Electrophysiology of dentate gyrus granule cells

The orthodromic synaptic responses, membrane properties, and responses of dentate gyrus granule cells (DGCs) to several convulsant agents were studied in the in vitro hippocampal slice preparation. Orthodromic stimulation via the perforant pathway (PP) evoked excitatory-inhibitory postsynaptic potentials (EPSP-IPSP) sequences in 27 of 34 DGCs studied. In the majority, only one action potential could be evoked by supramaximal orthodromic stimulation. In recordings from DGC somata, overshooting spikes could be evoked either orthodromically or by current injections. Small-amplitude, fast transients were seen in 5 of 34 DGCs. The current/voltage (I-V) characteristic of most DGCs was linear throughout a range of membrane potentials between 15 and 20 mV negative and 5 and 15 mV positive to the resting potential. At the extremes of this range nonohmic behavior was noted. Exposure of slices to agents that block IPSPs, such as penicillin, bicuculline, picrotoxin, and media containing low Cl- concentrations, eliminated PP-evoked hyperpolarizations in DGCs and prolonged the repolarizing phase of the PP EPSP. In contrast to findings in hippocampal pyramidal cells and neocortical neurons, blockade of IPSPs did not lead to the development of orthodromically evoked slow depolarizations and burst discharges. After slices were exposed to 5 mM tetraethylammonium, current pulses evoked slow spikes, which were resistant to tetrodotoxin and presumably mediated by Ca2+. Spontaneous burst discharges or bursts evoked by brief depolarizing pulses did not occur under these conditions. Substitution of Ba2+ for Ca2+ in the perfusion solution resulted in development of spontaneous slow membrane depolarizations and burst discharges in DGCs. Burst discharges could be directly evoked and spikes were prolonged and resistant to tetrodotoxin (TTX). After hyperpolarizations lasting 200-1,000 ms, associated with a conductance increase and presumably due to a Ca2+-activated K+ conductance, followed directly evoked spike trains in 5 of 20 DGCs. These data suggest that Ca2+ conductances may be evoked in DGCs under certain circumstances but are not prominent during activation of DGCs under standard in vitro recording conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Newborn granule cells in the ageing dentate gyrus

The dentate gyrus of the hippocampus generates neurons throughout life, but adult neurogenesis exhibits a marked age-dependent decline. Although the decrease in the rate of neurogenesis has been extensively documented in the ageing hippocampus, the specific characteristics of dentate granule cells born in such a continuously changing environment have received little attention. We have used retroviral labelling of neural progenitor cells of the adult mouse dentate gyrus to study morphological properties of neurons born at different ages. Dendritic spine density was measured to estimate glutamatergic afferent connectivity. Fully mature neurons born at the age of 2 months display ∼2.3 spines μm⁻¹ and maintain their overall morphology and spine density in 1-year-old mice. Surprisingly, granule cells born in 10-month-old mice, at which time the rate of neurogenesis has decreased by ∼40-fold, reach a density of dendritic spines similar to that of neurons born in young adulthood. Therefore, in spite of the sharp decline in cell proliferation, differentiation and overall neuronal number, the ageing hippocampus presents a suitable environment for new surviving neurons to reach a high level of complexity, comparable to that of all other dentate granule cells.     

Bidirectional modulation of GABAergic transmission by cholecystokinin in hippocampal dentate gyrus granule cells of juvenile rats

Cholecystokinin (CCK) interacts with two types of G protein-coupled receptors in the brain: CCK-A and CCK-B receptors. Both CCK and CCK-B receptors are widely distributed in the hippocampal formation, but the functions of CCK there have been poorly understood. In the present study, we initially examined the effects of CCK on GABA_A receptor-mediated synaptic transmission in the hippocampal formation and then explored the underlying cellular mechanisms by focusing on the dentate gyrus region, where the highest levels of CCK-binding sites have been detected. Our results indicate that activation of CCK-B receptors initially and transiently increased spontaneous IPSC (sIPSC) frequency, followed by a persistent reduction. The effects of CCK were more evident in juvenile rats, suggesting that they are developmentally regulated. Cholecystokinin failed to modulate the miniature IPSCs recorded in the presence of TTX and the amplitude of the evoked IPSCs, but produced a transient increase followed by a reduction in action potential firing frequency recorded from GABAergic interneurons, suggesting that CCK acts by modulating the excitability of the interneurons to regulate GABA release. Cholecystokinin reduced the amplitude of the after-hyperpolarization of the action potentials, and application of paxilline or charybdotoxin considerably reduced CCK-mediated modulation of sIPSC frequency, suggesting that the effects of CCK are related to the inhibition of Ca²⁺-activated K⁺ currents ( I _K(Ca)). The effects of CCK were independent of the functions of phospholipase C, intracellular Ca²⁺ release, protein kinase C or phospholipase A₂, suggesting a direct coupling between the G proteins of CCK-B receptors and I _K(Ca). Our results provide a novel mechanism underlying CCK-mediated modulation of GABA release.     

Neonatally born granule cells numerically dominate adult mice dentate gyrus

Hippocampal granule cells (GCs) are continuously generated in the subgranular zone of the dentate gyrus (DG) and functionally incorporated to dentate neural circuits even in adulthood. This raises a question about the fate of neonatally born GCs in adult DG. Do they exist until adulthood or are they largely superseded by adult-born GCs? To investigate this question, we examined the contributions of postnatally born GCs to the adult mouse DG. C57BL/6 mice were grouped in three different postnatal (P) ages (group 1: P0, group 2: P7, and group 3: P35) and received a daily bromodeoxyuridine (BrdU) injection for three consecutive days (P0/1/2, P7/8/9, and P35/36/37, respectively) to label dividing cells. At 6 months old, hippocampal sections were prepared from the animals and immunostained with anti-BrdU antibody and an antibody against the homeobox prospero-like protein Prox1, a marker of GCs. We defined BrdU- and Prox1-double positive cells as newborn GCs and analyzed their density and distribution in the granule cell layer (gcl), revealing that newborn GCs of each group still existed 6 months after BrdU injections and that the density of GCs born during P0-2 (group 1) was significantly higher compared with the other groups. Although the density of newborn GCs in the each group did not differ between male and female, the radial distribution of them in gcl showed some differences, that is, male newborn GCs localized toward the molecular layer compared with female ones in group 1, while to the hilus in group 2. These results suggest that GCs born in early postnatal days numerically dominate adult DG and that there exist sex differences in GC localizations which depend on the time when they were born.     

Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus

Throughout adulthood, new neurons are continuously added to the dentate gyrus, a hippocampal subregion that is important in spatial learning. Whether these adult-generated granule cells become functionally integrated into memory networks is not known. We used immunohistochemical approaches to visualize the recruitment of new neurons into circuits supporting water maze memory in intact mice. We show that as new granule cells mature, they are increasingly likely to be incorporated into circuits supporting spatial memory. By the time the cells are 4 or more weeks of age, they are more likely than existing granule cells to be recruited into circuits supporting spatial memory. This preferential recruitment supports the idea that new neurons make a unique contribution to memory processing in the dentate gyrus.     

Analysis of single K(ATP) channels in mammalian dentate gyrus granule cells

ATP-sensitive potassium (K(ATP)) channels are heteromultimer complexes of subunits from members of the inwardly rectifying K(+) channel and the ATP-binding cassette protein superfamilies. K(ATP) channels couple metabolic state to membrane excitability, are distributed widely, and participate in a variety of physiological functions. Understood best in pancreatic beta cells, where their activation inhibits insulin release, K(ATP) channels have been implicated also in postischemia cardio- and neuroprotection. The dentate gyrus (DG) is a brain region with a high density of K(ATP) channels and is relatively resistant to ischemia/reperfusion-induced cell death. Therefore we were interested in describing the characteristics of single K(ATP) channels in DG granule cells. We recorded single K(ATP) channels in 59/105 cell-attached patches from DG granule cells in acutely prepared hippocampal slices. Single-channel openings had an E(K) close to 0 mV (symmetrical K(+)) and were organized in bursts with a duration of 19.3 +/- 1.6 (SE) ms and a frequency of 3.5 +/- 0.8 Hz, a unitary slope conductance of 27 pS, and a low, voltage-independent, probability of opening (P(open), 0.04 +/- 0.01). Open and closed dwell-time histograms were fitted best with one (tau(open) = 1.3 +/- 0.2 ms) and the sum of two (tau(closed,fast) = 2.6 +/- 0.9 ms, tau(closed,slow) = 302.7 +/- 67. 7 ms) exponentials, respectively, consistent with a kinetic model having at least a single open and two closed states. The P(open) was reduced ostensibly to zero by the sulfonylureas, glybenclamide (500 nM, 2/6; 10 microM,11/14 patches) and tolbutamide (20 microM, 4/6; 100 microM, 4/4 patches). The blocking dynamics for glybenclamide included transition to a subconductance state (43.3 +/- 2.6% of control I(open channel)). Unlike glybenclamide, the blockade produced by tolbutamide was reversible. In 5/5 patches, application of diazoxide (100 microM) increased significantly P(open) (0.12 +/- 0.02), which was attributable to a twofold increase in the frequency of bursts (8.3 +/- 2.0 Hz). Diazoxide was without effect on tau(open) and tau(closed,fast) but decreased significantly tau(closed,slow) (24.4 +/- 2.6 ms). We observed similar effects in 6/7 patches after exposure to hypoxia/hypoglycemia, which increased significantly P(open) (0.09 +/- 0.03) and the frequency of bursts (7.1 +/- 1.7 Hz) and decreased significantly tau(closed,slow) (29.5 +/- 1.8 ms). We have presented convergent evidence consistent with single K(ATP) channel activity in DG granule cells. The subunit composition of K(ATP) channels native to DG granule cells is not known; however, the characteristics of the channel activity we recorded are representative of Kir6.1/SUR1, SUR2B-based channels.     

Microglia-associated granule cell death in the normal adult dentate gyrus

Microglial cells are constantly monitoring the central nervous system for sick or dying cells and pathogens. Previous studies showed that the microglial cells in the dentate gyrus have a heterogeneous morphology with multipolar cells in the hilus and fusiform cells apposed to the granule cell layer both at the hilar and at the molecular layer borders. Although previous studies showed that the microglia in the dentate gyrus were not activated, the data in the present study show dying granule cells apposed by Iba1-immunolabeled microglial cell bodies and their processes both at hilar and at molecular layer borders of the granule cell layer. Initially, these Iba1-labeled microglial cells surround individual, intact granule cell bodies. When small openings in the plasma membrane of granule cells are observed, microglial cells are apposed to these openings. When larger openings in the plasma membrane occur at this site of apposition, the granule cells display watery perikaryal cytoplasm, watery nucleoplasm and damaged organelles. Such morphological features are characteristic of neuronal edema. The data also show that following this localized disintegration of the granule cell’s plasma membrane, the Iba1-labeled microglial cell body is found within the electron-lucent perikaryal cytoplasm of the granule cell, where it is adjacent to the granule cell’s nucleus which is deformed. We propose that granule cells are dying by a novel microglia-associated mechanism that involves lysis of their plasma membranes followed by neuronal edema and nuclear phagocytosis. Based on the morphological evidence, this type of cell death differs from either apoptosis or necrosis.     

Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus.

Temporal lobe epilepsy is known to be associated with hyperactivity that is likely to be generated or amplified in the hippocampal formation. The majority of granule cells, the principal cells of the dentate gyrus, are found to be resistant to damage in epilepsy, and may serve as generators of seizures if their inhibition is impaired. Therefore, the parvalbumin-containing subset of interneurons, known to provide the most powerful inhibitory input to granule cell somata and axon initial segments, were examined in human control and epileptic dentate gyrus. A strong reduction in the number of parvalbumin-containing cells was found in the epileptic samples especially in the hilar region, although in some patches of the granule cell layer parvalbumin-positive terminals that form vertical clusters characteristic of axo-axonic cells were more numerous than in controls. Analysis of the postsynaptic target elements of parvalbumin-positive axon terminals showed that they form symmetric synapses with somata, dendrites, axon initial segments and spines as in the control, but the ratio of axon initial segment synapses was increased in the epileptic tissue (control: 15.9%, epileptic: 31.3%). Furthermore, the synaptic coverage of granule cell axon initial segments increased more than three times (control: 0.52, epileptic: 2.10 microm synaptic length/100 microm axon initial segment membrane) in the epileptic samples, whereas the amount of somatic symmetric synapses did not change significantly. Although the number of parvalbumin-positive interneurons is decreased, the perisomatic inhibitory input of dentate granule cells is preserved in temporal lobe epilepsy. Basket and axo-axonic cell terminals - whether positive or negative for parvalbumin - are present, moreover, the axon collaterals targeting axon initial segments sprout in the epileptic dentate gyrus.We suggest that perisomatic inhibitory interneurons survive in epilepsy, but their somadendritic compartment and partly the axon loses parvalbumin or immunoreactivity for parvalbumin. The hyperinnervation of axon initial segments might be a compensatory change in the inhibitory network, but at the same time may lead to a more effective synchronization of granule cell firing that could contribute to the generation or amplification of epileptic seizures.     

Measurements of dendritic conductance changes to GABA in granule cells of the rat dentate gyrus

The magnitude of dendritic conductance changes occurring distantly from the somatic site of recording can be difficult to measure. We have used measurements of the neuronal time constant, tau 0, instead of the neuronal input resistance, RN, to estimate the resistance decrease that accompanies the depolarizing response of the dendrites of granule cells when GABA is applied. From the changes in tau 0, we estimated the reversal potential of the response and found that the conductance change accompanying a given GABA-mediated voltage response as measured at the cell body was the same regardless of where in the dendritic tree the drug was applied. On the other hand, RN changes underestimated the increase in conductance of the GABA responses in the distal dendrites and were not accurate for determining the reversal potential.     

Tetanus toxin blocks inhibition of granule cells in the dentate gyrus of the urethane-anaesthetized rat

Field potentials of dentate granule cells in response to stimulation of the perforant path have been studied before and after injecting tetanus toxin (200 mouse LD50; or phosphate-buffered saline in controls) into the hilus of the dentate gyrus of rats under urethane anaesthesia. Within 1 h of toxin injection, the population spike, but not the slope of the excitatory postsynaptic potential, had increased markedly in amplitude and double or treble population spikes appeared in response to perforant path stimulation. Both paired pulse inhibition (15-ms interval between conditioning and test stimuli) and commissural inhibition (10-ms interval) were substantially reduced by the toxin. Neither multiple spikes nor the reduction in inhibition were seen in controls. Apparent inhibition of the excitatory postsynaptic potential, seen with paired stimuli to the perforant path, was not affected by the toxin. At later times after the injections, a progressive increase in the size of the spikes was seen in the controls while in the toxin animals there was often a secondary decrease in size. It is concluded that tetanus toxin can block both feed-back and feed-forward inhibitory components acting on dentate granule cells. The results are discussed with respect to the role of inhibitory processes in the control of epileptogenesis.     

The GABAergic phenotype of the "glutamatergic" granule cells of the dentate gyrus

The granule cells of the dentate gyrus (DG), origin of the mossy fibers (MFs), have been considered to be glutamatergic. However, data obtained with different experimental approaches in recent years may be calling for a redefinition of their phenotype. Although they indeed release glutamate for fast neurotransmission, immunohistological and molecular biology evidence has revealed that these glutamatergic cells also express GABAergic markers. The granule cell expression of a GABAergic phenotype is developmentally regulated. Electrophysiological studies reveal that during the first 3 weeks of age, mossy fiber stimulation provokes monosynaptic fast inhibitory transmission mediated by GABA, besides the monosynaptic excitatory glutamatergic transmission, onto their targets in CA3. After this age, mossy fiber GABAergic transmission abruptly disappears and the GABAergic markers are undetected. In the adult, the GABAergic markers are upregulated and GABA-mediated transmission emerges after induction of hyperexcitability. The simultaneous glutamate- and GABA-mediated signals share the same plastic and pharmacological characteristics that correspond to neurotransmission of mossy fiber origin. This intriguing evidence gives rise to two fundamental points of discussion. The first is the plausible fact that glutamate and GABA, two neurotransmitters of opposing actions, are coreleased from the mossy fibers. The second relates to its functional implications that can be immediately inferred, as the dentate gyrus can exert direct GABA-mediated excitatory actions early in life and inhibitory actions in young and adult hippocampus. This evidence poses the need to reevaluate and reinterpret some aspects of the physiology of the mossy fiber pathway under normal and pathological conditions. This work reviews the recent evidence that supports the assumption that glutamate and GABA can be coreleased from a single pathway, the mossy fibers, and makes some considerations about its functional implications.     

Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections

Summary Fully mature rats were injected intraperitoneally with ³H-TdR on postnatal day (P) 100. After an additional 28–32 days, a retrograde fluorescent tracer, either FB or DY, was injected into the regio inferior of the hippocampal formation to label granule cells of the dentate gyrus through their mossy fiber axons. Examination of autoradiographs from these brains reveals that ³H-TdR labeled cells within the granule cell layer of the dentate gyrus are often labeled with the retrograde tracer as well. This indicates that within the mature hippocampal formation, newly generated dentate granule cells are capable of extending axonal projections for considerable distances.Abbreviations FB fast blue - DY diamidino yellow dihydrochloride - ³H-TdR tritiated thymidine - UV ultraviolet     

Activation of NMDA receptors in rat dentate gyrus granule cells by spontaneous and evoked transmitter release

Activation of N-methyl-D-aspartate (NMDA) receptors by synaptically released glutamate in the nervous system is usually studied using evoked events mediated by a complex mixture of AMPA, kainate, and NMDA receptors. Here we have characterized pharmacologically isolated spontaneous NMDA receptor-mediated synaptic events and compared them to stimulus evoked excitatory postsynaptic currents (EPSCs) in the same cell to distinguish between various modes of activation of NMDA receptors. Spontaneous NMDA receptor-mediated EPSCs recorded at 34 degrees C in dentate gyrus granule cells (DGGC) have a frequency of 2.5 +/- 0.3 Hz and an average peak amplitude of 13.2 +/- 0.8 pA, a 10-90% rise time of 5.4 +/- 0.3 ms, and a decay time constant of 42.1 +/- 2.1 ms. The single-channel conductance estimated by nonstationary fluctuation analysis was 60 +/- 5 pS. The amplitudes (46.5 +/- 6.4 pA) and 10-90% rise times (18 +/- 2.3 ms) of EPSCs evoked from the entorhinal cortex/subiculum border are significantly larger than the same parameters for spontaneous events (paired t-test, P < 0.05, n = 17). Perfusion of 50 microM D(-)-2-amino-5-phosphonopentanoic acid blocked all spontaneous activity and caused a significant baseline current shift of 18.8 +/- 3.0 pA, thus identifying a tonic conductance mediated by NMDA receptors. The NR2B antagonist ifenprodil (10 microM) significantly reduced the frequency of spontaneous events but had no effect on their kinetics or on the baseline current or variance. At the same time, the peak current and charge of stimulus-evoked events were significantly diminished by ifenprodil. Thus spontaneous NMDA receptor-mediated events in DGGC are predominantly mediated by NR2A or possibly NR2A/NR2B receptors while the activation of NR2B receptors reduces the excitability of entorhinal afferents either directly or through an effect on the entorhinal cells.     

Membrane properties of dentate gyrus granule cells: comparison of sharp microelectrode and whole-cell recordings.

1. Whole-cell and sharp electrode recordings from adult rat dentate gyrus GCs were performed in the 400-microns-thick hippocampal slice preparation maintained at 34 +/- 1 degrees C. Intrinsic membrane properties of granule cells (GCs) were evaluated with the use of a switching current-clamp amplifier. 2. With the whole-cell technique, the average resting membrane potential (RMP) of GCs was -85 mV when a potassium gluconate electrode solution was used versus -74 mV measured with potassium acetate-filled sharp microelectrodes. The membrane voltage response to injected current was linear over two membrane potential ranges, greater than 10 mV hyperpolarized from RMP and between 10 mV more negative than RMP and -62 mV. The average input resistances (RN) calculated over these ranges were 107 and 228 M omega in the whole-cell recordings versus 37 and 54 M omega in the sharp electrode recordings. There was no correlation between RMP and RN with either recording technique. The membrane time constant (tau m) determined at the RMP was 26.9 ms for whole-cell recordings and 13.9 ms for sharp electrode recordings. 3. There was no evidence of time-dependent changes in RMP, RN, and tau m in whole-cell recordings, although the slow inward rectification seen at hyperpolarized potentials decreased over 30-60 min. Addition of calcium buffers to the whole-cell recording solution did not result in a significant change in the average RMP, the average RN, or the average tau m. 4. Action potential threshold was comparable in whole-cell (-49 mV) and sharp electrode (-52 mV) recordings, but action potential amplitude was larger in whole-cell (126 mV) than in sharp electrode (106 mV) recordings. Spike frequency adaptation was present in the whole-cell recordings and could be abolished by addition of calcium buffers to the electrode solution. 5. We estimated rho, the ratio of dendritic to somatic conductance, to be 5.1 for the whole-cell records and 2.1 for sharp electrode recordings. The electrotonic length of the equivalent cylinder representing the cell processes was estimated to be 0.49 from the whole-cell data and 0.79 from the sharp electrode recordings. This implies that at rest there is only a 10% decrement in steady-state membrane voltage along the length of the dendrite due to shunting across the membrane resistance; small synaptic events occurring in the distal dendritic tree will therefore have a more substantial influence on the soma than previous analyses suggested.(ABSTRACT TRUNCATED AT 400 WORDS)     

一种获得小鼠背侧齿状回健康颗粒细胞的脑片制备方法

目的:研究获得背侧齿状回健康颗粒细胞的制片方法。方法:成年小鼠。(1)控制切片角度,尽量保留颗粒细胞顶树突的完整沿水平20~30°切片角度获取海马横向(transverse)脑片;(2)注意区分脑片的正、反面选择海马脑片反面的背侧齿状回上片;(3)合并使用其它保护脑片细胞活性的措施,如采用无钠切片液,切片液中添加抗氧化剂等。结果:本方法所得特定角度的海马横向脑片,其上有大量颗粒细胞表现出轮廓清晰、柔和、胞体圆润的健康状态,且记录出颗粒细胞兴奋性微小突触后电流(mEPSCs)的频率和幅度,均较传统海马横向脑片方法有明显增加。结论:适当的切片角度可保持颗粒细胞远端树突末梢的完整,提高细胞的活性,获得大量健康颗粒细胞。提示在制备脑片时要考虑感兴趣细胞整体的走行,保证切片时细胞整体的完整性。     

FGF2 modulates the voltage-dependent K current and changes excitability of rat dentate gyrus granule cells

Fibroblast growth factor 2 (FGF2) is involved in hippocampus-dependent learning. In this study, the effects of FGF2 on the excitability were investigated in granule cells of rat dentate gyrus. Hippocampal slices were used to perform patch clamp recordings in granule cells. Extracellularly applied FGF2 early quenched the depolarization-induced repetitive firing, suggesting a decreased excitability under these conditions. Consistently, transient and sustained voltage-gated K⁺ currents decreased in a dose-dependent manner, repolarization phase of action potential was slowed down, afterhyperpolarization was reduced, and membrane resistance was decreased. These effects were not mediated by tyrosine kinase FGF2 receptors. Moreover, an involvement of G protein signaling was ruled out, as well as an intracellular action of FGF2. Considering the relationship between FGF2 and hippocampal functions, the modulation of neuron excitability by activity-driven FGF2 release may be regarded as a part of a homeostatic mechanism of self-regulation of hippocampal activity.     

Properties of two voltage-activated potassium currents in acutely isolated juvenile rat dentate gyrus granule cells.

1. The properties of outward currents were investigated in acutely isolated dentate gyrus granule cells at postnatal ages of day 5-7, 10-14, 18-24 (P5-7, P10-14, P18-24) and at adulthood (2-3 mo), with the use of the whole-cell patch-clamp technique. 2. Kinetic analysis and pharmacological properties showed that an A-type K+ current (IA) and a delayed rectifier current (IK) were present in these cells. 3. IA in P10-14 cells activated and inactivated rapidly with a decay time constant of 7.5 +/- 2.1 (SD) ms with command pulses to +30 mV. The removal of inactivation was monoexponential with a time constant of 23.1 ms (holding potential, -50 mV; conditioning voltage steps of varying duration to -110 mV). V 1/2 of the Boltzmann function describing steady-state inactivation was -65.1 +/- 1.8 mV with a slope factor of -6.0. IA was sensitive to 5 mM 4-aminopyridine (4-AP) but not to 10 mM tetraethylammonium (TEA). 4. IK in P10-14 cells displayed a voltage-dependent activation time constant (4.3 +/- 0.8 ms for command pulses to +30 mV and 16.2 +/- 2.4 for command pulses to -10 mV) and a double-exponential decay (time constants 194 +/- 21 and 1,625 +/- 254 ms). The rate constant of removal of inactivation was 332.1 ms. IK showed a reduction by 61.4 +/- 5.3% with 10 mM TEA and was partially blocked by 5 mM 4-AP in a subpopulation of cells. 5. Whereas IA remained stable over time, IK showed a substantial reduction of current amplitude by 67% after 30 min of cell perfusion through the patch pipette. The time course of this reduction was monoexponential with a time constant of 6.9 min and was partly due to a shift in V1/2 of the steady-state inactivation from -79.2 to -99.6 mV. 6. IA and IK remained stable with respect to kinetic properties during ontogenesis. However, the relative contribution and pharmacological properties of the investigated K+ currents varied with age. Although IA dominated in P5-7 cells, IK was prominent in most older cells. Five millimolars 4-AP reduced IA by 40.7 +/- 26.7% in P5-7 cells and blocked IA completely in 80% of investigated P10-14 cells. Similar changes were observed for the effects of 4-AP on IK (18.7% depression in the age group P5-8, 46.1% in the age group P10-14, and 45.7% in adult animals).