Pharmacological Blockade Of Gap Junctions Research Articles

Gap junctions regulate the activity of AgRP neurons and diet-induced obesity in male mice.

Recent studies indicated an important role of connexins, gap junction proteins, in the regulation of metabolism. However, most of these studies focused on the glial expression of connexins, whereas the actions of connexins in neurons are still poorly investigated. Thus, the present study had the objective to investigate the possible involvement of gap junctions, and in particular connexin 43 (CX43), for the central regulation of energy homeostasis. Initially, we demonstrated that hypothalamic CX43 expression was suppressed in fasted mice. Using whole-cell patch-clamp recordings, we showed that pharmacological blockade of gap junctions induced hyperpolarization and decreased the frequency of action potentials in 50-70% of agouti-related protein (AgRP)-expressing neurons, depending on the blocker used (carbenoxolone disodium, TAT-Gap19 or Gap 26). When recordings were performed with a biocytin-filled pipette, this intercellular tracer was detected in surrounding cells. Then, an AgRP-specific CX43 knockout (AgRPΔCX43) mouse was generated. AgRPΔCX43 mice exhibited no differences in body weight, adiposity, food intake, energy expenditure and glucose homeostasis. Metabolic responses to 24 h fasting or during refeeding were also not altered in AgRPΔCX43 mice. However, AgRPΔCX43 male, but not female mice, exhibited a partial protection against high-fat diet-induced obesity, even though no significant changes in energy intake or expenditure were detected. In summary, our findings indicate that gap junctions regulate the activity of AgRP neurons, and AgRP-specific CX43 ablation is sufficient to mildly prevent diet-induced obesity specifically in males.

Gap junctions and connexin hemichannels both contribute to the electrical properties of retinal pigment epithelium.

Gap junctions are intercellular channels that permit the transfer of ions and small molecules between adjacent cells. These cellular junctions are particularly dense in the retinal pigment epithelium (RPE), and their contribution to many retinal diseases has been recognized. While gap junctions have been implicated in several aspects of RPE physiology, their role in shaping the electrical properties of these cells has not been characterized in mammals. The role of gap junctions in the electrical properties of the RPE is particularly important considering the growing appreciation of RPE as excitable cells containing various voltage-gated channels. We used a whole-cell patch clamp to measure the electrical characteristics and connectivity between RPE cells, both in cultures derived from human embryonic stem cells and in the intact RPE monolayers from mouse eyes. We found that the pharmacological blockade of gap junctions eliminated electrical coupling between RPE cells, and that the blockade of gap junctions or Cx43 hemichannels significantly increased their input resistance. These results demonstrate that gap junctions function in the RPE not only as a means of molecular transport but also as a regulator of electrical excitability.

Influence of gap junctions upon Ca2+ propagation from stimulated keratinocytes to DRG neurons.

Epidermal cells, such as keratinocytes, are regarded as the first sensory cells to transmit nociception and mechanoreception to free nerve endings extended from the dorsal root ganglion (DRG). Previous studies suggested that this transmission occurs as Ca2+ propagation via ATP receptors. Conversely, the influence of gap junctions on this Ca2+ propagation is largely unknown. Thus, we examined the localization and the role of connexin 43 among keratinocytes and DRG neurons. We co-cultured keratinocytes and DRG neurons and investigated the effect of pharmacological blockade of gap junctions on Ca2+ propagation upon stimulation of a single keratinocyte. Immunocytochemical experiments showed that connexin 43 is localized between keratinocytes and between keratinocytes and DRG neurons. Octanol, a gap junction inhibitor, significantly suppressed the concentrical Ca2+ propagation. Therefore, we conclude that the Ca2+ propagation mechanism via gap junctions from stimulated keratinocytes to free nerve endings should be taken into account.

Neuroprotection of the Inner Retina Also Prevents Secondary Outer Retinal Pathology in a Mouse Model of Glaucoma.

PurposeWe examined structural and functional changes in the outer retina of a mouse model of glaucoma. We examined whether these changes are a secondary consequence of damage in the inner retina and whether neuroprotection of the inner retina also prevents outer retinal changes.MethodsWe used an established microbead occlusion model of glaucoma whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to label rod and cone bipolar cells (BCs), horizontal cells (HCs), and retinal ganglion cells (RGCs), as well as synaptic components in control and glaucomatous eyes, to assess structural damage and cell loss. ERG recordings were made to assess outer retina function.ResultsWe found structural and functional damage of BCs, including significant cell loss and dendritic/axonal remodeling of HCs, following IOP elevation. The first significant loss of both BCs occurred at 4 to 5 weeks after microbead injection. However, early changes in the dendritic structure of RGCs were observed at 3 weeks, but significant changes in the rod BC axon terminal structure were not seen until 4 weeks. We found that protection of inner retinal neurons in glaucomatous eyes by pharmacological blockade of gap junctions or genetic ablation of connexin 36 largely prevented outer retinal damage.ConclusionsTogether, our results indicate that outer retinal impairments in glaucoma are a secondary sequalae of primary damage in the inner retina. The finding that neuroprotection of the inner retina can also prevent outer retinal damage has important implications with regard to the targets for effective neuroprotective therapy.

Propagating Activity in Neocortex, Mediated by Gap Junctions and Modulated by Extracellular Potassium.

Parvalbumin-expressing interneurons in cortical networks are coupled by gap junctions, forming a syncytium that supports propagating epileptiform discharges, induced by 4-aminopyridine. It remains unclear, however, whether these propagating events occur under more natural states, without pharmacological blockade. In particular, we investigated whether propagation also happens when extracellular K+ rises, as is known to occur following intense network activity, such as during seizures. We examined how increasing [K+]o affects the likelihood of propagating activity away from a site of focal (200–400 μm) optogenetic activation of parvalbumin-expressing interneurons. Activity was recorded using a linear 16-electrode array placed along layer V of primary visual cortex. At baseline levels of [K+]o (3.5 mm), induced activity was recorded only within the illuminated area. However, when [K+]o was increased above a threshold level (50th percentile = 8.0 mm; interquartile range = 7.5–9.5 mm), time-locked, fast-spiking unit activity, indicative of parvalbumin-expressing interneuron firing, was also recorded outside the illuminated area, propagating at 59.1 mm/s. The propagating unit activity was unaffected by blockade of GABAergic synaptic transmission, but it was modulated by glutamatergic blockers, and was reduced, and in most cases prevented altogether, by pharmacological blockade of gap junctions, achieved by any of the following three different drugs: quinine, mefloquine, or carbenoxolone. Washout of quinine rapidly re-established the pattern of propagating activity. Computer simulations show qualitative differences between propagating discharges in high [K+]o and 4-aminopyridine, arising from differences in the electrotonic effects of these two manipulations. These interneuronal syncytial interactions are likely to affect the complex electrographic dynamics of seizures, once [K+]o is raised above this threshold level.

Abstract P2-01-09: ESR1 mutations drive breast cancer metastasis by context-dependent alterations in adhesive and migratory properties

Abstract Background: Estrogen receptor alpha (ERα/ESR1) is mutated in 30-40% of endocrine resistant ER+ breast cancer. These mutations, primarily located in the ligand binding domain, are associated with worse outcome in patients, and preclinical studies have shown that they cause ligand independent growth. An open question is whether these mutations contribute to actual metastatic process, or merely endocrine resistance. Methods: Using Y537S and D538G genome-edited MCF7 and T47D cells, 3D growth was assessed in ultralow attachment plates. Cell-cell adhesion was determined using calcein-labelled adhesion assay and quantitative microfluidic fluorescence microscope (qMFM). Collagen-based adhesion and spheroid invasion assays were used to test adhesive and invasive properties. Wound scratching, spheroid collective migration and Boyden chamber transwell assays were applied to monitor cell migratory phenotypes. Mutated ER cistromes were profiled using ChIP-sequencing. ESR1 mutations in clinical samples were characterized using ddPCR. Results: Visual inspection of cells grown in suspension culture revealed more compressed multicellular spheroids in ESR1 mutant cells, indicative of increased cell-cell interactions. This observation was confirmed in both static and microfluidic conditions. This effect was more pronounced in MCF7 than T47D cells, correlating with increased expression of desmosome and gap junction genes. Pharmacological blockade of gap junctions decreased cell-cell adhesion. Decreased attachment and increased invasion to collagen were discerned in all mutant cell types. Further functional analysis identified alterations in the TIMP3-MMP axis causing these phenotypes. The cell-cell adhesion phenotypes were restricted to MCF7-Y537S/D538G and T47D-Y537S, whereas T47D-D538G cells showed significantly increased migration. A GSEA screen identified Wnt signaling as uniquely induced in this context, and combination treatment using the Wnt inhibitor LGK974 and Fulvestrant led to synergistic inhibition of migration. ChIP-seq identified mutation-specific cistromes with an overall increased ligand-independent ER binding. However, it did not reveal binding sites in any candidate metastases genes, suggesting secondary epigenetic mechanisms. The motif analysis revealed the enrichment of FOXA1 motifs in mutated ER cistromes except T47D-D538G cells. However, knockdown of FOXA1 induced significantly higher inhibition of T47D-D538G migration than Fulvestrant treatment alone, indicating a FOXA1-dominated mechanism. Collectively, these data show that ESR1 mutant cells gain metastatic properties, in addition to endocrine resistance. To prove this using clinical samples, we measured ESR1 mutations in a well-defined cohort of endocrine resistant local or distant recurrence. Significant enrichment of ESR1 mutations in distant (9/55) vs local (0/27) recurrences confirms critical role of mutant ERα in metastases. Conclusion: Further analysis of context dependent changes in cell-cell adhesion and migration of ESR1 mutant cells might guide the design and development of drugs targeting ERα-mutant tumors, such as inhibitors of gap junction, FOXA1, MMP, and Wnt signaling pathways. Disclosure: The authors declare no conflict of interest. Citation Format: Li Z, Bahreini A, Levine KM, Wang P, Tasdemir N, Montanez MA, Sundd P, Wallace CT, Watkins SC, Chu D, Park BH, Hou W, Mooring MS, Zhu L, Tseng GC, Carroll JS, Atkinson JM, Lee AV, Oesterreich S. ESR1 mutations drive breast cancer metastasis by context-dependent alterations in adhesive and migratory properties [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-09.

Burst Firing in Bee Gustatory Neurons Prevents Adaptation

Animals detect changes in the environment using modality-specific, peripheral sensory neurons. The insect gustatory system encodes tastant identity and concentration through the independent firing of gustatory receptor neurons (GRNs) that spike rapidly at stimulus onset and quickly adapt. Here, we show the first evidence that concentrated sugar evokes a temporally structured burst pattern of spiking involving two GRNs within the gustatory sensilla of bumblebees. Bursts of spikes resulted when a sucrose-activated GRN was inhibited by another GRN at a frequency of ∼22Hz during the first 1s of stimulation. Pharmacological blockade of gap junctions abolished bursting, indicating that bee GRNs have electrical synapses that produce a temporal pattern of spikes when one GRN is activated by a sugar ligand. Bursting permitted bee GRNs to maintain a high rate of spiking and to exhibit the slowest rate of adaptation of any insect species. Feeding bout duration correlated with coherent bursting; only sugar concentrations that produced bursting evoked the bumblebee's feeding reflex. Volume of solution imbibed was a direct function of time in contact with food. We propose that gap junctions among GRNs enable a sustained rate of GRN spiking that is necessary to drive continuous feeding by the bee proboscis.

Gap junctional coupling between retinal amacrine and ganglion cells underlies coherent activity integral to global object perception

Coherent spike activity occurs between widely separated retinal ganglion cells (RGCs) in response to a large, contiguous object, but not to disjointed objects. Since the large spatial separation between the RGCs precludes common excitatory inputs from bipolar cells, the mechanism underlying this long-range coherence remains unclear. Here, we show that electrical coupling between RGCs and polyaxonal amacrine cells in mouse retina forms the synaptic mechanism responsible for long-range coherent activity in the retina. Pharmacological blockade of gap junctions or genetic ablation of connexin 36 (Cx36) subunits eliminates the long-range correlated spiking between RGCs. Moreover, we find that blockade of gap junctions or ablation of Cx36 significantly reduces the ability of mice to discriminate large, global objects from small, disjointed stimuli. Our results indicate that synchronous activity of RGCs, derived from electrical coupling with amacrine cells, encodes information critical to global object perception.

Targeting neuronal gap junctions in mouse retina offers neuroprotection in glaucoma.

The progressive death of retinal ganglion cells and resulting visual deficits are hallmarks of glaucoma, but the underlying mechanisms remain unclear. In many neurodegenerative diseases, cell death induced by primary insult is followed by a wave of secondary loss. Gap junctions (GJs), intercellular channels composed of subunit connexins, can play a major role in secondary cell death by forming conduits through which toxic molecules from dying cells pass to and injure coupled neighbors. Here we have shown that pharmacological blockade of GJs or genetic ablation of connexin 36 (Cx36) subunits, which are highly expressed by retinal neurons, markedly reduced loss of neurons and optic nerve axons in a mouse model of glaucoma. Further, functional parameters that are negatively affected in glaucoma, including the electroretinogram, visual evoked potential, visual spatial acuity, and contrast sensitivity, were maintained at control levels when Cx36 was ablated. Neuronal GJs may thus represent potential therapeutic targets to prevent the progressive neurodegeneration and visual impairment associated with glaucoma.

Short-term depression of gap junctional coupling in reticular thalamic neurons of absence epileptic rats.

Gap junctional electrical coupling between neurons of the reticular thalamic nucleus (RTN) is critical for hypersynchrony in the thalamo-cortical network. This study investigates the role of electrical coupling in pathological rhythmogenesis in RTN neurons in a rat model of absence epilepsy. Rhythmic activation resulted in a Ca(2+) -dependent short-term depression (STD) of electrical coupling between pairs of RTN neurons in epileptic rats, but not in RTN of a non-epileptic control strain. Pharmacological blockade of gap junctions in RTN in vivo induced a depression of seizure activity. The STD of electrical coupling represents a mechanism of Ca(2+) homeostasis in RTN aimed to counteract excessive synchronization. Neurons in the reticular thalamic nucleus (RTN) are coupled by electrical synapses, which play a major role in regulating synchronous activity. This study investigates electrical coupling in RTN neurons from a rat model of childhood absence epilepsy, genetic absence epilepsy rats from Strasbourg (GAERS), compared with a non-epileptic control (NEC) strain, to assess the impact on pathophysiological rhythmogenesis. Whole-cell recordings were obtained from pairs of RTN neurons of GAERS and NEC in vitro. Coupling was determined by injection of hyperpolarizing current steps in one cell and monitoring evoked voltage responses in both activated and coupled cell. The coupling coefficient (cc) was compared under resting condition, during pharmacological interventions and repeated activation using a series of current injections. The effect of gap junctional coupling on seizure expression was investigated by application of gap junctional blockers into RTN of GAERS in vivo. At resting conditions, cc did not differ between GAERS and NEC. During repeated activation, cc declined in GAERS but not in NEC. This depression in cc was restored within 25s and was prevented by intracellular presence of BAPTA in the activated but not in the coupled cell. Local application of gap junctional blockers into RTN of GAERS in vivo resulted in a decrease of spike wave discharge (SWD) activity. Repeated activation results in a short-term depression (STD) of gap junctional coupling in RTN neurons of GAERS, depending on intracellular Ca(2+) mechanisms in the activated cell. As blockage of gap junctions in vivo results in a decrease of SWD activity, the STD observed in GAERS is considered a compensatory mechanism, aimed to dampen SWD activity.

Gap Junctions and Epileptogenesis: No Laughing Matter

Gap Junctions and Epileptogenesis: No Laughing Matter

Gap Junctions Contribute to Ictal/Interictal Genesis in Human Hypothalamic Hamartomas

Human hypothalamic hamartoma (HH) is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36) and connexin-43 (Cx43) in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA) neurons observed with whole-cell patch-clamp recordings from the same patient's HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients.

GnRH Episodic Secretion Is Altered by Pharmacological Blockade of Gap Junctions: Possible Involvement of Glial Cells.

Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 μM 18-α-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-α-glycyrrhetinic acid washout.

Pharmacological blockade of gap junctions induces repetitive surging of extracellular potassium within the locust CNS

The maintenance of cellular ion homeostasis is crucial for optimal neural function and thus it is of great importance to understand its regulation. Glial cells are extensively coupled by gap junctions forming a network that is suggested to serve as a spatial buffer for potassium (K+) ions. We have investigated the role of glial spatial buffering in the regulation of extracellular K+ concentration ([K+]o) within the locust metathoracic ganglion by pharmacologically inhibiting gap junctions. Using K+-sensitive microelectrodes, we measured [K+]o near the ventilatory neuropile while simultaneously recording the ventilatory rhythm as a model of neural circuit function. We found that blockade of gap junctions with either carbenoxolone (CBX), 18β-glycyrrhetinic acid (18β-GA) or meclofenamic acid (MFA) reliably induced repetitive [K+]o surges and caused a progressive impairment in the ability to maintain baseline [K+]o levels throughout the treatment period. We also show that a low dose of CBX that did not induce surging activity increased the vulnerability of locust neural tissue to spreading depression (SD) induced by Na+/K+-ATPase inhibition with ouabain. CBX pre-treatment increased the number of SD events induced by ouabain and hindered the recovery of [K+]o back to baseline levels between events. Our results suggest that glial spatial buffering through gap junctions plays an essential role in the regulation of [K+]o under normal conditions and also contributes to a component of [K+]o clearance following physiologically elevated levels of [K+]o.

Blockade of gap junctions markedly alters hypoglossal motor discharge in arterially‐perfused adult rat

In the arterially‐perfused adult rat, pharmacological blockade of gap junctions increases frequency, decreases amplitude, and in some cases, alters the pattern of phrenic bursts. The role of functional gap junction coupling in hypoglossal (XII) motor output in this preparation, however, remains to be identified. Therefore, we examined the effects of pharmacological blockade of gap junctions using carbenoxolone (CBX; 100 μM; n=20) on phrenic and XII nerve discharges in arterially‐perfused adult rats; experiments were also conducted using the inert analog glycyrrhzic acid (GZA; 100 μM, n=6) as a control for non‐specific effects of CBX. We found that perfusion with CBX (1) increased phrenic and XII burst frequency by significantly reducing both burst duration and the duration between bursts, (2) decreased phrenic and XII burst amplitude, and (3) shifted the time‐to‐peak to an earlier time point in the phrenic and XII bursts. The effects of CBX on XII burst timing and patterning were greater that those observed for phrenic bursts, with pre‐I XII discharge being markedly reduced or absent in most, but not all, XII bursts. In contrast, GZA was ineffective in similarly altering phrenic or XII activity. These data lend further support to a role of gap junctions in central respiratory control in adult rat, and suggest that the role of gap junction coupling in phrenic and XII motor control may not be identical. Supported by NS045321

Olfactory ensheathing cell membrane properties are shaped by connectivity

Olfactory ensheathing cells (OECs) have been repeatedly implicated in mediating plasticity, particularly in situ in the olfactory nerve in which they support the extension of olfactory sensory neuron (OSN) axons from the olfactory epithelium to the olfactory bulb (OB). OECs are specialized glia whose processes surround OSN axon fascicles within the olfactory nerve and across the OB surface. Despite their purported importance in promoting axon extension, and following transplants, little is known about either morphology or biophysical properties of OECs in situ. In particular, cell-cell interactions that may influence OEC function are largely unexplored. Here, we studied OEC connectivity and morphology in slice preparations, preserving tissue structure and cell-cell interactions. Our analyses showed that OECs form a matrix of cellular projections surrounding axons, unique among glia, and express high levels of connexin-43. Lucifer Yellow injections revealed selective dye coupling among small subgroups of OECs. Two types of OECs were biophysically distinguished with whole-cell voltage-clamp recordings: (1) with low-input resistance (R(i)), linear current profiles, and frequently dye coupled; and (2) with high R(i), nonlinear current profiles, and infrequent dye coupling. Pharmacological blockade of gap junctions changed OEC membrane properties such that linear OECs became nonlinear. Double recordings indicated that the appearance of the nonlinear current profile was associated with the loss of electrical coupling between OECs. We conclude that the diversity of OEC current profiles can be explained by differences in gap-junction connectivity and discuss implications of this diversity for OEC influences on axon growth and excitability.

Electrical coupling synchronises spinal motoneuron activity during swimming in hatchling Xenopus tadpoles

The role of electrical coupling between neurons in the swimming rhythm generator of Xenopus embryos has been studied using pharmacological blockade of gap junctions. A conspicuous effect of 18beta-glycyrrhetinic acid (18beta-GA) and carbenoxolone, which have been shown to block electrical coupling in this preparation, was to increase the duration of ventral root bursts throughout the spinal cord during swimming. The left-right coordination, the swimming frequency and the duration of swimming episodes were not affected by concentrations of 18beta-GA which significantly increased burst durations. However, the longitudinal coupling was affected such that 18beta-GA led to a significant correlation between rostrocaudal delays and cycle periods, which is usually only present in older larval animals. Patch clamp recordings from spinal motoneurons tested whether gap junction blockers affect the spike timing and/or firing pattern of motoneurons during fictive swimming. In the presence of 18beta-GA motoneurons continued to fire a single, but broader action potential in each cycle of swimming, and the timing of their spikes relative to the ventral root burst became more variable. 18beta-GA had no detectable effect on the resting membrane potential of motoneurons, but led to a significant increase in input resistance, consistent with the block of gap junctions. This effect did not result in increased firing during swimming, despite the fact that multiple spikes can occur in response to current injection. Applications of 18beta-GA at larval stage 42 had no discernible effect on locomotion. The results, which suggest that electrical coupling primarily functions to synchronize activity in synergistic motoneurons during embryo swimming, are discussed in the context of motor system development.

Blockade of neuronal gap junctions modifies slow and fast oscillatory rhythms in phrenic nerve discharge in arterially‐perfused adult rat

We have previously demonstrated that pharmacological blockade of gap junctions alters respiratory rhythm, phrenic burst pattern, and inspiratory‐phase synchronization. The pharmacological agents used to block functional gap junction coupling, however, were not specific to gap junctions composed of a specific connexin (Cx) isoform, and therefore may have exerted their effects on both neuronal and astrocytic gap junctions. To further delineate the role of neuronal gap junctions in central respiratory control, we examined the effects of mefloquine (MEQ; 0.5‐1.0 μM), which blocks neuron‐specific Cx36‐containing gap junctions, on phrenic nerve discharge in 5 arterially‐perfused adult rat preparations. We found that perfusion with MEQ increased phrenic burst frequency by 35±4% (P<0.001) due to decreases in both TI (P=0.001) and TE (P<0.001). No significant changes in burst amplitude were observed; however, time‐to‐peak activity (Tpeak/TI) was shifted from 85±2 to 72±5% (P<0.01). Perfusion with MEQ also produced a small increase in HFO spectral power (P=0.07), shifted HFO onset to an earlier time point in the phrenic burst (from 62±3 to 36±7% TI; P<0.01), and prolonged the duration of significant HFO activity. No changes in MFO activity were seen. These data suggest that neuronal gap junctions participate in both the slow and fast oscillatory rhythms underlying phrenic nerve discharge. Supported by NS045321

Fluctuations of Contraction Rhythm During Simulated Ischemia/Reperfusion in Cultured Cardiac Myocytes from Neonatal Rats

Cardiac ischemia results in a rapid decrease of intracellular pH and in the rise of intracellular Ca 2+, changes that have been shown to reduce intercellular communication via gap junctions (GJ) between cardiac myocytes. Ischemia also results in electrical instability probably caused by the reduced GJ permeability contributing to an increased vulnerability to arrhythmias. This study aims at elucidating whether the fluctuations of contraction rhythm of spontaneously beating cardiac myocytes in culture changes during simulated ischemia/reperfusion. The coefficient of variation (CV) of contraction intervals, reflecting the fluctuation of contraction rhythm, increased significantly during simulated ischemia/reperfusion. However, the contraction rhythm of the cardiac myocytes in an aggregate remained synchronized during simulated ischemia/reperfusion. In contrast, pharmacological blockade of GJ with 12-doxyl stearic acid, a blocker of GJ permeability, resulted in the de-synchronization of contraction rhythm and in an increase in the CV of contraction intervals in normoxic conditions. The present findings lead to the suggestion that GJ remained open during simulated ischemia/reperfusion, and that a mechanism other than electrical uncoupling between myocytes contributed to the observed increase in the fluctuation of beating rhythm during ischemia.