Optogenetic action potentials and intrinsic pacemaker interplay in retrogradely identified midbrain dopamine neurons.

Dissecting the diversity of midbrain dopamine (DA) neurons by optotagging is a promising addition to better identify their functional properties and contribution to motivated behavior. Retrograde molecular targeting of DA neurons with specific axonal projection allows further refinement of this approach. Here, we focus on adult mouse DA neurons in the substantia nigra pars compacta (SNc) projecting to dorsal striatum (DS) by demonstrating the selectivity of a floxed AAV9-based retrograde channelrhodopsin-eYFP (ChR-eYFP) labeling approach in DAT-cre mice. Furthermore, we show the utility of a sparse labeling version for anatomical single-cell reconstruction and demonstrate that ChR-eYFR expressing DA neurons retain intrinsic functional properties indistinguishable from conventionally retrogradely red-beads-labeled neurons. We systematically explore the properties of optogenetically evoked action potentials (oAPs) and their interaction with intrinsic pacemaking in this defined subpopulation of DA neurons. We found that the shape of the oAP and its first derivative, as a proxy for extracellularly recorded APs, is highly distinct from spontaneous APs (sAPs) of the same neurons and systematically varies across the pacemaker duty cycle. The timing of the oAP also affects the backbone oscillator of the intrinsic pacemaker by introducing transient "compensatory pauses". Characterizing this systematic interplay between oAPs and sAPs in defined DA neurons will also facilitate a refinement of DA neuron optotagging in vivo.

The human channel gating-modifying A749G CACNA1D (Cav1.3) variant induces a neurodevelopmental syndrome-like phenotype in mice.

Germline de novo missense variants of the CACNA1D gene, encoding the pore-forming α1 subunit of Cav1.3 L-type Ca2+ channels (LTCCs), have been found in patients with neurodevelopmental and endocrine dysfunction, but their disease-causing potential is unproven. These variants alter channel gating, enabling enhanced Cav1.3 activity, suggesting Cav1.3 inhibition as a potential therapeutic option. Here we provide proof of the disease-causing nature of such gating-modifying CACNA1D variants using mice (Cav1.3AG) containing the A749G variant reported de novo in a patient with autism spectrum disorder (ASD) and intellectual impairment. In heterozygous mutants, native LTCC currents in adrenal chromaffin cells exhibited gating changes as predicted from heterologous expression. The A749G mutation induced aberrant excitability of dorsomedial striatum-projecting substantia nigra dopamine neurons and medium spiny neurons in the dorsal striatum. The phenotype observed in heterozygous mutants reproduced many of the abnormalities described within the human disease spectrum, including developmental delay, social deficit, and pronounced hyperactivity without major changes in gross neuroanatomy. Despite an approximately 7-fold higher sensitivity of A749G-containing channels to the LTCC inhibitor isradipine, oral pretreatment over 2 days did not rescue the hyperlocomotion. Cav1.3AG mice confirm the pathogenicity of the A749G variant and point toward a pathogenetic role of altered signaling in the dopamine midbrain system.

Cav1.3 calcium channels are full-range linear amplifiers of firing frequencies in lateral DA SN neurons.

The low-threshold L-type calcium channel Cav1.3 accelerates the pacemaker rate in the heart, but its functional role for the extended dynamic range of neuronal firing is still unresolved. Here, we show that Cav1.3 calcium channels act as unexpectedly simple, full-range linear amplifiers of firing rates for lateral dopamine substantia nigra (DA SN) neurons in mice. This means that they boost in vitro or in vivo firing frequencies between 2 and 50 hertz by about 30%. Furthermore, we demonstrate that clinically relevant, low nanomolar concentrations of the L-type channel inhibitor isradipine selectively reduce the in vivo firing activity of these nigrostriatal DA SN neurons at therapeutic plasma concentrations. Thus, our study identifies the pacemaker function of neuronal Cav1.3 channels and provides direct evidence that repurposing dihydropyridines such as isradipine is feasible to selectively modulate the in vivo activity of highly vulnerable DA SN subpopulations in Parkinson's disease.

Dendritic Architecture Predicts in vivo Firing Pattern in Mouse Ventral Tegmental Area and Substantia Nigra Dopaminergic Neurons.

The firing activity of ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) dopaminergic (DA) neurons is an important factor in shaping DA release and its role in motivated behavior. Dendrites in DA neurons are the main postsynaptic compartment and, along with cell body and axon initial segment, contribute to action potential generation and firing pattern. In this study, the organization of the dendritic domain in individual VTA and SNc DA neurons of adult male mice, and their relationship to in vivo spontaneous firing, are described. In comparison with dorsal VTA DA neurons, ventrally located VTA neurons (as measured by cell body location) possess a shorter total dendritic length and simpler dendritic architecture, and exhibit the most irregular in vivo firing patterns among DA neurons. In contrast, for DA neurons in the SNc, the higher irregularity of firing was related to a smaller dendritic domain, as measured by convex hull volumes. However, firing properties were also related to the specific regional distribution of the dendritic tree. Thus, VTA DA neurons with a larger extension of their dendritic tree within the parabrachial pigmented (PBP) nucleus fired more regularly compared with those with relatively more dendrites extending outside the PBP. For DA neurons in the SNc, enhanced firing irregularity was associated with a smaller proportion of dendrites penetrating the substantia nigra pars reticulata. These results suggest that differences in dendritic morphology contribute to the in vivo firing properties of individual DA neurons, and that the existence of region-specific synaptic connectivity rules that shape firing diversity.

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity.

Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

Inactivation mode of sodium channels defines the different maximal firing rates of conventional versus atypical midbrain dopamine neurons.

Two subpopulations of midbrain dopamine (DA) neurons are known to have different dynamic firing ranges in vitro that correspond to distinct projection targets: the originally identified conventional DA neurons project to the dorsal striatum and the lateral shell of the nucleus accumbens, whereas an atypical DA population with higher maximum firing frequencies projects to prefrontal regions and other limbic regions including the medial shell of nucleus accumbens. Using a computational model, we show that previously identified differences in biophysical properties do not fully account for the larger dynamic range of the atypical population and predict that the major difference is that originally identified conventional cells have larger occupancy of voltage-gated sodium channels in a long-term inactivated state that recovers slowly; stronger sodium and potassium conductances during action potential firing are also predicted for the conventional compared to the atypical DA population. These differences in sodium channel gating imply that longer intervals between spikes are required in the conventional population for full recovery from long-term inactivation induced by the preceding spike, hence the lower maximum frequency. These same differences can also change the bifurcation structure to account for distinct modes of entry into depolarization block: abrupt versus gradual. The model predicted that in cells that have entered depolarization block, it is much more likely that an additional depolarization can evoke an action potential in conventional DA population. New experiments comparing lateral to medial shell projecting neurons confirmed this model prediction, with implications for differential synaptic integration in the two populations.

Developmental HCN channelopathy results in decreased neural progenitor proliferation and microcephaly in mice.

The development of the cerebral cortex relies on the controlled division of neural stem and progenitor cells. The requirement for precise spatiotemporal control of proliferation and cell fate places a high demand on the cell division machinery, and defective cell division can cause microcephaly and other brain malformations. Cell-extrinsic and -intrinsic factors govern the capacity of cortical progenitors to produce large numbers of neurons and glia within a short developmental time window. In particular, ion channels shape the intrinsic biophysical properties of precursor cells and neurons and control their membrane potential throughout the cell cycle. We found that hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits are expressed in mouse, rat, and human neural progenitors. Loss of HCN channel function in rat neural stem cells impaired their proliferation by affecting the cell-cycle progression, causing G1 accumulation and dysregulation of genes associated with human microcephaly. Transgene-mediated, dominant-negative loss of HCN channel function in the embryonic mouse telencephalon resulted in pronounced microcephaly. Together, our findings suggest a role for HCN channel subunits as a part of a general mechanism influencing cortical development in mammals.

The gating pore blocker 1-(2,4-xylyl)guanidinium selectively inhibits pacemaking of midbrain dopaminergic neurons.

Although several ionic mechanisms are known to control rate and regularity of the slow pacemaker in dopamine (DA) neurons, the core mechanism of pacing is controversial. Here we tested the hypothesis that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidinium (XG), an established blocker of gating pore currents, selectively inhibits pacemaking of DA neurons. The compound inhibited all slow pacemaking DA neurons that were tested, both in the substantia nigra pars compacta, and in the ventral tegmental area. Interestingly, bursting behavior was not affected by XG. Furthermore, the drug did not affect fast pacemaking of GABAergic neurons from substantia nigra pars reticulata neurons or slow pacemaking of noradrenergic neurons. In DA neurons, current-clamp analysis revealed that XG did not appear to affect ion channels involved in the action potential. Its inhibitory effect persisted during blockade of all ion channels previously suggested to contribute to pacemaking. RNA sequencing and voltage-clamp recordings yielded no evidence for a gating pore current to underlie the conductance. However, we could isolate a small subthreshold XG-sensitive current, which was carried by both Na+ and Cl- ions. Although the molecular target of XG remains to be defined, these observations represent a step towards understanding pacemaking in DA neurons.

α-Synuclein-induced Kv4 channelopathy in mouse vagal motoneurons drives nonmotor parkinsonian symptoms.

No disease-modifying therapy is currently available for Parkinson's disease (PD), the second most common neurodegenerative disease. The long nonmotor prodromal phase of PD is a window of opportunity for early detection and intervention. However, we lack the pathophysiological understanding to develop selective biomarkers and interventions. By using a mutant α-synuclein selective-overexpression mouse model of prodromal PD, we identified a cell-autonomous selective Kv4 channelopathy in dorsal motor nucleus of the vagus (DMV) neurons. This functional remodeling of intact DMV neurons leads to impaired pacemaker function in vitro and in vivo, which, in turn, reduces gastrointestinal motility, a common early symptom of prodromal PD. We identify a chain of events from α-synuclein via a biophysical dysfunction of a specific neuronal population to a clinically relevant prodromal symptom. These findings will facilitate the rational design of clinical biomarkers to identify people at risk for developing PD.

Sex-dependent alterations in behavior, drug responses and dopamine transporter expression in heterozygous DAT-Cre mice.

Heterozygous mice that express Cre-recombinase under the dopamine transporter promoter (DAT-Cre knock in mice, or KI) are widely used for targeting midbrain dopamine neurons, under the assumption that their constitutive physiology is not affected. We report here that these mice display striking sex-dependent behavioral and molecular differences in relation to wildtypes (WT). Male and female KI mice were constitutively hyperactive, and male KI mice showed attenuated hyperlocomotor responses to amphetamine. In contrast, female KIs displayed a marked reduction in locomotion ("calming" effect) in response to the same dose of amphetamine. Furthermore, male and female DAT-Cre KI mice showed opposing differences in reinforcement learning, with females showing faster conditioning and males showing slower extinction. Other behavioral variables, including working memory and novelty preference, were not changed compared to WT. These effects were paralleled by differences in striatal DAT expression that disproportionately affected female KI mice. Our findings reveal clear limitations of the DAT-Cre line that must be considered when using this model.

Patterns of Domain-Specific Learning Among Medical Undergraduate Students in Relation to Confidence in Their Physiology Knowledge: Insights From a Pre-post Study.

RESEARCH FOCUS: The promotion of domain-specific knowledge is a central goal of higher education and, in the field of medicine, it is particularly essential to promote global health. Domain-specific knowledge on its own is not exhaustive; confidence regarding the factual truth of this knowledge content is also required. An increase in both knowledge and confidence is considered a necessary prerequisite for making professional decisions in the clinical context. Especially the knowledge of human physiology is fundamental and simultaneously critical to medical decision-making. However, numerous studies have shown difficulties in understanding and misconceptions in this area of knowledge. Therefore, we investigate (i) how preclinical medical students acquire knowledge in physiology over the course of their studies and simultaneously gain confidence in the correctness of this knowledge as well as (ii) the interrelations between these variables, and (iii) how they affect the development of domain-specific knowledge. METHOD: In a pre-post study, 169 medical students' development of physiology knowledge and their confidence related to this knowledge were assessed via paper-pencil questionnaires before and after attending physiology seminars for one semester. Data from a longitudinal sample of n = 97 students were analyzed using mean comparisons, regression analyses, and latent class analyses (LCAs). In addition, four types of item responses were formed based on confidence and correctness in the knowledge test. RESULTS: We found a significant and large increase in the students' physiology knowledge, with task-related confidence being the strongest predictor (apart from learning motivation). Moreover, a significantly higher level of confidence at t2 was confirmed, with the level of prior confidence being a strong predictor (apart from knowledge at t2). Furthermore, based on the students' development of knowledge and confidence levels between measurement points, three empirically distinct groups were distinguished: knowledge gainers, confidence gainers, and overall gainers. The students whose confidence in incorrect knowledge increased constituted one particularly striking group. Therefore, the training of both knowledge and the ability to critically reflect on one's knowledge and skills as well as an assessment of their development in education is required, especially in professions such as medicine, where knowledge-based decisions made with confidence are of vital importance.

In vivo patch-clamp recordings reveal distinct subthreshold signatures and threshold dynamics of midbrain dopamine neurons.

The in vivo firing patterns of ventral midbrain dopamine neurons are controlled by afferent and intrinsic activity to generate sensory cue and prediction error signals that are essential for reward-based learning. Given the absence of in vivo intracellular recordings during the last three decades, the subthreshold membrane potential events that cause changes in dopamine neuron firing patterns remain unknown. To address this, we established in vivo whole-cell recordings and obtained over 100 spontaneously active, immunocytochemically-defined midbrain dopamine neurons in isoflurane-anaesthetized adult mice. We identified a repertoire of subthreshold membrane potential signatures associated with distinct in vivo firing patterns. Dopamine neuron activity in vivo deviated from single-spike pacemaking by phasic increases in firing rate via two qualitatively distinct biophysical mechanisms: 1) a prolonged hyperpolarization preceding rebound bursts, accompanied by a hyperpolarizing shift in action potential threshold; and 2) a transient depolarization leading to high-frequency plateau bursts, associated with a depolarizing shift in action potential threshold. Our findings define a mechanistic framework for the biophysical implementation of dopamine neuron firing patterns in the intact brain.

Genetic Architecture of Parkinson's Disease in the Indian Population: Harnessing Genetic Diversity to Address Critical Gaps in Parkinson's Disease Research.

Over the past two decades, our understanding of Parkinson's disease (PD) has been gleaned from the discoveries made in familial and/or sporadic forms of PD in the Caucasian population. The transferability and the clinical utility of genetic discoveries to other ethnically diverse populations are unknown. The Indian population has been under-represented in PD research. The Genetic Architecture of PD in India (GAP-India) project aims to develop one of the largest clinical/genomic bio-bank for PD in India. Specifically, GAP-India project aims to: (1) develop a pan-Indian deeply phenotyped clinical repository of Indian PD patients; (2) perform whole-genome sequencing in 500 PD samples to catalog Indian genetic variability and to develop an Indian PD map for the scientific community; (3) perform a genome-wide association study to identify novel loci for PD and (4) develop a user-friendly web-portal to disseminate results for the scientific community. Our "hub-spoke" model follows an integrative approach to develop a pan-Indian outreach to develop a comprehensive cohort for PD research in India. The alignment of standard operating procedures for recruiting patients and collecting biospecimens with international standards ensures harmonization of data/bio-specimen collection at the beginning and also ensures stringent quality control parameters for sample processing. Data sharing and protection policies follow the guidelines established by local and national authorities.We are currently in the recruitment phase targeting recruitment of 10,200 PD patients and 10,200 healthy volunteers by the end of 2020. GAP-India project after its completion will fill a critical gap that exists in PD research and will contribute a comprehensive genetic catalog of the Indian PD population to identify novel targets for PD.

Neuronal activity triggers uptake of hematopoietic extracellular vesicles in vivo.

Communication with the hematopoietic system is a vital component of regulating brain function in health and disease. Traditionally, the major routes considered for this neuroimmune communication are by individual molecules such as cytokines carried by blood, by neural transmission, or, in more severe pathologies, by the entry of peripheral immune cells into the brain. In addition, functional mRNA from peripheral blood can be directly transferred to neurons via extracellular vesicles (EVs), but the parameters that determine their uptake are unknown. Using varied animal models that stimulate neuronal activity by peripheral inflammation, optogenetics, and selective proteasome inhibition of dopaminergic (DA) neurons, we show that the transfer of EVs from blood is triggered by neuronal activity in vivo. Importantly, this transfer occurs not only in pathological stimulation but also by neuronal activation caused by the physiological stimulus of novel object placement. This discovery suggests a continuous role of EVs under pathological conditions as well as during routine cognitive tasks in the healthy brain.

Inhibition of histone deacetylation rescues phenotype in a mouse model of Birk-Barel intellectual disability syndrome.

Mutations in the actively expressed, maternal allele of the imprinted KCNK9 gene cause Birk-Barel intellectual disability syndrome (BBIDS). Using a BBIDS mouse model, we identify here a partial rescue of the BBIDS-like behavioral and neuronal phenotypes mediated via residual expression from the paternal Kcnk9 (Kcnk9pat) allele. We further demonstrate that the second-generation HDAC inhibitor CI-994 induces enhanced expression from the paternally silenced Kcnk9 allele and leads to a full rescue of the behavioral phenotype suggesting CI-994 as a promising molecule for BBIDS therapy. Thus, these findings suggest a potential approach to improve cognitive dysfunction in a mouse model of an imprinting disorder.

Undergraduate Students' Critical Online Reasoning-Process Mining Analysis.

To successfully learn using open Internet resources, students must be able to critically search, evaluate and select online information, and verify sources. Defined as critical online reasoning (COR), this construct is operationalized on two levels in our study: (1) the student level using the newly developed Critical Online Reasoning Assessment (CORA), and (2) the online information processing level using event log data, including gaze durations and fixations. The written responses of 32 students for one CORA task were scored by three independent raters. The resulting score was operationalized as "task performance," whereas the gaze fixations and durations were defined as indicators of "process performance." Following a person-oriented approach, we conducted a process mining (PM) analysis, as well as a latent class analysis (LCA) to test whether-following the dual-process theory-the undergraduates could be distinguished into two groups based on both their process and task performance. Using PM, the process performance of all 32 students was visualized and compared, indicating two distinct response process patterns. One group of students (11), defined as "strategic information processers," processed online information more comprehensively, as well as more efficiently, which was also reflected in their higher task scores. In contrast, the distributions of the process performance variables for the other group (21), defined as "avoidance information processers," indicated a poorer process performance, which was also reflected in their lower task scores. In the LCA, where two student groups were empirically distinguished by combining the process performance indicators and the task score as a joint discriminant criterion, we confirmed these two COR profiles, which were reflected in high vs. low process and task performances. The estimated parameters indicated that high-performing students were significantly more efficient at conducting strategic information processing, as reflected in their higher process performance. These findings are so far based on quantitative analyses using event log data. To enable a more differentiated analysis of students' visual attention dynamics, more in-depth qualitative research of the identified student profiles in terms of COR will be required.

In vivo functional diversity of midbrain dopamine neurons within identified axonal projections.

Functional diversity of midbrain dopamine (DA) neurons ranges across multiple scales, from differences in intrinsic properties and connectivity to selective task engagement in behaving animals. Distinct in vitro biophysical features of DA neurons have been associated with different axonal projection targets. However, it is unknown how this translates to different firing patterns of projection-defined DA subpopulations in the intact brain. We combined retrograde tracing with single-unit recording and labelling in mouse brain to create an in vivo functional topography of the midbrain DA system. We identified differences in burst firing among DA neurons projecting to dorsolateral striatum. Bursting also differentiated DA neurons in the medial substantia nigra (SN) projecting either to dorsal or ventral striatum. We found differences in mean firing rates and pause durations among ventral tegmental area (VTA) DA neurons projecting to lateral or medial shell of nucleus accumbens. Our data establishes a high-resolution functional in vivo landscape of midbrain DA neurons.

An ER Assembly Line of AMPA-Receptors Controls Excitatory Neurotransmission and Its Plasticity.

Excitatory neurotransmission and its activity-dependent plasticity are largely determined by AMPA-receptors (AMPARs), ion channel complexes whose cell physiology is encoded by their interactome. Here, we delineate the assembly of AMPARs in the endoplasmic reticulum (ER) of native neurons as multi-state production line controlled by distinct interactome constituents: ABHD6 together with porcupine stabilizes pore-forming GluA monomers, and the intellectual-disability-related FRRS1l-CPT1c complexes promote GluA oligomerization and co-assembly of GluA tetramers with cornichon and transmembrane AMPA-regulatory proteins (TARP) to render receptor channels ready for ER exit. Disruption of the assembly line by FRRS1l deletion largely reduces AMPARs in the plasma membrane, impairs synapse formation, and abolishes activity-dependent synaptic plasticity, while FRRS1l overexpression has the opposite effect. As a consequence, FRSS1l knockout mice display severe deficits in learning tasks and behavior. Our results provide mechanistic insight into the stepwise biogenesis of AMPARs in native ER membranes and establish FRRS1l as a powerful regulator of synaptic signaling and plasticity.

Axonal projection-specific differences in somatodendritic α2 autoreceptor function in locus coeruleus neurons.

The locus coeruleus (LC) contains the majority of central noradrenergic neurons sending wide projections throughout the entire CNS. The LC is considered to be essential for multiple key brain functions including arousal, attention and adaptive stress responses as well as higher cognitive functions and memory. Electrophysiological studies of LC neurons have identified several characteristic functional features such as low-frequency pacemaker activity with broad action potentials, transient high-frequency burst discharges in response to salient stimuli and an apparently homogeneous inhibition of firing by activation of somatodendritic α2 autoreceptors (α2AR). While stress-mediated plasticity of the α2AR response has been described, it is currently unclear whether different LC neurons projecting to distinct axonal targets display differences in α2AR function. Using fluorescent beads-mediated retrograde tracing in adult C57Bl6/N mice, we compared the anatomical distributions and functional in vitro properties of identified LC neurons projecting either to medial prefrontal cortex, hippocampus or cerebellum. The functional in vitro analysis of LC neurons confirmed their mostly uniform functional properties regarding action potential generation and pacemaker firing. However, we identified significant differences in tonic and evoked α2AR-mediated responses. While hippocampal-projecting LC neurons were partially inhibited by endogenous levels of norepinephrine and almost completely silenced by application of saturating concentrations of the α2 agonist clonidine, prefrontal-projecting LC neurons were not affected by endogenous levels of norepinephrine and only partially inhibited by saturating concentrations of clonidine. Thus, we identified a limited α2AR control of electrical activity for prefrontal-projecting LC neurons indicative of functional heterogeneity in the LC-noradrenergic system.