ABSTRACT
Amyotrophic Lateral Sclerosis (ALS) involves protracted pre-symptomatic periods of abnormal motor neuron (MN) excitability occurring in parallel with central and peripheral synaptic perturbations. Focusing on inhibitory control of MNs, we first compared longitudinal changes in pre-synaptic terminal proteins for GABA and glycine neurotransmitters around the soma of retrogradely identified trigeminal jaw closer (JC) MNs and ChAT-labeled midbrain extraocular (EO) MNs in the SOD1G93A mouse model for ALS. Fluorescence immunocytochemistry and confocal imaging were used to quantify GAD67 and GlyT2 synaptic bouton density (SBD) around MN soma at pre-symptomatic ages â¼P12 (postnatal), â¼P50 (adult) and near disease end-stage (â¼P135) in SOD1G93A mice and age-matched wild-type (WT) controls. We noted reduced GAD67 innervation in the SOD1G93A trigeminal jaw closer MNs around P12, relative to age-matched WT and no significant difference around P50 and P135. In contrast, both GAD67 and GlyT2 innervation were elevated in the SOD1G93A EO MNs at the pre-symptomatic time points. Considering trigeminal MNs are vulnerable in ALS while EO MNs are spared, we suggest that upregulation of inhibition in the latter might be compensatory. Notable contrast also existed in the innate co-expression patterns of GAD67 and GlyT2 with higher mutual information (co-dependency) in EO MNs compared to JC in both SOD1G93A and WT mice, especially at adult stages (P50 and P135). Around P12 when GAD67 terminals expression was low in the mutant, we further tested for persistent GABA inhibition in those MNs using in vitro patch-clamp electrophysiology. Our results show that SOD1G93A JC MNs have reduced persistent GABA inhibition, relative to WT. Pharmacological blocking of an underlying tonically active GABA conductance using the GABA-α5 subunit inverse agonist, L-655-708, disinhibited WT JC MNs and lowered their recruitment threshold, suggesting its role in the control of intrinsic MN excitability. Quantitative RT-PCR in laser dissected JC MNs further supported a reduction in GABA-α5 subunit mRNA expression in the mutant. In light of our previous report that JC MNs forming putative fast motor units have lower input threshold in the SOD1G93A mice, we suggest that our present result on reduced GABA-α5 tonic inhibition provides for a mechanism contributing to such imbalance. In parallel with reduced GABA inhibition, we noted an increase in voltage-gated L-type Ca2+ currents in the mutant JC MNs around P12. Together these results support that, early modifications in intrinsic properties of vulnerable MNs could be an adaptive response to counter synaptic deficits.
Subject(s)
Amyotrophic Lateral Sclerosis , Animals , Mice , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Disease Models, Animal , Drug Inverse Agonism , gamma-Aminobutyric Acid/metabolism , Mice, Transgenic , Motor Neurons/metabolism , Spinal Cord/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , Calcium Channels, L-Type/metabolismABSTRACT
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons throughout the brain and spinal cord progressively degenerate resulting in muscle atrophy, paralysis and death. Recent studies using animal models of ALS implicate multiple cell-types (e.g., astrocytes and microglia) in ALS pathogenesis in the spinal motor systems. To ascertain cellular vulnerability and cell-type specific mechanisms of ALS in the brainstem that orchestrates oral-motor functions, we conducted parallel single cell RNA sequencing (scRNA-seq) analysis using the high-throughput Drop-seq method. We isolated 1894 and 3199 cells from the brainstem of wildtype and mutant SOD1 symptomatic mice respectively, at postnatal day 100. We recovered major known cell types and neuronal subpopulations, such as interneurons and motor neurons, and trigeminal ganglion (TG) peripheral sensory neurons, as well as, previously uncharacterized interneuron subtypes. We found that the majority of the cell types displayed transcriptomic alterations in ALS mice. Differentially expressed genes (DEGs) of individual cell populations revealed cell-type specific alterations in numerous pathways, including previously known ALS pathways such as inflammation (in microglia), stress response (ependymal and an uncharacterized cell population), neurogenesis (astrocytes, oligodendrocytes, neurons), synapse organization and transmission (microglia, oligodendrocyte precursor cells, and neuronal subtypes), and mitochondrial function (uncharacterized cell populations). Other cell-type specific processes altered in SOD1 mutant brainstem include those from motor neurons (axon regeneration, voltage-gated sodium and potassium channels underlying excitability, potassium ion transport), trigeminal sensory neurons (detection of temperature stimulus involved in sensory perception), and cellular response to toxic substances (uncharacterized cell populations). DEGs consistently altered across cell types (e.g., Malat1), as well as cell-type specific DEGs, were identified. Importantly, DEGs from various cell types overlapped with known ALS genes from the literature and with top hits from an existing human ALS genome-wide association study (GWAS), implicating the potential cell types in which the ALS genes function with ALS pathogenesis. Our molecular investigation at single cell resolution provides comprehensive insights into the cell types, genes and pathways altered in the brainstem in a widely used ALS mouse model.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Brain Stem/metabolism , Brain Stem/pathology , Superoxide Dismutase-1/metabolism , Amyotrophic Lateral Sclerosis/genetics , Animals , Female , Mice, Transgenic , Mutation , Neurons/metabolism , Neurons/pathology , Sequence Analysis, RNA , Signal Transduction , Single-Cell Analysis , Superoxide Dismutase-1/genetics , TranscriptomeABSTRACT
Neuropeptide Y (NPY) is one of a number of neuropeptides with powerful orexigenic effects. Intracerebroventricular administration of NPY induces increases in food intake and alters feeding rate. Besides it role in feeding behavior, NPY also has significant effects on neuronal systems related to other spontaneous behaviors such as rearing and grooming. In the present study, we examined the direct effects of NPY on mesencephalic V neurons (Mes V), which are important sensory neurons involved in oral motor reflexes and rhythmical jaw movements, as well as masticatory proprioception. Coronal brain slices were prepared from neonatal Sprague-Dawley rats (P3-17) and whole-cell patch clamp recordings were obtained from Mes V neurons. Bath application of NPY depolarized the membrane potential and induced inward current in most neurons. Application of NPY shortened the duration of the afterhyperpolarization following an action potential, and increased the mean spike frequency during repetitive discharge. In those neurons which exhibited rhythmical burst discharge in response to maintained current injection, the bursting frequency was also increased. These effects were mediated predominately by both Y1 and Y5 receptors.
Subject(s)
Action Potentials/drug effects , Neurons/drug effects , Neuropeptide Y/pharmacology , Animals , Animals, Newborn , Membrane Potentials/drug effects , Patch-Clamp Techniques , Rats , Rats, Sprague-DawleyABSTRACT
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in muscle atrophy, paralysis, and fatality. Studies using mouse models of ALS indicate a protracted period of disease development with progressive motor neuron pathology, evident as early as embryonic and postnatal stages. Key missing information includes concomitant alterations in the sensorimotor circuit essential for normal development and function of the neuromuscular system. Leveraging unique brainstem circuitry, we show in vitro evidence for reflex circuit-specific postnatal abnormalities in the jaw proprioceptive sensory neurons in the well-studied SOD1G93A mouse. These include impaired and arrhythmic action potential burst discharge associated with a deficit in Nav1.6 Na+ channels. However, the mechanoreceptive and nociceptive trigeminal ganglion neurons and the visual sensory retinal ganglion neurons were resistant to excitability changes in age-matched SOD1G93A mice. Computational modeling of the observed disruption in sensory patterns predicted asynchronous self-sustained motor neuron discharge suggestive of imminent reflexive defects, such as muscle fasciculations in ALS. These results demonstrate a novel reflex circuit-specific proprioceptive sensory abnormality in ALS.SIGNIFICANCE STATEMENT Neurodegenerative diseases have prolonged periods of disease development and progression. Identifying early markers of vulnerability can therefore help devise better diagnostic and treatment strategies. In this study, we examined postnatal abnormalities in the electrical excitability of muscle spindle afferent proprioceptive neurons in the well-studied SOD1G93A mouse model for neurodegenerative motor neuron disease, amyotrophic lateral sclerosis. Our findings suggest that these proprioceptive sensory neurons are exclusively afflicted early in the disease process relative to sensory neurons of other modalities. Moreover, they presented Nav1.6 Na+ channel deficiency, which contributed to arrhythmic burst discharge. Such sensory arrhythmia could initiate reflexive defects, such as muscle fasciculations in amyotrophic lateral sclerosis, as suggested by our computational model.
Subject(s)
Amyotrophic Lateral Sclerosis/physiopathology , Proprioception/physiology , Sensory Receptor Cells/physiology , Tegmentum Mesencephali/physiology , Action Potentials , Animals , Disease Models, Animal , Female , Jaw/innervation , Jaw/physiopathology , Male , Mechanoreceptors/physiology , Mice, Transgenic , Models, Neurological , Nociception/physiology , Superoxide Dismutase-1/geneticsABSTRACT
Neurons utilize bursts of action potentials as an efficient and reliable way to encode information. It is likely that the intrinsic membrane properties of neurons involved in burst generation may also participate in preserving its temporal features. Here we examined the contribution of the persistent and resurgent components of voltage-gated Na+ currents in modulating the burst discharge in sensory neurons. Using mathematical modeling, theory and dynamic-clamp electrophysiology, we show that, distinct from the persistent Na+ component which is important for membrane resonance and burst generation, the resurgent Na+ can help stabilize burst timing features including the duration and intervals. Moreover, such a physiological role for the resurgent Na+ offered noise tolerance and preserved the regularity of burst patterns. Model analysis further predicted a negative feedback loop between the persistent and resurgent gating variables which mediate such gain in burst stability. These results highlight a novel role for the voltage-gated resurgent Na+ component in moderating the entropy of burst-encoded neural information.
Subject(s)
Models, Neurological , Neurons/physiology , Sodium Channels/physiology , Action Potentials/physiology , Animals , Computational Biology , Feedback, Physiological , MiceABSTRACT
The ability to critically evaluate neuroscientific findings is a skill that is rapidly becoming important in non-science professions. As neuroscience research is increasingly being used in law, business, education, and politics, it becomes imperative to educate future leaders in all areas of society about the brain. Undergraduate general education courses are an ideal way to expose students to issues of critical importance, but non-science students may avoid taking a neuroscience course because of the perception that neuroscience is more challenging than other science courses. A recently developed general education cluster course at UCLA aims to make neuroscience more palatable to undergraduates by pairing neuroscientific concepts with philosophy and history, and by building a learning community that supports the development of core academic skills and intellectual growth over the course of a year. This study examined the extent to which the course was successful in delivering neuroscience education to a broader undergraduate community. The results indicate that a majority of students in the course mastered the basics of the discipline regardless of their major. Furthermore, 77% of the non-life science majors (approximately two-thirds of students in the course) indicated that they would not have taken an undergraduate neuroscience course if this one was not offered. The findings also demonstrate that the course helped students develop core academic skills and improved their ability to think critically about current events in neuroscience. Faculty reported that teaching the course was highly rewarding and did not require an inordinate amount of time.
ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative motoneuron disease with presently no cure. Motoneuron (MN) hyperexcitability is commonly observed in ALS and is suggested to be a precursor for excitotoxic cell death. However, it is unknown whether hyperexcitability also occurs in MNs that are resistant to degeneration. Second, it is unclear whether all the MNs within homogeneous motor pools would present similar susceptibility to excitability changes since high-threshold MNs innervating fast fatigable muscle fibers selectively degenerate compared with low-threshold MNs innervating fatigue resistant slow muscle fibers. Therefore, we concurrently examined the excitability of ALS-vulnerable trigeminal motoneurons (TMNs) controlling jaw musculature and ALS-resistant oculomotor neurons (OMNs) controlling eye musculature in a well studied SOD1(G93A) ALS mouse model using in vitro patch-clamp electrophysiology at presymptomatic ages P8-P12. Our results show that hyperexcitability is not a global change among all the MNs, although mutant SOD1 is ubiquitously expressed. Instead, complex changes occur in ALS-vulnerable TMNs based on motor unit type and discharge characteristics. Firing threshold decreases among high-threshold TMNs and increases in a subpopulation of low-threshold TMNs. The latter group was identified based on their linear frequency-current responses to triangular ramp current injections. Such complex changes in MN recruitment were absent in ALS-resistant OMNs. We simulated the observed complex changes in TMN excitability using a computer-based jaw closer motor pool model. Model results suggest that hypoexcitability may indeed represent emerging disease symptomology that causes resistance in muscle force initiation. Identifying the cellular and molecular properties of these hypoexcitable cells may guide effective therapeutic strategies in ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/physiopathology , Membrane Potentials , Motor Neurons/physiology , Oculomotor Nerve/physiopathology , Trigeminal Nerve/physiopathology , Amyotrophic Lateral Sclerosis/genetics , Animals , Homeostasis , Masticatory Muscles/innervation , Mice , Superoxide Dismutase/genetics , Superoxide Dismutase-1ABSTRACT
The properties of neurons participating in masticatory rhythmogenesis are not clearly understood. Neurons within the dorsal trigeminal principal sensory nucleus (dPrV) are potential candidates as components of the masticatory central pattern generator (CPG). The present study examines in detail the ionic mechanisms controlling burst generation in dPrV neurons in rat (postnatal day 8-12) brain stem slices using whole cell and perforated patch-clamp methods. Nominal extracellular Ca(2+) concentration transformed tonic discharge in response to a maintained step pulse of current into rhythmical bursting in 38% of nonbursting neurons. This change in discharge mode was suppressed by riluzole, a persistent Na(+) current (INaP) antagonist. Veratridine, which suppresses the Na(+) channel inactivation mechanism, induced rhythmical bursting in nonbursting neurons in normal artificial cerebrospinal fluid, suggesting that INaP contributes to burst generation. Nominal extracellular Ca(2+) exposed a prominent afterdepolarizing potential (ADP) following a single spike induced by a 3-ms current pulse, which was suppressed, but not completely blocked, by riluzole. Application of BAPTA, a Ca(2+) chelator, intracellularly, or flufenamic acid, a Ca(2+)-activated nonspecific cationic channel (ICAN) antagonist, extracellularly to the bath, suppressed rhythmical bursting and the postspike ADP. Application of drugs to alter Ca(2+) release from endoplasmic reticulum also suppressed bursting. Finally, voltage-clamp methods demonstrated that nominal Ca(2+) facilitated INaP and induced ICAN. These data demonstrate for the first time that the previously observed induction in dPrV neurons of rhythmical bursting in nominal Ca(2+) is mediated by enhancement of INaP and onset of ICAN, which are dependent on intracellular Ca(2+).
Subject(s)
Action Potentials , Calcium/metabolism , Sensory Receptor Cells/physiology , Sodium/metabolism , Trigeminal Nuclei/physiology , Animals , Calcium Signaling , Endoplasmic Reticulum/metabolism , Membrane Potentials , Rats , Rats, Sprague-Dawley , Riluzole/pharmacology , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/metabolism , Trigeminal Nuclei/drug effects , Veratridine/pharmacologyABSTRACT
The function and biophysical properties of low threshold Kv1 current in control of membrane resonance, subthreshold oscillations, and bursting in mesencephalic V neurons (Mes V) were examined in rat brain stem slices (P8-P12) using whole cell current and voltage patch-clamp methods. alpha-dendrotoxin application, a toxin with high specificity for Kv1.1, 1.2, and 1.6 channels, showed the presence of a low-threshold K(+) current that activated rapidly around -50 mV and was relatively noninactivating over a 1-s period and had a V(1/2)max of -36.2 mV. Other toxins, specific for individual channels containing either Kv 1.1, 1.2, or 1.3 alpha-subunits, were applied individually, or in combination, and showed that Kv1 channels are heteromeric, composed of combinations of subunits. In current-clamp mode, toxin application transformed the high-frequency resonant properties of the membrane into a low-pass filter and concomitantly reduced the frequency of the subthreshold membrane oscillations. During this period, rhythmical bursting was transformed into low-frequency tonic discharge. Interestingly, in a subset of neurons that did not show bursting, low doses of alpha-dendrotoxin (alpha-DTX) sufficient to block 50% of the low threshold Kv1 channels induced bursting and increased the resonant peak impedance and subthreshold oscillations, which was replicated with computer simulation. This suggests that a critical balance between inward and outward currents is necessary for bursting. This was replicated with computer simulation. Single cell RT-PCR and immunohistochemical methods confirmed the presence of Kv1.1, 1.2, and 1.6 alpha-subunits in Mes V neurons. These data indicate that low threshold Kv1 channels are responsible for membrane resonance, contribute to subthreshold oscillations, and are critical for burst generation.
Subject(s)
Action Potentials/physiology , Biophysical Phenomena/physiology , Mesencephalon/cytology , Neurons/physiology , Shaker Superfamily of Potassium Channels/metabolism , Action Potentials/drug effects , Animals , Animals, Newborn , Biophysical Phenomena/drug effects , Biophysics , Computer Simulation , Dose-Response Relationship, Drug , Elapid Venoms/pharmacology , Electric Stimulation/methods , In Vitro Techniques , Models, Neurological , Patch-Clamp Techniques , Peptides/pharmacology , Potassium Channel Blockers/pharmacology , Rats , Rats, Sprague-Dawley , Scorpion Venoms/pharmacology , Shaker Superfamily of Potassium Channels/geneticsABSTRACT
The membrane properties and morphological features of interneurons in the supratrigeminal area (SupV) were studied in rat brain slices using whole-cell patch clamp recording techniques. We classified three morphological types of neurons as fusiform, pyramidal, and multipolar and four physiological types of neurons according to their discharge pattern in response to a 1-sec depolarizing current pulse from -80 mV. Single-spike neurons responded with a single spike, phasic neurons showed an initial burst of spikes and were silent during the remainder of the stimulus, delayed-firing (DF) neurons exhibited a slow depolarization and delay to initial spike onset, and tonic (T) neurons showed maintained a discharge throughout the stimulus pulse. In a subpopulation of neurons (10%), membrane depolarization to around -44 mV produced a rhythmic burst discharge (RB) that was associated with voltage-dependent subthreshold membrane oscillations. Both these phenomena were blocked by the sodium channel blocker riluzole at a concentration that did not affect the fast transient spike. Low doses of 4-AP, which blocks low-threshold K+ currents, transformed bursting into low-frequency tonic discharge. In contrast, bursting occurred with exposure to cadium, a calcium-channel blocker. This suggests that persistent sodium currents and low-threshold K+ currents have a role in intrinsic burst generation. Importantly, RB cells were most often associated with multipolar neurons that exhibited either a DF or a T discharge. Thus, the SupV contains a variety of physiological cell types with unique morphologies and discharge characteristics. Intrinsic bursting neurons form a unique group in this region. .
Subject(s)
Action Potentials/physiology , Cell Membrane/physiology , Interneurons/physiology , Pons/physiology , Trigeminal Nuclei/physiology , Animals , Animals, Newborn , Biological Clocks/physiology , Cell Shape/physiology , Dendrites/physiology , Dendrites/ultrastructure , Electric Stimulation , Interneurons/cytology , Organ Culture Techniques , Patch-Clamp Techniques , Pons/cytology , Potassium Channel Blockers/pharmacology , Potassium Channels/drug effects , Potassium Channels/physiology , Rats , Sodium Channel Blockers/pharmacology , Sodium Channels/drug effects , Sodium Channels/physiology , Trigeminal Nuclei/cytologyABSTRACT
Previous studies using pharmacological methods suggest that subthreshold sodium currents are critical for rhythmical burst generation in mesencephalic trigeminal neurons (Mes V). In this study, we characterized transient (I(NaT)), persistent (I(N)(aP)), and resurgent (I(res)) sodium currents in Na(v)1.6-null mice (med mouse, Na(v)1.6(-/-)) lacking expression of the sodium channel gene Scn8a. We found that peak transient, persistent, and resurgent sodium currents from med (Na(v)1.6(-/-)) mice were reduced by 18, 39, and 76% relative to their wild-type (Na(v)1.6(+/+)) littermates, respectively. Current clamp recordings indicated that, in response to sinusoidal constant amplitude current (ZAP function), all neurons exhibited membrane resonance. However, Mes V neurons from med mice had reduced peak amplitudes in the impedance-frequency relationship (resonant Q-value) and attenuated subthreshold oscillations despite the similar passive membrane properties compared with wild-type littermates. The spike frequency-current relationship exhibited reduced instantaneous discharge frequencies and spike block at low stimulus currents and seldom showed maintained spike discharge throughout the stimulus in the majority of med neurons compared with wild-type neurons. Importantly, med neurons never exhibited maintained stimulus-induced rhythmical burst discharge unlike those of wild-type littermates. The data showed that subthreshold sodium currents are critical determinants of Mes V electrogenesis and burst generation and suggest a role for resurgent sodium currents in control of spike discharge.
Subject(s)
Membrane Potentials/genetics , Mesencephalon/cytology , Nerve Tissue Proteins/deficiency , Neurons/physiology , Sodium Channels/deficiency , Trigeminal Nuclei/physiology , Animals , Animals, Newborn , Differential Threshold/physiology , Dose-Response Relationship, Radiation , Electric Stimulation , Fourier Analysis , In Vitro Techniques , Membrane Potentials/physiology , Mice , Mice, Knockout , NAV1.6 Voltage-Gated Sodium Channel , Patch-Clamp Techniques , Pyrimidines/pharmacologyABSTRACT
Subthreshold sodium currents are important in sculpting neuronal discharge and have been implicated in production and/or maintenance of subthreshold membrane oscillations and burst generation in mesencephalic trigeminal neurons (Mes V). Moreover, recent data suggest that, in some CNS neurons, resurgent sodium currents contribute to production of high-frequency burst discharge. In the present study, we sought to determine more directly the participation of these currents during Mes V electrogenesis using the action potential-clamp method. In postnatal day 8-14 rats, the whole-cell patch-clamp method was used to record sodium currents by subtraction in response to application of TTX in voltage-clamp mode using the action potential waveform as the command protocol. We found that TTX-sensitive sodium current is the main inward current flowing during the interspike interval, compared with the h-current (Ih) and calcium currents. Furthermore, in addition to the transient sodium current that flows during the upstroke of action potential, we show that resurgent sodium current flows at the peak of afterhyperpolarization and persistent sodium current flows in the middle of the interspike interval to drive high-frequency firing. Additionally, transient, resurgent, and persistent sodium current components showed voltage- and time-dependent slow inactivation, suggesting that slow inactivation of these currents can contribute to burst termination. The data suggest an important role for these components of the sodium current in Mes V neuron electrogenesis.
Subject(s)
Action Potentials/physiology , Biological Clocks/physiology , Cell Membrane/physiology , Membrane Potentials/physiology , Mesencephalon/physiology , Neurons/physiology , Trigeminal Nuclei/physiology , Animals , Animals, Newborn , Cells, Cultured , Differential Threshold/physiology , Excitatory Postsynaptic Potentials/physiology , Rats , Rats, Sprague-DawleyABSTRACT
In rat mesencephalic trigeminal (Mes V) neurons, persistent sodium currents in conjunction with low-threshold potassium currents are critical for generation of subthreshold membrane oscillations and onset of burst behavior. Here we demonstrate that the cAMP/protein kinase A (PKA) signaling pathway modulates persistent sodium currents. In particular, we show that elevation of cAMP suppresses a low-threshold I(NaP) via a PKA intracellular pathway. Bath application of forskolin (20 microM), a stimulant for the production of cAMP, reduced the peak I(NaP). 1,9-Dideoxy-forskolin (20 microM), an inactive form of forskolin, produced minimal effects on I(NaP), and the membrane-permeable cAMP analogue 8-bromo-cAMP (500 microM) mimicked the effect of forskolin. Additionally, preapplication of H89 (2 microM), a specific PKA inhibitor, suppressed the effect of forskolin, suggesting the involvement of the cAMP/PKA intracellular signaling pathway in this modulation. 5-HT receptor stimulation (20 microM) also mimicked the modulation of I(NaP) by forskolin via the cAMP/PKA-dependent signaling pathway. Current clamp analysis demonstrated that voltage-dependent membrane resonance in response to a ZAP input current at depolarized holding potentials (approximately -50 mV) was specifically suppressed by forskolin or 5-HT. Moreover, drug application enhanced frequency adaptation in response to a 1-sec current pulse. These results indicate that modulation of persistent sodium currents by a cAMP/PKA pathway can significantly alter the membrane excitability and discharge characteristics of Mes V neurons.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Mesencephalon/metabolism , Neurons, Afferent/metabolism , Serotonin/metabolism , Sodium Channels/metabolism , Trigeminal Nuclei/metabolism , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Colforsin/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Electric Stimulation , Enzyme Inhibitors/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mesencephalon/cytology , Neurons, Afferent/drug effects , Organ Culture Techniques , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Receptors, Serotonin/drug effects , Receptors, Serotonin/metabolism , Serotonin/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Sodium Channels/drug effects , Trigeminal Nuclei/cytology , Trigeminal Nuclei/drug effects , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that affects primarily the striatum and cerebral cortex. A search for the factors that increase the vulnerability of striatal neurons will lead to a better understanding of the pathological cascades of this disease. A current hypothesis for neurodegeneration of striatal medium-sized spiny neurons in HD is an alteration in N-methyl-D-aspartate (NMDA) receptor function. In the present study we examined electrophysiological properties of NMDA receptors in the R6/2 transgenic mouse model. These animals express exon 1 of the human HD gene and present an overt behavioral phenotype at about 5 weeks of age. Whole-cell voltage clamp recordings from acutely dissociated striatal neurons were obtained from three different age groups of transgenic mice (15, 21, and 40 days old) and their littermate controls (WT). In transgenic animals, two groups of neurons were found with respect to NMDA and Mg2+ sensitivity. One group of R6/2 cells displayed responses similar to those of WT, whereas the other showed increased responses to NMDA and decreased Mg2+ sensitivity. These cells were encountered in all age groups. The abnormal sensitivity to NMDA and Mg2+ indicates that NMDA receptor alterations occur very early in development and suggest the presence of constitutively abnormal NMDA receptors. These alterations may contribute to an enhancement of NMDA responses at hyperpolarized membrane potentials that may be a key factor in striatal neuronal dysfunction.
Subject(s)
Corpus Striatum/metabolism , Drug Resistance/genetics , Huntington Disease/metabolism , Magnesium/pharmacology , Neurons/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Cell Death/genetics , Cells, Cultured , Corpus Striatum/physiopathology , Disease Models, Animal , Excitatory Amino Acid Agonists/pharmacology , Humans , Huntingtin Protein , Huntington Disease/genetics , Huntington Disease/physiopathology , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice , Mice, Transgenic , N-Methylaspartate/pharmacology , Nerve Degeneration/genetics , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Patch-Clamp Techniques , Receptors, N-Methyl-D-Aspartate/drug effects , Synaptic Transmission/drug effects , Synaptic Transmission/geneticsABSTRACT
Trigeminal motoneurons relay the final output signals generated within the oral-motor pattern generating circuit(s) to muscles for execution of various motor patterns. In recent years, these motoneurons were shown to possess voltage dependent nonlinear membrane properties that allow them to actively participate in sculpting their final output. A complete understanding of the factors controlling trigeminal motoneuronal (TMN) discharge during oral-motor activity requires, at a minimum, a detailed understanding of the palette of ion channels responsible for membrane excitability and a determination of whether these ion channels are targets for modulation. Toward that end, we studied in detail the properties of calcium channels in TMNs and their susceptibility to modulation by 5-HT in rat brain slices. We found that based on pharmacological and voltage-dependent properties, high-voltage-activated (HVA) N-type [omega-conotoxin GVIA (omega-CgTX)]-sensitive, and to a lesser extent P/Q-type [omega-agatoxin IVA (omega-Aga IVA)]-sensitive, calcium channels make up the majority of the whole cell calcium current. 5-HT (5.0 microM) decreased HVA current by 31.3 +/- 2.2%, and the majority of this suppression resulted from reduction of current flow through N- and P/Q-type calcium channels. In contrast, 5-HT had no effect on low-voltage-activated (LVA) current amplitude in TMNs. HVA calcium current inhibition was mimicked by 5-CT, a 5-HT1 receptor agonist, and by R(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), a specific 5-HT1A agonist. The effects of 5-HT were blocked by the 5-HT1A antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide (NAN-190) but not by ketanserin, a 5-HT(2/1C) antagonist. Under current clamp, omega-CgTX and 5-HT were most effective in suppressing the mAHP and both increased the spike frequency and input/output gain in response to current injection. Calcium current modulation by 5-HT1A receptors likely is an important mechanism to fine tune the input/output gain of TMNs in response to small incoming synaptic inputs and accounts for some of the previously reported effects of 5-HT on TMN excitability during tonic and burst activity during oral-motor behavior.
Subject(s)
Calcium Channels/physiology , Motor Neurons/physiology , Receptors, Serotonin/physiology , Trigeminal Nuclei/cytology , 8-Hydroxy-2-(di-n-propylamino)tetralin/pharmacology , Amphetamines/pharmacology , Analysis of Variance , Animals , Animals, Newborn , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Calcium Channels/radiation effects , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Drug Interactions , Electric Stimulation/methods , In Vitro Techniques , Ketanserin/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Motor Neurons/drug effects , Motor Neurons/radiation effects , Neural Inhibition/drug effects , Neural Inhibition/physiology , Neural Inhibition/radiation effects , Patch-Clamp Techniques/methods , Piperazines/pharmacology , Rats , Serotonin/pharmacology , Serotonin Antagonists/pharmacology , Serotonin Receptor Agonists/pharmacologyABSTRACT
Huntington's disease (HD) is a neurodegenerative disorder that mainly affects the projection neurons of the striatum and cerebral cortex. Genetic mouse models of HD have shown that neurons susceptible to the mutation exhibit morphological and electrophysiological dysfunctions before and during development of the behavioral phenotype. We used HD transgenic mouse models to examine inwardly and outwardly rectifying K+ conductances, as well as expression of some related K+ channel subunits. Experiments were conducted in slices and dissociated cells from two mouse models, the R6/2 and TgCAG100, at the beginning and after full development of overt behavioral phenotypes. Striatal medium-sized spiny neurons (MSNs) from symptomatic transgenic mice had increased input resistances, depolarized resting membrane potentials, and reductions in both inwardly and outwardly rectifying K+ currents. These changes were more dramatic in the R6/2 model than in the TgCAG100. Parallel immunofluorescence studies detected decreases in the expression of K+ channel subunit proteins, Kir2.1, Kir2.3, and Kv2.1 in MSNs, which contribute to the formation of the channel ionophores for these currents. Attenuation in K+ conductances and channel subunit expression contribute to altered electrophysiological properties of MSNs and may partially account for selective cellular vulnerability in the striatum.
Subject(s)
Huntington Disease/physiopathology , Neostriatum/cytology , Neostriatum/physiopathology , Neurons/physiology , Potassium Channels/physiology , Age Factors , Analysis of Variance , Animals , Cesium/pharmacology , Disease Models, Animal , Dose-Response Relationship, Radiation , Electric Stimulation , Gene Expression Regulation/physiology , Huntington Disease/genetics , Huntington Disease/metabolism , Immunohistochemistry/methods , In Vitro Techniques , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Mice , Mice, Transgenic , Neostriatum/metabolism , Neurons/drug effects , Neurons/radiation effects , Patch-Clamp Techniques/methods , Potassium Channel Blockers/pharmacology , Potassium Channels/drug effects , Potassium Channels/radiation effects , Tetraethylammonium/pharmacologyABSTRACT
The functional and biophysical properties of a persistent sodium current (I(NaP)) previously proposed to participate in the generation of subthreshold oscillations and burst discharge in mesencephalic trigeminal sensory neurons (Mes V) were investigated in brain stem slices (rats, p7-p12) using whole cell patch-clamp methods. I(NaP) activated around -76 mV and peaked at -48 mV, with V1/2 of -58.7 mV. Ramp voltage-clamp protocols showed that I(NaP) undergoes time- as well as voltage-dependent inactivation and recovery from inactivation in the range of several seconds (tau(onset) = 2.04 s, tau(recov) = 2.21 s). Riluzole (< or =5 microM) substantially reduced I(NaP), membrane resonance, postinhibitory rebound (PIR), and subthreshold oscillations, and completely blocked bursting, but produced modest effects on the fast transient Na+ current (I(NaT)). Before complete cessation, burst cycle duration was increased substantially, while modest and inconsistent changes in burst duration were observed. The properties of the I(NaT) were obtained and revealed that the amplitude and voltage dependence of the resulting "window current" were not consistent with those of the observed I(NaP) recorded in the same neurons. This suggests an additional mechanism for the origin of I(NaP). A neuronal model was constructed using Hodgkin-Huxley parameters obtained experimentally for Na+ and K+ currents that simulated the experimentally observed membrane resonance, subthreshold oscillations, bursting, and PIR. Alterations in the model g(NaP) parameters indicate that I(NaP) is critical for control of subthreshold and suprathreshold Mes V neuron membrane excitability and burst generation.
Subject(s)
Membrane Potentials/physiology , Mesencephalon/cytology , Neurons/physiology , Sodium Channels/physiology , Action Potentials/drug effects , Action Potentials/physiology , Action Potentials/radiation effects , Animals , Animals, Newborn , Biophysics/methods , Cadmium/pharmacology , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Electric Stimulation/methods , Excitatory Amino Acid Antagonists/pharmacology , In Vitro Techniques , Ion Channel Gating/drug effects , Ion Channel Gating/radiation effects , Membrane Potentials/drug effects , Membrane Potentials/radiation effects , Models, Neurological , Neurons/drug effects , Neurons/radiation effects , Patch-Clamp Techniques/methods , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Riluzole/pharmacology , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacology , Time FactorsABSTRACT
The present study was performed to assess the postnatal development and functional roles of inward rectifying currents in rat mesencephalic trigeminal (Mes V) neurons, which are involved in the genesis and control of oral-motor activities. Whole cell voltage-clamp recordings obtained from Mes V neurons in brain stem slices identified fast (I(KIR)) and slow (I(h)) inward rectifying currents, which were specifically blocked by BaCl(2) (300-500 microM) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD 7288, 10 microM), respectively. The whole cell current density for these channels increased between postnatal days 2 to 12 (P2-P12), and the time courses for I(h) activation and deactivation were each well described by two time constants. Application of ZD 7288 produced membrane hyperpolarization in the majority of cells and prolonged afterhyperpolarization repolarization. Additionally, in the presence of ZD 7288, spike frequency was decreased and adaptation was more pronounced. Interestingly, these neurons exhibited a voltage-dependent membrane resonance (<10 Hz) that was prominent around resting potential and more negative to rest and was blocked by ZD 7288. These results suggest that I(h) contributes to stabilizing resting membrane potential and controlling cell excitability. The presence of I(h) imparts the neuron with the unique property of low-frequency membrane resonance; the ability to discriminate between synaptic inputs based on frequency content.
Subject(s)
Mesencephalon/physiology , Neurons/physiology , Trigeminal Nuclei/physiology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Barium Compounds/pharmacology , Cardiotonic Agents/pharmacology , Chlorides/pharmacology , Mesencephalon/cytology , Mesencephalon/growth & development , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/physiology , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Trigeminal Nuclei/cytology , Trigeminal Nuclei/growth & developmentABSTRACT
N-methyl-D-aspartate (NMDA) receptors play an important role in the production of rhythmical trigeminal motor activity resembling suckling and chewing. The developmental relationship between the expression of NMDA receptor subunits and the function of neurons comprising brainstem oral-motor circuitry is not clear. We conducted receptor immunohistochemistry studies to demonstrate the expression of NR2A and NR2B subunits in trigeminal motoneurons (Mo5) and mesencephalic trigeminal neurons (Me5) during the first 2 weeks of development. During this time period, rats begin the transition from suckling to chewing, two distinct motor behaviors. In Mo5, NR2A and NR2B immunoreactivity was observed throughout the time frame sampled. A significant increase in the NR2A:NR2B ratio occurred between P3-4 and P11 due to a reduction in the number of NR2B immunoreactive neurons. The temporal and spatial expression of NR2A and NR2B was differentially regulated between caudal and rostral regions of Me5. In contrast to Mo5, the NR2A:NR2B ratio decreased between P0-1 and P11 in caudal Me5 due to a concurrent increase in the number of NR2A and NR2B immunoreactive neurons. In rostral Me5, NR2A and NR2B immunoreactivity emerged at P3 and P11, respectively. Our data provides further insight into the molecular changes of trigeminal neurons during the transition from suckling to chewing behaviors. The differences in the NR2A:NR2B ratio between Mo5 and Me5 suggest functional differences in these neurons during NMDA-mediated neurotransmission.