Phasic Firing Research Articles

Dopamine errors drive excitatory and inhibitory components of backward conditioning in an outcome-specific manner

For over two decades, phasic activity in midbrain dopamine neurons was considered synonymous with the prediction error in temporal-difference reinforcement learning.1-4 Central to this proposal is the notion that reward-predictive stimuli become endowed with the scalar value of predicted rewards. When these cues are subsequently encountered, their predictive value is compared to the value of the actual reward received, allowing for the calculation of prediction errors.5,6 Phasic firing of dopamine neurons was proposed to reflect this computation,1,2 facilitating the backpropagation of value from the predicted reward to the reward-predictive stimulus, thus reducing future prediction errors. There are two critical assumptions of this proposal: (1) that dopamine errors can only facilitate learning about scalar value and not more complex features of predicted rewards, and (2) that the dopamine signal can only be involved in anticipatory cue-reward learning in which cues or actions precede rewards. Recent work7-15 has challenged the first assumption, demonstrating that phasic dopamine signals across species are involved in learning about more complex features of the predicted outcomes, in a manner that transcends this value computation. Here, we tested the validity of the second assumption. Specifically, we examined whether phasic midbrain dopamine activity would be necessary for backward conditioning-when a neutral cue reliably follows a rewarding outcome.16-20 Using a specific Pavlovian-to-instrumental transfer (PIT) procedure,21-23 we show rats learn both excitatory and inhibitory components of a backward association, and that this association entails knowledge of the specific identity of the reward and cue. We demonstrate that brief optogenetic inhibition of VTADA neurons timed to the transition between the reward and cue reduces both of these components of backward conditioning. These findings suggest VTADA neurons are capable of facilitating associations between contiguously occurring events, regardless of the content of those events. We conclude that these data may be in line with suggestions that the VTADA error acts as a universal teaching signal. This may provide insight into why dopamine function has been implicated in myriad psychological disorders that are characterized by very distinct reinforcement-learning deficits.

Targeted ubiquitination of sensory neuron calcium channels reduces the development of neuropathic pain

Neuropathic pain caused by lesions to somatosensory neurons due to injury or disease is a widespread public health problem that is inadequately managed by small-molecule therapeutics due to incomplete pain relief and devastating side effects. Genetically encoded molecules capable of interrupting nociception have the potential to confer long-lasting analgesia with minimal off-target effects. Here, we utilize a targeted ubiquitination approach to achieve a unique posttranslational functional knockdown of high-voltage-activated calcium channels (HVACCs) that are obligatory for neurotransmission in dorsal root ganglion (DRG) neurons. CaV-aβlator comprises a nanobody targeted to CaV channel cytosolic auxiliary β subunits fused to the catalytic HECT domain of the Nedd4-2 E3 ubiquitin ligase. Subcutaneous injection of adeno-associated virus serotype 9 encoding CaV-aβlator in the hind paw of mice resulted in the expression of the protein in a subset of DRG neurons that displayed a concomitant ablation of CaV currents and also led to an increase in the frequency of spontaneous inhibitory postsynaptic currents in the dorsal horn of the spinal cord. Mice subjected to spare nerve injury displayed a characteristic long-lasting mechanical, thermal, and cold hyperalgesia underlain by a dramatic increase in coordinated phasic firing of DRG neurons as reported by in vivo Ca2+ spike recordings. CaV-aβlator significantly dampened the integrated Ca2+ spike activity and the hyperalgesia in response to nerve injury. The results advance the principle of targeting HVACCs as a gene therapy for neuropathic pain and demonstrate the therapeutic potential of posttranslational functional knockdown of ion channels achieved by exploiting the ubiquitin-proteasome system.

P532. Smoking as a Common Modulator of Sensory Gating and Reward Learning in Individuals with Psychotic Disorders

Motivational and perceptual disturbances co-occur in psychosis and have been linked to aberra-tions in reward learning and sensory gating, respectively. Although traditionally studied inde-pendently, when viewed through a predictive coding framework these processes can both be linked to dysfunction in striatal dopaminergic prediction error signaling. This study examined whether reward learning and sensory gating are correlated in individuals with psychotic disor-ders, and whether nicotine – a psychostimulant that amplifies phasic striatal dopamine firing – is a common modulator of these two processes.

Gq neuromodulation of BLA parvalbumin interneurons induces burst firing and mediates fear-associated network and behavioral state transition in mice

Patterned coordination of network activity in the basolateral amygdala (BLA) is important for fear expression. Neuromodulatory systems play an essential role in regulating changes between behavioral states, however the mechanisms underlying this neuromodulatory control of transitions between brain and behavioral states remain largely unknown. We show that chemogenetic Gq activation and α1 adrenoreceptor activation in mouse BLA parvalbumin (PV) interneurons induces a previously undescribed, stereotyped phasic bursting in PV neurons and time-locked synchronized bursts of inhibitory postsynaptic currents and phasic firing in BLA principal neurons. This Gq-coupled receptor activation in PV neurons suppresses gamma oscillations in vivo and in an ex vivo slice model, and facilitates fear memory recall, which is consistent with BLA gamma suppression during conditioned fear expression. Thus, here we identify a neuromodulatory mechanism in PV inhibitory interneurons of the BLA which regulates BLA network oscillations and fear memory recall.

Phasic firing of dopaminergic neurons in the ventral tegmental area triggers peripheral immune responses

Dopaminergic neurons in the ventral tegmental area (VTA) play a crucial role in the processing of reward-related information. Recent studies with pharmacological manipulations of VTA neuronal activity demonstrated a VTA-induced immunoenhancement in peripheral organs. Here, to examine the detailed physiological dynamics, we took an optogenetic approach in which VTA dopaminergic neurons were selectively activated with millisecond precision. Optogenetic phasic, rather than tonic, stimulation of VTA dopaminergic neurons increased serum cytokine levels, such as IL-2, IL-4 and TNF-α. These results provide direct evidence to link dopaminergic neuronal phasic firing to peripheral immunity. Next, we tested whether cytokine induction in male mice was boosted by female encounters, a natural condition that induces increased active VTA neurons and gamma power. Female encounters increased serum IL-2 levels, which were abolished by pharmacological inhibition of VTA neuronal activity. Taken together, our results highlight the importance of the brain reward system in the treatment and management of immune-related disorders.

Pupillometry and the vigilance decrement: Task-evoked but not baseline pupil measures reflect declining performance in visual vigilance tasks.

Baseline and task‐evoked pupil measures are known to reflect the activity of the nervous system's central arousal mechanisms. With the increasing availability, affordability and flexibility of video‐based eye tracking hardware, these measures may one day find practical application in real‐time biobehavioural monitoring systems to assess performance or fitness for duty in tasks requiring vigilant attention. But real‐world vigilance tasks are predominantly visual in their nature and most research in this area has taken place in the auditory domain. Here, we explore the relationship between pupil size—both baseline and task‐evoked—and behavioural performance measures in two novel vigilance tasks requiring visual target detection: (1) a traditional vigilance task involving prolonged, continuous and uninterrupted performance (n = 28) and (2) a psychomotor vigilance task (n = 25). In both tasks, behavioural performance and task‐evoked pupil responses declined as time spent on task increased, corroborating previous reports in the literature of a vigilance decrement with a corresponding reduction in task‐evoked pupil measures. Also in line with previous findings, baseline pupil size did not show a consistent relationship with performance measures. Our data offer novel insights into the complex interplay of brain systems involved in vigilant attention and question the validity of the assumption that baseline (prestimulus) pupil size and task‐evoked (poststimulus) pupil measures reflect the tonic and phasic firing modes of the locus coeruleus.

Smoking as a Common Modulator of Sensory Gating and Reward Learning in Individuals with Psychotic Disorders.

Motivational and perceptual disturbances co-occur in psychosis and have been linked to aberrations in reward learning and sensory gating, respectively. Although traditionally studied independently, when viewed through a predictive coding framework, these processes can both be linked to dysfunction in striatal dopaminergic prediction error signaling. This study examined whether reward learning and sensory gating are correlated in individuals with psychotic disorders, and whether nicotine—a psychostimulant that amplifies phasic striatal dopamine firing—is a common modulator of these two processes. We recruited 183 patients with psychotic disorders (79 schizophrenia, 104 psychotic bipolar disorder) and 129 controls and assessed reward learning (behavioral probabilistic reward task), sensory gating (P50 event-related potential), and smoking history. Reward learning and sensory gating were correlated across the sample. Smoking influenced reward learning and sensory gating in both patient groups; however, the effects were in opposite directions. Specifically, smoking was associated with improved performance in individuals with schizophrenia but impaired performance in individuals with psychotic bipolar disorder. These findings suggest that reward learning and sensory gating are linked and modulated by smoking. However, disorder-specific associations with smoking suggest that nicotine may expose pathophysiological differences in the architecture and function of prediction error circuitry in these overlapping yet distinct psychotic disorders.

Mechanism and function of phasic firing in vasopressin-releasing magnocellular neurosecretory cells.

Magnocellular neurosecretory cells that release vasopressin (MNCVP ) from axon terminals in the neurohypophysis display a unique pattern of action potential firing termed phasic firing. Under basal conditions, only a small proportion of MNCVP display spontaneous phasic firing. However, acute and chronic conditions that stimulate vasopressin release, such as hemorrhage and dehydration, greatly enhance the number of MNCVP that fire phasically. Phasic firing optimizes VP neurosecretion at axon terminals by allowing action potential broadening to promote calcium-dependent frequency-facilitation, at the same time as preventing the secretory fatigue caused by spike inactivation that occurs during prolonged continuous stimulation. This review provides an update on our mechanistic understanding of these processes and highlights important gaps in our knowledge that must be addressed in future experiments.

Dopaminergic Projection from Ventral Tegmental Area to Substantia Nigra Pars Reticulata Mediates Chronic Social Defeat Stress-Induced Hypolocomotion.

Numerous human clinical studies have suggested that decreased locomotor activity is a common symptom of major depressive disorder (MDD), as well as other psychiatric diseases. In MDD, the midbrain ventral tegmental area (VTA) dopamine (DA) neurons are closely related to regulate the information processing of reward, motivation, cognition, and aversion. However, the neural circuit mechanism that underlie the relationship between VTA-DA neurons and MDD-related motor impairments, especially hypolocomotion, is still largely unknown. Herein, we investigate how the VTA-DA neurons contribute to the hypolocomotion performance in chronic social defeat stress (CSDS), a mouse model of depression-relevant neurobehavioral states. The results show that CSDS could affect the spontaneous locomotor activity of mice, but not the grip strength and forced locomotor ability. Chemogenetic activation of VTA-DA neurons alleviated CSDS-induced hypolocomotion. Subsequently, quantitative whole-brain mapping revealed decreased projections from VTA-DA neurons to substantia nigra pars reticulata (SNr) after CSDS treatment. Optogenetic activation of dopaminergic projection from VTA to SNr with the stimulation of phasic firing, but not tonic firing, could significantly increase the locomotor activity of mice. Moreover, chemogenetic activation of VTA-SNr dopaminergic circuit in CSDS mice could also rescued the decline of locomotor activity. Taken together, our data suggest that the VTA-SNr dopaminergic projection mediates CSDS-induced hypolocomotion, which provides a theoretical basis and potential therapeutic target for MDD.

Time‐of‐Day Variation in Learning, Reward‐Associated Behaviors, and Rapid Dopamine Release

Research has shown that circadian rhythms in physiology and behavior play a role in a number of psychiatric disorders, like depression and substance use disorder (SUD). For example, time-of-day variation has been observed in craving for nicotine, heroin, and cocaine in abstinent individuals. However, we have yet to fully delineate how these rhythms mediate variations in motivated behavior, learning, and reward seeking. This is critical as aberrant motivation and learning are hallmarks in these disorders. Additionally, the mesolimbic dopamine (DA) system is implicated in the neurobiology of motivation and learning, and research has shown time-of-day variations in DA tone. Less work has focused on rapid DA signals that are critical for motivation and learning. Thus, the purpose of this study was to examine time-of-day variations in incentive motivational properties of reward-associated cues and learning and the degree to which the magnitude of mesolimbic DA release in the nucleus accumbens (NAc) corresponded to dynamic shifts in motivation and learning across time-of-day. Our central hypothesis is that there are critical times within the day that confer increased sensitivity to reward-associated cues, which are mediated by time-of-day variations in the magnitude of phasic DA signaling. Initially, we examined the magnitude of NAc DA signaling to stimulations that mimic both tonic and phasic firing of mesolimbic DA neurons in rats. We used ex vivo fast scan cyclic voltammetry (FSCV) at four different time points across the 12:12 light/dark cycle. Our data demonstrate robust time-of-day differences in DA release magnitude following stimulations that model both phasic (≥20 Hz) and tonic (5 Hz) firing. Given that the phasic/tonic ratio of DA release magnitude was greatest midway through the dark cycle and lowest midway through the light cycle, we examined whether behavioral paradigms sensitive to DA release magnitude would fluctuate consistently with these DA oscillations. We therefore utilized Pavlovian paradigms in rodents to measure incentive motivational aspects and conditioned approach towards a reward-associated cue, during midway through the light cycle and midway through the dark cycle. Furthermore, since DA release and motivated behavior is modulated by striatal cholinergic interneuron (CIN) activity via nicotinic receptors (nAChRs) located on DA axons, we examined whether both DA release and incentive motivation was altered following nicotine exposure. To unmask the circuitry underlying these variations, we utilized ex vivo optogentics. We found a robust increase in DA release magnitude without the contribution of CIN activity midway through the dark cycle. In contrast, DA release was significantly higher in DA release magnitude modulated by CIN activity midway through the light cycle. Lastly, we are currently using in vivo optogenetics to mediate the magnitude of DA release during Pavlovian paradigms at times throughout the light cycles. Collectively, our data suggest that rats midway through their dark cycle exhibit increased sensitivity to reward-associated cues, which may be mediated by rapid DA release that is modulated by oscillations in CIN activity throughout the day.

Fast A-type currents shape a rapidly adapting form of delayed short latency firing of excitatory superficial dorsal horn neurons that express the neuropeptide Y Y1 receptor.

Neuropeptide Y Y1 receptor-expressing neurons in the dorsal horn of the spinal cord contribute to chronic pain. For the first time, we characterized the firing patterns of Y1-expressing neurons in Y1eGFP reporter mice. Under hyperpolarized conditions, most Y1eGFP neurons exhibited fast A-type potassium currents and delayed, short-latency firing (DSLF). Y1eGFP DSLF neurons were almost always rapidly adapting and often exhibited rebound spiking, characteristics of spinal pain neurons under the control of T-type calcium channels. These results will inspire future studies to determine whether tissue or nerve injury downregulates the channels that underlie A-currents, thus unmasking membrane hyperexcitability in Y1-expressing dorsal horn neurons, leading to persistent pain. Neuroanatomical and behavioural evidence indicates that neuropeptide Y Y1 receptor-expressing interneurons (Y1-INs) in the superficial dorsal horn (SDH) are predominantly excitatory and contribute to chronic pain. Using an adult ex vivo spinal cord slice preparation from Y1eGFP reporter mice, we characterized firing patterns in response to steady state depolarizing current injection of GFP-positive cells in lamina II, the great majority of which expressed Y1 mRNA (88%). Randomly sampled (RS) and Y1eGFP neurons exhibited five firing patterns: tonic, initial burst, phasic, delayed short-latency <180ms (DSLF) and delayed long-latency >180ms (DLLF). When studied at resting membrane potential, most RS neurons exhibited delayed firing, while most Y1eGFP neurons exhibited phasic firing. A preconditioning membrane hyperpolarization produced only subtle changes in the firing patterns of RS neurons, but dramatically shifted Y1eGFP neurons to DSLF (46%) and DLLF (24%). In contrast to RS DSLF neurons, which rarely exhibited spike frequency adaptation, Y1eGFP DSLF neurons were almost always rapidly adapting, a characteristic of nociceptive-responsive SDH neurons. Rebound spiking was more prevalent in Y1eGFP neurons (6% RS vs. 32% Y1eGFP), indicating enrichment of T-type calcium currents. Y1eGFP DSLF neurons exhibited fast A-type potassium currents that are known to delay or limit action potential firing and exhibited smaller current density as compared to RS DSLF neurons. Our results will inspire future studies to determine whether tissue or nerve injury downregulates channels that contribute to A-currents, thus potentially unmasking T-type calcium channel activity and membrane hyperexcitability in Y1-INs, leading to persistent pain.

Locus Coeruleus Activation Patterns Differentially Modulate Odor Discrimination Learning and Odor Valence in Rats.

ABSTRACTThe locus coeruleus (LC) produces phasic and tonic firing patterns that are theorized to have distinct functional consequences. However, how different firing modes affect learning and valence encoding of sensory information are unknown. Here, we show bilateral optogenetic activation of rat LC neurons using 10-Hz phasic trains of either 300 ms or 10 s accelerated acquisition of a similar odor discrimination. Similar odor discrimination learning was impaired by noradrenergic blockade in the piriform cortex (PC). However, 10-Hz phasic light-mediated learning facilitation was prevented by a dopaminergic antagonist in the PC, or by ventral tegmental area (VTA) silencing with lidocaine, suggesting a LC–VTA–PC dopamine circuitry involvement. Ten-hertz tonic stimulation did not alter odor discrimination acquisition, and was ineffective in activating VTA DA neurons. For valence encoding, tonic stimulation at 25 Hz induced conditioned odor aversion, whereas 10-Hz phasic stimulations produced an odor preference. Both conditionings were prevented by noradrenergic blockade in the basolateral amygdala (BLA). Cholera Toxin B retro-labeling showed larger engagement of nucleus accumbens-projecting neurons in the BLA with 10-Hz phasic activation, and larger engagement of central amygdala projecting cells with 25-Hz tonic light. These outcomes argue that the LC activation patterns differentially influence both target networks and behavior.

Neuromodulatory effects of GnRH on the caudal neurosecretory Dahlgren cells in female olive flounder

Gonadotropin-releasing hormone (GnRH) is considered a key player in reproduction. The caudal neurosecretory system (CNSS) is a unique neurosecretory structure of fish that may be involved in osmoregulation, nutrition, reproduction, and stress-related responses. However, a direct effect of GnRH on Dahlgren cells remains underexplored. Here, we examined the electrophysiological response of Dahlgren cell population of the CNSS to GnRH analog LHRH-A2 and the transcription of related key genes of CNSS. We found that GnRH increased overall firing frequency and may be changed the firing pattern from silent to burst or phasic firing in a subpopulation of Dahlgren cells. The effect of GnRH on a subpopulation of Dahlgren cells firing activity was blocked by the GnRH receptor (GnRH-R) antagonist cetrorelix. A positive correlation was observed between the UII and GnRH-R mRNA levels in CNSS or gonadosomatic index (GSI) during the breeding season. These findings are the first demonstration of the ability of GnRH acts as a modulator within the CNSS and add to our understanding of the physiological role of the CNSS in reproduction and seasonal adaptation.

Phasic Neuronal Firing in the Rodent Nucleus of the Solitary Tract ex vivo.

Phasic pattern of neuronal activity has been previously described in detail for magnocellular vasopressin neurons in the hypothalamic paraventricular and supraoptic nuclei. This characteristic bistable pattern consists of alternating periods of electrical silence and elevated neuronal firing, implicated in neuropeptide release. Here, with the use of multi-electrode array recordings ex vivo, we aimed to study the firing pattern of neurons in the nucleus of the solitary tract (NTS) – the brainstem hub for homeostatic, cardio-vascular, and metabolic processes. Our recordings from the mouse and rat hindbrain slices reveal the phasic activity pattern to be displayed by a subset of neurons in the dorsomedial NTS subjacent to the area postrema (AP), with the inter-spike interval distribution closely resembling that reported for phasic magnocellular vasopressin cells. Additionally, we provide interspecies comparison, showing higher phasic frequency and firing rate of phasic NTS cells in mice compared to rats. Further, we describe daily changes in their firing rate and pattern, peaking at the middle of the night. Last, we reveal these phasic cells to be sensitive to α2 adrenergic receptors activation and to respond to electrical stimulation of the AP. This study provides a comprehensive description of the phasic neuronal activity in the rodent NTS and identifies it as a potential downstream target of the AP noradrenergic system.

Suppression of delayed rectifier K+ channels by gentamicin induces membrane hyperexcitability through JNK and PKA signaling pathways in vestibular ganglion neurons

Aminoglycoside antibiotics, such as gentamicin, are known to have vestibulotoxic effects, including ataxia and disequilibrium. To date, however, the underlying cellular and molecular mechanisms are still unclear. In this study, we determined the role of gentamicin in regulating the sustained delayed rectifier K+ current (IDR) and membrane excitability in vestibular ganglion (VG) neurons in mice. Our results showed that the application of gentamicin to VG neurons decreased the IDR in a concentration-dependent manner, while the transient outward A-type K+ current (IA) remained unaffected. The decrease in IDR induced by gentamicin was independent of G-protein activity and led to a hyperpolarizing shift of the inactivation Vhalf. The analysis of phospho-c-Jun N-terminal kinase (p-JNK) revealed that gentamicin significantly stimulated JNK, while p-ERK and p-p38 remained unaffected. Blocking Kv1 channels with α-dendrotoxin or pretreating VG neurons with the JNK inhibitor II abrogated the gentamicin-induced decrease in IDR. Antagonism of JNK signaling attenuated the gentamicin-induced stimulation of PKA activity, whereas PKA inhibition prevented the IDR response induced by gentamicin. Moreover, gentamicin significantly increased the number of action potentials fired in both phasic and tonic firing type neurons; pretreating VG neurons with the JNK inhibitor II and the blockade of the IDR abolished this effect. Taken together, our results demonstrate that gentamicin decreases the IDR through a G-protein-independent but JNK and PKA-mediated signaling pathways. This gentamicin-induced IDR response mediates VG neuronal hyperexcitability and might contribute to its pharmacological vestibular effects.

Intrinsic properties of avian interaural level difference sound localizing neurons

Intrinsic properties of neurons are one major determinant for how neurons respond to their synaptic inputs and shape their outputs in neural circuits. Here, we studied the intrinsic properties of neurons in the chicken posterior portion of the dorsal nucleus of the lateral lemniscus (LLDp), the first interaural level difference (ILD) encoder of the avian auditory pathway. Using whole-cell recordings in brain slices, we revealed that the LLDp is composed of a heterogeneous neuron population based on their firing patterns. LLDp neurons were broadly classified as either phasic or tonic firing neurons, with further classification applied to tonic firing neurons, such as regular (most dominant, n = 82 out of 125 cells, 65.6%), pauser, or adaptive firing. Neurons with different firing patterns were distributed about evenly across the dorsoventral as well as mediolateral axis of LLDp. Phasic firing neurons were of faster membrane time constant, and lower excitability than tonic firing neurons. The action potentials (APs) elicited at the current thresholds displayed significant differences in first spike latency, AP peak amplitude, half-width, and maximal rising and falling rates. Interestingly, for APs elicited at suprathreshold currents (400 pA above thresholds), some of the differences diminished while a few others emerged. Remarkably, most parameters of the APs at thresholds were significantly different from those of APs at suprathresholds. Combined with our previous study (Curry and Lu, 2016), the results lend support to the two-cell type model for ILD coding in the avian system.

Locus coeruleus in memory formation and Alzheimer's disease.

Catecholamine neurons of the locus coeruleus (LC) in the dorsal pontine tegmentum innervate the entire neuroaxis, with signaling actions implicated in the regulation of attention, arousal, sleep–wake cycle, learning, memory, anxiety, pain, mood, and brain metabolism. The co‐release of norepinephrine (NE) and dopamine (DA) from LC terminals in the hippocampus plays a role in all stages of hippocampal‐memory processing. This catecholaminergic regulation modulates the encoding, consolidation, retrieval, and reversal of hippocampus‐based memory. LC neurons in awake animals have two distinct firing modes: tonic firing (explorative) and phasic firing (exploitative). These two firing modes exert different modulatory effects on post‐synaptic dendritic spines. In the hippocampus, the firing modes regulate long‐term potentiation (LTP) and long‐term depression, which differentially regulate the mRNA expression and transcription of plasticity‐related proteins (PRPs). These proteins aid in structural alterations of dendritic spines, that is, structural long‐term potentiation (sLTP), via expansion and structural long‐term depression (sLTD) via contraction of post‐synaptic dendritic spines. Given the LC's role in all phases of memory processing, the degeneration of 50% of the LC neuron population occurring in Alzheimer's disease (AD) is a clinically relevant aspect of disease pathology. The loss of catecholaminergic regulation contributes to dysfunction in memory processes along with impaired functions associated with attention and task completion. The multifaceted role of the LC in memory and general task performance and the close correlation of LC degeneration with neurodegenerative disease progression together implicate it as a target for new clinical assessment tools.

Polymer Modified Carbon Fiber Multielectrode Arrays for Precision Neurotransmitter Measurements

Fast scan cyclic voltammetry (FSCV) and carbon-fiber microelectrodes (CFMEs) have been utilized used to detect several important neurochemicals in vivo. However, this method is limited due to the ability to discriminate dopamine from several of its metabolites. PEDOT, Nafion, and Polyethyleneimine modified microelectrodes will be utilized to detect physiologically low levels of neurotransmitters that also resist surface fouling and have high temporal resolution to detect fast changes of neurotransmitters. Furthermore, novel electrode coatings and waveforms will also be utilized to detect several neurotransmitter metabolites such as dopamine, norepinephrine, normetanephrine, 3-methoxytyramine (3-MT), homovanillic acid (HVA), 3,4 dihydroxyphenylacetic acid (DOPAC), and other metabolites. Currently, dopamine is thought to be an important neurotransmitter concerning several disease states such Parkinson’s disease, drug abuse (amphetamine, cocaine, etc.), and even for gambling and sex-disorders. However, dopamine is metabolized on a subsecond timescale, and studies have pointed to the importance of neurotransmitter metabolites in these disease states apart from dopamine. Presently, there is no method to selectively co-detect these neurotransmitter metabolites of dopamine utilizing FSCV. Through several waveform modifications and polymer electrode coatings, we develop a novel method to tune the detection of dopamine and said metabolites, which will help differentiate dopamine and respective metabolites through the shapes and positions of their respective CVs. Preliminary measurements have also been made in zebrafish retina showing the application of this technique in biological tissue. The co-detection and differentiation of dopamine metabolites and dopamine will have many implications in better understanding complex disease, behavioral, and pharmacological states.The brain is by far the most heterogeneous organ, and it is critically important to monitor various brain regions simultaneously in order to understand complex pharmacological, drug, and behavioral states. Therefore, it is critically important to make high temporal resolution neurochemical measurements to study the phasic firing of neurons in several brain regions concurrently. FSCV allows for high temporal measurements (< 100 msec) of neurotransmitter levels. Some of the many applications of this technique could be measuring neurochemical changes during epileptic seizures with simultaneous EEG measurements in rodents, drug abuse studies, measuring robust dopamine increases during deep brain stimulation in Parkinsonian models, and possibly many others.This work will also discuss the development of multielectrode arrays (MEAs) for neurotransmitter detection with fast scan cyclic voltammetry in multiple brain regions simultaneously. Parylene and silicon insulated carbon fiber microelectrode arrays were shown to be able to measure neurochemicals in multiple brain regions simultaneously when coupled with multichannel potentiostats. Moreover, we have utilized techniques such as plasma enhanced chemical vapor deposition (PECVD) to deposit conductive carbon nanospikes onto the surface of existing metal multielectrode arrays to give them dual functionality as neurotransmitter sensors with FSCV in addition to being used primarily for electrical stimulation and recording. Other assays have shown the utility of electrodepositing carbon nanotubes and polymers such as PEDOT to coat metal arrays with carbon to give them dual sensing capabilities.

Three-Week Treadmill Exercise Enhances Persistent Inward Currents, Facilitates Dendritic Plasticity, and Upregulates the Excitability of Dorsal Raphe Serotonin Neurons in ePet-EYFP Mice.

Exercise plays a key role in preventing or treating mental or motor disorders caused by dysfunction of the serotonergic system. However, the electrophysiological and ionic channel mechanisms underlying these effects remain unclear. In this study, we investigated the effects of 3-week treadmill exercise on the electrophysiological and channel properties of dorsal raphe nucleus (DRN). Serotonin (5-HT) neurons in ePet-EYFP mice, using whole-cell patch clamp recording. Treadmill exercise was induced in ePet-EYFP mice of P21–24 for 3 weeks, and whole-cell patch clamp recording was performed on EYFP-positive 5-HT neurons from DRN slices of P42–45 mice. Experiment data showed that 5-HT neurons in the DRN were a heterogeneous population with multiple firing patterns (single firing, phasic firing, and tonic firing). Persistent inward currents (PICs) with multiple patterns were expressed in 5-HT neurons and composed of Cav1.3 (Ca-PIC) and sodium (Na-PIC) components. Exercise hyperpolarized the voltage threshold for action potential (AP) by 3.1 ± 1.0 mV (control: n = 14, exercise: n = 18, p = 0.005) and increased the AP amplitude by 6.7 ± 3.0 mV (p = 0.031) and firing frequency by more than 22% especially within a range of current stimulation stronger than 70 pA. A 3-week treadmill exercise was sufficient to hyperpolarize PIC onset by 2.6 ± 1.3 mV (control: −53.4 ± 4.7 mV, n = 28; exercise: −56.0 ± 4.7 mV, n = 25, p = 0.050) and increase the PIC amplitude by 28% (control: 193.6 ± 81.8 pA; exercise: 248.5 ± 105.4 pA, p = 0.038). Furthermore, exercise hyperpolarized Na-PIC onset by 3.8 ± 1.8 mV (control: n = 8, exercise: n = 9, p = 0.049) and increased the Ca-PIC amplitude by 23% (p = 0.013). The exercise-induced enhancement of the PIC amplitude was mainly mediated by Ca-PIC and hyperpolarization of PIC onset by Na-PIC. Moreover, exercise facilitated dendritic plasticity, which was shown as the increased number of branch points by 1.5 ± 0.5 (p = 0.009) and dendritic branches by 2.1 ± 0.6 (n = 20, p = 0.001) and length by 732.0 ± 100.1 μm (p < 0.001) especially within the range of 50–200 μm from the soma. Functional analysis suggested that treadmill exercise enhanced Na-PIC for facilitation of spike initiation and Ca-PIC for regulation of repetitive firing. We concluded that PICs broadly existed in DRN 5-HT neurons and could influence serotonergic neurotransmission in juvenile mice and that 3-week treadmill exercise induced synaptic adaptations, enhanced PICs, and thus upregulated the excitability of the 5-HT neurons.

Discharge and Role of GABA Pontomesencephalic Neurons in Cortical Activity and Sleep-Wake States Examined by Optogenetics and Juxtacellular Recordings in Mice.

The cholinergic neurons in the pontomesencephalic tegmentum have been shown to discharge in association with and promote cortical activation during active or attentive waking and paradoxical or rapid eye movement sleep. However, GABA neurons lie intermingled with the cholinergic neurons and may contribute to or oppose this activity and role. Here we investigated in vitro and in vivo the properties, activities, and role of GABA neurons within the laterodorsal tegmental and sublaterodorsal tegmental nuclei (LDT/SubLDT) using male and female transgenic mice expressing channelrhodopsin-(ChR2)-EYFP in vesicular GABA transporter (VGAT)-expressing neurons. Presumed GABA (pGABA) neurons were identified by response to photostimulation and verified by immunohistochemical staining following juxtacellular labeling in vivo pGABA neurons were found to be fast-firing neurons with the capacity to burst when depolarized from a hyperpolarized membrane potential. When stimulated in vivo in urethane-anesthetized or unanesthetized mice, the pGABA neurons fired repetitively at relatively fast rates (∼40 Hz) during a continuous light pulse or phasically in bursts (>100 Hz) when driven by rhythmic light pulses at theta (4 or 8 Hz) frequencies. pNon-GABA, which likely included cholinergic, neurons were inhibited during each light pulse to discharge rhythmically in antiphase to the pGABA neurons. The reciprocal rhythmic bursting by the pGABA and pNon-GABA neurons drove rhythmic theta activity in the EEG. Such phasic bursting by GABA neurons also occurred in WT mice in association with theta activity during attentive waking and paradoxical sleep.SIGNIFICANCE STATEMENT Neurons in the pontomesencephalic tegmentum, particularly cholinergic neurons, play an important role in cortical activation, which occurs during active or attentive waking and paradoxical or rapid eye movement sleep. Yet the cholinergic neurons lie intermingled with GABA neurons, which could play a similar or opposing role. Optogenetic stimulation and recording of these GABA neurons in mice revealed that they can discharge in rhythmic bursts at theta frequencies and drive theta activity in limbic cortex. Such phasic burst firing also occurs during natural attentive waking and paradoxical sleep in association with theta activity and could serve to enhance sensory-motor processing and memory consolidation during these states.