Compensatory enhancement of intrinsic spiking upon NKCC1 disruption in neonatal hippocampus.

Depolarizing and excitatory GABA actions are thought to be important in cortical development. We show here that GABA has no excitatory action on CA3 pyramidal neurons in hippocampal slices from neonatal NKCC1(-/-) mice that lack the Na-K-2Cl cotransporter isoform 1. Strikingly, NKCC1(-/-) slices generated endogenous network events similar to giant depolarizing potentials (GDPs), but, unlike in wild-type slices, the GDPs were not facilitated by the GABA(A) agonist isoguvacine or blocked by the NKCC1 inhibitor bumetanide. The developmental upregulation of the K-Cl cotransporter 2 (KCC2) was unperturbed, whereas the pharmacologically isolated glutamatergic network activity and the intrinsic excitability of CA3 pyramidal neurons were enhanced in the NKCC1(-/-) hippocampus. Hence, developmental expression of KCC2, unsilencing of AMPA-type synapses, and early network events can take place in the absence of excitatory GABAergic signaling in the neonatal hippocampus. Furthermore, we show that genetic as well as pharmacologically induced loss of NKCC1-dependent excitatory actions of GABA results in a dramatic compensatory increase in the intrinsic excitability of glutamatergic neurons, pointing to powerful homeostatic regulation of neuronal activity in the developing hippocampal circuitry.

Depolarizing GABA acts on intrinsically bursting pyramidal neurons to drive giant depolarizing potentials in the immature hippocampus.

Spontaneous periodic network events are a characteristic feature of developing neuronal networks, and they are thought to play a crucial role in the maturation of neuronal circuits. In the immature hippocampus, these types of events are seen in intracellular recordings as giant depolarizing potentials (GDPs) during the stage of neuronal development when GABA(A)-mediated transmission is depolarizing. However, the precise mechanism how GABAergic transmission promotes GDP occurrence is not known. Using whole-cell, cell-attached, perforated-patch, and field-potential recordings in hippocampal slices, we demonstrate here that CA3 pyramidal neurons in the newborn rat generate intrinsic bursts when depolarized. Furthermore, the characteristic rhythmicity of GDP generation is not based on a temporally patterned output of the GABAergic interneuronal network. However, GABAergic depolarization plays a key role in promoting voltage-dependent, intrinsic pyramidal bursting activity. The present data indicate that glutamatergic CA3 neurons have an instructive, pacemaker role in the generation of GDPs, whereas both synaptic and tonic depolarizing GABAergic mechanisms exert a temporally nonpatterned, facilitatory action in the generation of these network events.

GABA uptake via GABA transporter-1 modulates GABAergic transmission in the immature hippocampus.

GABA uptake limits GABA actions during synaptic responses when the density of active release sites is high or multiple axons are synchronously activated. GABA transporter-1 (GAT-1) is the main neuronal GABA transporter subtype and is already expressed in the early postnatal rat hippocampus. However, previous studies have demonstrated little functional role for the transporter during this developmental period. We used whole-cell voltage-clamp and field-potential recordings in hippocampal slices of neonatal rats (postnatal day 4-5) to study whether GAT-1 plays a role in GABAergic transmission during spontaneous population oscillations, which are seen as "giant depolarizing potentials" (GDPs) in intracellular recordings. We show that the GDP-associated GABAergic current observed in CA3 pyramidal neurons is strongly enhanced by the GAT-1-specific blocker NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride). Our results indicate a novel role for GAT-1 in the control of endogenous activity of the immature hippocampus.

Carbonic anhydrase isoform VII acts as a molecular switch in the development of synchronous gamma-frequency firing of hippocampal CA1 pyramidal cells.

Identification of the molecular mechanisms that enable synchronous firing of CA1 pyramidal neurons is central to the understanding of the functional properties of this major hippocampal output pathway. Using microfluorescence measurements of intraneuronal pH, in situ hybridization, as well as intracellular, extracellular, and K+-sensitive microelectrode recordings, we show now that the capability for synchronous gamma-frequency (20-80 Hz) firing in response to high-frequency stimulation (HFS) emerges abruptly in the rat hippocampus at approximately postnatal day 12. This was attributable to a steep developmental upregulation of intrapyramidal carbonic anhydrase isoform VII, which acts as a key molecule in the generation of HFS-induced tonic GABAergic excitation. These results point to a crucial role for the developmental expression of intrapyramidal carbonic anhydrase VII activity in shaping integrative functions, long-term plasticity and susceptibility to epileptogenesis.

Intrinsic bursting of immature CA3 pyramidal neurons and consequent giant depolarizing potentials are driven by a persistent Na+ current and terminated by a slow Ca2+ -activated K+ current.

The CA3 area of the mature hippocampus is known for its ability to generate intermittent network activity both in physiological and in pathological conditions. We have recently shown that in the early postnatal period, the intrinsic bursting of interconnected CA3 pyramidal neurons generates network events, which were originally called giant depolarizing potentials (GDPs). The voltage-dependent burst activity of individual pyramidal neurons is promoted by the well-known depolarizing action of endogenous GABA on immature neurons. In the present work, we show that a persistent Na+ current, I-Nap, accounts for the slow regenerative depolarization that triggers the intrinsic bursts in the neonatal rat CA3 pyramidal neurons (postnatal day 3-6), while a slow Ca2+ -activated K+ current, sI-K(Ca), is primarily responsible for the postburst slow afterhyperpolarization and consequent burst termination. In addition, we exploited pharmacological data obtained from intracellular recordings to study the mechanisms involved in network events recorded with field potential recordings. The data as a whole indicate that I-Nap and sI-K(Ca) are involved in the initiation and termination, respectively, of the pyramidal bursts and consequent network events underlying GDPs.

Distinct properties of functional KCC2 expression in immature mouse hippocampal neurons in culture and in acute slices.

A hallmark in the development of GABAergic neurotransmission is the switch in GABA(A)-mediated responses from depolarizing to hyperpolarizing. This occurs due to a gradual decrease in the intracellular concentration of chloride caused by the functional expression of the neuron-specific K-Cl cotransporter KCC2. Whether a mere increase in the amount of KCC2 protein is the rate-limiting step in vivo, or a further activation of the otherwise nonfunctional cotransporter is required, is not clear. Imposing a fixed Cl(-) load via patch pipette we measured the resultant somato-dendritic gradients in reversal potential of GABAergic currents to determine the time course of functional maturation of KCC2-mediated Cl(-) extrusion in two preparations: cultured mouse hippocampal neurons plated at embryonic day 17 and CA1 pyramidal cells in acute slices. We found that in immature neurons in both preparations the gradient is initially small or not detectable. It undergoes an abrupt increase at around days 13-14 in culture, while a more gradual increase occurs between postnatal days 5-14 in slices. Consistent with the presence of a nonfunctional form of KCC2 in immature hippocampal neurons grown in culture, application of the broad-spectrum kinase inhibitor staurosporine produces a rapid and potent up-regulation of KCC2 function in these cultured neurons, but not in neonatal slices. Taken together with our previously published data, these results indicate that the functional activity of KCC2 in vivo parallels the developmental expression of the protein, whereas cultured neurons require an additional activation step (mimicked by staurosporine) for KCC2 to become functional.