Enrico Cherubini

GABA: an excitatory transmitter in early postnatal life

In the adult mammalian CNS, GABA is the main inhibitory transmitter. It inhibits neuronal firing by increasing a Cl- conductance. Bicuculline blocks this effect and induces interictal discharges. A different picture is present in neonatal hippocampal neurones, where synaptically released or exogenously applied GABA depolarizes and excites neuronal membranes--an effect that is due to a different Cl- gradient. In fact, during the early neonatal period, GABA acting on GABAA receptors provides most of the excitatory drive, whereas excitatory glutamatergic synapses are quiescent. It is suggested that during development GABA exerts mainly a trophic action through membrane depolarization and a rise in intracellular Ca2+.

The GABA Excitatory/Inhibitory Shift in Brain Maturation and Neurological Disorders

Ionic currents and the network-driven patterns they generate differ in immature and adult neurons: The developing brain is not a “small adult brain.” One of the most investigated examples is the developmentally regulated shift of actions of the transmitter GABA that inhibit adult neurons but excite immature ones because of an initially higher intracellular chloride concentration [Cl−]i, leading to depolarizing and often excitatory actions of GABA instead of hyperpolarizing and inhibitory actions. The levels of [Cl−]i are also highly labile, being readily altered transiently or persistently by enhanced episodes of activity in relation to synaptic plasticity or a variety of pathological conditions, including seizures and brain insults. Among the plethora of channels, transporters, and other devices involved in controlling [Cl−]i, two have emerged as playing a particularly important role: the chloride importer NKCC1 and the chloride exporter KCC2. Here, the authors stress the importance of determining how [Cl−]i is dynamically regulated and how this affects brain operation in health and disease. In a clinical perspective, agents that control [Cl−]i and reinstate inhibitory actions of GABA open novel therapeutic perspectives in many neurological disorders, including infantile epilepsies, autism spectrum disorders, and other developmental disorders.

Generating diversity at GAB Aergic synapses

Abstract GABA-mediated transmission is characterized by high variability of synaptic responses. Major contributors to this variability are: presynaptic factors, including release probability and number of release sites; factors that determine synaptic GABA transients in the cleft, including diffusion and the actions of GABA transporters; and postsynaptic factors, including GABA A receptors subtypes, their location and number, their modulation by endogenous and exogenous factors, and their interactions with postsynaptic-anchoring proteins.

BK potassium channels control transmitter release at CA3–CA3 synapses in the rat hippocampus

Large conductance calcium‐ and voltage‐activated potassium channels (BK channels) activate in response to calcium influx during action potentials and contribute to the spike repolarization and fast afterhyperpolarization. BK channels targeted to active zones in presynaptic nerve terminals have been shown to limit calcium entry and transmitter release by reducing the duration of the presynaptic spike at neurosecretory nerve terminals and at the frog neuromuscular junction. However, their functional role in central synapses is still uncertain. In the hippocampus, BK channels have been proposed to act as an ‘emergency brake’ that would control transmitter release only under conditions of excessive depolarization and accumulation of intracellular calcium. Here we demonstrate that in the CA3 region of hippocampal slice cultures, under basal experimental conditions, the selective BK channel blockers paxilline (10 μm) and iberiotoxin (100 nm) increase the frequency, but not the amplitude, of spontaneously occurring action potential‐dependent EPSCs. These drugs did not affect miniature currents recorded in the presence of tetrodotoxin, suggesting that their action was dependent on action potential firing. Moreover, in double patch‐clamp recordings from monosynaptically interconnected CA3 pyramidal neurones, blockade of BK channels enhanced the probability of transmitter release, as revealed by the increase in success rate, EPSC amplitude and the concomitant decrease in paired‐pulse ratio in response to pairs of presynaptic action potentials delivered at a frequency of 0.05 Hz. BK channel blockers also enhanced the appearance of delayed responses, particularly following the second action potential in the paired‐pulse protocol. These results are consistent with the hypothesis that BK channels are powerful modulators of transmitter release and synaptic efficacy in central neurones.

Alterations of GABAergic Signaling in Autism Spectrum Disorders

Autism spectrum disorders (ASDs) comprise a heterogeneous group of pathological conditions, mainly of genetic origin, characterized by stereotyped behavior, marked impairment in verbal and nonverbal communication, social skills, and cognition. Interestingly, in a small number of cases, ASDs are associated with single mutations in genes encoding for neuroligin-neurexin families. These are adhesion molecules which, by regulating transsynaptic signaling, contribute to maintain a proper excitatory/inhibitory (E/I) balance at the network level. Furthermore, GABA, the main inhibitory neurotransmitter in adult life, at late embryonic/early postnatal stages has been shown to depolarize and excite targeted cell through an outwardly directed flux of chloride. The depolarizing action of GABA and associated calcium influx regulate a variety of developmental processes from cell migration and differentiation to synapse formation. Here, we summarize recent data concerning the functional role of GABA in building up and refining neuronal circuits early in development and the molecular mechanisms regulating the E/I balance. A dysfunction of the GABAergic signaling early in development leads to a severe E/I unbalance in neuronal circuits, a condition that may account for some of the behavioral deficits observed in ASD patients.

Responses of intracellularly recorded cortical neurons to the iontophoretic application of dopamine

Considering that a well-defined dopaminergic projection from the mesencephalic structures to the rat frontal cortex has been demonstrated, the purpose of this research was to study the action of iontophoretically applied dopamine (DA) on intracellularly recorded rat frontal neurons. The stimulation of the substantia nigra (SN) and the ventral tegmental area (VTA) evoked EPSP-IPSP sequences in these cells. About 50% of the tested neurons, widely distributed in all the frontal cortex, responded to DA application and no difference in the response to DA was observed between neurons with monosynaptic inputs and neurons with polysynaptic inputs. The catecholamine depolarized the cell membrane and decreased the firing rate, generally without significant changes in membrane resistance, as already observed in rat and cat striatal cells. In some neurons the decrease of the spikes preceded the membrane depolarization. Considering the complex effect of DA on the electrical properties of these neurons, these results seem to be indicative of a mechanism of action dependent on metabolic changes.

Control of GABA Release at Mossy Fiber-CA3 Connections in the Developing Hippocampus

In this review some of the recent work carried out in our laboratory concerning the functional role of GABAergic signalling at immature mossy fibres (MF)-CA3 principal cell synapses has been highlighted. While in adulthood MF, the axons of dentate gyrus granule cells release onto CA3 principal cells and interneurons glutamate, early in postnatal life they release GABA, which exerts into targeted cells a depolarizing and excitatory action. We found that GABAA-mediated postsynaptic currents (MF-GPSCs) exhibited a very low probability of release, were sensitive to L-AP4, a group III metabotropic glutamate receptor agonist, and revealed short-term frequency-dependent facilitation. Moreover, MF-GPSCs were down regulated by presynaptic GABAB and kainate receptors, activated by spillover of GABA from MF terminals and by glutamate present in the extracellular medium, respectively. Activation of these receptors contributed to the low release probability and in some cases to synapses silencing. By pairing calcium transients, associated with network-driven giant depolarizing potentials or GDPs (a hallmark of developmental networks thought to represent a primordial form of synchrony between neurons), generated by the synergistic action of glutamate and GABA with MF activation increased the probability of GABA release and caused the conversion of silent synapses into conductive ones suggesting that GDPs act as coincident detector signals for enhancing synaptic efficacy. Finally, to compare the relative strength of CA3 pyramidal cell output in relation to their MF glutamatergic or GABAergic inputs in adulthood or in postnatal development, respectively, a realistic model was constructed taking into account different biophysical properties of these synapses.

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.

‘Deaf, mute and whispering’ silent synapses: their role in synaptic plasticity

Mechanisms of long‐term potentiation (LTP) maintenance are discussed in the light of the phenomenon of silent synapses. Evidence that LTP is associated with the insertion of new AMPA receptors (AMPARs) in postsynaptically silent (deaf) synapses expressing only NMDA receptors (NMDARs) before LTP induction has led to the assumption that the debate on pre‐ versus postsynaptic locus of LTP expression has been resolved in favour of the latter. However, recent data indicate that these synapses are mainly presynaptically silent (mute or whispering), because the probability of glutamate release (Pr) or glutamate concentration in the cleft is too low to activate AMPARs. In this case LTP could be explained by an increase in Pr or enhanced glutamate concentration to activate low affinity AMPARs. Optical methods to probe calcium transients in dendritic spines have revealed an increase in Pr during LTP with concomitant postsynaptic modifications. A hypothesis is considered that accounts for the differences in both the initial failure rates between AMPAR‐ and NMDAR‐mediated responses, and the LTP‐associated decrease in failures of AMPAR‐mediated responses. According to this hypothesis, glutamate release is potentiated by the strong postsynaptic depolarization used to identify NMDAR‐mediated responses. We suggest that the expression of LTP may depend on coordinated pre‐ and postsynaptic modifications whose relative contributions vary according to the initial state of the synapse, the experimental protocol and time after induction.

Nicotine activates immature “silent” connections in the developing hippocampus

In the hippocampus at birth, most glutamatergic synapses are immature and functionally “silent” either because the neurotransmitter is released in insufficient amount to activate low-affinity α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors or because the appropriate receptor system is missing or nonfunctional. Here we show that, in the newborn rat, a brief application of nicotine at immature Schaffer collateral-CA1 connections strongly enhances neurotransmitter release and converts presynaptically silent synapses into conductive ones. This effect is persistent and can be mimicked by endogenous acetylcholine released from cholinergic fibers. Thus, during a critical period of postnatal development, activation of nicotinic acetylcholine receptors contributes to the maturation of functional synaptic contacts and the wiring of adult hippocampal circuitry.