Abstract The neuronal mechanisms driving progression in neuroinflammatory disorders from early relapse-remitting phases to later neurodegenerative phases remain largely elusive. Functional brain state shifts towards hyperactivity, persisting beyond relapses, represent an early maladaptive response. Here, in remission stage of an experimental autoimmune encephalitis (EAE) mouse model of RRMS, we identified a reduced excitability upon optogenetic stimulation in the brain stem, the area of active disease, while in the cortex a persistent cortical neuronal hyperactivity and synaptic remodeling emerged, accompanied with an increase of markers of early apoptosis. In contrast, hippocampal circuits, which undergo a functional state shift without hyperactivity, do not show increased apoptosis. Visual cortical networks showed a deterioration of the accuracy of encoding visual information and a decrease in the behavioural visual discrimination ability in mice. In RRMS patients in remission, we identified a reduced visual colour discrimination, indicating both the presence and the clinical relevance of early brain state maladaptation that may contribute to progression independent from relapse activity (PIRA). Summary In a RRMS model and in patients, impaired visual processing was reported, indicating brain state maladaptations, associated with persistent cortical hyperactivity, brain stem hypoactivity, synaptic remodeling, and apoptosis. These maladaptations might contribute to relapse-independent disease progression through sustained network dysfunction.

To investigate the effect of endogenously released dopamine on the stimulus-induced blood oxygen level-dependent (BOLD) responses, we used rats expressing inhibitory designer receptors exclusively activated by designer drugs (DREADDs) in neurons of the ventral tegmental area (VTA) and electrically stimulated the fimbria/fornix. This stimulation activates multiple components of the mesolimbic dopamine system, as demonstrated by the BOLD signal changes during functional magnetic resonance imaging (fMRI) and dopamine release in the nucleus accumbens (NAcc) as detected byin vivofast-scan cyclic voltammetry. Activation of inhibitory DREADDs by clozapineN-oxide (CNO) significantly reduced stimulus-induced dopamine release and the BOLD response in the NAcc. In contrast, the concurrently induced BOLD response in the medial prefrontal cortex (mPFC) was not significantly reduced after CNO administration, but the hemodynamic response was shifted to the left. Specifically, the Granger causality test showed that the temporal relationship between the BOLD signal changes in the hippocampus and the mPFC, changed. Under control conditions (i.e., in the absence of CNO), the BOLD signal changes in the mPFC and NAcc clearly preceded the BOLD signal changes in the right hippocampus, whereas in the presence of CNO this was only the case for the BOLD signal changes in the NAcc. In the control rats, that is, the rats that received a control virus and thus did not express DREADDs in the VTA, this CNO-mediated effect was not present. Our results indicate that activation of the endogenous dopaminergic system has region-specific effects on the stimulus-induced BOLD responses, so there is no generally applicable fMRI parameter that clearly indicates increased activity of the dopaminergic system.

Why some of us remember events more clearly than others and why memory loses precision over time is a major focus in memory research. Here, we show that the recruitment of specific neuroanatomical pathways within the medial temporal lobe (MTL) of the brain defines the precision of the memory recalled over the life span. Using optogenetics, neuronal activity mapping, and studying recent to very remote memories, we report that the hippocampal subfield CA1 is necessary for retrieving the gist of events and receives maximal support from MTL cortical areas (MEC, LEC, PER, and POR) for recalling the most remote memories. In contrast, reduction of CA3's activity alone coincides with the loss of memory precision over time. We propose that a shift between specific MTL subnetworks over time might be a fundamental mechanism of memory consolidation.

Objective. In tetrode recordings, the cell types of the recorded units are difficult to determine based on electrophysiological characteristics alone. Optotagging, the use of optogenetic stimulation to precisely identify cells, is a method to overcome this challenge. However, recording from many different cells requires advancing electrodes and light sources slowly through the brain with a microdrive. Existing designs suffer from a number of drawbacks, such as limited stability and precision, high cost, complex assembly, or excessive size and weight. Approach. We designed TetrODrive as a microdrive that can be 3D printed on an inexpensive desktop resin printer, has minimal parts, assembly time, and cost. The microdrive can be assembled in 15 min and the price for all materials, including the 3D printer, is lower than a single commercial microdrive. To maximize recording stability, we mechanically decoupled the drive mechanism from the electrical and optical connectors. Main results. The developed microdrive is small and light enough (<1.5 g) to be carried effortlessly by a mouse. It allows reliable recordings from single units and optogenetically identified units, even across recording sessions. In contrast to previous designs, it provides a decoupling of plugging forces from the main drive body for enhanced stability. Owing to its moveable optical fiber, our microdrive can also be used for fiber photometry. The cost of a single drive is below 20 €. We evaluated our microdrive by recording single units and calcium signals in the ventral tegmental area of mice and confirmed cell identity via optotagging. Thereby we found units not following the classical reward prediction error model. Significance. TetrODrive is a tiny, lightweight, and affordable microdrive for optophysiology in mice. Its open design, price, and built-in characteristics can significantly expand the use of microdrives in mice.

A well–accepted view in memory research is that retrieving the gist of a memory over time depends on the cortex, typically the prefrontal cortex, while retrieving its precision relies on the hippocampus. More recent advances indicate that the hippocampal subfield CA1, as opposed to CA3, remains engaged even for retrieving very remote memories and that this engagement coincides with a maximal recruitment of parahippocampal cortical areas (LEC, MEC, PER and POR) 1 . Using a time-window comparable to that used in human long-term memory studies, here we show that CA1 is necessary for retrieving the gist of a memory independently of its age while memory precision specifically depends on CA3 in a time-dependent manner. The precision for the memory of a context-footshock association was tested in mice after one day or very remotely (i.e. after 6 months or one year) allowing for the natural fading of the memory trace. Retrieving recent memories engaged both CA1 and CA3 in control mice as revealed by high levels of RNA of the immediate-early gene Arc , strongly tied to synaptic plasticity and memory function. Optogenetic inhibition of CA3 cell firing led to the loss of memory precision, i.e. the retrieval of the gist memory selectively supported by CA1. In contrast, CA1 inhibition abolished memory retrieval and reduced both CA1 and CA3’s activity. At very remote tests, controls retrieved only the gist of the event by recruiting CA1 and parahippocampal areas. Retrieving this gist was selectively abrogated upon CA1 optogenetic inactivation that dramatically reduced parahippocampal activity. Our findings indicate that the hippocampus, specifically CA1, is required for gist memory retrieval even for very remote memories that were previously reported to be hippocampal-independent, while CA3 is necessary for recalling precise memories in a time-dependent manner.

Episodic memories are hippocampus dependent. Over time such memories undergo consolidation and are thought to transition from precise to gist memories and additionally engage neocortical areas. Whether remote memories still depend on the hippocampus remains a controversy that might stem from the difficulties to distinguish activity from CA1 and CA3 subregions in the hippocampus. CA3 is thought to perform computations that are more time sensitive than CA1 such as pattern completion (completion of a full memory representation based on details). Since details fade over time, we predicted that CA3 would no longer contribute to memory retrieval for very remote memory traces. Further, the contribution of parahippocampal cortical areas which tightly interact with the hippocampus is not well understood. We recently published that indeed CA3 plays a time-limited role in the retrieval of memory while CA1 remains engaged for up to 1-year (equivalent to 40-years-old memory in humans) and that parahippocampal cortical areas were increasingly engaged over time. Here we study the contribution of CA1 and CA3 to the nature (gist/precise) of memory retrieval for recent and up to 1-year-old remote memories using a causal approach. We combined optogenetic cell firing inhibition in CA1 and CA3 with a contextual fear conditioning task to investigate memory precision and measured the neuroanatomical correlates using Arc imaging. While CA3 is required for memory precision at recent time points CA1 contributed to retrieving gist memories independently of the age of the memory trace with the support of the parahippocampal areas for the most remote memories.

Objective. A number of tissue penetrating opto-electrodes to simultaneously record and optogenetically influence brain activity have been developed. For experiments at the surface of the brain, such as electrocorticogram (ECoG) recordings and surface optogenetics, fewer devices have been described and no device has found widespread adoption for neuroscientific experiments. One issue slowing adoption is the complexity and fragility of existing devices, typically based on transparent electrode materials like graphene and indium-tin oxide (ITO). We focused here on improving existing processes based on metal traces and polyimide (PI), which produce more robust and cost-effective devices, to develop a multi-electrode array for optophysiology. Approach. The most widely used substrate material for surface electrodes, PI, has seen little use for optophysiologicalμECoG/ECoG arrays. This is due to its lack of transparency at optogenetically relevant short wavelengths. Here we use very thin layers of PI in combination with chrome-gold-platinum electrodes to achieve the necessary substrate transparency and high mechanical flexibility in a device that still rejects light artifacts well. Main results. The manufactured surface arrays have a thickness of only 6.5 µm, resulting in 80% transparency for blue light. We demonstrate immunity against opto-electric artifacts, long term stability and biocompatibility as well as suitability for optical voltage imaging. The biocompatible arrays are capable of recording stable ECoGs over months without any measurable degradation and can be used to map the tonotopic organization of the curved rodent auditory cortex. Significance. Our novel probes combine proven materials and processing steps to create optically near-transparent electrode arrays with superior longevity. In contrast to previous opto-electrodes, our probes are simple to manufacture, robust, offer long-term stability, and are a practical engineering solution for optophysiological experiments not requiring transparency of the electrode sites themselves.

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

BACKGROUND:Optogenetic stimulation has grown into a popular brain stimulation method in basic neuroscience while electrical stimulation predominates in clinical applications. In order to explain the effects of electrical stimulation on a cellular level and evaluate potential advantages of optogenetic therapies, comparisons between the two stimulation modalities are necessary. This comparison is hindered, however, by the difficulty of effectively matching the two fundamentally different modalities.OBJECTIVE:Comparison of brain-wide activation patterns in response to intensity-matched electrical and optogenetic VTA stimulation.METHODS:We mapped optogenetic and electrical self-stimulation rates in the same mice over stimulation intensity and determined iso-behavioral intensities. Using functional 99mTc-HMPAO SPECT imaging of cerebral blood flow in awake animals, we obtained brain-wide activation patterns for both modalities at these iso-behavioral intensities. We performed these experiments in two mouse lines commonly used for optogenetic VTA stimulation, DAT::Cre and TH::Cre mice.RESULTS:We find iso-behavioral intensity matching of stimulation gives rise to similar brain activation patterns. Differences between mouse lines were more pronounced than differences between modalities.CONCLUSIONS:Previously found large differences of electrical and optogenetic stimulation might be due to unmatched stimulation intensity, particularly relative electrical overstimulation. These findings imply that therapeutic electrical VTA stimulation might be relatively specific if employed with optimized parameters.

Time locking between neocortical sleep slow oscillations, thalamo-cortical spindles, and hippocampal sharp-wave ripples has convincingly been shown to be a key element of systems consolidation. Here we investigate the role of monosynaptic projections from ventral/intermediate hippocampus to medial prefrontal cortex (mPFC) in sleep-dependent memory consolidation in male mice. Following acquisition learning in the Barnes maze, we optogenetically silenced the axonal terminals of hippocampal projections within mPFC during slow-wave sleep. This silencing during SWS selectively impaired recent but not remote memory in the absence of effects on error rate and escape latencies. Furthermore, it prevented the development of the most efficient search strategy and sleep spindle time-locking to slow oscillation. An increase in post-learning sleep sharp-wave ripple (SPWR) density and reduced time locking of learning-associated SPWR activity to sleep spindles may be a less specific response. Our results demonstrate that monosynaptic projections from hippocampus to mPFC contribute to sleep-dependent memory consolidation, potentially by affecting the temporal coupling of sleep-associated electrophysiological events.SIGNIFICANCE STATEMENT Convincing evidence supports the role of slow-wave sleep (SWS), and the relevance of close temporal coupling of neuronal activity between brain regions for systems consolidation. Less attention has been paid so far to the specific neuronal pathways underlying these processes. Here, we optogenetically silenced the direct monosynaptic projection from ventral/intermediate hippocampus (HC) to medial prefrontal cortex (mPFC) during SWS in male mice following repeated learning trials in a weakly aversive spatial task. Our results confirm the concept that the monosynaptic projection between HC and mPFC contributes to memory consolidation and support an important functional role of this pathway in shaping the temporal precision among sleep-associated electrophysiological events.

Reward associations during auditory learning induce cortical plasticity in the primary auditory cortex. A prominent source of such influence is the ventral tegmental area (VTA), which conveys a dopaminergic teaching signal to the primary auditory cortex.Yet, it is unknown, how the VTA influences cortical frequency processing and spectral integration. Therefore, we investigated the temporal effects of direct optogenetic stimulation of the VTA onto spectral integration in the auditory cortex on a synaptic circuit level by current-source-density analysis in anesthetized Mongolian gerbils. While auditory lemniscal input predominantly terminates in the granular input layers III/IV, we found that VTA-mediated modulation of spectral processing is relayed by a different circuit, namely enhanced thalamic inputs to the infragranular layers Vb/VIa. Activation of this circuit yields a frequency-specific gain amplification of local sensory input and enhances corticocortical information transfer, especially in supragranular layers I/II. This effects persisted over more than 30 minutes after VTA stimulation. Altogether, we demonstrate that the VTA exhibits a long-lasting influence on sensory cortical processing via infragranular layers transcending the signaling of a mere reward-prediction error. We thereby demonstrate a cellular and circuit substrate for the influence of reinforcement-evaluating brain systems on sensory processing in the auditory cortex.

Recording from single neurons in the brain for long periods of time has been a central goal in both basic neuroscience and translational neurology, in order to understand mechanisms underlying brain processes such as learning and to understand the pathogenesis of neurodynamic disease states 1 . Recent advances in materials engineering, digital signal acquisition, and analysis algorithms have brought us closer to achieving this goal, and the possibility has gathered much public attention 2,3 . However, it remains a challenge to record from the same units for weeks to months. Here, we record many high-quality tetrode neuronal signals reliably over long periods of time in both deep and superficial areas of the brain. We achieve this by combining electrochemical roughening and carbon nanotube coating of a flexible platinum/iridium substrate, with materials, packaging, and insertion optimized to minimize tip movement with brain pulsation. This “Magdeburger” probe enables recordings with long-term signal stability and high signal-to-noise ratio at a reasonable cost in both rodent brains and in substantially larger primate brains. Robust tetrode tracking of identified neurons over longer time periods, in multiple independently targeted areas of the brain, will allow fundamental advances in the study of cognitive learning, aging, and pathogenesis, and opens new possibilities for brain interfaces in humans.

Mapping the activity of the human mesolimbic dopamine system by BOLD-fMRI is a tempting approach to non-invasively study the action of the brain reward system during different experimental conditions. However, the contribution of dopamine release to the BOLD signal is disputed. To assign the actual contribution of dopaminergic and non-dopaminergic VTA neurons to the formation of BOLD responses in target regions of the mesolimbic system, we used two optogenetic approaches in rats. We either activated VTA dopaminergic neurons selectively, or dopaminergic and mainly glutamatergic projecting neurons together. We further used electrical stimulation to non-selectively activate neurons in the VTA. All three stimulation conditions effectively activated the mesolimbic dopaminergic system and triggered dopamine releases into the NAcc as measured by in vivo fast-scan cyclic voltammetry. Furthermore, both optogenetic stimulation paradigms led to indistinguishable self-stimulation behavior. In contrast to these similarities, however, the BOLD response pattern differed greatly between groups. In general, BOLD responses were weaker and sparser with increasing stimulation specificity for dopaminergic neurons. In addition, repetitive stimulation of the VTA caused a progressive decoupling of dopamine release and BOLD signal strength, and dopamine receptor antagonists were unable to block the BOLD signal elicited by VTA stimulation. To exclude that the sedation during fMRI is the cause of minimal mesolimbic BOLD in response to specific dopaminergic stimulation, we repeated our experiments using CBF SPECT in awake animals. Again, we found activations only for less-specific stimulation. Based on these results we conclude that canonical BOLD responses in the reward system represent mainly the activity of non-dopaminergic neurons. Thus, the minor effects of projecting dopaminergic neurons are concealed by non-dopaminergic activity, a finding which highlights the importance of a careful interpretation of reward-related human fMRI data.

Calcium imaging in freely behaving rodents using head-mounted miniature microscopes is currently becoming an increasingly popular technique in neuroscience. Due to the large amounts of complex data that the technique produces, user friendly software is needed for quick and efficient processing. Here, we present a new tool for analyzing calcium imaging data from head-mounted microscopes together with simultaneously acquired behavioral data: CAVE (Calcium ActiVity Explorer). CAVE bundles a unique set of algorithms specifically tailored to the analysis of single-photon imaging data from awake behaving animals including efficient motion correction and automatic ROI selection with manual audit and refinement. For behavioral analysis, CAVE can automatically track animal position and orientation. Individual behavioral epochs and external events can then be analyzed in correlation to calcium imaging and tracking data. Our program is written in MATLAB, the source code is open source and particularly focuses on providing a streamlined workflow for novice users while also retaining detailed configuration options for advanced users. We evaluate the performance of CAVE by investigating neural activity in hippocampus and somatosensory cortex. The fast analysis provided by CAVE allowed us to track activity in a large set of animals over the course of several months during exploration behavior, detailing the properties of onset and offset of observable activity and the visible cells per imaging location.

Recent developments in the field of neuroprosthetics have created a demand for cost-effective advanced bonding techniques to mount polymer-based, thin-film, multi-electrode arrays onto PCBs. Low thicknesses of polymer substrates present challenges for common packaging technologies. Here we implemented a cost-effective direct bonding process of polyimide thin-film electrode arrays using vapor phase soldering. Our polymer foil is composed of PI-2611 and contains embedded gold/platinum/chromium traces. Contact pads are platinum coated and no under bump metallization is required. Instead of using wire bonding techniques, the contact pads are flip-chip bonded directly onto the PCB using a lead free solder paste. Transfer of the foil is carried out by vacuum placement. Soldering is subsequently performed in a vapor phase soldering oven. In contrast to wire bonding on flexible substrate materials, vapor phase soldering results in enhanced contact yield of approximately 99%. The landing area of the thin-film electrode, containing the soldered contact pads, is then passivated with a low-shrinkage epoxy in a pin-transfer process. Using such a low-shrinkage material is paramount to achieve sufficient long-term stability of the solder connections and to stabilize the thin polyimide substrate.

The locus coeruleus (LC) is the sole source of noradrenergic projections to the cortex and essential for attention-dependent cognitive processes. In this study we used unilateral optogenetic silencing of the LC in an attentional set-shifting task (ASST) to evaluate the influence of the LC on prefrontal cortex-dependent functions in mice. We expressed the halorhodopsin eNpHR3.0 to reversibly silence LC activity during task performance, and found that silencing selectively impaired learning of those parts of the ASST that most strongly rely on cognitive flexibility. In particular, extra-dimensional set-shifting (EDS) and reversal learning was impaired, suggesting an involvement of the medial prefrontal cortex (mPFC) and the orbitofrontal cortex. In contrast, those parts of the task that are less dependent on cognitive flexibility, i.e. compound discrimination (CD) and the intra-dimensional shifts (IDS) were not affected. Furthermore, attentional set formation was unaffected by LC silencing. Our results therefore suggest a modulatory influence of the LC on cognitive flexibility, mediated by different frontal networks.

Optogenetics is one of the most important techniques in neurophysiology, with potential clinical applications. However, the strong light needed may cause harmful temperature rises. So far, there are no methods to reliably estimate brain heating and safe limits in actual optogenetic experiments. We used thermal imaging to directly measure such temperature rises at the surface of live mouse brains during laser illumination with wavelengths and intensities typical for optogenetics. We then modeled the temperature rise with a simple logarithmic model. Our results indicate that previous finite-element models can underestimate temperature increases by an order of magnitude. We validate our empirical model by predicting the temperature rise caused by pulsed stimulation paradigms. These predictions fit closely to the empirical data and constitute a better estimate of real temperature increases. Additionally, we provide a web-based app for easy calculation that can be used as a tool for safe design of optogenetic experiments.