Giant Depolarizing Potentials Research Articles

Comparison of extracellular Giant depolarizing potentials in vitro and early sharp waves in vivo in the CA1 hippocampus of neonatal rats

Giant Depolarizing Potentials (GDPs) and Early Sharp Waves (eSPWs) are the major patterns of neuronal network activity in the developing hippocampus of neonatal rodents in vitro and in vivo, respectively. Because of certain similarities in their electrographic traits, GDPs and eSPWs were originally considered as homologous patterns. Here, we compared electrographic features and current density profiles of field GDPs (fGDPs) and eSPWs using extracellular multisite silicon probe recordings from neonatal rat CA1 hippocampus. We found that fGDPs in hippocampal slices were much less in amplitude than eSPWs, and were characterized by electronegativity and current sinks in CA1 pyramidal cell layer and stratum radiatum, and positive waves/sources in stratum lacunosum-moleculare. eSPWs in vivo showed a remarkably different depth profile, with positivity and current source in the CA1 pyramidal cell layer, and negativity/sinks in stratum radiatum and stratum lacunosum-moleculare. Current sinks of CA3-evoked responses corresponded to sinks of fGDPs and eSPWs in the stratum radiatum. However, current sinks of entorhinal inputs - evoked responses in stratum lacunosum-moleculare, which were characteristic of eSPWs, were absent in fGDPs. In addition, fGDPs more strongly modulated neuronal firing in CA1 compared to eSPWs. Thus, we show important differences in the electrographic properties of GDPs and eSPWs that challenge the homology of these two activity patterns.

On the Origin of Paroxysmal Depolarization Shifts: The Contribution of Cav1.x Channels as the Common Denominator of a Polymorphous Neuronal Discharge Pattern

Since their discovery in the 1960s, the term paroxysmal depolarization shift (PDS) has been applied to a wide variety of reinforced neuronal discharge patterns. Occurrence of PDS as cellular correlates of electrographic spikes during latent phases of insult-induced rodent epilepsy models and their resemblance to giant depolarizing potentials (GDPs) nourished the idea that PDS may be involved in epileptogenesis. Both GDPs and – in analogy – PDS may lead to progressive changes of neuronal properties by generation of pulsatile intracellular Ca2+ elevations. Herein, a key element is the gating of L-type voltage gated Ca2+ channels (LTCCs, Cav1.x family), which may convey Ca2+ signals to the nucleus. Accordingly, the present study investigates various insult-associated neuronal challenges for their propensities to trigger PDS in a LTCC-dependent manner. Our data demonstrate that diverse disturbances of neuronal function are variably suited to induce PDS-like events, and the contribution of LTCCs is essential to evoke PDS in rat hippocampal neurons that closely resemble GDPs. These PDS appear to be initiated in the dendritic sub-compartment. Their morphology critically depends on the position of recording electrodes and on their rate of occurrence. These results provide novel insight into induction mechanisms, origin, variability, and co-existence of PDS with other discharge patterns and thereby pave the way for future investigations regarding the role of PDS in epileptogenesis.

Effects of Homocysteine and its Derivatives on Spontaneous Network Activity in the Hippocampus of Neonatal Rat Pups

Homocysteine is a sulfur-containing amino acid, which at high concentrations has neurotoxic effects and induces impairments to the development of the nervous system. Homocysteine is rapidly oxidized in the plasma, forming disulfide bonds with proteins and other low molecular weight thiols; it also undergoes transformation into the into homocysteine thiolactone. On chronic exposure, the neurotoxicity of homocysteine is therefore mediated mainly by its derivatives. The aim of the present work was to investigate the effects of homocysteine and its derivatives – homocystine and homocysteine thiolactone – on spontaneous network activity in the hippocampus of rats in the first week after birth. Giant depolarizing potentials (GDP) and multiple action potentials (MAP) were recorded using an extracellular electrode in hippocampal field CA3. All three study compounds were found to induce increases in the frequency of GDP and MAP at concentrations of 100 and 500 μM, homocystine producing the most significant increase in neuron network activity. The effects of homocysteine, homocystine, and homocysteine thiolactone on the spontaneous network activity of neurons were completely eliminated on blockade of NMDA and AMPA receptors. Thus, homocysteine and its derivatives lead to increased spontaneous network activity of hippocampal neurons in neonatal rats, which can induce impairments to the formation of the neural networks of the hippocampus in conditions of chronic hyperhomocysteinemia, and could also induce hyperexcitability and the risk of developing epilepsy in the postnatal period.

Sonic Hedgehog Signaling Agonist (SAG) Triggers BDNF Secretion and Promotes the Maturation of GABAergic Networks in the Postnatal Rat Hippocampus.

Sonic hedgehog (Shh) signaling plays critical roles during early central nervous system development, such as neural cell proliferation, patterning of the neural tube and neuronal differentiation. While Shh signaling is still present in the postnatal brain, the roles it may play are, however, largely unknown. In particular, Shh signaling components are found at the synaptic junction in the maturing hippocampus during the first two postnatal weeks. This period is characterized by the presence of ongoing spontaneous synaptic activity at the cellular and network levels thought to play important roles in the onset of neuronal circuit formation and synaptic plasticity. Here, we demonstrate that non-canonical Shh signaling increases the frequency of the synchronized electrical activity called Giant Depolarizing Potentials (GDP) and enhances spontaneous GABA post-synaptic currents in the rodent hippocampus during the early postnatal period. This effect is mediated specifically through the Shh co-receptor Smoothened via intracellular Ca2+ signal and the activation of the BDNF-TrkB signaling pathway. Given the importance of these spontaneous events on neuronal network maturation and refinement, this study opens new perspectives for Shh signaling on the control of early stages of postnatal brain maturation and physiology.

Developmental Switch of Leptin Action on Network Driven Activity in the Neonatal Rat Hippocampus.

The adipose-derived circulating hormone leptin plays a pivotal role in the control of energy balance and body weight. Sound data indicate that this hormone also acts as an important developmental signal impacting a number of brain regions during fetal and postnatal stages. Leptin levels surge during the two first postnatal weeks of life in rodents. This period is characterized by the presence of early network driven activity in the immature hippocampus, the so-called Giant Depolarizing Potentials (GDPs). GDPs are thought to contribute to the wiring of the hippocampal network. We therefore tested the effect of leptin on GDPs. Leptin increased GDPs frequency between the postnatal days (P) 1 and 3 via a calcium/Calmodulin-dependent kinase (CaMK) and extracellular signal-related kinase (ERK) dependent pathways. Between P6 and P7, leptin inhibited the frequency of GDPs through the activation of large conductance Ca2+ activated K+ (BK) channels driven by a phosphoinositol-3 kinase (PI3K) dependent pathway. These results show that leptin exerts a bi-directional and age-dependent control of GDPs and extends the scope of leptin’s action in the developing brain.

Role of CX3CR1 Signaling on the Maturation of GABAergic Transmission and Neuronal Network Activity in the Neonate Hippocampus

In the developing brain, microglial cells play an important role in shaping neuronal circuits. These immune cells communicate with neurons through fractalkine (CX3CL1), a neuronal cytokine that acts on microglial CX3CR1 receptor. Among various functions, this signaling pathway has been implicated in the postnatal maturation of glutamatergic synapses. Although microglial cells are present in the neonate hippocampus when GABA receptor-mediated synaptic transmission and synchronized oscillatory events take place, it remains unknown whether microglial cells tune the establishment of these activities. Using CX3CR1-deficient mice and electrophysiological means, we investigated in CA3 pyramidal neurons the role of the fractalkine signaling in the maturation of GABAA receptor-mediated synaptic currents and giant depolarizing potentials (GDPs), a network activity important for shaping synaptic connections. In CX3CR1-deficient mice, GABAergic currents were slightly altered, whereas the developmental changes of these currents were comparable with wild-type animals. Despite these minor changes in GABAergic transmission, the GDP frequency was strikingly reduced in CX3CR1-deficient mice compared to wild-type, with no change in the GDP shape and ending period. Collectively, it emerges that, in the neonate hippocampus, the fractalkine signaling pathway tunes GDP activities and is marginally involved in the maturation of GABAergic synapses, suggesting that microglial cells have distinct impact on maturing GABAergic, glutamatergic, and network functions.

Влияние гомоцистеина и его производных на спонтанную сетевую активность в гиппокампе новорожденных крысят

Homocysteine is a sulfur-containing amino acid, which at high concentrations induces neurotoxic effects and causes impairments in the development of the nervous system. In the blood homocysteine is rapidly oxidized, forming disulfide bonds with proteins and low-molecular thiols, and is also converted into homocysteine-thiolactone. Therefore during chronic exposure, homocysteine neurotoxicity is mediated by its derivatives. Our work aimed to study the effects of homocysteine, homocystine, and homocysteine-thiolactone on the network spontaneous activity of rat hippocampal neurons during the first postnatal week. Giant depolarizing potentials (GDP) and multiple-unit activities (MUA) were recorded using the extracellular electrode in the CA3 zone of the hippocampus. It was shown that all three thiol compounds induced an increase of the frequency of GDP and MUA in concentrations of 100 and 500 μM, while homocystine exerted the most profound effects on the network activity. Inhibition of NMDA and AMPA receptors completely prevented the effects of homocysteine and its derivatives on the spontaneous neuronal activity. Thus, homocysteine and its derivatives lead to the increase of network activity of hippocampal neurons of neonatal rats, which may impair the formation of hippocampal neuronal networks in chronic hyperhomocysteinemia; and may cause hyperexcitability and the risk of epilepsy development in the postnatal period.

Giant Depolarizing Potentials Trigger Transient Changes in the Intracellular Cl- Concentration in CA3 Pyramidal Neurons of the Immature Mouse Hippocampus.

Giant depolarizing potentials (GDPs) represent a typical spontaneous activity pattern in the immature hippocampus. GDPs are mediated by GABAergic and glutamatergic synaptic inputs and their initiation requires an excitatory GABAergic action, which is typical for immature neurons due to their elevated intracellular Cl- concentration ([Cl-]i). Because GABAA receptors are ligand-gated Cl- channels, activation of these receptors can potentially influence [Cl-]i. However, whether the GABAergic activity during GDPs influences [Cl-]i is unclear. To address this question we performed whole-cell and gramicidin-perforated patch-clamp recordings from visually identified CA3 pyramidal neurons in immature hippocampal slices of mice at postnatal days 4–7. These experiments revealed that the [Cl-]i of CA3 neurons displays a considerable heterogeneity, ranging from 13 to 70 mM (average 38.1 ± 3.2 mM, n = 36). In accordance with this diverse [Cl-]i, GDPs induced either Cl--effluxes or Cl--influxes. In high [Cl-]i neurons with a negative Cl--driving force (DFCl) the [Cl-]i decreased after a GDP by 12.4 ± 3.4 mM (n = 10), while in low [Cl-]i neurons with a positive DFCl [Cl-]i increased by 4.4 ± 0.9 mM (n = 6). Inhibition of GDP activity by application of the AMPA receptor antagonist CNQX led to a [Cl-]i decrease to 24.7 ± 2.9 mM (n = 8). We conclude from these results, that Cl--fluxes via GABAA receptors during GDPs induced substantial [Cl-]i changes and that this activity-dependent ionic plasticity in neuronal [Cl-]i contributes to the functional consequences of GABAergic responses, emphasizing the concept that [Cl-]i is a state- and compartment-dependent parameter of individual cells.

Bumepamine, a brain-permeant benzylamine derivative of bumetanide, does not inhibit NKCC1 but is more potent to enhance phenobarbital's anti-seizure efficacy

Based on the potential role of Na-K-Cl cotransporters (NKCCs) in epileptic seizures, the loop diuretic bumetanide, which blocks the NKCC1 isoforms NKCC1 and NKCC2, has been tested as an adjunct with phenobarbital to suppress seizures. However, because of its physicochemical properties, bumetanide only poorly penetrates through the blood-brain barrier. Thus, concentrations needed to inhibit NKCC1 in hippocampal and neocortical neurons are not reached when using doses (0.1–0.5 mg/kg) in the range of those approved for use as a diuretic in humans. This prompted us to search for a bumetanide derivative that more easily penetrates into the brain. Here we show that bumepamine, a lipophilic benzylamine derivative of bumetanide, exhibits much higher brain penetration than bumetanide and is more potent than the parent drug to potentiate phenobarbital's anticonvulsant effect in two rodent models of chronic difficult-to-treat epilepsy, amygdala kindling in rats and the pilocarpine model in mice. However, bumepamine suppressed NKCC1-dependent giant depolarizing potentials (GDPs) in neonatal rat hippocampal slices much less effectively than bumetanide and did not inhibit GABA-induced Ca2+ transients in the slices, indicating that bumepamine does not inhibit NKCC1. This was substantiated by an oocyte assay, in which bumepamine did not block NKCC1a and NKCC1b after either extra- or intracellular application, whereas bumetanide potently blocked both variants of NKCC1. Experiments with equilibrium dialysis showed high unspecific tissue binding of bumetanide in the brain, which, in addition to its poor brain penetration, further reduces functionally relevant brain concentrations of this drug. These data show that CNS effects of bumetanide previously thought to be mediated by NKCC1 inhibition can also be achieved by a close derivative that does not share this mechanism. Bumepamine has several advantages over bumetanide for CNS targeting, including lower diuretic potency, much higher brain permeability, and higher efficacy to potentiate the anti-seizure effect of phenobarbital.

Persistent Sodium Current Drives Excitability of Immature Renshaw Cells in Early Embryonic Spinal Networks.

Spontaneous network activity (SNA) emerges in the spinal cord (SC) before the formation of peripheral sensory inputs and central descending inputs. SNA is characterized by recurrent giant depolarizing potentials (GDPs). Because GDPs in motoneurons (MNs) are mainly evoked by prolonged release of GABA, they likely necessitate sustained firing of interneurons. To address this issue we analyzed, as a model, embryonic Renshaw cell (V1R) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1R are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1R already produce GABA in E12.5 embryo, and that V1R make synaptic-like contacts with MNs and have putative extrasynaptic release sites, while paracrine release of GABA occurs at this developmental stage. In addition, we discovered that V1R are spontaneously active during SNA and can already generate several intrinsic activity patterns including repetitive-spiking and sodium-dependent plateau potential that rely on the presence of persistent sodium currents (INap). This is the first demonstration that INap is present in the embryonic SC and that this current can control intrinsic activation properties of newborn interneurons in the SC of mammalian embryos. Finally, we found that 5 μm riluzole, which is known to block INaP, altered SNA by reducing episode duration and increasing inter-episode interval. Because SNA is essential for neuronal maturation, axon pathfinding, and synaptogenesis, the presence of INaP in embryonic SC neurons may play a role in the early development of mammalian locomotor networks.SIGNIFICANCE STATEMENT The developing spinal cord (SC) exhibits spontaneous network activity (SNA) involved in the building of nascent locomotor circuits in the embryo. Many studies suggest that SNA depends on the rhythmic release of GABA, yet intracellular recordings of GABAergic neurons have never been performed at the onset of SNA in the SC. We first discovered that embryonic Renshaw cells (V1R) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents (INaP) in driving plateau potential in V1R and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for INaP in the excitability of V1R and the developing SC.

Developmental excitatory-to-inhibitory GABA polarity switch is delayed in Ts65Dn mice, a genetic model of Down syndrome

Down syndrome (DS) is the most frequent genetic cause of developmental abnormalities leading to intellectual disability. One notable phenomenon affecting the formation of nascent neural circuits during late developmental periods is developmental switch of GABA action from depolarizing to hyperpolarizing mode. We examined properties of this switch in DS using primary cultures and acute hippocampal slices from Ts65Dn mice, a genetic model of DS. Cultures of DIV3–DIV13 Ts65Dn and control normosomic (2 N) neurons were loaded with FURA-2 AM, and GABA action was assessed using local applications. In 2 N cultures, the number of GABA-activated cells dropped from ~100% to 20% between postnatal days 3–13 (P3–P13) reflecting the switch in GABA action polarity. In Ts65Dn cultures, the timing of this switch was delayed by 2–3 days. Next, microelectrode recordings of multi-unit activity (MUA) were performed in CA3 slices during bath application of the GABAA agonist isoguvacine. MUA frequency was increased in P8–P12 and reduced in P14–P22 slices reflecting the switch of GABA action from excitatory to inhibitory mode. The timing of this switch was delayed in Ts65Dn by approximately 2 days. Finally, frequency of giant depolarizing potentials (GDPs), a form of primordial neural activity, was significantly increased in slices from Ts65Dn pups at P12 and P14. These experimental evidences show that GABA action polarity switch is delayed in Ts65Dn model of DS, and that these changes lead to a delay in maturation of nascent neural circuits. These alterations may affect properties of neural circuits in adult animals and, therefore, represent a prospective target for pharmacotherapy of cognitive impairment in DS.

Early Correlated Network Activity in the Hippocampus: Its Putative Role in Shaping Neuronal Circuits

Synchronized neuronal activity occurring at different developmental stages in various brain structures represents a hallmark of developmental circuits. This activity, which differs in its specific patterns among animal species may play a crucial role in de novo formation and in shaping neuronal networks. In the rodent hippocampus in vitro, the so-called giant depolarizing potentials (GDPs) constitute a primordial form of neuronal synchrony preceding more organized forms of activity such as oscillations in the theta and gamma frequency range. GDPs are generated at the network level by the interaction of the neurotransmitters glutamate and GABA which, immediately after birth, exert both a depolarizing and excitatory action on their targets. GDPs are triggered by GABAergic interneurons, which in virtue of their extensive axonal branching operate as functional hubs to synchronize large ensembles of cells. Intrinsic bursting activity, driven by a persistent sodium conductance and facilitated by the low expression of Kv7.2 and Kv7.3 channel subunits, responsible for IM, exerts a permissive role in GDP generation. Here, we discuss how GDPs are generated in a probabilistic way when neuronal excitability within a local circuit reaches a certain threshold and how GDP-associated calcium transients act as coincident detectors for enhancing synaptic strength at emerging GABAergic and glutamatergic synapses. We discuss the possible in vivo correlate of this activity. Finally, we debate recent data showing how, in several animal models of neuropsychiatric disorders including autism, a GDPs dysfunction is associated to morphological alterations of neuronal circuits and behavioral deficits reminiscent of those observed in patients.

Postsynaptic GABA(B) Receptors Contribute to the Termination of Giant Depolarizing Potentials in CA3 Neonatal Rat Hippocampus.

During development, hippocampal CA3 network generates recurrent population bursts, so-called Giant Depolarizing Potentials (GDPs). GDPs are characterized by synchronous depolarization and firing of CA3 pyramidal cells followed by afterhyperpolarization (GDP-AHP). Here, we explored the properties of GDP-AHP in CA3 pyramidal cells using gramicidin perforated patch clamp recordings from neonatal rat hippocampal slices. We found that GDP-AHP occurs independently of whether CA3 pyramidal cells fire action potentials (APs) or remain silent during GDPs. However, the amplitude of GDP-AHP increased with the number of APs the cells fired during GDPs. The reversal potential of the GDP-AHP was close to the potassium equilibrium potential. During voltage-clamp recordings, current-voltage relationships of the postsynaptic currents activated during GDP-AHP were characterized by reversal near the potassium equilibrium potential and inward rectification, similar to the responses evoked by the GABA(B) receptor agonists. Finally, the GABA(B) receptor antagonist CGP55845 strongly reduced GDP-AHP and prolonged GDPs, eventually transforming them to the interictal and ictal-like discharges. Together, our findings suggest that the GDP-AHP involves two mechanisms: (i) postsynaptic GABA(B) receptor activated potassium currents, which are activated independently on whether the cell fires or not during GDPs; and (ii) activity-dependent, likely calcium activated potassium currents, whose contribution to the GDP-AHP is dependent on the amount of firing during GDPs. We propose that these two complementary inhibitory postsynaptic mechanisms cooperate in the termination of GDP.

Hydrogen sulfide inhibits giant depolarizing potentials and abolishes epileptiform activity of neonatal rat hippocampal slices

Hydrogen sulfide (H2S) is an endogenous gasotransmitter with neuroprotective properties that participates in the regulation of transmitter release and neuronal excitability in various brain structures. The role of H2S in the growth and maturation of neural networks however remains unclear. The aim of the present study is to reveal the effects of H2S on neuronal spontaneous activity relevant to neuronal maturation in hippocampal slices of neonatal rats. Sodium hydrosulfide (NaHS) (100μM), a classical donor of H2S produced a biphasic effect with initial activation and subsequent concentration-dependent suppression of network-driven giant depolarizing potentials (GDPs) and neuronal spiking activity. Likewise, the substrate of H2S synthesis l-cysteine (1mM) induced an initial increase followed by an inhibition of GDPs and spiking activity. Our experiments indicate that the increase in initial discharge activity by NaHS is evoked by neuronal depolarization which is partially mediated by a reduction of outward K+ currents. The subsequent decrease in the neuronal activity by H2S appears to be due to the rightward shift of activation and inactivation of voltage-gated Na+ currents, thus preventing network activity. NaHS also reduced N-methyl-d-aspartate (NMDA)-mediated currents, without essential effect on AMPA/kainate or GABAA-mediated currents. Finally, H2S abolished the interictal-like events induced by bicuculline. In summary, our results suggest that through the inhibitory action on voltage-gated Na+ channels and NMDA receptors, H2S prevents the enhanced neuronal excitability typical to early hippocampal networks.

GABA and glycine in the developing brain.

GABA and glycine are major inhibitory neurotransmitters in the CNS and act on receptors coupled to chloride channels. During early developmental periods, both GABA and glycine depolarize membrane potentials due to the relatively high intracellular Cl(-) concentration. Therefore, they can act as excitatory neurotransmitters. GABA and glycine are involved in spontaneous neural network activities in the immature CNS such as giant depolarizing potentials (GDPs) in neonatal hippocampal neurons, which are generated by the synchronous activity of GABAergic interneurons and glutamatergic principal neurons. GDPs and GDP-like activities in the developing brains are thought to be important for the activity-dependent functiogenesis through Ca(2+) influx and/or other intracellular signaling pathways activated by depolarization or stimulation of metabotropic receptors. However, if GABA and glycine do not shift from excitatory to inhibitory neurotransmitters at the birth and in maturation, it may result in neural disorders including autism spectrum disorders.

Interneurons Differentially Contribute to Spontaneous Network Activity in the Developing Hippocampus Dependent on Their Embryonic Lineage.

During nervous system development, immature circuits internally generate rhythmic patterns of electrical activity that promote the establishment of synaptic connections. Immature interneurons are excitatory rather than inhibitory and actively contribute to the generation of these spontaneous network events, referred to as giant depolarizing potentials (GDPs) in the hippocampus. Interneurons can be generally separated into two distinct groups based on their origin in the embryo from the medial or caudal ganglionic eminences (MGE and CGE). Here we show that MGE interneurons play a dominant role in generating GDPs compared with their CGE counterparts. They accomplish this due to their high synaptic connectivity within the local circuitry. Finally, they can control network activity across large regions of the developing hippocampus.

Dynamic Changes from Depolarizing to Hyperpolarizing GABAergic Actions during Giant Depolarizing Potentials in the Neonatal Rat Hippocampus.

During development GABA exerts a depolarizing action on immature neurons. However, at the network level the effects of GABA are complex involving both excitatory and inhibitory actions. Here we show that GABA actions critically depend on the network state. Although GABA depolarizes neurons at rest and at the onset of population bursts, it transiently becomes hyperpolarizing at the peak of the population bursts. These dynamic changes in GABA actions enable GABAergic interneurons not only to initiate the network discharge but also to control excitation to prevent epileptiform synchronization.

Bidirectional global spontaneous network activity precedes the canonical unidirectional circuit organization in the developing hippocampus

Spontaneous network activity is believed to sculpt developing neural circuits. Spontaneous giant depolarizing potentials (GDPs) were first identified with single-cell recordings from rat CA3 pyramidal neurons, but here we identify and characterize a large-scale spontaneous network activity we term global network activation (GNA) in the developing mouse hippocampal slices, which is measured macroscopically by fast voltage-sensitive dye imaging. The initiation and propagation of GNA in the mouse is largely GABA-independent and dominated by glutamatergic transmission via AMPA receptors. Despite the fact that signal propagation in the adult hippocampus is strongly unidirectional through the canonical trisynaptic circuit (dentate gyrus [DG] to CA3 to CA1), spontaneous GNA in the developing hippocampus originates in distal CA3 and propagates both forward to CA1 and backward to DG. Photostimulation-evoked GNA also shows prominent backward propagation in the developing hippocampus from CA3 to DG. Mouse GNA is strongly correlated to electrophysiological recordings of highly localized single-cell and local field potential events. Photostimulation mapping of neural circuitry demonstrates that the enhancement of local circuit connections to excitatory pyramidal neurons occurs over the same time course as GNA and reveals the underlying pathways accounting for GNA backward propagation from CA3 to DG. The disappearance of GNA coincides with a transition to the adult-like unidirectional circuit organization at about 2 weeks of age. Taken together, our findings strongly suggest a critical link between GNA activity and maturation of functional circuit connections in the developing hippocampus.

Intracellular blockade of GABAA receptors in the rat hippocampal neurons

The intracellular blockade of GABAA-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABAA receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABAA receptor channels substitute for Cl− and in which Mg2+ and ATP are omitted to induce rundown of the GABAA receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg2+- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABAA-receptor-mediated synaptic currents and responses induced by the GABAA receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg2+, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities.

Electrophysiological characterization of granule cells in the dentate gyrus immediately after birth

Granule cells (GCs) in the dentate gyrus are generated mainly postnatally. Between embryonic day 10 and 14, neural precursors migrate from the primary dentate matrix to the dentate gyrus where they differentiate into neurons. Neurogenesis reaches a peak at the end of the first postnatal week and it is completed at the end of the first postnatal month. This process continues at a reduced rate throughout life. Interestingly, immediately after birth, GCs exhibit a clear GABAergic phenotype. Only later they integrate the classical glutamatergic trisynaptic hippocampal circuit. Here, whole cell patch clamp recordings, in current clamp mode, were performed from immature GCs, intracellularly loaded with biocytin (in hippocampal slices from P0 to P3 old rats) in order to compare their morphological characteristics with their electrophysiological properties. The vast majority of GCs were very immature with small somata, few dendritic branches terminating with small varicosities and growth cones. In spite of their immaturity their axons reached often the cornu ammonis 3 area. Immature GCs generated, upon membrane depolarization, either rudimentary sodium spikes or more clear overshooting action potentials that fired repetitively. They exhibited also low threshold calcium spikes. In addition, most spiking neurons showed spontaneous synchronized network activity, reminiscent of giant depolarizing potentials (GDPs) generated in the hippocampus by the synergistic action of glutamate and GABA, both depolarizing and excitatory. This early synchronized activity, absent during adult neurogenesis, may play a crucial role in the refinement of local neuronal circuits within the developing dentate gyrus.