Optogenetic Excitation Research Articles

A prefrontal-periaqueductal gray pathway differentially engages autonomic, hormonal, and behavioral features of the stress coping response.

Activation of autonomic and hypothalamo-pituitary-adrenal (HPA) systems occur interdependently with behavioral adjustments under varying environmental demands. Nevertheless, laboratory rodent studies examining the neural bases of stress responses have generally attributed increments in these systems to be monolithic, regardless of whether an active or passive coping strategy is employed. Using the shock probe defensive burying test (SPDB) to measure stress-coping features naturalistically in male and female rats, we identify a neural pathway whereby activity changes may promote distinctive response patterns of hemodynamic and HPA indices typifying active and passive coping phenotypes. Optogenetic excitation of the rostral medial prefrontal cortex (mPFC) input to the ventrolateral periaqueductal gray (vlPAG) decreased passive behavior (immobility), attenuated the glucocorticoid hormone response, but did not prevent arterial pressure and heart rate increases associated with rats' active behavioral (defensive burying) engagement during the SPDB. By contrast, inhibition of the same pathway increased behavioral immobility and attenuated hemodynamic output but did not affect glucocorticoid increases. Correlational analyses confirmed that hemodynamic increments occurred preferentially during active behaviors, and decrements during immobility epochs, whereas pathway manipulations, regardless of the directionality of effect, weakened the correlational relationship. Finally, neuroanatomical evidence indicated that the influence of the rostral mPFC-vlPAG pathway on coping response patterns are mediated predominantly through GABAergic neurons within vlPAG. These data highlight the importance of this prefrontal-midbrain connection in organizing stress-coping responses, and in coordinating bodily systems with behavioral output for adaptation to aversive experiences.Significance statement Organisms maximize fitness by exhibiting distinct stress-coping responses that are specific to a particular challenge. However, the neurobiology underlying cortical control over coping styles is poorly understood. We reveal a novel role for a prefrontal-to-ventrolateral periaqueductal gray pathway in regulating active versus passive stress-coping response patterns in rats. Optogenetic excitation of this pathway decreased behavioral passivity, attenuated stress-induced glucocorticoid increases, but did not prevent associated increases in autonomic output. Pathway inhibition increased behavioral passivity, attenuated autonomic output, but did not affect glucocorticoid increases. These data highlight the importance of this prefrontal-midbrain connection in organizing stress-coping responses, and in coordinating bodily systems with behavioral output for adaptation to aversive experiences.

Galanin receptor 1 expressing neurons in hippocampal-prefrontal circuitry modulate goal directed attention and impulse control.

While amino acid neurotransmitters are the main chemical messengers in the brain, they are co-expressed with neuropeptides which are increasingly recognized as modulators of cognitive pathways. For example, the neuropeptide galanin has been implicated in a wide range of pathological conditions in which frontal and temporal structures are compromised. In a recent study in rats, we discovered that direct pharmacological stimulation of galanin receptor type 1 (GalR1) in the ventral prefrontal cortex (vPFC) and ventral hippocampus (vHC) led to opposing effects on attention and impulse control behavior. In the present study, we investigate how subtypes of neurons expressing GalR1 in these two areas differentially contribute to these behaviors. We first establish that GalR1 is predominantly expressed in glutamatergic neurons in both the vPFC and HC. We develop a novel viral approach to gain genetic access to GalR1-expressing neurons and demonstrate that optogenetic excitation of GalR1 expressing neurons in the vPFC, but not vHC, selectively disrupts attention in a complex behavioral task. Finally, using fiber photometry, we measure the bulk calcium dynamics in GalR1-expressing neurons during the same task to demonstrate opposing activity in vPFC and vHC. These results are consistent with our previous work demonstrating differential behavioral effects induced by GalR1 activating in vPFC and vHC. These results indicate the distinct neuromodulatory and behavioral contributions of galanin mediated by subclasses of neurons in the hippocampal and prefrontal circuitry.

Effect of optogenetic excitation of non-orexinergic neurons in the hypothalamic perifornical area on motor behaviors and cardiovascular parameters in rats

Central command, a motor volition originating in the rostral part of the brain, plays a pivotal role in the precise regulation of autonomic nervous and cardiovascular systems. Central neuronal substrates responsible for transmitting central command signals remain incompletely understood. This study aimed to investigate the effect of optogenetic excitation of non-orexinergic (NOrx) neurons in the hypothalamic perifornical area (PeFA), where orexinergic neurons are densely distributed, on motor behaviors and cardiovascular parameters in rats. An adeno-associated viral serotype 2 vector carrying the human synapsin promoter encoding channelrhodopsin 2 (ChR2) fused to EYFP was injected into the PeFA of Sprague-Dawley rats, resulting in selective expression of ChR2-EYFP in NOrx PeFA neurons. In conscious rats, optogenetic excitation of NOrx PeFA neurons rapidly elicited walking or biting behavior, simultaneously causing pressor and tachycardiac responses regardless of the observed behavioral patterns. Under anesthesia, this excitation rapidly increased renal sympathetic nerve activity, immediately followed by sympathoinhibition. These findings suggest that NOrx PeFA neurons transmit central command signals, concurrently regulating somatomotor and autonomic nervous systems for locomotor exercise or biting behavior.

Hippocampal PACAP signaling activation triggers a rapid antidepressant response

BackgroundThe development of ketamine-like rapid antidepressants holds promise for enhancing the therapeutic efficacy of depression, but the underlying cellular and molecular mechanisms remain unclear. Implicated in depression regulation, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is investigated here to examine its role in mediating the rapid antidepressant response.MethodsThe onset of antidepressant response was assessed through depression-related behavioral paradigms. The signaling mechanism of PACAP in the hippocampal dentate gyrus (DG) was evaluated by utilizing site-directed gene knockdown, pharmacological interventions, or optogenetic manipulations. Overall, 446 mice were used for behavioral and molecular signaling testing. Mice were divided into control or experimental groups randomly in each experiment, and the experimental manipulations included: chronic paroxetine treatments (4, 9, 14 d) or a single treatment of ketamine; social defeat or lipopolysaccharides-injection induced depression models; different doses of PACAP (0.4, 2, 4 ng/site; microinjected into the hippocampal DG); pharmacological intra-DG interventions (CALM and PACAP6-38); intra-DG viral-mediated PACAP RNAi; and opotogenetics using channelrhodopsins 2 (ChR2) or endoplasmic natronomonas halorhodopsine 3.0 (eNpHR3.0). Behavioral paradigms included novelty suppressed feeding test, tail suspension test, forced swimming test, and sucrose preference test. Western blotting, ELISA, or quantitative real-time PCR (RT-PCR) analysis were used to detect the expressions of proteins/peptides or genes in the hippocampus.ResultsChronic administration of the slow-onset antidepressant paroxetine resulted in an increase in hippocampal PACAP expression, and intra-DG blockade of PACAP attenuated the onset of the antidepressant response. The levels of hippocampal PACAP expression were reduced in both two distinct depression animal models and intra-DG knockdown of PACAP induced depression-like behaviors. Conversely, a single infusion of PACAP into the DG region produced a rapid and sustained antidepressant response in both normal and chronically stressed mice. Optogenetic intra-DG excitation of PACAP-expressing neurons instantly elicited antidepressant responses, while optogenetic inhibition induced depression-like behaviors. The longer optogenetic excitation/inhibition elicited the more sustained antidepressant/depression-like responses. Intra-DG PACAP infusion immediately facilitated the signaling for rapid antidepressant response by inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII)-eukaryotic elongation factor 2 (eEF2) and activating the mammalian target of rapamycin (mTOR). Pre-activation of CaMKII signaling within the DG blunted PACAP-induced rapid antidepressant response as well as eEF2-mTOR-brain-derived neurotrophic factor (BDNF) signaling. Finally, acute ketamine treatment upregulated hippocampal PACAP expression, whereas intra-DG blockade of PACAP signaling attenuated ketamine’s rapid antidepressant response.ConclusionsActivation of hippocampal PACAP signaling induces a rapid antidepressant response through the regulation of CaMKII inhibition-governed eEF2-mTOR-BDNF signaling.

Sex differences in neural projections of fear memory processing in mice and humans.

It remains unexplored in the field of fear memory whether functional neuronal connectivity between two brain areas is necessary for one sex but not the other. Here, we show that chemogenetic silencing of centromedial (CeM)-Tac2 fibers in the lateral posterior BNST (BNSTpl) decreased fear memory consolidation in male mice but not females. Optogenetic excitation of CeM-Tac2 fibers in the BNSTpl exhibited enhanced inhibitory postsynaptic currents in males compared to females. In vivo calcium imaging analysis revealed a sex-dimorphic fear memory engram in the BNSTpl. Furthermore, in humans, the single-nucleotide polymorphism (SNP) in the Tac2 receptor (rs2765) (TAC3R) decreased CeM-BNST connectivity in a fear task, impaired fear memory consolidation, and increased the expression of the TAC3R mRNA in AA-carrier men but not in women. These sex differences in critical neuronal circuits underlying fear memory formation may be relevant to human neuropsychiatric disorders with fear memory alterations such as posttraumatic stress disorder.

A Cocaine-Activated Ensemble Exerts Increased Control Over Behavior While Decreasing in Size

BackgroundSubstance use disorder is characterized by long-lasting changes in reward-related brain regions, such as the nucleus accumbens. Previous work has shown that cocaine exposure induces plasticity in broad, genetically defined cell types in the nucleus accumbens; however, in response to a stimulus, only a small percentage of neurons are transcriptionally active—termed an ensemble. Here, we identify an Arc-expressing neuronal ensemble that has a unique trajectory of recruitment and causally controls drug self-administration after repeated, but not acute, cocaine exposure. MethodsUsing Arc-CreERT2 transgenic mice, we expressed transgenes in Arc+ ensembles activated by cocaine exposure (either acute [1 × 10 mg/kg intraperitoneally] or repeated [10 × 10 mg/kg intraperitoneally]). Using genetic, optical, and physiological recording and manipulation strategies, we assessed the contribution of these ensembles to behaviors associated with substance use disorder. ResultsRepeated cocaine exposure reduced the size of the ensemble while simultaneously increasing its control over behavior. Neurons within the repeated cocaine ensemble were hyperexcitable, and their optogenetic excitation was sufficient for reinforcement. Finally, lesioning the repeated cocaine, but not the acute cocaine, ensemble blunted cocaine self-administration. Thus, repeated cocaine exposure reduced the size of the ensemble while simultaneously increasing its contributions to drug reinforcement. ConclusionsWe showed that repeated, but not acute, cocaine exposure induced a physiologically distinct ensemble characterized by the expression of the immediate early gene Arc, which was uniquely capable of modulating reinforcement behavior.

Crosstalk amongst oxytocin and vasopressin synthesizing neurons of the paraventricular nucleus of the hypothalamus increases blood pressure in mice

The paraventricular nucleus of the hypothalamus (PVH) orchestrates neuroendocrine release and autonomic outflow to maintain body fluid homeostasis and cardiovascular function. Emerging evidence supports the notion that neuropeptides synthesized by PVH neurons create paracrine signals that alter physiology and behavior by affecting the activity of neighboring neurons. Here, we test the hypothesis that PVH neurons synthesizing oxytocin (hereafter referred to a PVHOT) create a paracrine signal that regulates blood pressure. Mice with the expression of Cre-recombinase directed to the OT gene were bred that mice that express a CAG-driven LoxP-flanked stop-codon preceding a channelrhodopsin-2 (ChR2) and eYFP fusion gene at the Rosa26 locus. This produced mice expressing only Cre-recombinase (controls) or mice with ChR2-eYFP expression directed to cells that express oxytocin (OT-ChR2). Confocal image analysis found that ~90% of eYFP expressing neurons were also immunolabeled for OT and electrophysiological recordings obtained from these neurons revealed that pulses of blue light elicited the firing of action potentials. These results confirm directed and functional expression of ChR2 within PVHOT. In vivo optogenetic excitation of PVHOT increased blood pressure and decreased heart rate in OT-ChR2 mice relative to controls. Ganglionic blockade with hexamethonium had no effect on the increased blood pressure elicited by optogenetic excitation of PVHOT but it completely abolished the bradycardia that was previously observed. These results suggest that excitation of PVHOT recruits a neuroendocrine signal that promotes vasoconstriction, which elicits the baroreflex to induce bradycardia by engaging the autonomic nervous system. Consistent with this interpretation, optogenetic excitation of PVHOT increased circulating levels of OT; however, the elevated blood pressure persisted after systemic administration of an OT receptor antagonist. Intriguingly, in vitro optogenetic excitation of PVHOT evoked Ca2+ flux in CHO cells expressing OT or vasopressin receptors (V1aR), suggesting that robust firing of PVHOT is followed by local release of OT. Optogenetic excitation of PVHOT augmented firing of neurons of the PVH that synthesize vasopressin and tended to increase circulating levels of this neuropeptide. Remarkably, systemic administration of a V1aR antagonist completely abolished the increase in blood pressure and bradycardia that were previously observed with in vivo optogenetic excitation of PVHOT. Collectively, these results reveal that robust firing of PVHOT creates an oxytocinergic paracrine signal that increases blood pressure by activating V1aR(s) and increasing circulating vasopressin. AHA 23POST1020034 to KE. NHLBI F31HL1162540 to CBH. NHLBI R01HL136595, R35HL150750, R01HL145028 and NCCIH R65AT012142 to ADK & EGK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparison of unitary synaptic currents generated by indirect and direct pathway neurons of the mouse striatum.

Two subtypes of striatal spiny projection neurons, iSPNs and dSPNs, whose axons form the "indirect" and "direct" pathways of the basal ganglia, respectively, both make synaptic connections in the external globus pallidus (GPe) but are usually found to have different effects on behavior. Activation of the terminal fields of iSPNs or dSPNs generated compound currents in almost all GPe neurons. To determine whether iSPNs and dSPNs have the same or different effects on pallidal neurons, we studied the unitary synaptic currents generated in GPe neurons by action potentials in single striatal neurons. We used optogenetic excitation to elicit repetitive firing in a small number of nearby SPNs, producing sparse barrages of inhibitory postsynaptic currents (IPSCs) in GPe neurons. From these barrages, we isolated sequences of IPSCs with similar time courses and amplitudes, which presumably arose from the same SPN. There was no difference between the amplitudes of unitary IPSCs generated by the indirect and direct pathways. Most unitary IPSCs were small, but a subset from each pathway were much larger. To determine the effects of these unitary synaptic currents on the action potential firing of GPe neurons, we drove SPNs to fire as before and recorded the membrane potential of GPe neurons. Large unitary potentials from iSPNs and dSPNs perturbed the spike timing of GPe neurons in a similar way. Most SPN-GPe neuron pairs are weakly connected, but a subset of pairs in both pathways are strongly connected.NEW & NOTEWORTHY This is the first study to record the synaptic currents generated by single identified direct or indirect pathway striatal neurons on single pallidal neurons. Each GPe neuron receives synaptic inputs from both pathways. Most striatal neurons generate small synaptic currents that become influential when occurring together, but a few are powerful enough to be individually influential.

Evidence for a role of the basolateral amygdala in regulating regional metabolism in the stressed brain.

The brain regulates every physiological process in the body, including metabolism. Studies investigating brain metabolism have shown that stress can alter major metabolic processes, and that these processes can vary between regions. However, no study has investigated how metabolic pathways may be altered by stressor perception, or whether stress-responsive brain regions can also regulate metabolism. The basolateral amygdala (BLA), a region important for stress and fear, has reciprocal connections to regions responsible for metabolic regulation. In this study, we investigated how BLA influences regional metabolic profiles within the hippocampus (HPC) and medial prefrontal cortex (mPFC), regions involved in regulating the stress response and stress perception, using optogenetics in male C57BL/6 mice during footshock presentation in a yoked shuttlebox paradigm based on controllable (ES) and uncontrollable (IS) stress. RNA extracted from HPC and mPFC were loaded into NanoString® Mouse Neuroinflammation Panels, which also provides a broad view of metabolic processes, for compilation of gene expression profiles. Results showed differential regulation of carbohydrate and lipid metabolism, and insulin signaling gene expression pathways in HPC and mPFC following ES and IS, and that these differences were altered in response to optogenetic excitation or inhibition of the BLA. These findings demonstrate for the first time that individual brain regions can utilize metabolites in a way that are unique to their needs and function in response to a stressor, and that vary based on stressor controllability and influence by BLA.

Direct contribution of the sensory cortex to the judgment of stimulus duration

Decision making frequently depends on monitoring the duration of sensory events. To determine whether, and how, the perception of elapsed time derives from the neuronal representation of the stimulus itself, we recorded and optogenetically modulated vibrissal somatosensory cortical activity as male rats judged vibration duration. Perceived duration was dilated by optogenetic excitation. A second set of rats judged vibration intensity; here, optogenetic excitation amplified the intensity percept, demonstrating sensory cortex to be the common gateway both to time and to stimulus feature processing. A model beginning with the membrane currents evoked by vibrissal and optogenetic drive and culminating in the representation of perceived time successfully replicated rats’ choices. Time perception is thus as deeply intermeshed within the sensory processing pathway as is the sense of touch itself, suggesting that the experience of time may be further investigated with the toolbox of sensory coding.

Orexinergic neurons contribute to autonomic cardiovascular regulation for locomotor exercise.

While the hypothalamic orexinergic nervous system is established as having a pivotal role in the long-term regulation of various organismic functions, including wakefulness, metabolism and hypertensive states, whether this system contributes to the rapid autonomic cardiovascular regulation during physical activity remains elusive. This study aimed to elucidate the role of the orexinergic nervous system in transmitting volitional motor signals, i.e. central command, to drive somatomotor and sympathetic cardiovascular responses. We first found that this system is activated by voluntary locomotor exercise as evidenced by an increased expression of Fos, a marker of neural activation, in the orexinergic neurons of Sprague-Dawley rats engaged in spontaneous wheel running. Next, using transgenic Orexin-Cre rats for optogenetic manipulation of orexinergic neurons, we found that optogenetic excitation of orexinergic neurons caused sympathoexcitation on a subsecond timescale under anaesthesia. In freely moving conscious rats, this excitatory stimulation rapidly elicited exploration-like behaviours, predominantly locomotor activity, along with pressor and tachycardiac responses. Meanwhile, optogenetic inhibition of orexinergic neurons during spontaneous wheel running immediately suppressed locomotor activities and blood pressure elevation without affecting basal cardiovascular homeostasis. Taken together, these findings demonstrate the essential role of the orexinergic nervous system in the central circuitry that transmits central command signals for locomotor exercise. This study not only offers insights into the brain circuit mechanisms precisely regulating autonomic cardiovascular systems during voluntary exercise but also likely contributes to our understanding of brain mechanisms underlying abnormal cardiovascular adjustments to exercise in pathological conditions, such as hypertension. KEY POINTS: The hypothalamic orexinergic nervous system plays various roles in the long-term regulation of autonomic and endocrine functions, as well as motivated behaviours. We present a novel, rapid role of the orexinergic nervous system, revealing its significance as a crucial substrate in the brain circuit mechanisms that coordinate somatomotor and autonomic cardiovascular controls for locomotor exercise. Our data demonstrate that orexinergic neurons relay volitional motor signals, playing a necessary and sufficient role in the autonomic cardiovascular regulation required for locomotor exercise in rats. The findings contribute to our understanding of how the brain precisely regulates autonomic cardiovascular systems during voluntary exercise, providing insights into the central neural mechanisms that enhance physical performance moment-by-moment during exercise.

Engineered red Opto-mGluR6 Opsins, a red-shifted optogenetic excitation tool, an in vitro study.

Degenerative eye diseases cause partial or complete blindness due to photoreceptor degeneration. Optogenetic gene therapy is a revolutionary technique combining genetics and optical methods to control the function of neurons. Due to the inherent risk of photochemical damage, the light intensity necessary to activate Opto-mGluR6 surpasses the safe threshold for retinal illumination. Conversely, red-shifted lights pose a significantly lower risk of inducing such damage compared to blue lights. We designed red-shifted Opto-mGluR6 photopigments with a wide, red-shifted working spectrum compared to Opto-mGluR6 and examined their excitation capability in vitro. ROM19, ROM18 and ROM17, red-shifted variants of Opto-mGluR6, were designed by careful bioinformatics/computational studies. The predicted molecules with the best scores were selected, synthesised and cloned into the pAAV-CMV-IRES-EGFP vector. Expression of constructs was confirmed by functional assessment in engineered HEK-GIRK cells. Spectrophotometry and patch clamp experiments demonstrated that the candidate molecules were sensitive to the desired wavelengths of the light and directly coupled light stimuli to G-protein signalling. Herein, we introduce ROM17, ROM18 and ROM19 as newly generated, red-shifted variants with maximum excitation red-shifted of ~ 40nm, 70 nm and 126 nm compared to Opto-mGluR6.

Characterization of Anxiety-Like Behaviors and Neural Circuitry following Chronic Moderate Noise Exposure in Mice.

Commonly encountered nontraumatic, moderate noise is increasingly implicated in anxiety; however, the neural substrates underlying this process remain unclear. We investigated the neural circuit mechanism through which chronic exposure to moderate-level noise causes anxiety-like behaviors. Mice were exposed to chronic, moderate white noise [85 decibel (dB) sound pressure level (SPL)], 4 h/d for 4 wk to induce anxiety-like behaviors, which were assessed by open field, elevated plus maze, light-dark box, and social interaction tests. Viral tracing, immunofluorescence confocal imaging, and brain slice patch-clamp recordings were used to characterize projections from auditory brain regions to the lateral amygdala. Neuronal activities were characterized by invivo multielectrode and fiber photometry recordings in awake mice. Optogenetics and chemogenetics were used to manipulate specific neural circuitry. Mice chronically (4 wk) exposed to moderate noise (85 dB SPL, 4 h/d) demonstrated greater neuronal activity in the lateral amygdala (LA), and the LA played a critical role in noise-induced anxiety-like behavior in these model mice. Viral tracing showed that the LA received monosynaptic projections from the medial geniculate body (MG) and auditory cortex (ACx). Optogenetic excitation of the or circuits acutely evoked anxiety-like behaviors, whereas their chemogenetic inactivation abolished noise-induced anxiety-like behavior. Moreover, mice chronically exposed to moderate noise were more susceptible to acute stress, with more neuronal firing in the LA, even after noise withdrawal. Mice exposed to 4 wk of moderate noise (85 dB SPL, 4 h/d) demonstrated behavioral and physiological differences compared to controls. The neural circuit mechanisms involved greater excitation from glutamatergic neurons of the MG and ACx to LA neurons under chronic, moderate noise exposure, which ultimately promoted anxiety-like behaviors. Our findings support the hypothesis that nontraumatic noise pollution is a potentially serious but unrecognized public health concern. https://doi.org/10.1289/EHP12532.

Purkinje cell microzones mediate distinct kinematics of a single movement

The classification of neuronal subpopulations has significantly advanced, yet its relevance for behavior remains unclear. The highly organized flocculus of the cerebellum, known to fine-tune multi-axial eye movements, is an ideal substrate for the study of potential functions of neuronal subpopulations. Here, we demonstrate that its recently identified subpopulations of 9+ and 9- Purkinje cells exhibit an intermediate Aldolase C expression and electrophysiological profile, providing evidence for a graded continuum of intrinsic properties among PC subpopulations. By identifying and utilizing two Cre-lines that genetically target these floccular domains, we show with high spatial specificity that these subpopulations of Purkinje cells participate in separate micromodules with topographically organized connections. Finally, optogenetic excitation of the respective subpopulations results in movements around the same axis in space, yet with distinct kinematic profiles. These results indicate that Purkinje cell subpopulations integrate in discrete circuits and mediate particular parameters of single movements.

Basal forebrain parvalbumin neurons modulate vigilant attention and rescue deficits produced by sleep deprivation.

Attention is impaired in many neuropsychiatric disorders, as well as by sleep disruption, leading to decreased workplace productivity and increased risk of accidents. Thus, understanding the neural substrates is important. Here we test the hypothesis that basal forebrain neurons that contain the calcium-binding protein parvalbumin modulate vigilant attention in mice. Furthermore, we test whether increasing the activity of basal forebrain parvalbumin neurons can rescue the deleterious effects of sleep deprivation on vigilance. A lever release version of the rodent psychomotor vigilance test was used to assess vigilant attention. Brief and continuous low-power optogenetic excitation (1 s, 473 nm @ 5 mW) or inhibition (1 s, 530 nm @ 10 mW) of basal forebrain parvalbumin neurons was used to test the effect on attention, as measured by reaction time, under control conditions and following 8 hr of sleep deprivation by gentle handling. Optogenetic excitation of basal forebrain parvalbumin neurons that preceded the cue light signal by 0.5 s improved vigilant attention as indicated by quicker reaction times. By contrast, both sleep deprivation and optogenetic inhibition slowed reaction times. Importantly, basal forebrain parvalbumin excitation rescued the reaction time deficits in sleep-deprived mice. Control experiments using a progressive ratio operant task confirmed that optogenetic manipulation of basal forebrain parvalbumin neurons did not alter motivation. These findings reveal for the first time a role for basal forebrain parvalbumin neurons in attention, and show that increasing their activity can compensate for disruptive effects of sleep deprivation.

Optogenetic Generation of Neural Firing Patterns with Temporal Shaping of Light Pulses

The fundamental process of information processing and memory formation in the brain is associated with complex neuron firing patterns, which can occur spontaneously or be triggered by sensory inputs. Optogenetics has revolutionized neuroscience by enabling precise manipulation of neuronal activity patterns in specified neural populations using light. However, the light pulses used in optogenetics have been primarily restricted to square waveforms. Here, we present a detailed theoretical analysis of the temporal shaping of light pulses in optogenetic excitation of hippocampal neurons and neocortical fast-spiking interneurons expressed with ultrafast (Chronos), fast (ChR2), and slow (ChRmine) channelrhodopsins. Optogenetic excitation has been studied with light pulses of different temporal shapes that include square, forward-/backward ramps, triangular, left-/right-triangular, Gaussian, left-/right-Gaussian, positive-sinusoidal, and left-/right-positive sinusoidal. Different light shapes result in significantly different photocurrent amplitudes and kinetics, spike-timing, and spontaneous firing rate. For short duration stimulations, left-Gaussian pulse results in larger photocurrent in ChR2 and Chronos than square pulse of the same energy density. Time to peak photocurrent in each opsin is minimum at right-Gaussian pulse. The optimal pulse width to achieve peak photocurrent for non-square pulses is 10 ms for Chronos, and 50 ms for ChR2 and ChRmine. The pulse energy to evoke spike in hippocampal neurons can be minimized on choosing square pulse with Chronos, Gaussian pulse with ChR2, and positive-sinusoidal pulse with ChRmine. The results demonstrate that non-square waveforms generate more naturalistic spiking patterns compared to traditional square pulses. These findings provide valuable insights for the development of new optogenetic strategies to better simulate and manipulate neural activity patterns in the brain, with the potential to improve our understanding of cognitive processes and the treatment of neurological disorders.

Structure and Function of Neuronal Circuits Linking Ventrolateral Preoptic Nucleus and Lateral Hypothalamic Area.

To understand how sleep-wakefulness cycles are regulated, it is essential to disentangle structural and functional relationships between the preoptic area (POA) and lateral hypothalamic area (LHA), since these regions play important yet opposing roles in the sleep-wakefulness regulation. GABA- and galanin (GAL)-producing neurons in the ventrolateral preoptic nucleus (VLPO) of the POA (VLPOGABA and VLPOGAL neurons) are responsible for the maintenance of sleep, while the LHA contains orexin-producing neurons (orexin neurons) that are crucial for maintenance of wakefulness. Through the use of rabies virus-mediated neural tracing combined with in situ hybridization (ISH) in male and female orexin-iCre mice, we revealed that the vesicular GABA transporter (Vgat, Slc32a1)- and galanin (Gal)-expressing neurons in the VLPO directly synapse with orexin neurons in the LHA. A majority (56.3 ± 8.1%) of all VLPO input neurons connecting to orexin neurons were double-positive for Vgat and Gal Using projection-specific rabies virus-mediated tracing in male and female Vgat-ires-Cre and Gal-Cre mice, we discovered that VLPOGABA and VLPOGAL neurons that send projections to the LHA received innervations from similarly distributed input neurons in many brain regions, with the POA and LHA being among the main upstream areas. Additionally, we found that acute optogenetic excitation of axons of VLPOGABA neurons, but not VLPOGAL neurons, in the LHA of male Vgat-ires-Cre mice induced wakefulness. This study deciphers the connectivity between the VLPO and LHA, provides a large-scale map of upstream neuronal populations of VLPO→LHA neurons, and reveals a previously uncovered function of the VLPOGABA→LHA pathway in the regulation of sleep and wakefulness.SIGNIFICANCE STATEMENT We identified neurons in the ventrolateral preoptic nucleus (VLPO) that are positive for vesicular GABA transporter (Vgat) and/or galanin (Gal) and serve as presynaptic partners of orexin-producing neurons in the lateral hypothalamic area (LHA). We depicted monosynaptic input neurons of GABA- and galanin-producing neurons in the VLPO that send projections to the LHA throughout the entire brain. Their input neurons largely overlap, suggesting that they comprise a common neuronal population. However, acute excitatory optogenetic manipulation of the VLPOGABA→LHA pathway, but not the VLPOGAL→LHA pathway, evoked wakefulness. This study shows the connectivity of major components of the sleep/wake circuitry in the hypothalamus and unveils a previously unrecognized function of the VLPOGABA→LHA pathway in sleep-wakefulness regulation. Furthermore, we suggest the existence of subpopulations of VLPOGABA neurons that innervate LHA.

The Orienting Reflex Reveals Behavioral States Set by Demanding Contexts: Role of the Superior Colliculus.

Sensory stimuli can trigger an orienting reflex (response) by which animals move the head to position their sensors (e.g., eyes, pinna, whiskers). Orienting responses may be important to evaluate stimuli that call for action (e.g., approach, escape, ignore), but little is known about the dynamics of orienting responses in the context of goal-directed actions. Using mice of either sex, we found that, during a signaled avoidance action, the orienting response evoked by the conditioned stimulus (CS) consisted of a fast head movement containing rotational and translational components that varied substantially as a function of the behavioral and underlying brain states of the animal set by different task contingencies. Larger CS-evoked orienting responses were associated with high-intensity auditory stimuli, failures to produce the appropriate signaled action, and behavioral states resulting from uncertain or demanding situations and the animal's ability to cope with them. As a prototypical orienting neural circuit, we confirmed that the superior colliculus controls and codes the direction of spontaneous exploratory orienting movements. In addition, superior colliculus activity correlated with CS-evoked orienting responses, and either its optogenetic inhibition or excitation potentiated CS-evoked orienting responses, which are likely generated downstream in the medulla. CS-evoked orienting responses may be a useful probe to assess behavioral and related brain states, and state-dependent modulation of orienting responses may involve the superior colliculus.SIGNIFICANCE STATEMENT Humans and other animals produce an orienting reflex (also known as orienting response) by which they rapidly orient their head and sensors to evaluate novel or salient stimuli. Spontaneous orienting movements also occur during exploration of the environment in the absence of explicit, salient stimuli. We monitored stimulus-evoked orienting responses in mice performing signaled avoidance behaviors and found that these responses reflect the behavioral state of the animal set by contextual demands and the animal's ability to cope with them. Various experiments involving the superior colliculus revealed a well-established role in spontaneous orienting but only an influencing effect over orienting responses. Stimulus-evoked orienting responses may be a useful probe of behavioral and related brain states.

Hemodynamic transient and functional connectivity follow structural connectivity and cell type over the brain hierarchy

The neural circuit of the brain is organized as a hierarchy of functional units with wide-ranging connections that support information flow and functional connectivity. Studies using MRI indicate a moderate coupling between structural and functional connectivity at the system level. However, how do connections of different directions (feedforward and feedback) and regions with different excitatory and inhibitory (E/I) neurons shape the hemodynamic activity and functional connectivity over the hierarchy are unknown. Here, we used functional MRI to detect optogenetic-evoked and resting-state activities over a somatosensory pathway in the mouse brain in relation to axonal projection and E/I distribution. Using a highly sensitive ultrafast imaging, we identified extensive activation in regions up to the third order of axonal projections following optogenetic excitation of the ventral posteriomedial nucleus of the thalamus. The evoked response and functional connectivity correlated with feedforward projections more than feedback projections and weakened with the hierarchy. The hemodynamic response exhibited regional and hierarchical differences, with slower and more variable responses in high-order areas and bipolar response predominantly in the contralateral cortex. Electrophysiological recordings suggest that these reflect differences in neural activity rather than neurovascular coupling. Importantly, the positive and negative parts of the hemodynamic response correlated with E/I neuronal densities, respectively. Furthermore, resting-state functional connectivity was more associated with E/I distribution, whereas stimulus-evoked effective connectivity followed structural wiring. These findings indicate that the structure-function relationship is projection-, cell-type- and hierarchy-dependent. Hemodynamic transients could reflect E/I activity and the increased complexity of hierarchical processing.

A neural circuit from the dorsal CA3 to the dorsomedial hypothalamus mediates balance between risk exploration and defense.

An appropriate balance between explorative and defensive behavior is essential for the survival and reproduction of prey animals in risky environments. However, the neural circuit and mechanism that allow for such a balance remains poorly understood. Here, we use a semi-naturalistic predator threat test (PTT) to observe and quantify the defense-exploration balance, especially risk exploration behavior in mice. During the PTT, the activity of the putative dorsal CA3 glutamatergic neurons (dCA3Glu) is suppressed by predatory threat and risk exploration, whereas the neurons are activated during contextual exploration. Moreover, optogenetic excitation of these neurons induces a significant increase in risk exploration. A circuit, comprising the dorsal CA3, dorsal lateral septal, and dorsomedial hypothalamic (dCA3Glu-dLSGABA-DMH) areas, may be involved. Moreover, activation of the dCA3Glu-dLSGABA-DMH circuit promotes the switch from defense to risk exploration and suppresses threat-induced increase in arousal.