Excitability In Slices Research Articles

Hippocampal Network Hyperactivity After Selective Reduction of Tonic Inhibition in GABAA Receptor α5 Subunit–Deficient Mice

Functionally, gamma-aminobutyric acid receptor (GABAR)-mediated inhibition can be classified as phasic (synaptic) and tonic (extrasynaptic). The GABARs underlying tonic inhibition assemble from subunits different from those responsible for phasic inhibition. We wanted to assess the excitability of hippocampal pyramidal cell (PC) networks following a selective impairment of tonic inhibition. This is difficult to accomplish by pharmacological means. Because the GABAR alpha5 subunits mostly mediate the tonic inhibition in CA1 and CA3 PCs, we quantified changes in tonic inhibition and examined network excitability in slices of adult gabra5-/- mice. In gabra5-/- CA1 and CA3 PCs tonic inhibitory currents were 60 and 53%, respectively, of those recorded in wild type (WT), with no alterations in phasic inhibition. The amount of tonic inhibition recorded in slices was significantly affected by the method of slice storage (interface or submerged chamber). Field recordings in gabra5-/- CA3 pyramidal layer showed an increased network excitability that was decreased by the GABAR agonist muscimol at a concentration that restored the tonic inhibition of gabra5-/- PCs to the WT level without altering phasic inhibition. Through a battery of pharmacological experiments, we have identified delta subunit-containing GABARs as the mediators of the residual tonic inhibition in gabra5-/- PCs. Our study is consistent with an important role of tonic inhibition in the control of hippocampal network excitability and highlights selective enhancers of tonic inhibition as promising therapeutic approaches for diseases involving network hyperexcitability.

Methylphenidate Increases Cortical Excitability via Activation of Alpha-2 Noradrenergic Receptors

Although methylphenidate (MPH), a catecholaminergic reuptake blocker, is prescribed for attention-deficit/hyperactivity disorder, there is a dearth of information regarding the cellular basis of its actions. To address this issue, we used whole-cell patch-clamp recordings to investigate the roles of various catecholamine receptors in MPH-induced changes in cortical neuron excitability. We bath-applied dopamine or noradrenaline receptor antagonists in combination with MPH to pyramidal cells located in deep layers of the infralimbic and prelimbic prefrontal cortices. Application of MPH (10 microM) by itself increased cortical cell excitability in slices obtained from juvenile rats. This MPH-mediated increase in excitability was lost when catecholamines were depleted with reserpine prior to recording, demonstrating the requirement for a presynaptic monoamine component. Antagonist studies further revealed that stimulation of alpha-2 noradrenergic receptors mediates the MPH-induced increase in intrinsic excitability. Dopamine D1 receptors played no observable role in the actions of MPH. We therefore propose that MPH is acting to increase catecholaminergic tone in the PFC, and thereby increases cortical excitability by mediating the disinhibition of pyramidal cells through mechanisms that may include activation of alpha-2 adrenoreceptors located in interneurons.

Nitrous Oxide (Laughing Gas) Facilitates Excitability in Rat Hippocampal Slices through γ-Aminobutyric Acid A Receptor-mediated Disinhibition

Nitrous Oxide (Laughing Gas) Facilitates Excitability in Rat Hippocampal Slices through γ-Aminobutyric Acid A Receptor-mediated Disinhibition

Nicotinic modulation of area postrema neuronal excitability in rat brain slices

We investigated the functions of nicotinic receptor activation on area postrema neurons by making whole-cell recordings in rat brainstem slices. Excitatory responses to nicotine application were found in approximately 78% (35/45) of all cells tested. Responsive cells included both the cells that display the hyperpolarization-activated cation current ( I h) and cells that do not display I h. An inhibitory effect of nicotine was never seen. Current-clamp recordings showed the nicotine-induced depolarization of a cell's membrane potential that could be sufficient to cause spontaneous firing. In voltage-clamp recordings, many cells showed nicotine-induced inward currents (18.3±3.2 pA, n=6) that persisted during pharmacological blockade of synaptic transmission (e.g., zero [Ca 2+] out and 5 mM [Mg 2+] out, n=6/8). Other two cells, however, showed increases in the frequency of excitatory postsynaptic currents (EPSCs), which were blocked by CNQX ( n=2/8). We analyzed miniature EPSCs (mEPSCs) recorded from cells that showed no inward currents but marked increases in the frequency of mEPSCs (0.8±0.2 to 4.8±1.7 Hz, n=4) during nicotine application. Nicotine augmented mEPSC amplitude ( n=4); however, amplitude distribution was not significantly changed in two of four cells tested. We conclude that nicotinic receptors in the rat area postrema can excite cells via (1) a direct post- and/or extrasynaptic mechanism; and (2) an indirect enhancement of glutamate release.

Dopamine-glutamate interactions controlling prefrontal cortical pyramidal cell excitability involve multiple signaling mechanisms.

Although the importance of dopamine (DA) for prefrontal cortical (PFC) cognitive functions is widely recognized, the nature of DA actions in the PFC remains controversial. A critical component in DA actions is its modulation of glutamate transmission, which can be different when specific receptors are activated. To obtain a clear picture of cellular mechanisms involved in these interactions, we studied the effects of DA-glutamate coactivation on pyramidal cell excitability in brain slices obtained from developmentally mature rats using whole-cell patch-clamp recordings. Bath application of NMDA, AMPA, and the D1 agonist SKF38393 induced concentration-dependent excitability increases, whereas bath application of the D2 receptor agonist quinpirole induced a concentration-dependent excitability decrease. The NMDA-mediated response was potentiated by SKF38393. This NMDA-D1 synergism required postsynaptic intracellular Ca2+ and protein kinase A (PKA) and was independent of membrane depolarization. On the other hand, the excitatory effects of both NMDA and AMPA were attenuated by a D2 agonist. Surprisingly, the D2-NMDA interaction was also blocked by the GABA(A) antagonists bicuculline and picrotoxin, suggesting that the inhibitory action of D2 receptors on NMDA-induced responses in the PFC may be mediated by GABAergic interneurons. In contrast, the D2-AMPA interaction involves inhibition of PKA and activation of phospholipase lipase C-IP3 and intracellular Ca2+ at a postsynaptic level. Thus, the modulatory actions of D1 and D2 receptors on PFC pyramidal cell excitability are mediated by multiple intracellular mechanisms and by activation of GABA(A) receptors, depending on the glutamate receptor subtypes involved.

Effects of uniform extracellular DC electric fields on excitability in rat hippocampal slices in vitro.

The effects of uniform steady state (DC) extracellular electric fields on neuronal excitability were characterized in rat hippocampal slices using field, intracellular and voltage-sensitive dye recordings. Small electric fields (</40/ mV mm(-1)), applied parallel to the somato-dendritic axis, induced polarization of CA1 pyramidal cells; the relationship between applied field and induced polarization was linear (0.12 +/- 0.05 mV per mV mm(-1) average sensitivity at the soma). The peak amplitude and time constant (15-70 ms) of membrane polarization varied along the axis of neurons with the maximal polarization observed at the tips of basal and apical dendrites. The polarization was biphasic in the mid-apical dendrites; there was a time-dependent shift in the polarity reversal site. DC fields altered the thresholds of action potentials evoked by orthodromic stimulation, and shifted their initiation site along the apical dendrites. Large electric fields could trigger neuronal firing and epileptiform activity, and induce long-term (>1 s) changes in neuronal excitability. Electric fields perpendicular to the apical-dendritic axis did not induce somatic polarization, but did modulate orthodromic responses, indicating an effect on afferents. These results demonstrate that DC fields can modulate neuronal excitability in a time-dependent manner, with no clear threshold, as a result of interactions between neuronal compartments, the non-linear properties of the cell membrane, and effects on afferents.

Age-related effects of Ginkgo biloba extract on synaptic plasticity and excitability

EGb 761 is a standardized extract from the Ginkgo biloba leaf and is purported to improve age-related memory impairment. The acute and chronic effect of EGb 761 on synaptic transmission and plasticity in hippocampal slices from young adult (8–12 weeks) and aged (18–24 months) C57Bl/6 mice was tested because hippocampal plasticity is believed to be a key component of memory. Acutely applied EGb 761 significantly increased neuronal excitability in slices from aged mice by reducing the population spike threshold and increased the early phase of long-term potentiation, though there was no effect in slices from young adults. In chronically treated mice fed for 30 days with an EGb 761-supplemented diet, EGb 761 significantly increased the population spike threshold and long-term potentiation in slices from aged animals, but had no effect on slices from young adults. The rapid effects of EGb 761 on plasticity indicate a direct interaction with the glutamatergic system and raise interesting implications with respect to a mechanism explaining its effect on cognitive enhancement in human subjects experiencing dementia.

Inhibition of neuronal firing in murine striatal slices by cyclodiene insecticides is mediated by release of dopamine and not GABA antagonism

This study investigated the effects of dieldrin and heptachlor epoxide, members of the cyclodiene class of insecticides, on neuronal firing in murine brain slices. The cyclodienes are environmentally persistent and human exposure to these compounds still occurs. Major physiological effects of cyclodienes include: (1) blockage of 3 GABA A receptors and (2) facilitation of neurotransmitter release with specificity for release of dopamine. Extracellular recordings were used to compare the effects of the cyclodienes to those of the prototypical GABA antagonist, picrotoxinin, on firing rates of striatal neurons. Recordings were made from cells that were inhibited by exogenously applied dopamine, which is an ideal neural substrate for differentiating effects on dopamine release from GABA antagonism. Low micromolar concentrations of cyclodiene caused depression of firing, inconsistent with GABA antagonism. Alternatively, application of picrotoxinin produced a consistent neuronal excitation in slices. The inhibitory action of dieldrin was blocked by the dopamine receptor antagonist fluphenazine, verifying the fact that cyclodiene-released dopamine was mediating the observed depression of striatal neurons. These results suggest that the ability of cyclodienes to evoke neurotransmitter release, especially dopamine, may significantly contribute to the neurotoxicity of these compounds, in vivo.

Effect of halothane on neuronal excitation in the superficial dorsal horn of rat spinal cord slices: evidence for a presynaptic action.

The action of the volatile anaesthetic halothane on optically recorded neuronal excitation in juvenile rat spinal cord slices was investigated. Prolonged neuronal excitation lasting approximately 100 ms was evoked in the superficial dorsal horn after single-pulse dorsal root stimulation that activated both A- and C-fibres. Halothane depressed the neuronal excitation in a concentration-dependent manner (IC(50) 0.21 mm, I(max) 28%). In Ca(2+)-free solution, dorsal root stimulation induced excitation with a short duration of several tens of milliseconds, in which the excitation of the postsynaptic component was largely eliminated. Under these conditions, halothane also depressed the excitation concentration-dependently (IC(50) 0.46 mm, I(max) 60%). Most of the suppression occurred within 5 min of halothane application, and the effect of halothane was fully reversible upon washout of the anaesthetic. Application of bicuculline and strychnine or picrotoxin, or reduction of extracellular Cl(-) concentration ([Cl(-)](o)), had no effect on halothane inhibition. Applications of K(+) channel blockers tetraethyl ammonium, 4-aminopyridine, Cs(+) or Ba(2+) either had no effect or augmented the inhibitory effect of halothane. On the other hand, the degree of inhibition by halothane was found to be dependent on [K(+)](o); the higher [K(+)](o), the larger the depression. In addition, decreases in [Na+]o and [Mg(2+)](o) reduced the excitation similar to that of halothane treatment, and the degree of halothane inhibition became larger with lower [Mg(2+)](o). These results lead to a hypothesis that halothane suppresses the excitation of presynaptic elements by inhibiting presynaptic Na(+) channels by shifting the steady-state inactivation curve in the hyperpolarizing direction.

Sex differences in hippocampal slice excitability: role of testosterone

In vivo fluctuations in gonadal hormones alter hippocampal excitability and modulate both physiological and pathological hippocampal processes. To assess hormonal effects on excitability within a functional hippocampal circuit, extracellular CA1 field responses were compared in slices from intact male, intact female, orchidectomized male, and ovariectomized female rats. Secondly, the effects of in vitro applications of 17-β estradiol, progesterone, or testosterone on baseline excitability of slices from gonadectomized rats were assessed versus pre-hormone baseline measures. Finally, using the in vitro kindling model of slice epileptogenesis, steroid hormone effects on interictal-like activity were also examined. Significant sex differences in excitatory postsynaptic potential amplitude were observed, with slices from males having larger excitatory postsynaptic potential amplitudes than those from females. Gonadectomy significantly decreased excitatory postsynaptic potential amplitude in slices from male rats. Slices from gonadectomized male and female rats also showed a decreased dendritic excitatory postsynaptic potential slope relative to slices from intact male and females rats. In vitro application of testosterone significantly increased excitatory postsynaptic potential amplitudes in slices from both orchidectomized males and ovariectomized females and the population spike amplitude of slices from ovariectomized females. Following in vitro kindling, slices from intact males showed greater spontaneous burst rates than slices from intact females, further suggesting an excitatory effect of testosterone. These results suggest: (1) a sex difference in the level of baseline excitability between slices from intact males and females as measured by excitatory postsynaptic potential amplitudes, (2) testosterone has excitatory effects on baseline physiology and kindled hippocampal responses, and (3) slices from males show a greater level of excitability than those from females in the in vitro kindling model.

Effects of the atypical antidepressant trimipramine on neuronal excitability and long-term potentiation in guinea pig hippocampal slices

Acute effects of the atypical tricyclic antidepressant (TCA) trimipramine on long-term potentiation (LTP) were investigated in this study. Electrically evoked population spikes (PS) were tested under the influence of trimipramine in the CA1 region of the hippocampus. A concentration of 10 μM trimipramine reduced PS amplitudes to 85.1±8.8%. They were reduced to 12.2±8.5% with 50 μM trimipramine. In experiments with LTP, trimipramine 10 μM was applied. Only 8 of 10 tested slices showed LTP. These potentiations were smaller than in the control experiments. LTP could not be induced with 50 μM trimipramine. In conclusion, the influence of trimipramine on LTP is similar to other TCA. It is contended that this contributes to the antidepressant effect of the drug.

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

The HIV glycoproteins gp41 and gp120 cause rapid excitation in rat cortical slices

Inflammation and associated excitotoxicity may play important roles in various neurodegenerative diseases including AIDS dementia. Here we show that exposure of rat parietal cortical slices to the HIV glycoproteins gp120 and gp41 triggered very rapid releases of the neurotransmitters glutamate and [3H]noradrenaline (NA), and of the neuromodulator adenosine. Gp41 was more effective than gp120 at releasing glutamate and [3H]NA, while both glycoproteins were equi-effective at releasing adenosine. The responses to gp120 and gp41 declined rapidly to basal levels following their removal. It seems possible that rapid, inappropriate excitation may occur in the immediate vicinity of HIV infections in the brain, possibly producing some of the transient neurological and psychiatric symptoms associated with AIDS dementia.

Neuronal excitability and conduction velocity changes in hippocampal slices from streptozotocin-treated diabetic rats

The effects of streptozotocin-induced diabetes on the electrophysiological properties of central neurones were investigated in rat hippocampal slices. No differences in membrane potential, input resistance or action potential parameters could be detected in pyramidal or granule cells. However, a slowing of conduction velocity in granule cells and an increase in excitability of fibre volleys in both the perforant pathway and granule cell mossy fibre projections was observed. The lack of effect on evoked fEPSPs in these pathways is also consistent with diabetes-dependent changes in the voltage-dependence of synaptic strength.

Influence of lamotrigine on synaptic transmission, neuronal excitability and LTP in guinea pig hippocampal slices

Influence of lamotrigine on synaptic transmission, neuronal excitability and LTP in guinea pig hippocampal slices

Functional circuitry for the induction of prolonged excitation in the rat spinal dorsal horn.

The neuronal circuitry through which prolonged excitation is generated in the spinal dorsal horn was investigated using optical imaging of neuronal excitation in transverse slices of rat spinal cords. It is known that tetanic stimulation (20 Hz for 1 s) of the dorsal root that activates both A and C primary afferent fibres elicits slow intrinsic optical signals (IOS) in the dorsal horn, seen most intensely in the substantia gelatinosa (SG), lamina II, and that IOS expresses in part the slow synaptic response recorded intracellularly in dorsal horn neurons. We here report that the slow IOS within the SG were completely abolished after an incision was made at the border between the SG and the deeper laminae, but not after an incision within the deeper dorsal horn of the laminae III-V. The result demonstrates directly that, in order to generate prolonged excitation in the SG, the neuronal elements in the deeper dorsal horn must be intact. Thus, the afferent information might be received first by the deeper elements and then transmitted to the SG region, and/or collaboration between the SG and deeper elements is necessary to maintain prolonged excitation in the SG.

Thapsigargin inhibits bicuculline-induced epileptiform excitability in rat hippocampal slices

Evoked field potentials were recorded in the CA3 region of rat hippocampal slices to detect whether intracellular Ca 2+ stores are involved in the epileptiform effects of the two prototypic GABA A antagonists, bicuculline methiodide (BMI) and gabazine (SR-95531; GBZ). Field population spikes gradually increased and became repetitive (epileptiform bursting) in the presence of either BMI (5 μM), or GBZ (5 μM). Thapsigargin (2 μM), a depletor of intracellular Ca 2+ stores, reduced the epileptiform effect of BMI, but had no significant effect on the GBZ-induced hyperexcitability. These data suggest that Ca 2+ release from intracellular stores participates in the epileptiform response of hippocampal CA3 neurons to BMI, but not in the response to GBZ.

Acute and chronic increases in excitability in rat hippocampal slices after perinatal hypoxia In vivo.

We have previously shown that hypoxia induces both acute and chronic epileptogenic effects that are age dependent. Global hypoxia (3-4% O2) induces seizure activity in the developing brain [postnatal day (P)10-12] but not at younger or older ages. Adult rats with prior seizures induced by hypoxia at P10 show increased seizure susceptibility to chemical convulsants compared with controls. In the present study, we tested the hypothesis that acute and chronic epileptogenic effects of hypoxia are demonstrable in hippocampus both in vivo and in vitro. Depth electrode recordings confirmed the presence of ictal activity within hippocampus in P10 rats during global hypoxia. Hippocampal slices prepared from P10 pups killed at 10 min after recovery from hypoxia showed evidence of increased excitability. Extracellular field recordings revealed that the amplitude and duration of long-term potentiation (LTP) was increased significantly in area CA1 of hippocampal slices removed from hypoxic pups. In addition, extracellular recordings within areas CA1 and CA3 showed significantly longer afterdischarge durations in response to kindling stimuli in slices from hypoxic pups compared with controls. To evaluate whether there were also long-term changes in hippocampal excitability, hippocampal slices were prepared from adult rats that had underwent hypoxia at P10 and compared with slices from adult litter-mate controls. A Mg2+-free medium was superfused to induce epileptiform activity within the slices. Extracellular recordings from stratum pyramidale of area CA1 showed that Mg2+-free media induced significantly more frequent ictal discharges in slices from previously hypoxic rats compared with controls. These results provide evidence that the naturally occurring stimulus of hypoxia can result in both acute and chronic changes in the excitability of the CA1 neuronal network. These results parallel our previous in vivo studies demonstrating that global hypoxia acutely increases excitability in the immature brain and that hypoxia during the age window approximately P10 results in long-lasting increases in seizure susceptibility within hippocampus. Our results suggest that the age-dependent epileptogenic effects of hypoxia are in part mediated by a direct and permanent effect on neuronal excitability within hippocampal neuronal networks.

Visualization of neuronal form and function in brain slices by infrared videomicroscopy.

As a standard preparation for neurophysiological experiments, brain slices were introduced some 20 years ago. Although this technique has greatly advanced our understanding of brain physiology, the utility of this preparation has been limited to some extent by the difficulty of visualizing individual neurons in standard thick slices. The use of infrared videomicroscopy has solved this problem. It is now possible to visualize neurons in slices in great detail, and neuronal processes can be patch-clamped under visual control. Infrared videomicroscopy has also been applied successfully to other fields of neuroscience, such as neuronal development and neurotoxicity. A further development of infrared videomicroscopy allows the visualization of the spread of excitation in slices, making the technique a tool for investigating neuronal function and the pharmacology of synaptic transmission.

Interaction between 3 alpha-hydroxy-5 alpha-pregnan-20-one and carbachol in the control of neuronal excitability in hippocampal slices of female rats in defined phases of the oestrus.

The effects of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and carbachol on CA1 and dentate gyrus action potentials were studied in hippocampus slices in premature, follicular and luteal phase rats. A 0.5 nL droplet of allopregnanolone (12.5 mumol L-1), carbachol (5 mumol L-1) or a mixed solution of 12.5 mumol L-1 allopregnanolone and 5 mumol L-1 carbachol was applied locally onto the stratum oriens-pyramidale or granular layer. The amplitude of CA1 population spike (POPSP) was reduced by allopregnanolone (-38 +/- 3%) and carbachol (-21 +/- 4%) in the luteal phase slices. The mixture of allopregnanolone and carbachol doubled this inhibition (-77 +/- 6%). The inhibition caused by allopregnanolone and the mixture of allopregnanolone and carbachol in CA1 was significantly larger in the luteal phase than in the follicular phase (P = 0.02 and 0.0002). In the granular layer of the dentate gyrus, these inhibitions showed no significant difference between the phases. Neither in CA1 nor in the dentate gyrus did the carbachol inhibition differ between the phases. Perfusion with 5-10 mumol L-1 carbachol caused an increasing inhibition of the POPSP during the first few minutes. Thereafter the inhibition gradually diminished and was replaced by a facilitation. The local allopregnanolone inhibition was enhanced by simultaneous carbachol perfusion. Picrotoxin (100 mumol L-1) substantially reduced the allopregnanolone but not the carbachol inhibition. Atropine (10 mumol L-1) blocked the carbachol response, but not the allopregnanolone inhibition. Perfusion with a mixed solution of picrotoxin and atropine reduced, but did not block, the inhibition caused by local application of allopregnanolone or by the mixture of allopregnanolone and carbachol. Our data suggest that neuroprogestine modulators of the GABAA-receptor-mediated inhibition may play a significant role in the control of the cholinergic excitation in the hippocampus.