The synaptic proteome during development and plasticity of the mouse visual cortex

During brain development, the neocortex shows periods of enhanced plasticity, which enables the acquisition of knowledge and skills that we use and build on in adult life. Key to persistent modifications of neuronal connectivity and plasticity of the neocortex are molecular changes occurring at the synapse. Here we used isobaric tag for relative and absolute quantification to measure levels of 467 synaptic proteins in a well-established model of plasticity in the mouse visual cortex and the regulation of its critical period. We found that inducing visual cortex plasticity by monocular deprivation during the critical period increased levels of kinases and proteins regulating the actin-cytoskeleton and endocytosis. Upon closure of the critical period with age, proteins associated with transmitter vesicle release and the tubulin- and septin-cytoskeletons increased, whereas actin-regulators decreased in line with augmented synapse stability and efficacy. Maintaining the visual cortex in a plastic state by dark rearing mice into adulthood only partially prevented these changes and increased levels of G-proteins and protein kinase A subunits. This suggests that in contrast to the general belief, dark rearing does not simply delay cortical development but may activate signaling pathways that specifically maintain or increase the plasticity potential of the visual cortex. Altogether, this study identified many novel candidate plasticity proteins and signaling pathways that mediate synaptic plasticity during critical developmental periods or restrict it in adulthood.     

NMDA receptors‐dependent plasticity in the phototaxis preference behavior induced by visual deprivation in young and adult flies

Adult mammals have experience‐dependent plasticity in visual system, but it is unclear whether adult insects also have this plasticity after the critical period of visual development. Here, we have established a modified Y‐maze apparatus for investigating experience‐dependent plasticity in Drosophila. Using this setup we demonstrate that flies after the critical period have bidirectional modifications of the phototaxis preference behavior (PPB) induced by visual deprivation and experience: Visual deprivation decreases the preference of flies for visible light, while visual experience exerts the opposite effect. We also found an age‐dependent PPB plasticity induced by visual deprivation. Molecular and cellular studies suggest that the N‐methyl‐ d ‐aspartate receptors (NMDARs) mediate ocular dominance plasticity in visual cortex in mammals, but direct behavioral evidence is lacking. Here, we used the genetic approaches to demonstrate that NMDAR1, which is NMDARs subunit in Drosophila, can mediate PPB plasticity in young and adult flies. These findings provide direct behavioral evidence that NMDAR1 mediates PPB plasticity in Drosophila. Our results suggest that mammals and insects have analogous mechanisms for experience‐dependent plasticity and its regulation by NMDAR signaling.     

BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex.

Maturation of the visual cortex is influenced by visual experience during an early postnatal period. The factors that regulate such a critical period remain unclear. We examined the maturation and plasticity of the visual cortex in transgenic mice in which the postnatal rise of brain-derived neurotrophic factor (BDNF) was accelerated. In these mice, the maturation of GABAergic innervation and inhibition was accelerated. Furthermore, the age-dependent decline of cortical long-term potentiation induced by white matter stimulation, a form of synaptic plasticity sensitive to cortical inhibition, occurred earlier. Finally, transgenic mice showed a precocious development of visual acuity and an earlier termination of the critical period for ocular dominance plasticity. We propose that BDNF promotes the maturation of cortical inhibition during early postnatal life, thereby regulating the critical period for visual cortical plasticity.     

Sensitivity profile for orientation selectivity in the visual cortex of goggle-reared mice

It has been widely accepted that ocular dominance in the responses of visual cortical neurons can change depending on visual experience in a postnatal period. However, experience-dependent plasticity for orientation selectivity, which is another important response property of visual cortical neurons, is not yet fully understood. To address this issue, using intrinsic signal imaging and two-photon calcium imaging we attempted to observe the alteration of orientation selectivity in the visual cortex of juvenile and adult mice reared with head-mounted goggles, through which animals can experience only the vertical orientation. After one week of goggle rearing, the density of neurons optimally responding to the exposed orientation increased, while that responding to unexposed orientations decreased. These changes can be interpreted as a reallocation of preferred orientations among visually responsive neurons. Our obtained sensitivity profile for orientation selectivity showed a marked peak at 5 weeks and sustained elevation at 12 weeks and later. These features indicate the existence of a critical period between 4 and 7 weeks and residual orientation plasticity in adult mice. The presence of a dip in the sensitivity profile at 10 weeks suggests that different mechanisms are involved in orientation plasticity in childhood and adulthood.     

Elimination of inhibitory synapses is a major component of adult ocular dominance plasticity

During development, cortical plasticity is associated with the rearrangement of excitatory connections. While these connections become more stable with age, plasticity can still be induced in the adult cortex. Here we provide evidence that structural plasticity of?inhibitory synapses onto pyramidal neurons is?a major component of plasticity in the adult neocortex. In?vivo two-photon imaging was used to monitor the formation and elimination of fluorescently labeled inhibitory structures on pyramidal neurons. We find that ocular dominance plasticity in the adult visual cortex is associated with rapid inhibitory synapse loss, especially of those present on dendritic spines. This occurs not only with monocular deprivation but also with subsequent restoration of binocular vision. We propose that in the adult visual cortex the experience-induced loss of inhibition may effectively strengthen specific visual inputs with limited need for rearranging the excitatory circuitry.     

Time to change: retina sends a messenger to promote plasticity in visual cortex

Maturation of GABA inhibitory circuitry in primary visual cortex activates the critical period of plasticity, but the underlying mechanisms are not well understood. In the August 8th issue of Cell, Sugiyama et al. demonstrate that visual experience promotes the passage of a retina-derived homeoprotein along the visual pathway, which nurtures subclasses of cortical interneurons implicated in regulating critical period plasticity.     

Plasticity in the visual system: role of neurotrophins and electrical activity

We report recent results concerning the action of neurotrophins on the development and plasticity of the visual system of mammals and in particular of their visual cortex. It has been demonstrated that NGF prevents all the effects of monocular deprivation during the critical period. BDNF, that in part also prevents the effects of monocular deprivation, has the interesting additional property of accelerating the development of inhibitory processes. In transgenic mice overexpressing BDNF only in the cortex, the critical period for plasticity initiates a week earlier and presents a precocious closure. Visual acuity also develops much before than in normal animals. These phenomenological observations are paralleled by a precocious increase of inhibitory synapses and inhibitory currents in pyramidal neurons. LTP, tested by stimulation of the white matter, recording in layers 2 and 3 of the visual cortex, presents modifications correlated with the alterations observed in the critical period. Last we report the finding from in vitro and in vivo experiments that MAPkase (Erg 1 and 2) is the molecular chain of events driven both by light and neurotrophins, likely at the bases of the phenomena of plasticity observed during the critical period.     

Stimulus timing-dependent plasticity in cortical processing of orientation.

The relative timing of presynaptic and postsynaptic spikes plays a critical role in activity-induced synaptic modification. Here we examined whether plasticity of orientation selectivity in the visual cortex depends on stimulus timing. Repetitive pairing of visual stimuli at two orientations induced a shift in orientation tuning of cat cortical neurons, with the direction of the shift depending on the temporal order of the pair. Induction of a significant shift required that the interval between the pair fall within +/-40 ms, reminiscent of the temporal window for spike timing-dependent synaptic plasticity. Mirroring the plasticity found in cat visual cortex, similar conditioning also induced a shift in perceived orientation by human subjects, further suggesting functional relevance of this phenomenon. Thus, relative timing of visual stimuli can play a critical role in dynamic modulation of adult cortical function, perhaps through spike timing-dependent synaptic plasticity.     

Environmental enrichment promotes plasticity and visual acuity recovery in adult monocular amblyopic rats

Loss of visual acuity caused by abnormal visual experience during development (amblyopia) is an untreatable pathology in adults. In some occasions, amblyopic patients loose vision in their better eye owing to accidents or illnesses. While this condition is relevant both for its clinical importance and because it represents a case in which binocular interactions in the visual cortex are suppressed, it has scarcely been studied in animal models. We investigated whether exposure to environmental enrichment (EE) is effective in triggering recovery of vision in adult amblyopic rats rendered monocular by optic nerve dissection in their normal eye. By employing both electrophysiological and behavioral assessments, we found a full recovery of visual acuity in enriched rats compared to controls reared in standard conditions. Moreover, we report that EE modulates the expression of GAD67 and BDNF. The non invasive nature of EE renders this paradigm promising for amblyopia therapy in adult monocular people.     

Development and plasticity-related changes in protein expression patterns in cat visual cortex: a fluorescent two-dimensional difference gel electrophoresis approach

During early postnatal brain development, changes in visual input can lead to specific alteration of function and connectivity in mammalian visual cortex. In cat, this so-called critical period exhibits maximal sensory-driven adaptations around postnatal day 30 (P30), and ceases toward adulthood. We examined the molecular framework that directs age- and experience-dependent plasticity in cat visual cortex, by comparing protein expression profiles at eye opening (postnatal day 10 (P10), when experience-dependent plasticity starts), the peak of the critical period (P30), and in adulthood. Using 2-D DIGE, we performed comparisons of P10-P30 and P30-adult brain protein samples. Sixty protein spots showed statistically significant intensity changes in at least one comparison. Fifty-one spots were identified using quadrupole-TOF MS/MS or LC-MS/MS, containing 37 different proteins. The progressive increase or decrease in protein expression levels could be correlated to age-dependent postnatal brain development. Four spots containing transferrin, 14-3-3 alpha/beta and cypin, showed maximal protein expression levels at P30, thereby showing a positive correlation to critical period plasticity. Western analysis indeed revealed a clear effect of visual deprivation on cypin expression in cat visual cortex. Our results therefore demonstrate the power of 2-D DIGE as a tool toward understanding the molecular basis of nervous system development and plasticity.     

Optimization of somatic inhibition at critical period onset in mouse visual cortex

Local GABAergic circuits trigger visual cortical plasticity in early postnatal life. How these diverse connections contribute to critical period onset was investigated by nonstationary fluctuation analysis following laser photo-uncaging of GABA onto discrete sites upon individual pyramidal cells in slices of mouse visual cortex. The GABA(A) receptor number decreased on the soma-proximal dendrite (SPD), but not at the axon initial segment, with age and sensory deprivation. Benzodiazepine sensitivity was also higher on the immature SPD. Too many or too few SPD receptors in immature or dark-reared mice, respectively, were adjusted to critical period levels by benzodiazepine treatment in vivo, which engages ocular dominance plasticity in these animal models. Combining GAD65 deletion with dark rearing from birth confirmed that an intermediate number of SPD receptors enable plasticity. Site-specific optimization of perisomatic GABA response may thus trigger experience-dependent development in visual cortex.     

Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity

Neural circuits are shaped by experience in early postnatal life. Distinct GABAergic connections within visual cortex determine the timing of the critical period for rewiring ocular dominance to establish visual acuity. We find that maturation of the parvalbumin (PV)-cell network that controls plasticity onset is regulated by a selective re-expression of the embryonic Otx2 homeoprotein. Visual experience promoted the accumulation of non-cell-autonomous Otx2 in PV-cells, and cortical infusion of exogenous Otx2 accelerated both PV-cell development and critical period timing. Conversely, conditional removal of Otx2 from non-PV cells or from the visual pathway abolished plasticity. Thus, the experience-dependent transfer of a homeoprotein may establish the physiological milieu for postnatal plasticity of a neural circuit.     

Experience-dependent plasticity in adult visual cortex

Experience-dependent plasticity is a prominent feature of the mammalian visual cortex. Although such neural changes are most evident during development, adult cortical circuits can be modified by a variety of manipulations, such as perceptual learning and visual deprivation. Elucidating the underlying mechanisms at the cellular and synaptic levels is an essential step in understanding neural plasticity in the mature animal. Although developmental and adult plasticity share many common features, notable differences may be attributed to developmental cortical changes at multiple levels. These range from shifts in the molecular profiles of cortical neurons to changes in the spatiotemporal dynamics of network activity. In this review, we will discuss recent progress and remaining challenges in understanding adult visual plasticity, focusing on the primary visual cortex.     

Critical periods during sensory development

Recent studies have made progress in characterizing the determinants of critical periods for experience-dependent plasticity. They highlight the role of neurotrophins, NMDA receptors and GABAergic inhibition. In particular, genetic manipulation of a single molecule, brain-derived neurotrophic factor (BDNF), has been shown to alter the timing of the critical period of plasticity in mouse visual cortex, establishing a causal relation between neurotrophin action, the development of visual function, and the duration of the critical period.     

Developmental regulation of spine and filopodial motility in primary visual cortex: reduced effects of activity and sensory deprivation

Dendritic protrusions are highly motile during postnatal development. Although spine morphological plasticity could be associated with synaptic plasticity, the function of rapid spine/filopodial motility is still unknown. To investigate the role of spine motility in the development of the visual cortex and its relation with critical periods, we used two-photon imaging of neurons from layers receiving visual input in developing mouse primary visual cortex and compared motility between control and visually deprived animals. Spine and filopodia motility was prominent during early synaptogenesis (P11-P13) but greatly decreased after P15. This "switch" was coincident with a 2.5-fold increase in protrusion density and spine formation. Spine motility was not regulated during the critical period for monocular deprivation (P19-P34). Moreover, delaying the critical period by dark rearing did not delay the normal developmental decrease of spine motility, but caused a modest further reduction in motility at P28-P35. Dark rearing and enucleation also mildly reduced spine motility before eye opening and dark rearing reduced the proportion of filopodia. We conclude that (1) rapid spine motility is not related to critical period plasticity, but is likely to play a role in early synaptogenesis, and (2) neuronal activity stimulates spine motility during synaptogenesis and promotes the appearance of dendritic filopodia.     

Otx2's incredible journey

A surprising new mechanism that regulates the plasticity of postnatal neurons is reported in this issue by Sugiyama et al. (2008). These authors show in mice that visual experience triggers cell-to-cell transfer of the homeoprotein Otx2 to cortical interneurons, where it promotes maturation of inhibitory neural circuitry and opens the critical period for plasticity in the visual cortex.     

Synaptotagmin-2 is a reliable marker for parvalbumin positive inhibitory boutons in the mouse visual cortex

Background

Inhibitory innervation by parvalbumin (PV) expressing interneurons has been implicated in the onset of the sensitive period of visual plasticity. Immunohistochemical analysis of the development and plasticity of these inhibitory inputs is difficult because PV expression is low in young animals and strongly influenced by neuronal activity. Moreover, the synaptic boutons that PV neurons form onto each other cannot be distinguished from the innervated cell bodies by immunostaining for this protein because it is present throughout the cells. These problems call for the availability of a synaptic, activity-independent marker for PV+ inhibitory boutons that is expressed before sensitive period onset. We investigated whether synaptotagmin-2 (Syt2) fulfills these properties in the visual cortex. Syt2 is a synaptic vesicle protein involved in fast Ca²⁺ dependent neurotransmitter release. Its mRNA expression follows a pattern similar to that of PV throughout the brain and is present in 30–40% of hippocampal PV expressing basket cells. Up to now, no quantitative analyses of Syt2 expression in the visual cortex have been carried out.

Methodology/Principal Findings

We used immunohistochemistry to analyze colocalization of Syt2 with multiple interneuron markers including vesicular GABA transporter VGAT, calbindin, calretinin, somatostatin and PV in the primary visual cortex of mice during development and after dark-rearing.

Conclusions/Significance

We show that in the adult visual cortex Syt2 is only found in inhibitory, VGAT positive boutons. Practically all Syt2 positive boutons also contain PV and vice versa. During development, Syt2 expression can be detected in synaptic boutons prior to PV and in contrast to PV expression, Syt2 is not down-regulated by dark-rearing. These properties of Syt2 make it an excellent marker for analyzing the development and plasticity of perisomatic inhibitory innervations onto both excitatory and inhibitory neurons in the visual cortex.     

CRE-mediated gene transcription in neocortical neuronal plasticity during the developmental critical period

Neuronal activity-dependent processes are believed to mediate the formation of synaptic connections during neocortical development, but the underlying intracellular mechanisms are not known. In the visual system, altering the pattern of visually driven neuronal activity by monocular deprivation induces cortical synaptic rearrangement during a postnatal developmental window, the critical period. Here, using transgenic mice carrying a CRE-lacZ reporter, we demonstrate that a calcium- and cAMP-regulated signaling pathway is activated following monocular deprivation. We find that monocular deprivation leads to an induction of CRE-mediated lacZ expression in the visual cortex preceding the onset of physiologic plasticity, and this induction is dramatically downregulated following the end of the critical period. These results suggest that CRE-dependent coordinate regulation of a network of genes may control physiologic plasticity during postnatal neocortical development.