Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy.

Voltage imaging with cellular specificity has been made possible by advances in genetically encoded voltage indicators. However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines the signal-to-noise ratio and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating while also maximizing signal detection efficiency. The resulting benefits in signal-to-noise ratio and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different genetically encoded voltage indicator classes.

Photothrombosis induced cortical stroke produces electrographic epileptic biomarkers in mice.

Objective: Interictal epileptiform spikes, high-frequency ripple oscillations, and their co-occurrence (spike ripples) in human scalp or intracranial voltage recordings are well-established epileptic biomarkers. While clinically significant, the neural mechanisms generating these electrographic biomarkers remain unclear. To reduce this knowledge gap, we introduce a novel photothrombotic stroke model in mice that reproduces focal interictal electrographic biomarkers observed in human epilepsy. Methods: We induced a stroke in the motor cortex of C57BL/6 mice unilaterally (N=7) using a photothrombotic procedure previously established in rats. We then implanted intracranial electrodes (2 ipsilateral and 2 contralateral) and obtained intermittent local field potential (LFP) recordings over several weeks in awake, behaving mice. We evaluated the LFP for focal slowing and epileptic biomarkers - spikes, ripples, and spike ripples - using both automated and semi-automated procedures. Results: Delta power (1-4 Hz) was higher in the stroke hemisphere than the non-stroke hemisphere in all mice ( p <0.001). Automated detection procedures indicated that compared to the non-stroke hemisphere, the stroke hemisphere had an increased spike ripple ( p =0.006) and spike rates ( p =0.039), but no change in ripple rate ( p =0.98). Expert validation confirmed the observation of elevated spike ripple rates ( p =0.008) and a trend of elevated spike rate ( p =0.055) in the stroke hemisphere. Interestingly, the validated ripple rate in the stroke hemisphere was higher than the non-stroke hemisphere ( p =0.031), highlighting the difficulty of automatically detecting ripples. Finally, using optimal performance thresholds, automatically detected spike ripples classified the stroke hemisphere with the best accuracy (sensitivity 0.94, specificity 0.94). Significance: Cortical photothrombosis-induced stroke in commonly used C57BL/6 mice produces electrographic biomarkers as observed in human epilepsy. This model represents a new translational cortical epilepsy model with a defined irritative zone, which can be broadly applied in transgenic mice for cell type specific analysis of the cellular and circuit mechanisms of pathologic interictal activity. Key Points: Cortical photothrombosis in mice produces stroke with characteristic intermittent focal delta slowing.Cortical photothrombosis stroke in mice produces the epileptic biomarkers spikes, ripples, and spike ripples.All biomarkers share morphological features with the corresponding human correlate.Spike ripples better lateralize to the lesional cortex than spikes or ripples.This cortical model can be applied in transgenic mice for mechanistic studies.

Theta and gamma rhythmic coding through two spike output modes in the hippocampus during spatial navigation.

Hippocampal CA1 neurons generate single spikes and stereotyped bursts of spikes. However, it is unclear how individual neurons dynamically switch between these output modes and whether these two spiking outputs relay distinct information. We performed extracellular recordings in spatially navigating rats and cellular voltage imaging and optogenetics in awake mice. We found that spike bursts are preferentially linked to cellular and network theta rhythms (3-12 Hz) and encode an animal's position via theta phase precession, particularly as animals are entering a place field. In contrast, single spikes exhibit additional coupling to gamma rhythms (30-100 Hz), particularly as animals leave a place field. Biophysical modeling suggests that intracellular properties alone are sufficient to explain the observed input frequency-dependent spike coding. Thus, hippocampal neurons regulate the generation of bursts and single spikes according to frequency-specific network and intracellular dynamics, suggesting that these spiking modes perform distinct computations to support spatial behavior.

Large-scale deep tissue voltage imaging with targeted illumination confocal microscopy.

Voltage imaging with cellular specificity has been made possible by the tremendous advances in genetically encoded voltage indicators (GEVIs). However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines signal-to-noise ratio (SNR) and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating, while also maximizing signal detection efficiency. The resulting benefits in SNR and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different GEVI classes.

A Selective Projection from the Subthalamic Nucleus to Parvalbumin-Expressing Interneurons of the Striatum.

The striatum and subthalamic nucleus (STN) are considered to be the primary input nuclei of the basal ganglia. Projection neurons of both striatum and STN can extensively interact with other basal ganglia nuclei, and there is growing anatomic evidence of direct axonal connections from the STN to striatum. There remains, however, a pressing need to elucidate the organization and impact of these subthalamostriatal projections in the context of the diverse cell types constituting the striatum. To address this, we conducted monosynaptic retrograde tracing from genetically-defined populations of dorsal striatal neurons in adult male and female mice, quantifying the connectivity from STN neurons to spiny projection neurons, GABAergic interneurons, and cholinergic interneurons. In parallel, we used a combination of ex vivo electrophysiology and optogenetics to characterize the responses of a complementary range of dorsal striatal neuron types to activation of STN axons. Our tracing studies showed that the connectivity from STN neurons to striatal parvalbumin-expressing interneurons is significantly higher (∼4- to 8-fold) than that from STN to any of the four other striatal cell types examined. In agreement, our recording experiments showed that parvalbumin-expressing interneurons, but not the other cell types tested, commonly exhibited robust monosynaptic excitatory responses to subthalamostriatal inputs. Taken together, our data collectively demonstrate that the subthalamostriatal projection is highly selective for target cell type. We conclude that glutamatergic STN neurons are positioned to directly and powerfully influence striatal activity dynamics by virtue of their enriched innervation of GABAergic parvalbumin-expressing interneurons.

Striatal cholinergic interneuron membrane voltage tracks locomotor rhythms in mice.

Rhythmic neural network activity has been broadly linked to behavior. However, it is unclear how membrane potentials of individual neurons track behavioral rhythms, even though many neurons exhibit pace-making properties in isolated brain circuits. To examine whether single-cell voltage rhythmicity is coupled to behavioral rhythms, we focused on delta-frequencies (1-4 Hz) that are known to occur at both the neural network and behavioral levels. We performed membrane voltage imaging of individual striatal neurons simultaneously with network-level local field potential recordings in mice during voluntary movement. We report sustained delta oscillations in the membrane potentials of many striatal neurons, particularly cholinergic interneurons, which organize spikes and network oscillations at beta-frequencies (20-40 Hz) associated with locomotion. Furthermore, the delta-frequency patterned cellular dynamics are coupled to animals' stepping cycles. Thus, delta-rhythmic cellular dynamics in cholinergic interneurons, known for their autonomous pace-making capabilities, play an important role in regulating network rhythmicity and movement patterning.

Deep brain stimulation creates informational lesion through membrane depolarization in mouse hippocampus.

Deep brain stimulation (DBS) is a promising neuromodulation therapy, but the neurophysiological mechanisms of DBS remain unclear. In awake mice, we performed high-speed membrane voltage fluorescence imaging of individual hippocampal CA1 neurons during DBS delivered at 40 Hz or 140 Hz, free of electrical interference. DBS powerfully depolarized somatic membrane potentials without suppressing spike rate, especially at 140 Hz. Further, DBS paced membrane voltage and spike timing at the stimulation frequency and reduced timed spiking output in response to hippocampal network theta-rhythmic (3-12 Hz) activity patterns. To determine whether DBS directly impacts cellular processing of inputs, we optogenetically evoked theta-rhythmic membrane depolarization at the soma. We found that DBS-evoked membrane depolarization was correlated with DBS-mediated suppression of neuronal responses to optogenetic inputs. These results demonstrate that DBS produces powerful membrane depolarization that interferes with the ability of individual neurons to respond to inputs, creating an informational lesion.

Neuropilin 2/Plexin-A3 Receptors Regulate the Functional Connectivity and the Excitability in the Layers 4 and 5 of the Cerebral Cortex.

The functions of cortical networks are progressively established during development by series of events shaping the neuronal connectivity. Synaptic elimination, which consists of removing the supernumerary connections generated during the earlier stages of cortical development, is one of the latest stages in neuronal network maturation. The semaphorin 3F coreceptors neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) may play an important role in the functional maturation of the cerebral cortex by regulating the excess dendritic spines on cortical excitatory neurons. Yet, the identity of the connections eliminated under the control of Nrp2/PlxnA3 signaling is debated, and the importance of this synaptic refinement for cortical functions remains poorly understood. Here, we show that Nrp2/PlxnA3 controls the spine densities in layer 4 (L4) and on the apical dendrite of L5 neurons of the sensory and motor cortices. Using a combination of neuroanatomical, ex vivo electrophysiology, and in vivo functional imaging techniques in Nrp2 and PlxnA3 KO mice of both sexes, we disprove the hypothesis that Nrp2/PlxnA3 signaling is required to maintain the ectopic thalamocortical connections observed during embryonic development. We also show that the absence of Nrp2/PlxnA3 signaling leads to the hyperexcitability and excessive synchronization of the neuronal activity in L5 and L4 neuronal networks, suggesting that this system could participate in the refinement of the recurrent corticocortical connectivity in those layers. Altogether, our results argue for a role of semaphorin-Nrp2/PlxnA3 signaling in the proper maturation and functional connectivity of the cerebral cortex, likely by controlling the refinement of recurrent corticocortical connections.SIGNIFICANCE STATEMENT The function of a neuronal circuit is mainly determined by the connections that neurons establish with one another during development. Understanding the mechanisms underlying the establishment of the functional connectivity is fundamental to comprehend how network functions are implemented, and to design treatments aiming at restoring damaged neuronal circuits. Here, we show that the cell surface receptors for the family of semaphorin guidance cues neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) play an important role in shaping the functional connectivity of the cerebral cortex likely by trimming the recurrent connections in layers 4 and 5. By removing the supernumerary inputs generated during early development, Nrp2/PlxnA3 signaling reduces the neuronal excitability and participates in the maturation of the cortical network functions.

Cholinergic midbrain afferents modulate striatal circuits and shape encoding of action strategies.

Assimilation of novel strategies into a consolidated action repertoire is a crucial function for behavioral adaptation and cognitive flexibility. Acetylcholine in the striatum plays a pivotal role in such adaptation, and its release has been causally associated with the activity of cholinergic interneurons. Here we show that the midbrain, a previously unknown source of acetylcholine in the striatum, is a major contributor to cholinergic transmission in the striatal complex. Neurons of the pedunculopontine and laterodorsal tegmental nuclei synapse with striatal cholinergic interneurons and give rise to excitatory responses. Furthermore, they produce uniform inhibition of spiny projection neurons. Inhibition of acetylcholine release from midbrain terminals in the striatum impairs the association of contingencies and the formation of habits in an instrumental task, and mimics the effects observed following inhibition of acetylcholine release from striatal cholinergic interneurons. These results suggest the existence of two hierarchically-organized modes of cholinergic transmission in the striatum, where cholinergic interneurons are modulated by cholinergic neurons of the midbrain.

Striatal cholinergic receptor activation causes a rapid, selective and state-dependent rise in cortico-striatal ß activity.

Cortico-basal ganglia-thalamic (CBT) ß oscillations (15-30 Hz) are elevated in Parkinson's disease and correlated with movement disability. To date, no experimental paradigm outside of loss of dopamine has been able to specifically elevate ß oscillations in the CBT loop. Here, we show that activation of striatal cholinergic receptors selectively increased ß oscillations in mouse striatum and motor cortex. In individuals showing simultaneous ß increases in both striatum and M1, ß partial directed coherence (PDC) increased from striatum to M1 (but not in the reverse direction). In individuals that did not show simultaneous ß increases, ß PDC increased from M1 to striatum (but not in the reverse direction), and M1 was characterized by persistent ß-high frequency oscillation phase-amplitude coupling. Finally, the direction of ß PDC distinguished between ß sub-bands. This suggests that (1) striatal cholinergic tone exerts state-dependent and frequency-selective control over CBT ß power and coordination; (2) ongoing rhythmic dynamics can determine whether elevated ß oscillations are expressed in striatum and M1; and (3) altered striatal cholinergic tone differentially modulates distinct ß sub-bands.

Striatal cholinergic interneurons generate beta and gamma oscillations in the corticostriatal circuit and produce motor deficits.

Cortico-basal ganglia-thalamic (CBT) neural circuits are critical modulators of cognitive and motor function. When compromised, these circuits contribute to neurological and psychiatric disorders, such as Parkinson's disease (PD). In PD, motor deficits correlate with the emergence of exaggerated beta frequency (15-30 Hz) oscillations throughout the CBT network. However, little is known about how specific cell types within individual CBT brain regions support the generation, propagation, and interaction of oscillatory dynamics throughout the CBT circuit or how specific oscillatory dynamics are related to motor function. Here, we investigated the role of striatal cholinergic interneurons (SChIs) in generating beta and gamma oscillations in cortical-striatal circuits and in influencing movement behavior. We found that selective stimulation of SChIs via optogenetics in normal mice robustly and reversibly amplified beta and gamma oscillations that are supported by distinct mechanisms within striatal-cortical circuits. Whereas beta oscillations are supported robustly in the striatum and all layers of primary motor cortex (M1) through a muscarinic-receptor mediated mechanism, gamma oscillations are largely restricted to the striatum and the deeper layers of M1. Finally, SChI activation led to parkinsonian-like motor deficits in otherwise normal mice. These results highlight the important role of striatal cholinergic interneurons in supporting oscillations in the CBT network that are closely related to movement and parkinsonian motor symptoms.

Optogenetics and deep brain stimulation neurotechnologies.

Brain neural network is composed of densely packed, intricately wired neurons whose activity patterns ultimately give rise to every behavior, thought, or emotion that we experience. Over the past decade, a novel neurotechnique, optogenetics that combines light and genetic methods to control or monitor neural activity patterns, has proven to be revolutionary in understanding the functional role of specific neural circuits. We here briefly describe recent advance in optogenetics and compare optogenetics with deep brain stimulation technology that holds the promise for treating many neurological and psychiatric disorders.

Novel 4-substituted-N,N-dimethyltetrahydronaphthalen-2-amines: synthesis, affinity, and in silico docking studies at serotonin 5-HT2-type and histamine H1 G protein-coupled receptors.

Syntheses were undertaken of derivatives of (2S,4R)-(-)-trans-4-phenyl-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (4-phenyl-2-dimethylaminotetralin, PAT), a stereospecific agonist at the serotonin 5-HT2C G protein-coupled receptor (GPCR), with inverse agonist activity at 5-HT2A and 5-HT2B GPCRs. Molecular changes were made at the PAT C(4)-position, while preserving N,N-dimethyl substitution at the 2-position as well as trans-stereochemistry, structural features previously shown to be optimal for 5-HT2 binding. Affinities of analogs were determined at recombinant human 5-HT2 GPCRs in comparison to the phylogenetically closely-related histamine H1 GPCR, and in silico ligand docking studies were conducted at receptor molecular models to help interpret pharmacological results and guide future ligand design. In most cases, C(4)-substituted PAT analogs exhibited the same stereoselectivity ([-]-trans>[+]-trans) as the parent PAT across 5-HT2 and H1 GPCRs, albeit, with variable receptor selectivity. 4-(4'-substituted)-PAT analogs, however, demonstrated reversed stereoselectivity ([2S,4R]-[+]-trans>[2S,4R]-[-]-trans), with absolute configuration confirmed by single X-ray crystallographic data for the 4-(4'-Cl)-PAT analog. Pharmacological affinity results and computational results herein support further PAT drug development studies and provide a basis for predicting and interpreting translational results, including, for (+)-trans-4-(4'-Cl)-PAT and (-)-trans-4-(3'-Br)-PAT that were previously shown to be more potent and efficacious than their corresponding enantiomers in rodent models of psychoses, psychostimulant-induced behaviors, and compulsive feeding ('binge-eating').

A novel aminotetralin-type serotonin (5-HT) 2C receptor-specific agonist and 5-HT2A competitive antagonist/5-HT2B inverse agonist with preclinical efficacy for psychoses.

Development of 5-HT2C agonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2C receptor agonist, (-)-trans-(2S,4R)-4-(3'[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (-)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (-)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (-)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (-)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (-)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2C receptor-specific agonist, such as (-)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders.

Molecular and behavioral pharmacology of two novel orally-active 5HT2 modulators: potential utility as antipsychotic medications.

BACKGROUND: Desired serotonin 5HT2 receptor pharmacology for treatment of psychoses is 5HT2A antagonism and/or 5HT2C agonism. No selective 5HT2A antagonist has been approved for psychosis and the only approved 5HT2C agonist (for obesity) also activates 5HT2A and 5HT2B receptors, which can lead to clinical complications. Studies herein tested the hypothesis that a dual-function 5HT2A antagonist/5HT2C agonist that does not activate 5HT2B receptors would be suitable for development as an antipsychotic drug, without liability for weight gain. METHODS: The novel compounds (+)- and (-)-trans-4-(4'-chlorophenyl)-N,N-dimethyl-2-aminotetralin (p-Cl-PAT) were synthesized, characterized in vitro for affinity and functional activity at human 5HT2 receptors, and administered by intraperitoneal (i.p.) and oral (gavage) routes to mice in behavioral paradigms that assessed antipsychotic efficacy and effects on feeding behavior. RESULTS: (+)- and (-)-p-Cl-PAT activated 5HT2C receptors, with (+)-p-Cl-PAT being 12-times more potent, consistent with its higher affinity across 5HT2 receptors. Neither p-Cl-PAT enantiomer activated 5HT2A or 5HT2B receptors at concentrations up to 300-times greater than their respective affinity (Ki), and (+)-p-Cl-PAT was shown to be a 5HT2A competitive antagonist. When administered i.p. or orally, (+)- and (-)-p-Cl-PAT attenuated the head-twitch response (HTR) in mice elicited by the 5HT2 agonist (-)-2,5-dimethoxy-4-iodoamphetamine (DOI) and reduced intake of a highly palatable food in non-food-deprived mice, with (+)-p-Cl-PAT being more potent across behavioral assays. CONCLUSIONS: The novel in vitro pharmacology of (+)-p-Cl-PAT (5HT2A antagonism/5HT2C agonism without activation of 5HT2B) translated in vivo to an orally-active drug candidate with preclinical efficacy to treat psychoses without liability for weight gain.

Molecular Determinants for Ligand Binding at Serotonin 5-HT2A and 5-HT2C GPCRs: Experimental Affinity Results Analyzed by Molecular Modeling and Ligand Docking Studies.

Ligands that activate the serotonin 5-HT2C G protein-coupled receptor (GPCR) may be therapeutic for psychoses, addiction, and other neuropsychiatric disorders. Ligands that are antagonists at the closely related 5-HT2A GPCR also may treat neuropsychiatric disorders; in contrast, 5-HT2A activation may cause hallucinations. 5-HT2C-specific agonist drug design is challenging because 5-HT2 GPCRs share 80% transmembrane (TM) homology, same second messenger signaling, and no crystal structures are reported. To help delineate molecular determinants underlying differential binding and activation of 5-HT2 GPCRs, 5-HT2A, and 5-HT2C homology models were built from the ß2-adrenergic GPCR crystal structure and equilibrated in a lipid phosphatidyl choline bilayer performing molecular dynamics simulations. Ligand docking studies at the 5-HT2 receptor models were conducted with the (2R, 4S)- and (2S, 4R)-enantiomers of the novel 5-HT2C agonist/5-HT2A/2B antagonist trans-4-phenyl-N,N-dimethyl-2-aminotetralin (PAT) and its 4'-chlorophenyl congners. Results indicate PAT-5-HT2 molecular interactions especially in TM domain V are important for the (2R, 4S) enantiomer, whereas, TM domain VI and VII interactions are more important for the (2S, 4R) enantiomer.

Poisoning severity score, APACHE II and GCS: effective clinical indices for estimating severity and predicting outcome of acute organophosphorus and carbamate poisoning.

Self-poisoning with organophosphorus (OP) compounds is a major cause of morbidity and mortality across South Asian countries. To develop uniform and effective management guidelines, the severity of acute OP poisoning should be assessed through scientific methods and a clinical database should be maintained. A prospective descriptive survey was carried out to assess the utility of severity scales in predicting the outcome of 71 organophosphate (OP) and carbamate poisoning patients admitted during a one year period at the Kasturba Hospital, Manipal, India. The Glasgow coma scale (GCS) scores, acute physiology and chronic health evaluation II (APACHE II) scores, predicted mortality rate (PMR) and Poisoning severity score (PSS) were estimated within 24h of admission. Significant correlation (P<0.05) between PSS and GCS and APACHE II and PMR scores were observed with the PSS scores predicting mortality significantly (P< or =0.001). A total of 84.5% patients improved after treatment while 8.5% of the patients were discharged with severe morbidity. The mortality rate was 7.0%. Suicidal poisoning was observed to be the major cause (80.2%), while other reasons attributed were occupational (9.1%), accidental (6.6%), homicidal (1.6%) and unknown (2.5%) reasons. This study highlights the application of clinical indices like GCS, APACHE, PMR and severity scores in predicting mortality and may be considered for planning standard treatment guidelines.