A deficit in face-voice integration in developing vervet monkeys exposed to ethanol during gestation.

Children with fetal alcohol spectrum disorders display behavioural and intellectual impairments that strongly implicate dysfunction within the frontal cortex. Deficits in social behaviour and cognition are amongst the most pervasive outcomes of prenatal ethanol exposure. Our naturalistic vervet monkey model of fetal alcohol exposure (FAE) provides an unparalleled opportunity to study the neurobehavioral outcomes of prenatal ethanol exposure in a controlled experimental setting. Recent work has revealed a significant reduction of the neuronal population in the frontal lobes of these monkeys. We used an intersensory matching procedure to investigate audiovisual perception of socially relevant stimuli in young FAE vervet monkeys. Here we show a domain-specific deficit in audiovisual integration of socially relevant stimuli. When FAE monkeys were shown a pair of side-by-side videos of a monkey concurrently presenting two different calls along with a single audio track matching the content of one of the calls, they were not able to match the correct video to the single audio track. This was manifest by their average looking time being equally spent towards both the matching and non-matching videos. However, a group of normally developing monkeys exhibited a significant preference for the non-matching video. This inability to integrate and thereby discriminate audiovisual stimuli was confined to the integration of faces and voices as revealed by the monkeys' ability to match a dynamic face to a complex tone or a black-and-white checkerboard to a pure tone, presumably based on duration and/or onset-offset synchrony. Together, these results suggest that prenatal ethanol exposure negatively affects a specific domain of audiovisual integration. This deficit is confined to the integration of information that is presented by the face and the voice and does not affect more elementary aspects of sensory integration.

Cross-modal recruitment of primary visual cortex by auditory stimuli in the nonhuman primate brain: a molecular mapping study.

Recent studies suggest that exposure to only one component of audiovisual events can lead to cross-modal cortical activation. However, it is not certain whether such crossmodal recruitment can occur in the absence of explicit conditioning, semantic factors, or long-term associations. A recent study demonstrated that crossmodal cortical recruitment can occur even after a brief exposure to bimodal stimuli without semantic association. In addition, the authors showed that the primary visual cortex is under such crossmodal influence. In the present study, we used molecular activity mapping of the immediate early gene zif268. We found that animals, which had previously been exposed to a combination of auditory and visual stimuli, showed increased number of active neurons in the primary visual cortex when presented with sounds alone. As previously implied, this crossmodal activation appears to be the result of implicit associations of the two stimuli, likely driven by their spatiotemporal characteristics; it was observed after a relatively short period of exposure (~45 min) and lasted for a relatively long period after the initial exposure (~1 day). These results suggest that the previously reported findings may be directly rooted in the increased activity of the neurons occupying the primary visual cortex.

Partial recovery of hemiparesis following hemispherectomy in infant monkeys.

Hemiparesis, unilateral weakness or partial paralysis, is a common outcome following hemispherectomy in humans. We use the non-human primate as an invaluable translational model for our understanding of developmental plasticity in response to hemispherectomy. Three infant vervet monkeys (Chlorocebus sabeus) underwent hemispherectomy at a median age of 9 weeks and two additional monkeys at 48 months. Gross motor assessment was conducted in a large open field that contained a horizontal bar spanning the width of the cage. Subjects were assessed yearly following surgery in infantile lesions for a period of 3 years. Adult-lesioned subjects were assessed 40 months following surgery. Shortly after surgery both infant and adult-lesioned subjects were unable to move the contralateral side of their body, but all subjects were able to walk within 6 months following surgery. At each time point the lower limb gait was normal in infant-lesioned subjects with no apparent limp or dragging, however the upper limb demonstrated significant impairment. Horizontal bar crossing was significantly impaired during the first 24 months following surgery. Adult-lesioned subjects also displayed upper limb movement impairments similar to infant-lesioned subjects. In addition the adult-lesioned subjects displayed a noticeable lower limb limp, which was not observed in the infant-lesioned group. Both groups at each time point showed a propensity for ipsiversive turning. The upper limb gait impairment and horizontal bar crossing of lesioned subjects are reminiscent of hemiparesis seen in hemisperectomized humans with the young-lesioned subjects showing a greater propensity for recovery.

Crossmodal recruitment of primary visual cortex following brief exposure to bimodal audiovisual stimuli.

Several lines of evidence suggest that exposure to only one component of typically audiovisual events can lead to crossmodal cortical activation. These effects are likely explained by long-term associations formed between the auditory and visual components of such events. It is not certain whether such crossmodal recruitment can occur in the absence of explicit conditioning, semantic factors, or long-term association; nor is it clear whether primary sensory cortices can be recruited in such paradigms. In the present study we tested the hypothesis that crossmodal cortical recruitment would occur even after a brief exposure to bimodal stimuli without semantic association. We used positron emission tomography, and an apparatus allowing presentation of spatially and temporally congruous audiovisual stimuli (noise bursts and light flashes). When presented with only the auditory or visual components of the bimodal stimuli, naïve subjects showed only modality-specific cortical activation, as expected. However, subjects who had previously been exposed to the audiovisual stimuli showed increased cerebral blood flow in the primary visual cortex when presented with sounds alone. Functional connectivity analysis suggested that the auditory cortex was the source of visual cortex activity. This crossmodal activation appears to be the result of implicit associations of the two stimuli, likely driven by their spatiotemporal characteristics; it was observed after a relatively short period of exposure (approximately 45 min), and lasted for a relatively long period after the initial exposure (approximately 1 day). The findings indicate that auditory and visual cortices interact with one another to a larger degree than typically assumed.

Dissecting the non-human primate brain in stereotaxic space.

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans(1-6). In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse(7). Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50 microm. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

Brain banking: making the most of your research specimens.

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest(1). To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20 degrees C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Batch immunostaining for large-scale protein detection in the whole monkey brain.

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

The gateway to the brain: dissecting the primate eye.

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea(1). Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

Knowing what counts: unbiased stereology in the non-human primate brain.

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections, generates reliable structural data for biological features in the mammalian brain. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

Whole-brain expression analysis of FMRP in adult monkey and its relationship to cognitive deficits in fragile X syndrome.

Fragile X syndrome (FXS) is one of the most prevalent forms of heritable mental retardation and developmental delay in males. The syndrome is caused by the silencing of a single gene (fragile X mental retardation-1; FMR1) and the lack of expression of its protein product (fragile X mental retardation-1 protein; FMRP). Recent work has linked the high expression levels of FMRP in the magnocellular layers of lateral geniculate nucleus (M-LGN) of the visual system to a specific reduction of perceptual function known to be mediated by that neural structure. This finding has given rise to the intriguing notion that FMRP expression level may be used as an index of susceptibility of specific brain regions to the observed perceptual and cognitive deficits in FXS. We undertook a comprehensive expression profiling study of FMRP in the monkey to obtain further insight into the link between FMPR expression and the behavioural impact of its loss in FXS. We report here the first 3D whole-brain map of FMRP expression in the Old-World monkey and show that certain brain structures display high FMRP levels, such as the cerebellum, striatum, and temporal lobe structures. This finding provides support for the notion that FMRP expression loss is linked to behavioural and cognitive impairment associated with these structures. We argue that whole-brain FMRP expression mapping may be used to formulate and test new hypotheses about other forms of impairments in FXS that were not specifically examined in this study.

Heterochrony and cross-species intersensory matching by infant vervet monkeys.

BACKGROUND: Understanding the evolutionary origins of a phenotype requires understanding the relationship between ontogenetic and phylogenetic processes. Human infants have been shown to undergo a process of perceptual narrowing during their first year of life, whereby their intersensory ability to match the faces and voices of another species declines as they get older. We investigated the evolutionary origins of this behavioral phenotype by examining whether or not this developmental process occurs in non-human primates as well. METHODOLOGY/PRINCIPAL FINDINGS: We tested the ability of infant vervet monkeys (Cercopithecus aethiops), ranging in age from 23 to 65 weeks, to match the faces and voices of another non-human primate species (the rhesus monkey, Macaca mulatta). Even though the vervets had no prior exposure to rhesus monkey faces and vocalizations, our findings show that infant vervets can, in fact, recognize the correspondence between rhesus monkey faces and voices (but indicate that they do so by looking at the non-matching face for a greater proportion of overall looking time), and can do so well beyond the age of perceptual narrowing in human infants. Our results further suggest that the pattern of matching by vervet monkeys is influenced by the emotional saliency of the Face+Voice combination. That is, although they looked at the non-matching screen for Face+Voice combinations, they switched to looking at the matching screen when the Voice was replaced with a complex tone of equal duration. Furthermore, an analysis of pupillary responses revealed that their pupils showed greater dilation when looking at the matching natural face/voice combination versus the face/tone combination. CONCLUSIONS/SIGNIFICANCE: Because the infant vervets in the current study exhibited cross-species intersensory matching far later in development than do human infants, our findings suggest either that intersensory perceptual narrowing does not occur in Old World monkeys or that it occurs later in development. We argue that these findings reflect the faster rate of neural development in monkeys relative to humans and the resulting differential interaction of this factor with the effects of early experience.

Patchy organization and asymmetric distribution of the neural correlates of face processing in monkey inferotemporal cortex.

BACKGROUND: It is believed that a face-specific system exists within the primate ventral visual pathway that is separate from a domain-general nonface object coding system. In addition, it is believed that hemispheric asymmetry, which was long held to be a distinct feature of the human brain, can be found in the brains of other primates as well. We show here for the first time by way of a functional imaging technique that face- and object-selective neurons form spatially distinct clusters at the cellular level in monkey inferotemporal cortex. We have used a novel functional mapping technique that simultaneously generates two separate activity profiles by exploiting the differential time course of zif268 mRNA and protein expression. RESULTS: We show that neurons activated by face stimulation can be visualized at cellular resolution and distinguished from those activated by nonface complex objects. Our dual-activity maps of face and object selectivity show that face-selective patches of various sizes (mean, 22.30 mm2; std, 32.76 mm2) exist throughout the IT cortex in the context of a large expanse of cortical territory that is responsive to visual objects. CONCLUSIONS: These results add to recent findings that face-selective patches of various sizes exist throughout area IT and provide the first direct anatomical evidence at cellular resolution for a hemispheric asymmetry in favor of the right hemisphere. Together, our results support the notion that human and monkey brains share a similarity in both anatomical organization and distribution of function with respect to high-level visual processing.

Cellular-resolution activity mapping of the brain using immediate-early gene expression.

Immediate-Early Genes are a class of genes that are rapidly up-regulated following neural stimulation. Due to their quality as potential activity markers in the CNS, they have been used extensively in functional mapping studies. At least three genes have been popularly used, including zif268 (Egr1, NGFI, Krox-24, or ZENK), c-fos and recently, Arc. A number of techniques have been developed in applying IEG labelling for the development of functional maps, thus overcoming some of the earlier limitations of this approach. Current developments highlight the future prospects of cellular-resolution functional activity mapping of the brain.

Differential impact of the FMR1 gene on visual processing in fragile X syndrome.

Fragile X syndrome (FXS) is the most common form of heritable mental retardation, affecting approximately 1 in 4000 males. The syndrome arises from expansion of a trinucleotide repeat in the 5'-untranslated region of the fragile X mental retardation 1 (FMR1) gene, leading to methylation of the promoter sequence and lack of the fragile X mental retardation protein (FMRP). Affected individuals display a unique neurobehavioural phenotype that includes striking visual-motor deficits. Here we provide neurobiological and behavioural evidence that supports the hypothesis that these visual-motor deficits are attributable to a magnocellular (M) visual pathway impairment. Immunohistochemical staining of a lateral geniculate nucleus (LGN) of a normal human male revealed high FMRP basal expression selectively within the M layers, suggesting an increased susceptibility of these neurons to the lack of FMRP as occurs in FXS. Similar staining of monkey LGNs for quantification purposes revealed that the difference is not an artefact of cell size differences between M and parvocellular (P) neurons. Further, Nissl staining of the LGNs of a male FXS patient revealed alaminar nuclei comprised of a homogenous population of small sized neurons, providing anatomical and morphological support for the idea that an M pathway pathology exists in FXS. Consistent with these neurobiological data, we have found that male patients with FXS have reduced sensitivity for psychophysical stimuli that probe the M pathway but not for those that probe the P pathway, a complementary visual stream that performs a separate set of early visual operations. Finally, male patients with FXS performed poorly on a global motion task but not on a form perception task, suggesting that the M pathway thalamic deficit may have a selective impact on cortical visual functioning in the parietal lobe, which is known to be a major recipient of M pathway afferents via the primary visual cortex. Together, these findings provide the first evidence that the loss of a single gene product, FMRP, in humans leads to abnormal neuroanatomical morphology of the LGN and a concomitant selective visual deficit of the M pathway.

Differential induction and decay curves of c-fos and zif268 revealed through dual activity maps.

The use of inducible transcription factors for mapping neural activity is now a common procedure. We have previously developed a double-labelling technique that allows visualization of activated neurons after two different stimulation sequences. The technique exploits the differential time course of mRNA versus protein expression of transcription factors. However, the precise details of the differential time course remained unknown. Here, we provide a complete up- and downregulation profile for both the c-fos and zif268 genes, as determined through combined in situ hybridization and immunocytochemical detection of the mRNA and protein products in primary visual cortex. The data presented here can be used in the design of future studies employing double-label mapping of neural activation following a compound stimulus.