Victoria F. Safiulina

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.

GABAergic signaling at mossy fiber synapses in neonatal rat hippocampus

In the adult rat hippocampus, granule cell mossy fibers (MFs) form excitatory glutamatergic synapses with CA3 principal cells and local inhibitory interneurons. However, evidence has been provided that, in young animals and after seizures, the same fibers can release in addition to glutamate GABA. Here we show that, during the first postnatal week, stimulation of granule cells in the dentate gyrus gave rise to monosynaptic GABAA-mediated responses in principal cells and in interneurons. These synapses were indeed made by MFs because they exhibited strong paired-pulse facilitation, high sensitivity to the metabotropic glutamate receptor agonist l-AP-4, and short-term frequency-dependent facilitation. MF responses were potentiated by blocking the plasma membrane GABA transporter GAT-1 with NO-711 or by allosterically modulating GABAA receptors with flurazepam. Chemical stimulation of granule cell dendrites with glutamate induced barrages of GABAA-mediated postsynaptic currents into target neurons. Furthermore, immunocytochemical experiments demonstrated colocalization of vesicular GABA transporter with vesicular glutamate transporter-1 and zinc transporter 3, suggesting that GABA can be taken up and stored in synaptic vesicles of MF terminals. Additional fibers releasing both glutamate and GABA into principal cells and interneurons were recruited by increasing the strength of stimulation. Both the GABAergic and the glutamatergic component of synaptic currents occurred with the same latency and were reversibly abolished by l-AP-4, indicating that they originated from the MFs. GABAergic signaling may play a crucial role in tuning hippocampal network during postnatal development. Low-threshold GABA-releasing fibers may undergo elimination, and this may occur when GABA shifts from the depolarizing to the hyperpolarizing direction.

At Immature Mossy Fibers-CA3 Connections, Activation of Presynaptic GABAB Receptors by Endogenously Released GABA Contributes to Synapses Silencing

Early in postnatal life correlated GABAergic activity in the hippocampus is thought to play a crucial role in synaptogenesis and in the development of adult neuronal networks. Unlike adulthood, at this developmental stage, mossy fibers (MF) which are the axons of granule cells, release GABA into CA3 principal cells and interneurons. Here, we tested the hypothesis that at MF-CA3 connections, tonic activation of GABAB autoreceptors by GABA is responsible for the low probability of release and synapse silencing. Blocking GABAB receptors with CGP55845 enhanced the probability of GABA release and switched on silent synapses while the opposite was observed with baclofen. Both these effects were presynaptic and were associated with changes in paired-pulse ratio and coefficient of variation. In addition, enhancing the extracellular GABA concentration by repetitive stimulation of MF or by blocking the GABA transporter GAT-1, switched off active synapses, an effect that was prevented by CGP55845. In the presence of CGP55845, stimulation of MF-induced synaptic potentiation. The shift of EGABA from the depolarizing to the hyperpolarizing direction with bumetanide, a blocker of the cation-chloride co-transporter NKCC1, prevented synaptic potentiation and caused synaptic depression, suggesting that the depolarizing action of GABA observed in the presence of CGP55845 is responsible for the potentiating effect. It is proposed that, activation of GABAB receptors by spillover of GABA from MF terminals reduces the probability of release and contributes to synapses silencing. This would act as a filter to prevent excessive activation of the auto-associative CA3 network and the emergence of seizures.

Nicotinic modulation of network and synaptic transmission in the immature hippocampus investigated with genetically modified mice.

The hippocampus, a key structure in learning and memory processes, receives a powerful cholinergic innervation from the septum and contains nicotinic acetylcholine receptors (nAChRs). Early in postnatal development, activation of nAChRs by nicotine or endogenous acetylcholine contributes to enhance synaptic signalling. Here, the patch‐clamp technique was used to assess the contribution of α7 and β2‐containing (α7* and β2*) nAChRs to nicotine‐elicited modulation of GABAergic and glutamatergic activity at the network and single‐cell level in the immature hippocampus of wild‐type (WT), α7−/− and β2−/− mice. We found that α7* and β2* nAChRs were sufficient to modulate nicotine‐induced increase in frequency of spontaneously occurring giant depolarizing potentials (GDPs), which are generated at the network level by the synergistic action of glutamate and depolarizing GABA, and thought to play a crucial role in neuronal wiring. However, α7* but not β2* receptors were essential in nicotine‐induced increase of interictal discharge frequency recorded after postnatal day 3 in the presence of bicuculline, when GABA shifted from the depolarizing to the hyperpolarizing direction. To correlate these observations with nicotine‐elicited changes in synaptic transmission, we recorded spontaneous GABAergic and glutamatergic postsynaptic currents in pyramidal cells and interneurons localized in stratum oriens, stratum pyramidale and stratum radiatum, in slices obtained from WT and knock‐out animals. We found that early in postnatal life α7* and β2* nAChRs exert a fine regional modulation of GABAergic and glutamatergic transmission that underlies nicotine‐elicited changes in network synchronization.

Up-regulation of GLT-1 severely impairs LTD at mossy fibre–CA3 synapses

Glutamate transporters are responsible for clearing synaptically released glutamate from the extracellular space. By this action, they maintain low levels of ambient glutamate, thus preventing excitotoxic damage, and contribute to shaping synaptic currents. We show that up‐regulation of the glutamate transporter GLT‐1 by ceftriaxone severely impaired mGluR‐dependent long‐term depression (LTD), induced at rat mossy fibre (MF)–CA3 synapses by repetitive stimulation of afferent fibres. This effect involved GLT‐1, since LTD was rescued by the selective GLT‐1 antagonist dihydrokainate (DHK). DHK per se produced a modest decrease in fEPSP amplitude that rapidly regained control levels after DHK wash out. Moreover, the degree of fEPSP inhibition induced by the low‐affinity glutamate receptor antagonist γ‐DGG was similar during basal synaptic transmission but not during LTD, indicating that in ceftriaxone‐treated rats LTD induction did not alter synaptic glutamate transient concentration. Furthermore, ceftriaxone‐induced GLT‐1 up‐regulation significantly reduced the magnitude of LTP at MF–CA3 synapses but not at Schaffer collateral–CA1 synapses. Postembedding immunogold studies in rats showed an increased density of gold particles coding for GLT‐1a in astrocytic processes and in mossy fibre terminals; in the latter, gold particles were located near and within the active zones. In both CEF‐treated and untreated GLT‐1 KO mice used for verifying the specificity of immunostaining, the density of gold particles in MF terminals was comparable to background levels. The enhanced expression of GLT‐1 at release sites may prevent activation of presynaptic receptors, thus revealing a novel mechanism by which GLT‐1 regulates synaptic plasticity in the hippocampus.

Low expression of Kv7/M channels facilitates intrinsic and network bursting in the developing rat hippocampus

Early in development, network activity in the hippocampus is characterized by recurrent synchronous bursts, whose cellular correlates are giant depolarizing potentials (GDPs). The propensity for generating GDPs is attributed to GABAergic synaptic transmission being depolarizing and excitatory in neonatal neurons. However, developmental regulation of intrinsic conductances may also influence GDPs generation. A likely candidate is the non‐inactivating, low‐threshold, muscarinic‐sensitive K+ current (M current; Im), which down‐regulates intrinsic bursting activity in adult hippocampal pyramidal neurons. Western blot analysis of homogenates of the CA3 hippocampal region showed that expression of the Kv7.2 subunit, one of the constituents of neuronal M channels, is weak in neonatal neurons, and markedly increases after the first postnatal week. Likewise, the density of Im was very low in neonatal CA3 pyramidal cells and increased later on. Spontaneously occurring intrinsic bursts in neonatal neurons were longer and more robust, and recurred more regularly, than in juvenile neurons. The Im blocker linopirdine only mildly affected intrinsic bursting in neonatal neurons, but strongly facilitated and regularized it in juvenile neurons. We conclude that the low expression of Kv7/M channels and the depolarizing action of GABA early after birth enhance intrinsic bursting and neuronal synchronization leading to generation of GDPs within the hippocampal network.

The Depolarizing Action of GABA Controls Early Network Activity in the Developing Hippocampus

Early in postnatal life γ-aminobutyric acid (GABA), the primary inhibitory transmitter in adults, excites targeted neurons by an outwardly directed flux of chloride which results from the unbalance between the cation–chloride cotransporters NKCC1 and KCC2, involved in chloride uptake and extrusion, respectively. This effect contributes to generate synchronized network activity or giant depolarizing potentials (GDPs) in the developing hippocampus. Here, we review some recent data concerning the mechanisms by which GDPs are generated and their functional role in enhancing synaptic efficacy at poorly developed GABAergic and glutamatergic synapses. In adulthood, reshaping neuronal circuits due to changes in chloride homeostasis and to the shift of GABA from hyperpolarizing to depolarizing, has been implicated in several neurological disorders, including epilepsy. Evidence has been recently provided that in chronically nerve growth factor-deprived mice expressing a progressive age-dependent neurodegenerative pathology resembling that observed in patients with Alzheimer’s disease, the reduced expression of mRNA encoding for the Kcc2 gene and the depolarizing action of GABA lead to the reorganization of the neuronal hippocampal network. This may represent a novel mechanism by which GABAergic signaling counterbalances the loss of synaptic activity in neurodegenerative diseases.

Reactive Oxygen Species Mediate the Potentiating Effects of ATP on GABAergic Synaptic Transmission in the Immature Hippocampus

Reactive oxygen species (ROS) constitute important signaling molecules in the central nervous system. They regulate a number of different functions both under physiological conditions and under pathological conditions. Here we tested the hypothesis that in the immature hippocampus ATP, the most diffuse neurotransmitter in the brain, modulates synaptic transmission via ROS. We show that ATP, acting on metabotropic P2Y1 receptors, increased the frequency of GABAA-mediated spontaneous postsynaptic currents (SPSCs) in CA3 principal cells, an effect that was prevented by the antioxidant N-acetyl-cysteine or by catalase, an enzyme that breaks down H2O2. The effect of ATP on SPSCs was mimicked by H2O2 or by the pro-oxidant, Fe2+, which, through the Fentol reaction, catalyzes the conversion of H2O2 into highly reactive hydroxyl radicals. MRS-2179, a P2Y1 receptor antagonist, removed the facilitatory action of Fe2+ on SPSCs, suggesting that endogenous ATP acting on P2Y1 receptors is involved in Fe2+-induced modulation of synaptic transmission. Imaging ROS with the H2O2-sensitive dye DCF revealed that ATP induces generation of peroxide in astrocytes via activation of P2Y1 receptors coupled to intracellular calcium rise. Neither N-acetyl-cysteine nor catalase prevented Ca2+ transients induced by ATP in astrocytes. Since a single hippocampal astrocyte can contact many neurons, ATP-induced ROS signaling may control thousands of synapses. This may be crucial for information processing in the immature brain when GABAergic activity is essential for the proper wiring of the hippocampal network.

PrPC Controls via Protein Kinase A the Direction of Synaptic Plasticity in the Immature Hippocampus

The cellular form of prion protein PrPC is highly expressed in the brain, where it can be converted into its abnormally folded isoform PrPSc to cause neurodegenerative diseases. Its predominant synaptic localization suggests a crucial role in synaptic signaling. Interestingly, PrPC is developmentally regulated and its high expression in the immature brain could be instrumental in regulating neurogenesis and cell proliferation. Here, PrPC-deficient (Prnp0/0) mice were used to assess whether the prion protein is involved in synaptic plasticity processes in the neonatal hippocampus. To this aim, calcium transients associated with giant depolarizing potentials, a hallmark of developmental networks, were transiently paired with mossy fiber activation in such a way that the two events were coincident. While this procedure caused long-term potentiation (LTP) in wild-type (WT) animals, it caused long-term depression (LTD) in Prnp0/0 mice. Induction of LTP was postsynaptic and required the activation of cAMP-dependent protein kinase A (PKA) signaling. The induction of LTD was presynaptic and relied on G-protein-coupled GluK1 receptor and protein lipase C. In addition, at emerging CA3-CA1 synapses in WT mice, but not in Prnp0/0 mice, pairing Schaffer collateral stimulation with depolarization of CA1 principal cells induced LTP, known to be PKA dependent. Postsynaptic infusion of a constitutively active isoform of PKA catalytic subunit Cα into CA1 and CA3 principal cells in the hippocampus of Prnp0/0 mice caused a persistent synaptic facilitation that was occluded by subsequent pairing. These data suggest that PrPC plays a crucial role in regulating via PKA synaptic plasticity in the developing hippocampus.

GABA is the Main Neurotransmitter Released from Mossy Fiber Terminals in the Developing Rat Hippocampus

Early in postnatal development, correlated activity in the hippocampus is characterized by giant depolarizing potentials (GDPs). GDPs are generated by the interplay between glutamate and GABA, which in the immediate postnatal period is depolarizing and excitatory. Here, we review some recent data obtained in our laboratory concerning neuronal signaling at immature MF connections. MF responses were identified on the basis of their strong paired-pulse facilitation, short-term frequency-dependent facilitation and sensitivity to group III mGluR agonist L-AP4. Unlike adulthood, during the first week of postnatal life minimal stimulation of MF evoked responses that were potentiated by flurazepam and abolished by picrotoxin indicating that they were GABAergic. In addition, using a pairing procedure we found that GDPs and associated calcium transients act as coincident detectors for enhancing synaptic efficacy at poorly developed MF-CA3 and MF-interneurons connections. This may be crucial for synaptogenesis and for establishing the adult neuronal circuit.