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Because at least some squirrel monkeys lack ocular dominance columns (ODCs) in the striate cortex (V1) that are detectable by cytochrome oxidase (CO) histochemistry, the functional importance of ODCs on stereoscopic 3-D vision has been questioned. However, conventional CO histochemistry or trans-synaptic tracer study has limited capacity to reveal cortical functional architecture, whereas the expression of immediate-early genes (IEGs), c-FOS and ZIF268, is more directly responsive to neuronal activity of cortical neurons to demonstrate ocular dominance (OD)-related domains in V1 following monocular inactivation. Thus, we wondered whether IEG expression would reveal ODCs in the squirrel monkey V1. In this study, we first examined CO histochemistry in V1 of five squirrel monkeys that were subjected to monocular enucleation or tetrodotoxin (TTX) treatment to address whether there is substantial cross-individual variation as reported previously. Then, we examined the IEG expression of the same V1 tissue to address whether OD-related domains are revealed. As a result, staining patterns of CO histochemistry were relatively homogeneous throughout layer 4 of V1. IEG expression was also moderate and homogeneous throughout layer 4 of V1 in all cases. On the other hand, the IEG expression was patchy in accordance with CO blobs outside layer 4, particularly in infragranular layers, although they may not directly represent OD clusters. Squirrel monkeys remain an exceptional species among anthropoid primates with regard to OD organization, and thus are potentially good subjects to study the development and function of ODCs.

Cytochrome oxidase (CO) histochemistry has been used to reveal the cytoarchitecture of the primate brain, including blobs/puffs/patches in the striate cortex (V1), and thick, thin and pale stripes in the middle layer of the secondary visual cortex (V2). It has been suggested that CO activity is coupled with the spiking activity of neurons, implying that neurons in these CO-rich subcompartments are more active than surrounding regions. However, we have discussed possibility that CO histochemistry represents the distribution of thalamo-cortical afferent terminals that generally use vesicular glutamate transporter 2 (VGLUT2) as their main glutamate transporter, and not the activity of cortical neurons. In this study, we systematically compared the labeling patterns observed between CO histochemistry and immunohistochemistry (IHC) for VGLUT2 from the system to microarchitecture levels in the visual cortex of squirrel monkeys. The two staining patterns bore striking similarities at all levels of the visual cortex, including the honeycomb structure of V1 layer 3Bβ (Brodmann's layer 4A), the patchy architecture in the deep layers of V1, the superficial blobs of V1, and the V2 stripes. The microarchitecture was more evident in VGLUT2 IHC, as expected. VGLUT2 protein expression that produced specific IHC labeling is thought to originate from the thalamus since the lateral geniculate nucleus (LGN) and the pulvinar complex both show high expression levels of VGLUT2 mRNA, but cortical neurons do not. These observations support our theory that the subcompartments revealed by CO histochemistry represent the distribution of thalamo-cortical afferent terminals in the primate visual cortex.

Ocular dominance plasticity has been extensively studied in various mammalian species. While robust ocular dominance (OD) shifts are typically observed after monocular eyelid suture, relatively poor OD plasticity is observed for early eye removal or after tetrodotoxin (TTX) injections in mice. Hence, abnormal binocular signal interactions in the visual cortex may play a critical role in eliciting OD plasticity. Here, we examined the histochemical changes in the lateral geniculate nucleus (LGN) and the striate cortex (V1) in macaque monkeys that experienced two different monocular sensory deprivations in the same eye beginning at 3 weeks of age: restricted laser lesions in macular or peripheral retina and form deprivation induced by wearing a diffuser lens during the critical period. The monkeys were subsequently reared for 5 years under a normal visual environment. In the LGN, atrophy of neurons and a dramatic increase of GFAP expression were observed in the lesion projection zones (LPZs). In V1, although no obvious shift of the LPZ border was found, the ocular dominance columns (ODCs) for the lesioned eye shrunk and those for the intact eye expanded over the entirety of V1. This ODC size change was larger in the area outside the LPZ and in the region inside the LPZ near the border compared to that in the LPZ center. These developmental changes may reflect abnormal binocular interactions in V1 during early infancy. Our observations provide insights into the nature of degenerative and plastic changes in the LGN and V1 following early chronic monocular sensory deprivations.

Ocular dominance columns (ODCs) exist in many primates and carnivores, but it is believed that they do not exist in rodents. Using a combination of transneuronal tracing, in situ hybridization for Zif268 and electrophysiological recordings, we show that inputs from both eyes are largely segregated in the binocular region of V1 in Long Evans rats. We also show that, interposed between this binocular region and the lateral border of V1, there lies a strip of cortex that is strongly dominated by the contralateral eye. Finally, we show that callosal connections colocalize primarily with ipsilateral eye domains in the binocular region and with contralateral eye input in the lateral cortical strip, mirroring the relationship between patchy callosal connections and specific sets of ODCs described previously in the cat. Our results suggest that development of cortical modular architecture is more conserved among rodents, carnivores, and primates than previously thought.

Ocular dominance columns (ODCs) have been well studied in the striate cortex (V1) of macaques, as well defined arrays of columnar structure that receive inputs from one eye or the other, whereas ODC expression seems more obscure in some New World primate species. ODCs have been identified by means of eye injections of transneuronal transporters and examination of cytochrome oxidase (CO) activity patterns after monocular enucleation. More recently, live-imaging techniques have been used to reveal ODCs. Here, we used the expression of immediate-early genes (IEGs), protooncogene, c-Fos, and zinc finger protein, Zif268, after monocular inactivation (MI) to identify ODCs in V1 of New World owl monkeys. Because IEG expression is more sensitive to activity changes than CO expression, it is capable of revealing activity maps in all layers throughout V1 and demonstrating brief activity changes within a couple of hours. Using IEGs, we not only revealed apparent ODCs in owl monkeys but also discovered a number of unique features of their ODCs. Distinct from those in macaques, these ODCs sometimes bridged to other columns in layer 4 (Brodmann layer 4C ). CO blobs straddled ODC borders in the central visual field, whereas they centered ODC patches in the peripheral visual field. In one case, the ODC pattern continued into V2. Finally, an elevation of IEG expression in layer 4 (4C) was observed along ODC borders after only brief MI. Our data provide insights into the structure and variability of ODCs in primates and revive debate over the functions and development of ODCs.

The extracellular matrix (ECM) plays important roles in the development and plasticity of the central nervous system, and it has been shown that it regulates reorganization of the neuronal network. We have found that expression of OCC1, testican-1, testican-2, testican-3, SPARC and SC1 mRNAs, which encode members of the OCC1-related family of ECM proteins, exhibits distinct activity-dependent expression patterns in the adult macaque visual cortex. This finding suggests that OCC1-related proteins play crucial roles in the visual processing pathway. In the present study, we examined mRNA expression patterns of OCC1-related genes in the dorsal lateral geniculate nucleus (dLGN) of macaques. The mRNAs of testican-1 and testican-2 were strongly expressed in both excitatory projection neurons and GABAergic interneurons in the dLGN. Expression of testican-3 mRNA, which is predominantly observed in GABAergic interneurons in the cortex, was restricted to excitatory projection neurons in the dLGN. SPARC mRNA was strongly, and exclusively, expressed in glial cells in the dLGN. Interestingly, neuronal SC1 mRNA expression was abundantly observed in intercalated, koniocellular layers of the dLGN, while it was preferentially observed in blob regions of the primary visual area that receives color coding K-pathway projection from dLGN koniocellular layers, suggesting a pathway preference of expression. Finally, monocular inactivation experiments demonstrated that expression of testican-1, testican-2 and testican-3 mRNAs in the dLGN is dependent on sensory activity. Given their differential expression patterns and activity dependence, products of OCC1-related genes may modulate visual processing and plasticity at the level of the dLGN and the visual cortex.

To study the molecular mechanism how cortical areas are specialized in adult primates, we searched for area-specific genes in macaque monkeys and found striking enrichment of serotonin (5-hydroxytryptamine, 5-HT) 1B receptor mRNA, and to a lesser extent, of 5-HT2A receptor mRNA, in the primary visual area (V1). In situ hybridization analyses revealed that both mRNA species were highly concentrated in the geniculorecipient layers IVA and IVC, where they were coexpressed in the same neurons. Monocular inactivation by tetrodotoxin injection resulted in a strong and rapid (

occ1/Follistatin-related protein (Frp) is strongly expressed in the primary visual cortex (V1) of macaque monkeys, and its expression is strongly down-regulated by intraocular tetrodotoxin (TTX) injection. The pronounced area selectivity of occ1/Frp mRNA expression occurs in macaques and marmosets, but not in mice, rabbits and ferrets, suggesting that occ1/Frp is an important clue to the evolution of the primate cerebral cortex. To further determine species differences, we examined the sensory-input dependency of occ1/Frp mRNA expression in mice in comparison with macaque V1. In macaque V1, occ1/Frp mRNA expression level significantly decreased with even 1-day monocular deprivation (MD) by TTX injection. In contrast to that in macaques, however, the occ1/Frp mRNA expression in the visual cortex in mice was not down-regulated by 1- to 7-day MD by TTX injection. Similarly, MD had no effect on occ1/Frp mRNA expression level in the dorsal lateral geniculate nucleus of mice. In addition, the extirpation of the cochlear or olfactory epithelium had no effect on occ1/Frp mRNA expression in either the cochlear nucleus or the olfactory bulb in mice. Thus, occ1/Frp mRNA expression is independent of sensory-input in mice. The results suggest that activity-dependent occ1/Frp mRNA expression is not common between mice and monkeys, and that primate V1 has acquired a unique gene regulatory mechanism that enables a rapid response to environmental changes. The characteristic feature of the activity dependency of occ1/Frp mRNA expression is discussed, in comparison with that of the expression of the immediate-early genes, c-fos and zif268.

occ1 is a gene whose expression is particularly abundant in neurons in the macaque primary visual cortex (V1). In the present study, we report that the expression of occ1 mRNA in the macaque neocortex can be classified into two modes. The first mode is associated with excitatory neurons distributed in the major thalamocortical recipient layers that exhibit strong cytochrome oxidase activity. This is highly prominent in V1. The second mode is associated with parvalbumin-positive GABAergic interneurons and is distributed across the macaque neocortex. In V1, monocular deprivation showed that occ1 mRNA expression in excitatory neurons was markedly dependent on afferent activity, whereas that in GABAergic interneurons was not. Cross-species comparison showed specific differences in expression. In marmosets, a strong expression was observed in V1 similarly to macaques. The occ1 mRNA expression, however, was generally weak in the mouse neocortex. In rabbit and ferret cortices, the strong expression was observed only in GABAergic interneurons. We conclude that activity-dependent occ1 mRNA expression in the excitatory neurons of V1 was caused by a novel mechanism acquired by primates after their separation from other lineages.

Vesicular transporter proteins are an essential component of the presynaptic machinery that regulates neurotransmitter storage and release. They also provide a key point of control for homeostatic signaling pathways that maintain balanced excitation and inhibition following changes in activity levels, including the onset of sensory experience. To advance understanding of their roles in the developing auditory forebrain, we tracked the expression of the vesicular transporters of glutamate (VGluT1, VGluT2) and GABA (VGAT) in primary auditory cortex (A1) and medial geniculate body (MGB) of developing mice (P7, P11, P14, P21, adult) before and after ear canal opening (~P11-P13). RNA sequencing, in situ hybridization, and immunohistochemistry were combined to track changes in transporter expression and document regional patterns of transcript and protein localization. Overall, vesicular transporter expression changed the most between P7 and P21. The expression patterns and maturational trajectories of each marker varied by brain region, cortical layer, and MGB subdivision. VGluT1 expression was highest in A1, moderate in MGB, and increased with age in both regions. VGluT2 mRNA levels were low in A1 at all ages, but high in MGB, where adult levels were reached by P14. VGluT2 immunoreactivity was prominent in both regions. VGluT1 (+) and VGluT2 (+) transcripts were co-expressed in MGB and A1 somata, but co-localization of immunoreactive puncta was not detected. In A1, VGAT mRNA levels were relatively stable from P7 to adult, while immunoreactivity increased steadily. VGAT (+) transcripts were rare in MGB neurons, whereas VGAT immunoreactivity was robust at all ages. Morphological changes in immunoreactive puncta were found in two regions after ear canal opening. In the ventral MGB, a decrease in VGluT2 puncta density was accompanied by an increase in puncta size. In A1, perisomatic VGAT and VGluT1 terminals became prominent around the neuronal somata. Overall, the observed changes in gene and protein expression, regional architecture, and morphology relate to-and to some extent may enable-the emergence of mature sound-evoked activity patterns. In that regard, the findings of this study expand our understanding of the presynaptic mechanisms that regulate critical period formation associated with experience-dependent refinement of sound processing in auditory forebrain circuits.

The primate neocortex consists of much more evolved areas than those in other mammals. To understand the molecular basis and physiological significance specific to the primate neocortex, we have been searching and characterizing the genes that are selectively and highly expressed in the macaque neocortex. Such gene can be classified into three groups: the primary visual area (V1)-selective genes, the association-area-selective genes, and the motor-area-selective genes (Yamamori, Progress Neurobiol 94:201–222, 2011). The V1-selective genes (OCC1/FSTL1, HTR1B, and HTR2A) may play roles in keeping visual homeostasis in primates to adjust to large changes in light amount in the natural environment. In this article, we report on SEMA7A as another V1-selective gene in macaque monkeys. The expression pattern of SEMA7A differs from those of the V1-selective genes we previously reported in that it is already expressed in the mid-embryonic stage (embryonic day 83) when the thalamocortical fibers are about to project. In addition, SEMA7A only shows weak activity-dependent expression upon monocular inhibition by TTX injection into one eye compared with other V1-selective genes. These findings suggest that there are at least two subgroups of genes that show high and V1-selective expression. The significance of these findings is discussed.

Pooja Balaram1, Toru Takahata1, Jon H Kaas1,21Department of Psychology, 2Department of Cell and Molecular Biology, Vanderbilt University, Nashville, TN, USAAbstract: Vesicular glutamate transporters (VGLUTs) control the storage and presynaptic release of glutamate in the central nervous system, and are involved in the majority of glutamatergic transmission in the brain. Two VGLUT isoforms, VGLUT1 and VGLUT2, are known to characterize complementary distributions of glutamatergic neurons in the rodent brain, which suggests that they are each responsible for unique circuits of excitatory transmission. In rodents, VGLUT2 is primarily utilized in thalamocortical circuits, and is strongly expressed in the primary sensory nuclei, including all areas of the visual thalamus. The distribution of VGLUT2 in the visual thalamus and midbrain has yet to be characterized in primate species. Thus, the present study describes the expression of VGLUT2 mRNA and protein across the visual thalamus and superior colliculus of prosimian galagos to provide a better understanding of glutamatergic transmission in the primate brain. VGLUT2 is strongly expressed in all six layers of the dorsal lateral geniculate nucleus, and much less so in the intralaminar zones, which correspond to retinal and superior collicular inputs, respectively. The parvocellular and magnocellular layers expressed VGLUT2 mRNA more densely than the koniocellular layers. A patchy distribution of VGLUT2 positive terminals in the pulvinar complex possibly reflects inputs from the superior colliculus. The upper superficial granular layers of the superior colliculus, with inputs from the retina, most densely expressed VGLUT2 protein, while the lower superficial granular layers, with projections to the pulvinar, most densely expressed VGLUT2 mRNA. The results are consistent with the conclusion that retinal and superior colliculus projections to the thalamus depend highly on the VGLUT2 transporter, as do cortical projections from the magnocellular and parvocellular layers of the lateral geniculate nucleus and neurons of the pulvinar complex.Keywords: lateral geniculate nucleus, superior colliculus, pulvinar, primate, glutamate

The vesicular glutamate transporters (VGLUTs) regulate the storage and release of glutamate in the brain. In adult animals, the VGLUT1 and VGLUT2 isoforms are widely expressed and differentially distributed, suggesting that neural circuits exhibit distinct modes of glutamate regulation. Studies in rodents suggest that VGLUT1 and VGLUT2 mRNA expression patterns are partly complementary, with VGLUT1 expressed at higher levels in the cortex and VGLUT2 prominent subcortically, but with overlapping distributions in some nuclei. In primates, VGLUT gene expression has not been previously studied in any part of the brain. The purposes of the present study were to document the regional expression of VGLUT1 and VGLUT2 mRNA in the auditory pathway through A1 in the cortex, and to determine whether their distributions are comparable to rodents. In situ hybridization with antisense riboprobes revealed that VGLUT2 was strongly expressed by neurons in the cerebellum and most major auditory nuclei, including the dorsal and ventral cochlear nuclei, medial and lateral superior olivary nuclei, central nucleus of the inferior colliculus, sagulum, and all divisions of the medial geniculate. VGLUT1 was densely expressed in the hippocampus and ventral cochlear nuclei, and at reduced levels in other auditory nuclei. In the auditory cortex, neurons expressing VGLUT1 were widely distributed in layers II-VI of the core, belt and parabelt regions. VGLUT2 was expressed most strongly by neurons in layers IIIb and IV, weakly by neurons in layers II-IIIa, and at very low levels in layers V-VI. The findings indicate that VGLUT2 is strongly expressed by neurons at all levels of the subcortical auditory pathway, and by neurons in the middle layers of the cortex, whereas VGLUT1 is strongly expressed by most if not all glutamatergic neurons in the auditory cortex and at variable levels among auditory subcortical nuclei. These patterns imply that VGLUT2 is the main vesicular glutamate transporter in subcortical and thalamocortical (TC) circuits, whereas VGLUT1 is dominant in corticocortical (CC) and corticothalamic (CT) systems of projections. The results also suggest that VGLUT mRNA expression patterns in primates are similar to rodents, and establish a baseline for detailed studies of these transporters in selected circuits of the auditory system.