Cerebellar state estimation enables resilient coupling across behavioural domains.

Cerebellar computations are necessary for fine behavioural control and may rely on internal models for estimation of behaviourally relevant states. Here, we propose that the central cerebellar function is to estimate how states interact with each other, and to use these estimates to coordinates extra-cerebellar neuronal dynamics underpinning a range of interconnected behaviours. To support this claim, we describe a cerebellar model for state estimation that includes state interactions, and link this model with the neuronal architecture and dynamics observed empirically. This is formalised using the free energy principle, which provides a dual perspective on a system in terms of both the dynamics of its physical-in this case neuronal-states, and the inferential process they entail. As a demonstration of this proposal, we simulate cerebellar-dependent synchronisation of whisking and respiration, which are known to be tightly coupled in rodents, as well as limb and tail coordination during locomotion. In summary, we propose that the ubiquitous involvement of the cerebellum in behaviour arises from its central role in precisely coupling behavioural domains.

Cerebro-cerebellar networks facilitate learning through feedback decoupling.

Behavioural feedback is critical for learning in the cerebral cortex. However, such feedback is often not readily available. How the cerebral cortex learns efficiently despite the sparse nature of feedback remains unclear. Inspired by recent deep learning algorithms, we introduce a systems-level computational model of cerebro-cerebellar interactions. In this model a cerebral recurrent network receives feedback predictions from a cerebellar network, thereby decoupling learning in cerebral networks from future feedback. When trained in a simple sensorimotor task the model shows faster learning and reduced dysmetria-like behaviours, in line with the widely observed functional impact of the cerebellum. Next, we demonstrate that these results generalise to more complex motor and cognitive tasks. Finally, the model makes several experimentally testable predictions regarding cerebro-cerebellar task-specific representations over learning, task-specific benefits of cerebellar predictions and the differential impact of cerebellar and inferior olive lesions. Overall, our work offers a theoretical framework of cerebro-cerebellar networks as feedback decoupling machines.

Histamine Release in the Prefrontal Cortex Excites Fast-Spiking Interneurons while GABA Released from the Same Axons Inhibits Pyramidal Cells.

We studied how histamine and GABA release from axons originating from the hypothalamic tuberomammillary nucleus (TMN) and projecting to the prefrontal cortex (PFC) influence circuit processing. We optostimulated histamine/GABA from genetically defined TMN axons that express the histidine decarboxylase gene (TMNHDC axons). Whole-cell recordings from PFC neurons in layer 2/3 of prelimbic, anterior cingulate, and infralimbic regions were used to monitor excitability before and after optostimulated histamine/GABA release in male and female mice. We found that histamine-GABA release influences the PFC through actions on distinct neuronal types: the histamine stimulates fast-spiking interneurons; and the released GABA enhances tonic (extrasynaptic) inhibition on pyramidal cells (PyrNs). For fast-spiking nonaccommodating interneurons, histamine released from TMNHDC axons induced additive gain changes, which were blocked by histamine H1 and H2 receptor antagonists. The excitability of other fast-spiking interneurons in the PFC was not altered. In contrast, the GABA released from TMNHDC axons predominantly produced divisive gain changes in PyrNs, increasing their resting input conductance, and decreasing the slope of the input-output relationship. This inhibitory effect on PyrNs was not blocked by histamine receptor antagonists but was blocked by GABAA receptor antagonists. Across the adult life span (from 3 to 18 months of age), the GABA released from TMNHDC axons in the PFC inhibited PyrN excitability significantly more in older mice. For individuals who maintain cognitive performance into later life, the increases in TMNHDC GABA modulation of PyrNs during aging could enhance information processing and be an adaptive mechanism to buttress cognition.SIGNIFICANCE STATEMENT The hypothalamus controls arousal state by releasing chemical neurotransmitters throughout the brain to modulate neuronal excitability. Evidence is emerging that the release of multiple types of neurotransmitters may have opposing actions on neuronal populations in key cortical regions. This study demonstrates for the first time that the neurotransmitters histamine and GABA are released in the prefrontal cortex from axons originating from the tuberomammillary nucleus of the hypothalamus. This work demonstrates how hypothalamic modulation of neuronal excitability is maintained throughout adult life, highlighting an unexpected aspect of the aging process that may help maintain cognitive abilities.

Refinements to rodent head fixation and fluid/food control for neuroscience.

The use of head fixation in mice is increasingly common in research, its use having initially been restricted to the field of sensory neuroscience. Head restraint has often been combined with fluid control, rather than food restriction, to motivate behaviour, but this too is now in use for both restrained and non-restrained animals. Despite this, there is little guidance on how best to employ these techniques to optimise both scientific outcomes and animal welfare. This article summarises current practices and provides recommendations to improve animal wellbeing and data quality, based on a survey of the community, literature reviews, and the expert opinion and practical experience of an international working group convened by the UK's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). Topics covered include head fixation surgery and post-operative care, habituation to restraint, and the use of fluid/food control to motivate performance. We also discuss some recent developments that may offer alternative ways to collect data from large numbers of behavioural trials without the need for restraint. The aim is to provide support for researchers at all levels, animal care staff, and ethics committees to refine procedures and practices in line with the refinement principle of the 3Rs.

Psilocybin reduces low frequency oscillatory power and neuronal phase-locking in the anterior cingulate cortex of awake rodents.

Psilocybin is a hallucinogenic compound that is showing promise in the ability to treat neurological conditions such as depression and post-traumatic stress disorder. There have been several investigations into the neural correlates of psilocybin administration using non-invasive methods, however, there has yet to be an invasive study of the mechanism of action in awake rodents. Using multi-unit extracellular recordings, we recorded local field potential and spiking activity from populations of neurons in the anterior cingulate cortex of awake mice during the administration of psilocybin (2 mg/kg). The power of low frequency bands in the local field potential was found to significantly decrease in response to psilocybin administration, whilst gamma band activity trended towards an increase. The population firing rate was found to increase overall, with just under half of individual neurons showing a significant increase. Psilocybin significantly decreased the level of phase modulation of cells with each neural frequency band except high-gamma oscillations, consistent with a desynchronization of cortical populations. Furthermore, bursting behavior was altered in a subset of cells, with both positive and negative changes in the rate of bursting. Neurons that increased their burst firing following psilocybin administration were highly likely to transition from a phase-modulated to a phase unmodulated state. Taken together, psilocybin reduces low frequency oscillatory power, increases overall firing rates and desynchronizes local neural activity. These findings are consistent with dissolution of the default mode network under psilocybin, and may be indicative of disruption of top-down processing in the acute psychedelic state.

Accounting for uncertainty: inhibition for neural inference in the cerebellum.

Sensorimotor coordination is thought to rely on cerebellar-based internal models for state estimation, but the underlying neural mechanisms and specific contribution of the cerebellar components is unknown. A central aspect of any inferential process is the representation of uncertainty or conversely precision characterizing the ensuing estimates. Here, we discuss the possible contribution of inhibition to the encoding of precision of neural representations in the granular layer of the cerebellar cortex. Within this layer, Golgi cells influence excitatory granule cells, and their action is critical in shaping information transmission downstream to Purkinje cells. In this review, we equate the ensuing excitation-inhibition balance in the granular layer with the outcome of a precision-weighted inferential process, and highlight the physiological characteristics of Golgi cell inhibition that are consistent with such computations.

Using Temporal Expectation to Assess Auditory Streaming in Mice.

Auditory streaming is the process by which environmental sound is segregated into discrete perceptual objects. The auditory system has a remarkable capability in this regard as revealed in psychophysical experiments in humans and other primates. However, little is known about the underlying neuronal mechanisms, in part because of the lack of suitable behavioural paradigms in non-primate species. The mouse is an increasingly popular model for studying the neural mechanisms of perception and action because of the range of molecular tools enabling precise manipulation of neural circuitry. Here we present a novel behavioural task that can be used to assess perceptual aspects of auditory streaming in head-fixed mice. Animals were trained to detect a target sound in a one of two simultaneously presented, isochronous pure tone sequences. Temporal expectation was manipulated by presenting the target sound in a particular stream either early (~2 s) or late (~4 s) with respect to trial onset in blocks of 25-30 trials. Animals reached high performance on this task (d' > 1 overall), and notably their false alarms were very instructive of their behavioural state. Indeed, false alarm timing was markedly delayed for late blocks compared to early ones, indicating that the animals associated a different context to an otherwise identical stimulus. More finely, we observed that the false alarms were timed to the onset of the sounds present in the target stream. This suggests that the animals could selectively follow the target stream despite the presence of a distractor stream. Extracellular electrophysiological recordings during the task revealed that sound processing is flexibly modulated in a manner consistent with the optimisation of behavioural outcome. Together, these results indicate that the perceptual streaming can be inferred via the timing of false alarms in mice, and provide a new paradigm with which to investigate neuronal mechanisms of selective attention.

Combining mGRASP and Optogenetics Enables High-Resolution Functional Mapping of Descending Cortical Projections.

We have applied optogenetics and mGRASP, a light microscopy technique that labels synaptic contacts, to map the number and strength of defined corticocollicular (CC) connections. Using mGRASP, we show that CC projections form small, medium, and large synapses, and both the number and the distribution of synapse size vary among the IC regions. Using optogenetics, we show that low-frequency stimulation of CC axons expressing channelrhodopsin produces prolonged elevations of the CC miniature EPSC (mEPSC) rate. Functional analysis of CC mEPSCs reveals small-, medium-, and large-amplitude events that mirror the synaptic distributions observed with mGRASP. Our results reveal that descending ipsilateral projections dominate CC feedback via an increased number of large synaptic contacts, especially onto the soma of IC neurons. This study highlights the feasibility of combining microscopy (i.e., mGRASP) and optogenetics to reveal synaptic weighting of defined projections at the level of single neurons, enabling functional connectomic mapping in diverse neural circuits.

ON-OFF receptive fields in auditory cortex diverge during development and contribute to directional sweep selectivity.

Neurons in the auditory cortex exhibit distinct frequency tuning to the onset and offset of sounds, but the cause and significance of ON and OFF receptive field (RF) organisation are not understood. Here we demonstrate that distinct ON and OFF frequency tuning is largely absent in immature mouse auditory cortex and is thus a consequence of cortical development. Simulations using a novel implementation of a standard Hebbian plasticity model show that the natural alternation of sound onset and offset is sufficient for the formation of non-overlapping adjacent ON and OFF RFs in cortical neurons. Our model predicts that ON/OFF RF arrangement contributes towards direction selectivity to frequency-modulated tone sweeps, which we confirm by neuronal recordings. These data reveal that a simple and universally accepted learning rule can explain the organisation of ON and OFF RFs and direction selectivity in the developing auditory cortex.

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology.

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum.

Serial processing of kinematic signals by cerebellar circuitry during voluntary whisking.

Purkinje cells (PCs) in Crus 1 represent whisker movement via linear changes in firing rate, but the circuit mechanisms underlying this coding scheme are unknown. Here we examine the role of upstream inputs to PCs-excitatory granule cells (GCs) and inhibitory molecular layer interneurons-in processing of whisking signals. Patch clamp recordings in GCs reveal that movement is accompanied by changes in mossy fibre input rate that drive membrane potential depolarisation and high-frequency bursting activity at preferred whisker angles. Although individual GCs are narrowly tuned, GC populations provide linear excitatory drive across a wide range of movement. Molecular layer interneurons exhibit bidirectional firing rate changes during whisking, similar to PCs. Together, GC populations provide downstream PCs with linear representations of volitional movement, while inhibitory networks invert these signals. The exquisite sensitivity of neurons at each processing stage enables faithful propagation of kinematic representations through the cerebellum.Cerebellar Purkinje cells (PCs) linearly encode whisker position but the precise circuit mechanisms that generate these signals are not well understood. Here the authors use patch clamp recordings to show that selective tuning of granule cell inputs and bidirectional tuning of interneuron inputs are required to generate the kinematic representations in PCs.

Comodulation Enhances Signal Detection via Priming of Auditory Cortical Circuits.

Acoustic environments are composed of complex overlapping sounds that the auditory system is required to segregate into discrete perceptual objects. The functions of distinct auditory processing stations in this challenging task are poorly understood. Here we show a direct role for mouse auditory cortex in detection and segregation of acoustic information. We measured the sensitivity of auditory cortical neurons to brief tones embedded in masking noise. By altering spectrotemporal characteristics of the masker, we reveal that sensitivity to pure tone stimuli is strongly enhanced in coherently modulated broadband noise, corresponding to the psychoacoustic phenomenon comodulation masking release. Improvements in detection were largest following priming periods of noise alone, indicating that cortical segregation is enhanced over time. Transient opsin-mediated silencing of auditory cortex during the priming period almost completely abolished these improvements, suggesting that cortical processing may play a direct and significant role in detection of quiet sounds in noisy environments. SIGNIFICANCE STATEMENT: Auditory systems are adept at detecting and segregating competing sound sources, but there is little direct evidence of how this process occurs in the mammalian auditory pathway. We demonstrate that coherent broadband noise enhances signal representation in auditory cortex, and that prolonged exposure to noise is necessary to produce this enhancement. Using optogenetic perturbation to selectively silence auditory cortex during early noise processing, we show that cortical processing plays a crucial role in the segregation of competing sounds.

The cerebellum linearly encodes whisker position during voluntary movement.

Active whisking is an important model sensorimotor behavior, but the function of the cerebellum in the rodent whisker system is unknown. We have made patch clamp recordings from Purkinje cells in vivo to identify whether cerebellar output encodes kinematic features of whisking including the phase and set point. We show that Purkinje cell spiking activity changes strongly during whisking bouts. On average, the changes in simple spike rate coincide with or slightly precede movement, indicating that the synaptic drive responsible for these changes is predominantly of efferent (motor) rather than re-afferent (sensory) origin. Remarkably, on-going changes in simple spike rate provide an accurate linear read-out of whisker set point. Thus, despite receiving several hundred thousand discrete synaptic inputs across a non-linear dendritic tree, Purkinje cells integrate parallel fiber input to generate precise information about whisking kinematics through linear changes in firing rate.

Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition.

Classical feed-forward inhibition involves an excitation-inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses.

Sensory-evoked synaptic integration in cerebellar and cerebral cortical neurons.

Neurons integrate synaptic inputs across time and space, a process that determines the transformation of input signals into action potential output. This article explores how synaptic integration contributes to the richness of sensory signalling in the cerebellar and cerebral cortices. Whether a neuron receives a few or a few thousand discrete inputs, most evoked synaptic activity generates only subthreshold membrane potential fluctuations. Sensory tuning of synaptic inputs is typically broad, but short-term dynamics and the interplay between excitation and inhibition restrict action potential firing to narrow windows of opportunity. We highlight the challenges and limitations of the use of somatic recordings in the study of synaptic integration and the importance of active dendritic mechanisms in sensory processing.

Tonic inhibition enhances fidelity of sensory information transmission in the cerebellar cortex.

Tonic inhibition is a key regulator of neuronal excitability and network function in the brain, but its role in sensory information processing remains poorly understood. The cerebellum is a favorable model system for addressing this question as granule cells, which form the input layer of the cerebellar cortex, permit high-resolution patch-clamp recordings in vivo, and are the only neurons in the cerebellar cortex that express the α6δ-containing GABA(A) receptors mediating tonic inhibition. We investigated how tonic inhibition regulates sensory information transmission in the rat cerebellum by using a combination of intracellular recordings from granule cells and molecular layer interneurons in vivo, selective pharmacology, and in vitro dynamic clamp experiments. We show that blocking tonic inhibition significantly increases the spontaneous firing rate of granule cells while only moderately increasing sensory-evoked spike output. In contrast, enhancing tonic inhibition reduces the spike probability in response to sensory stimulation with minimal effect on the spontaneous spike rate. Both manipulations result in a reduction in the signal-to-noise ratio of sensory transmission in granule cells and of parallel fiber synaptic input to downstream molecular layer interneurons. These results suggest that under basal conditions the level of tonic inhibition in vivo enhances the fidelity of sensory information transmission through the input layer of the cerebellar cortex.

How do neurons work together? Lessons from auditory cortex.

Recordings of single neurons have yielded great insights into the way acoustic stimuli are represented in auditory cortex. However, any one neuron functions as part of a population whose combined activity underlies cortical information processing. Here we review some results obtained by recording simultaneously from auditory cortical populations and individual morphologically identified neurons, in urethane-anesthetized and unanesthetized passively listening rats. Auditory cortical populations produced structured activity patterns both in response to acoustic stimuli, and spontaneously without sensory input. Population spike time patterns were broadly conserved across multiple sensory stimuli and spontaneous events, exhibiting a generally conserved sequential organization lasting approximately 100 ms. Both spontaneous and evoked events exhibited sparse, spatially localized activity in layer 2/3 pyramidal cells, and densely distributed activity in larger layer 5 pyramidal cells and putative interneurons. Laminar propagation differed however, with spontaneous activity spreading upward from deep layers and slowly across columns, but sensory responses initiating in presumptive thalamorecipient layers, spreading rapidly across columns. In both unanesthetized and urethanized rats, global activity fluctuated between "desynchronized" state characterized by low amplitude, high-frequency local field potentials and a "synchronized" state of larger, lower-frequency waves. Computational studies suggested that responses could be predicted by a simple dynamical system model fitted to the spontaneous activity immediately preceding stimulus presentation. Fitting this model to the data yielded a nonlinear self-exciting system model in synchronized states and an approximately linear system in desynchronized states. We comment on the significance of these results for auditory cortical processing of acoustic and non-acoustic information.

Methods for predicting cortical UP and DOWN states from the phase of deep layer local field potentials.

During anesthesia, slow-wave sleep and quiet wakefulness, neuronal membrane potentials collectively switch between de- and hyperpolarized levels, the cortical UP and DOWN states. Previous studies have shown that these cortical UP/DOWN states affect the excitability of individual neurons in response to sensory stimuli, indicating that a significant amount of the trial-to-trial variability in neuronal responses can be attributed to ongoing fluctuations in network activity. However, as intracellular recordings are frequently not available, it is important to be able to estimate their occurrence purely from extracellular data. Here, we combine in vivo whole cell recordings from single neurons with multi-site extracellular microelectrode recordings, to quantify the performance of various approaches to predicting UP/DOWN states from the deep-layer local field potential (LFP). We find that UP/DOWN states in deep cortical layers of rat primary auditory cortex (A1) are predictable from the phase of LFP at low frequencies (< 4 Hz), and that the likelihood of a given state varies sinusoidally with the phase of LFP at these frequencies. We introduce a novel method of detecting cortical state by combining information concerning the phase of the LFP and ongoing multi-unit activity.

The synaptic representation of sound source location in auditory cortex.

A key function of the auditory system is to provide reliable information about the location of sound sources. Here, we describe how sound location is represented by synaptic input arriving onto pyramidal cells within auditory cortex by combining free-field acoustic stimulation in the frontal azimuthal plane with in vivo whole-cell recordings. We found that subthreshold activity was panoramic in that EPSPs could be evoked from all locations in all cells. Regardless of the sound location that evoked the largest EPSP, we observed a slowing in the EPSP slope along the contralateral-ipsilateral plane that was reflected in a temporal sequence of peak EPSP times. Contralateral sounds evoked EPSPs with earlier peak times and consequently generated action potential firing with shorter latencies than ipsilateral sounds. Thus, whereas spiking probability reflected the region of space evoking the largest EPSP, across the population, synaptic inputs enforced a gradient of spike latency and precision along the horizontal axis. Therefore, within auditory cortex and regardless of preferred location, the time window of synaptic integration reflects sound source location and ensures that spatial acoustic information is represented by relative timings of pyramidal cell output.

High-fidelity transmission of sensory information by single cerebellar mossy fibre boutons.

Understanding the transmission of sensory information at individual synaptic connections requires knowledge of the properties of presynaptic terminals and their patterns of firing evoked by sensory stimuli. Such information has been difficult to obtain because of the small size and inaccessibility of nerve terminals in the central nervous system. Here we show, by making direct patch-clamp recordings in vivo from cerebellar mossy fibre boutons-the primary source of synaptic input to the cerebellar cortex-that sensory stimulation can produce bursts of spikes in single boutons at very high instantaneous firing frequencies (more than 700 Hz). We show that the mossy fibre-granule cell synapse exhibits high-fidelity transmission at these frequencies, indicating that the rapid burst of excitatory postsynaptic currents underlying the sensory-evoked response of granule cells can be driven by such a presynaptic spike burst. We also demonstrate that a single mossy fibre can trigger action potential bursts in granule cells in vitro when driven with in vivo firing patterns. These findings suggest that the relay from mossy fibre to granule cell can act in a 'detonator' fashion, such that a single presynaptic afferent may be sufficient to transmit the sensory message. This endows the cerebellar mossy fibre system with remarkable sensitivity and high fidelity in the transmission of sensory information.