Parvalbumin-expressing basal forebrain neurons mediate learning from negative experience.

Parvalbumin (PV)-expressing GABAergic neurons of the basal forebrain (BFPVNs) were proposed to serve as a rapid and transient arousal system, yet their exact role in awake behaviors remains unclear. We performed bulk calcium measurements and electrophysiology with optogenetic tagging from the horizontal limb of the diagonal band of Broca (HDB) while male mice were performing an associative learning task. BFPVNs responded with a distinctive, phasic activation to punishment, but showed slower and delayed responses to reward and outcome-predicting stimuli. Optogenetic inhibition during punishment impaired the formation of cue-outcome associations, suggesting a causal role of BFPVNs in associative learning. BFPVNs received strong inputs from the hypothalamus, the septal complex and the median raphe region, while they synapsed on diverse cell types in key limbic structures, where they broadcasted information about aversive stimuli. We propose that the arousing effect of BFPVNs is recruited by aversive stimuli to serve crucial associative learning functions.

Behavioral, neural and ultrastructural alterations in a graded-dose 6-OHDA mouse model of early-stage Parkinson's disease.

Studying animal models furthers our understanding of Parkinson's disease (PD) pathophysiology by providing tools to investigate detailed molecular, cellular and circuit functions. Different versions of the neurotoxin-based 6-hydroxydopamine (6-OHDA) model of PD have been widely used in rats. However, these models typically assess the result of extensive and definitive dopaminergic lesions that reflect a late stage of PD, leading to a paucity of studies and a consequential gap of knowledge regarding initial stages, in which early interventions would be possible. Additionally, the better availability of genetic tools increasingly shifts the focus of research from rats to mice, but few mouse PD models are available yet. To address these, we characterize here the behavioral, neuronal and ultrastructural features of a graded-dose unilateral, single-injection, striatal 6-OHDA model in mice, focusing on early-stage changes within the first two weeks of lesion induction. We observed early onset, dose-dependent impairments of overall locomotion without substantial deterioration of motor coordination. In accordance, histological evaluation demonstrated a partial, dose-dependent loss of dopaminergic neurons of substantia nigra pars compacta (SNc). Furthermore, electron microscopic analysis revealed degenerative ultrastructural changes in SNc dopaminergic neurons. Our results show that mild ultrastructural and cellular degradation of dopaminergic neurons of the SNc can lead to certain motor deficits shortly after unilateral striatal lesions, suggesting that a unilateral dose-dependent intrastriatal 6-OHDA lesion protocol can serve as a successful model of the early stages of Parkinson's disease in mice.

Cholinergic activity reflects reward expectations and predicts behavioral responses.

Basal forebrain cholinergic neurons (BFCNs) play an important role in associative learning, suggesting that BFCNs may participate in processing stimuli that predict future outcomes. However, the impact of outcome probabilities on BFCN activity remained elusive. Therefore, we performed bulk calcium imaging and recorded spiking of identified cholinergic neurons from the basal forebrain of mice performing a probabilistic Pavlovian cued outcome task. BFCNs responded more to sensory cues that were often paired with reward. Reward delivery also activated BFCNs, with surprising rewards eliciting a stronger response, whereas punishments evoked uniform positive-going responses. We propose that BFCNs differentially weigh predictions of positive and negative reinforcement, reflecting divergent relative salience of forecasting appetitive and aversive outcomes, partially explained by a simple reinforcement learning model of a valence-weighed unsigned prediction error. Finally, the extent of cue-driven cholinergic activation predicted subsequent decision speed, suggesting that the expectation-gated cholinergic firing is instructive to reward-seeking behaviors.

Micro-scale Experimental System Coupled with Fluorescence-based Estimation of Fungal Biomass to Study Utilisation of Plant Substrates.

The degradation capacity and utilisation of complex plant substrates are crucial for the functioning of saprobic fungi and different plant symbionts with fundamental functions in ecosystems. Measuring the growth capacity and biomass of fungi on such systems is a challenging task. We established a new micro-scale experimental setup using substrates made of different plant species and organs as media for fungal growth. We adopted and tested a reliable and simple titration-based method for the estimation of total fungal biomass within the substrates using fluorescence-labelled lectin. We found that the relationship between fluorescence intensity and fungal dry weight was strong and linear but differed among fungi. The effect of the plant organ (i.e. root vs. shoot) used as substrate on fungal growth differed among plant species and between root endophytic fungal species. The novel microscale experimental system is useful for screening the utilisation of different substrates, which can provide insight into the ecological roles and functions of fungi. Furthermore, our fungal biomass estimation method has applications in various fields. As the estimation is based on the fungal cell wall, it measures the total cumulative biomass produced in a certain environment.

Training protocol for probabilistic Pavlovian conditioning in mice using an open-source head-fixed setup.

High throughput, temporally controlled, reproducible quantitative behavioral assays are important for understanding the neural mechanisms underlying behavior. Here, we provide a step-by-step training protocol for a probabilistic Pavlovian conditioning task, where two auditory cues predict probabilistic outcomes with different contingencies. This protocol allows us to study the differential behavioral and neuronal correlates of expected and surprising outcomes. It has been tested in combination with chronic in vivo electrophysiological recordings and optogenetic manipulations in ChAT-Cre and PV-Cre mouse lines. For complete details on the use and execution of this protocol, please refer to Hegedüs et al. (2021).

Differential recruitment of ventral pallidal e-types by behaviorally salient stimuli during Pavlovian conditioning.

The ventral pallidum (VP) is interfacing striatopallidal and limbic circuits, conveying information about salience and valence crucial to adjusting behavior. However, how VP neuron populations with distinct electrophysiological properties (e-types) represent these variables is not fully understood. Therefore, we trained mice on probabilistic Pavlovian conditioning while recording the activity of VP neurons. Many VP neurons responded to punishment (54%), reward (48%), and outcome-predicting auditory stimuli (32%), increasingly differentiating distinct outcome probabilities through learning. We identified e-types based on the presence of bursts or fast rhythmic discharges and found that non-bursting, non-rhythmic neurons were the most sensitive to reward and punishment. Some neurons exhibited distinct responses of their bursts and single spikes, suggesting a multiplexed coding scheme in the VP. Finally, we demonstrate synchronously firing neuron assemblies, particularly responsive to reinforcing stimuli. These results suggest that electrophysiologically defined e-types of the VP differentially participate in transmitting reinforcement signals during learning.

Publisher Correction: Distinct synchronization, cortical coupling and behavioral function of two basal forebrain cholinergic neuron types.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

OPETH: Open Source Solution for Real-Time Peri-Event Time Histogram Based on Open Ephys.

Single cell electrophysiology remains one of the most widely used approaches of systems neuroscience. Decisions made by the experimenter during electrophysiology recording largely determine recording quality, duration of the project and value of the collected data. Therefore, online feedback aiding these decisions can lower monetary and time investment, and substantially speed up projects as well as allow novel studies otherwise not possible due to prohibitively low throughput. Real-time feedback is especially important in studies that involve optogenetic cell type identification by enabling a systematic search for neurons of interest. However, such tools are scarce and limited to costly commercial systems with high degree of specialization, which hitherto prevented wide-ranging benefits for the community. To address this, we present an open-source tool that enables online feedback during electrophysiology experiments and provides a Python interface for the widely used Open Ephys open source data acquisition system. Specifically, our software allows flexible online visualization of spike alignment to external events, called the online peri-event time histogram (OPETH). These external events, conveyed by digital logic signals, may indicate photostimulation time stamps for in vivo optogenetic cell type identification or the times of behaviorally relevant events during in vivo behavioral neurophysiology experiments. Therefore, OPETH allows real-time identification of genetically defined neuron types or behaviorally responsive populations. By allowing "hunting" for neurons of interest, OPETH significantly reduces experiment time and thus increases the efficiency of experiments that combine in vivo electrophysiology with behavior or optogenetic tagging of neurons.

Distinct synchronization, cortical coupling and behavioral function of two basal forebrain cholinergic neuron types.

Basal forebrain cholinergic neurons (BFCNs) modulate synaptic plasticity, cortical processing, brain states and oscillations. However, whether distinct types of BFCNs support different functions remains unclear. Therefore, we recorded BFCNs in vivo, to examine their behavioral functions, and in vitro, to study their intrinsic properties. We identified two distinct types of BFCNs that differ in their firing modes, synchronization properties and behavioral correlates. Bursting cholinergic neurons (Burst-BFCNs) fired synchronously, phase-locked to cortical theta activity and fired precisely timed bursts after reward and punishment. Regular-firing cholinergic neurons (Reg-BFCNs) were found predominantly in the posterior basal forebrain, displayed strong theta rhythmicity and responded with precise single spikes after behavioral outcomes. In an auditory detection task, synchronization of Burst-BFCNs to the auditory cortex predicted the timing of behavioral responses, whereas tone-evoked cortical coupling of Reg-BFCNs predicted correct detections. We propose that differential recruitment of two basal forebrain cholinergic neuron types generates behavior-specific cortical activation.

Brainstem nucleus incertus controls contextual memory formation.

Hippocampal pyramidal cells encode memory engrams, which guide adaptive behavior. Selection of engram-forming cells is regulated by somatostatin-positive dendrite-targeting interneurons, which inhibit pyramidal cells that are not required for memory formation. Here, we found that γ-aminobutyric acid (GABA)-releasing neurons of the mouse nucleus incertus (NI) selectively inhibit somatostatin-positive interneurons in the hippocampus, both monosynaptically and indirectly through the inhibition of their subcortical excitatory inputs. We demonstrated that NI GABAergic neurons receive monosynaptic inputs from brain areas processing important environmental information, and their hippocampal projections are strongly activated by salient environmental inputs in vivo. Optogenetic manipulations of NI GABAergic neurons can shift hippocampal network state and bidirectionally modify the strength of contextual fear memory formation. Our results indicate that brainstem NI GABAergic cells are essential for controlling contextual memories.

Open Source Tools for Temporally Controlled Rodent Behavior Suitable for Electrophysiology and Optogenetic Manipulations.

Understanding how the brain controls behavior requires observing and manipulating neural activity in awake behaving animals. Neuronal firing is timed at millisecond precision. Therefore, to decipher temporal coding, it is necessary to monitor and control animal behavior at the same level of temporal accuracy. However, it is technically challenging to deliver sensory stimuli and reinforcers as well as to read the behavioral responses they elicit with millisecond precision. Presently available commercial systems often excel in specific aspects of behavior control, but they do not provide a customizable environment allowing flexible experimental design while maintaining high standards for temporal control necessary for interpreting neuronal activity. Moreover, delay measurements of stimulus and reinforcement delivery are largely unavailable. We combined microcontroller-based behavior control with a sound delivery system for playing complex acoustic stimuli, fast solenoid valves for precisely timed reinforcement delivery and a custom-built sound attenuated chamber using high-end industrial insulation materials. Together this setup provides a physical environment to train head-fixed animals, enables calibrated sound stimuli and precisely timed fluid and air puff presentation as reinforcers. We provide latency measurements for stimulus and reinforcement delivery and an algorithm to perform such measurements on other behavior control systems. Combined with electrophysiology and optogenetic manipulations, the millisecond timing accuracy will help interpret temporally precise neural signals and behavioral changes. Additionally, since software and hardware provided here can be readily customized to achieve a large variety of paradigms, these solutions enable an unusually flexible design of rodent behavioral experiments.