Tetrodotoxin-sensitive Action Potentials Research Articles

Bladder infection with uropathogenic Escherichia coli increases the excitability of afferent neurons.

Urinary tract infections (UTIs) cause bladder hyperactivity and pelvic pain, but the underlying causes of these symptoms remain unknown. We investigated whether afferent sensitization contributes to the bladder overactivity and pain observed in mice suffering from experimentally induced bacterial cystitis. Inoculation of mouse bladders with the uropathogenic Escherichia coli strain UTI89 caused pelvic allodynia, increased voiding frequency, and prompted an acute inflammatory process marked by leukocytic infiltration and edema of the mucosa. Compared with controls, isolated bladder sensory neurons from UTI-treated mice exhibited a depolarized resting membrane potential, lower action potential threshold and rheobase, and increased firing in response to suprathreshold stimulation. To determine whether bacterial virulence factors can contribute to the sensitization of bladder afferents, neurons isolated from naïve mice were incubated with supernatants collected from bacterial cultures with or depleted of lipopolysaccharide (LPS). Supernatants containing LPS prompted the sensitization of bladder sensory neurons with both tetrodotoxin (TTX)-resistant and TTX-sensitive action potentials. However, bladder sensory neurons with TTX-sensitive action potentials were not affected by bacterial supernatants depleted of LPS. Unexpectedly, ultrapure LPS increased the excitability only of bladder sensory neurons with TTX-resistant action potentials, but the supplementation of supernatants depleted of LPS with ultrapure LPS resulted in the sensitization of both population of bladder sensory neurons. In summary, the results of our study indicate that multiple virulence factors released from UTI89 act on bladder sensory neurons to prompt their sensitization. These sensitized bladder sensory neurons mediate, at least in part, the bladder hyperactivity and pelvic pain seen in mice inoculated with UTI89.NEW & NOTEWORTHY Urinary tract infection (UTI) produced by uropathogenic Escherichia coli (UPEC) promotes sensitization of bladder afferent sensory neurons with tetrodotoxin-resistant and tetrodotoxin-sensitive action potentials. Lipopolysaccharide and other virulence factors produced by UPEC contribute to the sensitization of bladder afferents in UTI. In conclusion, sensitized afferents contribute to the voiding symptoms and pelvic pain present in mice bladder inoculated with UPEC.

Molecular determinants of afferent sensitization in a rat model of cystitis with urothelial barrier dysfunction.

The internal surface of the urinary bladder is covered by the urothelium, a stratified epithelium that forms an impermeable barrier to urinary solutes. Increased urothelial permeability is thought to contribute to symptom generation in several forms of cystitis by sensitizing bladder afferents. In this report we investigate the physiological mechanisms that mediate bladder afferent hyperexcitability in a rat model of cystitis induced by overexpression in the urothelium of claudin-2 (Cldn2), a tight junction-associated protein upregulated in bladder biopsies from patients with interstitial cystitis/bladder pain syndrome. Patch-clamp studies showed that overexpression of Cldn2 in the urothelium sensitizes a population of isolectin GS-IB4-negative [IB4(-)] bladder sensory neurons with tetrodotoxin-sensitive (TTX-S) action potentials. Gene expression analysis revealed a significant increase in mRNA levels of the delayed-rectifier voltage-gated K+ channel (Kv)2.2 and the accessory subunit Kv9.1 in this population of bladder sensory neurons. Consistent with this finding, Kv2/Kv9.1 channel activity was greater in IB4(-) bladder sensory neurons from rats overexpressing Cldn2 in the urothelium than in control counterparts. Likewise, current density of TTX-S voltage-gated Na+ (Nav) channels was greater in sensitized neurons than in control counterparts. Significantly, guangxitoxin-1E (GxTX-1E), a selective blocker of Kv2 channels, blunted the repetitive firing of sensitized IB4(-) sensory neurons. In summary, our studies indicate that an increase in the activity of TTX-S Nav and Kv2/Kv9.1 channels mediates repetitive firing of sensitized bladder sensory neurons in rats with increased urothelial permeability.NEW & NOTEWORTHY Hyperexcitability of sensitized bladder sensory neurons in a rat model of interstitial cystitis/bladder pain syndrome (IC/BPS) results from increased activity of tetrodotoxin-sensitive voltage-gated Na+ and delayed-rectifier voltage-gated K+ (Kv)2/Kv9.1 channels. Of major significance, our studies indicate that Kv2/Kv9.1 channels play a major role in symptom generation in this model of IC/BPS by maintaining the sustained firing of the sensitized bladder sensory neurons.

Urothelial Tight Junction Barrier Dysfunction Sensitizes Bladder Afferents.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic voiding disorder that presents with pain in the urinary bladder and surrounding pelvic region. A growing body of evidence suggests that an increase in the permeability of the urothelium, the epithelial barrier that lines the interior of the bladder, contributes to the symptoms of IC/BPS. To examine the consequence of increased urothelial permeability on pelvic pain and afferent excitability, we overexpressed in the urothelium claudin 2 (Cldn2), a tight junction (TJ)-associated protein whose message is significantly upregulated in biopsies of IC/BPS patients. Consistent with the presence of bladder-derived pain, rats overexpressing Cldn2 showed hypersensitivity to von Frey filaments applied to the pelvic region. Overexpression of Cldn2 increased the expression of c-Fos and promoted the activation of ERK1/2 in spinal cord segments receiving bladder input, which we conceive is the result of noxious stimulation of afferent pathways. To determine whether the mechanical allodynia observed in rats with reduced urothelial barrier function results from altered afferent activity, we examined the firing of acutely isolated bladder sensory neurons. In patch-clamp recordings, about 30% of the bladder sensory neurons from rats transduced with Cldn2, but not controls transduced with GFP, displayed spontaneous activity. Furthermore, bladder sensory neurons with tetrodotoxin-sensitive (TTX-S) action potentials from rats transduced with Cldn2 showed hyperexcitability in response to suprathreshold electrical stimulation. These findings suggest that as a result of a leaky urothelium, the diffusion of urinary solutes through the urothelial barrier sensitizes bladders afferents, promoting voiding at low filling volumes and pain.

Human Induced Pluripotent Cell-Derived Sensory Neurons for Fate Commitment of Bone Marrow-Derived Schwann Cells: Implications for Remyelination Therapy.

Strategies that exploit induced pluripotent stem cells (iPSCs) to derive neurons have relied on cocktails of cytokines and growth factors to bias cell‐signaling events in the course of fate choice. These are often costly and inefficient, involving multiple steps. In this study, we took an alternative approach and selected 5 small‐molecule inhibitors of key signaling pathways in an 8‐day program to induce differentiation of human iPSCs into sensory neurons, reaching ≥80% yield in terms of marker proteins. Continuing culture in maintenance medium resulted in neuronal networks immunopositive for synaptic vesicle markers and vesicular glutamate transporters suggestive of excitatory neurotransmission. Subpopulations of the derived neurons were electrically excitable, showing tetrodotoxin‐sensitive action potentials in patch‐clamp experiments. Coculture of the derived neurons with rat Schwann cells under myelinating conditions resulted in upregulated levels of neuronal neuregulin 1 type III in conjunction with the phosphorylated receptors ErbB2 and ErbB3, consistent with amenability of the neuritic network to myelination. As surrogates of embryonic dorsal root ganglia neurons, the derived sensory neurons provided contact‐dependent cues to commit bone marrow‐derived Schwann cell‐like cells to the Schwann cell fate. Our rapid and efficient induction protocol promises not only controlled differentiation of human iPSCs into sensory neurons, but also utility in the translation to a protocol whereby human bone marrow‐derived Schwann cells become available for autologous transplantation and remyelination therapy. Stem Cells Translational Medicine 2017;6:369–381

IPSC-derived sensory neurons for fate commitment of bone marrow-derived schwann cells: Implification for re-myelination therapy

Event Abstract Back to Event iPSC-derived sensory neurons for fate commitment of bone marrow-derived schwann cells: Implification for re-myelination therapy Daisy K. Shum1*, S Cai1, Q Ao2, L Han3 and Ying Shing Chan3 1 University of Hong Kong, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, China 2 Tsinghua University, Department of Neurosurgery, Yuquan Hospital, China 3 University of Hong Kong, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, China This is a sequel to our effort of deriving Schwann cells from bone marrow stromal cells (BMSCs) as a resource for translational research. Earlier studies succeeded as far as generating neurospheres from BMSCs of adult rats and demonstrating competence of the neurosphere-derived cells for directed differentiation to Schwann cell-like cells; these however reverted to a myofibroblast-like phenotype upon withdrawal of the differentiation-inducing factors. We took an alternative approach and selected five small-molecule inhibitors of key signaling pathways in an 8-day program to induce differentiation of human iPSCs into sensory neurons, reaching ≥80% yield in terms of marker proteins. Continuing culture in maintenance medium then found neuronal networks which were electrically excitable, showing tetrodotoxin-sensitive action potentials in patch-clamp experiments. Co-culturing the human iPSC-derived sensory neurons with BMSC-derived Schwann cell-like cells mediated juxtacrine signaling that effected the cell-intrinsic switch to the Schwann cell fate. Henceforth, the cell progeny maintained the Schwann cell phenotype without need for exogenous differentiation-inducing factors or neurons. These derived Schwann cells were functionally capable of myelination and neurotrophic support of neurite growth in in vitro assays, similar to sciatic nerve-derived Schwann cells. Acknowledgements HKRGC-GRF 777810, NSFC/RGC-Joint Research Scheme N_HKU741/11, HKITF (100/10), SK Yee Medical Research Fund, National NSF China (81000011, 81272080), and the SRT on Neuroscience (HKU) Keywords: Schwann Cells, neurospheres, fate determination, bone-marrow stromal cells, Juxtacrine signaling Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Symposium 11: Generating New Neurons: From Development to Cell-Based Therapy Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Shum DK, Cai S, Ao Q, Han L and Chan Y (2016). iPSC-derived sensory neurons for fate commitment of bone marrow-derived schwann cells: Implification for re-myelination therapy. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00045 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jul 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Daisy K Shum, University of Hong Kong, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, Hong Kong, Hong Kong SAR, China, shumdkhk@hku.hk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Daisy K Shum S Cai Q Ao L Han Ying Shing Chan Google Daisy K Shum S Cai Q Ao L Han Ying Shing Chan Google Scholar Daisy K Shum S Cai Q Ao L Han Ying Shing Chan PubMed Daisy K Shum S Cai Q Ao L Han Ying Shing Chan Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Optogenetic Probing of Fast Glutamatergic Transmission from Hypocretin/Orexin to Histamine NeuronsIn Situ

Hypothalamic hypocretin/orexin (hcrt/orx) neurons coordinate sleep-wake cycles, reward seeking, and body energy balance. Neurochemical data suggest that hcrt/orx cells contain several transmitters, but what hcrt/orx cells release onto their projection targets is unknown. A major pathway by which hcrt/orx neurons are thought to promote arousal is through projections to tuberomammillary histamine (HA) neurons. To study the impact of the electrical activity in hcrt/orx cells on HA neurons, we genetically targeted the light-activated excitatory ion channel channelrhodopsin-2 (ChR2) to the plasma membrane of hcrt/orx cells, and performed patch-clamp recordings from HA cells in acute mouse brain slices. Stimulation of ChR2-containing fibers with millisecond flashes of blue light produced fast postsynaptic currents in HA neurons, with a high connection probability (≈60% of HA cells were connected to ≈40% of hcrt/orx cells expressing ChR2). These inputs depended on tetrodotoxin-sensitive action potentials, had kinetics typical of glutamatergic responses mediated by AMPA receptors, were blocked by the AMPA receptor blocker CNQX, and displayed multiple forms of short-term plasticity (depression in ≈70% trials, facilitation in ≈30% trials, both often in the same cell). Furthermore, stimulation of hcrt/orx axons at physiological frequencies rapidly and reversibly increased action potential firing in HA cells, an effect that was abolished by blockade of AMPA receptors. These results provide the first functional evidence that hcrt/orx neurons are capable of fast glutamatergic control of their projection targets, and suggest that variations in electrical activity of hcrt/orx axons can induce rapid changes in long-range signals generated by HA neurons.

Neural differentiation of patient specific iPS cells as a novel approach to study the pathophysiology of multiple sclerosis

The recent introduction of technologies capable of reprogramming human somatic cells into induced pluripotent stem (iPS) cells offers a unique opportunity to study many aspects of neurodegenerative diseases in vitro that could ultimately lead to novel drug development and testing. Here, we report for the first time that human dermal fibroblasts from a patient with relapsing-remitting Multiple Sclerosis (MS) were reprogrammed to pluripotency by retroviral transduction using defined factors ( OCT4, SOX2, KLF4, and c-MYC). The MSiPS cell lines resembled human embryonic stem (hES) cell-like colonies in morphology and gene expression and exhibited silencing of the retroviral transgenes after four passages. MSiPS cells formed embryoid bodies that expressed markers of all three germ layers by immunostaining and Reverse Transcriptase (RT)-PCR. The injection of undifferentiated iPS cell colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. The MSiPS cells were successfully differentiated into mature astrocytes, oligodendrocytes and neurons with normal karyotypes. Although MSiPS-derived neurons displayed some differences in their electrophysiological characteristics as compared to the control cell line, they exhibit properties of functional neurons, with robust resting membrane potentials, large fast tetrodotoxin-sensitive action potentials and voltage-gated sodium currents. This study provides for the first time proof of concept that disease cell lines derived from skin cells obtained from an MS patient can be generated and successfully differentiated into mature neural lineages. This represents an important step in a novel approach for the study of MS pathophysiology and potential drug discovery.

Derivation of Neural Stem Cells from Mesenchymal Stem Cells: Evidence for a Bipotential Stem Cell Population

Neural stem cell (NSC) transplantation has been proposed as a future therapy for neurodegenerative disorders. However, NSC transplantation will be hampered by the limited number of brain donors and the toxicity of immunosuppressive regimens that might be needed with allogeneic transplantation. These limitations may be avoided if NSCs can be generated from clinically accessible sources, such as bone marrow (BM) and peripheral blood samples, that are suitable for autologous transplantation. We report here that NSCs can be generated from human BM-derived mesenchymal stem cells (MSCs). When cultured in NSC culture conditions, 8% of MSCs were able to generate neurospheres. These MSC-derived neurospheres expressed characteristic NSC antigens, such as nestin and musashi-1, and were capable of self-renewal and multilineage differentiation into neurons, astrocytes, and oligodendrocytes. Furthermore, when these MSC-derived neurospheres were cocultured with primary astrocytes, they differentiate into neurons that possess both dendritic and axonal processes, form synapses, and are able to fire tetrodotoxin-sensitive action potentials. When these MSC-derived NSCs were switched back to MSC culture conditions, a small fraction of NSCs (averaging 4-5%) adhered to the culture flasks, proliferated, and displayed the morphology of MSCs. Those adherent cells expressed the characteristic MSC antigens and regained the ability to differentiate into multiple mesodermal lineages. Data presented in this study suggest that MSCs contain a small fraction (averaging 4-5%) of a bipotential stem cell population that is able to generate either MSCs or NSCs depending on the culture conditions.

Roscovitine Triggers Excitotoxicity in Cultured Granule Neurons by Enhancing Glutamate Release

Cerebellar granule neurons are highly susceptible to injury in vivo and in vitro, and primary cultures are widely used to characterize relevant receptors and signaling pathways. However, there are problems associated with their use. In particular, cultures are typically grown in medium supplemented with elevated KCl levels because it improves survival, but accumulating evidence indicates that this causes profound neuroadaptations. For example, growth in elevated KCl levels renders neurons electrically silent. Thus, they cannot be used to examine excitotoxicity of synaptic origins. On the other hand, cultures grown in physiological medium are rarely studied because a proportion undergoes apoptosis. Herein, we provide evidence that mature neurons cultured in physiological KCl develop spontaneous action potentials that support survival through N-methyl-D-aspartate (NMDA) receptor-mediated mechanisms. Furthermore, the cdk inhibitor roscovitine enhances the coupling between tetrodotoxin-sensitive action potentials and P/Q-type voltage-dependent calcium channels (VDCCs), thereby converting this survival program to excitotoxicity of synaptic origin. Therefore, roscovitine-triggered necrosis requires spontaneous Na+-based action potentials (tetrodotoxin inhibits, (+/-)-2-amino-4-phosphonobutyric acid enhances), P/Q-type VDCC currents (omega-agatoxin-IVA and omega-conotoxin-MVIIC inhibit, but not omega-conotoxin-GVIA), intact vesicle fusion processes (tetanus toxin inhibits), and transmitter-filled vesicles (concanamycin and bafilomycin inhibit). From a postsynaptic standpoint, roscovitine-mediated excitotoxicity requires the functionally linked activation of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate/kainate (AMPA/KA) and NMDA receptors, which is consistent with evidence that activated AMPA/KA receptors relieve the voltage-dependent Mg2+ block of NMDA receptors, resulting in excitotoxic Ca2+ influx. In the end, NMDA receptor-linked pathways transduce excitotoxicity. On the other hand, L-type VDCC blockers are not protective. Further characterization of this new model is expected to provide important insights about excitotoxicity of synaptic origins and about roscovitine as a selective modulator of this process.

Derivation of midbrain dopamine neurons from human embryonic stem cells.

Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their potential to differentiate into any cell type of the human body. The challenge in using hES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential toward the derivation of a specific cell fate. Within the nervous system, hES cells have been shown to differentiate in vitro into neural progenitor cells, neurons, and astrocytes. However, to our knowledge, the selective derivation of any given neuron subtype has not yet been demonstrated. Here, we describe conditions to direct hES cells into neurons of midbrain dopaminergic identity. Neuroectodermal differentiation was triggered on stromal feeder cells followed by regional specification by means of the sequential application of defined patterning molecules that direct in vivo midbrain development. Progression toward a midbrain dopamine (DA) neuron fate was monitored by the sequential expression of key transcription factors, including Pax2, Pax5, and engrailed-1 (En1), measurements of DA release, the presence of tetrodotoxin-sensitive action potentials, and the electron-microscopic visualization of tyrosinehydroxylase-positive synaptic terminals. High-yield DA neuron derivation was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons is a first step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to probe the molecular mechanisms that control the development and function of human midbrain DA neurons.

A-type potassium currents dominate repolarisation of neonatal rat primary auditory neurones in situ

Spiral ganglion neurones provide the afferent innervation to cochlear hair cells. Little is known of the molecular physiological processes associated with the differentiation of these neurones, which occurs up to and beyond hearing onset. We have identified novel A-type (inactivating) potassium currents in neonatal rat spiral ganglion neurones in situ, which have not previously been reported from the mammalian cochlea, presumably as a consequence of altered protein expression associated with other preparations. Under whole-cell voltage clamp, voltage steps activated both A-type and non-inactivating outward currents from around −55 mV. The amplitude of the A-type currents was dependent on the holding potential, with steady-state inactivation relieved at hyperpolarised potentials. At −60 mV (close to the resting potential in situ) the currents were approximately 30% enabled. The inactivation kinetics and the degree of inactivation varied between cells, suggesting heterogeneous expression of multiple inactivating currents. A-type currents provided around 60% of total conductance activated by depolarising voltage steps from the resting potential, and were very sensitive to bath-applied 4-aminopyridine (0.01–1 mM). Tetraethylammonium (0.1–30 mM) also blocked the majority of the A-type currents, and the non-inactivating outward current, but left residual fast inactivating A-type current. Under current clamp, neurones fired single tetrodotoxin-sensitive action potentials. 4-Aminopyridine relieved the A-type current mediated stabilisation of membrane potential, resulting in periodic small amplitude action potentials. This study provides the first electrophysiological evidence for A-type potassium currents in neonatal spiral ganglion neurones and shows that these currents play an integral role in primary auditory neurone firing.

Effect of antipsychotic drugs and selective dopaminergic antagonists on dopamine-induced facilitatory activity in prelimbic cortical pyramidal neurons. An in vitro study

Intracellular recordings were obtained from 119 pyramidal neurons localized in prelimbic cortex, five in the dorsal cingulate cortex, one in the infralimbic cortex, one in the border of prelimbic and cingulate cortex and two in the border of prelimbic and infralimbic cortex. The passive membrane properties of these pyramidal neurons (i.e. resting membrane potential, input membrane resistance, shape of the tetrodotoxin-sensitive action potentials, spike frequency adaptation with a prominent postspike afterhyperpolarization, tetrodotoxin-sensitive inward rectification in the depolarizing direction and the absence of bursting) suggested that they resembled regular spiking or intrinsically bursting pyramidal neurons. Bath application of dopamine (EC 50 of 1.8 μM) produced a reversible facilitatory effect on all 119 pyramidal neurons localized in the middle layer of the prelimbic cortex. No consistent change in membrane potential was detected during the application of dopamine. No effect of dopamine was noted on the nine pyramidal neurons that were not localized in the prelimbic cortex. The facilitatory effect of dopamine in prelimbic cortex was concentration dependently antagonized by haloperidol, risperidone, quetiapine, clozapine and by the selective D 4 dopaminergic receptor antagonist L-745,870, but not by the selective D 2/D 3 dopaminergic receptor antagonist (−)-sulpiride. (+)-SCH 23390, which is a selective D 1/D 5 dopamine receptor antagonist, produced, similarly to dopamine, a facilitatory effect per se, and an additive effect when co-administered with dopamine. These results provide evidence that dopamine has a facilitatory effect specifically on pyramidal neurons localized in the middle layer of prelimbic cortex. Antipsychotic drugs and L-745,870 block this effect of dopamine.

Properties of Ectopic Neurons Induced byXenopusNeurogenin1 Misexpression

We have examined cells cultured from ectoderm-misexpressing Neurogenin1 (Ngn1) to describe better the extent to which this gene can control aspects of neuronal phenotype including motility, morphology, excitability, and synaptic properties. Like primary spinal neurons which normally express Ngn1, cells in Ngn1-misexpressing cultures exhibit a motility-correlated behavior called circus movements prior to neuritogenesis. Misexpression of NeuroD also causes circus movements and later neuronal differentiation. GSK3β, which inhibits NeuroD functionin vivo,blocks both Ngn1-induced and NeuroD-induced neuronal differentiation, while Notch signaling inhibits only Ngn1-induced neuronal differentiation, confirming that NeuroD is downstream of Ngn1 and insensitive to Notch inhibition. While interfering with NeuroD function in ventral ectoderm inhibits both circus movements and neuronal differentiation, such inhibition in the neural plate inhibits only neuronal differentiation, suggesting that additional factors regulate circus movements in the neural ectoderm. Ngn1-misexpressing cells extend N-tubulin-positive neurites and exhibit tetrodotoxin-sensitive action potentials. Unlike the majority of cultured spinal neurons, however, Ngn1-misexpressing cells do not respond to glutamate and do not form functional synapses with myocytes, suggesting that these cells are either like Rohon-Beard sensory neurons or are not fully differentiated.

Muscarinic receptor activation in guinea-pig chromaffin cells causes decreased membrane conductance and depolarization

Membrane potentials were recorded with conventional intracellular microelectrodes from chromaffin cells in isolated, bisected adrenal glands from guinea-pigs. The local pressure ejection of muscarinic agonists, acetylcholine (in the presence of hexamethonium) or bethanecol, caused a transient depolarization that was relatively slow (1–2 s) in onset compared with the depolarization associated with the activation of nicotinic receptors. Muscarinic receptor-induced depolarization was associated with an increase in input resistance and the firing of action potentials. Repetitive stimulation of splanchnic nerve fibers within the gland, in the presence of hexamethonium, caused a maintained depolarization that was slow in both onset and decay and in many cells caused the repetitive firing of action potentials. It is suggested that, in this species, the exocytosis of catecholamines caused by the activation of muscarinic receptors, described by others, may be due to the initiation of tetrodotoxin-sensitive action potentials and consequent opening of voltage sensitive Ca 2+ channels.

Presynaptic calcium channels and field-evoked transmitter exocytosis from cultured cerebellar granule cells

Regulated exocytosis from cultured rat cerebellar granule cells can be localized by the vesicle specific marker FM2-10 to specific sites, the highest density of which are at visible varicosities coinciding with neurite–neurite contacts. Exocytosis can be evoked by uniform electrical field pulses, which initiate tetrodotoxin-sensitive action potentials, or by elevated KCl. [ 3H] d-Aspartate is an authentic false transmitter in this preparation, judged by sensitivity of release to bafilomycin A1 and tetanus toxin. The coupling of presynaptic voltage-activated Ca 2+ channels to [ 3H] d-aspartate exocytosis was determined during field stimulation. The peak cytoplasmic free Ca 2+ concentration achieved in the varicosities was proportional to Ca 2+ entry during a 10 s train of pulses. L-type Ca 2+ channels did not contribute to either Ca 2+ entry or [ 3H] d-aspartate exocytosis. The P-type Ca 2+ channel antagonist ω-agatoxin-IVA (30 nM) only inhibited at 75% of the varicosities, although a mean 15% inhibition of Ca 2+ entry caused a 39% inhibition of exocytosis. In contrast the N-type Ca 2+ channel inhibitor ω-conotoxin-GVIA (1 μM), which inhibited at virtually all varicosities, caused mean inhibitions of Ca 2+ entry and exocytosis of 26% and 24% respectively. The toxin ω-conotoxin-MVIIC (5 μM), which inhibits N-, P- and Q-type Ca 2+ channels, was effective at all varicosities. The Q-type component of Ca 2+ entry was calculated to be only 5–10%; however, the additional inhibition of exocytosis was 30%. Thus P-type and particularly Q-type channels appear to be more closely coupled to exocytosis than N-type Ca 2+ channels. The residual Ca 2+ entry following 5 μM ω-conotoxin-MVIIC is scarcely coupled to release. The ω-agatoxin-IVA and ω-conotoxin-GVIA inhibitions of both Ca 2+ entry and exocytosis were additive and varied stochastically between individual varicosities. These results demonstrate that both Q- and P-type Ca 2+ channels are highly efficient in their coupling to amino acid exocytosis, with N-type less efficient, and L-type channels not at all. The Ca 2+ channel types coupled to exocytosis are also able to support exocytosis when evoked by either brief field-evoked action potentials or prolonged depolarization with KCl, indicating that these presynaptic channels, in contrast to those on the somata of the cells, can respond to widely different patterns of activation.

Myenteric neurons of the rat descending colon: electrophysiological and correlated morphological properties.

Conventional intracellular electrophysiological recordings were made from 502 myenteric neurons of the rat descending colon. Myenteric neurons could be classified into three groups on the basis of distinct electrophysiological properties. The first group of neurons (51% of all neurons) fired tetrodotoxin-sensitive action potentials in response to direct somal depolarization and the majority (98%) of this group generated fast cholinergic excitatory synaptic potentials in response to focal stimulation and were therefore designated S/Type 1 neurons. The second group (40%) of neurons fired tetrodotoxin-insensitive action potentials which were followed by long-lasting membrane afterhyperpolarizations, hence were termed AH neurons. These neurons did not receive fast cholinergic synaptic inputs but ionophoretic application of acetylcholine induced rapid nicotinic cholinoceptor-mediated depolarizations. The final group of neurons (9%), named Type 3 neurons, received fast cholinergic synaptic inputs but could never be made to fire action potentials. Rundown in amplitude of successive fast excitatory synaptic potentials evoked by a short train of presynaptic nerve stimuli was observed in only a small proportion of neurons (8/37; 22%) with the majority of neurons (29/37; 78%) showing no such decrease in amplitude, even at frequencies of stimulation as high as 10 Hz. Superfusion of 5-hydroxytryptamine could induce both an inhibition and a facilitation of cholinergic fast synaptic transmission. Evidence was adduced that these presynaptic inhibitory and facilitatory actions appeared to be mediated via 5-hydroxytryptamine 1A and 5-hydroxytryptamine 4 receptors, respectively. Muscarinic slow excitatory synaptic potentials were not detected (9/9 neurons tested) and non-cholinergic slow excitatory synaptic potentials following repetitive focal presynaptic nerve stimulation were observed in only 39/502 (8%) of all neurons. In those neurons in which a demonstrable change in membrane input resistance was detectable, slow excitatory potentials were accompanied by an increased input resistance. In addition, in a small subset (4%) of S/Type 1 neurons, slow membrane hyperpolarizations accompanied by an increased membrane input resistance were observed following tetanic presynaptic nerve stimulation. Superfusion of 5-hydroxytryptamine induced both membrane depolarizations and hyperpolarizations. Membrane depolarizations were observed in 40% of all neuronal types (34% of S/Type 1 neurons, 58% of AH neurons and 11% of Type 3 neurons) and were accompanied by an increased membrane input resistance and occasionally, in S/Type 1 and AH neurons, by anodal break excitation or spontaneous action potential firing. Membrane hyperpolarizations were observed in S/Type 1 neurons (5%) only and were accompanied, unexpectedly, by an increased membrane input resistance. In those neurons that responded both to application of 5-hydroxytryptamine and tetanic presynaptic nerve stimulation, 5-hydroxytryptamine always mimicked the slow synaptic response indicating that 5-hydroxytryptamine may function as a slow synaptic mediator in some myenteric neurons. Myenteric neurons identified by intracellular injection of the neuronal marker Neurobiotin TM were found to conform to the morphological classification schemes proposed for myenteric neurons of the guinea-pig and porcine intestine, that is, Dogiel Types I and II and Stach Type IV neurons were present. Simultaneous electrophysiological recording and intracellular staining techniques revealed that a correlation existed between the electrophysiological and morphological properties of myenteric neurons of the rat colon, with electrophysiological classified S/Type 1 neurons having Dogiel Type I morphologies (95/108 neurons; 88%) and electrophysiological classified AH neurons having Dogiel Type II morphologies (87/94 neurons; 93%)...

Calcium potentials and tetrodotoxin-resistant sodium potentials in unmyelinated C fibres of biopsied human sural nerve

Compound action potentials and electrotonic responses to 150 ms current pulses were recorded from isolated nerve fascicles of human sural nerve biopsies. Compound action potentials in normal bathing solution were characterized by previously described A beta, A delta and C fibre components. In addition, tetrodotoxin-resistant sodium- or calcium-dependent potential components were found when a mixture of tetrodotoxin and the potassium channel blockers 4-aminopyridine and tetraethylammonium was added to the bathing solution. In contrast to tetrodotoxin-sensitive action potentials, tetrodotoxin-resistant sodium- or calcium-dependent potentials could be recorded in the presence of high extracellular potassium concentrations (10-20 mM). Calcium action potentials were found to be sensitive to specific pharmacological antagonists or agonists of L-, N- and P-type calcium channels. Lidocaine, cadmium, verapamil and capsaicin showed unspecific blocking effects on calcium and tetrodotoxin-resistant potentials. Tetrodotoxin-resistant action potentials seem to originate from unmyelinated C fibres since a clear correlation was found between the number of C fibres and the amplitude of tetrodotoxin-resistant calcium and sodium spikes in preparations with different axon type composition. The evidence for tetrodotoxin-resistant Na+ and Ca2+ spikes in peripheral human axons offers new possibilities for a better understanding and/or treatment of abnormalities in the excitability of damaged or diseased peripheral nerves.

Tetrodotoxin-sensitive action potentials in smooth muscle of mouse vas deferens

Action potentials were recorded during impalements of some but not all smooth muscle cells of mouse vas deferens in response to both nerve stimulation and intracellular current injection. They were resistant to blockade by nifedipine (0.1–1.0 μM) but were blocked by tetrodotoxin (TTX, 0.2–1.0 μM) when this was added in the presence of nifedipine. It is suggested that voltage-dependent sodium (Na +) channels are present in mouse vas deferens that function to amplify calcium (Ca 2+) influx through voltage-dependent Ca 2+ channels.

An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.

Characterization of the neurons of the mouse hypogastric ganglion: morphology and electrophysiology

The somata of mouse hypogastric ganglion cells injected with Lucifer yellow were ovoid in shape, lacked dendritic processes but gave rise to a single axonal process. Antidromic activation demonstrated that some of the cells contained in this ganglion innervated the vas deferens. The passive and active membrane properties of the ganglion cells were determined in current clamp experiments. Cells fired tetrodotoxin-sensitive action potentials in response to intracellularly-applied depolarizing current. In voltage clamp evidence was obtained for both a persistent inward calcium current at potentials between −30 and −40 mV and a transient calcium current evoked by step depolarizations to around −20mV. In current clamp, however, cells did not fire calcium action potentials in the presence of tetrodotoxin. Three potassium currents, I M (blocked by 1 mM barium and by 30 μM bethanechol), I A (blocked by 2 mM 4-aminopyridine) and I K(Ca) fast (blocked by 100 μM cadmium and by 5 mM tetraethylammonium) were characterized in these neurons. In addition, I K(Ca) slow was observed in a small proportion of cells. Fast, all-or-nothing, excitatory synaptic potentials were recorded in response to single stimuli applied to the afferent fibres running to the ganglion. In most cells the excitatory synaptic potentials were suprathreshold for action potential initiation and were markedly reduced or abolished by 100 μM mecamylamine, 1 mM hexamethonium and following desensitization to 100 μM nicotine. Excitatory synaptic potentials arose from stimulation of a single presynaptic nerve process and are typical of strong synaptic inputs.