Gubbi Govindaiah

Circadian Rhythm of Redox State Regulates Excitability in Suprachiasmatic Nucleus Neurons

Diurnal metabolic changes in circadian clock neurons are coupled to changes in potassium channel activity. Daily rhythms of mammalian physiology, metabolism, and behavior parallel the day-night cycle. They are orchestrated by a central circadian clock in the brain, the suprachiasmatic nucleus (SCN). Transcription of clock genes is sensitive to metabolic changes in reduction and oxidation (redox); however, circadian cycles in protein oxidation have been reported in anucleate cells, where no transcription occurs. We investigated whether the SCN also expresses redox cycles and how such metabolic oscillations might affect neuronal physiology. We detected self-sustained circadian rhythms of SCN redox state that required the molecular clockwork. The redox oscillation could determine the excitability of SCN neurons through nontranscriptional modulation of multiple potassium (K+) channels. Thus, dynamic regulation of SCN excitability appears to be closely tied to metabolism that engages the clockwork machinery.

Low-Threshold Ca2+ Current Amplifies Distal Dendritic Signaling in Thalamic Reticular Neurons

The low-threshold transient calcium current (IT) plays a critical role in modulating the firing behavior of thalamic neurons; however, the role of IT in the integration of afferent information within the thalamus is virtually unknown. We have used two-photon laser scanning microscopy coupled with whole-cell recordings to examine calcium dynamics in the neurons of the strategically located thalamic reticular nucleus (TRN). We now report that a single somatic burst discharge evokes large-magnitude calcium responses, via IT, in distal TRN dendrites. The magnitude of the burst-evoked calcium response was larger than those observed in thalamocortical projection neurons under the same conditions. We also demonstrate that direct stimulation of distal TRN dendrites, via focal glutamate application and synaptic activation, can locally activate distal IT, producing a large distal calcium response independent of the soma/proximal dendrites. These findings strongly suggest that distally located IT may function to amplify afferent inputs. Boosting the magnitude ensures integration at the somatic level by compensating for attenuation that would normally occur attributable to passive cable properties. Considering the functional architecture of the TRN, elongated nature of their dendrites, and robust dendritic signaling, these distal dendrites could serve as sites of intense intra-modal/cross-modal integration and/or top-down modulation, leading to focused thalamocortical communication.

Metabotropic Glutamate Receptors Differentially Regulate GABAergic Inhibition in Thalamus

Thalamic interneurons and thalamic reticular nucleus (TRN) neurons provide inhibitory innervation of thalamocortical cells that significantly influence thalamic gating. The local interneurons in the dorsal lateral geniculate nucleus (dLGN) give rise to two distinct synaptic outputs: classical axonal and dendrodendritic. Activation of metabotropic glutamate receptors (mGluRs) by agonists or optic tract stimulation increases the output of these presynaptic dendrites leading to increased inhibition of thalamocortical neurons. The present study was aimed to evaluate the actions of specific mGluRs on inhibitory GABA-mediated signaling. We found that the group I mGluR (mGluR1,5) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) or optic tract stimulation produced a robust increase in spontaneous IPSCs (sIPSCs) in thalamocortical neurons that was attenuated by the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP). In contrast, the group II mGluR (mGluR2,3) agonists (2R, 4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC) or (2S,2′R,3′R)-2-(2′3′-dicarboxycyclopropyl)glycine (DCG-IV) suppressed the frequency of sIPSCs. In addition, mGluR1,5 agonist DHPG produced depolarizations and mGluR2/3 agonists APDC or L-CCG-I [(2S,1′S,2′S)-2-(carboxycyclopropyl)glycine] produced hyperpolarizations in dLGN interneurons. Furthermore, the enhanced sIPSC activity by optic tract stimulation was reduced when paired with corticothalamic fiber stimulation. The present data indicate that activation of specific mGluR subtypes differentially regulates inhibitory activity via different synaptic pathways. Our results suggest that activation of specific mGluR subtypes can upregulate or downregulate inhibitory activity in thalamic relay neurons, and these actions likely shape excitatory synaptic integration and thus regulate information transfer through thalamocortical circuits.

Heterogeneity of firing properties among rat thalamic reticular nucleus neurons

The thalamic reticular nucleus (TRN) provides inhibitory innervation to most thalamic relay nuclei and receives excitatory innervation from both cortical and thalamic neurons. Ultimately, information transfer through the thalamus to the neocortex is strongly influenced by TRN. In addition, the reciprocal synaptic connectivity between TRN with associated thalamic relay nuclei is critical in generating intrathalamic rhythmic activities that occur during certain arousal states and pathophysiological conditions. Despite evidence suggesting morphological heterogeneity amongst TRN neurons, the heterogeneity of intrinsic properties of TRN neurons has not been systematically examined. One key characteristic of virtually all thalamic neurons is the ability to produce action potentials in two distinct modes: burst and tonic. In this study, we have examined the prevalence of burst discharge within TRN neurons. Our intracellular recordings revealed that TRN neurons can be differentiated by their action potential discharge modes. The majority of neurons in the dorsal TRN (56%) lack burst discharge, and the remaining neurons (35%) show an atypical burst that consists of an initial action potential followed by small amplitude, long duration depolarizations. In contrast, most neurons in ventral TRN (82%) display a stereotypical burst discharge consisting of a transient, high frequency discharge of multiple action potentials. TRN neurons that lack burst discharge typically did not produce low threshold calcium spikes or produced a significantly reduced transient depolarization. Our findings clearly indicate that TRN neurons can be differentiated by differences in their spike discharge properties and these subtypes are not uniformly distributed within TRN. The functional consequences of such intrinsic differences may play an important role in modality‐specific thalamocortical information transfer as well as overall circuit level activities.

Patch clamp electrophysiology and capillary electrophoresis-mass spectrometry metabolomics for single cell characterization

The visual selection of specific cells within an ex vivo brain slice, combined with whole-cell patch clamp recording and capillary electrophoresis (CE)–mass spectrometry (MS)-based metabolomics, yields high chemical information on the selected cells. By providing access to a cell’s intracellular environment, the whole-cell patch clamp technique allows one to record the cell’s physiological activity. A patch clamp pipet is used to withdraw ∼3 pL of cytoplasm for metabolomic analysis using CE–MS. Sampling the cytoplasm, rather than an intact isolated neuron, ensures that the sample arises from the cell of interest and that structures such as presynaptic terminals from surrounding, nontargeted neurons are not sampled. We sampled the rat thalamus, a well-defined system containing gamma-aminobutyric acid (GABA)-ergic and glutamatergic neurons. The approach was validated by recording and sampling from glutamatergic thalamocortical neurons, which receive major synaptic input from GABAergic thalamic reticular nucleus neurons, as well as neurons and astrocytes from the ventral basal nucleus and the dorsal lateral geniculate nucleus. From the analysis of the cytoplasm of glutamatergic cells, approximately 60 metabolites were detected, none of which corresponded to the compound GABA. However, GABA was successfully detected when sampling the cytoplasm of GABAergic neurons, demonstrating the exclusive nature of our cytoplasmic sampling approach. The combination of whole-cell patch clamp with single cell cytoplasm metabolomics provides the ability to link the physiological activity of neurons and astrocytes with their neurochemical state. The observed differences in the metabolome of these neurons underscore the striking cell to cell heterogeneity in the brain.

Binding of amyloid β peptide to β2 adrenergic receptor induces PKA-dependent AMPA receptor hyperactivity

Progressive decrease in neuronal function is an established feature of Alzheimers disease (AD). Previous studies have shown that amyloid β (Aβ) peptide induces acute increase in spontaneous synaptic activity accompanied by neurotoxicity, and Aβ induces excitotoxic neuronal death by increasing calcium influx mediated by hyperactive α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) receptors. An in vivo study has revealed subpopulations of hyperactive neurons near Aβ plaques in mutant amyloid precursor protein (APP)‐transgenic animal model of Alzheimers disease (AD) that can be normalized by an AMPA receptor antagonist. In the present study, we aim to determine whether soluble Aβ acutely induces hyperactivity of AMPA receptors by a mechanism involving β2 adrenergic receptor (β2AR). We found that the soluble Aβ binds to β2AR, and the extracellular N terminus of β2AR is critical for the binding. The binding is required to induce G‐protein/cAMP/protein kinase A (PKA) signaling, which controls PKA‐dependent phosphorylation of GluR1 and β2AR, and AMPA receptor‐mediated excitatory postsynaptic currents (EPSCs). β2AR and GluR1 also form a complex comprising postsynaptic density protein 95 (PSD95), PKA and its anchor AKAP150, and protein phosphotase 2A (PP2A). Both the third intracellular (i3) loop and C terminus of β2AR are required for the β2AR/AMPA receptor complex. Aβ acutely induces PKA phosphorylation of GluR1 in the complex without affecting the association between two receptors. The present study reveals that non‐neurotransmitter Aβ has a binding capacity to β2AR and induces PKA‐dependent hyperactivity in AMPA receptors.—Wang, D., Govindaiah, G., Liu, R., De Arcangelis, V., Cox, C. L., Xiang, Y. K. Binding of amyloid β peptide to β2 adrenergic receptor induces PKA‐dependent AMPA receptor hyperactivity. FASEB J. 24, 3511–3521 (2010). www.fasebj.org

Modulation of thalamic neuron excitability by orexins

Orexins (hypocretins) are peptides of hypothalamic origin that play an important role in maintaining wakefulness. Reduced orexin levels have been associated with an increased incidence of narcolepsy. Considering thalamic nuclei are interconnected with virtually all neocortical regions and the thalamus has been found to produce distinct activities related to different levels of arousal, we have examined the actions of orexins on thalamic neurons using an in vitro thalamic slice preparation. The orexins (orexin-A and orexin-B) produced distinct actions within different intralaminar nuclei. Orexin-B strongly depolarized the majority of centrolateral nucleus (CL) neurons (71%), but depolarized a significantly smaller population of parafascicular nuclei (Pf) neurons (10%). In the mediodorsal thalamic nucleus (MD), orexin-B depolarized 21% of the neurons tested. Overall, orexin-B was found to be more potent than Orexin-A. Orexin-A depolarized a significantly smaller population of CL neurons (23%), but had no effect on Pf neurons. In addition, orexin-A produced a small depolarization in 28% of neurons in the thalamic reticular nucleus (TRN). Both orexin-A and orexin-B had no effect on neurons in the lateral posterior (LP), lateralodorsal (LD), posterior thalamic (Po), ventrobasal (VB) nucleus and lateral geniculate nucleus (LGN). The depolarizing actions of orexins were sufficient to alter the firing mode of these neurons from a burst- to tonic-firing mode. The excitatory actions of orexin-B result from a decrease in the apparent leak potassium current (Kleak). The orexin-B mediated excitation was also attenuated by bupivacaine suggesting the involvement of Kleak current. Further, the actions of orexin-B were occluded by the classical neurotransmitter dopamine, indicating the orexins may share similar ionic mechanisms. Thus, the depolarizing actions of orexins may play a key role in altering the firing mode of thalamic neurons associated with different states of consciousness.

Dopamine enhances the excitability of somatosensory thalamocortical neurons

The thalamus conveys sensory information from peripheral and subcortical regions to the neocortex in a dynamic manner that can be influenced by several neuromodulators. Alterations in dopamine (DA) receptor function in thalami of Schizophrenic patients have recently been reported. In addition, schizophrenia is associated with sensory gating abnormalities and sleep-wake disturbances, thus we examined the role of DA on neuronal excitability in somatosensory thalamus. The ventrobasal (VB) thalamus receives dopaminergic innervation and expresses DA receptors; however, the action of DA on VB neurons is unknown. In the present study, we performed whole cell current- and voltage-clamp recordings in rat brain slices to investigate the role of DA on excitability of VB neurons. We found that DA increased action potential discharge and elicited membrane depolarization via activation of different receptor subtypes. Activation of D2-like receptors (D(2R)) leads to enhanced action potential discharge, whereas the membrane depolarization was mediated by D1-like receptors (D(1R)). The D(2R-mediated) increase in spike discharge was mimicked and occluded by α-dendrotoxin (α-DTX), indicating the involvement of a slowly inactivating K(+) channels. The D1R-mediated membrane depolarization was occluded by barium, suggesting the involvement of a G protein-coupled K(+) channel or an inwardly rectifying K(+) channel. Our results indicate that DA produces dual modulatory effects acting on subtypes of DA receptors in thalamocortical relay neurons, and likely plays a significant role in the modulation of sensory information.

Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration

Organoid techniques provide unique platforms to model brain development and neurological disorders. Whereas several methods for recapitulating corticogenesis have been described, a system modeling human medial ganglionic eminence (MGE) development, a critical ventral brain domain producing cortical interneurons and related lineages, has been lacking until recently. Here, we describe the generation of MGE and cortex-specific organoids from human pluripotent stem cells that recapitulate the development of MGE and cortex domains, respectively. Population and single-cell RNA sequencing (RNA-seq) profiling combined with bulk assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses revealed transcriptional and chromatin accessibility dynamics and lineage relationships during MGE and cortical organoid development. Furthermore, MGE and cortical organoids generated physiologically functional neurons and neuronal networks. Finally, fusing region-specific organoids followed by live imaging enabled analysis of human interneuron migration and integration. Together, our study provides a platform for generating domain-specific brain organoids and modeling human interneuron migration and offers deeper insight into molecular dynamics during human brain development.

Regulation of Inhibitory Synapses by Presynaptic D4 Dopamine Receptors in Thalamus

Dopamine (DA) receptors are the principal targets of drugs used in the treatment of schizophrenia. Among the five DA receptor subtypes, the D(4) subtype is of particular interest because of the relatively high affinity of the atypical neuropleptic clozapine for D(4) compared with D(2) receptors. GABA-containing neurons in the thalamic reticular nucleus (TRN) and globus pallidus (GP) express D(4) receptors. TRN neurons receive GABAergic afferents from globus pallidus (GP), substantia nigra pars reticulata (SNr), and basal forebrain as well as neighboring TRN neuron collaterals. In addition, TRN receives dopaminergic innervations from substantia nigra pars compacta (SNc); however, the role of D(4) receptors in neuronal signaling at inhibitory synapses is unknown. Using whole cell recordings from in vitro pallido-thalamic slices, we demonstrate that DA selectively suppresses GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) evoked by GP stimulation. The D(2)-like receptor (D(2,3,4)) agonist, quinpirole, and selective D(4) receptor agonist, PD168077, mimicked the actions of DA. The suppressive actions of DA and its agonists were associated with alterations in paired pulse ratio and a decrease in the frequency of miniature IPSCs, suggesting a presynaptic site of action. GABA(A) receptor agonist, muscimol, induced postsynaptic currents in TRN neurons were unaltered by DA or quinpirole, consistent with the presynaptic site of action. Finally, DA agonists did not alter intra-TRN inhibitory signaling. Our data demonstrate that the activation of presynaptic D(4) receptors regulates GABA release from GP efferents but not TRN collaterals. This novel and selective action of D(4) receptor activation on GP-mediated inhibition may provide insight to potential functional significance of atypical antipsychotic agents. These findings suggest a potential heightened TRN neuron activity in certain neurological conditions, such as schizophrenia and attention deficit hyperactive disorders.