All-Optical Electrophysiology Reveals the Role of Lateral Inhibition in Sensory Processing in Cortical Layer 1.

Cortical layer 1 (L1) interneurons have been proposed as a hub for attentional modulation of underlying cortex, but the transformations that this circuit implements are not known. We combined genetically targeted voltage imaging with optogenetic activation and silencing to study the mechanisms underlying sensory processing in mouse barrel cortex L1. Whisker stimuli evoked precisely timed single spikes in L1 interneurons, followed by strong lateral inhibition. A mild aversive stimulus activated cholinergic inputs and evoked a bimodal distribution of spiking responses in L1. A simple conductance-based model that only contained lateral inhibition within L1 recapitulated the sensory responses and the winner-takes-all cholinergic responses, and the model correctly predicted that the network would function as a spatial and temporal high-pass filter for excitatory inputs. Our results demonstrate that all-optical electrophysiology can reveal basic principles of neural circuit function in vivo and suggest an intuitive picture for how L1 transforms sensory and modulatory inputs. VIDEO ABSTRACT.

Hox gene-specific cellular targeting using split intein Trojan exons.

The Trojan exon method, which makes use of intronically inserted T2A-Gal4 cassettes, has been widely used in Drosophila to create thousands of gene-specific Gal4 driver lines. These dual-purpose lines provide genetic access to specific cell types based on their expression of a native gene while simultaneously mutating one allele of the gene to enable loss-of-function analysis in homozygous animals. While this dual use is often an advantage, the truncation mutations produced by Trojan exons are sometimes deleterious in heterozygotes, perhaps by creating translation products with dominant negative effects. Such mutagenic effects can cause developmental lethality as has been observed with genes encoding essential transcription factors. Given the importance of transcription factors in specifying cell type, alternative techniques for generating specific Gal4 lines that target them are required. Here, we introduce a modified Trojan exon method that retains the targeting fidelity and plug-and-play modularity of the original method but mitigates its mutagenic effects by exploiting the self-splicing capabilities of split inteins. "Split Intein Trojan exons" (siTrojans) ensure that the two truncation products generated from the interrupted allele of the native gene are trans-spliced to create a full-length native protein. We demonstrate the efficacy of siTrojans by generating a comprehensive toolkit of Gal4 and Split Gal4 lines for the segmentally expressed Hox transcription factors and illustrate their use in neural circuit mapping by targeting neurons according to their position along the anterior-posterior axis. Both the method and the Hox gene-specific toolkit introduced here should be broadly useful.

Multimodal Interrogation of Ventral Pallidum Projections Reveals Projection-Specific Signatures and Effects on Cocaine Reward.

The ventral pallidum (VP) is a central hub in the reward circuitry with diverse projections that have different behavioral roles attributed mostly to the connectivity with the downstream target. However, different VP projections may represent, as in the striatum, separate neuronal populations that differ in more than just connectivity. In this study, we performed in mice of both sexes a multimodal dissection of four major projections of the VP-to the lateral hypothalamus (VP→LH), ventral tegmental area (VP→VTA), lateral habenula (VP→LHb), and mediodorsal thalamus (VP→MDT)-with physiological, anatomical, genetic, and behavioral tools. We also tested for physiological differences between VP neurons receiving input from nucleus accumbens medium spiny neurons (MSNs) that express either the D1 (D1-MSNs) or the D2 (D2-MSNs) dopamine receptor. We show that each VP projection (1) when inhibited during a cocaine conditioned place preference (CPP) test affects performance differently, (2) receives a different pattern of inputs using rabies retrograde labeling, (3) shows differentially expressed genes using RNA sequencing, and (4) has projection-specific characteristics in excitability and synaptic input characteristics using whole-cell patch clamp. VP→LH and VP→VTA projections have different effects on CPP and show low overlap in circuit tracing experiments, as VP→VTA neurons receive more striatal input, while VP→LH neurons receive more olfactory input. Additionally, VP→VTA neurons are less excitable, while VP→LH neurons are more excitable than the average VP neuron, a difference driven mainly by D2-MSN-responding neurons. Thus, VP→VTA and VP→LH neurons may represent largely distinct populations of VP neurons.

Functional and structural features of L2/3 pyramidal cells continuously covary with pial depth in mouse visual cortex.

Pyramidal cells of neocortical layer 2/3 (L2/3 PyrCs) integrate signals from numerous brain areas and project throughout the neocortex. These PyrCs show pial depth-dependent functional and structural specializations, indicating participation in different functional microcircuits. However, whether these depth-dependent differences result from separable PyrC subtypes or whether their features display a continuum correlated with pial depth is unknown. Here, we assessed the stimulus selectivity, electrophysiological properties, dendritic morphology, and excitatory and inhibitory connectivity across the depth of L2/3 in the binocular visual cortex of mice. We find that the apical, but not the basal dendritic tree structure, varies with pial depth, which is accompanied by variation in subthreshold electrophysiological properties. Lower L2/3 PyrCs receive increased input from L4, while upper L2/3 PyrCs receive a larger proportion of intralaminar input. In vivo calcium imaging revealed a systematic change in visual responsiveness, with deeper PyrCs showing more robust responses than superficial PyrCs. Furthermore, deeper PyrCs are more driven by contralateral than ipsilateral eye stimulation. Importantly, the property value transitions are gradual, and L2/3 PyrCs do not display discrete subtypes based on these parameters. Therefore, L2/3 PyrCs' multiple functional and structural properties systematically correlate with their depth, forming a continuum rather than discrete subtypes.

Organization of Cortical and Thalamic Input to Inhibitory Neurons in Mouse Motor Cortex.

Intracortical inhibition in motor cortex (M1) regulates movement and motor learning. If cortical and thalamic inputs target different inhibitory cell types in different layers, then these afferents may play different roles in regulating M1 output. Using mice of both sexes, we quantified input to two main classes of M1 interneurons, parvalbumin+ (PV+) cells and somatostatin+ (SOM+) cells, using monosynaptic rabies tracing. We then compared anatomic and functional connectivity based on synaptic strength from sensory cortex and thalamus. Functionally, each input innervated M1 interneurons with a unique laminar profile. Different interneuron types were excited in a distinct, complementary manner, suggesting feedforward inhibition proceeds selectively via distinct circuits. Specifically, somatosensory cortex (S1) inputs primarily targeted PV+ neurons in upper layers (L2/3) but SOM+ neurons in middle layers (L5). Somatosensory thalamus [posterior nucleus (PO)] inputs targeted PV+ neurons in middle layers (L5). In contrast to sensory cortical areas, thalamic input to SOM+ neurons was equivalent to that of PV+ neurons. Thus, long-range excitatory inputs target inhibitory neurons in an area and a cell type-specific manner, which contrasts with input to neighboring pyramidal cells. In contrast to feedforward inhibition providing generic inhibitory tone in cortex, circuits are selectively organized to recruit inhibition matched to incoming excitatory circuits.SIGNIFICANCE STATEMENT M1 integrates sensory information and frontal cortical inputs to plan and control movements. Although inputs to excitatory cells are described, the synaptic circuits by which these inputs drive specific types of M1 interneurons are unknown. Anatomical results with rabies tracing and physiological quantification of synaptic strength shows that two main classes of inhibitory cells (PV+ and SOM+ interneurons) both receive substantial cortical and thalamic input, in contrast to interneurons in sensory areas (where thalamic input strongly prefers PV+ interneurons). Further, each input studied targets PV+ and SOM+ interneurons in a different fashion, suggesting that separate, specific circuits exist for recruitment of feedforward inhibition.

Dendritic involvement in inhibition and disinhibition of vulnerable dopaminergic neurons in healthy and pathological conditions.

Dopaminergic neurons in the substantia nigra pars compacta (SNc) differentially degenerate in Parkinson's Disease, with the ventral region degenerating more severely than the dorsal region. Compared with the dorsal neurons, the ventral neurons in the SNc have distinct dendritic morphology, electrophysiological characteristics, and circuit connections with the basal ganglia. These characteristics shape information processing in the ventral SNc and structure the balance of inhibition and disinhibition in the striatonigral circuitry. In this paper, I review foundational studies and recent work comparing the circuitry of the ventral and dorsal SNc neurons and discuss how loss of the ventral neurons early in Parkinson's Disease could affect the overall balance of inhibition and disinhibition of dopamine signals.

Pathway-Specific Remodeling of Thalamostriatal Synapses in a Mouse Model of Parkinson's Disease.

BACKGROUND: The network pathophysiology underlying the motor symptoms of Parkinson's disease (PD) is poorly understood. In models of late-stage PD, there is significant cell-specific remodeling of corticostriatal, axospinous glutamatergic synapses on principal spiny projection neurons (SPNs). Neurons in the centrolateral nucleus (CLN) of the thalamus that relay cerebellar activity to the striatum also make axospinous synapses on SPNs, but the extent to which they are affected in PD has not been definitively characterized. OBJECTIVE: To fill this gap, transgenic mice in which CLN neurons express Cre recombinase were used in conjunction with optogenetic and circuit mapping approaches to determine changes in the CLN projection to SPNs in a unilateral 6-hydroxydopamine (6-OHDA) model of late-stage PD. METHODS: Adeno-associated virus vectors carrying Cre-dependent opsin expression constructs were stereotaxically injected into the CLN of Grp-KH288 mice in which CLN, but not parafascicular nucleus neurons, expressed Cre recombinase. The properties of this projection to identify direct pathway spiny projection neurons (dSPNs) and indirect pathway spiny projection neurons (iSPNs) were then studied in ex vivo brain slices of the dorsolateral striatum from control and 6-OHDA lesioned mice using anatomic, optogenetic, and electrophysiological approaches. RESULTS: Optogenetically evoked excitatory synaptic currents in both iSPNs and dSPNs were reduced in lesioned mice; however, the reduction was significantly greater in dSPNs. In iSPNs, the reduction in evoked responses was attributable to synaptic pruning, because synaptic channelrhodopsin assisted circuit mapping (sCRACm) revealed fewer synapses per cell after lesioning. In contrast, sCRACm mapping of CLN inputs to dSPNs failed to detect any change in synapse abundance in lesioned mice. However, the ratio of currents through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors to those through N-methyl-D-aspartate receptors was significantly reduced in dSPNs. Moreover, the distribution of currents evoked by optical stimulation of individual synapses shifted toward smaller amplitudes by lesioning, suggesting that they had undergone long-term depression. CONCLUSIONS: Taken together, our results demonstrate that the CLN projection to the striatum undergoes a pathway-specific remodeling that could contribute to the circuit imbalance thought to drive the hypokinetic features of PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Neural circuit mechanisms that govern inter-male attack in mice.

Individuals of many species fight with conspecifics to gain access to or defend critical resources essential for survival and reproduction. Such intraspecific fighting is evolutionarily selected for in a species-, sex-, and environment-dependent manner when the value of resources secured exceeds the cost of fighting. One such example is males fighting for chances to mate with females. Recent advances in new tools open up ways to dissect the detailed neural circuit mechanisms that govern intraspecific, particularly inter-male, aggression in the model organism Mus musculus (house mouse). By targeting and functional manipulating genetically defined populations of neurons and their projections, these studies reveal a core neural circuit that controls the display of reactive male-male attacks in mice, from sensory detection to decision making and action selection. Here, we summarize these critical results. We then describe various modulatory inputs that route into the core circuit to afford state-dependent and top-down modulation of inter-male attacks. While reviewing these exciting developments, we note that how the inter-male attack circuit converges or diverges with neural circuits that mediate other forms of social interactions remain not fully understood. Finally, we emphasize the importance of combining circuit, pharmacological, and genetic analysis when studying the neural control of aggression in the future.

Regulation of Lateral Hypothalamic Orexin Activity by Local GABAergic Neurons.

Orexin (also known as hypocretin) neurons are considered a key component of the ascending arousal system. They are active during wakefulness, at which time they drive and maintain arousal, and are silent during sleep. Their activity is controlled by long-range inputs from many sources, as well as by more short-range inputs, including from presumptive GABAergic neurons in the lateral hypothalamus/perifornical region (LH/PF). To characterize local GABAergic input to orexin neurons, we used channelrhodopsin-2-assisted circuit mapping in brain slices. We expressed channelrhodopsin-2 in GABAergic neurons (Vgat+) in the LH/PF and recorded from genetically identified surrounding orexin neurons (LH/PFVgat → Orx). We performed all experiments in mice of either sex. Photostimulation of LH/PF GABAergic neurons inhibited the firing of orexin neurons through the release of GABA, evoking GABAA-mediated IPSCs in orexin neurons. These photo-evoked IPSCs were maintained in the presence of TTX, indicating direct connectivity. Carbachol inhibited LH/PFVgat → Orx input through muscarinic receptors. By contrast, application of orexin was without effect on LH/PFVgat → Orx input, whereas dynorphin, another peptide produced by orexin neurons, inhibited LH/PFVgat → Orx input through κ-opioid receptors. Our results demonstrate that orexin neurons are under inhibitory control by local GABAergic neurons and that this input is depressed by cholinergic signaling, unaffected by orexin and inhibited by dynorphin. We propose that local release of dynorphin may, via collaterals, provides a positive feedback to orexin neurons and that, during wakefulness, orexin neurons may be disinhibited by acetylcholine and by their own release of dynorphin.SIGNIFICANCE STATEMENT The lateral hypothalamus contains important wake-promoting cell populations, including orexin-producing neurons. Intermingled with the orexin neurons, there are other cell populations that selectively discharge during nonrapid eye movement or rapid eye movement sleep. Some of these sleep-active neurons release GABA and are thought to inhibit wake-active neurons during rapid eye movement and nonrapid eye movement sleep. However, this hypothesis had not been tested. Here we show that orexin neurons are inhibited by a local GABAergic input. We propose that this local GABAergic input inhibits orexin neurons during sleep but that, during wakefulness, this input is depressed, possibly through cholinergically mediated disinhibition and/or by release of dynorphin from orexin neurons themselves.

Altered Excitability and Local Connectivity of mPFC-PAG Neurons in a Mouse Model of Neuropathic Pain.

The medial prefrontal cortex (mPFC) plays a major role in both sensory and affective aspects of pain. There is extensive evidence that chronic pain produces functional changes within the mPFC. However, our understanding of local circuit changes to defined subpopulations of mPFC neurons in chronic pain models remains unclear. A major subpopulation of mPFC neurons project to the periaqueductal gray (PAG), which is a key midbrain structure involved in endogenous pain suppression and facilitation. Here, we used laser scanning photostimulation of caged glutamate to map cortical circuits of retrogradely labeled cortico-PAG (CP) neurons in layer 5 (L5) of mPFC in brain slices prepared from male mice having undergone chronic constriction injury (CCI) of the sciatic nerve. Whole-cell recordings revealed a significant reduction in excitability for L5 CP neurons contralateral to CCI in the prelimbic (PL), but not infralimbic (IL), region of mPFC. Circuit mapping showed that excitatory inputs to L5 CP neurons in both PL and IL arose primarily from layer 2/3 (L2/3) and were significantly reduced in CCI mice. Glutamate stimulation of L2/3 and L5 elicited inhibitory inputs to CP neurons in both PL and IL, but only L2/3 input was significantly reduced in CP neurons of CCI mice. We also observed significant reduction in excitability and L2/3 inhibitory input to CP neurons ipsilateral to CCI. These results demonstrating region and laminar specific changes to mPFC-PAG neurons suggest that a unilateral CCI bilaterally alters cortical circuits upstream of the endogenous analgesic network, which may contribute to persistence of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a significant unresolved medical problem that is refractory to traditional analgesics and can negatively affect emotional health. The role of central circuits in mediating the persistent nature of chronic pain remains unclear. Local circuits within the medial prefrontal cortex (mPFC) process ascending pain inputs and can modulate endogenous analgesia via direct projections to the periaqueductal gray (PAG). However, the mechanisms by which chronic pain alters intracortical circuitry of mPFC-PAG neurons are unknown. Here, we report specific changes to local circuits of mPFC-PAG neurons in mice displaying chronic pain behavior after nerve injury. These findings provide evidence for a neural mechanism by which chronic pain disrupts the descending analgesic system via functional changes to cortical circuits.

The Relevance of AgRP Neuron-Derived GABA Inputs to POMC Neurons Differs for Spontaneous and Evoked Release.

Hypothalamic agouti-related peptide (AgRP) neurons potently stimulate food intake, whereas proopiomelanocortin (POMC) neurons inhibit feeding. Whether AgRP neurons exert their orexigenic actions, at least in part, by inhibiting anorexigenic POMC neurons remains unclear. Here, the connectivity between GABA-releasing AgRP neurons and POMC neurons was examined in brain slices from male and female mice. GABA-mediated spontaneous IPSCs (sIPSCs) in POMC neurons were unaffected by disturbing GABA release from AgRP neurons either by cell type-specific deletion of the vesicular GABA transporter or by expression of botulinum toxin in AgRP neurons to prevent vesicle-associated membrane protein 2-dependent vesicle fusion. Additionally, there was no difference in the ability of µ-opioid receptor (MOR) agonists to inhibit sIPSCs in POMC neurons when MORs were deleted from AgRP neurons, and activation of the inhibitory designer receptor hM4Di on AgRP neurons did not affect sIPSCs recorded from POMC neurons. These approaches collectively indicate that AgRP neurons do not significantly contribute to the strong spontaneous GABA input to POMC neurons. Despite these observations, optogenetic stimulation of AgRP neurons reliably produced evoked IPSCs in POMC neurons, leading to the inhibition of POMC neuron firing. Thus, AgRP neurons can potently affect POMC neuron function without contributing a significant source of spontaneous GABA input to POMC neurons. Together, these results indicate that the relevance of GABAergic inputs from AgRP to POMC neurons is state dependent and highlight the need to consider different types of transmitter release in circuit mapping and physiologic regulation.SIGNIFICANCE STATEMENT Agouti-related peptide (AgRP) neurons play an important role in driving food intake, while proopiomelanocortin (POMC) neurons inhibit feeding. Despite the importance of these two well characterized neuron types in maintaining metabolic homeostasis, communication between these cells remains poorly understood. To provide clarity to this circuit, we made electrophysiological recordings from mouse brain slices and found that AgRP neurons do not contribute spontaneously released GABA onto POMC neurons, although when activated with channelrhodopsin AgRP neurons inhibit POMC neurons through GABA-mediated transmission. These findings indicate that the relevance of AgRP to POMC neuron GABA connectivity depends on the state of AgRP neuron activity and suggest that different types of transmitter release should be considered when circuit mapping.

Functional Interrogation of the AgRP Neural Circuits in Control of Appetite, Body Weight, and Behaviors.

Neurons expressing agouti-related protein (AgRP), the so-called hunger neurons, protect mammals from starvation by promoting food-seeking behaviors (Trends Neurosci 36:504-512, 2013). Now an increasing amount of evidence show that these hunger-sensing neurons not only motivate animals to forage and ingest food but also help conserve energy by inhibiting innate processes that demand large amounts of energy such as growth, reproduction, and stress response. It has further been perceived that AgRP neurons transmit signals with negative valence to reward and cognitive centers so as to engage the motivational behavior toward seeking and obtaining foods (Physiol Behav 190:34-42, 2017). Recent advancement in genome editing and neurotechniques unleashed an escalated research of uniquely defined neuronal populations and neural circuits underlying the behavioral regulation of body weight and food responses (Nat Biotechnol 32:347-355, 2014; Proc Natl Acad Sci 113, 2016). In this chapter we will review literatures describing the functional organization of the AgRP circuit and its correlative signaling components that influence ingestive, foraging, motivational, and cognitive responses, a framework that reshaped our thinking toward the new hope and challenges in treatment of obesity and eating disorders.

Current Genetic Techniques in Neural Circuit Control of Feeding and Energy Metabolism.

The current epidemic of obesity and its associated metabolic syndromes imposes unprecedented challenges to our society. Despite intensive research focus on obesity pathogenesis, an effective therapeutic strategy to treat and cure obesity is still lacking. The obesity development is due to a disturbed homeostatic control of feeding and energy expenditure, both of which are controlled by an intricate neural network in the brain. Given the inherent complexity of brain networks in controlling feeding and energy expenditure, the understanding of brain-based pathophysiology for obesity development is limited. One key limiting factor in dissecting neural pathways for feeding and energy expenditure is unavailability of techniques that can be used to effectively reduce the complexity of the brain network to a tractable paradigm, based on which a strong hypothesis can be tested. Excitingly, emerging techniques have been involved to be able to link specific groups of neurons and neural pathways to behaviors (i.e., feeding and energy expenditure). In this chapter, novel techniques especially those based on animal models and viral vector approaches will be discussed. We hope that this chapter will provide readers with a basis that can help to understand the literatures using these techniques and with a guide to apply these exciting techniques to investigate brain mechanisms underlying feeding and energy expenditure.

Neuronal control of locomotor handedness in Drosophila.

Genetically identical individuals display variability in their physiology, morphology, and behaviors, even when reared in essentially identical environments, but there is little mechanistic understanding of the basis of such variation. Here, we investigated whether Drosophila melanogaster displays individual-to-individual variation in locomotor behaviors. We developed a new high-throughout platform capable of measuring the exploratory behavior of hundreds of individual flies simultaneously. With this approach, we find that, during exploratory walking, individual flies exhibit significant bias in their left vs. right locomotor choices, with some flies being strongly left biased or right biased. This idiosyncrasy was present in all genotypes examined, including wild-derived populations and inbred isogenic laboratory strains. The biases of individual flies persist for their lifetime and are nonheritable: i.e., mating two left-biased individuals does not yield left-biased progeny. This locomotor handedness is uncorrelated with other asymmetries, such as the handedness of gut twisting, leg-length asymmetry, and wing-folding preference. Using transgenics and mutants, we find that the magnitude of locomotor handedness is under the control of columnar neurons within the central complex, a brain region implicated in motor planning and execution. When these neurons are silenced, exploratory laterality increases, with more extreme leftiness and rightiness. This observation intriguingly implies that the brain may be able to dynamically regulate behavioral individuality.

Dual-channel circuit mapping reveals sensorimotor convergence in the primary motor cortex.

Cortical cells integrate synaptic input from multiple sources, but how these different inputs are distributed across individual neurons is largely unknown. Differences in input might account for diverse responses in neighboring neurons during behavior. We present a strategy for comparing the strengths of multiple types of input onto the same neuron. We developed methods for independent dual-channel photostimulation of synaptic inputs using ChR2 together with ReaChR, a red-shifted channelrhodopsin. We used dual-channel photostimulation to probe convergence of sensory information in the mouse primary motor cortex. Input from somatosensory cortex and thalamus converges in individual neurons. Similarly, inputs from distinct somatotopic regions of the somatosensory cortex are integrated at the level of single motor cortex neurons. We next developed a ReaChR transgenic mouse under the control of both Flp- and Cre-recombinases that is an effective tool for circuit mapping. Our approach to dual-channel photostimulation enables quantitative comparison of the strengths of multiple pathways across all length scales of the brain.

What genetic model organisms offer the study of behavior and neural circuits.

The past decade has witnessed the development of powerful, genetically encoded tools for manipulating and monitoring neuronal function in freely moving animals. These tools are most readily deployed in genetic model organisms and efforts to map the circuits that govern behavior have increasingly focused on worms, flies, zebrafish, and mice. The traditional virtues of these animals for genetic studies in terms of small size, short generation times, and ease of animal husbandry in a laboratory setting have facilitated rapid progress, and the neural basis of an increasing number of behaviors is being established at cellular resolution in each of these animals. The depth and breadth of this analysis should soon offer a significantly more comprehensive understanding of how the circuitry underlying behavior is organized in particular animals and promises to help answer long-standing questions that have waited for such a brain-wide perspective on nervous system function. The comprehensive understanding achieved in genetic model animals is thus likely to make them into paradigmatic examples that will serve as touchstones for comparisons to understand how behavior is organized in other animals, including ourselves.

Rationally subdividing the fly nervous system with versatile expression reagents.

The ability to image and manipulate specific cell populations in Drosophila enables the investigation of how neural circuits develop and coordinate appropriate motor behaviors. Gal4 lines give genetic access to many types of neurons, but the expression patterns of these reagents are often complex. Here, we present the generation and expression patterns of LexA lines based on the vesicular neurotransmitter transporters and Hox transcription factors. Intersections between these LexA lines and existing Gal4 collections provide a strategy for rationally subdividing complex expression patterns based on neurotransmitter or segmental identity.

Maturation of cortical input to dorsal raphe nucleus increases behavioral persistence in mice.

The ability to persist toward a desired objective is a fundamental aspect of behavioral control whose impairment is implicated in several behavioral disorders. One of the prominent features of behavioral persistence is that its maturation occurs relatively late in development. This is presumed to echo the developmental time course of a corresponding circuit within late-maturing parts of the brain, such as the prefrontal cortex, but the specific identity of the responsible circuits is unknown. Here, we used a genetic approach to describe the maturation of the projection from layer 5 neurons of the neocortex to the dorsal raphe nucleus in mice. Using optogenetic-assisted circuit mapping, we show that this projection undergoes a dramatic increase in synaptic potency between postnatal weeks 3 and 8, corresponding to the transition from juvenile to adult. We then show that this period corresponds to an increase in the behavioral persistence that mice exhibit in a foraging task. Finally, we used a genetic targeting strategy that primarily affected neurons in the medial prefrontal cortex, to selectively ablate this pathway in adulthood and show that mice revert to a behavioral phenotype similar to juveniles. These results suggest that frontal cortical to dorsal raphe input is a critical anatomical and functional substrate of the development and manifestation of behavioral persistence.

A deep-learning strategy to identify cell types across species from high-density extracellular recordings.

High-density probes allow electrophysiological recordings from many neurons simultaneously across entire brain circuits but don't reveal cell type. Here, we develop a strategy to identify cell types from extracellular recordings in awake animals, revealing the computational roles of neurons with distinct functional, molecular, and anatomical properties. We combine optogenetic activation and pharmacology using the cerebellum as a testbed to generate a curated ground-truth library of electrophysiological properties for Purkinje cells, molecular layer interneurons, Golgi cells, and mossy fibers. We train a semi-supervised deep-learning classifier that predicts cell types with greater than 95% accuracy based on waveform, discharge statistics, and layer of the recorded neuron. The classifier's predictions agree with expert classification on recordings using different probes, in different laboratories, from functionally distinct cerebellar regions, and across animal species. Our classifier extends the power of modern dynamical systems analyses by revealing the unique contributions of simultaneously-recorded cell types during behavior.

Long-term adaptation of prefrontal circuits in a mouse model of NMDAR hypofunction.

Pharmacological approaches to induce N-methyl-d-aspartate receptor (NMDAR) hypofunction have been intensively used to understand the aetiology and pathophysiology of schizophrenia. Yet, the precise cellular and molecular mechanisms that relate to brain network dysfunction remain largely unknown. Here, we used a set of complementary approaches to assess the functional network abnormalities present in male mice that underwent a 7-day subchronic phencyclidine (PCP 10 mg/kg, subcutaneously, once daily) treatment. Our data revealed that pharmacological intervention with PCP affected cognitive performance and auditory evoked gamma oscillations in the prefrontal cortex (PFC) mimicking endophenotypes of some schizophrenia patients. We further assessed PFC cellular function and identified altered neuronal intrinsic membrane properties, reduced parvalbumin (PV) immunostaining and diminished inhibition onto L5 PFC pyramidal cells. A decrease in the strength of optogenetically-evoked glutamatergic current at the ventral hippocampus to PFC synapse was also demonstrated, along with a weaker shunt of excitatory transmission by local PFC interneurons. On a macrocircuit level, functional ultrasound measurements indicated compromised functional connectivity within several brain regions particularly involving PFC and frontostriatal circuits. Herein, we reproduced a panel of schizophrenia endophenotypes induced by subchronic PCP application in mice. We further recapitulated electrophysiological signatures associated with schizophrenia and provided an anatomical reference to critical elements in the brain circuitry. Together, our findings contribute to a better understanding of the physiological underpinnings of deficits induced by subchronic NMDAR antagonist regimes and provide a test system for characterization of pharmacological compounds.