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Abstract Background In the adult nervous system, GABA acts as a major inhibitory neurotransmitter; however, at early stages of neurodevelopment, GABA receptor activation leads to membrane depolarization and accumulation of [Ca2+]i. The role of excitatory GABAergic neurotransmission in the development of the nervous system is not fully understood. In this study, we investigated the role of excitatory GABA-driven activity in regulating the dendritic morphology and network function in the developing chicken spinal cord. Results Both bicuculline, a GABA receptor antagonist, and muscimol, a GABA agonist, inhibit the generation of spontaneous network activity in the isolated spinal cord at E8 or E10, indicating that altering GABA receptor activation disrupts the generation of spontaneous network activity in the chicken spinal cord. Treatment of chicken embryos with bicuculline or muscimol between E5 and E8 (or between E8 and E10), inhibits the dendritic outgrowth of motoneurons when compared to vehicle-treated embryos. The inhibitory effect of bicuculline or muscimol on the dendritic morphology of motoneurons was likely due to inhibition of GABA-driven network activity since a similar effect was also observed following reduction of network activity by Kir2.1 overexpression in the spinal cord. The inhibitory effect of bicuculline or muscimol was not caused by an adverse effect on cell survival. Surprisingly, chronic treatment of chicken embryos with bicuculline or muscimol has no effect on the shape and duration of the episodes of spontaneous activity, suggesting that maturation of network activity is not altered by disruption of the dendritic outgrowth of motoneurons. Conclusions Taken together, these findings indicate that excitatory GABA receptor activation regulates the maturation of dendritic morphology in the developing spinal cord by an activity-dependent mechanism. However, inhibition of dendritic outgrowth caused by disruption of GABA-driven activity does not alter the maturation of spontaneous electrical activity generated by spinal cord networks, suggesting that compensatory mechanisms can reverse any adverse effect of dendritic morphology on network function.

During lytic or latent infection of sensory neurons with herpes simplex virus type 1 (HSV-1) there are significant changes in the expression of voltage-gated Na + channels, which may disrupt the transmission of pain information. HSV-1 infection can also evoke the secretion of various pro-inflammatory cytokines, including TNF-α and IL-6. In this work, we hypothesized that TNF-α regulates the expression of Na + channels during HSV-1 latency establishment in ND7/23 sensory-like neurons. Latency establishment was mimicked by culturing HSV-1 infected ND7/23 cells in the presence of acyclovir (ACV) for 3 days. Changes in the functional expression of voltage-gated Na + channels were assessed by whole-cell recordings. Our results demonstrate that infection of ND7/23 cells with the HSV-1 strain McKrae with GFP expression (M-GFP) causes a significant decrease in sodium currents during latency establishment. Exposure of ND7/23 cells to TNF-α during latency establishment reverses the effect of HSV-1, resulting in a significant increase in sodium current density. However, Na + currents were not restored by 3 day-treatment with IL-6. There were no changes in the pharmacological and biophysical properties of sodium currents promoted by TNF-α, including sensitivity to tetrodotoxin and the current-voltage relationship. TNF-α stimulation of ND7/23 cells increases p38 signaling. Inhibition of p38 signaling with SB203580 or SB202190 eliminates the stimulatory effect of TNF-α on sodium currents. These results indicate that TNF-α signaling in sensory neurons during latency establishment upregulates the expression of voltage-gated Na + channels in order to maintain the transmission of pain information., Competing Interests: Declarations. Conflicts of interest: The authors declare no potential conflicts of interest regarding the research, authorship, and/or publication of this article., (© 2024. The Author(s) under exclusive licence to The Journal of NeuroVirology, Inc.)

Androgen receptor signaling regulates the normal and pathological growth of the prostate. In particular, the growth and survival of prostate cancer cells is initially dependent on androgen receptor signaling. Exposure to androgen deprivation therapy leads to the development of castration-resistant prostate cancer. There is a multitude of molecular and cellular changes that occur in prostate tumor cells, including the expression of neuroendocrine features and various biomarkers, which promotes the switch of cancer cells to androgen-independent growth. These biomarkers include transcription factors (TP53, REST, BRN2, INSM1, c-Myc), signaling molecules (PTEN, Aurora kinases, retinoblastoma tumor suppressor, calcium-binding proteins), and receptors (glucocorticoid, androgen receptor-variant 7), among others. It is believed that genetic modifications, therapeutic treatments, and changes in the tumor microenvironment are contributing factors to the progression of prostate cancers with significant heterogeneity in their phenotypic characteristics. However, it is not well understood how these phenotypic characteristics and molecular modifications arise under specific treatment conditions. In this work, we summarize some of the most important molecular changes associated with the progression of prostate cancers and we describe some of the factors involved in these cellular processes.

The pro-inflammatory cytokine IL-6 has been associated with the progression of PCa to a castration-resistant phenotype. In this work, we characterized the biochemical changes evoked by IL-6 in three different models of PCa cells, including LNCaP, C4-2, and PC3. The effect of IL-6 on PCa cells was compared with the effect obtained by co-stimulation with the cAMP-inducing agent forskolin (FSK). Stimulation of LNCaP cells with IL-6 or IL-6 + FSK evoked increased expression of the neuroendocrine marker tubulin IIIβ and Cav3.2 T-type Ca 2+ channel subunit. PC3 cells, representing a more advanced state of PCa, had high levels of tubulin IIIβ expression without any further changes observed by treatment with IL-6 or IL-6 + FSK. Elevated expression of the glucocorticoid receptor was observed in PC3, but not in LNCaP or C4-2 cells. Glucocorticoid receptor expression was not regulated by IL-6 stimulation of LNCaP or C4-2 cells. IL-6 acting alone or together with FSK evoked a significant reduction in the expression of the transcription factor REST and retinoblastoma tumor suppressor protein Rb1. In LNCaP cells, IL-6 acting alone or together with FSK had no effect on the expression of several biological markers of advanced PCa, including Aurora kinase A, valosin-containing protein, calcium-sensing receptor, calreticulin, S100A protein, and Protein S. In PC3 cells, co-treatment with IL-6 + FSK evoked increased expression of REST and S100A proteins, as well as a reduction in Protein S levels. These findings reveal a complex pattern of biochemical changes in PCa cells under the influence of IL-6., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

HSV-1 is a neurotropic pathogen associated with severe encephalitis, excruciating orofacial sensation, and other chronic neuropathic complications. After the acute infection, the virus may establish a lifelong latency in the neurons of trigeminal ganglia (TG) and other sensory and autonomic ganglia, including the dorsal root ganglia (DRG), etc. The reactivation occurred periodically by a variety of physical or emotional stressors. We have been developing a human DRG neuronal cell-culture model HD10.6, which mimics the mature neurons for latency and reactivation with robust neuronal physiology. We found that miR124 overexpression without acyclovir (ACV) could maintain the virus in a quiescent infection, with the accumulation of latency-associate transcript (LAT). The immediate-early (IE) gene ICP0, on the other hand, was very low and the latent viruses could be reactivated by trichostatin A (TSA) treatment. Together, these observations suggested a putative role of microRNA in promoting HSV-1 latency in human neurons.

Trifluoromethylated N-benzamide enaminones have been identified as potential anticonvulsants for the treatment of drug-resistant epilepsy. T-type Ca 2+ channels are an important target for anti-seizure medications. Our laboratory has developed several fluorinated N-benzamide enaminone analogs that were evaluated by their ability to target T-type Ca 2+ channels. Using whole cell voltage-clamp recordings, we identified two meta-trifluoromethyl N-benzamide enaminones with a significant inhibitory effect on T-type Ca 2+ channels. These compounds had no effect on voltage-activated Na + channels. We also evaluated the effect of the fluorinated N-benzamide enaminone analogs on the T-type Ca 2+ channel subunits Cav3.2 and Cav3.3. The meta-trifluoromethyl N-benzamide enaminone lead analogs altered the steady-state inactivation of Cav3.2 T-type Ca 2+ channels, which resulted in a significant increase in the inactivation recovery time of the channels. There was no effect of fluorinated N-benzamide enaminone analogs on the gating mechanism of T-type Ca 2+ channels, as proven by the lack of effect on the activation and inactivation time constant of Ca 2+ currents. On the contrary, the meta-trifluoromethyl N-benzamide enaminone lead analogs altered the gating mechanism of Cav3.3 T-type Ca 2+ channels, as proven by the reduction in the activation and inactivation time constant of the channels. There was no effect on the inactivation kinetics of Cav3.3 T-type Ca 2+ channels. The present results demonstrate that meta-substituted trifluoromethyl N-benzamide enaminone analogs target T-type Ca 2+ channels by different mechanisms depending on the channel subunit. Meta-trifluoromethyl N-benzamide enaminone analogs can potentially lead to the design of more specific blockers of T-type Ca 2+ channels for the treatment of epileptic seizures., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galβ1-4(Fucα1-3)GlcNAcβ1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N-glycans between these cell types. O-glycans are highly differentiated, as RT4 cells synthesize core 2-based O-glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1-3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Herpesvirus is ranked as one of the grand old members of all pathogens. Of all the viruses in the superfamily, Herpes simplex virus type 1 (HSV-1) is considered as a model virus for a variety of reasons. In a permissive non-neuronal cell culture, HSV-1 concludes the entire life cycle in approximately 18-20 h, encoding approximately 90 unique transcriptional units. In latency, the robust viral gene expression is suppressed in neurons by a group of noncoding RNA. Historically the lesions caused by the virus can date back to centuries ago. As a neurotropic pathogen, HSV-1 is associated with painful oral lesions, severe keratitis and lethal encephalitis. Transmission of pain signals is dependent on the generation and propagation of action potential in sensory neurons. T-type Ca 2+ channels serve as a preamplifier of action potential generation. Voltage-gated Na + channels are the main components for action potential production. This review summarizes not only the voltage-gated ion channels in neuropathic disorders but also provides the new insights into HSV-1 induced pain., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

The molecular mechanisms of pain associated with alphaherpesvirus latency are not clear. We hypothesize that the voltage-gated sodium channels (VGSC) on the dorsal root ganglion (DRG) neurons controlling electrical impulses may have abnormal activity during latent viral infection and reactivation. We used herpes simplex virus 1 (HSV-1) to infect the human DRG-derived neuronal cell line HD10.6 in order to study the establishment and maintenance of viral latency, viral reactivation, and changes in the functional expression of VGSCs. Differentiated cells exhibited robust tetrodotoxin (TTX)-sensitive sodium currents, and acute infection significantly reduced the functional expression of VGSCs within 24 h and completely abolished VGSC activity within 3 days. A quiescent state of infection mimicking latency can be achieved in the presence of acyclovir (ACV) for 7 days followed by 5 days of ACV washout, and then the viruses can remain dormant for another 3 weeks. It was noted that during the establishment of HSV-1 latency, the loss of VGSC activity caused by HSV-1 infection could not be blocked by ACV treatment. However, neurons with continued ACV treatment for another 4 days showed a gradual recovery of VGSC functional expression. Furthermore, the latently infected neurons exhibited higher VGSC activity than controls. The overall regulation of VGSCs by HSV-1 during quiescent infection was proved by increased transcription and possible translation of Nav1.7. Together, these observations demonstrated a very complex pattern of electrophysiological changes during HSV infection of DRG neurons, which may have implications for understanding of the mechanisms of virus-mediated pain linked to latency and reactivation. IMPORTANCE The reactivation of herpesviruses, most commonly varicella-zoster virus (VZV) and pseudorabies virus (PRV), may cause cranial nerve disorder and unbearable pain. Clinical studies have also reported that HSV-1 causes postherpetic neuralgia and chronic occipital neuralgia in humans. The current work meticulously studies the functional expression profile changes of VGSCs during the processes of HSV-1 latency establishment and reactivation using human dorsal root ganglion-derived neuronal HD10.6 cells as an in vitro model. Our results indicated that VGSC activity was eliminated upon infection but steadily recovered during latency establishment and that latent neurons exhibited even higher VGSC activity. This finding advances our knowledge of how ganglion neurons generate uncharacteristic electrical impulses due to abnormal VGSC functional expression influenced by the latent virus., (Copyright © 2020 American Society for Microbiology.)

Herpes simplex virus-type 1 (HSV-1) infection of sensory neurons may lead to a significant reduction in the expression of voltage-activated Na + and Ca 2+ channels, which can disrupt the transmission of pain information. Viral infection also results in the secretion of various pro-inflammatory cytokines, including interleukin (IL)-6. In this work, we tested whether IL-6 regulates the expression of Na + and Ca 2+ channels post-HSV-1 infection in ND7/23 sensory-like neurons. Our results demonstrate that HSV-1 infection causes a significant decrease in the protein expression of the Cav3.2 T-type Ca 2+ channel subunit, despite increasing Cav3.2 mRNA synthesis. Neither Cav3.2 mRNA nor total protein content was affected by IL-6 treatment post-HSV-1 infection. In ND7/23 cells, HSV-1 infection caused a significant reduction in the expression of Na + and T-type Ca 2+ channels within 48 h. Exposure of ND7/23 cells to IL-6 for 24 h post-infection reverses the effect of HSV-1, resulting in a significant increase in T-type Ca 2+ current density. However, Na + currents were not restored by 24-h treatment with IL-6 post-HSV-1 infection of ND7/23 cells. The ability of IL-6 to increase the functional expression of T-type Ca 2+ channels on the membrane was blocked by the inhibition of protein trafficking with brefeldin-A and ERK1/2 activation. These results indicate that IL-6 release following HSV-1 infection regulates the expression of T-type Ca 2+ channels, which may alter the transmission of pain information., (© 2019 International Society for Neurochemistry.)

Although voltage-activated Ca 2+ channels are a common feature in excitable cells, their expression in cancer tissue is less understood. T-type Ca 2+ channels are particularly overexpressed in various cancers. Because of their activation profile at membrane potentials close to rest and the generation of a window current, T-type Ca 2+ channels may regulate a variety of Ca 2+ -dependent cellular processes, including cell proliferation, survival, and differentiation. The expression of T-type Ca 2+ channels is of special interest as a target for therapeutic interventions.

A small library of novel fluorinated N-benzamide enaminones were synthesized and evaluated in a battery of acute preclinical seizure models. Three compounds (GSA 62, TTA 35, and WWB 67) were found to have good anticonvulsant activity in the 6-Hz 'psychomotor' 44-mA rodent model. The focus of this study was to elucidate the active analogs' mode of action on seizure-related molecular targets. Electrophysiology studies were employed to evaluate the compounds' ability to inhibit neuronal activity in central olfactory neurons, mitral cells, and sensory-like ND7/23 cells, which express an assortment of voltage and ligand-gated ion channels. We did not find any significant effects of the three compounds on action potential generation in mitral cells. The treatment of ND7/23 cells with 50 µM of GSA 62, TTA 35, and WWB 67 generated a significant reduction in the amplitude of whole-cell sodium currents. Similar treatment of ND7/23 cells with these compounds had no effect on T-type calcium currents, indicating that fluorinated N-benzamide enaminone analogs may have a selective effect on voltage-gated sodium channels, but not calcium channels.

Therapies designed to reduce androgen production or receptor activation are effective in limiting prostate tumor growth. However, prolonged treatment with anti-androgen therapies results in the progression of prostate cancers into an androgen refractory state. Neuroendocrine differentiation (NED) has been associated with the progression of prostate cancers to an androgen resistant phenotype. In this work we investigated the effect of disrupting androgen receptor signaling in promoting NED of prostate carcinoma cells and whether it is accompanied by an increase in T-type Ca 2+ channel expression. The effect of disrupting androgen signaling was assessed in LNCaP and 22Rv1 prostate cancer cells following treatment with the androgen receptor blocker, bicalutamide, or hormone-depleted media. Treatment of LNCaP cells with bicalutamide or hormone-depleted media for 4-10 d evoked considerable morphological and biochemical changes consistent with NED including the development of long neurite-like processes and the expression of the neuronal marker, tubulin IIIβ. PCR analysis of bicalutamide-stimulated cells revealed no significant changes in Ca v 3.2 mRNA. However, stimulation of LNCaP cells with bicalutamide or hormone-depleted media for 10 d evoked a significant increase in Ca v 3.2 protein expression and the appearance of functional T-type Ca 2+ channels. Inhibition of T-type Ca 2+ channel function with various pharmacological blockers disrupted the morphological differentiation of LNCaP cells. Bicalutamide-evoked expression of functional T-type Ca 2+ channels in LNCaP cells promoted chemoresistance to docetaxel. These findings indicate that disruption of androgen receptor signaling in prostate cancer cells evokes increased expression of functional T-type Ca 2+ channels, which may result in significant morphological and biochemical changes., Competing Interests: None.

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca 2+ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca 2+ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca 2+ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca 2+ current densities. Increased T-type Ca 2+ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca 2+ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca 2+ channels is mediated by several factors, including decreased expression of Ca v 3.2 T-type Ca 2+ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca 2+ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca 2+ channels, which can results in morphological and functional changes in electrical excitability.

The review outlines data consistent with the hypothesis that inspiratory drive transmission that generates fetal breathing movements (FBMs) is essential for the developmental plasticity of phrenic motoneurons (PMNs) and diaphragm musculature prior to birth. A systematic examination during the perinatal period demonstrated a very marked transformation of PMN and diaphragm properties coinciding with the onset and strengthening of inspiratory drive and FBMs in utero. This included studies of age-dependent changes of: i) morphology, neuronal coupling, passive and electrophysiological properties of PMNs; ii) rhythmic inspiratory activity in vitro; iii) FBMs generated in vivo detected by ultrasonography; iv) contractile and end-plate potential properties of diaphragm musculature. We also propose how the hypothesis can be further evaluated with studies of perinatal hypoglossal motoneuron-tongue musculature and the use of Dbx1 null mice that provide an experimental model lacking descending inspiratory drive transmission in utero., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Herpes Simplex Virus Type -1 (HSV-1) infections can cause serious complications such as keratitis and encephalitis. The goal of this study was to identify any changes in the concentrations of volatile organic compounds (VOCs) produced during HSV-1 infection of epithelial cells that could potentially be used as an indicator of a response to stress. An additional objective was to study if any VOCs released from acute epithelial infection may influence subsequent neuronal infection to facilitate latency. To investigate these hypotheses, Vero cells were infected with HSV-1 and the emission of VOCs was analyzed using two-dimensional gas chromatograph/mass spectrometry (2D GC/MS). It was observed that the concentrations of gamma-butyrolactone (GBL) in particular changed significantly after a 24-hour infection. Since HSV-1 may establish latency in neurons after the acute infection, GBL was tested to determine if it exerts neuronal regulation of infection. The results indicated that GBL altered the resting membrane potential of differentiated LNCaP cells and promoted a non-permissive state of HSV-1 infection by repressing viral replication. These observations may provide useful clues towards understanding the complex signaling pathways that occur during the HSV-1 primary infection and establishment of viral latency.

Background: At early stages, the growth of prostate cancers is androgen dependent. At later stages, however, the growth of prostate cancers becomes androgen independent, which leads to an increase in mortality. The switch to an androgen-refractory state is associated with neuroendocrine differentiation (NED) of prostate cancer cells. Several factors including interleukin-6 (IL-6) and increased cAMP production promote NED of prostate cancer cells. In this work we investigated whether IL-6 evoked NED of LNCaP cells results in a significant change in T-type Ca(2+) channel expression in comparison to non-stimulated LNCaP cells., Methods: T-type Ca(2+) channel subunit Cav3.2 expression was studied using PCR analysis, western blot and whole cell recordings. Tubulin IIIβ expression and neurite-like morphology was assessed to investigate the role of T-type Ca(2+) channels in the differentiation of prostate cancer cells., Results: Treatment of LNCaP cells with IL-6 for 4days evokes considerable morphological and biochemical changes consistent with NED. Transcripts of the T-type Ca(2+) channel subunit Cav3.2 but not Cav3.1 or Cav3.3 are detected in IL-6 stimulated cells. Real time PCR analysis of IL-6 stimulated cells indicates no significant change in Cav3.2 mRNA expression in comparison to non-stimulated cells. LNCaP cells stimulated with IL-6 show a threefold increase in T-type Ca(2+) channel subunit Cav3.2 protein expression, suggesting that channel expression is upregulated by a posttranscriptional mechanism. Electrophysiological recordings reveal that increased Cav3.2 protein expression following IL-6 stimulation of LNCaP cells does not result in increased expression of functional channels in the membrane. Functional expression of Cav3.2 channels in LNCaP cells is facilitated by co-stimulation with IL-6 and the cAMP-stimulating agent, forskolin (FSK). Inhibition of T-type Ca(2+) channel activity in IL-6 stimulated LNCaP cells prevents the development of morphological characteristics consistent with NED., Conclusions: These results indicate that the functional expression of T-type Ca(2+) channels is regulated by the interplay of multiple factors in LNCaP cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Several cellular mechanisms contribute to the neuroendocrine differentiation of prostate cancer cells, including exposure to sodium butyrate (NaBu), a naturally occurring salt of the short chain fatty acid n-butyric acid. NaBu belongs to a class of histone deacetylase inhibitors with potential anticancer function. T-type calcium channel expression constitutes an important route for calcium influx in tumor cells that may trigger changes in cell proliferation and differentiation. In this work we investigated the role NaBu on the differentiation of lymph node carcinoma of the prostate (LNCaP) cells and its effect on T-type Ca(2+) channel expression. NaBu stimulates the morphological and molecular differentiation of LNCaP cells. Stimulation of LNCaP cells with NaBu evokes a significant increase in the expression of the Cav3.2 T-type channel subunits. Furthermore, the increased Cav3.2 expression promotes membrane insertion of T-type Ca(2+) channels capable of generating fast inactivating Ca(2+) currents, sensitive to 100μM Ni(2+) ions. Inhibition of T-type Ca(2+) channel function reduces the outgrowth of neurite-like processes in LNCaP cells. NaBu-evoked expression of T-type Ca(2+) channels is also involved in the regulation of cell viability. Inhibition of T-type Ca(2+) channels causes a significant reduction in the viability of LNCaP cells treated with 1mM NaBu, suggesting that Ca(2+) influx via T-type channels can promote cell proliferation. However, increased expression of T-type Ca(2+) channels enhanced the cytotoxic effect of thapsigargin and paclitaxel on cell proliferation. These findings demonstrate that NaBu stimulates T-type Ca(2+) channel expression, thereby regulating both the morphological differentiation and growth of prostate cancer cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

AMPA receptors lacking the GluA2 subunit allow a significant influx of Ca(2+) ions. Although Ca(2+)-permeable AMPA receptors are a familiar feature at early stages of development, the functional significance of these receptors during the maturation of the nervous system remains to be established. Chicken lumbar motoneurons express Ca(2+)-permeable AMPA receptors at E6 but the Ca(2+) permeability of AMPA receptors decreases ∼3-fold by E11. Considering that activity-dependent changes in intracellular Ca(2+) regulates dendritic outgrowth, in this study we investigated whether downregulation of GluA2 expression during a critical period of development alters the dendritic arborization of spinal motoneurons in ovo. We use an avian replication-competent retroviral vector RCASBP (B) carrying the marker red fluorescent protein (RFP) and a GluA2 RNAi construct to downregulate GluA2 expression. Chicken embryos were infected at E2 with one of the following constructs: RCASBP(B)-RFP, RCASBP(B)-RFP-scrambled RNAi, or RCASBP(B)-RFP-GluA2 RNAi. Infection of chicken embryos at E2 resulted in widespread expression of RFP throughout the spinal cord with ≥60% of Islet1/2-positive motoneurons infected, resulting in a significant reduction in GluA2 protein expression. Downregulation of GluA2 expression had no effect on the dendritic arborization of E6 motoneurons. However, downregulation of GluA2 expression caused a significant reduction in the dendritic arborization of E11 motoneurons. Neither motoneuron survival nor maturation of network activity was affected by changes in GluA2 expression. These findings demonstrate that increased GluA2 expression and changes in the Ca(2+) permeability of AMPA receptors regulate the dendritic arborization of spinal cord motoneurons during a critical period of development.

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels.

Glutamate receptor-mediated changes in intracellular Ca(2+) may have important implications for activity-dependent regulation of early embryonic development. NMDA receptors were originally considered to be the sole source of glutamate-mediated Ca(2+) influx. However, AMPA receptors lacking the GluR2 subunit also allow a significant influx of Ca(2+) ions. Although Ca(2+)-permeable AMPA receptors are a familiar feature in developing neurons, the developmental function of these receptors during the formation of the nervous system remains to be established. Previously, we have demonstrated that chicken lumbar motoneurons express Ca(2+)-permeable AMPA receptors at embryonic day (E) 6. The Ca(2+) permeability of AMPA receptors decreases three-fold by E11. In this study we explored the role of transiently expressed Ca(2+)-permeable AMPA receptors in regulating the dendritic morphology of developing motoneurons in ovo. The AMPA receptor blocker CNQX (1 mg/day), when applied between E5 and E8, causes a significant increase in dendritic outgrowth and branching as compared with vehicle-treated embryos. Inhibition of NMDA receptor activity with MK-801 (100 microg/day) during this period has no effect on dendritic morphology. Treatment of chicken embryos with CNQX between E8 and E11 (when most receptors become Ca(2+)-impermeable) has no significant effect on dendritic morphology. However, MK-801 application between E8 and E11 causes a significant reduction in dendritic length and branching. These findings indicate that AMPA receptor activation between E5 and E8 limits dendritic outgrowth in developing motoneurons, whereas NMDA receptor activation is involved in dendritic remodeling after the establishment of synaptic contacts with sensory afferents., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Culture of chicken nodose neurons with CNTF but not BDNF causes a significant increase in T-type Ca(2+) channel expression. CNTF-induced channel expression requires 12 h stimulation to reach maximal expression and is not affected by inhibition of protein synthesis, suggesting the involvement of a post-translational mechanism. In this study, we have investigated the biochemical mechanism responsible for the CNTF-dependent stimulation of T-type channel expression in nodose neurons. Stimulation of nodose neurons with CNTF evoked a considerable increase in signal transducer and activator of transcription (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation. CNTF-evoked ERK phosphorylation was transient whereas BDNF-evoked activation of ERK was sustained. Pre-treatment of nodose neurons with the Janus tyrosine kinase (JAK) inhibitor P6 blocked STAT3 and ERK phosphorylation, whereas the ERK inhibitor U0126 prevented ERK activation but not STAT3 phosphorylation. Both P6 and U0126 inhibited the stimulatory effect of CNTF on T-type channel expression. Inhibition of STAT3 activation by the selective blocker stattic has no effect on ERK phosphorylation and T-type channel expression. These results indicate that CNTF-evoked stimulation of T-type Ca(2+) channel expression in chicken nodose neurons requires JAK-dependent ERK signaling. A cardiac tissue extract derived from E20 chicken heart was also effective in promoting T-type Ca(2+) channel expression and STAT3 and ERK phosphorylation. The ability of the heart extract to stimulate JAK/STAT and ERK activation was developmentally regulated. These findings provide further support to the idea that CNTF or a CNTF-like factor mediates normal expression of T-type channels.

Network-driven spontaneous electrical activity in the chicken spinal cord regulates a variety of developmental processes including neuronal differentiation and formation of neuromuscular structures. In this study we have examined the effect of chronic inhibition of spinal cord activity on motoneuron survival and differentiation. Early spinal cord activity in chick embryos was blocked using an avian replication-competent retroviral vector RCASBP (B) carrying the inward rectifier potassium channel Kir2.1. Chicken embryos were infected with one of the following constructs: RCASBP(B), RCASBP(B)-Kir2.1, or RCASBP(B)-GFP. Infection of chicken embryos at E2 resulted in widespread expression of the viral protein marker p27 gag throughout the spinal cord. Electrophysiological recordings revealed the presence of functional Kir2.1 channels in RCASBP(B)-Kir2.1 but not in RCASBP(B)-infected embryos. Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo. Suppression of spontaneous electrical activity was not due to a reduction in the number of surviving motoneurons or the number of synapses in hindlimb muscle tissue. Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca(2+)-dependent K(+) channels. Reduction of spinal cord activity also generates a significant acceleration in the inactivation rate of A-type K(+) currents without any significant change in current density. Kir2.1 expression did not affect the expression of voltage-gated Na(+) channels or cell capacitance. These experiments demonstrate that chronic inhibition of chicken spinal cord activity causes a significant change in the electrical properties of developing motoneurons.

Functional expression of T-type Ca(2+) channels is developmentally regulated in chick nodose neurons. In this study we have tested the hypothesis that extrinsic factors regulate the expression of T-type Ca(2+) channels in vitro. Voltage-gated Ca(2+) currents were measured using whole-cell patch clamp recordings in E7 nodose neurons cultured under various conditions. Culture of E7 nodose neurons for 48 h with a heart extract induced the expression of T-type Ca(2+) channels without any significant effect on HVA currents. T-type Ca(2+) channel expression was not stimulated by survival promoting factors such as BDNF. The stimulatory effect of heart extract was mediated by a heat-labile, trypsin-sensitive factor. Various hematopoietic cytokines including CNTF and LIF mimic the stimulatory effect of heart extract on T-type Ca(2+) channel expression. The stimulatory effect of heart extract and CNTF requires at least 12 h continuous exposure to reach maximal expression and is not altered by culture of nodose neurons with the protein synthesis inhibitor anisomycin, suggesting that T-type Ca(2+) channel expression is regulated by a posttranslational mechanism. Disruption of the Golgi apparatus with brefeldin-A inhibits the stimulatory effect of heart extract and CNTF suggesting that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Heart extract- or CNTF-evoked stimulation of T-type Ca(2+) channel expression is blocked by the Jak/STAT and MAP kinase blockers, AG490 and U0126, respectively. This study provides new insights into the electrical differentiation of placode-derived sensory neurons and the role of extrinsic factors in regulating the functional expression of Ca(2+) channels.

In this study we have characterized the functional expression of T-type Ca(2+) channels in developing chick nodose neurons, a population of placode-derived sensory neurons innervating the heart and various visceral organs. Voltage-gated Ca(2+) currents were measured using whole cell patch clamp recordings in neurons acutely isolated between embryonic day (E) 7 and E20, prior to hatching. E7 nodose neurons express relatively large high voltage-activated (HVA) Ca(2+) currents. HVA current density progressively increases between E7 and E17. T-type Ca(2+) currents were restricted to a few nodose neurons between E7 and E10 but were present in approximately 60% of nodose neurons by E17. T-type Ca(2+) channels regulate the response of nodose neurons to injection of hyperpolarizing currents, but do not have any effect on the action potential waveform. Nickel ions blocked T-type Ca(2+) currents in a concentration-dependent manner with an IC(50) of 17 microM. The high sensitivity of T-type Ca(2+) channels to nickel blockade combined with sequencing of a partial cDNA suggests that T-type Ca(2+) currents are generated by alpha1H subunits in chick nodose neurons. Steady-state activation and inactivation kinetics were similar to those previously reported for other alpha1H channels in mammalian neurons. Semi-quantitative PCR analysis indicates that alpha1H mRNA was present in chick nodose neurons by E7, suggesting that the functional expression of T-type Ca(2+) channels involves a posttranscriptional mechanism. These findings demonstrate a distinct pattern of T-type Ca(2+) channel functional expression in placode-derived neurons when compared with CNS neurons.

Ca2+ fluxes through ionotropic glutamate receptors regulate a variety of developmental processes, including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors is regulated by the insertion of one or more edited GluR2 subunits. In this study, we tested the possibility that changes in GluR2 expression regulate the Ca2+ permeability of AMPA receptors during a critical period of neuronal development in chick lumbar motoneurons. GluR2 expression is absent between embryonic day (E) 5 and E7, but increases significantly by E8 in the chick ventral spinal cord. Increased GluR2 protein expression is correlated with parallel changes in GluR2 mRNA in the motoneuron pool. Electrophysiological recordings of kainate-evoked currents indicate a significant reduction in the Ca2(+)-permeability of AMPA receptors between E6 and E11. Kainate-evoked currents were sensitive to the AMPA receptor blocker GYKI 52466. Application of AMPA or kainate generates a significant increase in the intracellular Ca2+ concentration in E6 spinal motoneurons, but generates a small response in older neurons. Changes in the Ca(2+)-permeability of AMPA receptors are not mediated by age-dependent changes in the editing pattern of GluR2 subunits. These findings raise the possibility that Ca2+ influx through Ca(2+)-permeable AMPA receptors plays an important role during early embryonic development in chick spinal motoneurons., ((c) 2007 Wiley Periodicals, Inc.)

The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance K(Ca) channels in ciliary ganglion (CG) neurons evoked by beta-neuregulin-1 (NRG1) and transforming growth factor-beta1 (TGFbeta1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGF(beta)1. NRG1- and TGF(beta)1-evoked stimulation of K(Ca) is blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional K(Ca) channels. NRG1 and TGF(beta)1 evoked an Akt-dependent increase in cell-surface SLO alpha-subunits. These procedures have no effect on voltage-activated Ca2+ currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons.

Changes in the intrinsic spike discharge properties in one neuronal population can alter the functions and even the formation of an entire neuronal network. Therefore it is important to understand the factors that regulate acquisition of a mature electrophysiological phenotype. Here we focus on large-conductance K(Ca) channels, which shape the pattern of repetitive discharge and which are therefore likely to play a role in the refinement of neural networks during development. In the parasympathetic ciliary ganglion of chick, the developmental expression of K(Ca) channels coincides with stages at which ciliary cells form synapses with target tissues. Moreover, K(Ca) expression requires formation of synapses with target tissues, and with afferent preganglionic inputs. The trophic effect of targets is mediated by TGFbeta1, whereas the effect of the preganglionic input is mediated by an isoform of beta-neuregulin-1. These trophic factors act synergistically, and this appears to be a normal feature of their actions in vivo. The acute effects of TGFbeta1 entail translocation of preexisting K(Ca) channels from intracellular stores to the plasma membrane. This requires activation of the signaling enzymes Ras, Erk MAP kinase and PI3 kinase. TGFbeta1 also causes a more sustained increase in K(Ca) channels (i.e. for up to 2 weeks) that requires synthesis of new channel proteins. Inductive regulation of K(Ca) expression is also observed in CNS cells that form more complex networks. In lumbar motoneurons, the largest changes in K(Ca) expression coincide with the elimination of synapses with hindlimb targets. Interactions with target tissues play a key role in regulation of motoneuron K(Ca) expression, and this trophic effect of target muscle is mediated by GDNF or a closely related factor. In addition, K(Ca) expression in motoneurons is dependent on ongoing electrical activity both in vivo and in vitro. This provides an additional mechanism for use-dependent refinement of neural networks during embryonic development.

The functional expression of large-conductance Ca2+-activated K+ (K(Ca)) channels in lumbar motoneurons (LMNs) of the developing chick embryo is regulated in part by interactions with striated muscle target tissues. Here we show that the functional expression of K(Ca) channels in LMNs developing in vitro can be stimulated by application of a skeletal muscle extract (MEX) or by coculture with hindlimb myotubes. A similar stimulation of K(Ca) channels in vitro can be produced by the trophic factors glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor but not by neurotrophin (NT)-3 or NT-4. The actions of MEX and hindlimb myotubes are blocked by a GDNF-neutralizing antiserum. Moreover, injection of this same antiserum into the embryonic hindlimb reduced the functional expression of K(Ca) channels in vivo to levels seen in LMNs deprived of interactions with the hindlimb. The effects of GDNF on K(Ca) channel expression in LMNs require 24 hr of continuous exposure to reach maximum and are blocked by the translation inhibitor anisomycin, indicating the need for synthesis of new proteins. GDNF actions are also blocked by the farnesyl transferase inhibitor manumycin, suggesting a role for Ras in the actions of GDNF. Finally, the actions of GDNF are inhibited by PP2, an inhibitor of Src family tyrosine kinases, and by LY29003, an inhibitor of phosphatidylinositol 3 kinases, but not by PD98059, an inhibitor of the Erk signaling cascade. None of these treatments alter expression of voltage-activated Ca2+ channels. Thus, the actions of GDNF on LMN K(Ca) channel expression appear to use a transduction pathway similar to that used for regulation of apoptosis.

The functional expression of large-conductance (BK-type) Ca2+-activated K+ (K(Ca)) channels was examined in developing chick lumbar motoneurons (LMNs) between embryonic day 6 (E6) and E13 using patch-clamp recording techniques. The macroscopic K(Ca) current of E13 LMNs is inhibited by iberiotoxin and resistant to apamin. The average macroscopic K(Ca) density was low before E8 and increased 3.3-fold by E11, with an additional 1.8-fold increase occurring by E13. BK-type K(Ca) channels could not be detected in inside-out patches from E8 LMNs but were readily detected at E11. The density of voltage-activated Ca2+ currents did not change between E8 and E11. Surgical ablation of target tissues at E5 caused a significant reduction in average K(Ca) density in LMNs measured at E11. Conversely, chronic in ovo administration of d-tubocurarine, which causes an increase in motoneuron branching on the surface of the muscle target tissue, evoked a 1.8-fold increase in average LMN K(Ca) density measured at E11. Electrical activity also contributed to developmental regulation of LMN K(Ca) density. A significant reduction in E11 K(Ca) density was found after chronic in ovo treatment with the neuronal nicotinic antagonist mecamylamine or the GABA receptor agonist muscimol, agents that reduce activation of LMNs in ovo. Moreover, 3 d exposure to depolarizing concentrations of external K+ to LMNs cultured at E8 caused an increase in K(Ca) expression. Conversely, tetrodotoxin caused a decrease in K(Ca) expression in cultured E8 LMNs developing for 3 d in the presence of neurotrophic factors that promote neuronal survival in the absence of target tissues.

This study examined the ontogeny of voltage-sensitive calcium conductances in rat phrenic motoneurons (PMNs) and their role in regulating electrical excitability during the perinatal period. Specifically, we studied the period spanning from embryonic day (E)16 through postnatal day (P)1, when PMNs undergo fundamental transformation in their morphology, passive properties, ionic channel composition, synaptic inputs, and electrical excitability. Low voltage-activated (LVA) and high voltage-activated (HVA) conductances were measured using whole cell patch recordings utilizing a cervical slice-phrenic nerve preparation from perinatal rats. Changes between E16 and P0-1 included the following: an approximately 2-fold increase in the density of total calcium conductances, an approximately 2-fold decrease in the density of LVA calcium conductances, and an approximately 3-fold increase in the density of HVA conductances. The elevated expression of T-type calcium channels during the embryonic period lengthened the action potential and enhanced electrical excitability as evidenced by a hyperpolarization-evoked rebound depolarization. The reduction of LVA current density coupled to the presence of a hyperpolarizing outward A-type potassium current had a critical effect in diminishing the rebound depolarization in neonatal PMNs. The increase in HVA current density was concomitant with the emergence of a calcium-dependent "hump-like" afterdepolarization (ADP) and burst-like firing. Neonatal PMNs develop a prominent medium-duration afterhyperpolarization (mAHP) as the result of coupling between N-type calcium channels and small conductance, calcium-activated potassium channels. These data demonstrate that changes in calcium channel expression contribute to the maturation of PMN electrophysiological properties during the time from the commencement of fetal inspiratory drive to the onset of continuous breathing at birth.

Prior to the inception of inspiratory synaptic drive transmission from medullary respiratory centers, rat phrenic motoneurons (PMNs) have action potential and repetitive firing characteristics typical of immature embryonic motoneurons. During the period spanning from when respiratory bulbospinal and segmental afferent synaptic connections are formed at embryonic day 17 (E17) through to birth (gestational period is approximately 21 days), a pronounced transformation of PMN electrophysiological properties occurs. In this study, we test the hypothesis that the elaboration of action potential afterpotentials and the resulting changes in repetitive firing properties are due in large part to developmental changes in PMN potassium conductances. Ionic conductances were measured via whole cell patch recordings using a cervical slice-phrenic nerve preparation isolated from perinatal rats. Voltage- and current-clamp recordings revealed that PMNs expressed outward rectifier (I(KV)) and A-type potassium currents that regulated PMN action potential and repetitive firing properties throughout the perinatal period. There was an age-dependent leftward shift in the activation voltage and a decrease in the time-to-peak of I(KV) during the period from E16 through to birth. The most dramatic change during the perinatal period was the increase in calcium-activated potassium currents after the inception of inspiratory drive transmission at E17. Block of the maxi-type calcium-dependent potassium conductance caused a significant increase in action potential duration and a suppression of the fast afterhyperpolarizing potential. Block of the small conductance calcium-dependent potassium channels resulted in a marked suppression of the medium afterhyperpolarizing potential and an increase in the repetitive firing frequency. In conclusion, the increase in calcium-mediated potassium conductances are in large part responsible for the marked transformation in action potential shape and firing properties of PMNs from the time between the inception of fetal respiratory drive transmission and birth.

The following two hypotheses regarding diaphragm contractile properties in the perinatal rat were tested. First, there is a major transformation of contractile and fatigue properties during the period between the inception of inspiratory drive transmission in utero and birth. Second, the diaphragm muscle properties develop to functionally match changes occurring in phrenic motoneuron electrophysiological properties. Muscle force recordings and intracellular recordings of end-plate potentials were measured by using phrenic nerve-diaphragm muscle in vitro preparations isolated from rats on embryonic day 18 and postnatal days 0-1. The following age-dependent changes occurred: 1) twitch contraction and half relaxation times decreased approximately two- and threefold, respectively; 2) the tetanic force levels increased approximately fivefold; 3) the ratio of peak twitch force to maximum tetanic force decreased 2.3-fold; 4) the range of forces generated by the diaphragm in response to graded nerve stimulation increased approximately twofold; 5) the force-frequency curve was shifted to the right; and 6) the propensity for neuromuscular transmission failure decreased. In conclusion, the diaphragm contractile and phrenic motoneuron repetitive firing properties develop in concert so that the full range of potential diaphragm force recruitment can be utilized and problems associated with diaphragm fatigue are minimized.

In this overview, we outline what is known regarding the key developmental stages of phrenic nerve and diaphragm formation in perinatal rats. These developmental events include the following. Cervical axons emerge from the spinal cord during embryonic (E) day 11. At approximately E12.5, phrenic and brachial axons from the cervical segments merge at the brachial plexi. Subsequently, the two populations diverge as phrenic axons continue to grow ventrally toward the diaphragmatic primordium and brachial axons turn laterally to grow into the limb bud. A few pioneer axons extend ahead of the majority of the phrenic axonal population and migrate along a well-defined track toward the primordial diaphragm, which they reach by E13.5. The primordial diaphragmatic muscle arises from the pleuroperitoneal fold, a triangular protrusion of the body wall composed of the fusion of the primordial pleuroperitoneal and pleuropericardial tissues. The phrenic nerve initiates branching within the diaphragm at approximately E14, when myoblasts in the region of contact with the phrenic nerve begin to fuse and form distinct primary myotubes. As the nerve migrates through the various sectors of the diaphragm, myoblasts along the nerve's path begin to fuse and form additional myotubes. The phrenic nerve intramuscular branching and concomitant diaphragmatic myotube formation continue to progress up until E17, at which time the mature pattern of innervation and muscle architecture are approximated. E17 is also the time of the commencement of inspiratory drive transmission to phrenic motoneurons (PMNs) and the arrival of phrenic afferents to the motoneuron pool. During the period spanning from E17 to birth (gestation period of approximately 21 days), there is dramatic change in PMN morphology as the dendritic branching is rearranged into the rostrocaudal bundling characteristic of mature PMNs. This period is also a time of significant changes in PMN passive membrane properties, action-potential characteristics, and firing properties.

Past studies determined that there is a critical period at approximately embryonic day (E)17 during which phrenic motoneurons (PMNs) undergo a number of pivotal developmental events, including the inception of functional recruitment via synaptic drive from medullary respiratory centers, contact with spinal afferent terminals, the completion of diaphragm innervation, and a major transformation of PMN morphology. The objective of this study was to test the hypothesis that there would be a marked maturation of motoneuron electrophysiological properties occurring in conjunction with these developmental processes. PMN properties were measured via whole cell patch recordings with a cervical slice-phrenic nerve preparation isolated from perinatal rats. From E16 to postnatal day 1, there was a considerable transformation in a number of motoneuron properties, including 1) 10-mV increase in the hyperpolarization of the resting membrane potential, 2) threefold reduction in the input resistance, 3) 12-mV increase in amplitude and 50% decrease duration of action potential, 4) major changes in the shapes of potassium- and calcium-mediated afterpotentials, 5) decline in the prominence of calcium-dependent rebound depolarizations, and 6) increases in rheobase current and steady-state firing rates. Electrical coupling among PMNs was detected in 15-25% of recordings at all ages studied. Collectively, these data and those from parallel studies of PMN-diaphragm ontogeny describe how a multitude of regulatory mechanisms operate in concert during the embryonic development of a single mammalian neuromuscular system.

1. Photoactivated mechanical responses that resulted from exposure to 3-NO2-1,4-dihydropyridines (3-NO2-DHP5) or NO-donors were examined in rat isolated oesophageal smooth muscle with a view to determining the role of calcium and cyclic GMP. 2. Isometric contractile force was recorded in preparations bathed in normal Tyrode or 110 mM K(+)-depolarizing solution. Exposure to (+)-PN 202791, (+/-)-Bay K 8644 and (-)-PN 2020791 or the photodegradable NO-donors, sodium nitroprusside (SNP), streptozotocin (STZ) and sodium nitrite photosensitized precontracted tunica muscularis mucosae preparations in a concentration-dependent fashion. Photosensitizing potency followed the order: (+/-)-PN 202791 > (+/-)-Bay K 8644 > (-)-PN 202791 > SNP > STZ > NaNO2. 3. A low amplitude, slow photorelaxation (slope: 1 mg s-1) was obtained with the L-channel antagonists (-)-PN 202791 and (+)-Bay K 4407. Photosensitization by the agonist enantiomers (+)-PN 202 791 and (-)-Bay K 5407, as well as racemic Bay K 8644, was mimicked by NO donors and showed at least three different components, consisting of (i) a fast relaxation (slope: 140 mg s-1), (ii) a fast "off-contraction', and (iii) a delayed slow relaxation. The fast components, but not the delayed slow relaxation, were abolished by blockade of L-type voltage-operated calcium channels, chelation of extracellular calcium and skinning of the plasmalemma, suggesting their mediation by a process linked to calcium entry through L-channels. 4. Both cyclopiazonic acid (3-30 microM) and ryanodine (30 microM) inhibited the fast response. This inhibition was accelerated in the presence of extracellular calcium and resembled that seen in tissues exposed to the calcium ionophore A 23187 (1 microM). In calcium depleted tissues, cyclopiazonic acid (3 microM) prevented restoration of the cis-dioxolane-induced contraction following re-exposure to a calcium containing high K+ buffer, but failed to inhibit the photoresponse. 5. Both the fast and slow relaxations were potentiated by zaprinast (10 microM) and inhibited by LY B3583 (10 microM). However, in calcium-depleted, calyculin A-precontracted preparations only the slow relaxation was evident. 6. The present results support the conclusion that: (i) functional L-channels are required for the expression of the fast components of the 3-NO2-DHP- or NO-donor-induced photoresponse, (ii) NO photorelease followed by activation of soluble guanylyl cyclase is responsible for the photosensitizing activity of 3-NO2-DHPs and (iii) regulation of the contractile proteins via cyclic GMP-dependent phosphorylation may underlie the slow relaxation.