UNIVERSITY OF PISA
PhD Course in Basic and Developmental Neuroscience (M-PSI/02)
MIR-132/212 DELETION
PREVENTS EFFECTS OF
BINOCULAR VISUAL
EXPERIENCE IN MOUSE VISUAL
CORTEX
Tutors: Candidate: Prof Tommaso Pizzorusso Mazziotti Raffaele
Prof. Strettoi Enrica
A B S T R A C T
MicroRNA-132/212 (miR-132/212) is an experience and cAMP response element-binding protein (CREB) depen-dent MicroRNA (miRNA) that acts in the central ner-vous system and in peripheral tissue regulating impor-tant biological processes, such as circadian clock, spine maturation and neural inflammation. Recently miR-132/212 has been involved in Ocular Dominance (OD) plasticity during the critical period in mouse visual cortex. We have studied OD plasticity and binocular matching in MicroRNA-132/212 Knockout (miR-132/212 KO) mice, where the genomic locus of miR-132/212 is completely deleted.
To examine the role of miR-132/212 in visual cortical function, we analyzed Local Field Potentials (LFP) re-sponses to pattern Visual Evoked Potential (VEP) and measured single units activity to drifting sine gratings, in Wild Type (WT) and miR-132/212 KOmice.
We found that the preferred orientations of individ-ual cortical cells are mismatched through the two eyes at a significant higher level in animals that lack of miR-132/212 and monocularly deprived mice, respect to aged-matched WT subjects. Furthermore, as seen be-fore, three days of Monocular Deprivation (MD) were not sufficient to induce OD shift during the critical pe-riod in miR-132/212 KO mice, assessed using pattern VEP responses.
These results suggest a possible role of miR-132/212 in trigger adaptive rewiring of neuronal circuits following visually driven patterned activity.
C O N T E N T S
i e x p e r i e n c e-dependent plasticity 1
1 i n t r o d u c t i o n 3
1.1 The Visual System 4
1.2 Ocular Dominance Plasticity 5
1.3 The Mouse as Animal Model for Visual System Studies 7
1.4 Exploring the molecular mechanisms of OD plasticity 9
1.4.1 Glutamate receptors: the NR2B/NR2A switch 9
1.4.2 Inhibitory circuits maturation 10
1.4.3 Neuroactive released proteins 11
1.4.4 Extracellular influences 13
1.4.5 Environmental influences 14
1.5 Signaling Pathways 15
2 r e c e p t i v e f i e l d s d e v e l o p m e n t 19
2.1 Receptive Field 19
2.2 Receptive Fields Properties and Visual Experience 19
2.3 Binocular Orientation Preference 20
3 m i c r o r na 1 3 2/212 25
3.1 MicroRNAs 25
3.1.1 miR212/132: biogenesis and func-tion 25 4 a i m s o f t h i s t h e s i s 29 ii e x p e r i e n c e d e p e n d e n t p l a s t i c i t y i n m i r 1 3 2/212 k o m i c e 31 5 m at e r i a l s a n d m e t h o d s 33 5.1 Subjects 33 5.2 Surgery 33 5.3 Physiology 34 5.4 Visual Stimulation 36 5.5 Data Analysis 36 v
6 r e s u lt s 39
7 d i s c u s s i o n 45
L I S T O F F I G U R E S
Figure 1 OD Results 40
Figure 2 RF Selectivity Results 42
Figure 3 Representatives orientation tuning curves from two animals:WT (up-per side) and miR-132/212 KO (bot-tom side). 43
Figure 4 BMOP Results 44
L I S T O F TA B L E S
Table 1 Subjects & Cells 39
A C R O N Y M S
miRNA MicroRNA
miR-132/212 MicroRNA-132/212 miR-132 MicroRNA-132
miR-212 MicroRNA-212
miR-132/212 KO MicroRNA-132/212 Knockout KO Knockout
OD Ocular Dominance
ODP Ocular Dominance Plasticity
LFP Local Field Potentials
VEP Visually Evoked Potentials
WT Wild Type
MD Monocular Deprivation
BMOP Binocular Matching of Orientation Preference
DR Dark Rearing
CNS Central Nervous System
RGCs Retinal Ganglion Cells
SC Superior Culliculus V1 Primary Visual Cortex
dLGN Dorsal-Lateral Geniculate Nucleus
NMDA N-methyl- D-aspartate
AMPA alfa-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid
Ca2+ Calcium Ions
GABA Gamma-Aminobutyric Acid GAD65 Glutamic Acid Decarboxylase 65
BDNF Brain Derived Neurotrophic Factor
NT3 neurotrophin-3 NT4 neurotrophin-4
EE Environmental Enrichment
NGF Nerve Growth Factor
TNFa Tumor Necrosis Factor Alpha
a c r o n y m s ix
ECM ExtraCellular Matrix
tPA Tissue-Type Plasminogen Activator
CSPGs Chondroitin-Sulfate Proteo-Glycans PNNs Perineuronal Nets
ERK extracellular signal regulated kinase 1,2
LTP Long Term Potentiation LTD Long Term Depression
PKA cAMPdependent protein kinase
CaMKII calcium-calmodulin kinase II
CREB cAMP response element-binding protein
TTX Tetrodotoxin
CPP
(R,S)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid
IGF-1 Insulin-like growth factor 1
BDNF-OE BDNF overexpression
HDAC Histone deacetylase
PCR Polymerase Chain Reaction
VEP Visual Evoked Potential
OSI Orientation Selectivity Index DSI Direction Selectivity Index
RF Receptive Field
ODI Ocular Dominance Index P Postnatal
FMRP fragile X mental retardation protein
FXS Fragile X syndrome
AD Alzheimer s disease
ASD Autism Spectrum Disorders
Part I
E X P E R I E N C E - D E P E N D E N T
P L A S T I C I T Y
1
I N T R O D U C T I O NOne of the most fascinating properties of the brain is its capacity to modify neural circuits in relation to past experience. The nervous system continuously interact with its environment in an alterable feedback loop that seems to have mainly adaptive purposes. What seems to happen is a precise combination of molecu-lar, structural and functional changes at synaptic level. This aspect is known as synaptic plasticity: the activity-dependent modification of synaptic efficacy, that seems to encode persistent informations about past experi-ence.
This appear to be true in particular during develop-ment, where proper nervous system function critically depends on the precise assembly and maintenance of an intricate synaptic network. Once the initial scaffold of neuronal connections has been laid down, refine-ment processes continue to sculpt and transform mi-crocircuits into a mature brain and spinal cord [76]. This refinement is at its full strength during the early postnatal development, in which the brain undertakes intense morphological and functional rearrangement in response to sensory experience, with visual, audi-tory and somatosensory systems displaying sensitive phases of enhanced plasticity that have been called “sensitive or critical periods”[9]. Critical periods are specific windows of opportunity during which experi-ence provides information that is essential for normal development and permanently alters performance[44]. Plastic modifications of synapses are detectable not only during development, but also in adulthood [46], neuronal architecture and function is more stable but it remains subject to changes as part of an adaptive re-sponse to learning, aging, injury, or disease [48].
over is increasingly evident that synaptic abnormalities are correlated with many human brain disorders, in-cluding schizophrenia, autism, and various forms of mental disability, therefore an understanding of the neural and molecular basis of this phenomenon would inform not only classroom and educational policy, but also drug design, clinical therapy and strategies for improved learning into adulthood[44].
Clear evidence for critical periods has been found in the primary sensory systems of several species[42]. Animal models are now revealing, with greater resolu-tion, the molecular, cellular and structural events that underlie experience-dependent circuit refinement. In the primary visual cortex, which has been the premier model of critical period plasticity for 50 years[128], the principal experience dependent changes in synaptic ef-ficacy are called Ocular Dominance (OD) plasticity and Binocular Matching of Orientation Preference (BMOP).
1.1 t h e v i s ua l s y s t e m
In the mammalian visual system, visual informations are processed in the retina (that generates neural sig-nals representing the image reaching the eye) and sent to different structures of the Central Nervous System (CNS) through Retinal Ganglion Cells (RGCs) axons, which represent the output of the retina. RGCs project to the visual centers of the brain that are located in the midbrain and in the thalamus. The pattern of retinal projections varies from species to species. In ro-dents, the vast majority of RGCsproject to the Superior Culliculus (SC) and the pretectal nuclei, with about 30% of them sending collaterals to the Dorsal-Lateral Genic-ulate Nucleus (dLGN) in the thalamus [25]. RGCs axons from each eye project to both sides of the brain, how-ever the major afferents to the SC anddLGN arise from the contralateral eye and only 5% of optic axons project ipsilaterally. Within the dLGNganglion cell axons are not intermixed; in cats, ferrets and primates they
ter-1.2 ocular dominance plasticity 5
minate in a set of separate, alternate and eye-specific layers (three layers in cats and six layers in primates) that are strictly monocular [45]. In rodents there is not a proper lamination of the dLGN ; however, ipsilat-eral and contralatipsilat-eral retinal fibers are segregated in a patchy fashion originating two eye-specific territories in the dLGN : the ipsilateral portion or inner core and the contralateral portion or outer shell. The Primary Visual Cortex (V1), located occipitally in the brain, con-sists of six layers of cells between the pial surface and the underlying white matter. The dLGN projects to the visual cortex via thalamo-cortical connections that ter-minate in the layer IV of V1. The cortex is first site within the visual system where visual input from the two eyes converges onto single neurons. In carnivores and primates, afferents from the dLGNsegregate by eye within the cortical layer IV into alternating, equal-sized stripes called ocular dominance columns [101, 128]. The binocular zone is smaller in rodents than in cat and primates and it is not organized in ocular domi-nance columns, but in a salt and pepper fashion [81]. Finally, It is important to remember that the neurons of the rodent visual cortex are predominantly responsive to the inputs deriving from the contralateral eye, due to the higher ratio of RGCs fibers crossing at the optic chiasm.
1.2 o c u l a r d o m i na n c e p l a s t i c i t y
The expression “critical period” in the context of mam-malian visual system was introduced for the first time by Wiesel and Hubel [128]. They described the physio-logical shift in responsiveness of neurons in the visual cortex of cat to light stimulation when one eye was deprived of vision early in life. This form of plasticity is strongly robust during a specific developmental age and diminishes once the cat becomes older [54]. Wiesel and Hubel proposed that there was a period of develop-ment when changes in the external visual environdevelop-ment
can alter pre-existing neuronal connections. Proper sen-sory experience during this critical period is essential for shaping neuronal circuits and for the maintenance of appropriate synaptic connections. Neuronal activ-ity evoked by sensory experience allows maturation of important properties of visual system, such as vi-sual acuity (the spatial resolution of the vivi-sual system) and orientation preference [122,29, 131, 63]. Manipu-lations of visual experience through monocular depri-vation (MD) cause structural, functional and molecu-lar changes in the neuronal network of visual cortex [11]. MD consists in the closure of one eye resulting in an imbalance of the inputs from the two eyes with the consequent synaptic reorganization of V1circuitry. This form of plasticity is called Ocular Dominance Plasticity (ODP), and it represents a classic paradigm to study how experience-dependent activity models neuronal connections. MD(performed through eye lid suture) causes a loss of visual responses in the deprived eye resulting from a rearrangements in the visual in-puts of the closed eye which causes an irreversible reduction of the ability of that eye to drive neuronal responses in the cortex; therefore neurons in the binoc-ular zone of the contralateral V1, previously dominated by the deprived eye, shift their responsiveness toward the ipsilateral open eye. In rodents, two/three days of MD cause an initial decrease in deprived eye responses, due to the weakening of closed eye connections and a reorganization of intracortical horizontal connections in the superficial layers (II/III) of the binocular portion of V1[115]. ProlongedMD (five/seven days) shows an increase in the neuronal responses to the inputs from the ipsilateral open eye [35]. Architectural changes in thalamo-cortical arborization, terminating in layer IV, are evident only much later, more than a month in the mouse [4].
Another form of artificial manipulation of visual ex-perience is Dark Rearing (DR), a form of visual manip-ulation consisting in the complete absence of sensory
1.3 the mouse as animal model for visual system studies 7
input. DR from birth, has deep effects on the cortex: visual connections do not consolidate, remaining plas-tic well after the closure of normal criplas-tical period and visual acuity does not reach the adult level. In contrast to the classic notion of a critical period for experience-dependent plasticity, several studies have recently re-ported that OD shifts in mice can also be induced in adulthood [46, 108]. Nevertheless, the binocular cor-tical representation is still more sensitive in juvenile mice, in adults ODshifts require longer MD durations and are generally smaller. It remains unknown whether substantial structural rearrangements that accompany functional OD shifts in juvenile animals also occur in the mature cortex during MD [46]. Therefore, ODP in binocular visual cortex is most pronounced in young animals, reduced but present in adolescence and ab-sent in fully mature animals older than 110 days of age [66].
1.3 t h e m o u s e a s a n i m a l m o d e l f o r v i s ua l s y s -t e m s t u d i e s
Understanding the neural basis of visual perception is a long-standing fundamental goal of neuroscience. Studies of cortical visual processing have typically con-ducted on carnivores or primates, which are consid-ered to have a more refined visual system, including a much larger cortical region for visual processing, higher acuity, extensive visual behaviors, orientation and ocular dominance columns. Despite the low visual acuity and relatively small region of cortex devoted to visual processing, neurons in mouse V1 show se-lectivity for stimulus parameters and typical response properties that are near to that found in other species [79]. There are also practical reasons for using mice. First, the past 5 years have seen a shift toward the widespread availability of transgenic mice, such as Cre-expressor lines, as well as molecular tools for in vivo circuit labeling and manipulation (e.g. viral
transfec-tion agents). A second reason is that the smaller overall size of the mouse nervous system can be used to gather data over a large spatial scale; for instance, the total area of the mouse primary and extrastriate visual cor-tex spans only several millimeters across the cortical surface [123], potentially allowing the entire system to be visualized simultaneously. A third, and less obvious (but nonetheless influential) reason why many labs are now using mice to study vision is that mice are safer, simpler and less expensive to maintain than are cats or primates.
1.4 exploring the molecular mechanisms of od plasticity 9
1.4 e x p l o r i n g t h e m o l e c u l a r m e c h a n i s m s o f o d p l a s t i c i t y
To date, ODP remains the best studied experimental model for experience-dependent refinement of neu-ronal circuits because of the ease of manipulating vi-sual experience independently in the two eyes. How-ever, a complete understanding of critical period plas-ticity requires linking the systems-level change in cir-cuit function with the molecular mechanisms that make circuit changes possible. The cellular and molecular mechanisms that control the developmental plasticity of visual cortical connections and restrict experience-dependent plasticity to short critical periods are still lacking. The principals molecular and environmental mechanisms, non-mutually exclusive are:
1.4.1 Glutamate receptors: the NR2B/NR2A switch Experience provide primarily patterned activity, a cor-related series of signals that contains not only infor-mations about the percept, but also about structural and temporal circuit’s feature. Plasticity is gated by the activation of N-methyl- D-aspartate (NMDA) recep-tors, which respond to excitatory synaptic transmission by enabling calcium (Ca2+) influx into target synapse and its neuron. NMDA receptors are both transmitter and voltage-dependent, and their coupling via Calcium Ions (Ca2+) influx to plasticity-related intracellular sig-naling, has led to the notion that they might be a neu-ral implementation of Hebbian synapses, because they behave as coincidence detectors [13]. Involvement of NMDA receptors in developmental visual cortical plas-ticity has been initially suggested by the observation that block of NMDA receptors inhibits the effects ofMD [100]. However, the receptor’s capacity to drive plastic-ity depends on its subunit composition: some receptors are built from “NR2B” subunits, which enable a high Ca2+ permeability and thus enhanced plasticity, and
some are composed by “NR2A” subunits, that have a reduced Ca2+ influx, thus resulting in shortening of NMDA current. NMDA receptors are developmentally regulated: their subunit composition varies in the vi-sual cortex, from a dominant presence of receptors con-taining the subunit 2B to a high presence of receptors containing the subunit 2A, with a time course parallel-ing that of functional visual cortical development and the critical period: as animals are exposed to visual experience, the NR2B/NR2A ratio declines [91], thus reducing the capacity for further plasticity and suggest-ing that the 2B-to-2A switch is related to visual cortical development [9]. However, the initial suggestion that the shortening of NMDAR currents during develop-ment, by a subunit change from NR2B to NR2A, closes the critical period [19], appear not strictly tied to ODP, indeed animals lacking NR2A show normal sensitivity to MD during critical period. This results suggests that expression of the 2A subunit is not essential to delin-eate the time course of the critical period for ODP [30] and might be related to other features of visual cortical plasticity, like BMOP, that is also blocked by infusion of a NMDAreceptor antagonists [120].
1.4.2 Inhibitory circuits maturation
The development of inhibitory circuitry in the visual cortex seems to play a crucial role in controlling the onset and time course of critical periods [43]. Mice lacking the synaptic isoform of Gamma-Aminobutyric Acid (GABA) producing enzyme, Glutamic Acid Decar-boxylase 65 (GAD65), show noODP, an impairment that can be rescued by intracortical infusion of the GABA -A receptor agonist diazepam, demonstrating that a decrease in inhibition effectively abolished critical pe-riod plasticity [41]. Furthermore, an early enhancement of GABA-mediated inhibition by benzodiazepin treat-ment triggers the precocious onset of ODP [28]. It was demonstrated that GABA transmission mediated by the
1.4 exploring the molecular mechanisms of od plasticity 11
GABA-A receptor containing the ’a1’ subunit is required for the induction of critical period plasticity [31]. More recent data suggest a fundamental role for parvalbu-min positive basket interneurons maturation in the onset of critical period ODP, due to the optimization of GABA-A receptors number on the soma-proximal dendritic compartment of pyramidal cells [59]. Brain Derived Neurotrophic Factor (BDNF), an activity de-pendent molecule, is implicated inGABAergic synapses formation. Transgenic mice with precocious BDNF ex-pression have a premature onset and closure of ODP correlated with a marked increase in perisomatic in-hibitory innervation in the visual cortex [51]. Further-more, the administration of the antidepressant fluox-etine reduces GABA content in the adult visual cortex and enhancesODP[72].ODP enhancement by fluoxetine was blocked by the administration of the GABA ago-nist benzodiazepines suggesting that fluoxetine effect was mediated by a reduction of GABA transmission. Recently has been demonstrated that a homeoprotein produced by the retina, OTX2, is transferred in the visual cortex in an activity-dependent fashion. Once in the cortex, OTX2, promotes critical period plasticity by triggering the maturation of GABAergic inhibition [106]. Furthermore the transplantation of embryonic inhibitory interneurons in the visual cortex of adult mice induces ODP. These findings suggest that ODP is regulated by the execution of a maturational program intrinsic to inhibitory neurons [105].
1.4.3 Neuroactive released proteins
Neurotrophic factors consisting of Nerve Growth Factor (NGF),BDNF, neurotrophin-3 (NT3) and neurotrophin-4 (NT4) and are part of Neurotrophin family, they sup-port neuronal survival and differentiation by binding to activating tyrosine kinase receptor of the TRK family. Neuronal activity increases the synthesis and secretion of neurotrophins, indeed they are also implicated in
ac-tivity dependent neuronal plasticity [50]. It was shown that exogenous supply of neurotrophins in the visual cortex strongly affects the ODPinduced by MD [73] and that these factors regulators of normal visual cortical development and plasticity [8].BDNF and its receptor trkB is widespread in the visual cortex. BDNF levels are regulated by visual stimulation through retinal activity [67]. BDNFincreasing after eye opening during postna-tal development is prevented by DR [18] and MD [67]. Infusion of BDNFinto the rats’ visual cortex blocks the physiological effect ofMD [68]; conversely, intracortical administration ofBDNF restores ODP in adult rats [72]. Mutant mice overexpressing BDNF in the visual cortex are characterized by a precocious maturation of the vi-sual system and an accelerated critical period plasticity [51]. These effects seem to be mediated by a precocious maturation of intracortical inhibition and suggest that BDNF may regulate the onset of plasticity influencing GABAergic interneuron circuits formation [9,51]. Sev-eral studies on neurotrophin receptors have indicated that different neurotrophins act on different neuronal targets. Therefore, the synergy between neurotrophins and activity has to be considered to be specific for each neurotrophic factor and the neuronal populations that are its targets [10]. Astrocytes are capable of releasing neuroactive molecules, such as Tumor Necrosis Factor Alpha (TNFa), and thus have the potential to be not only supportive but also signaling cells in the brain. A report shows that TNFa-mediated synaptic scaling is involved in ODP. After MD, neurons in the binocu-lar region of the visual cortex decrease their response to the closed eye and increase their responsiveness to the open eye. Using transgenic mice, this study sug-gests that the increase in the open eye response is a homeostatic process mediated by TNFa [57]. Given that astrocytes are a major source ofTNFa, that they respond to visual stimulation, and that they are able to secrete permissive factors for ODP, these results suggest that astrocytes have the potential to be key elements in the
1.4 exploring the molecular mechanisms of od plasticity 13
control of neuronal network function and plasticity in vivo , and add TNFa to the repertoire of released proteins involved inODP regulation.
1.4.4 Extracellular influences
Extracellular space, and in particular the ExtraCellular Matrix (ECM), has a role in controlling spine dynam-ics and visual cortical plasticity. During development, Chondroitin-Sulfate Proteo-Glycans (CSPGs) condense at high concentration in lattice-like structures, called Perineuronal Nets (PNNs), which completely surround visual cortical neurons. The process begins during late development and complete after the end of the critical period [11]. The degradation ofPNNs, injecting intracor-tically chondroitinase ABC, reopens ODPin the adult rat visual cortex [87]; furthermore the same treatment recover visual acuity and OD in adult amblyopic rats [88]. PNNs represents a mechanical obstacle to struc-tural and functional plasticity. The formation ofPNNsis triggered by neuronal production of cartilage link pro-tein Crtl1. Mice lacking Crtl1 have attenuatedPNNs, but the overall levels of CSPGsand their pattern of glycan sulphation are unchanged. Interestingly, Crtl1 KO ani-mals retain juvenile levels ofODPand their visual acuity remains sensitive to visual deprivation [20]. PNNs pref-erentially wrap inhibitory interneurons, albeit CSPGs -containing nets were found also around pyramidal neurons and their spines [11]. The inhibitory nature of the mature ECMcould be one of the factors at the basis of spines remarkable stability [11]. The extracellular protease Tissue-Type Plasminogen Activator (tPA) has been shown to be highly expressed at periods of maxi-mal plasticity [71] and plays a crucial permissive role in enabling circuit remodeling during critical period [70, 83]. The released tPA increases extracellular prote-olysis directly or by the activation of plasmin from the zymogen plasminogen. These proteases have a wide spectrum of targets, including CSPGs, growth factors,
neurotrophins etc. Past results demonstratedtPA pro-teolytic activity as a key regulator of dendritic spines dynamics in the visual cortex, through the generation of a permissive plastic environment enabling spines motility, protrusion or pruning [11]. Similarly, it has been demonstrated that fear memories in adult mice can be made susceptible to erasure via degradation of PNNs (Gogolla, et al., 2009). In conclusion, ECM pro-vides a form of “hard wiring” that can be dissolved to allow structural plasticity and to modulate circuits remodeling in the mature cortex.
1.4.5 Environmental influences
Experience is a strong determinant for the duration and of critical periods: total lack of experience usually prolongs critical periods and delays development of sensory functions. The clearest example of this come from studies showing that DR prolongs the critical pe-riod for ODP. Another approach to investigate the in-fluence of experience on the brain is to manipulate the pattern of environmental stimulation to which an-imals are exposed. Environmental Enrichment (EE) is an experimental protocol specifically devoted to in-vestigate the influence of environment on brain and behavior, showing that the morphology, chemistry and physiology of the brain can be artificially altered by modifying the quality and intensity of environmental stimulation [98]. Many studies have been performed showing that EE can elicit various plastic responses in the brain, ranging from molecular to anatomical and functional changes [80]. Rearing animals in EE has profound effects on the development of the nervous system, leading to an acceleration of visual system de-velopment at the behavioral, electrophysiological and molecular level [17,22]. It has been shown that EE pro-motes a complete recovery of visual acuity and ODin adult amblyopic animals [99].
1.5 signaling pathways 15
1.5 s i g na l i n g pat h way s
Electrophysiological changes are the results of intracel-lular molecular pathways activation that determines and gates the “dynamic status” of synapses, cells and circuits. Recent experiments identified protein kinases implicated in ODP.
• ERK/MAPK pathway. Electrical activity and neu-rotrophins are among the strongest activators of extracellular signal regulated kinase 1,2 (ERK) (also called p42/44 mitogen- activated protein kinase) [37,86]. The activation ofERKis required for white matter Long Term Potentiation (LTP) and for ODP in the visual cortex during the critical period .ERK is also implicated in activity-dependent plastic-ity as demonstrated by studies of learning and memory [2].ERK downstream targets include im-portant plasticity triggers such as CREB [55], Arc [130], and transcription factors that regulate the ex-pression of immediate early genes [129]. Therefore, ERK signaling cascade activation leads to the mod-ulation of activity of crucial plasticity molecules such as synaptic proteins and ion channels, thus promoting coherent integration of inputs between single neuron and networks.ERK phosphorylation is induced by activation of different signaling cas-cade. A well known upstream regulator ofERK is the protein Ras, that results extremely interesting for ODP . Indeed, a constitutively active form of H-ras (HrasG12V), expressed presynaptically at excitatory synapses in mice, accelerates and en-hances multiple, mechanistically distinct forms of plasticity in the developing visual cortex. In vivo, H-rasG12V increases the rate ofOD change in re-sponse to MD and accelerates the recovery from deprivation by reverse occlusion [58].
• cAMP-dependent protein kinase cascade. Pharmaco-logical block of cAMPdependent protein kinase
(PKA) inhibits theODshift induced by MD during the critical period [6]. Investigations exploring the connection ofPKA in Long Term Depression (LTD) and OD have also been used to study a possible role of LTD in ODP. Loss of one PKA regulatory subunit disruptsLTD, but notOD[41], while loss of a different subunit leavesLTD intact but disrupts ODP [92]. Alternatively, a study of the predomi-nant cortical regulatory subunit of PKAindicates that the subunit RII beta is required for OD plas-ticity and LTD, though LTP is not disrupted . The disparity in the cited results could be explained by the fact that differentPKA regulatory subunits are known to localize this enzyme to distinct sub-cellular domains and that the expression of these subunits may vary among the different types of cortical neurons [49].
• Calcium-Calmodulin kinase II signaling. Calcium en-try at synaptic sites leads to the activation of calcium-calmodulin kinase II (CaMKII). This kinase is spa-tially positioned at synaptic spines to directly cap-ture NMDA mediated calcium fluxes [5] and re-sponds by favoring the localization of AMPA recep-tors to synapse [39]. a-CaMKII has the interesting properties of autophosphorylation, which allows it to undergo long-term modification and activation. The process of autophosphotylation maintains a-CaMKII activation independently of intracellular Ca2+ concentration. In this way, the transient ac-tivation produced by the coincidence detection operated by NMDA receptors is converted into a longer-lasting molecular signal [9]. Genetic sup-pression of a-CaMKIIautophosphorylation, blocks the OD shift that normally follows MD [109]. In-triguingly, a-CaMKII seems to be critical for consoli-dation of synaptic plasticity without impacting the architecture of sensory cortex [36]. Interfering with PKA, ERKor CaMKII pathways causes the same out-come: the suppression of ODP during the critical
1.5 signaling pathways 17
period. This result is not surprising because of the crosstalk and complex overlapping interactions of these three signaling cascades, so that the block-ade of a single kinase reverberate on the entire network.
2
R E C E P T I V E F I E L D S D E V E L O P M E N T2.1 r e c e p t i v e f i e l d
Historically the term receptive field was originally coined by Sherrington (1906) to describe an area of the body surface where a stimulus elicits a reflex, then the concept was extended to sensory neurons defin-ing a restricted region of visual space-time where a stimulus could drive electrical responses. The receptive fields of striate neurons are well defined, restricted to small regions of space, and in the case of simple cells, highly structured [56]. Most neurons in acv1 respond to lines and are selective to orientation and direction of motion. Some neurons are sharply tuned to orientation and fail to respond to lines that are just a bit tilted from their preferred orientation while other cortical neurons are broadly tuned and respond to a broad range of orientations. The receptive field structure determines the selectivity of each neuron to line orientation and other parameters. The receptive fields of visual cortical neurons that receive direct input from the thalamus can be modeled with Gabor functions [56].
2.2 r e c e p t i v e f i e l d s p r o p e r t i e s a n d v i s ua l e x -p e r i e n c e
On the basis of the selective properties of neurons recorded in very young, visually inexperienced cats and neonatal monkeys, Hubel and Wiesel concluded that visual experience was not necessary for the for-mation of selective receptive fields or the organization of functional architecture, and therefore that ‘‘innate’’ mechanisms determine the organization of receptive fields and cortical columns[53]. Many neurons are
lective around the time of natural eye opening, but the responses are typically weaker than in older animals [125]. Binocular visual deprivation by DRor MD allows responses to become stronger and more selective for a few weeks as the animal matures [24], indicating that most neurons develop selectivity without visual experience. In contrast, blockade of cortical activity by infusion of Tetrodotoxin (TTX) prevents the maturation of orientation selectivity [21, 125]. The development of orientation selectivity and orientation columns thus appears to require neural activity in the cortex but is modestly influenced, if at all, by deprivation of visual experience before the beginning of the critical period for ocular dominance plasticity [27]. However, a recent study in mice provided evidence that the orientation selectivity of some neurons may be altered by rear-ing mice with astigmatic lenses that focus a limited range of orientations; while a loss of responsive neu-rons in the upper half of layer 2/3 could account for the overrepresentation of the experienced orientation there, it could not account for the effects in the lower half [63]. Many neurons in V1 are direction selective as well as orientation selective, but the development and plasticity of direction selectivity is different. Direction selectivity in retinal ganglion cells makes the study of its cortical organization and development difficult [27]. In mice, direction as well as orientation selective neurons were present at eye opening and developed normally even when animals were reared in darkness [96].
2.3 b i n o c u l a r o r i e n tat i o n p r e f e r e n c e
The necessity of experience in neural systems devel-opment is often studied by depriving or manipulating sensory experiences. In the visual system, for example, following a period of MD in juvenile animals, cortical neurons lose their responses to the deprived eye and become more responsive to the non-deprived eye [128].
2.3 binocular orientation preference 21
Decades of studies have made ODPand its critical pe-riod a classical model of experience dependent neural development [42]. ODP is only induced by an imbal-ance of inputs from the two eyes, a condition that does not exist in normal visual system development. In fact, the degree of cortical OD does not change during the critical period unless the system is manipulated experi-mentally [100]. In other words, while the critical period marks a time window of increased cortical plasticity during development, functional cortical changes that normally take place during this time window are not known [120]. Two major transformations occur when visual information reaches the cortex. In addition to binocularity, cortical cells are also selective for stimulus orientation [34,52]. Binocular cells in the cortex must then match their orientation tuning through the two eyes in order, for the animal, to perceive coherently. Indeed, in cats and primates, the preferred orienta-tions of cortical neurons are similar through the two eyes [52, 15]. Activity-dependent changes induced by normal visual experience during the critical period serve to match eye-specific inputs in the cortex [120]. Recently, Wang, et al [120], measured through single-unit recordings that mice in adulthood show, like other mammals, that orientation tuning properties of each neuron, determined separately for each eye in response to drifting sinusoidal gratings, were tuned to nearly identical orientations through the two eyes. These re-sults indicate that the orientation preference of indi-vidual cortical neurons is closely matched between the two eyes, consistent with studies in cats and primates. Although the orientation preferences are well matched binocularly in adult animals, they found also that they are mismatched early during postnatal development (at the onset of the critical period for OD P20–P23). Many cells were tuned to quite different orientations through the two eyes, indicating that orientation pref-erence is significantly mismatched binocularly in the young mice. The mismatch decreases with age and
reaches adult levels by P31–P36 (at the offset of the critical period for OD). To determine if BMOP requires normal visual experience, they used two types of ma-nipulations: DRandMD. A short period of DRduring the critical period deprives the animal of any visual input, which is known to have no effect on OD. In con-trast, 7 days MD alters the balance of input between the two eyes and induces OD plasticity. Both manip-ulations blocked the developmental decrease of ΔO
(the absolute difference between preferred orientation of each eye). Therefore, normal visual experience be-tween P24–P31 is required for the BMOP. To further investigate on activity required for the correct develop-ment of BMOPthey used a competitive NMDAreceptor antagonist, (R,S)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). The NMDAreceptor is known as a coincidence detector in synaptic plasticity [13] and binocular matching process is presumably driven by the correlated neuronal activity between the eye spe-cific inputs to individual cortical neurons. Mice treated with CPP show, at P31–33, ΔO close to those of P20–23
mice, but intact selectivity parameters. They demon-strated also thatDR, as for the critical period ofODP de-laysBMOP. Animals recorded soon afterDRhave a high
ΔO, whereas 6–7 days of vision afterDRwas enough to
decrease ΔO to normal adult levels. In other words, the
critical period of the matching process is delayed by DR from birth, like that ofODPplasticity, leaving selectivity parameters like controls. They conclude that both ODP and BMOP take place during a similar period of critical time in early life, important conceptual differences ex-ist between them:ODP is a visual manipulation-induced plasticity, where cells lose responses to the deprived eye as an adaptation to pathological conditions, in contrast, BMOPis a normal vision-induced plasticity and leads to a beneficial outcome, where cortical cells match their ori-entation preference to the two eyes. Changes in ODare induced only by an imbalance between eye-specific in-puts, such as inMDor strabismus, rather than by visual
2.3 binocular orientation preference 23
manipulations that deprive both eyes, such asDRand binocular deprivation [36], in contrastBMOPoccurs dur-ing normal development and is blocked by any visual manipulation that disrupts binocular vision. Mechanis-tically, BMOPandODP should also be different. ODPis a competitive process wherein eye-specific inputs ‘‘fight’’ for postsynaptic targets based on their activity levels [127]. Hebbian plasticity, such asLTPandLTD[102], and homeostatic plasticity [57,77] have been suggested to mediate components of ODP.BMOP, on the other hand, is presumably mediated by correlation-based processes wherein the precise patterns of input activity instruct changes in eye-specific synaptic connections. Accord-ing to the feed forward model proposed originally by Hubel and Wiesel (1962)[52], orientation selectivity arises from specific arrangement of geniculate inputs, and the preferred orientation of individual cortical cells is determined by the layout of the elongated ON and OFF subregions in their receptive field [93,33]. An alter-native series of models, the feedback models, propose that orientation selectivity is an emergent property of intracortical circuitry [104, 7, 3]. In this case, binocu-lar matching should be mediated binocu-largely by synaptic changes of intracortical connections. Regardless of the exact mechanisms of orientation selectivity, however, changes in synaptic connections must be under the precise and delicate control of the synchronized inputs from the two eyes evoked by normal visual experience. In another study, Wang, et al.(2013) [121] examined whether and how altering critical period timing affects normal visual development. In particular, in animals with precocious critical period due to accelerated inhi-bition maturation in BDNF overexpression (BDNF-OE) mice , binocular matching process does not start earlier and fails to reach adult level,resulting in permanent mismatch in complex cells. This suggest that additional factors are needed to initiate the matching process, pre-sumably due to the precocious closure of cortical plas-ticity. They also investigated the role of environmental
factor such as EE, finding thatBDNF-OE mice exposed to EE advance BMOP, indicating that EE during early postnatal development must trigger certain factors to initiate binocular matching. They point to the role of Insulin-like growth factor 1 (IGF-1) that increases in EE conditions inducing histone modifications in the de-veloping visual cortex. Administration of an Histone deacetylase (HDAC) inhibitor, indeed, mimics EE’s ef-fects on binocular matching in both WT andBDNF-OE mice. Histone acetylation is an epigenetic mechanism of gene regulation and has been implicated in synap-tic plassynap-ticity in adult learning and memory [107, 84]. For example, histone acetylation is important for the expression of CREB-regulated genes [32, 117]. Because some of these genes are regulated by visual experience during the critical period [90,16], EE-induced acetyla-tion of histone H4 would activate potential ‘‘plasticity genes’’ responsible for binocular matching [121]. These discoveries give additional support to the notion that there are multiple critical periods in visual system de-velopment [30], these periods are temporally organized in a hierarchical manner, wherein the neural circuits in the earlier stages of visual processing mature sooner [121].
3
M I C R O R N A 1 3 2 / 2 1 23.1 m i c r o r na s
miRNAs are endogenous evolutionarily conserved short non-coding RNAs that interact with specific target mR-NAs based on sequence imperfect complementarity in the 3’ untranslated region (UTR) of the target mR-NAs, referred to as the miRNA ‘seed’ region, resulting in translational repression or mRNA deadenylation and degradation [112]. Recently, a flurry of papers reported important physiological roles of miR-132/212 family. Moreover, miRNAs could also positively regu-late gene expression by enhancing mRNA translation and inducing gene expression via target gene promoter binding [116, 89], thereby adding more complexity to their initially described mode of action [119].miRNAs are believed to control the expression of one to two third of human genes, which explains their involve-ment in most, if not all, physiological processes as well as their association with numerous diseases when their expression is deregulated[61].
3.1.1 miR212/132: biogenesis and function
The miR-132/212 gene has an equivalent structure in all species wherein the two miRNAs genes are arrayed in tandem on chromosome 10 in rat, 11 in mouse and 17 in humans. The miR-132/212 gene cluster produces four mature miRNAs: MicroRNA-132 (miR-132),miR-132* (or star), MicroRNA-212 (miR-212) andmiR-212*. In humans miR-212* is not found possibly because it is quickly degraded. In primary cortical neurons miR-132 is ex-pressed at much higher levels thanmiR-132*, whereas miR-212 and miR-212* have similar expression levels,
beit much lower than miR-132 [94].miR-132 expression is enriched in the brain, while miR-212expression is very low in all tissues tested [94, 118]. Studies performed in neurons demonstrated that miR-132andmiR-212are CREB regulated miRNAs [118, 47, 94]. Mature miR-212 and miR-132 follow the canonical pathway of miRNA biogenesis: into the nucleus, the pri-miRNA is cleaved by the RNAse III family enzyme Drosha in a ~70 nu-cleotides precursor hairpin RNA (pre-miRNA) and is exported to the cytoplasm. Here, Dicer (a RNAse III enzyme) processes the pre-miRNA in a 20 bp miRNA duplex. One strand of the duplex, the mature miRNA, is loaded into the miRNA induced silencing complex (RISC), whereas the star strand (also called passenger strand) is degraded [64].
Overexpression of miR-132 in neuronal cultures in-duced a marked increase in primary neurite outgrowth [118]. Synaptic activity rapidly inducedCREB-dependent miR-132 expression and its induction was required for activity-dependent dendritic growth in vitro. These ef-fects on dendrite morphology were mediated bymiR-132 translational inhibition of its target protein p250GAP, a Rho family GTPase activating protein, resulting in increased Rac signalling cascade activity [124]. Condi-tional knock down ofmiR-132/212locus in vivo caused a significant decrease in total dendritic length, arboriza-tion and spine density in newborn hippocampal neu-rons of adult mice [69]. Considering the much lower expression of miR-212 with respect to miR-132, the au-thors suggested that miR-132 is the predominant regu-lator of this morphological plasticity. Transgenic mice overexpressing miR-132 in forebrain neurons showed a marked increase in dendritic spine density and im-pairments in a novel object recognition memory test [38]. The pharmacological blockade ofNMDA receptors enhanced the LTP-dependent induction of these miRNAs whereas the selective blockade of mGlur1 receptor in-hibited the enhancement of mature miRNAs expression in response to LTP-inducing stimuli [126]. These data
3.1 micrornas 27
suggest that different signaling pathways might specif-ically regulate miR-132 and miR-212 expression by act-ing at transcriptional and possibly at biogenesis or decay level. Recent studies for the first time provide evidence for the role ofmiR-132 as mediator of plasticity of neuronal responses of developmental experience-dependent plasticity [74, 113]. In these papers, visual experience was shown to dynamically regulate miR-132levels in the visual cortex. Moreover, Tognini et al. [113] observed that visual experience results in histone methylation and phosphoacetylation on a CRE locus important for miR-132/212 gene cluster transcription [94], suggesting that histone posttranslational modi-fications play an important role in visual experience regulation of miR-132 levels. Strikingly, counteracting miR-132 downregulation through the infusion of chemi-cally modified miRNA mimic in the cortex, or reducing miR-132 through injection of a lentivirus expressing a miR-132-sequestering sponge, completely blocked OD plasticity in MD mice. These data show that an opti-mal concentration of miR-132 is necessary for plasticity during the critical period. Interestingly, miR-132mimic treatment increased the fraction of mushroom stubby dendritic spines representing the mature, more stable form of spines [113]. Conversely, miR-132 inhibition re-sulted in more immature spine, mainly increasing the percentage of filopodia [74]. Taken together, these two results suggest that the miR-132 decrease induced by MD could be necessary to make dendritic spines less stable, allowing the occurrence of the structural plas-ticity mechanisms underlying OD plasticity, whereas the strong miR-132reduction obtained by viral transduc-tion would make spines too unstable to consolidate OD plasticity.
4
A I M S O F T H I S T H E S I SRecent data indicate that the miR-132, that is induced in the visual cortex of light-exposed mice, would regu-late the ocular dominance plasticity possibly through action on dendritic spine morphology. Both Mellios and Tognini (2011) [113, 74] demonstrated that per-turbations of miR-132 concentrations, during develop-ment, are not permissive for synaptic reorganization due to imbalance in structured eyes stimulation. This seems to be related with some role of miR-132 in the transduction about effects of sensory experience at cortical level. Both reduction and enhancement of miR-132 are able to block ODP thus preventing the effects of MD. Given these evidences, in my PhD experience, I’ve investigated the role of miR-132/212 during normal and abnormal sensory development, through a rela-tively new electrophysiological approach. Using silicon probes and multichannel electrophysiologic devices, I’ve tryied to characterize the sensory development, studying how receptive fields and ODdevelop, in trans-genic mice that constitutively lack of miR-132/212, both in binocular and inMDconditions. The rationale for this work is that: two major transformations occur when visual information reaches the cortex, in addition to binocularity, cortical cells are also selective for stimulus orientation [34,52]. Binocular cells in the cortex must then match their orientation tuning through the two eyes in order for the animal to perceive coherently [120]. The heightened plasticity during the critical period al-lows visual experience to drive the binocular matching of orientation preference during normal development. So we expect to find deficits in BMOP in these animals also in absence of visual experience manipulations and low or effects on monocular Receptive Field (RF)
erties. We hypothesize that miR-132acts as a experience transducer in V1.
Part II
E X P E R I E N C E D E P E N D E N T
P L A S T I C I T Y I N M I R 1 3 2 / 2 1 2 K O
5
M AT E R I A L S A N D M E T H O D S5.1 s u b j e c t s
miR-132/212 knockout mice were generated using the strategy described in Remenyi, et al. (2013) [95] in which the targeting vector was designed to introduce LoxP sites either side of the region encoding miR-132 and miR-212. A neomycin resistance cassette was in-cluded for positive selection and a TK cassette for negative selection. The sequence of this vector is pro-vided as supplemental data. Targeting was carried out in ES cells derived from C57Bl/6N mice using stan-dard protocols. Correctly targeted clones were identi-fied by Southern blotting of Kpn I digested genomic DNA using a probe external to the targeting vector. Tar-geted ES cells were injected into blastocysts to generate chimeric mice. Germline transmitting chimeric mice were crossed to Flpe transgenic mice (also on a C57Bl/6 background) to delete the neomycin cassette, resulting in mice with a conditional allele for miR-132 miR-212. The deletion of miR-132 and miR-212 was achieved by crossing these mice to transgenic mice expressing Cre recombinase under a constitutive promoter (Taconis Artemis), following deletion mice were crossed away from the Cre transgene before experimental mice were generated. Routine genotyping of the mice was car-ried out by Polymerase Chain Reaction (PCR) using tail biopsy tissue . Each comparison were performed with WTlittermates .
5.2 s u r g e r y
Recordings were made from WT and miR-132/212 KO mice at P28-P30 of age. For surgery, mice were sedated
with isoflurane and then anesthetized with urethane (0.75 ml/100g, i.p., at 20% in saline). Additionally, dex-amethasone (2 mg/kg) were administered subcuta-neously to reduce secretions and edema. The animal was maintained at 37°C by a feedback-controlled heat-ing pad. After retractheat-ing the scalp, we performed a small craniotomy, 1 mm in diameter, and nicked a slit in the dura to allow insertion of the multisite elec-trode. The electrode was lowered into the brain to an appropriate depth and was allowed to settle for 20-30 min before the beginning of recording. The eyes were rinsed with saline and a thin layer of silicone oil was applied to prevent drying while allowing clear optical transmission.
5.3 p h y s i o l o g y
Recordings were made with silicon microprobes from NeuroNexus Technologies. Two configurations were used: a linear probe with 16 sites spaced at 100μm
inter-vals (model a1x16-3mm-100-177), which could be used to span across multiple layers of cortex; and a tetrode configuration, with four tetrode clusters, each consist-ing of four sites separated by 25 μm a side (model
a2x2-tet-3mm-150-121), which was used primarily to provide better isolation of units in layers 4 and 5. The shanks of the probes were 15 μm thick and 3 mm long,
with a maximum width at the top of the shank of 94
μm (tetrode) or 123 μm (linear). Signals were acquired
using Cheetah 5 (Neuralynx) and analyzed with cus-tom software in Matlab (MathWorks). For local field potential (LFP) recording, the extracellular signal was filtered from 1 to 275 Hz and sampled at 33 kHz. Cur-rent source density (CSD) was computed from the average LFP by taking the discrete second derivative across the electrode sites and interpolated to produce a smooth CSD map.
For single-unit recording, the extracellular signal was filtered from 0.6 to 6 kHz and sampled at 33 kHz.
Spik-5.3 physiology 35
ing events were detected on-line by voltage threshold crossing, and a 1 ms waveform sample was acquired around the time of threshold crossing. To improve isolation of single units, recordings from groups of four neighboring sites were linked, so that a waveform was acquired on all four sites in response to a thresh-old crossing on any of the four (Tetrode). Tetrododic acquisition had two primary benefits: improved dis-criminability when a waveform appeared on more than one site, and commonmode noise rejection of signals shared on all four sites. Whereas the larger amplitude were sometimes recorded simultaneously on adjacent sites at the 50 μm spacing of the linear electrode,
neu-rons often appeared predominantly on one site, even at the 25 μm spacing of the tetrode configuration. In
both cases, many units had signals on nondominant sites that were below the voltage trigger threshold; however, the simultaneous acquisition allowed this low amplitude information to be integrated to improve dis-criminability [79]. The individual waveform samples were aligned by their most negative time point. To iden-tify single units, the spike waveforms from the four sites together were loaded in “OffLine Sorter” (Plexon), then clusters were identified using a semiautomated procedure. First, cross channel artifacts are removed. Second, spikes are visually inspected for anomalous waveforms (overlapping spikes, saturated spikes, arti-facts... ), discovered with principal component analysis were rejectet from subsequent analysis. Then wave-forms are processed using valley-seeking algorithm to find clusters, in the final stage every cluster is manu-ally checked to verify the validity of clusterization. As measere of goodness of clusterization we used Dunn’s metric.
5.4 v i s ua l s t i m u l at i o n VEP
VEP response to square wave vertical patterns, with abrupt phase inversion, were used to measure ODP (as the amplitude ratio, in time domain, between con-tralateral versus ipsilateral stimulation). We used com-puter controlled mechanical shutters (LEGO MINDOS-TORMS) to collect data from each eye, this reduced possible effects due to changes inbehavioral states, and adaptation.
Single-Units
A challenge involved in our approach of unbiased recording from a number of neurons simultaneously is that stimuli cannot be tailored to individual neurons. So we used random series of stimuli that provide a good mean to describe response tuning curves of most of visually driven neurons.
Stimuli were generated in Matlab using the Psy-chophysics Toolbox extensions [14,85] and displayed with gamma correction on a monitor (Sony Trinitron G500, 60 Hz refresh rate, 32 cd/m2 mean luminance) placed 20 cm from the mouse, subtending 60-75° of visual space. Episodic stimuli were repeated five times, with stimulus conditions randomly interleaved, and a gray blank condition (mean luminance) was included in all stimulus sets to estimate the spontaneous firing rate. Episodic stimuli included drifting sinusoidal grat-ings [1.5 s duration, temporal frequency of 2 Hz, 12 directions, spatial frequency of 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, and 0 cyc/deg (cpd).
5.5 d ata a na ly s i s
The average spontaneous rate for each unit was cal-culated by averaging the rate over all blank condition presentations. For drifting gratings, responses at each
5.5 data analysis 37
orientation and spatial frequency were calculated by averaging the spike rate during the 1.5 s presentation and subtracting the spontaneous rate. The preferred orientation was determined by averaging the response across all spatial frequencies and finding the peak. The orientation tuning curve was constructed for the spa-tial frequency that gave peak response at this orienta-tion. Given this fixed preferred orientation θ-pref, the
tuning curve was fitted as the sum of two Gaussians centered onθpref and θpref+π , of different amplitudes
A1 and A2 but equal width, with a constant baseline B. From this fit, we calculated: Orientation Selectivity Index (OSI) representing the ratio of the tuned versus untuned component of the response, and the width of the tuned component. OSI was calculated as the depth of modulation from the preferred orientation to its orthogonal orientation θortho= θpref+ π/2, as
(Rpref-Rortho)/(Rpref+Rortho). Tuning width was the halfwidth at half-maximum (HWHM) of the fit above the baseline, Rortho. In addition, Direction Selectivity Index (DSI) was calculated from the fitted function as (Rpref-Ropposite)/(Rpref+Ropposite). Statistical signif-icance was determined by Kolmogorov-Smirnov , ex-cept when otherwise stated. Error bars represent mean ± SEM (*pval < 0.05,**pval < 0.01, ***pval < 0.001).
6
R E S U LT SMirKO mice do not shift Ocular Dominance in function to visual experience
We have first examined if deletion of miR-132/212 locus from birth is able to interfere with plastic changes due to manipulation of visual experience. So we collected VEP responses to contrast reversal sine gratings from LFP activity and single unit responses to drifting sine gratings in animals, normal reared and monocularly deprived for 3 days. At VEPlevel, we found, coherently with literature [113, 74], that 3 days of MD do not pro-duce significant plastic changes inOD (Fig.1a), so these
animals do not show plastic susceptibility to visual experience and these data seem to be in favour of an abnormal or absent critical period. To complete and extend this analysis we examined responses at a cel-lular level through recording of single unit responses between the two eyes. What we have found is that Ocular Dominance Index (ODI) inWTand miR-132/212 KO mice, normal reared, is not significantly different (Fig.1b) and that 3 days of monocular deprivation in
miR-132/212 KO are not sufficient to shift or alter
binocu-g r o u p s s u b j e c t s c e l l s
WT 6 132
miR-132/212 KO 7 186
WT-MD 5 119
miR-132/212 KO-MD 5 169
Table 1: Data Summary
lar response magnitude (Fig.1c). So we can conclude
that plastic dynamic regulation of binocularity, seems to be impeared in these animals, at single unit and network level.
(a) (b)
(c) (d)
Figure 1: 1a: Ratios between contra and ipsi amplitude of
VEPresponses in time domain. Three days ofMD are sufficient to shiftODinWTmice (pval=>0.001), but do not alter the balance of OD in miR-132/212 KO mice (pval=>0.7). 1b: OD-score of responsive
single unit collected inWTandmiR-132/212 KOmice (pval=>0.127).1c: Comparison betweenOD-score
collected inWTmice in binocular vision and with 3 days of MD. There is a significant reduction of contralateral bias in animals with 3 days of MD (pval=>0.001).1d: The same comparison as in1c,
but with 3 days ofMDinmiR-132/212 KOmice. No significant changes are detected (pval=0.66).
r e s u lt s 41
MiR-132/212 KO mice have no impairements in orientation and direction selectivity
Given this aberration of plastic responses applying manipulation on the visual sistem in miR-132/212 KO mice, we tried to examine in depth, what happen to RF in absence of miR-132/212 during normal binocular development. We analyzed RF from these animals, be-tween P28-P30, at the end of the critical period for ODP through single-unit recordings made with tetrodes in the binocular zone of V1. The orientation tuning prop-erties of each neuron were determined separately for each eye in response to drifting sinusoidal gratings of varying orientations and spatial frequency. Figure ??
shows a representative orientation tuning curves of WT and miR-132/212 KO subjects, fitted on responses profile to sine gratings stimulation.
The results show that the vast majority of binocular neurons in adult mice are orientation selective, in fact, 87% of the cells had an OSI greater than 0.33 for both eyes and 64% had an OSI greater than 0.5 in WT simi-larly to that of miR-132/212 KO, in which 81% are greater than 0.33 and 58% greater than 0.5. Moreover the mean OSI was equally high through the two eyes (contralat-eral: 0.67 ± 0.02, ipsilat(contralat-eral: 0.69 ± 0.02 inWT,p=0.49 and contralateral: 0.65 ± 0.02, ipsilateral: 0.65 ± 0.02 in miR-132/212 KO,p=.42) and between groups (contralateral pval:0.22 ipsilateral pval:0.35). These values are similar to those in recent reports of monocular and binocular neurons in adult mice [120, 79]. We used also the tun-ing width as another measure of selectivity, findtun-ing no differences between eyes in WT (contralateral: 40.2 deg ± 1.97 deg and ipsilateral: 37 deg ± 1.95deg, pval=0.23), miR-132/212 KO (contralateral: 35.2 deg ± 1.5 deg and ipsilateral: 36 deg ± 1.95deg, pval=0.61) and between groups (contralateral pval: 0.13 ,ipsilateral pval: .47 ).
We also calculated the DSI, inWT (contralateral: 0.36 ± 0.02 and ipsilateral:0.38 ± 0.02, pval=0.91) and miR-132/212 KO (contralateral: 0.34 ± 0.02 and ipsilateral:
(a) (b)
(c)
Figure 2: OSI[2a] , tuning Width[2b] andDSI[2c] in WTand
miR-132/212 KO
0.36 ± 0.02, pval=0.61), detecting no differences be-tween groups (contralateral p-val: 0.44 ipsilateral p-val: 0.42). Given this results, RFin animals that lack of miR-132/212 develops normally with comparable monocular selectivity charachteristics.
Orientation Tunings of Cortical Neurons Are Mismatched Binocularly in MiR-132/212 KO Mice
AdultWT shows orientation selectivity tuning that is matched between the two eyes, these alignment is the result of refinement processes that occur during de-velopment in coincidence with patterned binocular visual stimulation. In line with the recently discovered BMOP, we asked whether plasticity processes that oc-cur during normal binocular matching are influenced by absence of miR-132/212. So we used drifting sine grating stimulation between the two eyes to measure the degree of mismatching of contralateral and
ipsilat-r e s u lt s 43
Figure 3: Representatives orientation tuning curves from two animals: WT(upper side) and miR-132/212 KO (bottom side).
eral RF, first determining the preferred orientation of RF of each eye and then comparing the absolute dis-tance between preferred orientations. We found that WT andmiR-132/212 KO mice significantly differ in this parameter. The mean alignment ofmiR-132/212 KO mice is greater than that of WT, and they have a degree of mismatching that is not significantly different from that of WT or miR-132/212 KOmonocularly deprived for three days (Fig.4e). This results is in favour with the
hypothesis that the presence of miR-132/212is a molecu-lar bottleneck that act as an experience transducer and drive functional and anatomical changes necessary for environment correct perception and adaptation.
(c) (d)
(e) (f)
(g)
Figure 4: 4a: OSI matching between eyes (correlation
coef-ficient r = 0.56 and p < 0.0001). 4b: alignment of
orientation preference (r=0.78 and p < 0.0001) in WT.4c: Distribution of distances between preferred
orientation of each eye in WT and miR-132/212 KO [4d] mice. 4e: Mean BMOP in all groups. 4f:
Em-pirical cumulative distribution function of each group. ??: Venn diagrams comparing differentially expressed transcripts betweenmiR-132/212 KOand WT-MD groups from TopHat analyses. 4g : time
superposition between expression ofmiR-132 and critical period forBMOP. Red line represents stan-dard reared animals, blue line Dark reared animals. Adapted from [27, 114]
7
D I S C U S S I O NIn this study, we have shown that ODPand BMOP are influenced by miR-132/212 presence. In fact, both these experience dependent plastic phenomena of cortical circuitry seem to be under some permissive control of this miRNA. Accordingly,MD is not effective in trigger adaptive functional rearrangement at both large scale and cellular level in mice miR-132/212 KO. These data suggest that miR-132/212 KO is a crucial molecular trans-ducer of the action of visual experience on plasticity, in developing visual cortical circuits. MicroRNA (miRNA)s are a recently discovered, extensive class of small non-coding RNAs that act as post-transcriptional regulators of genes and represent good candidates in the field of specific and local control of gene expression pat-tern. miR-132/212 is an activity dependent miRNA and in vitro studies showed that miR-132/212 is induced by synaptic activation, such as BDNFor bicuculline treat-ment, through induction of ERK-MSK pathway and CREB [94, 124,118]. It was noted also that visual stim-ulation itself can enhance miR-132/212levels, in fact as demonstrated by Tognini, et al. (2011)[113], DR from birth dramatically alter visual system maturation and miR-132/212 concentrations. In normal reared animals, around Postnatal (P)15 there is a developmental in-crease in miR-132/212, in coincidence with eyes opening, in agreement with activity-dependent expression of this miRNA. Interestingly, maximal expression is at P25 -30during the peak of critical period forODP. Before the opening of the critical period, synapses are immature and highly dynamic filopodia represent the prevalent form of spines. In this time-windowmiR-132/212 expres-sion is very low. We could speculate that miR-132/212 increase after P15-P20is implicated in the processes
derlying synaptic stabilization and pruning occurring at maximal level during the critical period. Conversely, in dark reared animals the levels of primary and ma-ture miR-132/212 remain very low, without any develop-mental change in expression possibly contributing to the abnormal maturation of visual cortical circuits in DR animals. In another work, Mellios,et al. (2011)[74] show that an optimal concentration of miR-132/212 is necessary for plasticity during the critical period. Inter-estingly, miR-132-mimic treatment increased the fraction of mushroom/stubby dendritic spines representing the mature, more stable form of spines [113]. Con-versely, miR-132inhibition resulted in more immature spine, mainly increasing the percentage of filopodia [74]. Taken together, these two results suggest that the miR-132/212 decrease induced by MD could be neces-sary to make dendritic spines less stable, allowing the occurrence of the structural plasticity mechanisms un-derlyingODP, whereas the strongmiR-132/212reduction obtained by viral transduction would make spines too unstable to consolidate ODP.
An important question is: which target proteins me-diate the effect of miR-132/212on ODP? In the central ner-vous system the best candidates proteins are Methyl CpG Binding Protein 2 (MeCP2) and p250GAP.
MeCP2is a DNA methyl binding protein implicated in transcriptional regulation, and its gene mutations cause Rett Syndrome, a pervasive developmental dis-order characterized by dendritic spines abnormalities. P250GAP is a Rho family GTPase activating protein. Previously, It was demonstrated that p250GAP is a bona fide miR-132 target [118, 124] and it was shown that this protein is enriched in the post-synaptic den-sity [78]. P250GAP interacts with multiple synaptic pro-teins that are effectors of synaptic plasticity including the NMDA NR2B receptor subunit, the scaffold protein PSD-95 [78], the NR2B kinase Fyn[111], and the signal-ing intermediate beta-catenin [82]. P250GAP activity is also regulated by CaM kinase II phosphorylation and
d i s c u s s i o n 47
its localization at the post-synaptic density may be reg-ulated by NMDA receptor signaling [78]. Therefore, we can speculate that regulation of p250GAP expression could be a mechanism by which miR-132/212 modulates structural plasticity at the synapse, in particular dy-namic changes in spines associated with ODP. Recently, a genetic cell-type based analysis of miRNA profiles in the mouse brain demonstrated that miR-132/212 is predominantly expressed in excitatory neurons and in somatostatin interneurons, innervating the more distal dendrites and controlling the input and plasticity of pyramidal neurons [40]. Thus, we can speculate the effect of miR-132/212 on neuronal plasticity could be due not only to an action on dendritic spines dynamic but also to a modulation of molecular pathway im-portant for inhibitory circuits function. Furthermore, miR-132 levels are altered in models of Rett syndrome [62] and MeCP2-Knockout (KO) mice display abnormali-ties in dendritic spines density and morphology [12]. Interestingly, miR-132 action on spine morphology in-volves its interaction with fragile X mental retardation protein (FMRP) , a protein acting as translational repres-sor of specific mRNAs [26]. Transcriptional silencing of FMRP expression causes Fragile X syndrome (FXS), the most common inherited cause of mental retardation. Strikingly, in both human patients and mouse model of FXS, cortical neurons have an excess of dendritic spines, as well as thin, long and immature spines [97]. Thus, we are tempting to speculate a possible role ofmiR-132 as molecular transducer of plasticity in neuronal cir-cuits, acting through modulation of dendritic spines dy-namics and maturation. Moreover, our observation that experience-dependent fine tuning of miR-132levels is re-quired for activity-dependent plasticity of brain circuits during critical periods of development raises the pos-sibility that alterations of miR-132 biogenesis, function and decay could play an important role in the patho-genesis of these neurodevelopmental disorders. Clin-ical investigations showed expression changes in
