Opto-seq reveals input-specific immediate-early gene induction in ventral tegmental area cell types.

The ventral tegmental area (VTA) is a critical node in circuits governing motivated behavior and is home to diverse populations of neurons that release dopamine, gamma-aminobutyric acid (GABA), glutamate, or combinations of these neurotransmitters. The VTA receives inputs from many brain regions, but a comprehensive understanding of input-specific activation of VTA neuronal subpopulations is lacking. To address this, we combined optogenetic stimulation of select VTA inputs with single-nucleus RNA sequencing (snRNA-seq) and highly multiplexed in situ hybridization to identify distinct neuronal clusters and characterize their spatial distribution and activation patterns. Quantification of immediate-early gene (IEG) expression revealed that different inputs activated select VTA subpopulations, which demonstrated cell-type-specific transcriptional programs. Within dopaminergic subpopulations, IEG induction levels correlated with differential expression of ion channel genes. This new transcriptomics-guided circuit analysis reveals the diversity of VTA activation driven by distinct inputs and provides a resource for future analysis of VTA cell types.

The role of artificial intelligence and convolutional neural networks in the management of melanoma: a clinical, pathological, and radiological perspective.

Clinical dermatoscopy and pathological slide assessment are essential in the diagnosis and management of patients with cutaneous melanoma. For those presenting with stage IIC disease and beyond, radiological investigations are often considered. The dermatoscopic, whole slide and radiological images used during clinical care are often stored digitally, enabling artificial intelligence (AI) and convolutional neural networks (CNN) to learn, analyse and contribute to the clinical decision-making. A keyword search of the Medline database was performed to assess the progression, capabilities and limitations of AI and CNN and its use in diagnosis and management of cutaneous melanoma. Full-text articles were reviewed if they related to dermatoscopy, pathological slide assessment or radiology. Through analysis of 95 studies, we demonstrate that diagnostic accuracy of AI/CNN can be superior (or at least equal) to clinicians. However, variability in image acquisition, pre-processing, segmentation, and feature extraction remains challenging. With current technological abilities, AI/CNN and clinicians synergistically working together are better than one another in all subspecialty domains relating to cutaneous melanoma. AI has the potential to enhance the diagnostic capabilities of junior dermatology trainees, primary care skin cancer clinicians and general practitioners. For experienced clinicians, AI provides a cost-efficient second opinion. From a pathological and radiological perspective, CNN has the potential to improve workflow efficiency, allowing clinicians to achieve more in a finite amount of time. Until the challenges of AI/CNN are reliably met, however, they can only remain an adjunct to clinical decision-making.

Temporal scaling of dopamine neuron firing and dopamine release by distinct ion channels shape behavior.

Dopamine is broadly implicated in reinforcement learning, but how patterns of dopamine activity are generated is poorly resolved. Here, we demonstrate that two ion channels, Kv4.3 and BKCa1.1, regulate the pattern of dopamine neuron firing and dopamine release on different time scales to influence separate phases of reinforced behavior in mice. Inactivation of Kv4.3 in VTA dopamine neurons increases ex vivo pacemaker activity and excitability that is associated with increased in vivo firing rate and ramping dynamics before lever press in a learned instrumental paradigm. Loss of Kv4.3 enhances performance of the learned response and facilitates extinction. In contrast, loss of BKCa1.1 increases burst firing and phasic dopamine release that enhances learning of an instrumental response and enhances extinction burst lever pressing in early extinction that is associated with a greater change in activity between reinforced and unreinforced actions. These data demonstrate that disruption of intrinsic regulators of neuronal activity differentially affects dopamine dynamics during reinforcement and extinction learning.

Circuit coordination of opposing neuropeptide and neurotransmitter signals.

Fast-acting neurotransmitters and slow, modulatory neuropeptides are co-released from neurons in the central nervous system, albeit from distinct synaptic vesicles1. The mechanisms of how co-released neurotransmitters and neuropeptides that have opposing actions-for example, stimulatory versus inhibitory-work together to exert control of neural circuit output remain unclear. This has been difficult to resolve owing to the inability to selectively isolate these signalling pathways in a cell- and circuit-specific manner. Here we developed a genetic-based anatomical disconnect procedure that utilizes distinct DNA recombinases to independently facilitate CRISPR-Cas9 mutagenesis2 of neurotransmitter- and neuropeptide-related genes in distinct cell types in two different brain regions simultaneously. We demonstrate that neurons within the lateral hypothalamus that produce the stimulatory neuropeptide neurotensin and the inhibitory neurotransmitter GABA (γ-aminobutyric acid) utilize these signals to coordinately activate dopamine-producing neurons of the ventral tegmental area. We show that GABA release from lateral hypothalamus neurotensin neurons inhibits GABA neurons within the ventral tegmental area, disinhibiting dopamine neurons and causing a rapid rise in calcium, whereas neurotensin directly generates a slow inactivating calcium signal in dopamine neurons that is dependent on the expression of neurotensin receptor 1 (Ntsr1). We further show that these two signals work together to regulate dopamine neuron responses to maximize behavioural responding. Thus, a neurotransmitter and a neuropeptide with opposing signals can act on distinct timescales through different cell types to enhance circuit output and optimize behaviour.

The potassium channel auxiliary subunit Kvß2 (Kcnab2) regulates Kv1 channels and dopamine neuron firing.

Ion channel complexes typically consist of both pore-forming subunits and auxiliary subunits that do not directly conduct current but can regulate trafficking or alter channel properties. Isolating the role of these auxiliary subunits in neurons has proved difficult due to a lack of specific pharmacological agents and the potential for developmental compensation in constitutive knockout models. Here, we use cell-type-specific viral-mediated CRISPR/Cas9 mutagenesis to target the potassium channel auxiliary subunit Kvß2 (Kcnab2) in dopamine neurons in the adult mouse brain. We find that mutagenesis of Kcnab2 reduces surface expression of Kv1.2, the primary Kv1 pore-forming subunit expressed in dopamine neurons, and shifts the voltage dependence of inactivation of potassium channel currents toward more hyperpolarized potentials. Loss of Kcnab2 broadens the action potential waveform in spontaneously firing dopamine neurons recorded in slice, reduces the afterhyperpolarization amplitude, and increases spike timing irregularity and excitability, all of which is consistent with a reduction in potassium channel current. Similar effects were observed with mutagenesis of the pore-forming subunit Kv1.2 (Kcna2). These results identify Kv1 currents as important contributors to dopamine neuron firing and demonstrate a role for Kvß2 subunits in regulating the trafficking and gating properties of these ion channels. Furthermore, they demonstrate the utility of CRISPR-mediated mutagenesis in the study of previously difficult to isolate ion channel subunits.NEW & NOTEWORTHY Here, we utilize CRISPR/Cas9-mediated mutagenesis in dopamine neurons in mice to target the gene encoding Kvß2, an auxiliary subunit that forms a part of Kv1 channel complexes. We find that the absence of Kvß2 alters action potential properties by reducing surface expression of pore-forming subunits and shifting the voltage dependence of channel inactivation. This work establishes a new function for Kvß2 subunits and Kv1 complexes in regulating dopamine neuron activity.

Distinct Encoding of Reward and Aversion by Peptidergic BNST Inputs to the VTA.

Neuropeptides play an important role in modulating mesolimbic system function. However, while synaptic inputs to the ventral tegmental area (VTA) have been extensively mapped, the sources of many neuropeptides are not well resolved. Here, we mapped the anatomical locations of three neuropeptide inputs to the VTA: neurotensin (NTS), corticotrophin releasing factor (CRF), and neurokinin B (NkB). Among numerous labeled inputs we identified the bed nucleus of the stria terminalis (BNST) as a major source of all three peptides, containing similar numbers of NTS, CRF, and NkB VTA projection neurons. Approximately 50% of BNST to VTA inputs co-expressed two or more of the peptides examined. Consistent with this expression pattern, analysis of calcium dynamics in the terminals of these inputs in the VTA revealed both common and distinct patterns of activation during appetitive and aversive conditioning. These data demonstrate additional diversification of the mesolimbic dopamine system through partially overlapping neuropeptidergic inputs.

Diabetic ankle fractures: a review of the literature and an introduction to the Adelaide fracture in the diabetic ankle algorithm and score.

Diabetics who have acquired an ankle fracture may be easily missed given their atypical presentation. As such, it is not infrequently seen that these patients are either initially misdiagnosed or ineffectively managed resulting in unnecessary hospital length of stay and procedures. Multiple review articles and retrospective studies have been previously published in the literature, but complete guidelines to assist in accurate diagnosis and cost-effective management for this complex problem do not currently exist. Through a critical analysis of the current literature, a proposed diagnostic and management algorithm and scoring system that can be used to quantify risks in the surgical management are presented for consideration.