Immature Rat Hippocampal Slices Research Articles

NKCC-1 mediated Cl\u2212 uptake in immature CA3 pyramidal neurons is sufficient to compensate phasic GABAergic inputs

Activation of GABAA receptors causes in immature neurons a functionally relevant decrease in the intracellular Cl− concentration ([Cl−]i), a process termed ionic plasticity. Amount and duration of ionic plasticity depends on kinetic properties of [Cl−]i homeostasis. In order to characterize the capacity of Cl− accumulation and to quantify the effect of persistent GABAergic activity on [Cl−]i, we performed gramicidin-perforated patch-clamp recordings from CA3 pyramidal neurons of immature (postnatal day 4–7) rat hippocampal slices. These experiments revealed that inhibition of NKCC1 decreased [Cl−]i toward passive distribution with a time constant of 381 s. In contrast, active Cl− accumulation occurred with a time constant of 155 s, corresponding to a rate of 15.4 µM/s. Inhibition of phasic GABAergic activity had no significant effect on steady state [Cl−]i. Inhibition of tonic GABAergic currents induced a significant [Cl−]i increase by 1.6 mM, while activation of tonic extrasynaptic GABAA receptors with THIP significantly reduced [Cl−]i.. Simulations of neuronal [Cl−]i homeostasis supported the observation, that basal levels of synaptic GABAergic activation do not affect [Cl−]i. In summary, these results indicate that active Cl−-uptake in immature hippocampal neurons is sufficient to maintain stable [Cl−]i at basal levels of phasic and to some extent also to compensate tonic GABAergic activity.

Neurotoxicity of Unconjugated Bilirubin in Mature and Immature Rat Organotypic Hippocampal Slice Cultures

Background: The physiopathology of bilirubin-induced neurological disorders is not completely understood. Objectives: The aim of our study was to assess the effect on bilirubin neurotoxicity of the maturity or immaturity of exposed cells, the influence of different unconjugated bilirubin (UCB) and human serum albumin (HSA) concentrations, and time of UCB exposure. Methods: Organotypic hippocampal slices were exposed for 48 h to different UCB and HSA concentrations after 14 (mature) or 7 (immature) days of in vitro culture. Immature slices were also exposed to UCB and HSA for 72 h. The different effects of exposure time to UCB on neurons and astrocytes were evaluated. Results: We found that 48 h of UCB exposure was neurotoxic for mature rat organotypic hippocampal slices while 72 h of exposure was neurotoxic for immature slices. Forty-eight-hour UCB exposure was toxic for astrocytes but not for neurons, while 72-h exposure was toxic for both astrocytes and neurons. HSA prevented UCB toxicity when the UCB:HSA molar ratio was ≤1 in both mature and immature slices. Conclusions: We confirmed UCB neurotoxicity in mature and immature rat hippocampal slices, although immature ones were more resistant. HSA was effective in preventing UCB neurotoxicity in both mature and immature rat hippocampal slices.

Peroxynitrite alters GABAergic synaptic transmission in immature rat hippocampal slices

Increasing of peroxynitrite (ONOO−) production during ischemia in the immature brain was considered to be associated with impaired cognitive function. GABAergic synapses played an important role in memory formation including the induction of long-term potentiation (LTP) and long-term depression (LTD) in hippocampus. In the present study, we examined the effects of acute exposure of the ONOO− donor, SIN-1 on GABAergic synaptic transmission in immature rat hippocampal slices with whole-cell patch-clamp recordings. The results showed that SIN-1 increased the peak amplitude of evoked inhibitory postsynaptic currents (eIPSCs) and decreased paired pulse ratio via the formation of ONOO−. In addition, it also increased the frequency of spontaneous (but not miniature) IPSCs in a dose-dependent manner without altering amplitudes or rise and decay times of both (sIPSCs and mIPSCs). It further demonstrated that the presynaptic action of SIN-1 was external calcium dependent and was not related to the changes of interneuron excitability. This study provides electrophysiological evidences from developing hippocampal slices to support that SIN-1 enhances action potential-dependent GABA release. It suggests that the potentiation effect of ONOO− may contribute to hyperexcitability and seizures and may underlie one of the mechanisms by which ischemia increases seizure susceptibility in the immature brain.

K+ M‐Current Regulates the Transition to Seizures in Immature and Adult Hippocampus

Loss-of-function mutations in Kv7.2 or Kv7.3 K(+) channel subunits underlies the neonatal epilepsy benign familial neonatal convulsions (BFNC). These two subunits interact to form a functional K(+) channel that underlies the M-current (I(M)), a voltage-dependent noninactivating K(+) current. In BFNC, seizures begin shortly after birth, and spontaneously remit in the first few months of life. The nature of this window of vulnerability is unclear. We address this issue using a hippocampal slice model, to study the effects of I(M) blockade or augmentation on epileptiform activity. We used the Mg(+)(+)-free seizure model in adult and immature (P8-P15) acute rat hippocampal slices. We recorded from both CA1 and CA3 regions using extracellular and intracellular methods. When M-channels are blocked pharmacologically, the transition from interictal to ictal bursting becomes much more likely, especially in immature brain. We also show augmentation of I(M) is effective in stopping ictal events in immature brain, at the developmental age that approximates a human newborn in cortical development. I(M) appears to counter the sustained N-methyl-D-aspartate (NMDA) receptor-mediated depolarizations needed to trigger an ictal event. The increased likelihood of ictal bursting by I(M) blockade is not shared by other selective K(+) channel blockers that increase hippocampal excitability. Voltage-dependent M-channels are activated during interictal bursts and contribute to burst termination. When these channels are compromised, interictal burst duration becomes sufficient to trigger the sustained depolarizations that underlie ictal bursts. This transition to ictal bursts upon I(M) blockade is especially likely to occur in immature hippocampus. This selective function of M-channels likely contributes to the transient window of vulnerability to seizures that occurs with BFNC.

Intracellular energy failure does not underlie hyperthermic spreading depressions in immature rat hippocampal slice

Hyperthermic spreading depression (HSD) in immature rat hippocampal slices is mediated by Na +/K +-ATPase failure. Here, we test whether depleting intracellular ATP serves as a possible mechanism for HSD genesis. Results indicate that (1) pre-incubation with 3 mM creatine for 3 h failed to prevent hyperthermic spreading depression occurrence; and (2) intracellular ATP concentration doubled during experimental hyperthermia. This study suggests that HSD is not be mediated by depletion of intracellular ATP during hyperthermia.

Structural changes at dendritic spine synapses during long-term potentiation.

Two key hypotheses about the structural basis of long-term potentiation (LTP) are evaluated in light of new findings from immature rat hippocampal slices. First, it is shown why dendritic spines do not split during LTP. Instead a small number of spine-like dendritic protrusions may emerge to enhance connectivity with potentiated axons. These 'same dendrite multiple synapse boutons' provide less than a 3% increase in connectivity and do not account for all of LTP or memory, as they do not accumulate during maturation. Second, polyribosomes in dendritic spines served to identify which of the existing synapses enlarged to sustain more than a 30% increase in synaptic strength. Thus, both enhanced connectivity and enlarged synapses result during LTP, with synapse enlargement being the greater effect.

Gamma oscillation underlies hyperthermia-induced epileptiform-like spikes in immature rat hippocampal slices

BackgroundRecently a hyperthermic rat hippocampal slice model system has been used to investigate febrile seizure pathophysiology. Our previous data indicates that heating immature rat hippocampal slices from 34 to 41°C in an interface chamber induced epileptiform-like population spikes accompanied by a spreading depression (SD). This may serve as an in vitro model of febrile seizures.ResultsIn this study, we further investigate cellular mechanisms of hyperthermia-induced initial population spike activity. We hypothesized that GABAA receptor-mediated 30–100 Hz γ oscillations underlie some aspects of the hyperthermic population spike activity. In 24 rat hippocampal slices, the hyperthermic population spike activity occurred at an average frequency of 45.9 ± 14.9 Hz (Mean ± SE, range = 21–79 Hz, n = 24), which does not differ significantly from the frequency of post-tetanic γ oscillations (47.1 ± 14.9 Hz, n = 34) in the same system. High intensity tetanic stimulation induces hippocampal neuronal discharges followed by a slow SD that has the magnitude and time course of the SD, which resembles hyperthermic responses. Both post-tetanic γ oscillations and hyperthermic population spike activity can be blocked completely by a specific GABAA receptor blocker, bicuculline (5–20 μM). Bath-apply kynurenic acid (7 mM) blocks synaptic transmission, but fails to prevent hyperthermic population spikes, while intracellular diffusion of QX-314 (30 mM) abolishes spikes and produces a smooth depolarization in intracellular recording.ConclusionThese results suggest that the GABAA receptor-governed γ oscillations underlie the hyperthermic population spike activity in immature hippocampal slices.

Activation of 5-HT1A receptors inhibits carbachol-stimulated inositol 1,4,5-trisphosphate mass accumulation in the rodent hippocampus.

We have previously demonstrated that 5-HT1A receptor agonists partially prevent the stimulation by carbachol of [3H]-phosphoinositide hydrolysis in immature rat hippocampal slices. This negative modulation has been investigated further by measuring, using a radioreceptor assay, the mass accumulation of IP3. In hippocampal slices from developing rats and in hippocampal neurons, carbachol enhanced the accumulation of IP3 and this response was partially inhibited by 8-OH-DPAT with a potency compatible with the affinity of this agonist for 5-HT1A receptors. The inhibition of the carbachol response by 8-OH-DPAT was non-competitive in nature and 8-OH-DPAT did not affect the inhibitory potency of pirenzepine. The inhibitory effect of 8-OH-DPAT was maintained after washing the slices preincubated with this compound but was not observed on the carbachol-stimulated PIP2 hydrolysis in hippocampal membranes, suggesting that this compound induces long lasting changes of muscarinic receptors and/or their effector mechanism by an indirect action.

Neuroprotective effects of riluzole on [formula omitted] or veratridine-induced neurotoxicity in rat hippocampal slices

The neuroprotective activity of riluzole has been studied on N- methyl- d-aspartate (NMDA)- or veratridine-induced toxicity in immature rat hippocampal slices. Neurodegeneration was assessed by the measurement of LDH release and histology. Veratridine-induced LDH release can be inhibited by 100 μM riluzole (−90%) and by tetrodotoxin (1 μM). Riluzole markedly reduced (−59%) the NMDA-induced LDH release and this protective effect was confirmed by histology. Riluzole inhibited the NMDA-induced LDH release in the presence of tetrodotoxin. Moreover, a pretreatment with pertussis toxin (1 μg/ml) abolished the effect of riluzole against NMDA-induced neurotoxicity. These results support the view that the neuroprotective properties of riluzole could be exerted via two distinct mechanisms of action.

218 The protective effect of NMDA receptor antagonist on the anoxic damage in the immature and mature rat hippocampal slices

218 The protective effect of NMDA receptor antagonist on the anoxic damage in the immature and mature rat hippocampal slices

CPP, an NMDA-receptor antagonist, blocks 4-aminopyridine-induced spreading depression episodes but not epileptiform activity in immature rat hippocampal slices.

Spontaneous episodes of spreading depression (SD) were observed in the CA3 subfield of immature or young (2-30 days postnatally) hippocampal slices perfused with medium containing 4-aminopyridine (4-AP, 50 microM). SD appeared in 34% of the hippocampal slices examined and was more frequently observed in slices obtained from 11 to 20-day-old animals. SD studied with extracellular field potential recordings consisted of large amplitude (18.7 +/- 1.1 mV, mean +/- S.E.M.) negative DC shifts that lasted 30-250 s. Unlike the epileptiform activity that was concomitantly seen during 4-AP application, SD was blocked by the NMDA receptor antagonist 3-((RS)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP, 2-10 microM). In contrast, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 5 microM), a non-NMDA-type receptor antagonist, blocked the epileptiform activity but only increased the interval between SD episodes. These results demonstrate that immature hippocampal tissue is susceptible to SD episodes, when perfused with 4-AP-containing medium, and that the occurrence of these episodes presumably depends on the activation of the NMDA receptor. In addition these findings indicate that SD shows a sensitivity to excitatory amino acid receptor antagonists that differs from that of the epileptiform activity recorded simultaneously.

Development of GABA‐mediated, chloride‐dependent inhibition in CA1 pyramidal neurones of immature rat hippocampal slices.

1. gamma-Aminobutyric acid (GABA)-mediated, Cl(-)-dependent inhibitory postsynaptic potentials (IPSPs) and GABA currents in immature rat hippocampal CA1 neurones were studied using the whole-cell recording technique in brain slices. 2. IPSPs evoked by electrical stimulation were observed in postnatal 2- to 5- (PN2-5), 8- to 13-(PN8-13) and 15- to 20-(PN15-20)day-old CA1 neurones. In the presence of glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonovaleric acid (APV), the reversal potential for the IPSP (EIPSP) was near the resting membrane potential (RMP) in the PN2-5 neurones, but 13 and 25 mV more negative than the RMP in PN8-13 and PN15-20 neurones respectively. IPSPs and GABA currents were blocked by the GABAA-receptor antagonists bicuculline or picrotoxin. 3. The reversal potential for somatic GABA currents (EGABA) was examined in the presence of tetrodotoxin (TTX). There was a strong dependence of the EGABA upon the patch pipette [Cl-] ([Cl-]p). indicating that the GABA currents were mediated by a Cl- conductance. In PN2-5 neurones, EGABA agreed with the value predicted by the Goldman-Hodgkin-Katz equation at given concentrations of internal and external anions permeable through GABA-activated Cl- channels, whereas EGABA in older neurones was 8-18 mV more negative. 4. Examination of the relations between EGABA, holding potential, [Cl-]p and resting conductance indicated that the membrane of the PN2-5 neurones was readily permeable to Cl- which followed a passive Donnan equilibrium. Passive distribution of Cl- played a decreasing role in PN8-13 neurones and in PN15-20 neurones. 5. To assess the contribution of outward Cl- co-transport, bath applications of high K+ or furosemide were performed. High K+ and furosemide caused a reversible positive shift of EGABA in PN15-20 neurones. Raising the temperature moved EGABA to a more negative potential, with a Q10 of 5 mV. A similar change of EGABA in response to high K+, but not to furosemide, was found in PN8-13 neurones. 6. The present data indicate the existence of GABAA-mediated inhibitory synaptic connections in CA1 neurones at the earliest stages of postnatal life. During the first postnatal week, Cl- ions are passively distributed and the EIPSP and EGABA are near the RMP.(ABSTRACT TRUNCATED AT 400 WORDS)

NMDA induces protein kinase C translocation in hippocampal slices of immature rat brain

N- Methyl- d-aspartate (NMDA)-induced translocation of protein kinase C (PKC) from cytosol to membrane fractions was examined by the methods of [ 3H]phorbol 12,13-dibutyrate binding and western blotting in rat hippocampal slices. NMDA and l-glutamate induced translocation of PKC from cytosol to membrane fractions in immature rat hippocampal slices, but not in mature ones. The NMDA-induced translocation of PKC was dependent on Ca 2+. It was inhibited by the NMDA receptor antagonists, 2-amino-5-phosphonovaleric acid and ketamine, but not by Mg 2+ and Zn 2+. These results suggest that stimulation of NMDA receptors enhances Ca 2+ influx and thereby induces translocation of PKC in immature rat hippocampus.

Potential Mechanisms Involved in the Negative Coupling Between Serotonin 5‐HT1A Receptors and Carbachol‐Stimulated Phosphoinositide Turnover in the Rat Hippocampus

Serotonin 5-HT1A receptors have been reported to be negatively coupled to muscarinic receptor-stimulated phosphoinositide turnover in the rat hippocampus. In the present study, we have investigated further the pharmacological specificity of this negative control and attempted to elucidate the mechanism whereby 5-HT1A receptor activation inhibits the carbachol-stimulated phosphoinositide response in immature or adult rat hippocampal slices. Various 5-HT1A receptor agonists were found to inhibit carbachol (10 microM)-stimulated formation of total inositol phosphates in immature rat hippocampal slices with the following rank order of potency (IC50 values in nM): 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (11) greater than ipsapirone (20) greater than gepirone (120) greater than RU 24969 (140) greater than buspirone (560) greater than 1-(m-trifluoromethylphenyl)piperazine (1,500) greater than methysergide (5,644); selective 5-HT1B, 5-HT2, and 5-HT3 receptor agonists were inactive. The potency of the 5-HT1A receptor agonists investigated as inhibitors of the carbachol response was well correlated (r = 0.92) with their potency as inhibitors of the forskolin-stimulated adenylate cyclase in guinea pig hippocampal membranes. 8-OH-DPAT (10 microM) fully inhibited the carbachol-stimulated formation of inositol di-, tris-, and tetrakisphosphate but only partially antagonized (-40%) inositol monophosphate production. The effect of 8-OH-DPAT on carbachol-stimulated phosphoinositide turnover was not prevented by addition of tetrodotoxin (1 microM), by prior destruction of serotonergic afferents, by experimental manipulations causing an increase in cyclic AMP levels (addition of 10 microM forskolin), or by changes in membrane potential (increase in K+ concentration or addition of tetraethylammonium). Prior intrahippocampal injection of pertussis toxin also failed to alter the ability of 8-OH-DPAT to inhibit the carbachol response. Carbachol-stimulated phosphoinositide turnover in immature rat hippocampal slices was inhibited by the protein kinase C activators phorbol 12-myristate 13-acetate (10 microM) and arachidonic acid (100 microM). Moreover, the inhibitory effect of 8-OH-DPAT on the carbachol response was blocked by 10 microM quinacrine (a phospholipase A2 inhibitor) but not by BW 755C (100 microM), a cyclooxygenase and lipoxygenase inhibitor. These results collectively suggest that 5-HT1A receptor activation inhibits carbachol-stimulated phosphoinositide turnover by stimulating a phospholipase A2 coupled to 5-HT1A receptors, leading to arachidonic acid release. Arachidonic acid could in turn activate a gamma-protein kinase C with as a consequence an inhibition of carbachol-stimulated phosphoinositide turnover. This inhibition may be the consequence of a phospholipase C phosphorylation and/or a direct effect on the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Whole-cell patch study of GABAergic inhibition in CA1 neurons of immature rat hippocampal slices

Inhibitory processes mediated by γ-aminobutyric acid (GABA) were studied in immature rat hippocampal slices using the whole-cell patch clamp technique. Orthodromically evoked hyperpolarizing inhibitory postsynaptic potentials (IPSPs) were observed in CA1 neurons of postnatal 2–5 (P2–5) and 7–13 (P7–13) day old rats under conditions of low internal [Cl −]. In the whole-cell voltage-clamp mode, applications of GABA evoked outward currents which reversed at −55 mV and −62 mV in P2–5 and P7–13 CA1 neurons, respectively, with comparable reversal potentials for the IPSPs for each age. An increase of internal [Cl −] caused a depolarizing shift of the GABA reversal potential which followed the Nernst equation. In both groups of neurons, the IPSPs and GABA currents were blocked with the bath applications of bicuculline (10 μM) and picrotoxin (100 μM). We conclude that the GABA A-mediated inhibitory synaptic process exists in P2–5 CA1 neurons and hypothesize that the absence of such IPSPs noted in previous studies of immature CA1 neurons was likely due to higher internal [Cl −] in the more immature neurons.

Developmental changes in serotonin actions in rat hippocampus

Intracellular activity was recorded from neurons in immature rat hippocampal slices. The presence of intrinsic inhibitory synaptic potentials as well as responses to serotonin were assessed in slices of 1, 2 or 3 postnatal weeks of age. Young (1 week) cells had only a marginal hyperpolarizing response to serotonin and no detectable intrinsic inhibitory synaptic potentials. At 2 weeks of age neurons already expressed a fast IPSP (inhibitory postsynaptic potential). The responses to serotonin were different from those of adult cells in that they involved primarily a large decrease in input resistance with only small potential changes. In cells of this age serotonin caused a marked increase in spontaneous IPSP discharges and a blockade of a slow afterhyperpolarization. In 3-week-old rats the fast and slow components of the IPSP were present as in adult and the responses to serotonin included a large hyperpolarization associated with an increase in K conductance, a blockade of slow afterhyperpolarization and a blockade of a slow IPSP, as seen in adult cells. These results indicate that the complex pattern of reactivity to serotonin is differentially regulated in the developing brain.

Calcium dependency of n-methyl- d-aspartate toxicity in slices from the immature rat hippocampus

The literature on ionic requirements for excitotoxity is largely contradictory. Depending on the experimental paradigms, it has been concluded that either Ca 2+ or Na + and Cl − mediate excitotoxicity. In the present study, the dependency on Ca 2+ of N-methyl- d-aspartate-induced damage to neurons in immature rat hippocampal slices was investigated with light microscopy. In addition N-methyl- d-aspartate-induced cell damage was followed by measurement of release of lactate dehydrogenase from slices. When incubated in N-methyl- d-aspartate-containing (100 μM) buffer for 30 min, hippocampal neurons displayed fine chromatin aggregation and swelling of neuronal nuclei and neuropil. Slices incubated in standard medium for 90 min after exposure to N-methyl- d-aspartate-contained a large number of neurons that failed to recover from the initial lesion. The acute edema was at least as severe in slices incubated in N-methyl- d-aspartate-containing, Ca 2+-free buffer. In contrast, clumping of the chromatin could not be observed. CA1 neurons recovered completely from the acute changes, and granule cells recovered to some extent. While omission of Ca 2+ had no obvious morphological effects on the tissue in its own right, the efflux of lactate dehydrogenase was significantly increased after incubation in Ca 2+-free medium. Slices exposed to N-methyl- d-aspartate released approximately twice as much lactate dehydrogenase as controls 1–5 h after the exposure, and the same rate of release was seen if Ca 2+ was absent during N-methyl- d-aspartate treatment. The morphological results suggest that N-methyl- d-aspartate toxicity is Ca 2+dependent in pyramidal cells whereas the toxicity in granule cells is partly Ca 2+ -independent. As the effects on lactate dehydrogenase release of Ca 2+-free medium and N-methyl- d-aspartate in Ca 2+-free medium were not additive, the biochemical data indicate the N-methyl- d-aspartate toxicity is partially Ca 2+-dependent. However, the relation between pathological changes and lactate dehydrogenase release is not entirely clear since slices exposed to N-methyl- d-aspartate in Ca 2+-free buffer release more lactate dehydrogenase than could be expected from the light microscopic observations. Therefore, there is a certain divergence between the morphological and biochemical data.