Laura Cancedda

Excitatory GABA action is essential for morphological maturation of cortical neurons in vivo

GABA exerts excitatory actions on embryonic and neonatal cortical neurons, but the in vivo function of this GABA excitation is essentially unknown. Using in utero electroporation, we eliminated the excitatory action of GABA in a subpopulation of rat ventricular progenitors and cortical neurons derived from these progenitors by premature expression of the Cl− transporter KCC2, as confirmed by the changes in the reversal potential of GABA-induced currents and the resting membrane potential after GABAA receptor blockade. We found that radial migration to layer II/III of the somatosensory cortex of neurons derived from the transfected progenitors was not significantly affected, but their morphological maturation was markedly impaired. Furthermore, reducing neuronal excitability of cortical neurons in vivo by overexpressing an inward-rectifying K+ channel, which lowered the resting membrane potential, mimicked the effect of premature KCC2 expression. Thus, membrane depolarization caused by early GABA excitation is critical for morphological maturation of neonatal cortical neurons in vivo.

LKB1/STRAD promotes axon initiation during neuronal polarization.

Axon/dendrite differentiation is a critical step in neuronal development. In cultured hippocampal neurons, the accumulation of LKB1 and STRAD, two interacting proteins critical for establishing epithelial polarity, in an undifferentiated neurite correlates with its subsequent axon differentiation. Downregulation of either LKB1 or STRAD by siRNAs prevented axon differentiation, and overexpression of these proteins led to multiple axon formation. Furthermore, interaction of STRAD with LKB1 promoted LKB1 phosphorylation at a PKA site S431 and elevated the LKB1 level, and overexpressing LKB1 with a serine-to-alanine mutation at S431 (LKB1(S431A)) prevented axon differentiation. In developing cortical neurons in vivo, downregulation of LKB1 or overexpression of LKB1(S431A) also abolished axon formation. Finally, local exposure of the undifferentiated neurite to brain-derived neurotrophic factor or dibutyryl-cAMP promoted axon differentiation in a manner that depended on PKA-dependent LKB1 phosphorylation. Thus local LKB1/STRAD accumulation and PKA-dependent LKB1 phosphorylation represents an early signal for axon initiation.

Reducing Intracortical Inhibition in the Adult Visual Cortex Promotes Ocular Dominance Plasticity

Experience-dependent plasticity in the cortex is often higher during short critical periods in postnatal development. The mechanisms limiting adult cortical plasticity are still unclear. Maturation of intracortical GABAergic inhibition is suggested to be crucial for the closure of the critical period for ocular dominance (OD) plasticity in the visual cortex. We find that reduction of GABAergic transmission in the adult rat visual cortex partially reactivates OD plasticity in response to monocular deprivation (MD). This is accompanied by an enhancement of activity-dependent potentiation of synaptic efficacy but not of activity-dependent depression. We also found a decrease in the expression of chondroitin sulfate proteoglycans in the visual cortex of MD animals with reduced inhibition, after the reactivation of OD plasticity. Thus, intracortical inhibition is a crucial limiting factor for the induction of experience-dependent plasticity in the adult visual cortex.

Acceleration of Visual System Development by Environmental Enrichment

Thus far, the developmental plasticity of the visual system has been studied by altering or reducing visual experience. Here, we investigated whether a complex sensory-motor stimulation, provided by rearing animals in an enriched environment, affects visual system development. We found that raising mice in this condition causes an earlier eye opening, a precocious development of visual acuity, and an accelerated decline of white matter-induced long-term potentiation. These effects are accompanied by a precocious cAMP response element-mediated gene expression and a significant increase of BDNF protein and GAD65/67 expression in enriched pups. In addition, we showed that enriched pups experienced higher levels of licking behavior provided by adult females. Thus, rearing mice from birth in an enriched environment leads to a conspicuous acceleration of visual system development as ascertained at behavioral, electrophysiological, and molecular level.

Modulation of GABAergic Transmission by Activity via Postsynaptic Ca2+-Dependent Regulation of KCC2 Function

Activity-induced modification of GABAergic transmission contributes to the plasticity of neural circuits. In the present work we found that prolonged postsynaptic spiking of hippocampal neurons led to a shift in the reversal potential of GABA-induced Cl- currents (E(Cl)) toward positive levels in a duration- and frequency-dependent manner. This effect was abolished by blocking cytosolic Ca2+ elevation and mimicked by releasing Ca2+ from internal stores. Activity- and Ca2+-induced E(Cl) shifts were larger in mature neurons, which express the K-Cl cotransporter KCC2 at high levels, and inhibition of KCC2 occluded the shifts. Overexpression of KCC2 in young cultured neurons, which express lower levels of KCC2 and have a more positive E(Cl), resulted in hyperpolarized E(Cl) similar to that of mature cells. Importantly, these young KCC2-expressing neurons became responsive to neuronal spiking and Ca2+ elevation by showing positive E(Cl) shifts. Thus, repetitive postsynaptic spiking reduces the inhibitory action of GABA through a Ca2+-dependent downregulation of KCC2 function.

Local and Long-Range Reciprocal Regulation of cAMP and cGMP in Axon/Dendrite Formation

Promoting Axon Formation How do neurons initiate one axon and lots of dendrites? Using an in vitro assay involving stripes of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), Shelly et al. (p. 547) show that an increase in cAMP initiates axon formation while an increase in cGMP initiates dendrites. Moreover, cAMP and cGMP reciprocally inhibit each other via the activation of specific phosphodiesterases, as well as protein kinase A and protein kinase G. Finally, long-range self-inhibition of cAMP can explain why only one axon, yet multiple dendrites, is initiated in single hippocampal neurons in culture. Locally increasing cAMP in one neurite causes long-range cAMP reduction in the rest of the neurites, accompanied by corresponding reciprocal changes in cGMP. Cyclic adenosine monophosphate and cyclic guanosine monophosphate regulate axon formation and exert opposite actions on dendrite formation. Cytosolic cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) often mediate antagonistic cellular actions of extracellular factors, from the regulation of ion channels to cell volume control and axon guidance. We found that localized cAMP and cGMP activities in undifferentiated neurites of cultured hippocampal neurons promote and suppress axon formation, respectively, and exert opposite effects on dendrite formation. Fluorescence resonance energy transfer imaging showed that alterations of the amount of cAMP resulted in opposite changes in the amount of cGMP, and vice versa, through the activation of specific phosphodiesterases and protein kinases. Local elevation of cAMP in one neurite resulted in cAMP reduction in all other neurites of the same neuron. Thus, local and long-range reciprocal regulation of cAMP and cGMP together ensures coordinated development of one axon and multiple dendrites.

Developmental Downregulation of Histone Posttranslational Modifications Regulates Visual Cortical Plasticity

The action of visual experience on visual cortical circuits is maximal during a critical period of postnatal development. The long-term effects of this experience are likely mediated by signaling cascades regulating experience-dependent gene transcription. Developmental modifications of these pathways could explain the difference in plasticity between the young and adult cortex. We studied the pathways linking experience-dependent activation of ERK to CREB-mediated gene expression in vivo. In juvenile mice, visual stimulation that activates CREB-mediated gene transcription also induced ERK-dependent MSK and histone H3 phosphorylation and H3-H4 acetylation, an epigenetic mechanism of gene transcription activation. In adult animals, ERK and MSK were still inducible; however, visual stimulation induced weak CREB-mediated gene expression and H3-H4 posttranslational modifications. Stimulation of histone acetylation in adult animals by means of trichostatin promoted ocular dominance plasticity. Thus, differing, experience-dependent activations of signaling molecules might be at the basis of the differences in experience-dependent plasticity between juvenile and adult cortex.

Enriched environment and acceleration of visual system development.

Rearing mice from birth in an enriched environment leads to a conspicuous acceleration of visual system development appreciable at behavioral, electrophysiological and molecular level. Little is known about the possible mechanisms of action through which enriched environment affects visual system development. It has been suggested that differences in maternal behavior between enriched and non-enriched conditions could contribute to the earliest effects of enriched environment on visual development and that neurotrophins, BDNF in particular, might be involved. Here, we examined Brain Derived Neurotrophic Factor (BDNF) levels in the visual cortex during development and showed that an increase occurs in the first week of life in enriched pups compared to standard reared pups; BDNF levels at birth were equal in the two groups. This suggests a postnatal rather than a prenatal effect of environment on BDNF. A detailed analysis of maternal care behavior showed that pups raised in a condition of social and physical enrichment experienced higher levels of licking behavior and physical contact compared to standard reared pups and that enhanced levels of licking were also provided to pups in an enriched environment where no adult females other than the mother were present. Thus, different levels of maternal care in different environmental conditions could act as indirect mediator for the earliest effects of enrichment on visual system development. Some of the effects of different levels of maternal care on the offspring behavior are long lasting. We measured the visual acuity of differentially reared mice at the end of the period of visual acuity development (postnatal day 45) and at 12 months of age, using a behavioral discrimination task. We found better learning abilities and higher visual acuity in enriched compared to standard reared mice at both ages.

Refuting the challenges of the developmental shift of polarity of GABA actions: GABA more exciting than ever!

During brain development, there is a progressive reduction of intracellular chloride associated with a shift in GABA polarity: GABA depolarizes and occasionally excites immature neurons, subsequently hyperpolarizing them at later stages of development. This sequence, which has been observed in a wide range of animal species, brain structures and preparations, is thought to play an important role in activity-dependent formation and modulation of functional circuits. This sequence has also been considerably reinforced recently with new data pointing to an evolutionary preserved rule. In a recent “Hypothesis and Theory Article,” the excitatory action of GABA in early brain development is suggested to be “an experimental artefact” (Bregestovski and Bernard, 2012). The authors suggest that the excitatory action of GABA is due to an inadequate/insufficient energy supply in glucose-perfused slices and/or to the damage produced by the slicing procedure. However, these observations have been repeatedly contradicted by many groups and are inconsistent with a large body of evidence including the fact that the developmental shift is neither restricted to slices nor to rodents. We summarize the overwhelming evidence in support of both excitatory GABA during development, and the implications this has in developmental neurobiology.

GABAergic control of neurite outgrowth and remodeling during development and adult neurogenesis: general rules and differences in diverse systems.

During development, Gamma-aminobutyric acidergic (GABAergic) neurons mature at early stages, long before excitatory neurons. Conversely, GABA reuptake transporters become operative later than glutamate transporters. GABA is therefore not removed efficiently from the extracellular domain and it can exert significant paracrine effects. Hence, GABA-mediated activity is a prominent source of overall neural activity in developing CNS networks, while neurons extend dendrites and axons, and establish synaptic connections. One of the unique features of GABAergic functional plasticity is that in early development, activation of GABAA receptors results in depolarizing (mainly excitatory) responses and Ca2+ influx. Although there is strong evidence from several areas of the CNS that GABA plays a significant role in neurite growth not only during development but also during adult neurogenesis, surprisingly little effort has been made into putting all these observations into a common framework in an attempt to understand the general rules that regulate these basic and evolutionary well-conserved processes. In this review, we discuss the current knowledge in this important field. In order to decipher common, universal features and highlight differences between systems throughout development, we compare findings about dendritic proliferation and remodeling in different areas of the nervous system and species, and we also review recent evidence for a role in axonal elongation. In addition to early developmental aspects, we also consider the GABAergic role in dendritic growth during adult neurogenesis, extending our discussion to the roles played by GABA during dendritic proliferation in early developing networks versus adult, well established networks.