Mouse Neocortical Slices Research Articles

The March of Epileptic Activity across Cortex is Limited (for a While) by the Powerful Forces of Surrounding Inhibition

Modular Propagation of Epileptiform Activity: Evidence for an Inhibitory Veto in Neocortex. Trevelyan AJ, Sussillo D, Watson BO, Yuste R. J Neurosci 2006;26(48):12447–12455. What regulates the spread of activity through cortical circuits? We present here data indicating a pivotal role for a vetoing inhibition restraining modules of pyramidal neurons. We combined fast calcium imaging of network activity with whole-cell recordings to examine epileptiform propagation in mouse neocortical slices. Epileptiform activity was induced by washing Mg2+ ions out of the slice. Pyramidal cells receive barrages of inhibitory inputs in advance of the epileptiform wave. The inhibitory barrages are effectively nullified at low doses of picrotoxin (2.5–5 μM). When present, however, these inhibitory barrages occlude an intense excitatory synaptic drive that would normally exceed action potential threshold by approximately a factor of 10. Despite this level of excitation, the inhibitory barrages suppress firing, thereby limiting further neuronal recruitment to the ictal event. Pyramidal neurons are recruited to the epileptiform event once the inhibitory restraint fails and are recruited in spatially clustered populations (150–250 μm diameter). The recruitment of the cells within a given module is virtually simultaneous, and thus epileptiform events progress in intermittent (0.5–1 Hz) steps across the cortical network. We propose that the interneurons that supply the vetoing inhibition define these modular circuit territories. Feedforward Inhibition Contributes to the Control of Epileptiform Propagation Speed Trevelyan AJ, Sussillo D, Yuste R. J Neurosci 2007;27(13):3383–3387. It is still poorly understood how epileptiform events can recruit cortical circuits. Moreover, the speed of propagation of epileptiform discharges in vivo and in vitro can vary over several orders of magnitude (0.1–100 mm/s), a range difficult to explain by a single mechanism. We previously showed how epileptiform spread in neocortical slices is opposed by a powerful feedforward inhibition ahead of the ictal wave. When this feedforward inhibition is intact, epileptiform spreads very slowly (100 μm/s). We now investigate whether changes in this inhibitory restraint can also explain much faster propagation velocities. We made use of a very characteristic pattern of evolution of ictal activity in the zero magnesium (0 Mg2+) model of epilepsy. With each successive ictal event, the number of preictal inhibitory barrages dropped, and in parallel with this change, the propagation velocity increased. There was a highly significant correlation ( p < 0.001) between the two measures over a 1,000-fold range of velocities, indicating that feedforward inhibition was the prime determinant of the speed of epileptiform propagation. We propose that the speed of propagation is set by the extent of the recruitment steps, which in turn is set by how successfully the feedforward inhibitory restraint contains the excitatory drive. Thus, a single mechanism could account for the wide range of propagation velocities of epileptiform events observed in vitro and in vivo.

Second Harmonic Generation in Neurons: Electro-Optic Mechanism of Membrane Potential Sensitivity

Second harmonic generation (SHG) from membrane-bound chromophores can be used to image membrane potential in neurons. We investigate the biophysical mechanism responsible for the SHG voltage sensitivity of the styryl dye FM 4-64 in pyramidal neurons from mouse neocortical slices. SHG signals are exquisitely sensitive to the polarization of the incident laser light. Using this polarization sensitivity in two complementary approaches, we estimate a ∼36° tilt angle of the chromophore to the membrane normal. Changes in membrane potential do not affect the polarization of the SHG signal. The voltage response of FM 4-64 is faster than 1 ms and does not reverse sign when imaged at either side of its absorption peak. We conclude that FM 4-64 senses membrane potential through an electro-optic mechanism, without significant chromophore membrane reorientation, redistribution, or spectral shift.

Modular Propagation of Epileptiform Activity: Evidence for an Inhibitory Veto in Neocortex

What regulates the spread of activity through cortical circuits? We present here data indicating a pivotal role for a vetoing inhibition restraining modules of pyramidal neurons. We combined fast calcium imaging of network activity with whole-cell recordings to examine epileptiform propagation in mouse neocortical slices. Epileptiform activity was induced by washing Mg2+ ions out of the slice. Pyramidal cells receive barrages of inhibitory inputs in advance of the epileptiform wave. The inhibitory barrages are effectively nullified at low doses of picrotoxin (2.5-5 microM). When present, however, these inhibitory barrages occlude an intense excitatory synaptic drive that would normally exceed action potential threshold by approximately a factor of 10. Despite this level of excitation, the inhibitory barrages suppress firing, thereby limiting further neuronal recruitment to the ictal event. Pyramidal neurons are recruited to the epileptiform event once the inhibitory restraint fails and are recruited in spatially clustered populations (150-250 microm diameter). The recruitment of the cells within a given module is virtually simultaneous, and thus epileptiform events progress in intermittent (0.5-1 Hz) steps across the cortical network. We propose that the interneurons that supply the vetoing inhibition define these modular circuit territories.

Role of Persistent Sodium Current in Bursting Activity of Mouse Neocortical Networks In Vitro

Most types of electrographic epileptiform activity can be characterized by isolated or repetitive bursts in brain electrical activity. This observation is our motivation to determine mechanisms that underlie bursting behavior of neuronal networks. Here we show that the persistent sodium (Na(P)) current in mouse neocortical slices is associated with cellular bursting and our data suggest that these cells are capable of driving networks into a bursting state. This conclusion is supported by the following observations. 1) Both low concentrations of tetrodotoxin (TTX) and riluzole reduce and eventually stop network bursting while they simultaneously abolish intrinsic bursting properties and sensitivity levels to electrical stimulation in individual intrinsically bursting cells. 2) The sensitivity levels of regular spiking neurons are not significantly affected by riluzole or TTX at the termination of network bursting. 3) Propagation of cellular bursting in a neuronal network depended on excitatory connectivity and disappeared on bath application of CNQX (20 microM) + CPP (10 microM). 4) Voltage-clamp measurements show that riluzole (20 microM) and very low concentrations of TTX (50 nM) attenuate Na(P) currents in the neural membrane within a 1-min interval after bath application of the drug. 5) Recordings of synaptic activity demonstrate that riluzole at this concentration does not affect synaptic properties. 6) Simulations with a neocortical network model including different types of pyramidal cells, inhibitory interneurons, neurons with and without Na(P) currents, and recurrent excitation confirm the essence of our experimental observations that Na(P) conductance can be a critical factor sustaining slow population bursting.

5-HT uptake blockade prevents the increasing effect of K ATP channel blockers on electrically evoked [ 3H]-5-HT release in rat and mouse neocortical slices

To explore if prolonged – as opposed to acute – 5-HT uptake blockade can lead to changes in the function of ATP-dependent potassium (K ATP) channels, we investigated in rat and mouse neocortical slices the effects of K ATP channel blockers on electrically evoked [ 3H]-serotonin ([ 3H]-5-HT) release after short- and long-term exposure to 5-HT uptake blockers. Glibenclamide (1 μM), a K ATP channel blocker, enhanced the electrically evoked [ 3H]-5-HT release by 66 and by 77%, respectively, in rat and in mouse neocortex slices. This effect was confirmed in the rat by tolbutamide (1 μM), another K ATP channel antagonist. After short-term blockade (45 min) of 5-HT uptake, glibenclamide still increased the release of [ 3H]-5-HT in the rat. Glibenclamide, however, failed to enhance [ 3H]-5-HT release after long-term uptake blockade (210 min). In the mouse, however, both short- and long-term inhibition of 5-HT reuptake by citalopram (1 μM) prevented the facilitatory effect of glibenclamide. The Na +/K +-ATPase inhibitor ouabain (3.2 μM) abolished the glibenclamide-induced increase in [ 3H]-5-HT release in both rat and mouse, suggesting that an operative Na +/K +-ATPase is a prerequisite for activation of K ATP channels. The terminal 5-HT 1B autoreceptor-mediated feedback control was involved in the glibenclamide-induced increase in [ 3H]-5-HT release only in mouse neocortical tissue, as evident from the use of the 5-HT 1B autoreceptor ligands metitepin (1 μM) and cyanopindolol (1 μM). These results suggest that in the rat long-term uptake blockade leads to an impaired activity of the Na +/K +-ATPase, which increases intracellular ATP and consequently closes K ATP channels. In the mouse, however, short-term uptake blockade seems to already reduce the activity of the Na +/K +-ATPase and thereby the consumption of ATP. Blockade of 5-HT transporters thus may close K ATP channels through increased intracellular ATP. The following slight depolarisation seems to facilitate 5-HT release. These results may contribute to a better understanding of the mechanisms involved in the clinical time latency of antidepressant efficacy of monoamine uptake blockers.

Dopamine release in human neocortical slices: Characterization of inhibitory autoreceptors and of nicotinic acetylcholine receptor-evoked release

The autoinhibitory control of electrically evoked release of [ 3H]-dopamine and the properties of that induced by nicotinic receptor (nAChR) stimulation were studied in slices of the human neocortex. In both models [ 3H]-dopamine release was action potential-induced and exocytotic. The selective dopamine D 2 receptor agonist (−)-quinpirole reduced electrically evoked release of [ 3H]-dopamine, yielding IC 50 and I max values of 23 nM and 76%, respectively. Also, the effects of several other subtype-selective dopamine receptor ligands confirmed that the terminal dopamine autoreceptor belongs to the D 2 subtype. The autoinhibitory feedback control was slightly operative under stimulation conditions of 90 pulses and 3 Hz, with a biophase concentration of endogenous dopamine of 3.6 nM, and was enhanced under blockade of dopamine reuptake. [ 3H]-dopamine release evoked in an identical manner in mouse neocortical slices was not inhibited by (−)-quinpirole, suggesting the absence of dopamine autoreceptors in this tissue and underlining an important species difference. Also, nAChR stimulation-induced release of [ 3H]-dopamine revealed a species difference: [ 3H]-dopamine release was evoked in human, but not in rat neocortical slices. The nAChRs inducing [ 3H]-dopamine release most probably belong to the α 3/β 2-subtype, according to the potencies and efficacies of subtype-selective nAChR ligands. Part of these receptors may be located on glutamatergic neurons.

Rapid and reactive nitric oxide production by astrocytes in mouse neocortical slices

Nitric oxide (NO), a cellular signaling molecule, is produced in the brain by both neurons and astrocytes. While neurons are capable of rapid release of small amounts of NO serving as neurotransmitter, astrocytic NO production has been demonstrated mainly as a slow reaction to various stress stimuli. Little is known about the role of astrocyte-produced NO. Using the NO indicator 4,5-diaminofluorescein-2 diacetate (DAF-2DA) and acute slices from mouse brain, we distinguished neurons from astrocytes based on their different fluorescence kinetics and pattern, cellular morphology, electrophysiology, and responses to selective nitric oxide synthase (NOS) inhibitors. Typically, astrocytic fluorescence followed neuronal fluorescence with a delay of 1-2 min and was dependent on the inducible NOS isoform (iNOS) activity. Western blot analysis established the presence of functional iNOS in the neocortex. An assay for cell death revealed that most DAF-2DA-positive neurons, but not astrocytes, were damaged. Whole cell recordings from astrocytes confirmed that these cells maintained their membrane potential and passive properties during illumination and afterward. Induction of excitotoxicity by brief application of glutamate triggered an immediate and intense astrocytic response, while high-frequency electrical stimulation failed to do so. The present study demonstrates, for the first time, rapid and massive iNOS-dependent NO production by astrocytes in situ, which appears to be triggered by acute neuronal death. These data may bear important implications for our theoretical understanding and practical management of acute brain insults.

Emergent epileptiform activity in neural networks with weak excitatory synapses

Brain electrical activity recorded during an epileptic seizure is frequently associated with rhythmic discharges in cortical networks. Current opinion in clinical neurophysiology is that strongly coupled networks and cellular bursting are prerequisites for the generation of epileptiform activity. Contrary to expectations, we found that weakly coupled cortical networks can create synchronized cellular activity and seizure-like bursting. Evaluation of a range of synaptic parameters in a detailed computational model revealed that seizure-like activity occurs when the excitatory synapses are weakened. Guided by this observation, we confirmed experimentally that, in mouse neocortical slices, a pharmacological reduction of excitatory synaptic transmission elicited sudden onset of repetitive network bursting. Our finding provides powerful evidence that onset of seizures can be associated with a reduction in synaptic transmission. These results open a new avenue to explore network synchrony and may ultimately lead to a rational approach to treatment of network pathology in epilepsy.

Electrical Coupling among Irregular-Spiking GABAergic Interneurons Expressing Cannabinoid Receptors

Anatomical studies have shown that the G-protein-coupled cannabinoid receptor-1 (CB1) is selectively expressed in a subset of GABAergic interneurons. It has been proposed that these cells regulate rhythmic activity and play a key role mediating the cognitive actions of marijuana and endogenous cannabinoids. However, the physiology, anatomy, and synaptic connectivity of neocortical CB1-expressing interneurons remain poorly studied. We identified a population of CB1-expressing interneurons in layer II/III in mouse neocortical slices. These cells were multipolar or bitufted, had a widely extending axon, and exhibited a characteristic pattern of irregular spiking (IS) in response to current injection. CB1-expressing-IS (CB1-IS) cells were inhibitory, establishing GABAA receptor-mediated synapses onto pyramidal cells and other CB1-IS cells. Recently, electrical coupling among other classes of cortical interneurons has been shown to contribute to the generation of rhythmic synchronous activity in the neocortex. We therefore tested whether CB1-IS interneurons are interconnected via electrical synapses using paired recordings. We found that 90% (19 of 21 pairs) of simultaneously recorded pairs of CB1-IS cells were electrically coupled. The average coupling coefficient was 6%. Signaling through electrical synapses promoted coordinated firing among CB1-IS cells. Together, our results identify a population of electrically coupled CB1-IS GABAergic interneurons in the neocortex that share a unique morphology and a characteristic pattern of irregular spiking in response to current injection. The synaptic interactions of these cells may play an important role mediating the cognitive actions of cannabinoids and regulating coherent neocortical activity.

Synchrony levels during evoked seizure-like bursts in mouse neocortical slices.

Slices (n = 45) from the somatosensory cortex of mouse (P8-13) generated spontaneous bursts of activity (0.10 +/- 0.05 Hz) that were recorded extracellularly. Multiunit action potential (AP) activity was integrated and used as an index of population activity. In this experimental model, seizure-like activity (SLA) was evoked with bicuculline (5-10 microM) or N-methyl-d-aspartate (NMDA, 5 microM). SLA was an episode with repetitive bursting at a frequency of 0.50 +/- 0.06 Hz. To evaluate whether SLA was associated with a change in synchrony, we obtained simultaneous intracellular and extracellular recordings (n = 40) and quantified the relationship between individual cells and the surrounding population of neurons. During the SLA there was an increase in population activity and bursting activity was observed in neurons and areas that were previously silent. We defined synchrony as cellular activity that is consistently locked with the population bursts. Signal-averaging techniques were used to determine this component. To quantitatively assess change in synchronous activity at SLA onset, we estimated the entropy of the single cell's spike trains and subdivided this measure into network burst-related information and noise-related entropy. The burst-related information was not significantly altered at the onset of NMDA-evoked SLA and slightly increased when evoked with bicuculline. The signal-to-noise ratio determined from the entropy estimates showed a significant decrease (instead of an expected increase) during SLA. We conclude that the increased population activity during the SLA is attributed to recruitment of neurons rather than to increased synchrony of each of the individual elements.

Multiple Ways to Enhance Neurotransmitter Release

Neurotransmitter release is dynamic and is influenced by the prior history of neuronal firing and the subsequent entry of Ca 2+ into the nerve terminal. The precise mechanisms linking neuronal activity to various short-term processes that modulate neurotransmitter release and thus synaptic efficacy, however, have remained unclear. Two papers shed light on different aspects of Ca 2+ -dependent regulation of short-term synaptic plasticity. Chi et al. investigated use-dependent modulation of synaptic vesicle mobilization through synapsin I, which regulates vesicle availability through phosphorylation-dependent association with the cytoskeleton. They discovered regulation through different signaling pathways at different frequencies of stimulation. The authors used confocal microscopy to monitor synaptic vesicles simultaneously with synapsin I in cultured rat hippocampal neurons and found that synapsin I dispersal correlated with vesicle turnover across a range of stimulation frequencies. Immunofluorescent analysis together with measurements of vesicle turnover and the dispersal of synapsin I mutants with alterations to their phosphorylation sites indicated that sites phosphorylated by Ca 2+ -calmodulin-dependent protein kinase II regulated mobilization at low stimulation frequencies. In contrast, frequency-dependent phosphorylation and dephosphorylation of sites phosphorylated by mitogen-activated protein kinase and dephosphorylated by calcineurin-modulated mobilization across a range of frequencies. Thus, synapsin I appeared to modulate vesicle availability in response to activation of distinct signaling pathways at different stimulation frequencies. Blatow et al. investigated the effects of endogenous and exogenous Ca 2+ buffers on paired-pulse facilitation, the briefest form of use-dependent enhancement of synaptic efficacy, in synaptically connected neurons in mouse neocortical and hippocampal slices. By using a combination of conditions-including washout of endogenous buffers, mutant mice lacking the endogenous buffer calbindin-D28k (CB), and loading with exogenous buffers or CB-the authors implicated local saturation of CB in paired-pulse facilitation in CB-containing nerve terminals. P. Chi, P. Greengard, T. A. Ryan, Synaptic vesicle mobilization is regulated by distinct synapsin I phosphorylation pathways at different frequencies. Neuron 38 , 69-78 (2003). [Online Journal] M. Blatow, A. Caputi, N. Burnashev, H. Monyer, A. Rozov, Ca 2+ buffer saturation underlies paired pulse facilitation in calbindin-D28k-containing terminals. Neuron 38 , 79-88 (2003). [Online Journal]

High potassium-induced activation of choline-acetyltransferase in human neocortex: implications and species differences

The role of electrical and potassium (K +)-induced depolarisation on choline-acetyltransferase (ChAT) activity in human and mouse neocortical slices was studied. When [ 3 H ]-ACh release was evoked by two K + stimulations in human neocortex, the mean S 2/ S 1 ratio was significantly below unity. ChAT inhibitors, like bromo-acetylcholine and ocadaic acid, raised this ratio by 79 and 63%, respectively, suggesting that the diminished S 2/ S 1 value in the absence of ChAT inhibitors reflected an increased ChAT activity at S 2 following K + depolarisation at S 1. When stimulated electrically, however, the S 2/ S 1 ratio in human neocortex was near unity and ocadaic acid remained without effect. In parallel experiments on mouse neocortical slices, the S 2/ S 1 ratio was near unity in both electrically or K +-evoked [ 3 H ]-ACh release and was not altered by ChAT inhibition. ChAT activity following K + depolarisation was also determined directly. ChAT activation in human neocortical slices was highest at 10 and 20 mM K +. ChAT activity in mouse neocortical tissue was not altered by K + depolarisation. These results suggest that in human, but not in mouse, neocortex ChAT activity may be increased due to ongoing K + depolarisation. This increase of ChAT activity supports a cholinergic degeneration hypothesis which has been entitled “autocannibalism” by Wurtman [TINS 15 (1992) 177].

Activation of protein kinase C increases neuronal excitability by regulating persistent Na+ current in mouse neocortical slices.

Effects of the protein kinase C activating phorbol ester, phorbol 12-myristate 13-acetate (PMA), were studied in whole cell recordings from layer V neurons in slices of mouse somatosensory neocortex. PMA was applied intracellularly (100 nM to 1 microM) to restrict its action to the cell under study. In current-clamp recordings, it enhanced neuronal excitability by inducing a 10- to 20-mV decrease in voltage threshold for action-potential generation. Because spike threshold in neocortical neurons critically depends on the properties of persistent Na+ current (INaP), effects of PMA on this current were studied in voltage clamp. After blocking K+ and Ca2+ currents, INaP was revealed by applying slow depolarizing voltage ramps from -70 to 0 mV. Intracellular PMA induced a decrease in INaP at very depolarized membrane potentials. It also shifted activation of INaP in the hyperpolarizing direction, however, such that there was a significant increase in persistent inward current at potentials more negative than -45 mV. When tetrodotoxin (TTX) was added to the bath, blocking INaP and leaving only an outward nonspecific cationic current (Icat), PMA had no apparent effect on responses to voltage ramps. Thus PMA did not affect Icat, and it did not induce any additional current. Intracellular application of the inactive PMA analogue, 4 alpha-PMA, did not affect INaP. The specific protein kinase C inhibitors, chelerythrine (20 microM) and calphostin C (10 microM), blocked the effect of PMA on INaP. The data suggest that PMA enhances neuronal excitability via a protein kinase C-mediated increase in INaP at functionally critical subthreshold voltages. This novel effect would modulate all neuronal functions that are influenced by INaP, including synaptic integration and active backpropagation of action potential from the soma into the dendrites.

Pattern and pharmacology of propagating epileptiform activity in mouse cerebral cortex.

Multiple extracellular recording electrodes were used to study the intra- and interhemispheric spread of stimulus-evoked epileptiform responses in adult mouse neocortical slices. Bath application of 20 microM bicuculline methiodide induced epileptiform activity that propagated at approximately 0.08 m/s over several millimeters in rostro-caudal and medio-lateral direction within the ipsilateral hemisphere and across the corpus callosum to the contralateral hemisphere. A vertical incision from layer II to subcortical regions did not prevent the spread to remote cortical regions, indicating that layer I plays a major role in the lateral propagation of epileptiform activity. The intra- and interhemispheric spread was not influenced by application of an N-methyl-d-aspartate (NMDA) receptor antagonist, but blocked by an antagonist acting at the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor. The potential role of potassium channel activation in controlling the generation or spread of epileptiform activity was tested by applying the potassium channel opener cromakalim and the serotonin type 1A (5-HT1A) receptor agonist (+/-)-8-hydroxydipropylaminotetralin (8-OH-DPAT) to the disinhibited slices. Whereas cromakalim reduced the neuronal excitability and blocked all epileptiform responses, 8-OH-DAPT did not affect the activity pattern. Our results suggest that propagating epileptiform activity in disinhibited neocortical structures is predominantly mediated by activation of AMPA receptors and controllable by activation of a voltage-dependent potassium current.

Long-term cellular dysfunction after focal cerebral ischemia: in vitro analyses

The long-term (≤six months) functional consequences of permanent middle cerebral artery occlusion were studied with in vitro extra- and intracellular recording techniques in adult mouse neocortical slices. After survival times of one to three days, 28 days and six months, intracellular recordings from layers II/III pyramidal cells in the vicinity of the infarct did not reveal any statistically significant changes in the intrinsic membrane properties when compared to age-matched control animals. However, a pronounced hyperexcitability could be observed upon orthodromic synaptic stimulation in neocortical slices obtained from mice 28 days after induction of ischemia. Low-intensity electrical stimulation of the afferents elicited particularly in this group epileptiform extracellular field potential responses and intracellular excitatory postsynaptic potentials, that were longer in duration as compared to the controls. When the N-methyl- d-aspartate receptor-mediated excitatory postsynaptic potential was pharmacologically isolated in a bathing solution containing 0.1 mM Mg 2+ and 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione, the synaptic responses were longer and larger in the ischemic cortex as compared to the controls. Higher stimulus intensities evoked in normal medium a biphasic inhibitory postsynaptic potential, that contained in the 28 days post-ischemia group a prominent amino-phosphonovaleric acid-sensitive component, indicating a strong concurrent activation of a N-methyl- d-aspartate receptor-mediated excitatory postsynaptic potential. This pronounced co-activation could only be observed in the 28 days ischemic group, and neither after one to three days or six months post-ischemia nor in the controls. The quantitative analysis of the efficiency of stimulus- evoked inhibitory postsynaptic potentials recorded in amino-phosphono-valeric acid revealed a reduction of GABA-mediated inhibition in ischemic cortex. Although this reduction in intracortical inhibition may already contribute to an augmentation of N-methyl- d-aspartate receptor-mediated excitation, our results do also indicate that the function of N-methyl- d-aspartate receptors is transiently enhanced in the ischemic cortex. This transient hyperexcitability does not only cause cellular dysfunction in the vicinity of the infarct, but may also contribute to neuronal damage due to excitotoxicity.

Changes in neurotransmitter sensitivity in the mouse neocortical slice following propranolol and theophylline administration.

1. The mouse neocortical slice has been used to examine the sensitivity of neurones to isoprenaline, 5-hydroxytryptamine (5-HT) and adenosine acutely and following chronic treatment of animals with propranolol or theophylline. 2. While having little effect alone, all three agonists enhanced the d.c. depolarizing potential produced by N-methyl-D-aspartate (NMDA). The effect of (-)-isoprenaline (0.2 microM) was shared by (+)-isoprenaline at the much higher concentration of 10 microM. 3. Superfusion of slices with theophylline or 8-phenyltheophylline blocked responses to adenosine with evidence of selectivity. A single injection of theophylline 24 h before slice preparation did not alter agonist sensitivity, but when administered daily at 100 mg kg-1 for 14 days, the xanthine caused a loss of sensitivity to adenosine and (-)-isoprenaline but not 5-HT. The lower dose of theophylline, 10 mg kg-1 daily, also led to a loss of adenosine responses but no change of sensitivity to the amines. 4. Following the 14 day treatment with theophylline at 100 mg kg-1 daily in two groups of mice, responses to adenosine recovered to control levels after 20 days. 5. Propranolol superfusion blocked responses to both isomers of isoprenaline and 5-HT but did not affect sensitivity to adenosine. 6. Chronic treatment with propranolol at 25 mg kg-1 daily for 14 days induced a loss of sensitivity to (-)-isoprenaline and 5-HT but not adenosine. A lower dose of 5 mg kg-1 daily caused no change in responses to adenosine or 5-HT, but yielded an increased sensitivity to (-)-isoprenaline. 7. The results are discussed with respect to reports of receptor up-regulation in binding studies; caution is clearly required in extrapolating from such work to receptor activity in a functional system, especially in the case of theophylline and adenosine.

Effect of changing extracellular levels of magnesium on spontaneous activity and glutamate release in the mouse neocortical slice.

1. The mouse neocortical slice preparation, maintained in a two compartment, grease gap bath, exhibits spontaneous depolarizing activity (with or without rhythmic after potentials) after perfusion with magnesium-free artificial cerebrospinal fluid. 2. If the magnesium concentration is decrementally lowered over an extended time period, then incrementally raised following a similar time course, the spontaneous depolarizing shift activity shows a hysteresis (with regard to both frequency and amplitude), the depolarizing shifts being more resistant to magnesium during the incremental period. 3. The amino acid content of the perfusing fluid was analysed by high performance liquid chromatography (h.p.l.c.). Although a basal efflux of 6 amino acids was quantifiable, only glutamate levels increased following superfusion of the preparation with magnesium-free, artificial cerebrospinal fluid. 4. Glutamate release increased to 266% of the resting release in the presence of magnesium within the first 12 min of the change into magnesium-free artificial cerebrospinal fluid. This increase in release preceded the onset of spontaneous depolarising activity. The release of glutamate remained elevated at 182% of control up to 60 min after perfusion with magnesium-free buffer, when depolarizing activity was well established. 5. A model is presented and discussed for the genesis and maintenance of the spontaneous depolarizing shifts. It is suggested that the maintenance of this spontaneous activity reflects a long term enhancement of neocortical neurone excitability which may be related to long term potentiation in the hippocampus.

The mouse neocortical slice: Preparation and responses to excitatory amino acids

1. An improved method of preparing wedges of cerebral cortex and corpus callosum from 500 μM thick coronal sections using mouse brain is described. 2. The characteristics of mixing in the two compartment baths is fully reported. 3. Cortical tissue could be depolarized relative to callosal tissue by superfusion of N- methyl- d-aspartate (NMDA), quinolinate, kainate or quisqualate. 4. At higher concentrations of agonists the depolarization was followed by a small hyperpolarization. This hyperpolarization was abolished by 5 μM ouabain. 5. Neither TTX (1 μM) nor bicuculline (50–62.5 μM) significantly affected the amplitude of the responses to the excitatory amino acids. 6. Responses to NMDA and quinolinate were selectively reduced by magnesium ions, 2-amino-5-phosphonovalerate or ketamine.

Chemical Nature of Synaptic Transmission in Vertebrates

Chemical Nature of Synaptic Transmission in Vertebrates