A competitive inhibitory circuit for selection of active and passive fear responses.

When faced with threat, the survival of an organism is contingent upon the selection of appropriate active or passive behavioural responses. Freezing is an evolutionarily conserved passive fear response that has been used extensively to study the neuronal mechanisms of fear and fear conditioning in rodents. However, rodents also exhibit active responses such as flight under natural conditions. The central amygdala (CEA) is a forebrain structure vital for the acquisition and expression of conditioned fear responses, and the role of specific neuronal sub-populations of the CEA in freezing behaviour is well-established. Whether the CEA is also involved in flight behaviour, and how neuronal circuits for active and passive fear behaviour interact within the CEA, are not yet understood. Here, using in vivo optogenetics and extracellular recordings of identified cell types in a behavioural model in which mice switch between conditioned freezing and flight, we show that active and passive fear responses are mediated by distinct and mutually inhibitory CEA neurons. Cells expressing corticotropin-releasing factor (CRF+) mediate conditioned flight, and activation of somatostatin-positive (SOM+) neurons initiates passive freezing behaviour. Moreover, we find that the balance between conditioned flight and freezing behaviour is regulated by means of local inhibitory connections between CRF+ and SOM+ neurons, indicating that the selection of appropriate behavioural responses to threat is based on competitive interactions between two defined populations of inhibitory neurons, a circuit motif allowing for rapid and flexible action selection.

Compromised trigemino-coerulean coupling in migraine sensitization can be prevented by blocking beta-receptors in the locus coeruleus.

BACKGROUND: Migraine is a disabling neurological disorder, characterized by recurrent headaches. During migraine attacks, individuals often experience sensory symptoms such as cutaneous allodynia which indicates the presence of central sensitization. This sensitization is prevented by oral administration of propranolol, a common first-line medication for migraine prophylaxis, that also normalized the activation of the locus coeruleus (LC), considered as the main origin of descending noradrenergic pain controls. We hypothesized that the basal modulation of trigeminal sensory processing by the locus coeruleus is shifted towards more facilitation in migraineurs and that prophylactic action of propranolol may be attributed to a direct action in LC through beta-adrenergic receptors. METHODS: We used simultaneous in vivo extracellular recordings from the trigeminocervical complex (TCC) and LC of male Sprague-Dawley rats to characterize the relationship between these two areas following repeated meningeal inflammatory soup infusions. Von Frey Hairs and air-puff were used to test periorbital mechanical allodynia. RNAscope and patch-clamp recordings allowed us to examine the action mechanism of propranolol. RESULTS: We found a strong synchronization between TCC and LC spontaneous activities, with a precession of the LC, suggesting the LC drives TCC excitability. Following repeated dural-evoked trigeminal activations, we observed a disruption in coupling of activity within LC and TCC. This suggested an involvement of the two regions' interactions in the development of sensitization. Furthermore, we showed the co-expression of alpha-2A and beta-2 adrenergic receptors within LC neurons. Finally propranolol microinjections into the LC prevented trigeminal sensitization by desynchronizing and decreasing LC neuronal activity. CONCLUSIONS: Altogether these results suggest that trigemino-coerulean coupling plays a pivotal role in migraine progression, and that propranolol's prophylactic effects involve, to some extent, the modulation of LC activity through beta-2 adrenergic receptors. This insight reveals new mechanistic aspects of LC control over sensory processing.

Centralized gaze as an adaptive component of defensive states in humans.

Adequate defensive responding is crucial for mental health but scientifically not well understood. Specifically, it seems difficult to dissociate defense and approach states based on autonomic response patterns. We thus explored the robustness and threat-specificity of recently described oculomotor dynamics upon threat in anticipation of either threatening or rewarding stimuli in humans. While visually exploring naturalistic images, participants (50 per experiment) expected an inevitable, no, or avoidable shock (Experiment 1) or a guaranteed, no, or achievable reward (Experiment 2) that could be averted or gained by a quick behavioural response. We observed reduced heart rate (bradycardia), increased skin conductance, pupil dilation and globally centralized gaze when shocks were inevitable but, more pronouncedly, when they were avoidable. Reward trials were not associated with globally narrowed visual exploration, but autonomic responses resembled characteristics of the threat condition. While bradycardia and concomitant sympathetic activation reflect not only threat-related but also action-preparatory states independent of valence, global centralization of gaze seems a robust phenomenon during the anticipation of avoidable threat. Thus, instead of relying on single readouts, translational research in animals and humans should consider the multi-dimensionality of states in aversive and rewarding contexts, especially when investigating ambivalent, conflicting situations.

Midbrain circuits for defensive behaviour.

Survival in threatening situations depends on the selection and rapid execution of an appropriate active or passive defensive response, yet the underlying brain circuitry is not understood. Here we use circuit-based optogenetic, in vivo and in vitro electrophysiological, and neuroanatomical tracing methods to define midbrain periaqueductal grey circuits for specific defensive behaviours. We identify an inhibitory pathway from the central nucleus of the amygdala to the ventrolateral periaqueductal grey that produces freezing by disinhibition of ventrolateral periaqueductal grey excitatory outputs to pre-motor targets in the magnocellular nucleus of the medulla. In addition, we provide evidence for anatomical and functional interaction of this freezing pathway with long-range and local circuits mediating flight. Our data define the neuronal circuitry underlying the execution of freezing, an evolutionarily conserved defensive behaviour, which is expressed by many species including fish, rodents and primates. In humans, dysregulation of this 'survival circuit' has been implicated in anxiety-related disorders.

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity.

Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding "Sylite"-a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.

Induction of BDNF Expression in Layer II/III and Layer V Neurons of the Motor Cortex Is Essential for Motor Learning.

Motor learning depends on synaptic plasticity between corticostriatal projections and striatal medium spiny neurons. Retrograde tracing from the dorsolateral striatum reveals that both layer II/III and V neurons in the motor cortex express BDNF as a potential regulator of plasticity in corticostriatal projections in male and female mice. The number of these BDNF-expressing cortical neurons and levels of BDNF protein are highest in juvenile mice when adult motor patterns are shaped, while BDNF levels in the adult are low. When mice are trained by physical exercise in the adult, BDNF expression in motor cortex is reinduced, especially in layer II/III projection neurons. Reduced expression of cortical BDNF in 3-month-old mice results in impaired motor learning while space memory is preserved. These findings suggest that activity regulates BDNF expression differentially in layers II/III and V striatal afferents from motor cortex and that cortical BDNF is essential for motor learning.SIGNIFICANCE STATEMENT Motor learning in mice depends on corticostriatal BDNF supply, and regulation of BDNF expression during motor learning is highest in corticostriatal projection neurons in cortical layer II/III.

The evolution of dystonia-like movements in TOR1A rats after transient nerve injury is accompanied by dopaminergic dysregulation and abnormal oscillatory activity of a central motor network.

TOR1A is the most common inherited form of dystonia with still unclear pathophysiology and reduced penetrance of 30-40%. ∆ETorA rats mimic the TOR1A disease by expression of the human TOR1A mutation without presenting a dystonic phenotype. We aimed to induce dystonia-like symptoms in male ∆ETorA rats by peripheral nerve injury and to identify central mechanism of dystonia development. Dystonia-like movements (DLM) were assessed using the tail suspension test and implementing a pipeline of deep learning applications. Neuron numbers of striatal parvalbumin+, nNOS+, calretinin+, ChAT+ interneurons and Nissl+ cells were estimated by unbiased stereology. Striatal dopaminergic metabolism was analyzed via in vivo microdialysis, qPCR and western blot. Local field potentials (LFP) were recorded from the central motor network. Deep brain stimulation (DBS) of the entopeduncular nucleus (EP) was performed. Nerve-injured ∆ETorA rats developed long-lasting DLM over 12 weeks. No changes in striatal structure were observed. Dystonic-like ∆ETorA rats presented a higher striatal dopaminergic turnover and stimulus-induced elevation of dopamine efflux compared to the control groups. Higher LFP theta power in the EP of dystonic-like ∆ETorA compared to wt rats was recorded. Chronic EP-DBS over 3 weeks led to improvement of DLM. Our data emphasizes the role of environmental factors in TOR1A symptomatogenesis. LFP analyses indicate that the pathologically enhanced theta power is a physiomarker of DLM. This TOR1A model replicates key features of the human TOR1A pathology on multiple biological levels and is therefore suited for further analysis of dystonia pathomechanism.

Neuronal circuits for fear and anxiety.

Decades of research has identified the brain areas that are involved in fear, fear extinction, anxiety and related defensive behaviours. Newly developed genetic and viral tools, optogenetics and advanced in vivo imaging techniques have now made it possible to characterize the activity, connectivity and function of specific cell types within complex neuronal circuits. Recent findings that have been made using these tools and techniques have provided mechanistic insights into the exquisite organization of the circuitry underlying internal defensive states. This Review focuses on studies that have used circuit-based approaches to gain a more detailed, and also more comprehensive and integrated, view on how the brain governs fear and anxiety and how it orchestrates adaptive defensive behaviours.

Amygdala interneuron subtypes control fear learning through disinhibition.

Learning is mediated by experience-dependent plasticity in neuronal circuits. Activity in neuronal circuits is tightly regulated by different subtypes of inhibitory interneurons, yet their role in learning is poorly understood. Using a combination of in vivo single-unit recordings and optogenetic manipulations, we show that in the mouse basolateral amygdala, interneurons expressing parvalbumin (PV) and somatostatin (SOM) bidirectionally control the acquisition of fear conditioning--a simple form of associative learning--through two distinct disinhibitory mechanisms. During an auditory cue, PV(+) interneurons are excited and indirectly disinhibit the dendrites of basolateral amygdala principal neurons via SOM(+) interneurons, thereby enhancing auditory responses and promoting cue-shock associations. During an aversive footshock, however, both PV(+) and SOM(+) interneurons are inhibited, which boosts postsynaptic footshock responses and gates learning. These results demonstrate that associative learning is dynamically regulated by the stimulus-specific activation of distinct disinhibitory microcircuits through precise interactions between different subtypes of local interneurons.

A disinhibitory microcircuit for associative fear learning in the auditory cortex.

Learning causes a change in how information is processed by neuronal circuits. Whereas synaptic plasticity, an important cellular mechanism, has been studied in great detail, we know much less about how learning is implemented at the level of neuronal circuits and, in particular, how interactions between distinct types of neurons within local networks contribute to the process of learning. Here we show that acquisition of associative fear memories depends on the recruitment of a disinhibitory microcircuit in the mouse auditory cortex. Fear-conditioning-associated disinhibition in auditory cortex is driven by foot-shock-mediated cholinergic activation of layer 1 interneurons, in turn generating inhibition of layer 2/3 parvalbumin-positive interneurons. Importantly, pharmacological or optogenetic block of pyramidal neuron disinhibition abolishes fear learning. Together, these data demonstrate that stimulus convergence in the auditory cortex is necessary for associative fear learning to complex tones, define the circuit elements mediating this convergence and suggest that layer-1-mediated disinhibition is an important mechanism underlying learning and information processing in neocortical circuits.

Measuring Heart Rate in Freely Moving Mice.

Measuring autonomic parameters like heart rate in behaving mice is not only a standard procedure in cardiovascular research but is applied in many other interdisciplinary research fields. With an electrocardiogram (ECG), the heart rate can be measured by deriving the electrical potential between subcutaneously implanted wires across the chest. This is an inexpensive and easy-to-implement technique and particularly suited for repeated recordings of up to eight weeks. This protocol describes a step-by-step guide for manufacturing the needed equipment, performing the surgical procedure of electrode implantation, and processing of acquired data, yielding accurate and reliable detection of heartbeats and calculation of heart rate (HR). We provide MATLAB graphical user interface (GUI)-based tools to extract and start processing the acquired data without a lot of coding knowledge. Finally, based on an example of a data set acquired in the context of defensive reactions, we discuss the potential and pitfalls in analyzing HR data. Key features • Next to surgical steps, the protocol provides a detailed description of manufacturing custom-made ECG connectors and a shielded, light-weight patch cable. • Suitable for recordings in which signal quality is challenged by ambient noise or noise from other recording devices. • Described for 2-channel differential recording but easily expandable to record from more channels. • Includes a summary of potential analysis methods and a discussion on the interpretation of HR dynamics in the case study of fear states.

Cortagine infused into the medial prefrontal cortex attenuates predator-induced defensive behaviors and Fos protein production in selective nuclei of the amygdala in male CD1 mice.

Corticotropin-releasing factor (CRF) plays an essential role in coordinating the autonomic, endocrine and behavioral responses to stressors. In this study, we investigated the role of CRF within the medial prefrontal cortex (mPFC) in modulating unconditioned defensive behaviors, by examining the effects of microinfusing cortagine a selective type-1 CRF receptor (CRF1) agonist, or acidic-astressin a preferential CRF1 antagonist, into the mPFC in male CD-1 mice exposed to a live predator (rat exposure test--RET). Cortagine microinfusions significantly reduced several indices of defense, including avoidance and freezing, suggesting a specific role for CRF1 within the infralimbic and prelimbic regions of the mPFC in modulating unconditioned behavioral responsivity to a predator. In contrast, microinfusions of acidic-astressin failed to alter defensive behaviors during predator exposure in the RET. Cortagine microinfusions also reduced Fos protein production in the medial, central and basomedial, but not basolateral subnuclei of the amygdala in mice exposed to the rat predatory threat stimulus. These results suggest that CRF1 activation within the mPFC attenuates predator-induced unconditioned anxiety-like defensive behaviors, likely via inhibition of specific amygdalar nuclei. Furthermore, the present findings suggest that the mPFC represents a unique neural region whereby activation of CRF1 produces behavioral effects that contrast with those elicited following systemic administration of CRF1 agonists.

Cerebellar contribution to the regulation of defensive states.

Despite fine tuning voluntary movement as the most prominently studied function of the cerebellum, early human studies suggested cerebellar involvement emotion regulation. Since, the cerebellum has been associated with various mood and anxiety-related conditions. Research in animals provided evidence for cerebellar contributions to fear memory formation and extinction. Fear and anxiety can broadly be referred to as defensive states triggered by threat and characterized by multimodal adaptations such as behavioral and cardiac responses integrated into an intricately orchestrated defense reaction. This is mediated by an evolutionary conserved, highly interconnected network of defense-related structures with functional connections to the cerebellum. Projections from the deep cerebellar nucleus interpositus to the central amygdala interfere with retention of fear memory. Several studies uncovered tight functional connections between cerebellar deep nuclei and pyramis and the midbrain periaqueductal grey. Specifically, the fastigial nucleus sends direct projections to the ventrolateral PAG to mediate fear-evoked innate and learned freezing behavior. The cerebellum also regulates cardiovascular responses such as blood pressure and heart rate-effects dependent on connections with medullary cardiac regulatory structures. Because of the integrated, multimodal nature of defensive states, their adaptive regulation has to be highly dynamic to enable responding to a moving threatening stimulus. In this, predicting threat occurrence are crucial functions of calculating adequate responses. Based on its role in prediction error generation, its connectivity to limbic regions, and previous results on a role in fear learning, this review presents the cerebellum as a regulator of integrated cardio-behavioral defensive states.

Integrated cardio-behavioral responses to threat define defensive states.

Fear and anxiety are brain states that evolved to mediate defensive responses to threats. The defense reaction includes multiple interacting behavioral, autonomic and endocrine adjustments, but their integrative nature is poorly understood. In particular, although threat has been associated with various cardiac changes, there is no clear consensus regarding the relevance of these changes for the integrated defense reaction. Here we identify rapid microstates that are associated with specific behaviors and heart rate dynamics, which are affected by long-lasting macrostates and reflect context-dependent threat levels. In addition, we demonstrate that one of the most commonly used defensive behavioral responses-freezing as measured by immobility-is part of an integrated cardio-behavioral microstate mediated by Chx10+ neurons in the periaqueductal gray. Our framework for systematic integration of cardiac and behavioral readouts presents the basis for a better understanding of complex neural defensive states and their associated systemic functions.

Deep learning-enabled segmentation of ambiguous bioimages with deepflash2.

Bioimages frequently exhibit low signal-to-noise ratios due to experimental conditions, specimen characteristics, and imaging trade-offs. Reliable segmentation of such ambiguous images is difficult and laborious. Here we introduce deepflash2, a deep learning-enabled segmentation tool for bioimage analysis. The tool addresses typical challenges that may arise during the training, evaluation, and application of deep learning models on ambiguous data. The tool's training and evaluation pipeline uses multiple expert annotations and deep model ensembles to achieve accurate results. The application pipeline supports various use-cases for expert annotations and includes a quality assurance mechanism in the form of uncertainty measures. Benchmarked against other tools, deepflash2 offers both high predictive accuracy and efficient computational resource usage. The tool is built upon established deep learning libraries and enables sharing of trained model ensembles with the research community. deepflash2 aims to simplify the integration of deep learning into bioimage analysis projects while improving accuracy and reliability.

Current approaches to characterize micro- and macroscale circuit mechanisms of Parkinson's disease in rodent models.

Accelerating technological progress in experimental neuroscience is increasing the scale as well as specificity of both observational and perturbational approaches to study circuit physiology. While these techniques have also been used to study disease mechanisms, a wider adoption of these approaches in the field of experimental neurology would greatly facilitate our understanding of neurological dysfunctions and their potential treatments at cellular and circuit level. In this review, we will introduce classic and novel methods ranging from single-cell electrophysiological recordings to state-of-the-art calcium imaging and cell-type specific optogenetic or chemogenetic stimulation. We will focus on their application in rodent models of Parkinson's disease while also presenting their use in the context of motor control and basal ganglia function. By highlighting the scope and limitations of each method, we will discuss how they can be used to study pathophysiological mechanisms at local and global circuit levels and how novel frameworks can help to bridge these scales.

Rodent models for gait network disorders in Parkinson's disease - a translational perspective.

Gait impairments in Parkinson's disease remain a scientific and therapeutic challenge. The advent of new deep brain stimulation (DBS) devices capable of recording brain activity from chronically implanted electrodes has fostered new studies of gait in freely moving patients. The hope is to identify gait-related neural biomarkers and improve therapy using closed-loop DBS. In this context, animal models offer a wealth of opportunities to investigate gait network impairments at multiple biological scales and address unresolved questions from clinical research. Yet, the contribution of rodent models to the development of future neuromodulation therapies will rely on translational validity. In this review, we summarize the most effective strategies to model parkinsonian gait in rodents. We discuss how clinical observations have inspired targeted brain lesions in animal models, and whether resulting motor deficits and network oscillations match recent findings in humans. We conclude that future research should incorporate behavioral tests with increased cognitive demands to potentially uncover episodic gait impairments in rodents. Additionally, we expect that basic research will benefit from the implementation of evolving signal processing strategies from clinical research. This coevolution of translational research may contribute to the future optimization of gait therapy in Parkinson's disease.

Circuits for State-Dependent Modulation of Locomotion.

Brain-wide neural circuits enable bi- and quadrupeds to express adaptive locomotor behaviors in a context- and state-dependent manner, e.g., in response to threats or rewards. These behaviors include dynamic transitions between initiation, maintenance and termination of locomotion. Advances within the last decade have revealed an intricate coordination of these individual locomotion phases by complex interaction of multiple brain circuits. This review provides an overview of the neural basis of state-dependent modulation of locomotion initiation, maintenance and termination, with a focus on insights from circuit-centered studies in rodents. The reviewed evidence indicates that a brain-wide network involving excitatory circuit elements connecting cortex, midbrain and medullary areas appears to be the common substrate for the initiation of locomotion across different higher-order states. Specific network elements within motor cortex and the mesencephalic locomotor region drive the initial postural adjustment and the initiation of locomotion. Microcircuits of the basal ganglia, by implementing action-selection computations, trigger goal-directed locomotion. The initiation of locomotion is regulated by neuromodulatory circuits residing in the basal forebrain, the hypothalamus, and medullary regions such as locus coeruleus. The maintenance of locomotion requires the interaction of an even larger neuronal network involving motor, sensory and associative cortical elements, as well as defined circuits within the superior colliculus, the cerebellum, the periaqueductal gray, the mesencephalic locomotor region and the medullary reticular formation. Finally, locomotor arrest as an important component of defensive emotional states, such as acute anxiety, is mediated via a network of survival circuits involving hypothalamus, amygdala, periaqueductal gray and medullary premotor centers. By moving beyond the organizational principle of functional brain regions, this review promotes a circuit-centered perspective of locomotor regulation by higher-order states, and emphasizes the importance of individual network elements such as cell types and projection pathways. The realization that dysfunction within smaller, identifiable circuit elements can affect the larger network function supports more mechanistic and targeted therapeutic intervention in the treatment of motor network disorders.

Central amygdala micro-circuits mediate fear extinction.

Fear extinction is an adaptive process whereby defensive responses are attenuated following repeated experience of prior fear-related stimuli without harm. The formation of extinction memories involves interactions between various corticolimbic structures, resulting in reduced central amygdala (CEA) output. Recent studies show, however, the CEA is not merely an output relay of fear responses but contains multiple neuronal subpopulations that interact to calibrate levels of fear responding. Here, by integrating behavioural, in vivo electrophysiological, anatomical and optogenetic approaches in mice we demonstrate that fear extinction produces reversible, stimulus- and context-specific changes in neuronal responses to conditioned stimuli in functionally and genetically defined cell types in the lateral (CEl) and medial (CEm) CEA. Moreover, we show these alterations are absent when extinction is deficient and that selective silencing of protein kinase C delta-expressing (PKCδ) CEl neurons impairs fear extinction. Our findings identify CEA inhibitory microcircuits that act as critical elements within the brain networks mediating fear extinction.

Maternal separation modulates short-term behavioral and physiological indices of the stress response.

Early-life stress produces an anxiogenic profile in adulthood, presumably by activating the otherwise quiescent hypothalamic-pituitary-adrenal (HPA) axis during the vulnerable 'stress hyporesponsive period'. While the long-term effects of such early-life manipulations have been extensively characterized, little is known of the short-term effects. Here, we compared the short-term effects of two durations of maternal separation stress and one unseparated group (US) on behavioral and physiological indices of the stress response in rat pups. Separations included 3h on each of 12days, from postnatal day (PND) 2 to 13 (MS2-13) and 3days of daily, 6-h separation from PND11-13 (MS11-13). On PND14 (Experiment 1), both MS2-13 and MS11-13 produced marked reductions in freezing toward an adult male conspecific along with reduced levels of glucocorticoid type 2 (GR) and CRF type-1 (CRF(1)) receptor mRNA in the hippocampus. Group MS2-13 but not MS11-13 produced deficits in stressor-induced corticosterone secretion, accompanied by reductions in body weight. Our results suggest that GR and/or CRF(1) levels, not solely the magnitude of corticosterone secretion, may be involved in the modulation of freezing. In a second experiment, we aimed to extend these findings by testing male and female separated and unseparated pups' unconditioned defensive behaviors to cat odor on PND26, and subsequent cue+context conditioning and extinction throughout postnatal days 27-32. Our results show that maternal separation produced reductions in unconditioned freezing on PND26, with MS2-13 showing stronger deficits than MS11-13. However, separation did not affect any other defensive behaviors. Furthermore, separated rats failed to show conditioned freezing, although they did avoid the no-odor block conditioned cue. There were no sex differences other than weight. We suggest that maternal separation may have produced these changes by disrupting normal development of hippocampal regions involved in olfactory-mediated freezing, not in mechanisms of learning and memory per se. These findings may have direct relevance for understanding the mechanisms by which early-life adverse experiences produce short-term and lasting psychopathologies.