome, allowing for characterization of thousands of genomic perturbations on gene expression. Ambient gRNAs, which are contaminating gRNAs, are a major source of noise in perturb-seq experiments because they result in an excess of false-positive gRNA assignments. Here, we utilize CRISPR barnyard assays to characterize ambient gRNAs in perturb-seq screens. We use these datasets to develop CRISPR Library Evaluation and Ambient Noise Suppression for Enhanced single-cell RNA-seq (CLEANSER), a mixture model that filters ambient gRNAs. CLEANSER includes both gRNA and cell-specific normalization parameters, correcting for confounding technical factors that affect individual gRNAs and cells. The output of CLEANSER is the probability that a gRNA-cell assignment is in the native distribution over the ambient distribution. We find that ambient gRNA filtering methods impact differential gene expression analysis outcomes and that CLEANSER outperforms alternate approaches by increasing gRNA-cell assignment accuracy across multiple screen formats., Competing Interests: Declaration of interests C.A.G. is an inventor on patents and patent applications related to genome engineering and CRISPR screens, and is a co-founder and advisor to Tune Therapeutics, an advisor to Sarepta Therapeutics, and a co-founder of Locus Biosciences., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent technological developments in single-cell RNA-seq CRISPR screens enable high-throughput investigation of the genome. Through transduction of a gRNA library to a cell population followed by transcriptomic profiling by scRNA-seq, it is possible to characterize the effects of thousands of genomic perturbations on global gene expression. A major source of noise in scRNA-seq CRISPR screens are ambient gRNAs, which are contaminating gRNAs that likely originate from other cells. If not properly filtered, ambient gRNAs can result in an excess of false positive gRNA assignments. Here, we utilize CRISPR barnyard assays to characterize ambient gRNA noise in single-cell CRISPR screens. We use these datasets to develop and train CLEANSER, a mixture model that identifies and filters ambient gRNA noise. This model takes advantage of the bimodal distribution between native and ambient gRNAs and includes both gRNA and cell-specific normalization parameters, correcting for confounding technical factors that affect individual gRNAs and cells. The output of CLEANSER is the probability that a gRNA-cell assignment is in the native distribution over the ambient distribution. We find that ambient gRNA filtering methods impact differential gene expression analysis outcomes and that CLEANSER outperforms alternate approaches by increasing gRNA-cell assignment accuracy.

Alterations in vascular networks, including angiogenesis and capillary regression, play key roles in disease, wound healing, and development. The spatial structures of blood vessels can be captured through imaging, but effective characterization of network architecture requires both metrics for quantification and software to carry out the analysis in a high-throughput and unbiased fashion. We present Rapid Editable Analysis of Vessel Elements Routine (REAVER), an open-source tool that researchers can use to analyze high-resolution 2D fluorescent images of blood vessel networks, and assess its performance compared to alternative image analysis programs. Using a dataset of manually analyzed images from a variety of murine tissues as a ground-truth, REAVER exhibited high accuracy and precision for all vessel architecture metrics quantified, including vessel length density, vessel area fraction, mean vessel diameter, and branchpoint count, along with the highest pixel-by-pixel accuracy for the segmentation of the blood vessel network. In instances where REAVER's automated segmentation is inaccurate, we show that combining manual curation with automated analysis improves the accuracy of vessel architecture metrics. REAVER can be used to quantify differences in blood vessel architectures, making it useful in experiments designed to evaluate the effects of different external perturbations (eg, drugs or disease states)., (© 2020 The Authors. Microcirculation published by John Wiley & Sons Ltd.)

Diabetic retinopathy is a potentially blinding eye disease that threatens the vision of one-ninth of patients with diabetes. Progression of the disease has long been attributed to an initial dropout of pericytes that enwrap the retinal microvasculature. Revealed through retinal vascular digests, a subsequent increase in basement membrane bridges was also observed. Using cell-specific markers, we demonstrate that pericytes rather than endothelial cells colocalize with these bridges. We show that the density of bridges transiently increases with elevation of Ang-2, PDGF-BB, and blood glucose; is rapidly reversed on a timescale of days; and is often associated with a pericyte cell body located off vessel. Cell-specific knockout of KLF4 in pericytes fully replicates this phenotype. In vivo imaging of limbal vessels demonstrates pericyte migration off vessel, with rapid pericyte filopodial-like process formation between adjacent vessels. Accounting for off-vessel and on-vessel pericytes, we observed no pericyte loss relative to nondiabetic control retina. These findings reveal the possibility that pericyte perturbations in location and process formation may play a role in the development of pathological vascular remodeling in diabetic retinopathy., (© 2020 by the American Diabetes Association.)

Purpose: To establish Myh11 as a marker of a subset of corneal endothelial cells (CECs), and to demonstrate the feasibility of restoring the corneal endothelium with Myh11-lineage (Myh11-Lin[+]) adipose-derived stromal cells (ASCs)., Methods: Intraperitoneal administration of tamoxifen and (Z)-4-hydroxytamoxifen eyedrops were used to trace the lineage of Myh11-expressing cells with the Myh11-Cre-ERT2-flox-tdTomato mouse model. Immunostaining and Western blot characterized marker expression and spatial distribution of Myh11-Lin(+) cells in the cornea, and administration of 5-ethynyl-2'-deoxyuridine labeled proliferating cells. ASCs were isolated from epididymal adipose Myh11+ mural cells and treated with cornea differentiation media to evaluate corneal endothelial differentiation potential. Differentiated ASCs were injected into the anterior chamber to test for incorporation into corneal endothelium following scratch injury., Results: A subset of CECs express Myh11, a marker previously thought restricted to only mural cells. Myh11-Lin(+) CECs marked a stable subpopulation of cells in the cornea endothelium. Myh11-Lin(+) ASCs undergo CEC differentiation in vitro and incorporate into injured corneal endothelium., Conclusions: Dystrophy and dysfunction of the corneal endothelium accounts for almost half of all corneal transplants, the maintenance of the cornea endothelium is poorly understood, and there are a lack of mouse models to study specific CEC populations. We establish a mouse model that can trace the cell fate of a subpopulation of CECs based on Myh11 expression. A subset of ASCs that share this Myh11 transcriptional lineage are capable of differentiating into CECs that can incorporate into injured corneal endothelium, revealing a potential cell source for creating engineered transplant material.

Ivane Beritashvili ("Beritoff" in Russian, and often in Western languages) was a major figure in 20th-century neuroscience. Mastering the string galvanometer, he founded the electrophysiology of spinal cord reflexes, showing that inhibition is a distinctly different process from excitation, contrary to the concepts of his famous mentor, Wedensky. Work on postural reflexes with Magnus was cut short by World War I, but he later demonstrated that navigation in two-dimensional space without vision is a function solely of the vestibular system rather than of muscle proprioception. Persevering in his experiments despite postwar turmoil he founded an enduring Physiology Institute in Tbilisi, where he pursued an ingenious and extensive investigation of comparative memory in vertebrates. This revealed the unique nature of mammalian memory processes, which he forthrightly called "image driven," and distinguished them unequivocally from those underlying conditional reflexes. For some 30 years the Stalinist terror confined his publications to the Russian language. Work with his colleague, Chichinadze, discovering that memory confined to one cerebral hemisphere could be accessed by the other via a specific forebrain commissure, did reach the West, and ultimately led to recognition of the fascinating "split brain" condition. In the 1950s he was removed from his professorial position for 5 years as being "anti-Pavlovian." Restored to favor, he was honorary president of the "Moscow Colloquium" that saw the foundation of the International Brain Research Organization.

Without special examination the brain offers no clue that it is the organ of the mind. From the dawn of time man thus either ignored the problem as to the source of thought, or attributed it to a variety of anatomical structures, usually the heart. The brain held no place in such intuitions, and in most languages it is analogized to bone marrow. Furthermore, nothing in early medical systems claimed any intellectual capacity for the brain; and the Egyptians, so fastidious in care for their afterlife, heedlessly discarded the brain in funerary practice. It was thus a unique event in world history when Alkmaion of Kroton (Alcmaeon, ca. 500 bc), based on anatomical evidence, proposed that the brain was essential for perception. Although no writings of Alkmaion survived, it was probably via a fortuitous linkage that his idea of the mental primacy of the brain was transmitted to, and preserved within, the teachings of the Hippocratic school. Nothing, of course, was secure as to mechanism, two millennia unfolding until the search for mind passed from the ventricles to the cerebral cortex. Nonetheless, Alkmaion was the beginning, and the ensuing understanding that he initiated is still transforming humanity's perception of the natural world, and their place within it.

There have been four major pioneers from Eastern Europe in the neuroscientific study of memory and learning: Pavlov, Bekhterev, Beritashvili and Konorski. The thinking of each evolved with the progress of neuroscientific knowledge throughout the world, and save for Pavlov, each encountered governmental opposition to their views. Among the clues largely overlooked in their examination of conditional reflexes was the fact that the animal appreciates not only its own appetitive state but its immersion in the experimental setting. The latter in itself must require considerable, ongoing neuronal activity to sustain it. There is also the question as to whether "motivation" is an essential feature for the formation of conditional connections; and in cases where it is seemingly absent, as in recognition memory, the processes that underlie the astonishing efficacy of such memory formation remain almost wholly obscure. Finally, it is remarked that the cerebral cortex, as initially supposed, may indeed be the governing locus, even of such simple effects as habituation.

Macaques were trained to signal their detection of electrical stimulation applied by a movable microelectrode to perifoveal striate cortex. Trains of < or =100 cathodal, 0.2-ms, constant current pulses were delivered at 50 or 100 Hz. The minimum current that could be reliably detected was measured at successive depths along radial electrode penetrations through the cortex. The lowest detection thresholds were routinely encountered when the stimulation was applied to layer 3, particularly just at the juncture between layers 3 and 4A. On the average, there was a twofold variation in threshold along the penetrations, with the highest intracortical thresholds being in layers 4C and 6. Variations as high as 20-fold were obtained in some individual penetrations, whereas relatively little change was observed in others. The minimum detectable current was 1 muA at a site in layer 3, i.e., 10-100 times lower than that for surface stimulation. Because macaques, as do human subjects, find electrical stimulation of striate cortex to be highly similar at all loci (a phosphene in the human case), it is puzzling as to how such uniformity of effect evolves from the exceedingly intricate circuitry available to the effective stimuli. It is hypothesized that the stimulus captures the most excitable elements, which then suppress other functional moieties, producing only the luminance of the phosphene. Lowest thresholds presumably are encountered when the electrode lies among these excitable elements that can, with higher currents, be stimulated directly from some distance or indirectly by the horizontal bands of myelinated axons, the stria of Baillarger.

Macaques indicated their detection of onset or alteration of 0.2-ms pulses applied in various configurations through electrodes implanted in striate cortex. When microelectrodes were introduced and left in place, the threshold for detection of 100-Hz pulses nearly doubled within 24 h. However, for chronically implanted platinum-alloy macroelectrodes detection thresholds usually remained stable for many months, independently of location within striate cortex or its immediately subjacent white matter. Thresholds were unaffected by the visual conditions, such as light versus darkness, or movement of the eyes; but in one animal blind after acute glaucoma thresholds for loci in striate cortex were permanently decreased by about 50%. Learning to respond to electrical stimulation of the optic tract produced no tendency to respond to such stimulation of striate cortex. Onset of stimulation at a given locus could be detected even in the face of continuous supraliminal stimulation at four surrounding loci on a 3-mm radius. The surround stimulation did alter the threshold of the central locus, but such stimuli could not summate if they were subliminal by some 10%. Cessation of stimulation that had been continuing for 1 min to 1 h could be detected if it were being applied at a level 20-75% above that needed for detection of stimulus onset. Continuous stimulation had a pronounced "priming" effect, in that modulation of frequency or intensity of such stimulation by as little as 5% could be detected (e.g., 20 microA in a background of 500 microA, or <2-ms interpulse interval with pulses at 50 Hz). Using pulses inserted in various phase relations to ongoing pulses at 2-5 Hz, it could be determined that stimulus pulses were surrounded by a strong facilitatory period for about 30 ms, which was then replaced by refractoriness. Given the congruence of macaque and human visual anatomy and psychophysics, these results further encourage efforts to develop a cortical prosthesis for the blind.

The cerebral hemispheres differ in their capabilities and response to verbal versus nonverbal visual material. A priori, it might thus be expected that the right hemisphere would be best activated during a mnemonic task with fMRI when using nonverbalizable images, and the left hemisphere with verbal material. However, previous psychological tests had shown a high degree of similarity in measures of memory for these disparate items. It was thus hypothesized that extensive commonality in the areas activated would prevail when this previously tested material was employed with fMRI. Six subjects underwent fMRI with four types of trials in blocks: fixating; passively viewing 12 words and 12 nonverbalizable images; endeavoring to remember (encoding) another set of 12 words and images; endeavoring to recognize (retrieve) previously viewed words or images. Passive viewing produced small islands of activation in left versus right frontal cortex for words and images, respectively. Endeavoring to remember enlarged the areas of activation and produced some bilaterality. Retrieval greatly augmented activation as well as bilaterality, and some 20% of the activated frontal volume was shared by words and images. Thus, on the one hand, the distribution of activation upon retrieval differed substantially for words versus images, but on the other, as predicted, there was considerable commonality. Predominant laterality of activation in some areas shifted between encoding and retrieval (HERA), importantly involving different regions for words versus images. Of course, processes other than memory per se are undoubtedly involved in these distributions of fMRI activation in frontal cortex, yet the nature of the to-be-remembered items is clearly a major factor, in accord with the asymmetric lateralization in their basic representation.

When, in the primeval sea, creatures first began to crawl, "right" and "left" came into being, yielding neuronal nets to control response to the sidedness of stimuli. In the half billion years of moving and sensing, two brains have evolved, the right and the left; and human experience now shows them to be roughly equivalent, potentially independent, conscious entities. This dramatic fact is evidenced by "split-brain" patients and by numerous cases of therapeutic removal of either hemisphere. Equally dramatic, of course, is that there is not the slightest sign of this duality in everyday experience, the right and left visual fields are seamlessly knit, and cross purpose is absent in the moment to moment operation of the two cerebral hemispheres. This unity is constantly synthesized by the 100,000,000 fibers passing from each hemisphere to the other; the vastness of that interchange emphasized upon comparison with the mere 1,000,000 fibers conveying all the visual world from each eye. With the large distances in the human brain some 100+ ms may commonly transpire for one hemisphere to send to and receive a response from the other. Efficiency thus demands that most neuronal calculation occur within rather than between hemispheres, thereby promoting differences in the characteristic capabilities of each alone, i.e., "hemispheric specialization". Despite this there is a bewildering bilaterality of activation revealed by fMRI for most cognitive tasks. In the absence of the forebrain commissures brainstem systems can be shown, in macaques, also to participate in the unification of behavioral result from the actions of the separated hemispheres. The system favors synthesis from congruent (visual) input to the two hemispheres; but in the face of incompatible hemispheric input, the two hemispheres are able to work out an accommodation in their control of subcortical systems.

How well will one cerebral hemisphere recognize items viewed initially via the other? Nonverbalizable images or words were presented to one visual field and memory for them tested in the same or the other visual field. The initially viewing hemisphere subsequently had no secure advantage in accuracy, and only for images was there a 30-ms (ca 3%) penalty in reaction time for viewing with the "other" hemisphere. Interhemispheric mnemonic communication is thus highly reliable. At longer retention intervals (1-2 min vs. 4-30 s, with accumulating added stimuli), however, recognition of words was asymmetric as to hemisphere, in that initial viewing via the right hemisphere was subsequently (and paradoxically) much better recognized via the left (other) hemisphere than was the converse situation. This suggests that the initial engram with right hemispheric viewing of words ultimately becomes established in the left, and that the right has less accurate access to a previous "left hemispheric view".

These experiments explored the interactions remaining between the cerebral hemispheres in two split-brain macaques. The 'split' was earlier confirmed by showing that one hemisphere was incapable of identifying visual images seen by the other. The critical tests for residual interactions were intermingled with control trials in a continuous recognition task. These tests were of two kinds: 'parallel processing', to determine how simultaneous viewing by both hemispheres affected subsequent recognition by one of them alone; and 'conflict', where opposite responses were demanded from the two hemispheres, thus assessing the issue of metacontrol. Two types of stimuli were also employed: ART, in which each hemisphere saw essentially the same image; and BIPARTITE, in which images were entirely different for each hemisphere. Since, with either type of stimulus, performance was best when viewed by both hemispheres at both encoding and retrieval, 'parallel processing' was highly efficient. However, when both hemispheres viewed initially and only one was subsequently queried, performance was significantly worse than when each hemisphere acted alone on each occasion. It is thus reasoned that when both hemisphere view together, the resultant memory trace somehow reflects the bilaterality, a conclusion concordant with observations of Marcel on blindsight. Processing different images (BIPARTITE) was somewhat more disruptive in this regard than if the same image was viewed by each hemisphere. This was particularly true in the conflict situation, where for one hemisphere the item seen was NEW and for the other it was OLD. A response of 'OLD' was, at first, consistently rewarded. When this well-established protocol was changed, the hemispheres in each animal were gradually able to revise their joint behavior. This, together with the effect of disparate images, and the deficiency evoked when the animals were forced to recognize unilaterally an image first viewed under bilateral conditions, all manifest considerable, and complex, interaction between the hemispheres despite absence of the forebrain commissures. The superior colliculus seems a likely focal point for such interhemispheric effects.

These experiments measured the extra time required to respond when the type of stimulus was changed from one trial to the next. Heretofore, switching costs have been measured for a change in task, but we wished to isolate the cost of changing the sensorial component per se and its necessary analytical processing, as distinct from changing the question being posed or the type of response to be given. Thus, the task was identical throughout the experiments: continuous recognition in a single, 240-trial session, in which the subject was required to distinguish initial from repeat appearances of a stimulus, the single repetition of each stimulus occurring after 1-31 intervening trials. There were two categories of 200-ms stimuli, linguistic (words and non-words) and images (multiple-colored or gray scale panels, human faces, or butterflies); and two conditions of switching, predictable (alternating on each trial) or unpredictable, in which the switch occurred after three to eight trials of one kind. In the majority of cases, there was a robust switching cost, from 24 to 92 ms. The similarity of costs in the predictable and unpredictable modes suggests that this cost is derived at least as much from terminating the modus operandi for the preceding type of stimulus as from a reconfiguration of processing for the new type of stimulus. In the switch between words and images, the costs were "paradoxical" (asymmetrical), in that the switching from an image to a word was more costly than the reverse, that is, changing from the more to the less difficult required the greater time. This, too, is compatible with the idea that termination of the previous mode of processing is a major component of the cost. Thus, in contemplating the neuronal/cognitive events underlying visual memory, consideration must be given to the inertia of pre-existing linkages.

Accuracy of response and pattern of ocular fixations in three split-brain macaques were used to evaluate performance of each hemisphere in a continuous visual recognition task. The animal indicated by ocular fixation upon response points whether a displayed image or face was 'NEW' or 'OLD'. An inadvertent lesion of cingulate gyrus severely reduced contralateral fixations and impaired performance of the affected hemisphere in one animal, confirming the inferred relation between hemisphere and laterality of fixations. The hemispheres in the other two animals were initially remarkably similar in accuracy with human faces and with images; but the right hemisphere was significantly superior to the left for macaque faces. Parallel to this, in the one animal tested while simultaneously using both eyes/hemispheres, fixations were made primarily on the left half of human and macaque faces (right hemispheric control), whereas for images the ocular fixations were predominantly focused on the right half. However, after further, extensive training the left hemisphere performed with significantly greater accuracy than the right on all material and this shift was accompanied and further corroborated by a reversal of the fixational pattern to favor the right half of faces, as continued to be the case with images. Thus, over the long term both the pattern of ocular fixations and the accuracy of performance demonstrate a migration from right to left hemispheric dominance as familiarity with the task increased. Performance of the initially superior hemisphere actually diminished with this shift, presenting a uniquely puzzling question of hemispheric balance in the absence of the forebrain commissures.

While Western man has recognized for almost 2500 years that mind derives exclusively from brain, clothing this fact with explanatory detail still proves elusive. First, is consciousness per se, created by processes demonstrably limited to certain, but still unspecified, neuronal arrangements and activities. Then there is perception, its ineffable qualia, and the fact that it arises from neuronal activity widely dispersed in space and time within networks of vast complexity. Voluntary control is equally dispersed as to neuronal participation, and nescient as to origin. An often overlooked mystery is the unity of mind and behavior that prevails despite the potential for bihemispheric duplication of processes and experience. Finally, there is memory, which while credibly within grasp of understanding as a synaptic alteration maintained via activation of the nuclear genome, still wholly defies comprehension when viewed as commanded recall of myriad, randomly selectable details of the past, a largely effortless and 'instantaneous' flood of memories. For two centuries science has endeavored to demonstrate how these mysteries proceed from physics and chemistry, as indeed they do; but viewed from this direction alone, mind is but the babbling of a robot, chained ineluctably to crude causality. In a bold and revolutionary stroke, Roger Sperry has conceived a more credible paradigm, that the totality of neuronal action, as a richly intercommunicating system, gives rise to effects transcendent to the individual physicochemical elements that compose it. A major achievement of this position is that it is immediately consonant with everyday human experience and belief. While neither Sperry's vision. nor the reduction of the mysteries to a dance of ions can yet be proven, the vast advantage of Sperry's thesis is that it again imbues human thought and action with responsibility, and opens morality to the light of science, while the long wait for certainty unfolds.

Memory for visual and verbal material engages widely distributed systems, to a large degree focussed in different hemispheres. It might thus be expected that these disparate neuronal populations should display significantly different characteristics in regard to mnemonic performance. Visual memory, fundamental to all human beings, and whose characteristics are largely shared with macaques, was assayed using unique non-objective, colored images lacking ready verbal description and was contrasted with memory for four-letter non-offensive English words. The effects of memory loading, stimulus duration and long-term test intervals (1-2 weeks) were studied in regard to accuracy and reaction times for recognizing initial versus re-exposure to these two types of items. No effects of memory loading were apparent despite the incrementing memory load in the 240-item, running recognition sessions. Words were better remembered than images, both in the long and short term, but the detailed characteristics of reaction times and accuracy in relation to number of intervening items, and in long-term memory were strikingly similar. Given the wide and well-established disparity in cerebral loci participating in linguistic versus image analysis, these multiple similarities in the pattern of mnemonic performance indicate that the underlying neuronal processes must be comparable for remembering either images or words. Furthermore, the strong link manifested between individual items across a varying number of intervening intervals and added items suggests that a phenomenon highly similar to the "stimulus specific adaptation" (SSA), displayed by units in macaque inferotemporal cortex, occurs for each item to be recognized. Finally, the significant augmentation in accuracy both in short- and long-term memory for images when viewing time permits saccades is explained if each saccade and fixational pause recruits additional neurons into the pool displaying SSA, or its equivalent, for the item being viewed.

In cats, unanesthetized following transection of the brainstem at a level precluding painful sensation, and limiting ocular motility to a vertically oriented course (the pretrigeminal preparation), habituation of the orienting reflex, consisting of ocular fixation and smooth pursuit, readily transferred between moving visual stimuli directed first at one and then the other cerebral hemisphere. Under the same conditions, when the corpus callosum had been transected 2 weeks prior to the habituation, interhemispheric transfer was absent. Thus, despite substantial brainstem involvement and bilateral coordination of ocular motility the neocortex plays an essential role in this habituation, just as it does in the interhemispheric transfer of visual discrimination learning. This suggests that habituation is a fundamental form of learning in the mammalian forebrain.

(1) In macaques with the optic chiasm transected, and forebrain commissural communication limited to the anterior commissure or the posterior 5 mm of the splenium of the corpus callosum, visual patterns viewed initially by only one eye (hemisphere) are subsequently recognized by the other with normal accuracy. (2) The efficiency of these commissural paths is further indicated by the fact that even when as many as six "target" images are presented for memorization to only one hemisphere, it makes essentially no difference as to accuracy or latency of performance which hemisphere is then required to distinguish "target" from "non-target" images. (3) By electrically tetanizing structures in one or the other temporal lobe at various times in relation to visual input and/or mnemonic testing it could be shown: (a) that a memory trace restricted in its formation to a single hemisphere was available to the other via either forebrain commissure, and (b) that the memory is formed bilaterally despite unilateral input. (4) When the chiasm is split but the commissures are intact, simultaneous presentation of disparate images to each hemisphere severely perturbs performance, suggesting that the callosal system operates continuously to unify visual percepts; but when only the anterior commissure is intact, the two hemispheres accept incongruent images without perturbation. (5) In the fully "split-brain" condition, when one hemisphere cannot access memories held in the other, the accuracy of performance by each hemisphere is nevertheless burdened by the memory load of its neocortically disconnected partner. It can thus be inferred that the brainstem plays a critical, unifying role in this mnemonic process.

Tomasch (1954) and Aboitiz et al. (1992) found the majority of the fibers of the human corpus callosum are under 1 micron in diameter. Electron microscopic studies of Swadlow et al. (1980) and the detailed study of LaMantia and Rakic (1990a) on macaques show the average size of the myelinated callosal axons also to be less than 1 micron. In man, the average-sized myelinated fiber interconnecting the temporal lobes would have a one-way, interhemispheric delay of over 25 msec. Thus, finely detailed, time-critical neuronal computations (i.e., tasks that strain the capacity of the callosum and hence could not be handled by just the larger fibers) would be performed more quickly via shorter and faster intrahemispheric circuits. While one transit across the commissural system might yield tolerable delays, multiple passes as in a system involving "setting" would seem prohibitively slow. We suggest that these temporal limits will be avoided if the neural apparatus necessary to perform each high-resolution, time-critical task is gathered in one hemisphere. If the, presumably overlapping, neural assemblies needed to handle overlapping tasks are clustered together, this would lead to hemispheric specialization. The prediction follows that the large brains of mammals such as elephants and cetaceans will also manifest a high degree of hemispheric specialization.

Experiments comparing binocular with monocular abilities of monkeys working on visual mnemonic tasks were performed. First, it was shown that even in split-brain monkeys performance was more accurate when both hemispheres were utilized than when the task was performed with only the single (better) hemisphere. Some form of noncommissural integration is thus possible. However, when the forebrain commissures are present, as in four other animals (with only optic chiasm transected) it was shown that integration occurs via callosal mechanisms as well. This was demonstrated by the fact that here, too, binocular performance was normally more accurate than monocular performance, but when different images to be remembered were presented concurrently to the two eyes, the binocular advantage was lost. Finally, in three monkeys with only the anterior commissure allowing interhemispheric communication the superiority of binocular assessment remained even when the two hemispheres simultaneously received such differing images.

A simple procedure is described for rapidly inactivating areas 17 and 18 in the rat by superfusion of the immediately overlying dura mater with chilled, physiological saline solution. Unit recording indicates deep depression of cortical activity within 20 s or less, and equally rapid restoration upon rewarming. Repetition does not appear to be deleterious. During such inactivation of the "visual" cortex (VC) essentially all neurons in the visual portion of the thalamic reticular nucleus (vTRN) are significantly depressed in their background activity and/or in their response to photically or electrically elicited input over the optic tract (Table 1). Activity of neurons in the dorsal portion of the lateral geniculate nucleus (LGNd), on the other hand, is much less likely to be affected, although in occasional neurons the effects should be profound. No evidence of disinhibition was apparent. It is concluded that the vTRN in the rat is highly dependent upon the VC in its activity, and is, thus, likely to be primarily a tool of the VC in the modulation of thalamic events. Extensive work of others shows that the vTRN provides inhibitory input to the LGNd, but in the present experiments the loss of inhibition via this route seems to be balanced by a corresponding loss of general excitatory input when the VC is inactivated.

Macaques were trained to respond to electrical excitation applied through electrodes permanently implanted within or upon striate cortex. Threshold current for the animal to detect this stimulation was highly consistent from day to day and, in the absence of tissue encapsulation of the electrodes or deliberately inflicted damage, remained stable indefinitely, 38 months in the longest case so far. Stimulating continuously for 1-8h, however, produces an elevation of threshold, which may be permanent or temporary, depending upon a variety of conditions. A major cause of such injury is the hydrolysis commonly occurring consequent to passage of low-level currents between solutions and metal electrodes. Even when the hydrolytic reaction is eliminated by restricting the level of electrode polarization or by using capacitative stimulation with tantalum pentoxide electrodes, a rise in threshold often still occurs with protracted stimulation. With proper control in some instances, however, effective stimulation at 2-10 times the threshold level could be maintained indefinitely without apparent injury, e.g. in a blind monkey having a threshold of 290 muA that could respond immediately to an 80-muA diminution in 580-muA, 0.2-msec stimulus pulses which had been applied steadily for 1 h at 50 Hz.

1. Single units in striate cortex were studied in alert macaques while they viewed a ganzfeld. Of the 385 well-isolated units studied for 10 min to 2 h, 24% gave "luxotonic" responses, i.e., their rate of discharge for 1 min or more in diffuse, featureless, wideangle illumination (20-450 cd/m2) was at least double that during a comparable period in darkness, or vice versa, and not attributable to eye movements of blinking. Those discharging faster in the light, "photergic" units, outnumber those responding to darkness, "scotergic" units 1 by 4:1. 2. In the lateral geniculate nucleus, on the other hand, among 46 units studied, 28% were luxotonic, but scotergic units were the more common. Both types were present in both magno- and parvocellular laminae. 3. For striate cortex two-thirds of the luxotonic units were binocular. Some showed highly similar response for either eye alone, and essentially no summation binocularly; others had grossly differing responses from each eye, and complex binocular interaction. 4. Many units of all types at striate cortex showed significant modulation of their activity consequent to saccadic eye movements made in darkness, whereas comparable modulation was not observed at the lateral geniculate nucleus. 5. On the basis of these and other findings it is concluded that luxotonic cortical activity is prominent probably only in alert primates, and that this is a consequence of the fact that all retinal ganglion cells in primates synapse in the lateral geniculate nucleus (Ref. 9). Possible functions range from mere trophic input to providing a veridical image or a scaling factor for maintenance of perceptual constancy in the face of varying levels of general illumination.

The cross-sectional areas of the corpus callosum (CC) and anterior commissure (AC) were determined by computer-assisted morphometry in normal human brains obtained at autopsy. In addition, the shape of each CC was examined qualitatively by three "blind" observers. A two-fold variation was observed in the cross-sectional area of the CC. Surprisingly, callosal cross-sectional area was not significantly related to brain weight. Moreover, contrary to recent reports, neither simple inspection nor morphometry revealed structural variation related to sex. A striking, seven-fold, variation was observed in the cross-sectional area of the AC. However, AC cross-sectional area was not related either to brain weight or CC cross-sectional area. A trend toward sexual dimorphism in AC cross-sectional area was observed, with males having the larger AC's. Since the interhemispheric commissures are composed, to a large extent, of fibers that link the various cortical areas of the two hemispheres, these observations suggest that variation in the cross-sectional area of the interhemispheric commissures is not simply related to brain weight or sex but, rather, reflects a similar degree of variation in some aspect of cortical structure.

Six macaques and 20 right-handed human subjects, given identical test material, were asked to choose which of two composites, one consisting entirely of the left half of a monkey or a human face, the other of the right half, appeared most like the normal face. Confirming observations of others, the group of human subjects selected the right-half composite of the face (i.e. the left-half composite of the photograph) 68% of the time, thereby demonstrating a highly significant (P less than 0.01) bias in favor of the left visual field (right hemisphere). No such bias was present when human subjects viewed faces of monkeys, despite the fact that according to limited measurements, the faces of both species are asymmetrical. And most important, the macaques exhibited no consistent bias with either monkey or human faces. On the other hand, the monkeys did respond emotionally to these colored facial images, but not to scenery, when first presented. Also, like man, they found inverted faces but not scenery more difficult to identify than when right side up. Thus, the monkeys responded emotionally and perceptually to these images as faces, yet, unlike the human observers, displayed no hemispheric preference in making their analysis.

The primary purpose of these exploratory experiments was to determine: (1) whether the forebrain commissures can provide full accessibility of the mnemonic store to either hemisphere when the taks involves memory for 'events' (images) rather than, as in essentially all previous tests on split-brain animals, memory for 'rules' (discrimination habits); and (2) whether the anterior commissure (AC) alone is capable of such function. Macaques, with optic chiasm transected to allow limitation of direct visual input to one or the other hemisphere, were trained on tasks requiring recognition of previously viewed photographic slides. For one task, delayed-matching-to-sample (DMTS), the animal was presented with a 'sample' image, and then 0-15s later was required to choose that image in preference to a second image concurrently displayed. On the other task, running recognition (RR), a series of images was presented, some of which were repetitions of images previously seen in that session, and the animal was required to signal its recognition of these repetitions. For either task the initial presentation could be made to one eye and hemisphere, and subsequent recognition required of the other. In such circumstance, if all forebrain commissures were divided, such interhemispheric recognition was no longer possible. For the DMTS task if either the AC or 5 mm of the splenium of the corpus callosum were available, interhemispheric recognition was basically equivalent to that using the same eye and hemisphere. However, interhemispheric accuracy with the RR task, while well above chance levels, was consistently inferior to that achieved intrahemispherically when complex scenes or objects were viewed. This is probably a consequence mostly of the differing visual fields of the two eyes, since interhemispheric accuracy was greatly improved by use of images having approximately identical right and left halves. No consistent hemispheric specialization nor difference in direction of interhemispheric communication was observed despite the use of different types of material and the different mnemonic tasks. It is concluded that the AC in macaques can achieve full and continuously operative neural unification of the mnemonic traces of past experience.

Two macaques, shown a series of pictures, recognized 79% and 85% upon re-presentation after 45 other pictures intervened. Human subjects working with the identical pictures (chosen to avoid human linguistic and experiential connotations) averaged 83% correctly recognized. The human false 'recognition' rates were lower than the macaques', hence the average human accuracy was better, but the range of accuracy among the human subjects overlapped that of the macaques.

Horseradish peroxidase (HRP) was injected in relatively massive amounts to cover most, or portions, of opercular striate cortex in four macaques. Absence of transcallosal or circumventricular labelling, plus discrete and consistent retrograde labelling in other areas in the four cases, assured the validity and specificity of the observations. Numerous labelled cells in regions directly bordering striate cortex, however, were excluded from the analysis because of the possibility of uptake consequent to physical diffusion. With this exception, all labelled cells were counted at roughly 2-mm intervals for one case with extensive unilateral injection of HRP. Even excluding the closely circumstriate population, the totals indicate that more than 30% of the afferent input to striate cortex arises from nongeniculate sources. Four areas of neocortex together make up about one-fourth of the total afferents: superior temporal sulcus 17.1%; inferior occipital area, 6.1%; intraparietal sulcus, 0.4%; and parahippocampal gyrus, 0.3%. Other areas projecting to striate cortex include claustrum, pulvinar, nucleus paracentralis, raphé system, locus coeruleus, and the nucleus basalis of Meynert. Cells of the latter were particularly striking with their very heavy uptake of HRP, and, even in cases of minimal effective injection, were scattered throughout an extensive area from the posterior edge of the globus pallidus passing rostrally beyond the chiasm and into the nucleus of the diagonal band. On the basis of their distribution and known cholinergic affinity, it is argued that this group also includes the cells labelled in and around lateral hypothalamus and cerebral peduncle, and that as a whole the group constitutes a cholinergic counterpart of the diffusely projecting monoaminergic systems. It seems possible that the basalis projection at first follows a fornical-subcallosal pathway to reach striate cortex via callosoperforant fibers.

The loss of visual perception or "blankout" which occurs when a homogeneous field (Ganzfeld) is presented monocularly is prevented when the same field is viewed binocularly. Thus, blankout cannot be retinal; and contours or transients in time and space are unnecessary for the continuous maintenance of visual perception. Experiments are reported in which blankout ensues only if the two eyes receive luminance disparities ca 0.75 log I. Furthermore, blankout is only marginally affected by stimulus intensity, nor is it dependent on stimulus hue. However, equally luminant but disparate hues presented to the two eyes produce perceptions reminiscent of blankout, with the darkness of blankout replaced with that of color. It is hypothesized that the underlying mechanisms have a commonality in the phenomena of blankout and binocular rivalry but several noncongruent features require explanation.

Consciousness derives from a neural process that requires unceasing metabolic support, and probably involves only a select population of neocortical elements. The essential process must operate for roughly more than 100 ms for sensorial registration (Libet). It is highly unlikely that the essence of the process lies in its computational logic and hence it can never be produced by inanimate machines. Since the process is thus unique to neurons, and since the consciousness of the left hemisphere normally communicates with that of the right (and probably vice versa) via the forebrain cornmissures, at least some portion of the nerve impulse traffic across the commissures must possess a wholly mysterious property enabling its transcendent compilation into a unified conscious experience. Comprehending the nature of this property which couples ionic fluxions into mentality is the quintessential problem of science. The forebrain commissures may ultimately provide the clues for its solution.

Three pig-tailed macaques were trained to select ("match") from a pair of colored images that which they had seen ("sample") and responded to 5--15 s previously. The anterior commissure (AC) and/or its radiation, various loci in basal ganglia, hippocampal formation and "control" areas, (splenium of corpus callosum, precentral gyrus, insular cortex), totalling 40 loci, were each tetanized for 4 s during presentation of the "sample" image, during the delay period, or when the monkey was required to select the "matching" image. For several loci in the hippocampal formation tetanization at any phase of the task reduced "matching" to chance levels and gave evidence of electrical after-discharge; but other comparable hippocampal loci had little or no effect. Response to "sample" or "match" stimuli were absent during tetanization of basal ganglia or anterior commissure. When finally made, upon cessation of tetanization, responses were equally correct for basal ganglia and "control" sites, but for AC were at chance levels.

The firing frequency of a population of 213 units in striate and circumstriate cortex of the moderately restrained rabbit was studied under the influences of alternating 1-min periods of darkness versus steady, diffuse, featureless illumination. The intent was to determine whether luxotonic responses, so prominent in striate cortex of primates, are indeed absent in rabbits. Such was the case, there being only transient occurrences in three units where the continuing rate of discharge in darkness was double that in the light. There were, however, much more modest differences in rate of continuing discharge in light versus darkness, and for 46% of the units discharging greater than 1/s this difference exceeded 10% and/or 1/s. The rate of discharge in any case did not provide a reliable index as to the characteristics of a unit's receptive field in response to patterned visual stimuli. The nature and function of luxotonic activity in primates still not being understood, it cannot be decided whether its absence in rabbits represents a true qualitative or merely a quantitative difference between species.

After control studies, using electrodes permanently implanted in the central visual system, squirrel monkeys and macaques were in most instances blinded by acute glaucoma. This permitted subsequent observation of eye movements. Ocular nystagmus developed in all cases. Beginning immediately upon recovery from anesthesia, and persisting for at least 1 year, the EEG of the striate cortex was characterized by totally flat periods up to several seconds in duration which were ended abruptly by a sharp "spike" trailed in turn by a ragged high voltage, slow pattern for another second or two. The great majority of these "spikes" from the blind striate cortex occurred within 60-200 msec after a saccadic eye movement, made either in nystagmus or attempted fixation. They were not dependent upon proprioception from the extraocular muscles. It is suggested that they represent a "corollary discharge" for movement of the eyes. The blind striate cortex was judged to be hyperexcitable on the basis of these saccade-associated "spikes", not often observable in intact monkeys, and from the increase both in response evoked by electrical stimulation of optic radiation and amplitude of the EEG in sleep.

A type of unit discharge, termed "luxotonic," has been found in the striate cortex of unanesthetized squirrel monkeys and macaques.6, 21 The firing frequency of these units shows relatively little adaptation, continues indefinitely (hours), and reflects the level of diffuse illumination of the eye. The more numerous "photergic" units discharge more rapidly in the light, whereas "scotergic" units fire fastest in the dark (or at luminance levels below threshold for cones). Luxotonic activity is abolished by anesthesia and has not been described for striate cortex of other species. Primates also display a profound alteration in the EEG of striate cortex following elimination of all retinal input.32 Since this change is far more drastic than that produced by blindness in other species, it is natural to inquire whether it is related to the loss of the normally prominent luxotonic activity. When the blind monkey sleeps, the bizarre EEG is replaced by patterns wholly normal in appearance,32 indicating that some nonvisual system has extensive access to striate cortex in this state.

With its head steadied within a form-fitting mask, a macaque was first taught to signal when it detected the application of 0.2-ms electrical pulses at 50 Hz through electrodes chronically implanted within its striate cortex. Stimuli were then applied via a movable microelectrode and the threshold for the animal’s detection determined at intervals of 50-250 micrometers. With permanently implanted 130- 200-micrometers diameter electrodes such thresholds range between 50 and 250 microamperes (and are highly stable), whereas with the microelectrodes sites were encountered, estimated to be primarily within cortical layers V-VI, where the monkey could reliably detect as little as 2-4 microamperes. The threshold at most sites within striate cortex with the microelectrode, however, was 15-25 microamperes. Background unit activity recorded with the microelectrode varied greatly in different laminae and survived the microstimulation, but has so far provided no clear basis for predicting threshold. It is tentatively hypothesized that the relatively rare points where the threshold is as much as 10 times less than that in the surround arise because the giant, solitary cells of Meynert provide the exclusively effective output for the behavioral response. This hypothesis would also explain the singular uniformity of sensation (a "phosphene") evoked in human subjects by such stimuli, and the equivalence of all such stimuli in striate cortex found for the macaque.

Using permanently implanted electrodes in squirrel monkeys and macaques, transmission through the lateral geniculate nucleus (LGN) was assayed from the amplitude of potentials evoked in optic radiation by and electrical pulse applied to optic tract. Averaging of either individually or machine selected potentials, elicited at 0.3, 1.0, 20 or 50 HZ, in all cases showed a decrease in transmission ranging from 5-60% in the period after saccadic eye movements made ad libitum. The suppression was greater in a patterned visual environment than in diffuse illumination, which in turn was greater than that occurring following saccades in the dark. Demonstration of the effect in darkness always required data averaging and never exceeded 20%. The effect was consistently greater in the magnocellular than parvocellular component. Suppresion was often abruptly terminated and replaced by a facilitation of 5-15% about 100 msec after saccade detection. Comparable effects were observed for excitability of striate cortex tested by a stimulus pulse applied to optic radiation. In addition, sharply demarcated potentials inherently arising in LGN and striate cortex were found in association with saccades made even in total darkness. Neglecting a possible but dubious contribution from eye muscle proprioceptors, the experiments establish the existence of a centrally originating modulation of visual processing at both LGN and striate cortex in ralation to saccadic eye movement in primates. This modulation may partially underlie the phenomenon of "saccadic suppression" and hasten the acquistion of a meaningful visualsample immediately following an ocular saccade. It remains uncertain as to how it may relate to similar or greater effects accompanying changes in alertness, or to fluctuations of unknown origin occurring sometimes semirhythmically at 0.05-0.03 HZ (Fig 7).

While there are several studies documenting the enduring nature of memory for acquired discriminatory "habits' or "skills' in animals, comparable data on memory for visual scenes, i.e., "events', are essentially non-existent, and difficult to obtain even in man. An opportunity to assay this question in macaques arose in the early stages of training an animal on a running recognition task. It had previously been trained on trial-unique delayed matching to sample, and its past experience with this visual material was precisely known. When some of these images which had not been seen by the monkey for at least 6 months were intermingled with comparable material during its training on the running recognition task, with a high degree of statistical reliability (P less than 0.005) it distinguished about one-third of the earlier images, many of which had been seen for a total of only 30 s or less. A medical student, who had previously trained the animals and had had more exposure to the material than did this macaque, and certainly had more precise instruction on how to perform, recognized two-thirds of these same images, also after a hiatus of 6 months. It thus appears likely that the permanence of mnemonic storage for briefly encountered scenes is comparable for the central visual systems of macaque and man.

The evidence is convincing that each human cerebral hemisphere is capable of human mental activity. This being so, every normal human thought and action demands either a consensus between the two hemispheres, or a dominance of one over the other, in any event integrated into a unity of conscious mentation. How this is achieved remains wholly mysterious, but anatomical and behavioral data suggest that the two hemispheres, and their respective bilateral, anatomical-functional components, maintain a dynamic equilibrium through neural competition. While the forebrain commissures must contribute substantially to this competitive process, it is emphasized in this review that the serotonergic raphé nuclei of pons and mesencephalon are also participants in interhemispheric events. Each side of the raphé projects heavily to both sides of the forebrain, and each is in receipt of bilateral input from the forebrain and the habenulo-interpeduncular system. A multifarious loop thus exists between the two hemispheres, comprised of both forebrain commissural and brainstem paths. There are many reasons for believing that perturbation of this loop, by a variety of pathogenic agents or processes, probably including severe mental stress in susceptible individuals, underlies the extraordinarily diverse symptomatology of schizophrenia. Abnormality of features reflecting interhemispheric processes is common in schizophrenic patients; and the 'first rank' symptoms of delusions or hallucinations are prototypical of what might be expected were the two hemispheres unable to integrate their potentially independent thoughts. Furthermore, additional evidence suggests that the disorder lies within, or is focused primarily through, the raphé serotonergic system, that plays such a fundamental role in consciousness, in dreaming, in response to psychotomimetic drugs, and probably in movement, and even the trophic state of the neocortex. This system is also well situated to control the dopaminergic neurons of the ventral tegmental area, thus relating to the prominence of dopaminergic features in schizophrenia; and the lipofuscin loading and intimate relation with blood vessels and ependyma may make neurons of the raphé uniquely vulnerable to deleterious agents.