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2.
J Physiol ; 599(5): 1391-1420, 2021 03.
Article in English | MEDLINE | ID: mdl-33449375

ABSTRACT

The spatial and temporal balance of spinal α-motoneuron (αMN) intrinsic membrane conductances underlies the neural output of the final common pathway for motor commands. Although the complete set and precise localization of αMN K+ channels and their respective outward conductances remain unsettled, important K+ channel subtypes have now been documented, including Kv1, Kv2, Kv7, TASK, HCN and SK isoforms. Unique kinetics and gating parameters allow these channels to differentially shape and/or modify αMN firing properties, and recent immunohistochemical localization of K+ -channel complexes reveals a framework in which their spatial distribution and/or focal clustering within different surface membrane compartments is highly tuned to their physiological function. Moreover, highly evolved regulatory mechanisms enable specific channels to operate over variable levels of αMN activity and contribute to either state-dependent enhancement or diminution of firing. While recent data suggest an additional, non-conducting role for clustered Kv2.1 channels in the formation of endoplasmic reticulum-plasma membrane junctions postsynaptic to C-bouton synapses, electrophysiological evidence demonstrates that conducting Kv2.1 channels effectively regulate αMN firing, especially during periods of high activity in which the cholinergic C-boutons are engaged. Intense αMN activity or cell injury rapidly disrupts the clustered organization of Kv2.1 channels in αMNs and further impacts their physiological role. Thus, αMN K+ channels play a critical regulatory role in motor processing and are potential therapeutic targets for diseases affecting αMN excitability and motor output, including amyotrophic lateral sclerosis.


Subject(s)
Motor Neurons , Shab Potassium Channels , Animals , Electrophysiological Phenomena , Mammals , Synapses
3.
J Physiol ; 597(14): 3769-3786, 2019 07.
Article in English | MEDLINE | ID: mdl-31145471

ABSTRACT

KEY POINTS: Kv2 currents maintain and regulate motoneuron (MN) repetitive firing properties. Kv2.1 channel clustering properties are dynamic and respond to both high and low activity conditions. The enzyme calcineurin regulates Kv2.1 ion channel declustering. In patholophysiological conditions of high activity, Kv2.1 channels homeostatically reduce MN repetitive firing. Modulation of Kv2.1 channel kinetics and clustering allows these channels to act in a variable way across a spectrum of MN activity states. ABSTRACT: Kv2.1 channels are widely expressed in the central nervous system, including in spinal motoneurons (MNs) where they aggregate as distinct membrane clusters associated with highly regulated signalling ensembles at specific postsynaptic sites. Multiple roles for Kv2 channels have been proposed but the physiological role of Kv2.1 ion channels in mammalian spinal MNs is unknown. To determine the contribution of Kv2.1 channels to rat α-motoneuron activity, the Kv2 inhibitor stromatoxin was used to block Kv2 currents in whole-cell current clamp electrophysiological recordings in rat lumbar MNs. The results indicate that Kv2 currents permit shorter interspike intervals and higher repetitive firing rates, possibly by relieving Na+ channel inactivation, and thus contribute to maintenance of repetitive firing properties. We also demonstrate that Kv2.1 clustering properties in motoneurons are dynamic and respond to both high and low activity conditions. Furthermore, we show that the enzyme calcineurin regulates Kv2.1 ion channel clustering status. Finally, in a high activity state, Kv2.1 channels homeostatically reduce motoneuron repetitive firing. These results suggest that the activity-dependent regulation of Kv2.1 channel kinetics allows these channels to modulate repetitive firing properties across a spectrum of motoneuron activity states.


Subject(s)
Action Potentials/physiology , Motor Neurons/metabolism , Shab Potassium Channels/metabolism , Animals , Electrophysiological Phenomena/physiology , Female , Ion Channels/metabolism , Male , Patch-Clamp Techniques/methods , Rats , Rats, Sprague-Dawley , Spinal Cord/metabolism
4.
J Neurophysiol ; 118(5): 2687-2701, 2017 11 01.
Article in English | MEDLINE | ID: mdl-28814636

ABSTRACT

The characteristic signaling and intraspinal projections of muscle proprioceptors best described in the cat are often generalized across mammalian species. However, species-dependent adaptations within this system seem necessary to accommodate asymmetric scaling of length, velocity, and force information required by the physics of movement. In the present study we report mechanosensory responses and intraspinal destinations of three classes of muscle proprioceptors. Proprioceptors from triceps surae muscles in adult female Wistar rats anesthetized with isoflurane were physiologically classified as muscle spindle group Ia or II or as tendon organ group Ib afferents, studied for their firing responses to passive-muscle stretch, and in some cases labeled and imaged for axon projections and varicosities in spinal segments. Afferent projections and the laminar distributions of provisional synapses in rats closely resembled those found in the cat. Afferent signaling of muscle kinematics was also similar to reports in the cat, but rat Ib afferents fired robustly during passive-muscle stretch and Ia afferents displayed an exaggerated dynamic response, even after locomotor scaling was accounted for. These differences in mechanosensory signaling by muscle proprioceptors may represent adaptations for movement control in different animal species.NEW & NOTEWORTHY Muscle sensory neurons signal information necessary for controlling limb movements. The information encoded and transmitted by muscle proprioceptors to networks in the spinal cord is known in detail only for the cat, but differences in size and behavior of other species challenge the presumed generalizability. This report presents the first findings detailing specializations in mechanosensory signaling and intraspinal targets for functionally identified subtypes of muscle proprioceptors in the rat.


Subject(s)
Mechanoreceptors/physiology , Muscle, Skeletal/physiology , Proprioception , Spinal Cord/physiology , Synapses/physiology , Animals , Female , Muscle Contraction , Muscle, Skeletal/innervation , Rats , Rats, Wistar , Spinal Cord/cytology
5.
Physiol Rep ; 4(22)2016 11.
Article in English | MEDLINE | ID: mdl-27884958

ABSTRACT

Homeostatic plasticity occurs through diverse cellular and synaptic mechanisms, and extensive investigations over the preceding decade have established Kv2.1 ion channels as key homeostatic regulatory elements in several central neuronal systems. As in these cellular systems, Kv2.1 channels in spinal motoneurons (MNs) localize within large somatic membrane clusters. However, their role in regulating motoneuron activity is not fully established in vivo. We have previously demonstrated marked Kv2.1 channel redistribution in MNs following in vitro glutamate application and in vivo peripheral nerve injury (Romer et al., 2014, Brain Research, 1547:1-15). Here, we extend these findings through the novel use of a fully intact, in vivo rat preparation to show that Kv2.1 ion channels in lumbar MNs rapidly and reversibly redistribute throughout the somatic membrane following 10 min of electrophysiological sensory and/or motor nerve stimulation. These data establish that Kv2.1 channels are remarkably responsive in vivo to electrically evoked and synaptically driven action potentials in MNs, and strongly implicate motoneuron Kv2.1 channels in the rapid homeostatic response to altered neuronal activity.


Subject(s)
Motor Neurons/physiology , Shab Potassium Channels/metabolism , Spinal Nerves/physiology , Action Potentials/physiology , Animals , Female , Glutamic Acid/metabolism , Homeostasis/physiology , Ion Channel Gating/physiology , Ion Channels , Motor Neurons/metabolism , Rats , Rats, Sprague-Dawley , Spinal Nerves/metabolism
6.
Front Neural Circuits ; 8: 106, 2014.
Article in English | MEDLINE | ID: mdl-25278842

ABSTRACT

C-boutons are important cholinergic modulatory loci for state-dependent alterations in motoneuron firing rate. m2 receptors are concentrated postsynaptic to C-boutons, and m2 receptor activation increases motoneuron excitability by reducing the action potential afterhyperpolarization. Here, using an intensive review of the current literature as well as data from our laboratory, we illustrate that C-bouton postsynaptic sites comprise a unique structural/functional domain containing appropriate cellular machinery (a "signaling ensemble") for cholinergic regulation of outward K(+) currents. Moreover, synaptic reorganization at these critical sites has been observed in a variety of pathologic states. Yet despite recent advances, there are still great challenges for understanding the role of C-bouton regulation and dysregulation in human health and disease. The development of new therapeutic interventions for devastating neurological conditions will rely on a complete understanding of the molecular mechanisms that underlie these complex synapses. Therefore, to close this review, we propose a comprehensive hypothetical mechanism for the cholinergic modification of α-MN excitability at C-bouton synapses, based on findings in several well-characterized neuronal systems.


Subject(s)
Motor Neurons/physiology , Presynaptic Terminals/physiology , Spinal Cord/cytology , Swimming/physiology , Synapses/physiology , Vesicular Acetylcholine Transport Proteins/metabolism , Animals , Calcium Signaling , Humans , Nerve Net/physiology , Receptor, Muscarinic M2 , Small-Conductance Calcium-Activated Potassium Channels/genetics , Small-Conductance Calcium-Activated Potassium Channels/metabolism , Synapses/genetics , Vesicular Acetylcholine Transport Proteins/genetics
7.
Brain Res ; 1547: 1-15, 2014 Feb 14.
Article in English | MEDLINE | ID: mdl-24355600

ABSTRACT

Pathophysiological responses to peripheral nerve injury include alterations in the activity, intrinsic membrane properties and excitability of spinal neurons. The intrinsic excitability of α-motoneurons is controlled in part by the expression, regulation, and distribution of membrane-bound ion channels. Ion channels, such as Kv2.1 and SK, which underlie delayed rectifier potassium currents and afterhyperpolarization respectively, are localized in high-density clusters at specific postsynaptic sites (Deardorff et al., 2013; Muennich and Fyffe, 2004). Previous work has indicated that Kv2.1 channel clustering and kinetics are regulated by a variety of stimuli including ischemia, hypoxia, neuromodulator action and increased activity. Regulation occurs via channel dephosphorylation leading to both declustering and alterations in channel kinetics, thus normalizing activity (Misonou et al., 2004; Misonou et al., 2005; Misonou et al., 2008; Mohapatra et al., 2009; Park et al., 2006). Here we demonstrate using immunohistochemistry that peripheral nerve injury is also sufficient to alter the surface distribution of Kv2.1 channels on motoneurons. The dynamic changes in channel localization include a rapid progressive decline in cluster size, beginning immediately after axotomy, and reaching maximum within one week. With reinnervation, the organization and size of Kv2.1 clusters do not fully recover. However, in the absence of reinnervation Kv2.1 cluster sizes fully recover. Moreover, unilateral peripheral nerve injury evokes parallel, but smaller effects bilaterally. These results suggest that homeostatic regulation of motoneuron Kv2.1 membrane distribution after axon injury is largely independent of axon reinnervation.


Subject(s)
Motor Neurons/metabolism , Peripheral Nerve Injuries/metabolism , Shab Potassium Channels/metabolism , Animals , Female , Rats , Rats, Sprague-Dawley , Tibial Nerve/injuries , Tibial Nerve/metabolism
8.
J Physiol ; 591(4): 875-97, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23129791

ABSTRACT

Small-conductance calcium-activated potassium (SK) channels mediate medium after-hyperpolarization (AHP) conductances in neurons throughout the central nervous system. However, the expression profile and subcellular localization of different SK channel isoforms in lumbar spinal α-motoneurons (α-MNs) is unknown. Using immunohistochemical labelling of rat, mouse and cat spinal cord, we reveal a differential and overlapping expression of SK2 and SK3 isoforms across specific types of α-MNs. In rodents, SK2 is expressed in all α-MNs, whereas SK3 is expressed preferentially in small-diameter α-MNs; in cats, SK3 is expressed in all α-MNs. Function-specific expression of SK3 was explored using post hoc immunostaining of electrophysiologically characterized rat α-MNs in vivo. These studies revealed strong relationships between SK3 expression and medium AHP properties. Motoneurons with SK3-immunoreactivity exhibit significantly longer AHP half-decay times (24.67 vs. 11.02 ms) and greater AHP amplitudes (3.27 vs. 1.56 mV) than MNs lacking SK3-immunoreactivity. We conclude that the differential expression of SK isoforms in rat and mouse spinal cord may contribute to the range of medium AHP durations across specific MN functional types and may be a molecular factor distinguishing between slow- and fast-type α-MNs in rodents. Furthermore, our results show that SK2- and SK3-immunoreactivity is enriched in distinct postsynaptic domains that contain Kv2.1 channel clusters associated with cholinergic C-boutons on the soma and proximal dendrites of α-MNs. We suggest that this remarkably specific subcellular membrane localization of SK channels is likely to represent the basis for a cholinergic mechanism for effective regulation of channel function and cell excitability.


Subject(s)
Motor Neurons/physiology , Small-Conductance Calcium-Activated Potassium Channels/physiology , Spinal Cord/physiology , Synapses/physiology , Animals , Cats , Female , In Vitro Techniques , Lumbosacral Region , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Rats , Rats, Sprague-Dawley , Rats, Wistar
9.
Hear Res ; 277(1-2): 163-75, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21276842

ABSTRACT

The development of cochlear implants for the treatment of patients with profound hearing loss has advanced considerably in the last few decades, particularly in the field of speech comprehension. However, attempts to provide not only sound decoding but also spatial hearing are limited by our understanding of circuit adaptations in the absence of auditory input. Here we investigate the lateral superior olive (LSO), a nucleus involved in interaural level difference (ILD) processing in the auditory brainstem using a mouse model of congenital deafness (the dn/dn mouse). An electrophysiological investigation of principal neurons of the LSO from the dn/dn mouse reveals a higher than normal proportion of single spiking (SS) neurons, and an increase in the hyperpolarisation-activated I(h) current. However, inhibitory glycinergic input to the LSO appears to develop normally both pre and postsynaptically in dn/dn mice despite the absence of auditory nerve activity. In combination with previous electrophysiological findings from the dn/dn mouse, we also compile a simple Hodgkin and Huxley circuit model in order to investigate possible computational deficits in ILD processing resulting from congenital hearing loss. We find that the predominance of SS neurons in the dn/dn LSO may compensate for upstream modifications and help to maintain a functioning ILD circuit in the dn/dn mouse. This could have clinical repercussions on the development of stimulation paradigms for spatial hearing with cochlear implants.


Subject(s)
Auditory Pathways/physiopathology , Auditory Perception , Deafness/physiopathology , Olivary Nucleus/physiopathology , Animals , Auditory Pathways/metabolism , Carrier Proteins/metabolism , Cochlear Nerve/physiopathology , Deafness/congenital , Deafness/metabolism , Deafness/psychology , Disease Models, Animal , Electric Stimulation , Evoked Potentials, Auditory, Brain Stem , Excitatory Postsynaptic Potentials , Glutamine/metabolism , Glycine/metabolism , Immunohistochemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Mutant Strains , Models, Neurological , Mutation , Neural Inhibition , Olivary Nucleus/metabolism , Patch-Clamp Techniques , Time Factors
10.
Eur J Neurosci ; 32(10): 1658-67, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20946234

ABSTRACT

The auditory system provides a valuable experimental model to investigate the role of sensory activity in regulating neuronal membrane properties. In this study, we have investigated the role of activity directly by measuring changes in medial nucleus of the trapezoid body (MNTB) neurons in normal hearing mice subjected to 1-h sound stimulation. Broadband (4-12 kHz) chirps were used to activate MNTB neurons tonotopically restricted to the lateral MNTB, as confirmed by c-Fos-immunoreactivity. Following 1-h sound stimulation a substantial increase in Kv3.1b-immunoreactivity was measured in the lateral region of the MNTB, which lasted for 2 h before returning to control levels. Electrophysiological patch-clamp recordings in brainstem slices revealed an increase in high-threshold potassium currents in the lateral MNTB of sound-stimulated mice. Current-clamp and dynamic-clamp experiments showed that MNTB cells from the sound-stimulated mice were able to maintain briefer action potentials during high-frequency firing than cells from control mice. These results provide evidence that acoustically driven auditory activity can selectively regulate high-threshold potassium currents in the MNTB of normal hearing mice, likely due to an increased membrane expression of Kv3.1b channels.


Subject(s)
Acoustic Stimulation/methods , Action Potentials/physiology , Auditory Pathways/physiology , Brain Stem/cytology , Neurons, Afferent/metabolism , Shaw Potassium Channels/metabolism , Animals , Cell Membrane/metabolism , Female , Male , Mice , Neurons, Afferent/cytology , Patch-Clamp Techniques , Potassium/metabolism , Potassium Channel Blockers/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Tetraethylammonium/metabolism
11.
Biochem Biophys Res Commun ; 362(4): 940-5, 2007 Nov 03.
Article in English | MEDLINE | ID: mdl-17765873

ABSTRACT

MicroRNAs are known to regulate the expression of many mRNAs by binding to complementary target sequences at the 3'UTRs. Because of such properties, miRNAs may regulate tissue-specific mRNAs as a cell undergoes transdifferentiation during regeneration. We have tested this hypothesis during lens and hair cell regeneration in newts using microarray analysis. We found that distinct sets of miRNAs are associated with lens and hair cell regeneration. Members of the let-7 family are expressed in both events and they are regulated in a similar fashion. All the let-7 members are down regulated during the initiation of regeneration, which is characterized by dedifferentiation of terminally differentiated cells. This is the first report to correlate expression of miRNAs as novel regulators of vertebrate regeneration, alluding to a novel mechanism whereby transdifferentiation occurs.


Subject(s)
Hair Cells, Auditory, Inner/cytology , Hair Cells, Auditory, Inner/metabolism , Lens, Crystalline/cytology , Lens, Crystalline/metabolism , Proteome/metabolism , Regeneration/physiology , Salamandridae/metabolism , Animals , Cell Differentiation/physiology , Gene Expression Regulation/physiology , Lens, Crystalline/growth & development , MicroRNAs/genetics , Morphogenesis/physiology , Salamandridae/anatomy & histology , Salamandridae/growth & development
12.
J Physiol ; 584(Pt 1): 31-45, 2007 Oct 01.
Article in English | MEDLINE | ID: mdl-17640932

ABSTRACT

Renshaw cell properties have been studied extensively for over 50 years, making them a uniquely well-defined class of spinal interneuron. Recent work has revealed novel ways to identify Renshaw cells in situ and this in turn has promoted a range of studies that have determined their ontogeny and organization of synaptic inputs in unprecedented detail. In this review we illustrate how mature Renshaw cell properties and connectivity arise through a combination of activity-dependent and genetically specified mechanisms. These new insights should aid the development of experimental strategies to manipulate Renshaw cells in spinal circuits and clarify their role in modulating motor output.


Subject(s)
Interneurons/cytology , Animals , Calcium-Binding Proteins/metabolism , Interneurons/metabolism , Interneurons/physiology , Synapses/ultrastructure
13.
Cell Physiol Biochem ; 20(1-4): 121-30, 2007.
Article in English | MEDLINE | ID: mdl-17595522

ABSTRACT

BACKGROUND: The molecular mechanism of K-Cl cotransport (KCC) consists of at least 4 isoforms, KCC 1, 2, 3, and 4 which, in multiple combinations, exist in most cells, including erythrocytes and neuronal cells. METHODS: We utilized reverse-transcriptase-polymerase chain reaction (RT-PCR) and ion flux studies to characterize KCC activity in an immortalized in vitro cell model for fibrous astrocytes, the rat C6 glioblastoma cell. Isoform-specific sets of oligonucleotide primers were synthesized for NKCC1, KCC1, KCC2, KCC3, KCC4, and also for NKCC1 and actin. K-Cl cotransport activity was determined by measuring either the furosemide-sensitive, or the Cl(-)-dependent bumetanide-insensitive Rb(+) (a K(+) congener) influx in the presence of the Na/K pump inhibitor ouabain. Rb(+) influx was measured at a fixed external Cl concentrations, [Cl(-)](e), as a function of varying external Rb concentrations, [Rb(+)](e), and at a fixed [Rb(+)](e) as a function of varying [Cl(-)](e), and with equimolar Cl replacement by anions of the chaotropic series. RESULTS: RT-PCR of C6 glioblastoma (C6) cells identified mRNA for three KCC isoforms (1, 3, and 4). NKCC1 mRNA was also detected. The apparent K(m) for KCC-mediated Rb(+) influx was 15 mM [Rb(+)](e), and V(max) 12.5 nmol Rb(+) * mg protein(-1) * minute(-1). The calculated apparent K(m) for external Cl(-) was 13 mM and V(max) 14.4 nmol Rb(+) * mg protein(-1) * minute(-1). The anion selectivity sequence of the furosemide-sensitive Rb(+) influx was Cl(-)>>Br-=NO(3)(-)>I(-)=SCN(-)>>Sfm(-) (sulfamate). Established activators of K-Cl cotransport, hyposmotic shock and N-ethylmaleimide (NEM) pretreatment, stimulated furosemide-sensitive Rb(+) influx. A ñ50% NEM-induced loss of intracellular K(+) was prevented by furosemide. CONCLUSION: We have identified by RT-PCR the presence of three distinct KCC isoforms (1, 3, and 4) in rat C6 glioblastoma cells, and functionally characterized the anion selectivity and kinetics of their collective sodium-independent cation-chloride cotransport activity.


Subject(s)
Chlorides/metabolism , Neuroglia/metabolism , Potassium/metabolism , Symporters/genetics , Symporters/metabolism , Animals , Base Sequence , Bumetanide/pharmacology , Cell Line , DNA Primers/genetics , Ethylmaleimide/pharmacology , Furosemide/pharmacology , Hypotonic Solutions , Ion Transport/drug effects , Kinetics , Neuroglia/drug effects , Ouabain/pharmacology , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Rubidium/metabolism , Sodium-Potassium-Chloride Symporters/genetics , Sodium-Potassium-Chloride Symporters/metabolism , Solute Carrier Family 12, Member 2 , K Cl- Cotransporters
14.
Clin Exp Pharmacol Physiol ; 34(7): 566-73, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17581210

ABSTRACT

1. Ion gradients across the cell membrane are important for proper cellular communication and homeostasis. With the exception of erythrocytes, chloride (Cl), one of the most important free anions in animal cells, is not distributed at thermodynamic equilibrium across the plasma membrane. The K-Cl cotransporter (COT), consisting of at least four isoforms, utilizes the larger outwardly directed chemical driving force of K to expel Cl from the cell against its inwardly directed chemical gradient and has been implicated recently as one of the main Cl extruders in developing neurons. 2. Previous in situ hybridization studies have indicated widespread mRNA distribution of the neuronal-specific K-Cl COT isoform (KCC2) throughout the rat central nervous system (CNS). However, immunohistochemical studies have been limited owing to the availability of a more selective antibody to KCC2. The goal of the present study was to develop a new molecular tool for the immunohistochemical identification and neuronal distribution of KCC2. 3. Herein, we present evidence of immunohistochemical corroboration of the widespread KCC2 mRNA expression using a novel extracellular anti-peptide antibody directed against the second extracellular loop (ECL2) of KCC2. Immunoperoxidase and immunofluorescent labelling revealed widespread post-synaptic somatic and dendritic localization of KCC2 in multiple neuronal populations in the cerebral cortex, hippocampus, brainstem, lumbar spinal cord and cerebellum. We also demonstrate that binding of the antibody to an extracellular epitope within ECL2 does not alter cotransporter function. In essence, the present study reports on a new molecular tool for structural and functional studies of KCC2.


Subject(s)
Antibodies , Brain Chemistry , Epitope Mapping , Immunohistochemistry/methods , Spinal Cord/chemistry , Symporters/analysis , Animals , Antibodies/metabolism , Brain Stem/chemistry , Cerebellum/chemistry , Cerebral Cortex/chemistry , Enzyme-Linked Immunosorbent Assay , Glutamate Decarboxylase/analysis , Hippocampus/chemistry , Immunization , Isoenzymes/analysis , Microinjections , Neurons/chemistry , Oocytes , Rabbits , Rats , Rats, Sprague-Dawley , Symporters/genetics , Symporters/immunology , Symporters/metabolism , Synapses/chemistry , Xenopus laevis , K Cl- Cotransporters
15.
J Physiol ; 576(Pt 3): 849-64, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-16916913

ABSTRACT

The hyperpolarization-activated cation current (I(h)) may influence precise auditory processing by modulating resting membrane potential and cell excitability. We used electrophysiology and immunohistochemistry to investigate the properties of I(h) in three auditory brainstem nuclei in mice: the anteroventral cochlear nucleus (AVCN), the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO). I(h) amplitude varied considerably between these cell types, with the order of magnitude LSO > AVCN > MNTB. Kinetically, I(h) is faster in LSO neurons, and more active at rest, compared with AVCN and MNTB cells. The half-activation voltage is -10 mV more hyperpolarized for AVCN and MNTB cells compared with LSO neurons. HCN1 immunoreactivity strongly labelled AVCN and LSO neurons, while HCN2 staining was more diffuse in all nuclei. The HCN4 subunit displayed robust membrane staining in AVCN and MNTB cells but weak labelling of the LSO. We used a dynamic clamp, after blocking I(h), to reinsert I(h) to the different cell types. Our results indicate that the native I(h) for each cell type influences the resting membrane potential and can delay the generation of action potentials in response to injected current. Native I(h) increases rebound depolarizations following hyperpolarizations in all cell types, and increases the likelihood of rebound action potentials (particularly in multiple-firing LSO neurons). This systematic comparison shows that I(h) characteristics vary considerably between different brainstem nuclei, and that these differences significantly affect the response properties of cells within these nuclei.


Subject(s)
Brain Stem/physiology , Evoked Potentials, Auditory, Brain Stem/physiology , Ion Channels/physiology , Membrane Potentials/physiology , Action Potentials/physiology , Animals , Anterior Thalamic Nuclei/cytology , Anterior Thalamic Nuclei/physiology , Brain Stem/cytology , Hydrogen Cyanide/metabolism , Mice , Mice, Inbred CBA , Neurons, Afferent/physiology , Olivary Nucleus/cytology , Olivary Nucleus/physiology , Patch-Clamp Techniques
16.
J Physiol ; 572(Pt 2): 313-21, 2006 Apr 15.
Article in English | MEDLINE | ID: mdl-16469782

ABSTRACT

Neural activity plays an important role in regulating synaptic strength and neuronal membrane properties. Attempts to establish guiding rules for activity-dependent neuronal changes have led to such concepts as homeostasis of cellular activity and Hebbian reinforcement of synaptic strength. However, it is clear that there are diverse effects resulting from activity changes, and that these changes depend on the experimental preparation, and the developmental stage of the neural circuits under study. In addition, most experimental evidence on activity-dependent regulation comes from reduced preparations such as neuronal cultures. This review highlights recent results from studies of the intact mammalian auditory system, where changes in activity have been shown to produce alterations in synaptic and membrane properties at the level of individual neurons, and changes in network properties, including the formation of tonotopic maps.


Subject(s)
Auditory Pathways/physiology , Neurons, Afferent/physiology , Synapses/physiology , Animals , Auditory Pathways/anatomy & histology , Cats , Cochlea/innervation , Cochlea/physiopathology , Cochlear Implants , Deafness/congenital , Deafness/physiopathology , Evoked Potentials, Auditory, Brain Stem/physiology , Homeostasis/physiology , Mice , Synaptic Membranes/physiology
17.
J Physiol ; 571(Pt 3): 563-78, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16373385

ABSTRACT

There is an orderly topographic arrangement of neurones within auditory brainstem nuclei based on sound frequency. Previous immunolabelling studies in the medial nucleus of the trapezoid body (MNTB) have suggested that there may be gradients of voltage-gated currents underlying this tonotopic arrangement. Here, our electrophysiological and immunolabelling results demonstrate that underlying the tonotopic organization of the MNTB is a combination of medio-lateral gradients of low-and high-threshold potassium currents and hyperpolarization-activated cation currents. Our results also show that the intrinsic membrane properties of MNTB neurones produce a topographic gradient of time delays, which may be relevant to sound localization, following previous demonstrations of the importance of the timing of inhibitory input from the MNTB to the medial superior olive (MSO). Most importantly, we demonstrate that, in the MNTB of congenitally deaf mice, which exhibit no spontaneous auditory nerve activity, the normal tonotopic gradients of neuronal properties are absent. Our results suggest an underlying mechanism for the observed topographic gradient of neuronal firing properties in the MNTB, show that an intrinsic neuronal mechanism is responsible for generating a topographic gradient of time-delays, and provide direct evidence that these gradients rely on spontaneous auditory nerve activity during development.


Subject(s)
Auditory Pathways/physiology , Brain Mapping , Brain Stem/physiology , Deafness/physiopathology , Action Potentials , Animals , Cyclic Nucleotide-Gated Cation Channels , Deafness/congenital , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Ion Channels/metabolism , Kv1.1 Potassium Channel/metabolism , Mice , Mice, Inbred DBA , Models, Neurological , Neurons/metabolism , Patch-Clamp Techniques , Potassium/metabolism , RNA, Messenger/metabolism , Shaw Potassium Channels/genetics , Shaw Potassium Channels/metabolism
18.
Cell Physiol Biochem ; 16(1-3): 87-98, 2005.
Article in English | MEDLINE | ID: mdl-16121037

ABSTRACT

Sheep K-Cl cotransporter-1(shKCC1) cDNA was cloned from kidney by RT-PCR with an open reading frame of 3258 base pairs exhibiting 92%, 90%, 88% and 87% identity with pig, rabbit and human, rat and mouse KCC1 cDNAs, respectively, encoding an approximately 122 kDa polypeptide of 1086-amino acids. Hydropathy analysis reveals the familiar KCC1 topology with 12 transmembrane domains (TMDs) and the hydrophilic NH2-terminal (NTD) and COOH-terminal (CTD) domains both at the cytoplasmic membrane face. However, shKCC1 has two rather than one large extracellular loops (ECL): ECL3 between TMDs 5 and 6, and ECL6, between TMDs 11 and 12. The translated shKCC1 protein differs in 12 amino acid residues from other KCC1s, mainly within the NTD, ECL3, ICL4, ECL6, and CTD. Notably, a tyrosine residue at position 996 replaces aspartic acid conserved in all other species. Human embryonic kidney (HEK293) cells and mouse NIH/3T3 fibroblasts, transiently transfected with shKCCI-cDNA, revealed the glycosylated approximately 150 kDa proteins by Western blots and positive immunofluorescence-staining with polyclonal rabbit anti-ratKCC1 antibodies. ShKCC1 was functionally expressed in NIH/3T3 cells by an elevated basal Cl-dependent K influx measured with Rb as K-congener that was stimulated three-fold by the KCC-activator N-ethylmaleimide.


Subject(s)
Kidney/metabolism , Sheep/genetics , Symporters/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chlorides/metabolism , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression , Humans , In Vitro Techniques , Mice , Molecular Sequence Data , NIH 3T3 Cells , Potassium/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Sheep/metabolism , Species Specificity , Symporters/chemistry , Symporters/metabolism , K Cl- Cotransporters
19.
J Neurophysiol ; 94(3): 1688-98, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15917321

ABSTRACT

Inhibitory synaptic inputs to Renshaw cells are concentrated on the soma and the juxtasomatic dendrites. In the present study, we investigated whether this proximal bias leads to more effective inhibition under different neuronal operating conditions. Using compartmental models based on detailed anatomical measurements of intracellularly stained Renshaw cells, we compared the inhibition produced by glycine/gamma-aminobutyric acid-A (GABA(A)) synapses when distributed with a proximal bias to the inhibition produced when the same synapses were distributed uniformly (i.e., with no regional bias). The comparison was conducted in subthreshold and suprathreshold conditions. The latter were mimicked by voltage clamping the soma to -55 mV. The voltage clamp reduces nonlinear interactions between excitatory and inhibitory synapses. We hypothesized that for electrotonically compact cells such as Renshaw cells, the strength of the inhibition would become much less dependent on synaptic location in suprathreshold conditions. This hypothesis was not confirmed. The inhibition produced when inhibitory inputs were proximally distributed was always stronger than when the same inputs were uniformly distributed. In fact, the relative effectiveness of proximally distributed inhibitory inputs over uniformly distributed synapses was greater in suprathreshold conditions than that in subthreshold conditions. The somatic voltage clamp minimized saturation of inhibitory driving potentials. Because this effect was greatest near the soma, the current produced by more distal synapses suffered a greater loss because of saturation. Conversely, in subthreshold conditions, the effectiveness of proximal synapses was substantially reduced at high levels of background synaptic activity because of saturation. Our results suggest glycine/GABA(A) synapses on Renshaw cells are strategically distributed to block the powerful excitatory drive produced by recurrent collaterals from motoneurons.


Subject(s)
Differential Threshold/physiology , Interneurons/physiology , Models, Neurological , Neural Inhibition/physiology , Acetylcholine/metabolism , Animals , Glycine/metabolism , Humans , Membrane Potentials/drug effects , Membrane Potentials/physiology , Synapses/physiology , gamma-Aminobutyric Acid/metabolism
20.
Am J Physiol Cell Physiol ; 289(3): C564-75, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15843438

ABSTRACT

The cellular mechanism for Cl(-) and K(+) secretion in the colonic epithelium requires K(+) channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the K(+) channel proteins K(V)LQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current (I(sc)) and transepithelial conductance (G(t)) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin E(2) (PGE(2)), and carbachol (CCh). HMR1556 (10 microM), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Brüggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 3831-3837, 2001), partially (approximately 50%) inhibited Cl(-) secretory I(sc) and G(t) activated by PGE(2) and CCh in rat colon with an IC(50) of 55 nM, but in guinea pig distal colon Cl(-) secretory I(sc) and G(t) were unaltered. EPI-activated K(+)-secretory I(sc) and G(t) also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 K(+) channel is not an essential component for producing Cl(-) secretion. Other K(+) channels present in the basolateral membrane presumably must also contribute directly to the K(+) conductance necessary for K(+) exit during activation of Cl(-) secretion in the colonic mucosa.


Subject(s)
Chlorides/metabolism , Colon/metabolism , Intestinal Mucosa/metabolism , Potassium Channels, Voltage-Gated/metabolism , Animals , Biological Transport/drug effects , Biological Transport/physiology , Cell Polarity/physiology , Cholinergic Agonists/pharmacology , Chromans/pharmacology , Colon/cytology , Female , Guinea Pigs , Intestinal Mucosa/cytology , KCNQ Potassium Channels , KCNQ1 Potassium Channel , Male , Potassium/metabolism , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Stimulation, Chemical , Sulfonamides/pharmacology
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