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1.
Neuroscience ; 452: 78-97, 2021 01 01.
Article in English | MEDLINE | ID: mdl-33212215

ABSTRACT

Spinal muscular atrophy (SMA) is a devastating genetic neuromuscular disease. Diffuse neuropathology has been reported in SMA patients and mouse models, however, functional changes in brain regions have not been studied. In the SMNΔ7 mouse model, we identified three types of differences in neuronal function in the cerebellum and motor cortex from two age groups: P7-9 (P7) and P11-14 (P11). Microelectrode array studies revealed significantly lower spontaneous firing and network activity in the cerebellum of SMA mice in both age groups, but it was more profound in the P11 group. In the motor cortex, however, neural activity was not different in either age group. Whole-cell patch-clamp was used to study the function of output neurons in both brain regions. In cerebellar Purkinje cells (PCs) of SMA mice, the input resistance was larger at P7, while capacitance was smaller at P11. In the motor cortex, no difference was observed in the passive membrane properties of layer V pyramidal neurons (PN5s). The action potential threshold of both types of output neurons was depolarized in the P11 group. We also observed lower spontaneous excitatory and inhibitory synaptic activity in PN5s and PCs respectively from P11 SMA mice. Overall, these differences suggest functional alterations in the neural network in these motor regions that change during development. Our results also suggest that neuronal dysfunction in these brain regions may contribute to the pathology of SMA. Comprehensive treatment strategies may consider motor regions outside of the spinal cord for better outcomes.


Subject(s)
Motor Cortex , Muscular Atrophy, Spinal , Animals , Cerebellum , Disease Models, Animal , Mice , Motor Neurons , Spinal Cord , Survival of Motor Neuron 1 Protein
2.
J Control Release ; 279: 243-250, 2018 06 10.
Article in English | MEDLINE | ID: mdl-29673641

ABSTRACT

Hearing loss is the most prevalent sensory disability worldwide and may be caused by age, drugs or exposure to excessive noise. We have previously developed a minimally-invasive nanohydrogel drug delivery system that successfully delivers nanoparticles into the inner ear. We have substantially extended this technique by functionalizing the nanoparticles and introducing a targeting peptide which recognizes prestin, a transmembrane electromotile protein uniquely expressed in outer hair cells (OHCs) of the inner ear. We demonstrate the successful delivery of molecules and plasmids specifically to OHCs. When compared to untargeted nanoparticles, the delivery of a c-Jun N-terminal kinase (JNK) inhibitor, D-JNKi-1, to OHCs by targeted nanoparticles improved protection from noise induced hearing loss (NIHL). This is the first demonstration of a protection from NIHL using a novel safe and controllable delivery system which is minimally-invasive to the inner ear and, as such, is an extremely appealing technique for use in many clinical applications.


Subject(s)
Drug Delivery Systems , Hearing Loss, Noise-Induced/drug therapy , Nanoparticles , Peptides/administration & dosage , Animals , CHO Cells , Cell Line , Cricetulus , Ear, Inner/metabolism , Female , Hair Cells, Auditory, Outer/metabolism , Hearing Loss, Noise-Induced/physiopathology , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mice , Mice, Inbred CBA , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/pharmacology , Peptides/pharmacology
3.
J Neurophysiol ; 115(5): 2536-55, 2016 05 01.
Article in English | MEDLINE | ID: mdl-26936982

ABSTRACT

Firing patterns differ between subpopulations of vestibular primary afferent neurons. The role of sodium (NaV) channels in this diversity has not been investigated because NaV currents in rodent vestibular ganglion neurons (VGNs) were reported to be homogeneous, with the voltage dependence and tetrodotoxin (TTX) sensitivity of most neuronal NaV channels. RT-PCR experiments, however, indicated expression of diverse NaV channel subunits in the vestibular ganglion, motivating a closer look. Whole cell recordings from acutely dissociated postnatal VGNs confirmed that nearly all neurons expressed NaV currents that are TTX-sensitive and have activation midpoints between -30 and -40 mV. In addition, however, many VGNs expressed one of two other NaV currents. Some VGNs had a small current with properties consistent with NaV1.5 channels: low TTX sensitivity, sensitivity to divalent cation block, and a relatively negative voltage range, and some VGNs showed NaV1.5-like immunoreactivity. Other VGNs had a current with the properties of NaV1.8 channels: high TTX resistance, slow time course, and a relatively depolarized voltage range. In two NaV1.8 reporter lines, subsets of VGNs were labeled. VGNs with NaV1.8-like TTX-resistant current also differed from other VGNs in the voltage dependence of their TTX-sensitive currents and in the voltage threshold for spiking and action potential shape. Regulated expression of NaV channels in primary afferent neurons is likely to selectively affect firing properties that contribute to the encoding of vestibular stimuli.


Subject(s)
Ganglia, Sensory/cytology , NAV1.5 Voltage-Gated Sodium Channel/metabolism , NAV1.8 Voltage-Gated Sodium Channel/metabolism , Neurons, Afferent/metabolism , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacology , Vestibule, Labyrinth/innervation , Action Potentials , Animals , Cells, Cultured , Ganglia, Sensory/metabolism , Ganglia, Sensory/physiology , NAV1.5 Voltage-Gated Sodium Channel/genetics , NAV1.8 Voltage-Gated Sodium Channel/genetics , Neurons, Afferent/physiology , Rats , Rats, Long-Evans
4.
Cell ; 157(3): 689-701, 2014 Apr 24.
Article in English | MEDLINE | ID: mdl-24766812

ABSTRACT

Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. We conducted a screen for circadian-relevant neurons in the Drosophila brain and report here that cells of the pars intercerebralis (PI), a functional homolog of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GFP reconstitution across synaptic partners (GRASP) analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin-releasing factor (CRF) homolog, DH44, as a circadian output molecule that is specifically expressed by PI neurons and is required for normal rest:activity rhythms. Notably, selective activation or ablation of just six DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms.


Subject(s)
Circadian Clocks , Drosophila/physiology , Neurons/physiology , Animals , Animals, Genetically Modified , Brain/cytology , Brain/physiology , Circadian Rhythm , Drosophila/cytology , Neurons/cytology , Single-Cell Analysis , Transcriptome
5.
Neuron ; 75(5): 779-85, 2012 Sep 06.
Article in English | MEDLINE | ID: mdl-22958819

ABSTRACT

Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that carries the point mutation A227D. The fluorescence intensity of ArcLight A242 decreases by 35% in response to a 100 mV depolarization when measured in HEK293 cells, which is more than five times larger than the signals from previously reported fluorescent protein voltage sensors. We show that the combination of signal size and response speed of these new probes allows the reliable detection of single action potentials and excitatory potentials in individual neurons and dendrites.


Subject(s)
Action Potentials/physiology , Fluorescent Dyes/chemical synthesis , Green Fluorescent Proteins/chemistry , Luminescent Proteins/chemical synthesis , Neurons/physiology , Recombinant Fusion Proteins/chemical synthesis , Synaptic Potentials/physiology , Voltage-Sensitive Dye Imaging/methods , Animals , Biosensing Techniques/methods , Ciona intestinalis , HEK293 Cells , Humans , Luminescent Proteins/genetics , Microscopy, Fluorescence/methods , Point Mutation/genetics , Recombinant Fusion Proteins/genetics
6.
J Assoc Res Otolaryngol ; 9(3): 307-20, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18506528

ABSTRACT

Prestin is a membrane protein in the outer hair cell (OHC) that has been shown to be essential for electromotility. OHCs from prestin-null mice do not express prestin, do not have a nonlinear capacitance (the electrical signature of electromotility), and are smaller in size than wild-type OHCs. We sought to determine whether prestin-null OHCs can be transduced to incorporate functional prestin protein in a normal fashion. A recombinant helper-dependent adenovirus expressing prestin and green fluorescent protein (HDAd-prestin-GFP) was created and tested in human embryonic kidney cells (HEK cells). Transduced HEK cells demonstrated membrane expression of prestin and nonlinear capacitance. HDAd-prestin-GFP was then applied to cochlear sensory epithelium explants harvested from wild-type and prestin-null mice at postnatal days 2-3, the age at which native prestin is just beginning to become functional in wild-type mice. At postnatal days 4-5, we investigated transduced OHCs for (1) their prestin expression pattern as revealed by immunofluorescence; (2) their cell surface area as measured by linear capacitance; and (3) their prestin function as indicated by nonlinear capacitance. HDAd-prestin-GFP efficiently transduced OHCs of both genotypes and prestin protein localized to the plasma membrane. Whole-cell voltage clamp studies revealed a nonlinear capacitance in transduced wild-type and prestin-null OHCs, but not in non-transduced cells of either genotype. Prestin transduction did not increase the linear capacitance (cell surface area) for either genotype. In peak nonlinear capacitance, voltage at peak nonlinear capacitance, charge density of the nonlinear capacitance, and shape of the voltage-capacitance curves, the transduced cells of the two genotypes resembled each other and previously reported data from adult wild-type mouse OHCs. Thus, prestin introduced into prestin-deficient OHCs segregates normally to the cell membrane and generates a normal nonlinear capacitance, indicative of normal prestin function.


Subject(s)
Aging/metabolism , Hair Cells, Auditory, Outer/metabolism , Molecular Motor Proteins/metabolism , Organ of Corti/metabolism , Adenoviridae/genetics , Animals , Animals, Newborn , Cell Line , Cell Membrane/metabolism , Electrophysiology , Green Fluorescent Proteins , Hair Cells, Auditory, Outer/cytology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Motor Proteins/genetics , Organ Culture Techniques , Organ of Corti/cytology , Patch-Clamp Techniques , Transduction, Genetic
7.
J Neurophysiol ; 97(2): 1684-704, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17065252

ABSTRACT

Two kinds of sodium current (I(Na)) have been separately reported in hair cells of the immature rodent utricle, a vestibular organ. We show that rat utricular hair cells express one or the other current depending on age (between postnatal days 0 and 22, P0-P22), hair cell type (I, II, or immature), and epithelial zone (striola vs. extrastriola). The properties of these two currents, or a mix, can account for descriptions of I(Na) in hair cells from other reports. The patterns of Na channel expression during development suggest a role in establishing the distinct synapses of vestibular hair cells of different type and epithelial zone. All type I hair cells expressed I(Na,1), a TTX-insensitive current with a very negative voltage range of inactivation (midpoint: -94 mV). I(Na,2) was TTX sensitive and had less negative voltage ranges of activation and inactivation (inactivation midpoint: -72 mV). I(Na,1) dominated in the striola at all ages, but current density fell by two-thirds after the first postnatal week. I(Na,2) was expressed by 60% of hair cells in the extrastriola in the first week, then disappeared. In the third week, all type I cells and about half of type II cells had I(Na,1); the remaining cells lacked sodium current. I(Na,1) is probably carried by Na(V)1.5 subunits based on biophysical and pharmacological properties, mRNA expression, and immunoreactivity. Na(V)1.5 was also localized to calyx endings on type I hair cells. Several TTX-sensitive subunits are candidates for I(Na,2).


Subject(s)
Hair Cells, Auditory, Inner/growth & development , Hair Cells, Auditory, Inner/physiology , Saccule and Utricle/growth & development , Saccule and Utricle/physiology , Sodium Channels/physiology , Aging/metabolism , Aging/physiology , Algorithms , Animals , Cell Separation , Cesium/physiology , DNA Primers , Epithelial Cells/drug effects , Evoked Potentials/physiology , Hair Cells, Auditory, Inner/drug effects , Half-Life , Immunohistochemistry , NAV1.5 Voltage-Gated Sodium Channel , Neural Conduction/drug effects , Neural Conduction/physiology , Patch-Clamp Techniques , Rats , Rats, Long-Evans , Reverse Transcriptase Polymerase Chain Reaction , Saccule and Utricle/drug effects , Tetrodotoxin/pharmacology
8.
J Neurosci ; 26(40): 10253-69, 2006 Oct 04.
Article in English | MEDLINE | ID: mdl-17021181

ABSTRACT

Type I vestibular hair cells have large K+ currents that, like neuronal M currents, activate negative to resting potential and are modulatable. In rodents, these currents are acquired postnatally. In perforated-patch recordings from rat utricular hair cells, immature hair cells [younger than postnatal day 7 (P7)] had a steady-state K+ conductance (g(-30)) with a half-activation voltage (V1/2) of -30 mV. The size and activation range did not change in maturing type II cells, but, by P16, type I cells had added a K conductance that was on average fourfold larger and activated much more negatively. This conductance may comprise two components: g(-60) (V1/2 of -60 mV) and g(-80) (V1/2 of -80 mV). g(-80) washed out during ruptured patch recordings and was blocked by a protein kinase inhibitor. M currents can include contributions from KCNQ and ether-a-go-go-related (erg) channels. KCNQ and erg channel blockers both affected the K+ currents of type I cells, with KCNQ blockers being more potent at younger than P7 and erg blockers more potent at older than P16. Single-cell reverse transcription-PCR and immunocytochemistry showed expression of KCNQ and erg subunits. We propose that KCNQ channels contribute to g(-30) and g(-60) and erg subunits contribute to g(-80). Type I hair cells are contacted by calyceal afferent endings. Recordings from dissociated calyces and afferent endings revealed large K+ conductances, including a KCNQ conductance. Calyx endings were strongly labeled by KCNQ4 and erg1 antisera. Thus, both hair cells and calyx endings have large M-like K+ conductances with the potential to control the gain of transmission.


Subject(s)
Hair Cells, Vestibular/growth & development , Nerve Endings/physiology , Neurons, Afferent/physiology , Potassium Channels/physiology , Saccule and Utricle/growth & development , Animals , Animals, Newborn , Hair Cells, Vestibular/drug effects , In Vitro Techniques , KCNQ Potassium Channels/antagonists & inhibitors , KCNQ Potassium Channels/physiology , Nerve Endings/drug effects , Neurons, Afferent/drug effects , Potassium Channel Blockers/pharmacology , Rats , Rats, Long-Evans , Saccule and Utricle/drug effects
9.
J Neurosci ; 23(8): 3176-85, 2003 Apr 15.
Article in English | MEDLINE | ID: mdl-12716925

ABSTRACT

Although many psychopharmacological factors contribute to nicotine addiction, midbrain dopaminergic systems have received much attention because of their roles in reinforcement and associative learning. It is generally thought that the mesocorticolimbic dopaminergic system is important for the acquisition of behaviors that are reinforced by the salient drives of the environment or by the inappropriate stimuli of addictive drugs. Nicotine, as obtained from tobacco, can activate nicotinic acetylcholine receptors (nAChRs) and excite midbrain neurons of the mesocorticolimbic system. Using midbrain slices from rats, wild-type mice, and genetically engineered mice, we have found differences in the nAChR currents from the ventral tegmental area (VTA) and the substantia nigra compacta (SNc). Nicotinic AChRs containing the alpha7 subunit (alpha7* nAChRs) have a low expression density. Electrophysiological analysis of nAChR currents, autoradiography of [125I]-alpha-bungarotoxin binding, and in situ hybridization revealed that alpha7* nAChRs are more highly expressed in the VTA than the SNc. In contrast, beta2* nAChRs are move evenly distributed at a higher density in both the VTA and SNc. At the concentration of nicotine obtained by tobacco smokers, the slow components of current (mainly mediated by beta2* nAChRs) become essentially desensitized. However, the minority alpha7* component of the current in the VTA/SNc is not significantly desensitized by nicotine in the range < or =100 nm. These results suggest that nicotine, as obtained from tobacco, can have multiple effects on the midbrain areas by differentially influencing dopamine neurons of the VTA and SNc and differentially desensitizing alpha7* and non-alpha7 nAChRs.


Subject(s)
Dopamine/metabolism , Mesencephalon/metabolism , Neurons/metabolism , Nicotine/pharmacology , Receptors, Nicotinic/metabolism , Animals , In Vitro Techniques , Mesencephalon/cytology , Mesencephalon/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Neurons/drug effects , Nicotinic Antagonists/pharmacology , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Receptors, Nicotinic/deficiency , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/genetics , Substantia Nigra/cytology , Substantia Nigra/drug effects , Substantia Nigra/metabolism , Ventral Tegmental Area/cytology , Ventral Tegmental Area/drug effects , Ventral Tegmental Area/metabolism , alpha7 Nicotinic Acetylcholine Receptor
10.
Br J Pharmacol ; 137(1): 29-38, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12183328

ABSTRACT

1. Whole-cell currents were recorded from Xenopus laevis oocytes and human embryonic kidney cells expressing GABA(A) receptor beta3 subunit homomers to search for additional residues affecting Zn(2+) inhibition. These residues would complement the previously identified histidine (H267), present just within the external portal of the ion channel, which modulates Zn(2+) inhibition. 2. Zinc inhibited the pentobarbitone-gated current on beta3(H267A) homomers at pH 7.4, but this effect was abolished at pH 5.4. The Zn(2+)-sensitive spontaneous beta3 subunit-mediated conductance was also insensitive to block by Zn(2+) at pH 5.4. 3. Changing external pH enabled the titration of the Zn(2+) sensitive binding site or signal transduction domain. The pK(a) was estimated at 6.8 +/- 0.03 implying the involvement of histidine residues. 4. External histidine residues in the beta3 receptor subunit were substituted with alanine, in addition to the background mutation, H267A, to assess their sensitivity to Zn(2+) inhibition. The Zn(2+) IC(50) was unaffected by either the H119A or H191A mutations. 5. The remaining histidine, H107, the only other candidate likely to participate in Zn(2+) inhibition, was substituted with various residues. Most mutants were expressed at the cell surface but they disrupted functional expression of beta3 homomers. However, H107G was functional and demonstrated a marked reduction in sensitivity to Zn(2+). 6. GABA(A) receptor beta3 subunits form functional ion channels that can be inhibited by Zn(2+). Two histidine residues are largely responsible for this effect, H267 in the pore lining region and H107 residing in the extracellular N-terminal domain.


Subject(s)
Histidine/physiology , Receptors, GABA-A/metabolism , Zinc/metabolism , Amino Acid Sequence , Animals , Cell Line , Histidine/genetics , Humans , Hydrogen-Ion Concentration , Membrane Potentials , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Oocytes , Patch-Clamp Techniques , Protein Subunits , Receptors, GABA-A/genetics , Receptors, GABA-A/physiology , Xenopus laevis , Zinc/pharmacology
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