Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 72
Filter
1.
Neuropharmacology ; 197: 108739, 2021 10 01.
Article in English | MEDLINE | ID: mdl-34339750

ABSTRACT

BACKGROUND: Many neuroactive steroids induce sedation/hypnosis by potentiating γ-aminobutyric acid (GABAA) currents. However, we previously demonstrated that an endogenous neuroactive steroid epipregnanolone [(3ß,5ß)-3-hydroxypregnan-20-one] (EpiP) exerts potent peripheral analgesia and blocks T-type calcium currents while sparing GABAA currents in rat sensory neurons. This study seeks to investigate the behavioral effects elicited by systemic administration of EpiP and to characterize its use as an adjuvant agent to commonly used general anesthetics (GAs). METHODS: Here, we utilized electroencephalographic (EEG) recordings to characterize thalamocortical oscillations, as well as behavioral assessment and mouse genetics with wild-type (WT) and different knockout (KO) models of T-channel isoforms to investigate potential sedative/hypnotic and immobilizing properties of EpiP. RESULTS: Consistent with increased oscillations in slower EEG frequencies, EpiP induced an hypnotic state in WT mice when injected alone intra-peritoneally (i.p.) and effectively facilitated anesthetic effects of isoflurane (ISO) and sevoflurane (SEVO). The CaV3.1 (Cacna1g) KO mice demonstrated decreased sensitivity to EpiP-induced hypnosis when compared to WT mice, whereas no significant difference was noted between CaV3.2 (Cacna1h), CaV3.3 (Cacna1i) and WT mice. Finally, when compared to WT mice, onset of EpiP-induced hypnosis was delayed in CaV3.2 KO mice but not in CaV3.1 and CaV3.3 KO mice. CONCLUSION: We posit that EpiP may have an important role as novel hypnotic and/or adjuvant to volatile anesthetic agents. We speculate that distinct hypnotic effects of EpiP across all three T-channel isoforms is due to their differential expression in thalamocortical circuitry.


Subject(s)
Calcium Channels, T-Type/drug effects , Hypnotics and Sedatives/pharmacology , Pregnanolone/pharmacology , Adjuvants, Anesthesia/pharmacology , Anesthetics, Inhalation/pharmacology , Animals , Behavior, Animal/drug effects , Calcium Channels, T-Type/genetics , Electroencephalography/drug effects , Isoflurane/pharmacology , Isomerism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Sevoflurane/pharmacology
2.
J Med Chem ; 63(4): 1709-1716, 2020 02 27.
Article in English | MEDLINE | ID: mdl-31999455

ABSTRACT

A hybrid of dearomatized isoprenylated acylphloroglucinol (DIAP) and monoterpenoid, hypatone A (1), together with its biosynthetic analogues 2-4 is characterized from Hypericum patulum. Structurally, 1 possesses an unprecedented spiro[bicyclo[3.2.1]octane-6,1'-cyclohexan]-2',4',6'-trione core as elucidated by extensive spectroscopic and X-ray crystallographic analyses. Biological studies reveal that compounds 1 and 2-4 produce opposite effects on Cav3.1 low voltage-gated Ca2+ channel, with 1 and 4, respectively, being the most potent Cav3.1 agonist and antagonist from natural products. Further studies suggest that compound 1 and its biogenetical precursor, 2, have the same binding site on Cav3.1 and that the rigid cagelike moiety at C-5 and C-6 is a key structural feature responsible for 1 being an agonist. Furthermore, 1 can normalize the pathological gating of a mutant Cav3.1 channel found in spinocerebellar ataxia 42 (SCA42), a hereditary neurodegenerative disorder with no available therapy. Collectively, our findings provide valuable tools for future studies on Cav3.1 physiology and pathophysiology, as well as afford possible leads for developing new drugs against SCA42, epilepsy, and pain.


Subject(s)
Calcium Channel Agonists/pharmacology , Calcium Channels, T-Type/metabolism , Monoterpenes/pharmacology , Phloroglucinol/analogs & derivatives , Phloroglucinol/pharmacology , Animals , Calcium Channel Agonists/isolation & purification , Calcium Channels, T-Type/genetics , HEK293 Cells , Humans , Hypericum/chemistry , Ion Channel Gating/drug effects , Membrane Potentials/drug effects , Mice , Monoterpenes/isolation & purification , Mutation , Phloroglucinol/isolation & purification
3.
Brain ; 143(1): 161-174, 2020 01 01.
Article in English | MEDLINE | ID: mdl-31800012

ABSTRACT

Inborn errors of CACNA1A-encoded P/Q-type calcium channels impair synaptic transmission, producing early and lifelong neurological deficits, including childhood absence epilepsy, ataxia and dystonia. Whether these impairments owe their pathologies to defective channel function during the critical period for thalamic network stabilization in immature brain remains unclear. Here we show that mice with tamoxifen-induced adult-onset ablation of P/Q channel alpha subunit (iKOp/q) display identical patterns of dysfunction, replicating the inborn loss-of-function phenotypes and, therefore demonstrate that these neurological defects do not rely upon developmental abnormality. Unexpectedly, unlike the inborn model, the adult-onset pattern of excitability changes believed to be pathogenic within the thalamic network is non-canonical. Specifically, adult ablation of P/Q channels does not promote Cacna1g-mediated burst firing or T-type calcium current (IT) in the thalamocortical relay neurons; however, burst firing in thalamocortical relay neurons remains essential as iKOp/q mice generated on a Cacna1g deleted background show substantially diminished seizure generation. Moreover, in thalamic reticular nucleus neurons, burst firing is impaired accompanied by attenuated IT. Interestingly, inborn deletion of thalamic reticular nucleus-enriched, human childhood absence epilepsy-linked gene Cacna1h in iKOp/q mice reduces thalamic reticular nucleus burst firing and promotes rather than reduces seizure, indicating an epileptogenic role for loss-of-function Cacna1h gene variants reported in human childhood absence epilepsy cases. Together, our results demonstrate that P/Q channels remain critical for maintaining normal thalamocortical oscillations and motor control in the adult brain, and suggest that the developmental plasticity of membrane currents regulating pathological rhythmicity is both degenerate and age-dependent.


Subject(s)
Ataxia/genetics , Calcium Channels, N-Type/genetics , Cerebral Cortex/metabolism , Epilepsy, Absence/genetics , Neurons/metabolism , Thalamus/metabolism , Action Potentials , Age Factors , Animals , Ataxia/metabolism , Ataxia/physiopathology , Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Cerebral Cortex/physiopathology , Disease Models, Animal , Epilepsy, Absence/metabolism , Epilepsy, Absence/physiopathology , Excitatory Postsynaptic Potentials/genetics , Inhibitory Postsynaptic Potentials/genetics , Membrane Potentials/genetics , Mice , Mice, Knockout , Patch-Clamp Techniques , Thalamic Nuclei/cytology , Thalamus/physiopathology
4.
Elife ; 72018 11 22.
Article in English | MEDLINE | ID: mdl-30465523

ABSTRACT

Learning the spatial organization of the environment is essential for most animals' survival. This requires the animal to derive allocentric spatial information from egocentric sensory and motor experience. The neural mechanisms underlying this transformation are mostly unknown. We addressed this problem in electric fish, which can precisely navigate in complete darkness and whose brain circuitry is relatively simple. We conducted the first neural recordings in the preglomerular complex, the thalamic region exclusively connecting the optic tectum with the spatial learning circuits in the dorsolateral pallium. While tectal topographic information was mostly eliminated in preglomerular neurons, the time-intervals between object encounters were precisely encoded. We show that this reliable temporal information, combined with a speed signal, can permit accurate estimation of the distance between encounters, a necessary component of path-integration that enables computing allocentric spatial relations. Our results suggest that similar mechanisms are involved in sequential spatial learning in all vertebrates.


Subject(s)
Egocentrism , Electric Fish/physiology , Space Perception/physiology , Action Potentials/physiology , Animals , Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Models, Biological , Motion , Neurons/physiology , Thalamus/physiology , Time Factors , Visual Pathways/physiology
5.
Neuropharmacology ; 138: 232-244, 2018 08.
Article in English | MEDLINE | ID: mdl-29913186

ABSTRACT

Since Cav3.2 T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293 cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (µM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68-0.75 for Cav3.2 and 0.99-1.41 for Cav3.1. (2S)-6-PNG and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated NG108-15 cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.


Subject(s)
Analgesics, Non-Narcotic/pharmacology , Calcium Channel Blockers/pharmacology , Flavonoids/pharmacology , Neuralgia/drug therapy , Visceral Pain/drug therapy , Analgesics, Non-Narcotic/chemistry , Analgesics, Non-Narcotic/isolation & purification , Animals , Calcium Channel Blockers/chemistry , Calcium Channel Blockers/isolation & purification , Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Disease Models, Animal , Flavonoids/chemistry , Flavonoids/isolation & purification , HEK293 Cells , Humans , Humulus , Hyperalgesia/drug therapy , Hyperalgesia/metabolism , Male , Mice, Inbred C57BL , Mice, Transgenic , Neuralgia/metabolism , Phytochemicals/chemistry , Phytochemicals/isolation & purification , Phytochemicals/pharmacology , Plant Extracts/chemistry , Plant Extracts/pharmacology , Random Allocation , Rats, Wistar , Visceral Pain/metabolism
6.
Neuron ; 95(5): 1181-1196.e8, 2017 Aug 30.
Article in English | MEDLINE | ID: mdl-28858620

ABSTRACT

Basal ganglia (BG) circuits orchestrate complex motor behaviors predominantly via inhibitory synaptic outputs. Although these inhibitory BG outputs are known to reduce the excitability of postsynaptic target neurons, precisely how this change impairs motor performance remains poorly understood. Here, we show that optogenetic photostimulation of inhibitory BG inputs from the globus pallidus induces a surge of action potentials in the ventrolateral thalamic (VL) neurons and muscle contractions during the post-inhibitory period. Reduction of the neuronal population with this post-inhibitory rebound firing by knockout of T-type Ca2+ channels or photoinhibition abolishes multiple motor responses induced by the inhibitory BG input. In a low dopamine state, the number of VL neurons showing post-inhibitory firing increases, while reducing the number of active VL neurons via photoinhibition of BG input, effectively prevents Parkinson disease (PD)-like motor symptoms. Thus, BG inhibitory input generates excitatory motor signals in the thalamus and, in excess, promotes PD-like motor abnormalities. VIDEO ABSTRACT.


Subject(s)
Globus Pallidus/physiology , Motor Neurons/physiology , Neural Inhibition/physiology , Thalamus/physiology , Action Potentials/physiology , Alcohol Oxidoreductases/genetics , Animals , Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/physiology , Dopamine/metabolism , Dystonia/diet therapy , Dystonia/drug therapy , Dystonia/physiopathology , Female , Globus Pallidus/cytology , Globus Pallidus/metabolism , Levodopa/therapeutic use , Male , Metabolism, Inborn Errors/diet therapy , Metabolism, Inborn Errors/drug therapy , Metabolism, Inborn Errors/physiopathology , Mice , Mice, Knockout , Muscle Contraction/physiology , Neural Pathways/physiology , Neurons/physiology , Psychomotor Disorders/diet therapy , Psychomotor Disorders/drug therapy , Psychomotor Disorders/physiopathology , Thalamus/cytology
7.
Clin Epigenetics ; 9: 46, 2017.
Article in English | MEDLINE | ID: mdl-28469732

ABSTRACT

BACKGROUND: The CpG island methylator phenotype (CIMP) in stage III colon cancer (CRC) has been associated with improved survival after treatment with adjuvant irinotecan-based chemotherapy. In this analysis, we determine whether CIMP status in the primary CRC is concordant with the CIMP status of matched metastases in order to determine if assessment of CIMP status in the primary tumor can be used to predict CIMP status of metastatic disease, which is relevant for patient management as well as for understanding the biology of CIMP CRCs. METHODS: We assessed the CIMP status of 70 pairs of primary CRC and matched metastases using a CRC-specific panel of five markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) where CIMP positive was defined as 3/5 positive markers at a percent methylated reference threshold of ≥10%. Concordance was compared using the Fisher's exact test and P < 0.05 was considered significant. RESULTS: Sixty-nine of the pairs (98.6%) showed concordant CIMP status in the primary tumor and matched metastasis; five (7.0%) of the pairs were concordantly CIMP positive. Only one pair (1.4%) had divergent CIMP status, demonstrating CIMP positivity (4/5 markers positive) in the primary tumor, while the matched metastasis was CIMP negative (0 markers positive). CONCLUSIONS: CIMP status is generally concordant between primary CRCs and matched metastases. Thus, CIMP status in the primary tumor is maintained in matched metastases and can be used to inform CIMP-based therapy options for the metastases.


Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , DNA Methylation , Adult , Aged , Basic Helix-Loop-Helix Transcription Factors/genetics , Calcium Channels, T-Type/genetics , Colorectal Neoplasms/pathology , Core Binding Factor Alpha 3 Subunit/genetics , CpG Islands , Epigenesis, Genetic , Female , Humans , Insulin-Like Growth Factor II/genetics , Male , Middle Aged , Neoplasm Metastasis , Nerve Tissue Proteins/genetics , Phenotype , Suppressor of Cytokine Signaling 1 Protein/genetics
8.
Toxicol In Vitro ; 37: 169-177, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27647474

ABSTRACT

Cadmium (Cd), a potent toxic heavy metal, is a widespread environmental contaminant. Its cellular traffic via pathways dedicated to transition metals contributes to the toxicity mechanisms. Zinc (Zn) homeostasis is complex, involving both zinc importers (Zip) and zinc exporters (ZnT). Cellular signal transduction pathways are influenced by Zn and redox status of the cell. The aim of the present study is to examine if the accumulation of Cd in the human lymphocyte B cell line BJAB and its capacity to promote oxidative stress and adverse effects could result from changes in the mRNA expression pattern of Zn transporters and metallothioneins. Cells were exposed to 5, 10, 20 and 40µM of CdCl2 equivalent to 0.91, 1.83, 3.66 and 7.33µg/ml respectively, for 24h. Cd significantly reduced the viability of BJAB cells and induced a dose-dependent increase in DNA damage. Cd also induced the formation of 8-hydroxy-2'-deoxyguanosine adducts and augmented MTF1 expression in BJAB cells. We observed interesting responses in relative gene expression to Cd exposure among the seven transporters we analyzed. Cd exposure increased the expression of DMT1 and caused an up-regulation of ZnT1. However, the T calcium channel alpha1G subunit could not be detected. A change in expression of ZnTs and Zips in response to Cd exposure emphasizes the involvement of Zn transporters in Cd cellular metabolism and induced oxidative stress.


Subject(s)
Cation Transport Proteins/genetics , Metals, Heavy/toxicity , Oxidative Stress/drug effects , 8-Hydroxy-2'-Deoxyguanosine , Calcium Channels, T-Type/genetics , Catalase/metabolism , Cell Line, Tumor , Cell Survival/drug effects , DNA Damage , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Humans , Malondialdehyde/metabolism , Metals, Heavy/metabolism , Selenium/metabolism , Superoxide Dismutase/metabolism
9.
Channels (Austin) ; 10(6): 466-77, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27331657

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. In a recent study by Steinberg and colleagues, 2 recessive missense mutations were identified in the Cav3.2 T-type calcium channel gene (CACNA1H), in a family with an affected proband (early onset, long duration ALS) and 2 unaffected parents. We have introduced and functionally characterized these mutations using transiently expressed human Cav3.2 channels in tsA-201 cells. Both of these mutations produced mild but significant changes on T-type channel activity that are consistent with a loss of channel function. Computer modeling in thalamic reticular neurons suggested that these mutations result in decreased neuronal excitability of thalamic structures. Taken together, these findings implicate CACNA1H as a susceptibility gene in amyotrophic lateral sclerosis.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Mutation, Missense , Neurons/physiology , Thalamus/cytology , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/physiopathology , Cell Line , Humans , Thalamus/physiology , Transfection
10.
Sleep ; 39(4): 875-85, 2016 Apr 01.
Article in English | MEDLINE | ID: mdl-26612388

ABSTRACT

STUDY OBJECTIVES: Low-threshold voltage-gated T-type Ca(2+) channels (T-channels or CaV3 channels) sustain oscillatory discharges of thalamocortical (TC) and nucleus Reticularis thalami (nRt) cells. The CaV3.3 subtype dominates nRt rhythmic bursting and mediates a substantial fraction of spindle power in the NREM sleep EEG. CaV3.2 channels are also found in nRt, but whether these contribute to nRt-dependent spindle generation is unexplored. We investigated thalamic rhythmogenesis in mice lacking this subtype in isolation (CaV3.2KO mice) or in concomitance with CaV3.3 deletion (CaV3.double-knockout (DKO) mice). METHODS: We examined discharge characteristics of thalamic cells and intrathalamic evoked synaptic transmission in brain slices from wild-type, CaV3.2KO and CaV3.DKO mice through patch-clamp recordings. The sleep profile of freely behaving CaV3.2KO and CaV3.DKO mice was assessed by polysomnographic recordings. RESULTS: CaV3.2 channel deficiency left nRt discharge properties largely unaltered, but additional deletion of CaV3.3 channels fully abolished low-threshold whole-cell Ca(2+) currents and bursting, and suppressed burst-mediated inhibitory responses in TC cells. CaV3.DKO mice had more fragmented sleep, with shorter NREM sleep episodes and more frequent microarousals. The NREM sleep EEG power spectrum displayed a relative suppression of the σ frequency band (10-15 Hz), which was accompanied by an increase in the δ band (1-4 Hz). CONCLUSIONS: Consistent with previous findings, CaV3.3 channels dominate nRt rhythmogenesis, but the lack of CaV3.2 channels further aggravates neuronal, synaptic, and EEG deficits. Therefore, CaV3.2 channels can boost intrathalamic synaptic transmission, and might play a modulatory role adjusting the relative presence of NREM sleep EEG rhythms.


Subject(s)
Calcium Channels, T-Type/deficiency , Periodicity , Sleep/genetics , Sleep/physiology , Animals , Brain Waves , Calcium Channels, T-Type/genetics , Electroencephalography , Male , Mice , Mice, Knockout , Patch-Clamp Techniques , Polysomnography , Sleep Deprivation/physiopathology , Synaptic Transmission , Thalamus/cytology , Thalamus/physiology
11.
Proc Natl Acad Sci U S A ; 112(25): 7839-44, 2015 Jun 23.
Article in English | MEDLINE | ID: mdl-26056284

ABSTRACT

In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of CaV3.1 T-type Ca(2+) channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking CaV3.1 channels (CaV3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that CaV3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (CaV3.1 WT). Intriguingly, we also found that CaV3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, CaV3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in CaV3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of CaV3.1 WT mice, during unconsciousness. These results suggest that the CaV3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states.


Subject(s)
Calcium Channels, T-Type/physiology , Cerebral Cortex/physiology , Circadian Rhythm , Thalamus/physiology , Unconsciousness , Animals , Calcium Channels, T-Type/genetics , Mice
12.
J Neurophysiol ; 114(1): 323-31, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25948871

ABSTRACT

The thalamus acts as a conduit for sensory and other information traveling to the cortex. In response to continuous sensory stimulation in vivo, the firing rate of thalamocortical neurons initially increases, but then within a minute firing rate decreases and T-type Ca(2+) channel-dependent action potential burst firing emerges. While neuromodulatory systems could play a role in this inhibitory response, we instead report a novel and cell-autonomous inhibitory mechanism intrinsic to the thalamic relay neuron. Direct intracellular stimulation of thalamocortical neuron firing initially triggered a continuous and high rate of action potential discharge, but within a minute membrane potential (Vm) was hyperpolarized and firing rate to the same stimulus was decreased. This self-inhibition was observed across a wide variety of thalamic nuclei, and in a subset firing mode switched from tonic to bursting. The self-inhibition resisted blockers of intracellular Ca(2+) signaling, Na(+)-K(+)-ATPases, and G protein-regulated inward rectifier (GIRK) channels as implicated in other neuron subtypes, but instead was in part inhibited by an ATP-sensitive K(+) channel blocker. The results identify a new homeostatic mechanism within the thalamus capable of gating excitatory signals at the single-cell level.


Subject(s)
Cerebral Cortex/physiology , Neurons/physiology , Thalamus/physiology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Calcium/metabolism , Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Cerebral Cortex/drug effects , G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , KATP Channels/metabolism , Mice, Inbred C57BL , Mice, Knockout , Neural Pathways/drug effects , Neural Pathways/physiology , Neuronal Plasticity/drug effects , Neuronal Plasticity/physiology , Neurons/drug effects , Neurotransmitter Agents/pharmacology , Patch-Clamp Techniques , Potassium/metabolism , Sodium/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Thalamus/drug effects , Tissue Culture Techniques
13.
Pflugers Arch ; 467(6): 1367-82, 2015 Jun.
Article in English | MEDLINE | ID: mdl-24953239

ABSTRACT

Burst-firing in distinct subsets of thalamic relay (TR) neurons is thought to be a key requirement for the propagation of absence seizures. However, in the well-regarded Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model as yet there has been no link described between burst-firing in TR neurons and spike-and-wave discharges (SWDs). GAERS ventrobasal (VB) neurons are a specific subset of TR neurons that do not normally display burst-firing during absence seizures in the GAERS model, and here, we assessed the underlying relationship of VB burst-firing with Ih and T-type calcium currents between GAERS and non-epileptic control (NEC) animals. In response to 200-ms hyperpolarizing current injections, adult epileptic but not pre-epileptic GAERS VB neurons displayed suppressed burst-firing compared to NEC. In response to longer duration 1,000-ms hyperpolarizing current injections, both pre-epileptic and epileptic GAERS VB neurons required significantly more hyperpolarizing current injection to burst-fire than those of NEC animals. The current density of the Hyperpolarization and Cyclic Nucleotide-activated (HCN) current (Ih) was found to be increased in GAERS VB neurons, and the blockade of Ih relieved the suppressed burst-firing in both pre-epileptic P15-P20 and adult animals. In support, levels of HCN-1 and HCN-3 isoform channel proteins were increased in GAERS VB thalamic tissue. T-type calcium channel whole-cell currents were found to be decreased in P7-P9 GAERS VB neurons, and also noted was a decrease in CaV3.1 mRNA and protein levels in adults. Z944, a potent T-type calcium channel blocker with anti-epileptic properties, completely abolished hyperpolarization-induced VB burst-firing in both NEC and GAERS VB neurons.


Subject(s)
Action Potentials , Cerebral Cortex/physiopathology , Epilepsy, Absence/physiopathology , Interneurons/physiology , Thalamus/physiopathology , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Epilepsy, Absence/genetics , Epilepsy, Absence/metabolism , Female , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Interneurons/drug effects , Interneurons/metabolism , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Thalamus/cytology , Thalamus/metabolism
14.
Article in English | MEDLINE | ID: mdl-26733818

ABSTRACT

A reason why the thalamus is more than a passive gateway for sensory signals is that two-third of the synapses of thalamocortical neurons are directly or indirectly related to the activity of corticothalamic axons. While the responses of thalamocortical neurons evoked by sensory stimuli are well characterized, with ON- and OFF-center receptive field structures, the prevalence of synaptic noise resulting from neocortical feedback in intracellularly recorded thalamocortical neurons in vivo has attracted little attention. However, in vitro and modeling experiments point to its critical role for the integration of sensory signals. Here we combine our recent findings in a unified framework suggesting the hypothesis that corticothalamic synaptic activity is adapted to modulate the transfer efficiency of thalamocortical neurons during selective attention at three different levels: First, on ionic channels by interacting with intrinsic membrane properties, second at the neuron level by impacting on the input-output gain, and third even more effectively at the cell assembly level by boosting the information transfer of sensory features encoded in thalamic subnetworks. This top-down population control is achieved by tuning the correlations in subthreshold membrane potential fluctuations and is adapted to modulate the transfer of sensory features encoded by assemblies of thalamocortical relay neurons. We thus propose that cortically-controlled (de-)correlation of subthreshold noise is an efficient and swift dynamic mechanism for selective attention in the thalamus.


Subject(s)
Attention/physiology , Cerebral Cortex/physiology , Models, Neurological , Neurons/physiology , Thalamus/physiology , Action Potentials/physiology , Animals , Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Computer Simulation , Feedback , Guinea Pigs , Information Theory , Mice, Inbred C57BL , Mice, Knockout , Neural Pathways/physiology , Patch-Clamp Techniques , Perception/physiology , Rats, Wistar , Tissue Culture Techniques , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/metabolism
15.
Neuron ; 83(5): 1144-58, 2014 Sep 03.
Article in English | MEDLINE | ID: mdl-25189210

ABSTRACT

T-type calcium channels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Cav3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Cav3.2 ubiquitination, decreases Cav3.2 protein levels, and reduces Cav3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T-type channel activity and their role in nociceptive signaling.


Subject(s)
Calcium Channels, T-Type/metabolism , Endopeptidases/metabolism , Inflammation/physiopathology , Neuralgia/enzymology , Animals , Calcium Channels, T-Type/genetics , Cells, Cultured , Disease Models, Animal , Endopeptidases/genetics , Freund's Adjuvant/toxicity , Humans , Hyperalgesia/diagnosis , Hyperalgesia/physiopathology , In Vitro Techniques , Inflammation/chemically induced , Male , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Neuralgia/drug therapy , Pain Threshold/drug effects , Pain Threshold/physiology , Peptides/therapeutic use , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/physiology , Spinal Cord/cytology , Transfection , Ubiquitination/genetics , Ubiquitination/physiology
16.
Biochem Pharmacol ; 89(4): 536-44, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24726441

ABSTRACT

Activation of T-type Ca²âº channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca²âº channels in chronic pain is supported by gene knockdown studies showing that decreased Ca(v)3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca²âº channel blocker. ABT-639 blocks recombinant human T-type (Ca(v)3.2) Ca²âº channels in a voltage-dependent fashion (IC50 = 2 µM) and attenuates LVA currents in rat DRG neurons (IC50 = 8 µM). ABT-639 was significantly less active at other Ca²âº channels (e.g. Ca(v)1.2 and Ca(v)2.2) (IC50 > 30 µM). ABT-639 has high oral bioavailability (%F = 73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED50 = 2 mg/kg, p.o.). ABT-639 (10-100 mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced). [corrected]. ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100-300 mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Ca(v)3.2) channels in chronic pain states.


Subject(s)
Calcium Channel Blockers/therapeutic use , Calcium Channels, T-Type/metabolism , Disease Models, Animal , Heterocyclic Compounds, 2-Ring/therapeutic use , Nerve Tissue Proteins/antagonists & inhibitors , Neuralgia/drug therapy , Nociceptive Pain/drug therapy , Peripheral Nerves/drug effects , Sulfonamides/therapeutic use , Animals , Behavior, Animal/drug effects , Biological Availability , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Calcium Channel Blockers/adverse effects , Calcium Channel Blockers/pharmacokinetics , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/chemistry , Calcium Channels, T-Type/genetics , Cells, Cultured , Chronic Pain/drug therapy , Chronic Pain/metabolism , Dose-Response Relationship, Drug , Heterocyclic Compounds, 2-Ring/adverse effects , Heterocyclic Compounds, 2-Ring/pharmacokinetics , Heterocyclic Compounds, 2-Ring/pharmacology , Humans , Male , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuralgia/metabolism , Nociceptive Pain/metabolism , Peripheral Nerves/cytology , Peripheral Nerves/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sulfonamides/adverse effects , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology
17.
Pflugers Arch ; 466(4): 635-44, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24449276

ABSTRACT

Ca(2+) entry is indispensable part of intracellular Ca(2+) signaling, which is vital for most of cellular functions. Low voltage-activated (LVA or T-type) calcium channels belong to the family of voltage-gated calcium channels (VGCCs) which provide Ca(2+) entry in response to membrane depolarization. VGCCs are generally characterized by exceptional Ca(2+) selectivity combined with high permeation rate, thought to be determined by the presence in their selectivity filter of a versatile Ca(2+) binding site formed by four glutamate residues (EEEE motif). The subfamily of LVA channels includes three members, Cav3.1, Cav3.2 and Cav3.3. They all possess two aspartates instead of glutamates (i.e., EEDD motif) in their selectivity filter and are the least Ca(2+)-selective of all VGCCs. They also have the lowest conductance, weakly discriminate Ca(2+), Sr(2+) and Ba(2+) and demonstrate channel-specific sensitivity to divalent metal blockers, such as Ni(2+). The available data suggest that EEDD binding site of LVA channels is more rigid compared to EEEE one, and their selectivity permeation and block are determined by two supplementary low-affinity intrapore Ca(2+) binding sites located above and below EEDD locus. In addition, LVA channels have extracellular metal binding site that allosterically regulates channel's gating, permeation and block depending on trace metals concentration.


Subject(s)
Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Calcium/metabolism , Models, Molecular , Amino Acid Sequence , Animals , Calcium Channels, T-Type/chemistry , Humans , Molecular Sequence Data , Permeability , Protein Structure, Secondary
18.
Proc Natl Acad Sci U S A ; 110(50): 20266-71, 2013 Dec 10.
Article in English | MEDLINE | ID: mdl-24282303

ABSTRACT

T-type Ca(2+) channels in thalamocortical (TC) neurons have long been considered to play a critical role in the genesis of sleep spindles, one of several TC oscillations. A classical model for TC oscillations states that reciprocal interaction between synaptically connected GABAergic thalamic reticular nucleus (TRN) neurons and glutamatergic TC neurons generates oscillations through T-type channel-mediated low-threshold burst firings of neurons in the two nuclei. These oscillations are then transmitted from TC neurons to cortical neurons, contributing to the network of TC oscillations. Unexpectedly, however, we found that both WT and KO mice for CaV3.1, the gene for T-type Ca(2+) channels in TC neurons, exhibit typical waxing-and-waning sleep spindle waves at a similar occurrence and with similar amplitudes and episode durations during non-rapid eye movement sleep. Single-unit recording in parallel with electroencephalography in vivo confirmed a complete lack of burst firing in the mutant TC neurons. Of particular interest, the tonic spike frequency in TC neurons was significantly increased during spindle periods compared with nonspindle periods in both genotypes. In contrast, no significant change in burst firing frequency between spindle and nonspindle periods was noted in the WT mice. Furthermore, spindle-like oscillations were readily generated within intrathalamic circuits composed solely of TRN and TC neurons in vitro in both the KO mutant and WT mice. Our findings call into question the essential role of low-threshold burst firings in TC neurons and suggest that tonic firing is important for the generation and propagation of spindle oscillations in the TC circuit.


Subject(s)
Brain Waves/physiology , Models, Neurological , Neurons/metabolism , Periodicity , Sleep/physiology , Thalamus/metabolism , Animals , Calcium Channels, T-Type/genetics , Electroencephalography , Mice , Mice, Knockout
19.
Circ Arrhythm Electrophysiol ; 6(4): 799-808, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23884198

ABSTRACT

BACKGROUND: When complete atrioventricular block (AVB) occurs, infranodal escape rhythms are essential to prevent bradycardic death. The role of T-type Ca(2+) channels in pacemaking outside the sinus node is unknown. We investigated the role of T-type Ca(2+) channels in escape rhythms and bradycardia-related ventricular tachyarrhythmias after AVB in mice. METHODS AND RESULTS: Adult male mice lacking the main T-type Ca(2+) channel subunit Cav3.1 (Cav3.1(-/-)) and wild-type (WT) controls implanted with ECG telemetry devices underwent radiofrequency atrioventricular node ablation to produce AVB. Before ablation, Cav3.1(-/-) mice showed sinus bradycardia (mean±SEM; RR intervals, 148±3 versus 128±2 ms WT; P<0.001). Immediately after AVB, Cav3.1(-/-) mice had slower escape rhythms (RR intervals, 650±75 versus 402±26 ms in WT; P<0.01) but a preserved heart-rate response to isoproterenol. Over the next 24 hours, mortality was markedly greater in Cav3.1(-/-) mice (19/31; 61%) versus WT (8/26; 31%; P<0.05), and Torsades de Pointes occurred more frequently (73% Cav3.1(-/-) versus 35% WT; P<0.05). Escape rhythms improved in both groups during the next 4 weeks but remained significantly slower in Cav3.1(-/-). At 4 weeks after AVB, ventricular tachycardia was more frequent in Cav3.1(-/-) than in WT mice (746±116 versus 214±78 episodes/24 hours; P<0.01). Ventricular function remodeling was similar in Cav3.1(-/-) and WT, except for smaller post-AVB fractional-shortening increase in Cav3.1(-/-). Expression changes were seen post-AVB for a variety of genes; these tended to be greater in Cav3.1(-/-) mice, and overexpression of fetal and profibrotic genes occurred only in Cav3.1(-/-). CONCLUSIONS: This study suggests that T-type Ca(2+) channels play an important role in infranodal escape automaticity. Loss of T-type Ca(2+) channels worsens bradycardia-related mortality, increases bradycardia-associated adverse remodeling, and enhances the risk of malignant ventricular tachyarrhythmias complicating AVB.


Subject(s)
Atrioventricular Block/metabolism , Bradycardia/metabolism , Calcium Channels, T-Type/metabolism , Calcium Signaling , Heart Conduction System/metabolism , Heart Rate , Periodicity , Torsades de Pointes/metabolism , Action Potentials , Animals , Atrioventricular Block/diagnosis , Atrioventricular Block/genetics , Atrioventricular Block/physiopathology , Bradycardia/diagnosis , Bradycardia/genetics , Bradycardia/physiopathology , Bradycardia/prevention & control , Calcium Channels, T-Type/deficiency , Calcium Channels, T-Type/genetics , Disease Models, Animal , Electrocardiography, Ambulatory , Electrophysiologic Techniques, Cardiac , Gene Expression Regulation , Heart Conduction System/physiopathology , Male , Mice , Mice, Knockout , RNA, Messenger/metabolism , Telemetry , Time Factors , Torsades de Pointes/diagnosis , Torsades de Pointes/genetics , Torsades de Pointes/physiopathology , Torsades de Pointes/prevention & control , Ventricular Remodeling
20.
J Neurosci ; 33(2): 624-30, 2013 Jan 09.
Article in English | MEDLINE | ID: mdl-23303941

ABSTRACT

The T-type Ca(2+) channels encoded by the Ca(V)3 genes are well established electrogenic drivers for burst discharge. Here, using Ca(V)3.3(-/-) mice we found that Ca(V)3.3 channels trigger synaptic plasticity in reticular thalamic neurons. Burst discharge via Ca(V)3.3 channels induced long-term potentiation at thalamoreticular inputs when coactivated with GluN2B-containing NMDA receptors, which are the dominant subtype at these synapses. Notably, oscillatory burst discharge of reticular neurons is typical for sleep-related rhythms, suggesting that sleep contributes to strengthening intrathalamic circuits.


Subject(s)
Calcium Channels, T-Type/physiology , Neuronal Plasticity/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Synapses/physiology , Thalamus/physiology , Animals , Calcium Channels, T-Type/genetics , Electrophysiological Phenomena , Excitatory Postsynaptic Potentials/physiology , In Vitro Techniques , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuronal Plasticity/genetics , Receptors, AMPA/physiology , Receptors, N-Methyl-D-Aspartate/genetics , Reticular Formation/growth & development , Reticular Formation/physiology , Synaptic Transmission/physiology , Thalamus/growth & development
SELECTION OF CITATIONS
SEARCH DETAIL