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 humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. 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 50μm. 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

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 fovea1. 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 sections2, generates reliable structural data for biological features in the mammalian brain3. 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 space4. 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

Heterochrony and Cross-Species Intersensory Matching by Infant Vervet Monkeys

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.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.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

Bilateral distribution of face- and object-selective neurones in the adult vervet monkey inferotemporal cortex : a molecular mapping study

A series of studies is described here which explore the functional organisation of face- and object-processing neurones in the adult vervet monkey brain. This fundamental issue in high-level vision is addressed by the use of a novel molecular mapping technique that was developed for this purpose.In the first study, the temporal dynamics of c-fos and zif268 expression were delineated in detail in the rat visual cortex. Knowing the precise temporal parameters of up-regulation (after onset of sensory stimulation) and down-regulation (after offset of sensory stimulation) of these genes was integral to optimising the temporal aspects of the stimuli to be used for subsequent mapping experiments. This study provided the critical information for devising stimuli with corresponding temporal parameters to those of c-fos or zif268 so that one could take advantage of the disparity between the expression of their mRNA and protein products in order to visualise activated neurones.In the second study, the newly developed molecular mapping technique was validated in the rat auditory, visual and multisensory systems. First, bimodal audiovisual stimuli were designed using the data obtained from the first study. Then, through the combined histological detection of the mRNA and protein products of zif268, discrete populations of neurones responsive to either component of the bimodal stimulus were visualised. It was also observed that a third population of neurones was found that responded to the stimulation through both sensory modalities. The combined results from these two studies set the stage for addressing the issue of the organisation of face- and object-selective neurones of the inferior temporal cortex in the vervet monkey brain.In the third study, the functional organisation of face- and object-selective neurones was examined using the molecular mapping technique. Based on the data gathered from the first two studies, suitable stimuli containing two distinct object classes (conspecific faces and non-face familiar objects) were designed with appropriate temporal parameters.Finally, the last study provided an opportunity to address the issue of hemispheric asymmetry of function in the context of face processing in the non-human primate brain. Results support the notion that there may indeed be phylogenetic explanations for the hemispheric asymmetry observed in the human brain

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

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