Activity-Dependent Ectopic Spiking in Parvalbumin-Expressing Interneurons of the Neocortex.

Canonically, action potentials of most mammalian neurons initiate at the axon initial segment (AIS) and propagate bidirectionally: orthodromically along the distal axon and retrogradely into the soma and dendrites. Under some circumstances, action potentials may initiate ectopically, at sites distal to the AIS, and propagate antidromically along the axon. These "ectopic action potentials" (EAPs) have been observed in experimental models of seizures and chronic pain, and more rarely in nonpathological forebrain neurons. Here we report that a large majority of parvalbumin-expressing (PV+) interneurons in the upper layers of mouse neocortex, from both orbitofrontal and primary somatosensory areas, fire EAPs after sufficient activation of their somata. Somatostatin-expressing interneurons also fire EAPs, though less robustly. Ectopic firing in PV+ cells occurs in varying temporal patterns and can persist for several seconds. PV+ cells evoke strong synaptic inhibition in pyramidal neurons and interneurons and play critical roles in cortical function. Our results suggest that ectopic spiking of PV+ interneurons is common and may contribute to both normal and pathological network functions of the neocortex.

Activity-dependent ectopic action potentials in regular-spiking neurons of the neocortex.

Introduction: Action potentials usually travel orthodromically along a neuron's axon, from the axon initial segment (AIS) toward the presynaptic terminals. Under some circumstances action potentials also travel in the opposite direction, antidromically, after being initiated at a distal location. Given their initiation at an atypical site, we refer to these events as "ectopic action potentials." Ectopic action potentials (EAPs) were initially observed in pathological conditions including seizures and nerve injury. Several studies have described regular-spiking (RS) pyramidal neurons firing EAPs in seizure models. Under nonpathological conditions, EAPs were reported in a few populations of neurons, and our group has found that EAPs can be induced in a large proportion of parvalbumin-expressing interneurons in the neocortex. Nevertheless, to our knowledge there have been no prior reports of ectopic firing in the largest population of neurons in the neocortex, pyramidal neurons, under nonpathological conditions. Methods: We performed in vitro recordings utilizing the whole-cell patch clamp technique. To elicit EAPs, we triggered orthodromic action potentialswith either long, progressively increasing current steps, or with trains of brief pulses at 30, 60, or 100 Hz delivered in 3 different ways, varying in stimulus and resting period duration. Results: We found that a large proportion (72.7%) of neocortical RS cells from mice can fire EAPs after a specific stimulus in vitro, and that most RS cells (56.1%) are capable of firing EAPs across a broad range of stimulus conditions. Of the 37 RS neurons in which we were able to elicit EAPs, it took an average of 863.8 orthodromic action potentials delivered over the course of an average of ~81.4 s before the first EAP was seen. We observed that some cells responded to specific stimulus frequencies while less selective, suggesting frequency tuning in a subset of the cells. Discussion: Our findings suggest that pyramidal cells can integrate information over long time-scales before briefly entering a mode of self-generated firing that originates in distal axons. The surprising ubiquity of EAP generation in RS cells raises interesting questions about the potential roles of ectopic spiking in information processing, cortical oscillations, and seizure susceptibility.

Thalamic neuromodulation in epilepsy: A primer for emerging circuit-based therapies.

INTRODUCTION: Epilepsy is a common, often debilitating disease of hyperexcitable neural networks. While medically intractable cases may benefit from surgery, there may be no single, well-localized focus for resection or ablation. In such cases, approaching the disease from a network-based perspective may be beneficial. AREAS COVERED: Herein, the authors provide a narrative review of normal thalamic anatomy and physiology and propose general strategies for preventing and/or aborting seizures by modulating this structure. Additionally, they make specific recommendations for targeting the thalamus within different contexts, motivated by a more detailed discussion of its distinct nuclei and their respective connectivity. By describing important principles governing thalamic function and its involvement in seizure networks, the authors aim to provide a primer for those now entering this fast-growing field of thalamic neuromodulation for epilepsy. EXPERT OPINION: The thalamus is critically involved with the function of many cortical and subcortical areas, suggesting it may serve as a compelling node for preventing or aborting seizures, and so it has increasingly been targeted for the surgical treatment of epilepsy. As various thalamic neuromodulation strategies for seizure control are developed, there is a need to ground such interventions in a mechanistic, circuit-based framework.

Cortical spheroids display oscillatory network dynamics.

Three-dimensional brain cultures can facilitate the study of central nervous system function and disease, and one of the most important components that they present is neuronal activity on a network level. Here we demonstrate network activity in rodent cortical spheroids while maintaining the networks intact in their 3D state. Networks developed by nine days in culture and became more complex over time. To measure network activity, we imaged neurons in rat and mouse spheroids labelled with a calcium indicator dye, and in mouse spheroids expressing GCaMP. Network activity was evident when we electrically stimulated spheroids, was abolished with glutamatergic blockade, and was altered by GABAergic blockade or partial glutamatergic blockade. We quantified correlations and distances between somas with micron-scale spatial resolution. Spheroids seeded at as few as 4000 cells gave rise to emergent network events, including oscillations. These results are the first demonstration that self-assembled rat and mouse spheroids exhibit network activity consistent with in vivo network events. These results open the door to experiments on neuronal networks that require fewer animals and enable high throughput experiments on network-perturbing alterations in neurons and glia.

A Novel Experiential Quality Improvement Training Program During Residency Improves Quality Improvement Confidence and Knowledge: a Prospective Cohort Study.

OBJECTIVE: The Accreditation Council for Graduate Medical Education (ACGME) mandates resident quality improvement (QI) training to improve patient safety, cost control, and efficiency. Thus, understanding this topic is crucial for early career physicians. This manuscript describes an enhanced, experiential QI curriculum for psychiatry residents and its outcomes. METHODS: Two cohorts of 12 third-year residents completed the curriculum, which included didactics, external resources, and expert guidance through small group project design, implementation, and analysis/presentation. A survey on resident confidence in QI principles and the quality improvement knowledge assessment tool-revised (QIKAT-R) was used before and after curriculum participation. Data were analyzed using parametric descriptive tests and repeated measures general linear models with Benjamini-Hochberg correction for multiple comparisons. RESULTS: Resident confidence in performing seven of the ten steps of QI and QIKAT-R scores significantly improved for both cohorts (p = .011). Eighty-nine percent of residents felt that the curriculum met their goals. CONCLUSIONS: The QI curriculum effectively improved resident QI confidence and knowledge. Residents reported that experiential engagement in the design, implementation, and analysis/presentation of their project was crucial to these achievements. This experiential QI curriculum with resident-generated QI projects addressed ACGME training requirements while integrating QI training directly into the residents' clinical activities, making the QI efforts relevant and meaningful while also achieving ACGME goals.

Early Life Stress Drives Sex-Selective Impairment in Reversal Learning by Affecting Parvalbumin Interneurons in Orbitofrontal Cortex of Mice.

Poverty, displacement, and parental stress represent potent sources of early life stress (ELS). Stress disproportionately affects females, who are at increased risk for stress-related pathologies associated with cognitive impairment. Mechanisms underlying stress-associated cognitive impairment and enhanced risk of females remain unknown. Here, ELS is associated with impaired rule-reversal (RR) learning in females, but not males. Impaired performance was associated with decreased expression and density of interneurons expressing parvalbumin (PV+) in orbitofrontal cortex (OFC), but not other interneuron subtypes. Optogenetic silencing of PV+ interneuron activity in OFC of control mice phenocopied RR learning deficits observed in ELS females. Localization of reversal learning deficits to PV+ interneurons in OFC was confirmed by optogenetic studies in which neurons in medial prefrontal cortex (mPFC) were silenced and associated with select deficits in rule-shift learning. Sex-, cell-, and region-specific effects show altered PV+ interneuron development can be a driver of sex differences in cognitive dysfunction.

In vitro imaging using laser photostimulation with flavoprotein autofluorescence.

Imaging of 300-500 µm mouse brain slices by laser photostimulation with flavoprotein autofluorescence (LFPA) allows the rapid and sensitive mapping of neuronal connectivity. It is accomplished using UV laser-based photo-uncaging of glutamate and imaging neuronal activation by capturing changes in green light (∼520 nm) emitted under blue light (∼460 nm) excitation. This fluorescence is generated by the oxidized form of flavoprotein and is a measure of metabolic activity. LPFA offers several advantages over imaging techniques that rely on dye loading. First, as flavoprotein imaging measures endogenous signals, it avoids the use of heterogeneously loaded and potentially cytotoxic dyes. Second, flavoprotein signals are large (1-20% above baseline), obviating the need for averaging. Third, the use of photostimulation ensures orthodromic neuronal activation and permits the rapid interrogation of multiple stimulation sites of the slice with a high degree of precision (∼50 µm). Here we describe a step-by-step protocol for the incorporation of LPFA into virtually any slice rig, as well as how to do the experiment.

Network variability across human tissue samples in vitro: the problem and a solution.

Slices prepared from cortical tissue that is surgically removed from patients to treat their epilepsy provide an opportunity to directly study the mechanisms underlying ictal activity. However, human in vitro studies have several limitations. One problem that may severely compromise investigations of network properties in these slices relates to how difficult it is to cut the tissue at angles that optimally preserve columnar connectivity. To address this problem, the authors investigated the degree of network variability in human tissue across samples and, within a single tissue sample, across slices cut at different angles using a novel form of optical imaging based on flavoprotein autofluorescence. The authors found a high degree of variability in the spatial extent, degree, and patterning of activation in slices from different samples. They also found variability across the slices cut from a single tissue sample at different angles. Indeed, these results suggest that human tissue samples have disparate degrees of network activity and that abnormal tissue may be confined to clusters of synchronously oscillating domains. Assessing circuit connectivity in a slice a priori will allow investigators to control for the overall degree of slice connectivity and selectively target active (or inactive) areas, making for better-informed comparisons of data.

Specific and nonspecific thalamocortical connectivity in the auditory and somatosensory thalamocortical slices.

Two classes of thalamic nuclei project to either middle layers or upper layers, including layer 1, of the neocortex, and are referred to as 'specific' and 'nonspecific' nuclei, respectively. The electrophysiological properties of the nonspecific nuclei have not been investigated, largely because of the paucity of in vitro slice preparations containing intact nonspecific pathways. In this study, we used flavoprotein autofluorescence imaging to show intact thalamocortical connectivity of nonspecific nuclei in slice preparations of the somatosensory and auditory systems. These preparations will enable the elucidation of electrophysiological properties of nonspecific pathways.

The corticothalamocortical circuit drives higher-order cortex in the mouse.

An unresolved question in neuroscience relates to the extent to which corticothalamocortical circuits emanating from layer 5B are involved in information transfer through the cortical hierarchy. Using a new form of optical imaging in a brain slice preparation, we found that the corticothalamocortical pathway drove robust activity in higher-order somatosensory cortex. When the direct corticocortical pathway was interrupted, secondary somatosensory cortex showed robust activity in response to stimulation of the barrel field in primary somatosensory cortex (S1BF), which was eliminated after subsequently cutting the somatosensory thalamus, suggesting a highly efficacious corticothalamocortical circuit. Furthermore, after chemically inhibiting the thalamus, activation in secondary somatosensory cortex was eliminated, with a subsequent return after washout. Finally, stimulation of layer 5B in S1BF, and not layer 6, drove corticothalamocortical activation. These findings suggest that the corticothalamocortical circuit is a physiologically viable candidate for information transfer to higher-order cortical areas.