Carbenoxolone depresses spontaneous epileptiform activity in the CA1 region of rat hippocampal slices

An important contributor to the generation of epileptiform activity is the synchronization of burst firing in a group of neurons. The aim of this study was to investigate whether gap junctions are involved in this synchrony using an in vitro model of epileptiform activity. Hippocampal slices (400 μm) were prepared from female Sprague–Dawley rats (120–170 g). The perfusion of slices with a medium containing no added magnesium and 4-aminopyridine (50 μM) resulted in the generation of spontaneous bursts of population spikes of a fast frequency along with less frequent negative-going bursts. The frequency of the bursts produced was consistent over a 3 h period. Carbenoxolone (100 μM), a gap junction blocker and mineralocorticoid agonist, perfused for 75 min, reduced the frequency of both types of spontaneous burst activity. Perfusion of spironolactone (1 μM), a mineralocorticosteroid antagonist, for 15 min prior to and during carbenoxolone perfusion did not alter the ability of carbenoxolone to depress the frequency of spontaneous activity. The incubation of hippocampal slices in carbenoxolone prior to recording increased the time taken for the spontaneous activity to start on change to the zero magnesium/4-aminopyridine medium and decreased the total number of spontaneous bursts over the first 60 min period.

The ability of carbenoxolone to delay induction of epileptiform activity and reduce established epileptiform activity suggests that gap junctions contribute to the synchronization of neuronal firing in this model.     

Involvement of electrical coupling in the in vivo ictal epileptiform activity induced by 4-aminopyridine in the neocortex

In the present study we have investigated the possible role of gap junctions in the induction and manifestation of 4-aminopyridine-induced acute seizure activity both at the primary focus and at the mirror focus in anaesthetized rats by combining electrophysiological, pharmacological and molecular biological techniques. In the course of the intracellular recordings, unusual firing patterns that are assumed to be mediated by electrical coupling and appearing either randomly or in close time-locked manner with the ictal discharges were observed. In another series of experiments, a significant decrease in the intensity of seizure activity of the already active epileptic foci was detected when electrical synaptic transmission was blocked by carbenoxolone either at the primary focus or at the mirror focus. When electrical synaptic transmission was depressed relative to the initial baseline prior to the induction of epileptic focus, only a mild influence on the induction of seizure discharges occurred. The role of the gap junctional communication in the epileptiform activity was further investigated by following the expression pattern of two connexin genes. Both, connexin-32 and connexin-43 mRNA levels were significantly elevated at the primary focus as well as at the mirror focus, after 60 min of repeated ictal discharges.We conclude that gap junction communication probably became a part of the neuronal synchronization both in the primary and in the secondarily-induced acute epileptiform activity in the neocortex in vivo. These results, together with earlier observations, indicate a direction for the development of new drugs targeting gap junctions for therapeutic intervention.     

Evidence against a role of gap junctions in vestibular compensation

Vestibular compensation following unilateral labyrinthectomy is associated with modifications of the membrane and firing properties of central vestibular neurons. To determine whether gap junctions could be involved in this process, immunofluorescent detection of neuronal connexin 36 and astrocytic connexin 43 was performed in the medial vestibular nucleus (MVN) of rats. In non-lesioned animals, strong staining was observed with anti-connexin 43 antibodies, while moderate staining was obtained with the anti-connexin 36 antibody. However, the expression of either type of connexin was not modified following unilateral labyrinthectomy. These morphological observations were complemented by pharmacological tests performed during extracellular recordings of MVN neurons in guinea pig brainstem slices. In non-lesioned animals, the gap junction blocker carbenoxolone reversibly decreased or suppressed the spontaneous discharge of about 60% of MVN neurons. This reduction was often associated with a long-duration disruption of the regularity of spike discharge. Both effects were mimicked by several other gap junction blockers, but not by glycyrrhizic acid, an analog of carbenoxolone that does not block gap junctions but reproduces its non-specific effects, nor by the selective inhibitor of astrocytic connexin-based networks endothelin-1. Similar effects of carbenoxolone were obtained on the spontaneous activity of ipsilesional MVN neurons recorded in brainstem slices taken from labyrinthectomized animals. Altogether, these results suggest that neuronal gap junctions are involved in shaping the spontaneous activity of MVN neurons. However, unilateral labyrinthectomy does not affect the expression of gap junctions in vestibular nuclei nor their implication in the regulation of neuronal activity.     

Sharp wave-like activity in the hippocampus in vitro in mice lacking the gap junction protein connexin 36

Bath application of kainate (100-300 nM) induced a persistent gamma-frequency (30-80 Hz) oscillation that could be recorded in stratum radiatum of the CA3 region in vitro. We have previously described that in knockout mice lacking the gap junction protein connexin 36 (Cx36KO), gamma-frequency oscillations are reduced but still present. We now demonstrate that in the Cx36KO mice, but not in wild-type (WT), large population field excitatory postsynaptic potentials, or sharp wave-burst discharges, also occurred during the on-going gamma-frequency oscillation. These spontaneous burst discharges were not seen in WT mice. Burst discharges in the Cx36KO mice occurred with a mean frequency of 0.23 +/- 0.11 Hz and were accompanied by a series of fast (approximately 60-115 Hz) population spikes or "ripple" oscillations in many recordings. Intracellular recordings from CA3 pyramidal cells showed that the burst discharges consisted of a depolarizing response and presumed coupling potentials (spikelets) could occasionally be seen either before or during the burst discharge. The burst discharges occurring in Cx36KO mice were sensitive to gap junctions blockers as they were fully abolished by carbenoxolone (200 microM). In control mice we made several attempts to replicate this pattern of sharp wave activity/ripples occurring with the on-going kainate-evoked gamma-frequency oscillation by manipulating synaptic and electrical signaling. Partial disruption of inhibition, in control slices, by bath application of the gamma-aminobutyric acid-A (GABA(A)) receptor antagonist bicuculline (1-4 microM) completely abolished all gamma-frequency activity before any burst discharges occurred. Increasing the number of open gap junctions in control slices by using trimethylamine (TMA; 2-10 mM), in conjunction with kainate, failed to elicit any sharp wave bursts or fast ripples. However, bath application of the potassium channel blocker 4-aminopyridine (4-AP; 20-80 microM) produced a pattern of activity in control mice (13/16 slices), consisting of burst discharges occurring in conjunction with kainate-evoked gamma-frequency oscillations, that was similar to that seen in Cx36KO mice. In a few cases (n = 9) the burst discharges were accompanied by fast ripple oscillations. Carbenoxolone also fully blocked the 4-AP-evoked burst discharges (n = 5). Our results show that disruption of electrical signaling in the interneuronal network can, in the presence of kainate, lead to the spontaneous generation of sharp wave/ripple activity similar to that observed in vivo. This suggests a complex role for electrically coupled interneurons in the generation of hippocampal network activity.     

Coupling an HCN2-expressing cell to a myocyte creates a two-cell pacing unit

We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell–cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell–cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of ∼0.6 to 1.7 Hz (1.09 ± 0.05 Hz). Addition of carbenoxolone (200 μ m ), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 μ m 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.     

Synaptic and nonsynaptic contributions to giant ipsps and ectopic spikes induced by 4-aminopyridine in the hippocampus in vitro

Hippocampal slices bathed in 4-aminopyridine (4-AP, < or =200 microM) exhibit 1) spontaneous large inhibitory postsynaptic potentials (IPSPs) in pyramidal cells, which occur without the necessity of fast glutamatergic receptors, and which hence are presumed to arise from coordinated firing in populations of interneurons; 2) spikes of variable amplitude, presumed to be of antidromic origin, in some pyramidal cells during the large IPSP; 3) bursts of action potentials in selected populations of interneurons, occurring independently of fast glutamatergic and of GABA(A) receptors. We have used neuron pairs, and a large network model (3,072 pyramidal cells, 384 interneurons), to examine how these phenomena might be inter-related. Network bursts in electrically coupled interneurons have previously been shown to be possible with dendritic gap junctions, when the dendrites were capable of spike initiation, and when action potentials could cross from cell to cell via gap junctions; recent experimental data showing that dendritic gap junctions between cortical interneurons lead to coupling potentials of only about 0.5 mV argue against this mechanism, however. We now show that axonal gap junctions between interneurons could also lead to network bursts; this concept is consistent with the occurrence of spikelets and partial spikes in at least some interneurons in 4-AP. In our model, spontaneous antidromic action potentials can induce spikelets and action potentials in principal cells during the large IPSP. The probability of observing this type of activity increases significantly when axonal gap junctions also exist between pyramidal cells. Sufficient antidromic activity in the model can lead to epileptiform bursts, independent of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors, in some principal cells, preceded by IPSPs and spikelets. The model predicts that gap junction blockers should suppress large IPSPs observed in 4-AP and should also reduce the probability of observing antidromic activity, or bursting, in pyramidal cells. Experiments show that, indeed, the gap junction blocking compound carbenoxolone does suppress spontaneous large IPSCs, occurring in 4-AP plus ionotropic glutamate blockers, together with a GABA(B) receptor blocker; carbenoxolone also suppresses large, fast inward currents, corresponding to ectopic spikes, which occur in 4-AP. Carbenoxolone does not suppress large depolarizing IPSPs induced by tetanic stimulation. We conclude that in 4-AP, axonal gap junctions could, at least in principle, account in part for both the large IPSPs, and for the antidromic activity in pyramidal neurons.     

Connexin-47 and connexin-32 in gap junctions of oligodendrocyte somata, myelin sheaths, paranodal loops and Schmidt-Lanterman incisures: implications for ionic homeostasis and potassium siphoning

The subcellular distributions and co-associations of the gap junction-forming proteins connexin 47 and connexin 32 were investigated in oligodendrocytes of adult mouse and rat CNS. By confocal immunofluorescence light microscopy, abundant connexin 47 was co-localized with astrocytic connexin 43 on oligodendrocyte somata, and along myelinated fibers, whereas connexin 32 without connexin 47 was co-localized with contactin-associated protein (caspr) in paranodes. By thin-section transmission electron microscopy, connexin 47 immunolabeling was on the oligodendrocyte side of gap junctions between oligodendrocyte somata and astrocytes. By freeze-fracture replica immunogold labeling, large gap junctions between oligodendrocyte somata and astrocyte processes contained much more connexin 47 than connexin 32. Along surfaces of internodal myelin, connexin 47 was several times as abundant as connexin 32, and in the smallest gap junctions, often occurred without connexin 32. In contrast, connexin 32 was localized without connexin 47 in newly-described autologous gap junctions in Schmidt-Lanterman incisures and between paranodal loops bordering nodes of Ranvier. Thus, connexin 47 in adult rodent CNS is the most abundant connexin in most heterologous oligodendrocyte-to-astrocyte gap junctions, whereas connexin 32 is the predominant if not sole connexin in autologous ("reflexive") oligodendrocyte gap junctions. These results clarify the locations and connexin compositions of heterologous and autologous oligodendrocyte gap junctions, identify autologous gap junctions at paranodes as potential sites for modulating paranodal electrical properties, and reveal connexin 47-containing and connexin 32-containing gap junctions as conduits for long-distance intracellular and intercellular movement of ions and associated osmotic water. The autologous gap junctions may regulate paranodal electrical properties during saltatory conduction. Acting in series and in parallel, autologous and heterologous oligodendrocyte gap junctions provide essential pathways for intra- and intercellular ionic homeostasis.     

A single evoked afterdischarge produces rapid time-dependent changes in connexin36 protein expression in adult rat dorsal hippocampus

Gap junctions between neurons contribute to synchronous neuronal firing and may play a role in the pathophysiology of epilepsy. We examined the expression of a number of gap junction subunits, including the neuronal gap junction forming protein connexin36 (Cx36), in the hippocampus at various time points following an electrically stimulated afterdischarge (AD) in freely-moving animals. Once recovered from electrode implantation, animals were tested with an escalating series of stimulations until an AD was evoked. Suprathreshold stimulation produced a brief AD with no convulsion. Groups of animals were sacrificed at 3, 12, and 24h post-stimulation, and connexin expression was assessed via semiquantitative immunoblotting. Compared to implanted non-stimulated controls, a significant decrease in Cx36 expression was observed in the stimulated dorsal hippocampus at 3h post-stimulation, which returned to control levels by 24h. No changes were seen in the ventral hippocampus. As well, no changes were seen in other selected connexin proteins including Cx26, Cx32, and Cx43, thought to be expressed primarily in glia, in either dorsal or ventral hippocampus. These data suggest that a relatively brief hypersynchronous neuronal discharge can produce rapid and specific changes in Cx36 expression, which may have implications for both normal brain function and the pathophysiology of epilepsy.     

Endogenous adenosine can reduce epileptiform activity in the human epileptogenic cortex maintained in vitro

The effects induced by adenosine and some related compounds upon Mg2+-free epileptogenesis were studied in slices of human epileptogenic neocortex maintained in vitro. Extracellular recordings revealed stimulus-induced and spontaneous epileptiform activity within 1-2 h of perfusion with Mg2+-free medium. A 30-90% decrease of the frequency of occurrence of spontaneous epileptiform discharges was induced by 40-50 microM adenosine while the analog 2-Cl-adenosine exerted a depressant effect (greater than 75% reduction in frequency of occurrence) at 0.3-3 microM. 2-Cl-adenosine also depressed stimulus-induced epileptiform responses and often blocked spontaneous epileptiform activity. Similar effects were seen during bath application of the adenosine uptake inhibitor nitrobenzylthioinosine (10-50 microM) indicating that endogenous adenosine can by itself influence epileptogenicity. Our data demonstrate that in the human epileptogenic neocortex a purinergic mechanism can control Mg2+-free epileptiform activity.     

Coexpression of connexin45 and -32 in oligodendrocytes of rat brain

Connexin proteins are the subunits of gap junction channels, and are encoded by a gene family. Although several connexin mRNAs were detected in brain, only a few connexin-proteins have been localized to specific cell types in this tissue. Here we describe expression of connexin45 protein in oligodendrocytes in rat hippocampus. Double immunofluorescent staining using specific antibodies to connexin45 and connexin32 paired with cell-type specific marker proteins revealed that connexin45 and connexin32 were co-expressed and colocalized in oligodendrocytes. Each of the connexin antibodies gave rise to the same pattern of punctate fluorescence in the plasma membrane of cell bodies and proximal processes of oligodendrocytes. Connexins in the plasma membrane of oligodendrocytes may form gap junctions between oligodendrocytes, or between oligodendrocytes and astrocytes. Expression of connexin45 in oligodendrocytes may prevent dysmyelinating effects of connexin32 mutations in the central nervous system of Charcot-Marie-Tooth (X-type) patients.     

Prolonged epileptiform bursting induced by 0-Mg(2+) in rat hippocampal slices depends on gap junctional coupling

The transition from brief interictal to prolonged seizure, or 'ictal', activity is a crucial event in epilepsy. In vitro slice models can mimic many phenomena observed in the electroencephalogram of patients, including transition from interictal to ictaform or seizure-like activity. In field potential recordings, three discharge types can be distinguished: (1) primary discharges making up the typical interictal burst, (2) secondary bursts, lasting several hundred milliseconds, and (3) tertiary discharges lasting for seconds, constituting the ictal series of bursts. The roles of chemical synapses in these classes of burst have been explored in detail. Here we test the hypothesis that gap junctions are necessary for the generation of secondary bursts.In rat hippocampal slices, epileptiform activity was induced by exposure to 0-Mg(2+). Epileptiform discharges started in the CA3 subfield, and generally consisted of primary discharges followed by 4-13 secondary bursts. Three drugs that block gap junctions, halothane (5-10 mM), carbenoxolone (100 microM) and octanol (0.2-1.0 mM), abolished the secondary discharges, but left the primary bursts intact. The gap junction opener trimethylamine (10 mM) reversibly induced secondary and tertiary discharges. None of these agents altered intrinsic or synaptic properties of CA3 pyramidal cells at the doses used. Surgically isolating the CA3 subfield made secondary discharges disappear, and trimethylamine under these conditions was able to restore them.We conclude that gap junctions can contribute to the prolongation of epileptiform discharges.     

Connexin36 vs. connexin32, "miniature" neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

Suprachiasmatic nucleus (SCN) neurons generate circadian rhythms, and these neurons normally exhibit loosely-synchronized action potentials. Although electrotonic coupling has long been proposed to mediate this neuronal synchrony, ultrastructural studies have failed to detect gap junctions between SCN neurons. Nevertheless, it has been proposed that neuronal gap junctions exist in the SCN; that they consist of connexin32 or, alternatively, connexin36; and that connexin36 knockout eliminates neuronal coupling between SCN neurons and disrupts circadian rhythms. We used confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling to examine the distributions of connexin30, connexin32, connexin36, and connexin43 in rat and mouse SCN and used whole-cell recordings to re-assess electrotonic and tracer coupling. Connexin32-immunofluorescent puncta were essentially absent in SCN but connexin36 was relatively abundant. Fifteen neuronal gap junctions were identified ultrastructurally, all of which contained connexin36 but not connexin32, whereas nearby oligodendrocyte gap junctions contained connexin32. In adult SCN, one neuronal gap junction was >600 connexons, whereas 75% were smaller than 50 connexons, which may be below the limit of detectability by fluorescence microscopy and thin-section electron microscopy. Whole-cell recordings in hypothalamic slices revealed tracer coupling with neurobiotin in <5% of SCN neurons, and paired recordings (>40 pairs) did not reveal obvious electrotonic coupling or synchronized action potentials, consistent with few neurons possessing large gap junctions. However, most neurons had partial spikes or spikelets (often <1 mV), which remained after QX-314 [N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide] had blocked sodium-mediated action potentials within the recorded neuron, consistent with spikelet transmission via small gap junctions. Thus, a few "miniature" gap junctions on most SCN neurons appear to mediate weak electrotonic coupling between limited numbers of neuron pairs, thus accounting for frequent detection of partial spikes and hypothetically providing the basis for "loose" electrical or metabolic synchronization of electrical activity commonly observed in SCN neuronal populations during circadian rhythms.     

戊四唑致痫大鼠脑内缝隙连接的作用

目的: 研究缝隙连接(gap junction, GJ)在癫痫发病中的作用及机制。方法: 以戊四唑 (pentylene-tetrazol, PTZ)致痫大鼠模型为研究对象,采用免疫组织化学和实时定量RT-PCR技术,分别检测缝隙连接蛋白Cx32和Cx43在癫痫发作后不同时点皮层和海马神经元的表达。加用卡马西平(carbamazepine, CBZ)和甘珀酸(carbenoxolone, CBX)干预,观察二者对Cx32/43表达以及大鼠癫痫发作的影响。结果: 免疫组织化学染色显示PTZ致痫2 h后大鼠脑内Cx32/43阳性细胞开始增多,8 h后增多更为明显。实时定量RT-PCR示致痫2 h Cx32 mRNA迅速升高,5 h达高峰。Cx43 mRNA表达水平较低,但明显高于对照组。CBX显著抑制了Cx32/43的表达,CBZ对Cx32和Cx43的表达无明显影响。二者均抑制了大鼠的痫样发作。结论: GJ参与癫痫的发病过程,具有促进癫痫发作的作用。CBZ不影响Cx32/43的表达,表明其抗癫痫作用机制与阻断GJ 无关。     

Epileptiform activity in the nucleus accumbens induced by GABAA receptor antagonists in rat forebrain slices is of cortical origin

Extracellularly recorded field potentials, evoked by stimulation of cortico-nucleus accumbens border, were recorded in the nucleus accumbens (NAcc) in horizontal slices of rat ventral forebrain. The field excitatory postsynaptic potential (EPSP) event (N2) was calcium dependent, blocked by tetrodotoxin (1 microM), and reduced by over 70% by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM), the antagonist of AMPA-type glutamate receptors. The EPSP amplitude was enhanced by either of the GABA(A) receptor antagonists, picrotoxin (10 microM; by 252+/-33%, n=18) and bicuculline methiodide (20 microM; by 216+/-34%, n=4). Additionally, picrotoxin (3-50 microM) and bicuculline methiodide (20 microM) promoted epileptiform activity within the NAcc, manifest as the emergence of additional late components, N3, N4 and N5, in the evoked synaptic waveform. In slices with the frontal cortex removed, picrotoxin (10-50 microM) and bicuculline methiodide (20 microM) were unable to promote epileptiform activity within the NAcc, although a smaller increase in the peak amplitude of the field EPSP (163+/-18%, n=6) was observed at the highest concentrations of picrotoxin (50 microM). Histological examination of the slice demonstrated that in such decorticated slices, the piriform cortex (PC) had been removed. We propose that stimulation of the cortico-NAcc border not only evokes an orthodromic EPSP in the NAcc, but also causes antidromic activation of cortical tissue. Disinhibition by GABA(A) antagonists of circuits intrinsic to the cortex, possibly the piriform cortex, is the principal cause of the facilitation of the EPSP and of regenerative epileptiform activity in NAcc evoked by stimulation of cortical input.     

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus

Locus coeruleus neurons are strongly coupled during early postnatal development, and it has been proposed that these neurons are linked by extraordinarily abundant gap junctions consisting of connexin32 (Cx32) and connexin26 (Cx26), and that those same connexins abundantly link neurons to astrocytes. Based on the controversial nature of those claims, immunofluorescence imaging and freeze-fracture replica immunogold labeling were used to re-investigate the abundance and connexin composition of neuronal and glial gap junctions in developing and adult rat and mouse locus coeruleus. In early postnatal development, connexin36 (Cx36) and connexin43 (Cx43) immunofluorescent puncta were densely distributed in the locus coeruleus, whereas Cx32 and Cx26 were not detected. By freeze-fracture replica immunogold labeling, Cx36 was found in ultrastructurally-defined neuronal gap junctions, whereas Cx32 and Cx26 were not detected in neurons and only rarely detected in glia. In 28-day postnatal (adult) rat locus coeruleus, immunofluorescence labeling for Cx26 was always co-localized with the glial gap junction marker Cx43; Cx32 was associated with the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase); and Cx36 was never co-localized with Cx26, Cx32 or Cx43. Ultrastructurally, Cx36 was localized to gap junctions between neurons, whereas Cx32 was detected only in oligodendrocyte gap junctions; and Cx26 was found only rarely in astrocyte junctions but abundantly in pia mater. Thus, in developing and adult locus coeruleus, neuronal gap junctions contain Cx36 but do not contain detectable Cx32 or Cx26, suggesting that the locus coeruleus has the same cell-type specificity of connexin expression as observed ultrastructurally in other regions of the CNS. Moreover, in both developing and adult locus coeruleus, no evidence was found for gap junctions or connexins linking neurons with astrocytes or oligodendrocytes, indicating that neurons in this nucleus are not linked to the pan-glial syncytium by Cx32- or Cx26-containing gap junctions or by abundant free connexons composed of those connexins.     

Further characterization of muscarinic agonist-induced epileptiform bursting activity in immature rat piriform cortex, in vitro

The characteristics of muscarinic acetylcholine receptor agonist-induced epileptiform bursting seen in immature rat piriform cortex slices in vitro were further investigated using intracellular recording, with particular focus on its postnatal age-dependence (P+14-P+30), pharmacology, site(s) of origin and the likely contribution of the muscarinic acetylcholine receptor agonist-induced post-stimulus slow afterdepolarization and gap junction functionality toward its generation. The muscarinic agonist, oxotremorine-M (10 microM), induced rhythmic bursting only in immature piriform cortex slices; however, paroxysmal depolarizing shift amplitude, burst duration and burst incidence were inversely related to postnatal age. No significant age-dependent changes in neuronal membrane properties or postsynaptic muscarinic responsiveness accounted for this decline. Burst incidence was higher when recorded in anterior and posterior regions of the immature piriform cortex. In adult and immature neurones, oxotremorine-M effects were abolished by M1-, but not M2-muscarinic acetylcholine receptor-selective antagonists. Rostrocaudal lesions, between piriform cortex layers I and II, or layer III and endopiriform nucleus in adult or immature slices did not influence oxotremorine-M effects; however, the slow afterdepolarization in adult (but not immature) lesioned slices was abolished. Gap junction blockers (carbenoxolone or octanol) disrupted muscarinic bursting and diminished the slow afterdepolarization in immature slices, suggesting that gap junction connectivity was important for bursting. Our data show that neural networks within layers II-III function as primary oscillatory circuits for burst initiation in immature rat piriform cortex during persistent muscarinic receptor activation. Furthermore, we propose that muscarinic slow afterdepolarization induction and gap junction communication could contribute towards the increased epileptiform susceptibility of this brain area.     

Sequential changes in intercellular junctions between hepatocytes during the course of acute liver injury and restoration after thioacetamide treatment

Sequential changes of gap junctions (GJs), tight junctions (TJs) and desmosomes (DSs) between hepatocytes during restorative proliferation were studied in rats after a single intraperitoneal administration of 200 mg/kg thioacetamide (TAA). Antibody against connexin 32 was used to demonstrate GJs; simultaneously the changes in TJs and DSs were studied using antibodies against 7H6 protein and desmoplakins. Propidium iodide and bromodeoxyuridine were used to recognize necrotic and proliferative cells. GJs were evenly distributed in early necrotic hepatocytes at 16 h after TAA treatment, then disappeared from necrotic and surrounding cells at 24 h. At 48 h, GJs had disappeared completely from hepatocytes in whole liver lobules, while many hepatocytes were heavily labelled with BrdU. At 72 h, GJs reappeared, firstly in perinecrotic areas. At 96 h after treatment, when the injured areas had disappeared and restorative proliferation ceased, GJs were distributed evenly throughout the lobules. Immunohistochemical observation of GJs in centrilobular, perinecrotic and periportal areas after TAA-induced hepatic necrosis was confirmed by counting the number of connexin-32-positive spots in the respective areas. TJs and DSs disappeared from necrotic cells at 24 h, but then increased between 24 and 48 h in perinecrotic areas, though the increased intensity of these junctions was more evident at 48 h. At 72 h, localization of TJs and DSs returned to normal. These results suggest that during the course of acute hepatic injury, GJs (cell-cell communication) behave differently from other intercellular junctions.     

GABAB receptors in various in vitro and in vivo models of epilepsy: a study with the GABAB receptor blocker CGP 35348.

The effect of the GABAB receptor blocker CGP 35348 on epileptic processes in vitro and in vivo was studied. In hippocampal slices of the rat maintained in vitro, CGP 35348 (100 microM) induced a moderate increase in the frequency of extracellularly recorded spontaneous epileptiform burst discharges induced in CA3 by penicillin (1.2 mM), bicuculline (5 microM) and low Mg(2+) (0.1 mM). This effect was observed in 50-75% of the slices. A similar but less consistent increase was also observed in CA1 in bicuculline and low Mg2+. Data obtained by intracellular recordings from CA1 pyramidal cells in the presence of bicuculline (10 microM) demonstrated that CGP 35348 (100 microM) increased the duration of the paroxysmal depolarization underlying an evoked epileptiform burst and reduced the early component of the after hyperpolarization which followed the burst. In mice pretreated with isoniazid, CGP 35348 (300 mg/kg, i.p.) significantly increased the number of convulsing mice. However, convulsions induced by submaximal doses of pentylenetetrazol, picrotoxin or strychnine were not facilitated by CGP 35348. We conclude that GABAB receptors appear to exert a suppressant effect on various kinds of epileptiform discharges of hippocampal neurons in vitro. In vivo, however, the role of GABAB receptors in regulating convulsions is less prominent since only isoniazid-induced convulsions were facilitated by GABAB receptor blockade.     

Epileptiform activity induced by changes in extracellular potassium in hippocampus

Using extra- and intracellular recording techniques, we investigated the induction and frequency modulation of spontaneous epileptiform activity produced by changes in the concentration of extracellular potassium ([K+]o). This paper describes a quantitative relationship between [K+]o and the frequency of spontaneously occurring epileptiform events. Recordings were made from the CA3 subfield of the rat in vitro hippocampal slice preparation. Intracellular microelectrodes were filled with 2 M Cs2SO4 and connected to a 3-kHz, time-share, single-electrode current- and voltage-clamp device. The frequency of spontaneous epileptiform (interictal) discharges was determined from extracellular recordings as a function of [K+]o. Current- and voltage-clamp techniques were used to characterize the intracellular correlate of these epileptiform events. The frequency of bicuculline-induced spontaneous epileptiform discharges was dependent on [K+]o. Below 4 mM [K+]o, spontaneous discharges occurred sporadically in the presence of 10 microM bicuculline. Increasing [K+]o from 5 to 10 mM caused a fivefold increase in the rate of spontaneous discharges. Spontaneous epileptiform discharges also occurred in the absence of bicuculline when [K+]o was increased above 6.5 mM. The rate of these discharges was dependent on [K+]o in much the same way as the discharges induced by bicuculline. For any given [K+]o concentration greater than 6.5 mM, however, the resultant discharge rate was faster than that obtained when bicuculline was present in the bathing solution. Simultaneous intra- and extracellular recordings revealed that the spontaneous high-[K+]o-induced interictal discharge was accompanied by a large depolarization of the membrane potential that appeared similar to the paroxysmal depolarizing shift (PDS) seen with other convulsants. The intracellularly recorded event fulfilled the criteria for a synaptically mediated PDS. The waveform of the PDS was complex and dependent on the membrane potential. When the membrane potential was held at 0 mV, spontaneously occurring hyperpolarizing potentials were noted during the inter-PDS interval. These events were blocked by picrotoxin or bicuculline and were probably spontaneous inhibitory postsynaptic potentials. The complexity of the PDS waveform suggested that more than one synaptic conductance was involved in the generation of the PDS. The mean measured reversal potential of the depolarizing phase was -10.7 mV. Voltage-clamp techniques were used to measure the conductance underlying the depolarizing phase of the high-[K+]o-induced PDS. The mean measured conductance was 51.5 nS, with a reversal potential of -7.9 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of gap junctional intercellular communication, connexin43 expression, and subsequent differentiation in human fetal neuronal cells by stimulation of the cyclic AMP pathway

Expression of gap junction proteins and cell-cell communication was studied in the human neural-glial cell line, SVG, as a first step in defining whether the SVG cells could be used as a model system to study the role of gap junctions in neuronal precursor cells. SVG cells were found to express connexin43 protein that co-migrated with WB-F344 rat liver connexin43 and that reacted with connexin43-specific antibodies on Western blots. However, fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein-loaded cells failed to show significant dye coupling. Agents that stimulate the adenylyl cyclase/cAMP pathway were used to induce gap junctional intercellular communication in the SVG cultures. A 24-48 h treatment of SVG cells with 5 microM forskolin or 5 microM forskolin + 200 microM 3-isobutyl-1-methylxanthine increased the percentage of dye-coupled cells from 5-65%, using the fluorescent recovery after photobleaching method. The increase in dye coupling induced by forskolin or forskolin + 3-isobutyl-1-methylxanthine was inhibited by octanol, which is known to block gap junction-mediated cell communication. Western blot analysis of total protein extracts revealed the appearance of a higher molecular weight connexin43 protein band after treatment of SVG cells with forskolin or forskolin + 3-isobutyl-1-methylxanthine, that was not observed in vehicle-treated controls. Alkaline phosphatase treatment of total protein extracts from forskolin or forskolin + 3-isobutyl-1-methylxanthine-treated cells reduced the higher molecular weight band to approximately 41,000 the same as observed in the control extracts. The alkaline phosphatase treatment demonstrates that the higher molecular weight band was due to a phosphorylation event stimulated by forskolin or the forskolin + 3-isobutyl-1-methylxanthine combination. In addition, treatment of the SVG cells with the forskolin or forskolin + 3-isobutyl-l-methylxanthine stimulated outgrowth of neurite-like processes from the cell body which immunostained positive for the connexin43 protein as well as protein markers for neurons and oligodendrocytes. We hypothesize that the SVG cells may represent a neuronal progenitor cell population that has the ability to differentiate when exposed to the appropriate signals.