Regional Specificity of GABAergic Regulation of Cross-Modal Plasticity in Mouse Visual Cortex after Unilateral Enucleation.

In adult mice, monocular enucleation (ME) results in an immediate deactivation of the contralateral medial monocular visual cortex. An early restricted reactivation by open eye potentiation is followed by a late overt cross-modal reactivation by whiskers (Van Brussel et al., 2011). In adolescence (P45), extensive recovery of cortical activity after ME fails as a result of suppression or functional immaturity of the cross-modal mechanisms (Nys et al., 2014). Here, we show that dark exposure before ME in adulthood also prevents the late cross-modal reactivation component, thereby converting the outcome of long-term ME into a more P45-like response. Because dark exposure affects GABAergic synaptic transmission in binocular V1 and the plastic immunity observed at P45 is reminiscent of the refractory period for inhibitory plasticity reported by Huang et al. (2010), we molecularly examined whether GABAergic inhibition also regulates ME-induced cross-modal plasticity. Comparison of the adaptation of the medial monocular and binocular cortices to long-term ME or dark exposure or a combinatorial deprivation revealed striking differences. In the medial monocular cortex, cortical inhibition via the GABAA receptor α1 subunit restricts cross-modal plasticity in P45 mice but is relaxed in adults to allow the whisker-mediated reactivation. In line, in vivo pharmacological activation of α1 subunit-containing GABAA receptors in adult ME mice specifically reduces the cross-modal aspect of reactivation. Together with region-specific changes in glutamate acid decarboxylase (GAD) and vesicular GABA transporter expression, these findings put intracortical inhibition forward as an important regulator of the age-, experience-, and cortical region-dependent cross-modal response to unilateral visual deprivation. SIGNIFICANCE STATEMENT: In adult mice, vision loss through one eye instantly reduces neuronal activity in the visual cortex. Strengthening of remaining eye inputs in the binocular cortex is followed by cross-modal adaptations in the monocular cortex, in which whiskers become a dominant nonvisual input source to attain extensive cortical reactivation. We show that the cross-modal component does not occur in adolescence because of increased intracortical inhibition, a phenotype that was mimicked in adult enucleated mice when treated with indiplon, a GABAA receptor α1 agonist. The cross-modal versus unimodal responses of the adult monocular and binocular cortices also mirror regional specificity in inhibitory alterations after visual deprivation. Understanding cross-modal plasticity in response to sensory loss is essential to maximize patient susceptibility to sensory prosthetics.

Retinal lesions induce fast intrinsic cortical plasticity in adult mouse visual system.

Neuronal activity plays an important role in the development and structural-functional maintenance of the brain as well as in its life-long plastic response to changes in sensory stimulation. We characterized the impact of unilateral 15° laser lesions in the temporal lower visual field of the retina, on visually driven neuronal activity in the afferent visual pathway of adult mice using in situ hybridization for the activity reporter gene zif268. In the first days post-lesion, we detected a discrete zone of reduced zif268 expression in the contralateral hemisphere, spanning the border between the monocular segment of the primary visual cortex (V1) with extrastriate visual area V2M. We could not detect a clear lesion projection zone (LPZ) in areas lateral to V1 whereas medial to V2M, agranular and granular retrosplenial cortex showed decreased zif268 levels over their full extent. All affected areas displayed a return to normal zif268 levels, and this was faster in higher order visual areas than in V1. The lesion did, however, induce a permanent LPZ in the retinorecipient layers of the superior colliculus. We identified a retinotopy-based intrinsic capacity of adult mouse visual cortex to recover from restricted vision loss, with recovery speed reflecting the areal cortical magnification factor. Our observations predict incomplete visual field representations for areas lateral to V1 vs. lack of retinotopic organization for areas medial to V2M. The validation of this mouse model paves the way for future interrogations of cortical region- and cell-type-specific contributions to functional recovery, up to microcircuit level.

Cortical and subcortical projections to primary visual cortex in anophthalmic, enucleated and sighted mice.

The purpose of this study was to identify and compare the afferent projections to the primary visual cortex in intact and enucleated C57BL/6 mice and in ZRDCT/An anophthalmic mice. Early loss of sensory-driven activity in blind subjects can lead to activations of the primary visual cortex by haptic or auditory stimuli. This intermodal activation following the onset of blindness is believed to arise through either unmasking of already present cortical connections, sprouting of novel cortical connections or enhancement of intermodal cortical connections. Studies in humans have similarly demonstrated heteromodal activation of visual cortex following relatively short periods of blindfolding. This suggests that the primary visual cortex in normal sighted subjects receives afferents, either from multisensory association cortices or from primary sensory cortices dedicated to other modalities. Here cortical afferents to the primary visual cortex were investigated to determine whether the visual cortex receives sensory input from other modalities, and whether differences exist in the quantity and/or the structure of projections found in sighted, enucleated and anophthalmic mice. This study demonstrates extensive direct connections between the primary visual cortex and auditory and somatosensory areas, as well as with motor and association cortices in all three animal groups. This suggests that information from different sensory modalities can be integrated at early cortical stages and that visual cortex activations following visual deprivations can partly be explained by already present intermodal corticocortical connections.

Subcortical auditory input to the primary visual cortex in anophthalmic mice.

Anatomical and imaging studies show ample evidence for auditory activation of the visual cortex following early onset of blindness in both humans and animal models. Anatomical studies in animal models of early blindness clearly show intermodal pathways through which auditory information can reach the primary visual cortex. There is clear evidence for intermodal corticocortical pathways linking auditory and visual cortex and also novel connections between the inferior colliculus and the visual thalamus. A recent publication [L.K. Laemle, N.L. Strominger, D.O. Carpenter, Cross-modal innervation of primary visual cortex by auditory fibers in congenitally anophthalmic mice, Neurosci. Lett. 396 (2006) 108-112] suggested the presence of a direct reciprocal connection between the inferior colliculus and the primary visual cortex (V1) in congenitally anophthalmic ZRDCT/An mice. This implies that this mutant mouse would be the only known vertebrate having a direct tectal connection with a primary sensory cortex. The presence of this peculiar pathway was reinvestigated in the ZRDCT/An mouse with highly sensitive neuronal tracers. We found the connections normally described in the ZRDCT/An mouse between: (i) the inferior colliculus and the dorsal lateral geniculate nucleus, (ii) V1 and the superior colliculus, (iii) the lateral posterior nucleus and V1 and between (iv) the inferior colliculus and the medial geniculate nucleus. We also show unambiguously that the auditory subcortical structures do not connect the primary visual cortex in the anophthalmic mouse. In particular, we find no evidence of a direct projection from the auditory mesencephalon to the cortex in this animal model of blindness.

A Tool for Brain-Wide Quantitative Analysis of Molecular Data upon Projection into a Planar View of Choice.

Several techniques, allowing the reconstruction and visualization of functional, anatomical or molecular information from tissue and organ slices, have been developed over the years. Yet none allow direct comparison without reprocessing the same slices. Alternative methods using publicly available reference maps like the Allen Brain Atlas lack flexibility with respect to age and species. We propose a new approach to reconstruct a segmented region of interest from serial slices by projecting the optical density values representing a given molecular signal to a plane of view of choice, and to generalize the results into a reference map, which is built from the individual maps of all animals under study. Furthermore, to allow quantitative comparison between experimental conditions, a non-parametric pseudo t-test has been implemented. This new mapping tool was applied, optimized and validated making use of an in situ hybridization dataset that represents the spatiotemporal expression changes for the neuronal activity reporter gene zif268, in relation to cortical plasticity induced by monocular enucleation, covering the entire mouse visual cortex. The created top view maps of the mouse brain allow precisely delineating and interpreting 11 extrastriate areas surrounding mouse V1. As such, and because of the opportunity to create a planar projection of choice, these molecular maps can in the future easily be compared with functional or physiological imaging maps created with other techniques such as Ca2+, flavoprotein and optical imaging.

Congenital Anophthalmia and Binocular Neonatal Enucleation Differently Affect the Proteome of Primary and Secondary Visual Cortices in Mice.

In blind individuals, visually deprived occipital areas are activated by non-visual stimuli. The extent of this cross-modal activation depends on the age at onset of blindness. Cross-modal inputs have access to several anatomical pathways to reactivate deprived visual areas. Ectopic cross-modal subcortical connections have been shown in anophthalmic animals but not in animals deprived of sight at a later age. Direct and indirect cross-modal cortical connections toward visual areas could also be involved, yet the number of neurons implicated is similar between blind mice and sighted controls. Changes at the axon terminal, dendritic spine or synaptic level are therefore expected upon loss of visual inputs. Here, the proteome of V1, V2M and V2L from P0-enucleated, anophthalmic and sighted mice, sharing a common genetic background (C57BL/6J x ZRDCT/An), was investigated by 2-D DIGE and Western analyses to identify molecular adaptations to enucleation and/or anophthalmia. Few proteins were differentially expressed in enucleated or anophthalmic mice in comparison to sighted mice. The loss of sight affected three pathways: metabolism, synaptic transmission and morphogenesis. Most changes were detected in V1, followed by V2M. Overall, cross-modal adaptations could be promoted in both models of early blindness but not through the exact same molecular strategy. A lower metabolic activity observed in visual areas of blind mice suggests that even if cross-modal inputs reactivate visual areas, they could remain suboptimally processed.

Evaluation of the expression pattern of rAAV2/1, 2/5, 2/7, 2/8, and 2/9 serotypes with different promoters in the mouse visual cortex.

This study compared the expression pattern, laminar distribution, and cell specificity of several rAAV serotypes (2/1, 2/5, 2/7, 2/8, and 2/9) injected in the primary visual cortex (V1) of adult C57Bl/6J mice. In order to obtain specific expression in certain neuron subtypes, different promoter sequences were evaluated for excitatory cell specificity: a universal cytomegalovirus (CMV) promoter, and two versions of the excitatory neuron-specific Ca(2+) /calmodulin-dependent kinase subunit α (CaMKIIα) promoter, CaMKIIα 0.4 and CaMKIIα 1.3. The spatial distribution as well as the cell type specificity was immunohistochemically verified. Depending on the rAAV serotype used, the transduced volume expressing reporter protein differed substantially (rAAV2/5 ≫ 2/7 ≈ 2/9 ≈ 2/8 ≫ 2/1). Excitatory neuron-specific targeting was promoter-dependent, with a surprising difference between the 1.3 kb and 0.4 kb CaMKIIα promoters. While CaMKIIα 1.3 and CMV carrying vectors were comparable, with 78% of the transduced neurons being excitatory for CMV and 82% for CaMKIIα 1.3, the shorter CaMKIIα 0.4 version resulted in 95% excitatory specificity. This study therefore puts forward the CaMKIIα 0.4 promoter as the best choice to target excitatory neurons with rAAVs. Together, these results can be used as an aid to select the most optimal vector system to deliver transgenes into specific rodent neocortical circuits, allowing further elucidation of their functions.

Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex.

Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area.

Visual cortical areas of the mouse: comparison of parcellation and network structure with primates.

Brains have evolved to optimize sensory processing. In primates, complex cognitive tasks must be executed and evolution led to the development of large brains with many cortical areas. Rodents do not accomplish cognitive tasks of the same level of complexity as primates and remain with small brains both in relative and absolute terms. But is a small brain necessarily a simple brain? In this review, several aspects of the visual cortical networks have been compared between rodents and primates. The visual system has been used as a model to evaluate the level of complexity of the cortical circuits at the anatomical and functional levels. The evolutionary constraints are first presented in order to appreciate the rules for the development of the brain and its underlying circuits. The organization of sensory pathways, with their parallel and cross-modal circuits, is also examined. Other features of brain networks, often considered as imposing constraints on the development of underlying circuitry, are also discussed and their effect on the complexity of the mouse and primate brain are inspected. In this review, we discuss the common features of cortical circuits in mice and primates and see how these can be useful in understanding visual processing in these animals.

Primary visual cortex projections to extrastriate cortices in enucleated and anophthalmic mice.

In the mouse, visual extrastriate areas are located within distinct acallosal zones. It has been proposed that the striate-extrastriate and callosal projections are interdependent. In visually deprived mice, the normal patterns of callosal and striate-extrastriate projections are disrupted. It remains unknown whether visual deprivation affects the topography of V1-extrastriate projections and their relationship with callosal projections. Two anterograde tracers were injected in V1 and multiple retrograde tracer injections were performed in the contralateral hemisphere of intact and enucleated C57BL/6 mice and in ZRDCT/An mice to determine the effects of prenatal and postnatal afferent sensory activity on the topography of V1-extrastriate and callosal projections. Greater topographic anomalies were found in striate-extrastriate projections of anophthalmic than enucleated mice. In enucleated mice, the relationship between striate-extrastriate projections and callosal zones was highly variable. In anophthalmic mice, there was also a greater overlap between these projections. These results suggest that the prenatal afferent sensory activity regulates some aspects of the distribution of V1-extrastriate and callosal projections, in addition to the development of a normal topographic representation in extrastriate areas.

Strain differences of the effect of enucleation and anophthalmia on the size and growth of sensory cortices in mice.

Anophthalmia is a condition in which the eye does not develop from the early embryonic period. Early blindness induces cross-modal plastic modifications in the brain such as auditory and haptic activations of the visual cortex and also leads to a greater solicitation of the somatosensory and auditory cortices. The visual cortex is activated by auditory stimuli in anophthalmic mice and activity is known to alter the growth pattern of the cerebral cortex. The size of the primary visual, auditory and somatosensory cortices and of the corresponding specific sensory thalamic nuclei were measured in intact and enucleated C57Bl/6J mice and in ZRDCT anophthalmic mice (ZRDCT/An) to evaluate the contribution of cross-modal activity on the growth of the cerebral cortex. In addition, the size of these structures were compared in intact, enucleated and anophthalmic fourth generation backcrossed hybrid C57Bl/6J×ZRDCT/An mice to parse out the effects of mouse strains and of the different visual deprivations. The visual cortex was smaller in the anophthalmic ZRDCT/An than in the intact and enucleated C57Bl/6J mice. Also the auditory cortex was larger and the somatosensory cortex smaller in the ZRDCT/An than in the intact and enucleated C57Bl/6J mice. The size differences of sensory cortices between the enucleated and anophthalmic mice were no longer present in the hybrid mice, showing specific genetic differences between C57Bl/6J and ZRDCT mice. The post natal size increase of the visual cortex was less in the enucleated than in the anophthalmic and intact hybrid mice. This suggests differences in the activity of the visual cortex between enucleated and anophthalmic mice and that early in-utero spontaneous neural activity in the visual system contributes to the shaping of functional properties of cortical networks.