Mitochondrial Regulation of the Hippocampal Firing Rate Set Point and Seizure Susceptibility.

Maintaining average activity within a set-point range constitutes a fundamental property of central neural circuits. However, whether and how activity set points are regulated remains unknown. Integrating genome-scale metabolic modeling and experimental study of neuronal homeostasis, we identified mitochondrial dihydroorotate dehydrogenase (DHODH) as a regulator of activity set points in hippocampal networks. The DHODH inhibitor teriflunomide stably suppressed mean firing rates via synaptic and intrinsic excitability mechanisms by modulating mitochondrial Ca2+ buffering and spare respiratory capacity. Bi-directional activity perturbations under DHODH blockade triggered firing rate compensation, while stabilizing firing to the lower level, indicating a change in the firing rate set point. In vivo, teriflunomide decreased CA3-CA1 synaptic transmission and CA1 mean firing rate and attenuated susceptibility to seizures, even in the intractable Dravet syndrome epilepsy model. Our results uncover mitochondria as a key regulator of activity set points, demonstrate the differential regulation of set points and compensatory mechanisms, and propose a new strategy to treat epilepsy.

Imaging of Somatic Ca2+ Transients in Differentiated Human Neurons.

Calcium is a major regulator of neuronal activity and calcium signaling is critically important for normal neuronal function. Ca imaging is a well-established tool for studying neuronal function and ongoing spontaneous Ca2+ transients are a good indicator of neuronal maturity. There are various indicators available today, differing by their sensitivity, spectra, and loading method. Here we present a method for measurement of Ca2+ transients in neurons using two different Ca2+ indicators, Oregon Green BAPTA-1 and GCaMP6.

Secreted amyloid-ß precursor protein functions as a GABABR1a ligand to modulate synaptic transmission.

Amyloid-ß precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for secreted APP (sAPP). Here we show that the sAPP extension domain directly bound the sushi 1 domain specific to the γ-aminobutyric acid type B receptor subunit 1a (GABABR1a). sAPP-GABABR1a binding suppressed synaptic transmission and enhanced short-term facilitation in mouse hippocampal synapses via inhibition of synaptic vesicle release. A 17-amino acid peptide corresponding to the GABABR1a binding region within APP suppressed in vivo spontaneous neuronal activity in the hippocampus of anesthetized Thy1-GCaMP6s mice. Our findings identify GABABR1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABABR1a function to modulate synaptic transmission.

IGF-1 Receptor Differentially Regulates Spontaneous and Evoked Transmission via Mitochondria at Hippocampal Synapses.

The insulin-like growth factor-1 receptor (IGF-1R) signaling is a key regulator of lifespan, growth, and development. While reduced IGF-1R signaling delays aging and Alzheimer's disease progression, whether and how it regulates information processing at central synapses remains elusive. Here, we show that presynaptic IGF-1Rs are basally active, regulating synaptic vesicle release and short-term plasticity in excitatory hippocampal neurons. Acute IGF-1R blockade or transient knockdown suppresses spike-evoked synaptic transmission and presynaptic cytosolic Ca(2+) transients, while promoting spontaneous transmission and resting Ca(2+) level. This dual effect on transmitter release is mediated by mitochondria that attenuate Ca(2+) buffering in the absence of spikes and decrease ATP production during spiking activity. We conclude that the mitochondria, activated by IGF-1R signaling, constitute a critical regulator of information processing in hippocampal neurons by maintaining evoked-to-spontaneous transmission ratio, while constraining synaptic facilitation at high frequencies. Excessive IGF-1R tone may contribute to hippocampal hyperactivity associated with Alzheimer's disease.

GABAB receptor deficiency causes failure of neuronal homeostasis in hippocampal networks.

Stabilization of neuronal activity by homeostatic control systems is fundamental for proper functioning of neural circuits. Failure in neuronal homeostasis has been hypothesized to underlie common pathophysiological mechanisms in a variety of brain disorders. However, the key molecules regulating homeostasis in central mammalian neural circuits remain obscure. Here, we show that selective inactivation of GABAB, but not GABA(A), receptors impairs firing rate homeostasis by disrupting synaptic homeostatic plasticity in hippocampal networks. Pharmacological GABA(B) receptor (GABA(B)R) blockade or genetic deletion of the GB(1a) receptor subunit disrupts homeostatic regulation of synaptic vesicle release. GABA(B)Rs mediate adaptive presynaptic enhancement to neuronal inactivity by two principle mechanisms: First, neuronal silencing promotes syntaxin-1 switch from a closed to an open conformation to accelerate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly, and second, it boosts spike-evoked presynaptic calcium flux. In both cases, neuronal inactivity removes tonic block imposed by the presynaptic, GB(1a)-containing receptors on syntaxin-1 opening and calcium entry to enhance probability of vesicle fusion. We identified the GB(1a) intracellular domain essential for the presynaptic homeostatic response by tuning intermolecular interactions among the receptor, syntaxin-1, and the Ca(V)2.2 channel. The presynaptic adaptations were accompanied by scaling of excitatory quantal amplitude via the postsynaptic, GB(1b)-containing receptors. Thus, GABA(B)Rs sense chronic perturbations in GABA levels and transduce it to homeostatic changes in synaptic strength. Our results reveal a novel role for GABA(B)R as a key regulator of population firing stability and propose that disruption of homeostatic synaptic plasticity may underlie seizure's persistence in the absence of functional GABA(B)Rs.

Interplay between population firing stability and single neuron dynamics in hippocampal networks.

Neuronal circuits' ability to maintain the delicate balance between stability and flexibility in changing environments is critical for normal neuronal functioning. However, to what extent individual neurons and neuronal populations maintain internal firing properties remains largely unknown. In this study, we show that distributions of spontaneous population firing rates and synchrony are subject to accurate homeostatic control following increase of synaptic inhibition in cultured hippocampal networks. Reduction in firing rate triggered synaptic and intrinsic adaptive responses operating as global homeostatic mechanisms to maintain firing macro-stability, without achieving local homeostasis at the single-neuron level. Adaptive mechanisms, while stabilizing population firing properties, reduced short-term facilitation essential for synaptic discrimination of input patterns. Thus, invariant ongoing population dynamics emerge from intrinsically unstable activity patterns of individual neurons and synapses. The observed differences in the precision of homeostatic control at different spatial scales challenge cell-autonomous theory of network homeostasis and suggest the existence of network-wide regulation rules.

Compartmentalization of the GABAB receptor signaling complex is required for presynaptic inhibition at hippocampal synapses.

Presynaptic inhibition via G-protein-coupled receptors (GPCRs) and voltage-gated Ca(2+) channels constitutes a widespread regulatory mechanism of synaptic strength. Yet, the mechanism of intermolecular coupling underlying GPCR-mediated signaling at central synapses remains unresolved. Using FRET spectroscopy, we provide evidence for formation of spatially restricted (<100 Å) complexes between GABA(B) receptors composed of GB(1a)/GB(2) subunits, Gα(o)ß(1)γ(2) G-protein heterotrimer, and Ca(V)2.2 channels in hippocampal boutons. GABA release was not required for the assembly but for structural reorganization of the precoupled complex. Unexpectedly, GB(1a) deletion disrupted intermolecular associations within the complex. The GB(1a) proximal C-terminal domain was essential for association of the receptor, Ca(V)2.2 and Gßγ, but was dispensable for agonist-induced receptor activation and cAMP inhibition. Functionally, boutons lacking this complex-formation domain displayed impaired presynaptic inhibition of Ca(2+) transients and synaptic vesicle release. Thus, compartmentalization of the GABA(B1a) receptor, Gßγ, and Ca(V)2.2 channel in a signaling complex is required for presynaptic inhibition at hippocampal synapses.

Basal GABA regulates GABA(B)R conformation and release probability at single hippocampal synapses.

Presynaptic GABA(B) receptor (GABA(B)R) heterodimers are composed of GB(1a)/GB(2) subunits and critically influence synaptic and cognitive functions. Here, we explored local GABA(B)R activation by integrating optical tools for monitoring receptor conformation and synaptic vesicle release at individual presynaptic boutons of hippocampal neurons. Utilizing fluorescence resonance energy transfer (FRET) spectroscopy, we detected a wide range of FRET values for CFP/YFP-tagged GB(1a)/GB(2) receptors that negatively correlated with release probabilities at single synapses. High FRET of GABA(B)Rs associated with low release probability. Notably, pharmacological manipulations that either reduced or increased basal receptor activation decreased intersynapse variability of GB(1a)/GB(2) receptor conformation. Despite variability along axons, presynaptic GABA(B)R tone was dendrite specific, having a greater impact on synapses at highly innervated proximal branches. Prolonged neuronal inactivity reduced basal receptor activation, leading to homeostatic augmentation of release probability. Our findings suggest that local variations in basal GABA concentration are a major determinant of GB(1a)/GB(2) conformational variability, which contributes to heterogeneity of neurotransmitter release at hippocampal synapses.

Carvacrol is a novel inhibitor of Drosophila TRPL and mammalian TRPM7 channels.

Transient receptor potential (TRP) channels are essential components of biological sensors that detect changes in the environment in response to a myriad of stimuli. A major difficulty in the study of TRP channels is the lack of pharmacological agents that modulate most members of the TRP superfamily. Notable exceptions are the thermoTRPs, which respond to either cold or hot temperatures and are modulated by a relatively large number of chemical agents. In the present study we demonstrate by patch clamp whole cell recordings from Schneider 2 and Drosophila photoreceptor cells that carvacrol, a known activator of the thermoTRPs, TRPV3 and TRPA1 is an inhibitor of the Drosophila TRPL channels, which belongs to the TRPC subfamily. We also show that additional activators of TRPV3, thymol, eugenol, cinnamaldehyde and menthol are all inhibitors of the TRPL channel. Furthermore, carvacrol also inhibits the mammalian TRPM7 heterologously expressed in HEK cells and ectopically expressed in a primary culture of CA3-CA1 hippocampal brain neurons. This study, thus, identifies a novel inhibitor of TRPC and TRPM channels. Our finding that the activity of the non-thermoTRPs, TRPL and TRPM7 channels is modulated by the same compound as thermoTRPs, suggests that common mechanisms of channel modulation characterize TRP channels.

A novel immune-based therapy for stroke induces neuroprotection and supports neurogenesis.

The ability of the central nervous system to cope with stressful conditions was shown to be dependent on proper T-cell-mediated immune response. Because the therapeutic window for neuroprotection after acute insults such as stroke is relatively narrow, we searched for a procedure that would allow the relevant T cells to be recruited rapidly. Permanent middle cerebral artery occlusion was induced in adult rats. To facilitate a rapid poststroke T cell activity, rats were treated with poly-YE using different regimens. Control and poly-YE-treated rats were assessed for functional recovery using neurological severity score and Morris water maze. Neuroprotection, neurogenesis, growth factor expression, and microglial phenotype were assessed using histological and immunofluorescence methods. Administration of poly-YE as late as 24 hours after middle cerebral artery occlusion yielded a beneficial effect manifested by better neurological performance, reduced neuronal loss, attenuation of behavioral deficits, and increased hippocampal and cortical neurogenesis. This compound affected the subacute phase by modulating microglial response and by increasing local production of insulin-like growth factor-I, known to be a key player in neuronal survival and neurogenesis. The relative wide therapeutic window, coupled with its efficacy in attenuating further degeneration and enhancing restoration, makes poly-YE a promising immune-based candidate for stroke therapy.