Graziella Di Cristo

Experience and Activity-Dependent Maturation of Perisomatic GABAergic Innervation in Primary Visual Cortex during a Postnatal Critical Period

The neocortical GABAergic network consists of diverse interneuron cell types that display distinct physiological properties and target their innervations to subcellular compartments of principal neurons. Inhibition directed toward the soma and proximal dendrites is crucial in regulating the output of pyramidal neurons, but the development of perisomatic innervation is poorly understood because of the lack of specific synaptic markers. In the primary visual cortex, for example, it is unknown whether, and to what extent, the formation and maturation of perisomatic synapses are intrinsic to cortical circuits or are regulated by sensory experience. Using bacterial artificial chromosome transgenic mice that label a defined class of perisomatic synapses with green fluorescent protein, here we show that perisomatic innervation developed during a protracted postnatal period after eye opening. Maturation of perisomatic innervation was significantly retarded by visual deprivation during the third, but not the fifth, postnatal week, implicating an important role for sensory input. To examine the role of cortical intrinsic mechanisms, we developed a method to visualize perisomatic synapses from single basket interneurons in cortical organotypic cultures. Characteristic perisomatic synapses formed through a stereotyped process, involving the extension of distinct terminal branches and proliferation of perisomatic boutons. Neuronal spiking in organotypic cultures was necessary for the proliferation of boutons and the extension, but not the maintenance, of terminal branches. Together, our results suggest that although the formation of perisomatic synapses is intrinsic to the cortex, visual experience can influence the maturation and pattern of perisomatic innervation during a postnatal critical period by modulating the level of neural activity within cortical circuits.

GAD67-Mediated GABA Synthesis and Signaling Regulate Inhibitory Synaptic Innervation in the Visual Cortex

The development of GABAergic inhibitory circuits is shaped by neural activity, but the underlying mechanisms are unclear. Here, we demonstrate a novel function of GABA in regulating GABAergic innervation in the adolescent brain, when GABA is mainly known as an inhibitory transmitter. Conditional knockdown of the rate-limiting synthetic enzyme GAD67 in basket interneurons in adolescent visual cortex resulted in cell autonomous deficits in axon branching, perisomatic synapse formation around pyramidal neurons, and complexity of the innervation fields; the same manipulation had little influence on the subsequent maintenance of perisomatic synapses. These effects of GABA deficiency were rescued by suppressing GABA reuptake and by GABA receptor agonists. Germline knockdown of GAD67 but not GAD65 showed similar deficits, suggesting a specific role of GAD67 in the maturation of perisomatic innervation. Since intracellular GABA levels are modulated by neuronal activity, our results implicate GAD67-mediated GABA synthesis in activity-dependent regulation of inhibitory innervation patterns.

Activity-dependent PSA expression regulates inhibitory maturation and onset of critical period plasticity

Functional maturation of GABAergic innervation in the developing visual cortex is regulated by neural activity and sensory inputs and in turn influences the critical period of ocular dominance plasticity. Here we show that polysialic acid (PSA), presented by the neural cell adhesion molecule, has a role in the maturation of GABAergic innervation and ocular dominance plasticity. Concentrations of PSA significantly decline shortly after eye opening in the adolescent mouse visual cortex; this decline is hindered by visual deprivation. The developmental and activity-dependent regulation of PSA expression is inversely correlated with the maturation of GABAergic innervation. Premature removal of PSA in visual cortex results in precocious maturation of perisomatic innervation by basket interneurons, enhanced inhibitory synaptic transmission, and earlier onset of ocular dominance plasticity. The developmental and activity-dependent decline of PSA expression therefore regulates the timing of the maturation of GABAergic inhibition and the onset of ocular dominance plasticity.

Subcellular domain-restricted GABAergic innervation in primary visual cortex in the absence of sensory and thalamic inputs

Distinct classes of GABAergic synapses target restricted subcellular domains, thereby differentially regulating the input, integration and output of principal neurons, but the underlying mechanism for such synapse segregation is unclear. Here we show that the distributions of two major classes of GABAergic synapses along the perisomatic and dendritic domains of pyramidal neurons were indistinguishable between primary visual cortex in vivo and cortical organotypic cultures. Therefore, subcellular synapse targeting is independent of thalamic input and probably involves molecular labels and experience-independent forms of activity.

Mutations in SYNGAP1 cause intellectual disability, autism, and a specific form of epilepsy by inducing haploinsufficiency.

De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP‐activating protein, cause nonsyndromic intellectual disability (NSID). All disease‐causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID. Here, we report the identification of the first pathogenic missense mutations (c.1084T>C [p.W362R], c.1685C>T [p.P562L]) and three novel truncating mutations (c.283dupC [p.H95PfsX5], c.2212_2213del [p.S738X], and (c.2184del [p.N729TfsX31]) in SYNGAP1 in patients with NSID. A subset of these patients also showed ataxia, autism, and a specific form of generalized epilepsy that can be refractory to treatment. All of these mutations occurred de novo, except c.283dupC, which was inherited from a father who is a mosaic. Biolistic transfection of wild‐type SYNGAP1 in pyramidal cells from cortical organotypic cultures significantly reduced activity‐dependent phosphorylated extracellular signal‐regulated kinase (pERK) levels. In contrast, constructs expressing p.W362R, p.P562L, or the previously described p.R579X had no significant effect on pERK levels. These experiments suggest that the de novo missense mutations, p.R579X, and possibly all the other truncating mutations in SYNGAP1 result in a loss of its function. Moreover, our study confirms the involvement of SYNGAP1 in autism while providing novel insight into the epileptic manifestations associated with its disruption.

Transient neurites of retinal horizontal cells exhibit columnar tiling via homotypic interactions

Sensory neurons with common functions are often nonrandomly arranged and form dendritic territories that show little overlap, or tiling. Repulsive homotypic interactions underlie such patterns in cell organization in invertebrate neurons. It is unclear how dendro-dendritic repulsive interactions can produce a nonrandom distribution of cells and their spatial territories in mammalian retinal horizontal cells, as mature horizontal cell dendrites overlap substantially. By imaging developing mouse horizontal cells, we found that these cells transiently elaborate vertical neurites that form nonoverlapping columnar territories on reaching their final laminar positions. Targeted cell ablation revealed that the vertical neurites engage in homotypic interactions that result in tiling of neighboring cells before the establishment of their dendritic fields. This developmental tiling of transient neurites correlates with the emergence of a nonrandom distribution of the cells and could represent a mechanism that organizes neighbor relationships and territories of neurons before circuit assembly.

GABA Signaling Promotes Synapse Elimination and Axon Pruning in Developing Cortical Inhibitory Interneurons

Accumulating evidence indicates that GABA acts beyond inhibitory synaptic transmission and regulates the development of inhibitory synapses in the vertebrate brain, but the underlying cellular mechanism is not well understood. We have combined live imaging of cortical GABAergic axons across time scales from minutes to days with single-cell genetic manipulation of GABA release to examine its role in distinct steps of inhibitory synapse formation in the mouse neocortex. We have shown previously, by genetic knockdown of GABA synthesis in developing interneurons, that GABA signaling promotes the maturation of inhibitory synapses and axons. Here we found that a complete blockade of GABA release in basket interneurons resulted in an opposite effect, a cell-autonomous increase in axon and bouton density with apparently normal synapse structures. These results not only demonstrate that GABA is unnecessary for synapse formation per se but also uncover a novel facet of GABA in regulating synapse elimination and axon pruning. Live imaging revealed that developing GABAergic axons form a large number of transient boutons, but only a subset was stabilized. Release blockade led to significantly increased bouton stability and filopodia density, increased axon branch extension, and decreased branch retraction. Our results suggest that a major component of GABA function in synapse development is transmission-mediated elimination of subsets of nascent contacts. Therefore, GABA may regulate activity-dependent inhibitory synapse formation by coordinately eliminating certain nascent contacts while promoting the maturation of other nascent synapses.

GABAergic circuit dysfunctions in neurodevelopmental disorders.

GABAergic interneurons control neuronal excitability, integration, and plasticity. Further, they regulate the generation of temporal synchrony and oscillatory behavior among networks of pyramidal neurons. Such oscillations within and across neural systems are believed to serve various complex functions, such as perception, movement initiation, and memory. Alterations in the development of GABAergic circuits have been implicated in various brain diseases with neurodevelopmental origin. Here, we highlight recent studies suggesting a role for alterations of GABA transmission in the pathophysiology of two neurodevelopmental diseases, schizophrenia, and autism. We further discuss how manipulations of GABA signaling may be used for novel therapeutic interventions.

Bcl-2 overexpression per se does not promote regeneration of neonatal crushed optic fibers.

We have explored whether overexpression of the bcl‐2gene ‘per se’ can promote regeneration of retinal ganglion cells (RGCs) after optic nerve axotomy in developing transgenic mice. We have used newborn mice (postnatal day 5) because at this age the central nervous system environment is more permissive for regeneration than in adults, thus, maximizing the probability to detect a regeneration‐promoting role of bcl‐2. Thirty days postsurgery we found that in mice overexpressing bcl‐2, a high proportion of retinal ganglion cells survived and also that some fibers in the proximal stump of the optic nerve were preserved. However, the optic nerve of transgenic mice does not show signs of regeneration. On the contrary, in the presence of Schwann cell transplants, there are signs of fiber regrowth. Indeed, many axonal terminals cross the crush site and reach the chiasm in both wild type and transgenic mice nerves. These results suggest that bcl‐2 overexpression is not sufficient ‘per se’ to increase the regenerative potentiality of axotomized RGCs.

Differential effects of cortical neurotrophic factors on development of lateral geniculate nucleus and superior colliculus neurons: anterograde and retrograde actions.

Neurotrophins strongly affect visual system development and plasticity. However, the mode of delivery and targets of neurotrophin action are still under debate. For instance, cortical NT-4/5 (neurotrophin 4/5; Ntf4/5) was shown to rescue lateral geniculate nucleus (LGN) neurons from monocular deprivation-induced atrophy suggesting a retrograde action on thalamic afferents. It is still unclear whether LGN neurons respond to NT-4/5 and other neurotrophins during development in animals with normal vision. We now show that infusions of NT-4/5 and NGF (nerve growth factor) into visual cortex at the onset and the peak of the critical period accelerated LGN neuron growth. BDNF (brain-derived neurotrophic factor) was ineffective. The effects of neurotrophin on LGN development were clearly dissociated from the effects at cortical level because soma growth of cortical layer IV and VI neurons was strongly promoted by BDNF. NT-4/5 was only effective at the onset, but no longer at the peak of the critical period suggesting a switch in neurotrophin dependency for these cortical cell classes. To dissociate retrograde and anterograde effects of the TrkB ligands, we analyzed the stratum griseum superficiale (SGS) of the superior colliculus, a target of visual cortical efferents. Indeed, TrkB-expressing inhibitory SGS neurons responded to cortical NT-4/5 infusion with somatic growth. Strikingly, the TrkB-expressing excitatory tectothalamic calbindin neurons in the SGS did not respond. This demonstrated for the first time a selective cell type-specific anterograde action of NT-4/5 and suggested for the LGN that anterograde as well as retrograde effects contribute to soma size regulation. Strikingly, cortical infusion of the cytokine LIF, which affects development of visual cortex neurochemical architecture, transiently inhibited growth of neurons in LGN, cortical layer IV and VI and SGS. In summary, the study presents three important results. First, central neurons regulate soma size development in an age-and ligand-specific fashion. Second, NT-4/5 and NGF accelerate LGN development in rats with normal vision while LIF delays growth. Third, anterogradely transported NT-4/5 effectively promotes neuronal maturation. These differential actions on subcortical neurons may contribute to the different effects of neurotrophins on visual system development and plasticity.