The cerebral cortex receives an extensive serotonergic (5-hydroxytryptamine, 5-HT) input. Immunohistochemical studies suggest that inhibitory neurons are the main target of 5-HT innervation. In vivo extracellular recordings have shown that 5-HT generally inhibited cortical pyramidal neurons, whereas in vitro studies have shown an excitatory action. To determine the cellular mechanisms underlying the diverse actions of 5-HT in the cortex, we examined its effects on cortical inhibitory interneurons and pyramidal neurons. We found that 5-HT, through activation of 5-HT(2A) receptors, induced a massive enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal neurons, lasting for approximately 6 min. In interneurons, this 5-HT-induced enhancement of sIPSCs was much weaker. Activation of 5-HT(2A) receptors also increased spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons. This response desensitized less and at a slower rate. In contrast, 5-HT slightly decreased evoked IPSCs (eIPSCs) and eEPSCs. In addition, 5-HT via 5-HT(3) receptors evoked a large and rapidly desensitizing inward current in a subset of interneurons and induced a transient enhancement of sIPSCs. Our results suggest that 5-HT has widespread effects on both interneurons and pyramidal neurons and that a short pulse of 5-HT is likely to induce inhibition whereas the prolonged presence of 5-HT may result in excitation.

Chloride (Cl Ϫ ) homeostasis is critical for many cell functions including cell signaling and volume regulation. The action of GABA at GABA A receptors is primarily determined by the concentration of intracellular Cl Ϫ . Developmental regulation of intracellular Cl Ϫ results in a depolarizing response to GABA in immature neocortical neurons and a hyperpolarizing or shunting response in mature neocortical neurons. One protein that participates in Cl Ϫ homeostasis is the neuron-specific K (Krnjevic and Schwartz, 1967;Dreifuss et al., 1969). Subsequent studies in vitro showed that both GABA responses and IPSPs reverse near the expected chloride (Cl Ϫ ) equilibrium potential and are blocked by bicuculline, suggesting mediation by GABA A receptors (GABA A Rs) (Weiss and Hablitz, 1984;Howe et al., 1987). The main permeant ion of GABA A receptor channel complexes is Cl Ϫ , although permeability to bicarbonate ions has been demonstrated (Bormann et al., 1987;Kaila et al., 1993). The membrane potential and the transmembrane gradients of permeant ions determine ionic flux through the GABA A receptor. In most mature neurons, the resting potential is close to the Cl Ϫ equilibrium potential, and activation of GABA A Rs results in shunting inhibition (Andersen et al., 1980) , 1997;Rivera et al., 1999;Williams et al., 1999). The expression of this transporter increases with development and is believed to support the developmental changes in GABA A Rmediated signaling (Lu et al., 1999;Rivera et al., 1999). (Payne, 1997). A consequence of such a mechanism would be the accumulation of [Cl Ϫ ] i , a phenomenon consistent with activity-dependent decreases in GABAergic inhibition (Thompson and Gähwiler, 1989a;Ling and Benardo, 1995).In the present study, we tested the hypothesis that developmental changes in the expression of KCC2 result in the coupling of MATERIALS AND METHODSBrain slice preparation, maintenance, and electrophysiolog ical recording. Animals were housed and handled according to approved guidelines. Brain slices were prepared from postnatal day 3 (P3) to P6 and P18 to P28 animals. Rats were anesthetized with ketamine (100 mg/kg) before decapitation. The brain was rapidly removed and submerged in oxygenated (95% O 2 /5% CO 2 ) ice-cold saline with no added calcium [containing (in mM ):125 NaCl, 3.5 KCl, 26 NaHCO 3 , 10 D-glucose, and 4 MgCl 2 ]. Coronal sections (300 m) containing somatosensory cortex were cut with a Vibratome. Slices were stored in saline consisting of (in mM ): 125 NaCl, 5 KCl, 26 NaHCO 3 , 10 D-glucose, 2.5 CaCl 2 , and 1.3 MgCl 2 , bubbled with 95% O 2 /5% CO 2 .

Spinal cord astrocytes (SCA) have a high permeability to K+ and hence have hyperpolarized resting membrane potentials. The underlying K+ channels are believed to participate in the uptake of neuronally released K+. These K+ channels have been studied extensively with regard to their biophysics and pharmacology, but their molecular identity in spinal cord is currently unknown. Using a combination of approaches, we demonstrate that channels composed of the Kir4.1 subunit are responsible for mediating the resting K+ conductance in SCA. Biophysical analysis demonstrates astrocytic Kir currents as weakly rectifying, potentiated by increasing [K+]o, and inhibited by micromolar concentrations of Ba2+. These currents were insensitive to tolbutemide, a selective blocker of Kir6.x channels, and to tertiapin, a blocker for Kir1.1 and Kir3.1/3.4 channels. PCR and Western blot analysis show prominent expression of Kir4.1 in SCA, and immunocytochemistry shows localization Kir4.1 channels to the plasma membrane. Kir4.1 protein levels show a developmental upregulation in vivo that parallels an increase in currents recorded over the same time period. Kir4.1 is highly expressed throughout most areas of the gray matter in spinal cord in vivo and recordings from spinal cord slices show prominent Kir currents. Electrophysiological recordings comparing SCA of wild-type mice with those of homozygote Kir4.1 knockout mice confirm a complete and selective absence of Kir channels in the knockout mice, suggesting that Kir4.1 is the principle channel mediating the resting K+ conductance in SCA in vitro and in situ.

GABAergic inhibition in the brain can be classified as either phasic or tonic. gamma-Aminobutyric acid (GABA) uptake by GABA transporters (GATs) can limit the time course of phasic currents arising from endogenous and exogenous GABA, as well as decrease a tonically active GABA current. GABA transporter subtypes 1 and 3 (GAT-1 and GAT-3) are the most heavily expressed of the four known GAT subtypes. The role of GATs in shaping GABA currents in the neocortex has not been explored. We obtained patch-clamp recordings from layer II/III pyramidal cells and layer I interneurons in rat sensorimotor cortex. We found that selective GAT-1 inhibition with NO711 decreased the amplitude and increased the decay time of evoked inhibitory postsynaptic currents (IPSCs) but had no effect on the tonic current or spontaneous IPSCs (sIPSCs). GAT-2/3 inhibition with SNAP-5114 had no effect on IPSCs or the tonic current. Coapplication of NO711 and SNAP-5114 substantially increased tonic currents and synergistically decreased IPSC amplitudes and increased IPSC decay times. sIPSCs were not resolvable with coapplication of NO711 and SNAP-5114. The effects of the nonselective GAT antagonist nipecotic acid were similar to those of NO711 and SNAP-5114 together. We conclude that synaptic GABA levels in neocortical neurons are controlled primarily by GAT-1, but that GAT-1 and GAT-2/3 work together extrasynaptically to limit tonic currents. Inhibition of any one GAT subtype does not increase the tonic current, presumably as a result of increased activity of the remaining transporters. Thus neocortical GAT-1 and GAT-2/3 have distinct but overlapping roles in modulating GABA conductances.