Barrel Cortex Function

Amsterdam 2016

"a multidisciplinary, international meeting on sensory (sub)cortical circuits"

Barrel Cortex Function Amsterdam 2016 Version 23rd April 2016 19th of May, Day 1 08:45 – 09:15 Registration, Coffee and Poster mounting 09:15 – 09:30 Welcome & Opening (Christiaan de Kock) Session 1: “Development”, Chair Fritjof Helmchen 09:30 – 10:15 Gordon Fishell (Keynote)

“The integration of interneurons into brain circuits: a conversation involving pedigree and communications”

10:15 – 10:45 Heiko Luhmann “Role of spontaneous activity in early cortical development” 10:45 – 11.15 Coffee break & posters Session 2: “Inhibition”, Chair Jochen Staiger 11:15 – 12:00 Adam Kepecs (Keynote) “Behavioral role and impact of a VIP-mediated interneuron circuit” 12:00 – 12:30 Carl Petersen

“Neocortical cell-type-specific sensorimotor processing during goal-directed behavior”

12:30 – 13:00 Koen Vervaeke “Shining light on neural inhibition in the somatosensory cortex during tactile sensation”

13.00 – 14:15 Lunch (restaurant section F, top floor) Session 3: “Linking circuits to behavior”, Chair Christiaan de Kock 14:15 – 15:00 Anthony Holtmaat “Mechanism for functional and structural plasticity” 15:00 – 15:30 Cornelius Schwarz

“Linking the barrel column. What is the functional basis of learned associations?”

15:30 – 16.00 Fritjof Helmchen “Neural correlates of tactile discrimination based on whisker touch” 16:00 – 16:15 Junior 1: Naoya Takahashi (Larkum lab) “Dendritic dynamics in sensory perception” 16:15 – 16:30 Junior 2: Christian Ebbesen (Brecht lab)

“Vibrissa motor cortex activity suppresses contralateral whisker movement”

16:30 – 17:00 Coffee break & posters Session 4: – “From sparse to dense connectomics”, Chair Randy Bruno 17:00 – 17:45 Moritz Helmstaedter (Keynote) “High-resolution connectomics” 17:45 – 18:15 Dirk Feldmeyer

“Excitatory and inhibitory connections in the barrel cortex - new vistas” 18:15 – 18:45 Marcel Oberlaender “Cortical Drive by Deep Layer Networks” 18:45 – 19:00 Junior 3: Nora Jamann (Engelhardt lab) “Plasticity of the axon initial segment in the mouse barrel cortex 19:00 – 19:15 Junior 4: Zeinab Fazlali (Arabzadeh lab)

“Correlation between Cortical State and Locus Coeruleus Activity: Implications for Sensory Coding in Rat Barrel Cortex”

PLENARY BARBEQUE (to join, advance registration for both meeting and BBQ is mandatory)

Barrel Cortex Function Amsterdam 2016 20th of May, Day 2 Session 5: “Cortical processing and behavior”, chair Dirk Feldmeyer 09:00 – 09:45 Michael Stryker (Keynote)

“A neural circuit that regulates cortical responsiveness and plasticity.” 09:45 – 10:15 James Poulet “Sensorimotor transformation in the mouse forepaw system" 10:15 – 10:45 Michael Brecht “Sex, Touch & Tickle” 10:45 – 11:15 Coffee break & Posters Session 6: “Active somatosensation”, chair Heiko Luhmann 11:15 – 12.00 David Kleinfeld (Keynote)

“Cortical-trigeminal loops in the control of vibrissa movement” 12.00 – 12:30 Christiaan de Kock “Cortical representation of naïve active touch” 12:30 – 13:00 Randy Bruno

“The many layers of neocortex” 13:00 – 14:00 Lunch (restaurant section F, top floor) Session 7: chair Marcel Oberlaender 14:00 – 14:15 Junior 5: Hemanth Mohan (de Kock lab)

“Encoding of free whisking and object touch in the posterior parietal cortex of rodents”

14:15 – 14:30 Junior 6: Teresa Guillamon Vivancos (Jennifer Luebke lab) “Contribution of Distinct Progenitor Lineages to Neuronal Diversity in Layer 4 of the Mouse Barrel Cortex”

14:30 – 14:45 Junior 7: Alexander van der Bourg (Helmchen lab) “Laminar and columnar refinement of sensory coding in developing mouse barrel cortex”

14:45 – 15.00 Junior 8: Guanxiao Qi (Feldmeyer lab) “Inter-columnar synaptic transmission in thalamo-recipient layers 4 and 6A of rat barrel cortex”

15:00 – 15:15 Short break (no catering) Session 8: “Inhibition-Disinhibition-Excitation”, chair Michael Brecht 15:15 – 16:00 Josh Huang (Keynote) “Genetic dissection of cortical circuits: chandeliers light up

pyramids“ 16:00 – 16:30 Miguel Maravall “Exploring tactile temporal integration and sequence discrimination.” 16:30 – 17:00 Jochen Staiger

“The ins and outs of cortical VIP neurons” 17:00 concluding remarks (Christiaan de Kock)

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Barrel Cortex Function 2016, 19-20th of May 2016

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Our meeting is supported by generous contributions from:

BarrelC

ortexFunction

2016,19-20thofM

ay2016

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ittestoel

SAP:2961107

Posters Posters are available throughout the full meeting. The presenting author is requested to be at the poster during the indicated time window. Presenting author (lab) Presenting time Day 1: regular font Day 2: bold #1: Roel de Haan (de Kock) Day 1, 16:30 – 17:00 #2: Berat Sermet (Petersen) Day 2, 10.45 – 11.15 #3: Angeliki Vavladeli (Petersen) Day 1, 16:30 – 17:00 #4: Pierre Le Merre (Crochet) Day 2, 10.45 – 11.15 #5: Mike Guest (Oberlaender) Day 1, 16:30 – 17:00 #6: Mythreya Seetharama (Oberlaender) Day 2, 10.45 – 11.15 #7: Rajeevan Narayanan (Oberlaender) Day 1, 16:30 – 17:00 #8: Daniel Udvary (Oberlaender) Day 2, 10.45 – 11.15 #9: Jiali Tang (Feldmeyer) Day 1, 16:30 – 17:00 #10: Claudia Barz (Feldmeyer) Day 2, 10.45 – 11.15 #11: Vishalini Sivarajan (Feldmeyer) Day 1, 16:30 – 17:00 #12: Francois Pauzin (Krieger) Day 2, 10.45 – 11.15 #13: Guanxiao Qi (Feldmeyer) Day 1, 16:30 – 17:00 #14: Manuel Marx (Feldmeyer) Day 2, 10.45 – 11.15 #15: Matías Goldin (Shulz) Day 1, 16:30 – 17:00 #16: Alexander van der Bourg (Helmchen) Day 2, 10.45 – 11.15 #17: Lianne Klaver (Bosman) Day 1, 16:30 – 17:00 #18: Stéphanie Miceli (Schubert) Day 2, 10.45 – 11.15 #19: Teresa Guillamon-Vivancos (Luebke) Day 1, 16:30 – 17:00 #20: Georg Hafner (Staiger) Day 2, 10.45 – 11.15 #21: Keti Cohen-Kashi (Lampl) Day 1, 16:30 – 17:00 #22: Ana Parabucki (Lampl) Day 2, 10.45 – 11.15 #23: Desire Humanes-Valera (Krieger) Day 1, 16:30 – 17:00 #24: Christian Ebbesen (Brecht) Day 2, 10.45 – 11.15 #25: Mostafa Nashaat (Larkum) Day 1, 16:30 – 17:00 #26: Jorrit Montijn (Pennartz) Day 2, 10.45 – 11.15 #27: Anton Sumser (Groh) Day 1, 16:30 – 17:00 #28: Zeinab Fazlali (Arabzadeh) Day 2, 10.45 – 11.15 #29: Konrad Juczewski (Krieger, Hardingham) Day 1, 16:30 – 17:00

Poster #1 Probing the neurophysiological correlates of cognitive performance in rat medial prefrontal cortex Roel de Haan, Sven van der Burg, Anton Pieneman, Vinod Nigade, Huib Mansvelder, Bert Sakmann, Christiaan de Kock Attention, working memory and executive functions are strongly associated with activity in rat medial prefrontal cortex (mPFC). Similar to other cortical areas, the mPFC has a laminar architecture containing functionally different cell types and layers. However, the contribution of individual layers and cell-types in mPFC to cognitive behavior is only beginning to be understood. To reveal which components of the mPFC circuitry orchestrate the diverse repertoire of prefrontal functions, we recorded populations of neurons at specific cortical depths across layers of the rat mPFC during a whisker-based go/no-go task. In short, rats were trained to determine the location of a pole on the radius of the whisker, where the proximal location should be reported by licking to receive a water reward and in the distal location the rat should not lick to avoid a 5 second time-out. We find populations of neurons with correlates to most phases of the task. The strongest neurophysiological correlate involved reward consumption after the rat made a correct decision. Further, we find strong sub-threshold activity in the LFP when the rat is cued for the next trial-start, which could reflect a ‘reset’ of the working memory to prepare for upcoming task execution.

Poster #2 Layer-specific thalamocortical input onto excitatory neurons in mouse primary somatosensory barrel cortex Berat Semihcan Sermet, Carl C. H. Petersen In the mouse whisker system, sensory information is relayed to the primary somatosensory barrel cortex by two major thalamic nuclei, the ventral posterior medial nucleus (VPM) and the posterior medial nucleus (POM). While the axonal innervation pattern of these two nuclei has been studied anatomically in some detail, their synaptic input to distinct cell-types across different layers in barrel cortex is incompletely understood. We used the specificity of optogenetics to selectively stimulate axons from VPM or POM, and we measured the evoked excitatory postsynaptic potentials in vitro with whole-cell patch-clamp recordings. VPM or POM was infected in vivo with an adenoassociated virus encoding the light-gated cation channel channelrhodopsin (ChR2). Synaptic input onto individual neurons of the barrel cortex was recorded in brain slices in vitro by activating the ChR2-expressing thalamic axons with blue light. We measured thalamic inputs onto excitatory neurons across all layers of the barrel cortex, finding that the biggest inputs appeared to largely colocalise with the anatomical innervation pattern. Anatomically, VPM preferentially innervates L4, deep L3 and the L5B/6A border, and, functionally, we found that the biggest input was observed in L4, followed by L2/3. Anatomically, POM innervates L5A and L1, and, functionally, we found the biggest input in L5A, followed by L2/3. The onset latencies were shortest in L4 and L5A for VPM and POM input respectively. Our results begin to provide a more complete understanding of the distribution of thalamic input onto excitatory neurons across the layers of the mouse barrel cortex.

Poster #3 Two-photon calcium imaging of neocortical projection neurons in mouse whisker primary somatosensory cortex during goal-directed sensorimotor learning Angeliki Vavladeli, Carl Petersen How goal-directed sensorimotor learning induces changes in neuronal networks to alter the flow of information between cortical areas is not well understood. Here, using genetically-encoded calcium indicators in combination with retrograde tracers and two-photon laser scanning microscopy, we have begun to chronically image the activity of layer 2/3 neurons in primary somatosensory cortex (S1) that project to secondary somatosensory cortex (S2p) or primary motor cortex (M1p), while mice learn a simple goal-directed sensorimotor transformation. Thirsty mice were trained to lick for water reward in response to single-whisker deflections and auditory stimulation. We are analyzing how sensory input (whisker and auditory stimuli) and motor output (licking and whisking) are represented by projection neurons in S1 of mice performing the detection task and how their activity evolves during the learning process.

Poster #4 Large-scale sensory integration in the mouse cortex during a tactile detection task. Pierre Le Merre, Paul Salin, Carl Petersen and Sylvain Crochet Sensory perception leading to goal-directed behavior involves multiple, spatially-distributed cortical areas. It has been hypothesized that sensory information flows from primary sensory areas encoding mainly the properties of the stimulus, to higher-order, more frontal areas encoding the valence of the stimulus. To understand further the integration of sensory signals, we have recorded sensory evoked potentials (SEPs) simultaneously from different cortical areas, either in mice performing a whisker-based sensory detection task (DT) or in mice exposed to same whisker stimulus that was not associated with reward (Neutral Exposition, NE). In mice performing the DT, we observed SEPs in response to the whisker stimulus in all recorded areas with latencies increasing from the whisker primary somatosensory area (wS1) to the secondary somatosensory area (wS2), the whisker motor area (wM1), the parietal area (PtA), the dorsal hippocampus (dCA1) and the medial prefrontal cortex (mPFC). We found a reduction of SEPs during Miss trials compared to Hit trials in all areas except wS1, with strongest reduction in mPFC and dCA1. We also observed a selective increase of the SEPs in mPFC and dCA1 when the mice were trained to the DT but not during NE, suggesting that DT training induced plastic changes in the mPFC and hippocampus leading to increased SEPs in response to the conditioning stimulus. Our results support the idea that mPFC and dCA1 could signal the relevance of a sensory stimulus in the context of a well-defined behavior, whereas sensory areas would be more constrained by the nature of the stimulus.

Poster #5 Structural Basis for Sensory-Motor Whisker Control Mike Guest, Elizabeth Wendel, Peter Strick, Marcel Oberlaender The rodent vibrissal system offers an ideal model for studying sensory-motor pathways in the mammalian central nervous system. There has been much consideration to bring insight to the organization of the whisker sensory pathways throughout the rodent brain. However, it is poorly understood how brain-wide whisker motor pathways are organized and how, together with sensory pathways, they constitute a sensory-motor loop that can provide sensory feedback to the whiskers during tactile-based behaviors. It has been previously reported that whisker muscles are directly innervated by vibrissal motor neurons(vMN) located in the lateral area of the Facial Nucleus(FN) and that these vMNs are directly innervated by output neurons of the vibrissal motor cortex (Herfst and Brecht, 2007). It has also been suggested that this vibrissal motor area of the cortex is divided into two segregated regions. 1) A sensory input area called the Transitional Zone (TZ) and 2) a whisker motor output area called vM1 (Smith & Alloway, 2013; Matyas et al., 2010). In this study we inject wild type rabies virus into the mystacial pad targeting a single muscle that is wrapped around a whisker follicle. The virus is transported retrogradely across multiple synapses throughout the central nervous system in a time dependent manner (Kelly and Strick, 2000), first labeling vMNs located in the FN and subsequently the hierarchy of pathways that provide input to these motor neurons. Combining this injection method with custom-designed brain-wide imaging techniques and automated somata detection software (Oberlaender et al., 2009), we provide unprecedented quantitative insight to the organization of the whisker motor pathways, setting the stage to investigate how cortex provides sensory feedback to peripheral receptor organs during sensory-motor tasks.

Poster #6 Automated Detection of Putative Synaptic Contacts between in vivo Labelled Neurons Mythreya Seetharama, David Slabik, Alison Smyth, Marcel Oberlaender Understanding the structural organization of the neural networks requires reconstruction of the underlying neural circuitry in anatomical detail and mapping of the synaptic contacts. A typical in vivo labeled axon innervates a large volume and imaging such in vivo labeled pairs of neurons at high resolution yields a large set of images, typically in the order of Tera Bytes. Manual mapping of synaptic contacts between such cell pairs would be labor intensive and error prone. Here, we present a software pipeline that automatically detects putative synaptic contacts between the boutons and spines of in vivo labeled pairs of neurons. The pipeline has three phases. Firstly, the regions where the skeletons of axon and dendrites of different neurons come close to each other are detected. Secondly, in these proximity regions, the boutons along the axons and spines along the dendrites are detected. Thirdly, the overlaps between boutons and spines along with their locations are detected. The resulting putative contacts are visualized on the original image stack using a visualization tool and can be verified by the user. This semi-automated approach reduces the number of sites to be manually inspected for putative contacts from tens of thousands to tens of putative contact sites. Hence achieving about three orders of magnitude reduction in the manual effort required.

Poster #7 Organizational principles of rat vibrissal motor cortex, an in vivo study Rajeevan T Narayanan and Marcel Oberlaender Vibrissal somatosensory cortex (vS1) and vibrissal motor cortex (vM1) are the two key structures involved in somatosensation in rodents. Although we know the vertical and horizontal organizational principles of vS1, very little is known about the organizational principles of vM1 at single cell level. Using in vivo cell attached recording, biocytin filling and semi-automatic reconstruction pipeline; we investigated electrophysiological properties and morphological characteristics of motor cortex neurons. We found substantial similarities between these two areas, indicating that both cortices follow similar organizational principles.

Poster #8 Dense Statistical Connectome of Rat Barrel Cortex Daniel Udvary, Robert Egger, Vincent J. Dercksen, and Marcel Oberlaender Synaptic connectivity is one important constrain for cortical signal flow and function. Consequently, a complete synaptic connectivity map (i.e., connectome) of a cortical area across spatial scales would advance our understanding of cortex organization and function. We present a dense statistical connectome of the entire rat vibrissal cortex based on measured 3D distributions of axons/dendrites/somata of excitatory and inhibitory neurons. By calculating the structural overlap between pre- and postsynaptic cells our model provides quantitative estimates on connectivity measurements like connection probability and number of synapses on cell type, cellular, and subcellular levels. We found that our model reproduces connectivity measurements between thalamic and excitatory/inhibitory neurons reported in paired recordings and light- and electron-microscopic studies. Similarly, intracortical synaptic connectivity of our model matches most connectivity measurements. However, the location and distance between pre- and postsynaptic cells and - in case of slicing experiments - the degree of truncation strongly influences the connectivity. When reproducing electron-microscopic and in vitro slicing experiments in our model, we found that measurements obtained under the respective experimental conditions are in line with our model's results, but represent only a small fraction of the underlying distribution. The experimental conditions such as the small volume analyzed in electron-microscopic studies or the truncation of morphologies thus biases the conclusions that are drawn, e.g. an underestimation of the connection probability. Our approach can therefore be used to improve experimental design and seen as a starting point to simulate sensory-evoked signal flow and investigate structural and functional organization of the cortex.

Poster #9 Effects of Noradrenaline on Neuronal Networks in Rat Neocortex Jiali Tang, Gabriele Radnikow, Dirk Feldmeyer Previous studies on the effects of noradrenaline (NA) in the neocortex have demonstrated its pivotal role in the regulation of brain function. Here, patch clamp recordings from single and synaptically coupled neurons were used to investigate the effects of NA on neocortical neuronal microcircuits. In neocortical layer 4 (L4), NA induces a hyperpolarization of spiny neurons by activation of inwardly rectifying K+ channels via α2 adrenoceptors, but enhances the action potential firing rate by blocking hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels via α2 adrenoceptors in response to suprathreshold stimuli. Furthermore, NA decreases synaptic efficacy in L4 excitatory microcircuits by suppressing presynaptic neurotransmitters release via activation of presynaptic α2 adrenoceptors. Moreover, NA modulates the excitability of excitatory neurons in the neocortex in a layer- and cell-type specific way: NA hyperpolarizes neurons in layers 4 and 5A, and depolarizes neurons in L5B and L6A. For L2/3 pyramidal neurons, NA hyperpolarizes neurons with broad apical dendritic tufts, but depolarizes those with slender apical dendritic tuft. In addition, NA causes a depolarization of both fast-spiking and non fast-spiking interneurons, thereby increasing the inhibitory tone and thus an overall suppression of L4 excitatory microcircuit activity. Here we have demonstrated that NA affects signal processing in the neocortex via several different mechanisms, including a bi-directionally regulation on the excitability of single excitatory neurons, the layer and cell-type specific modulation of excitatory neurons throughout cortical layers, and a homogenous facilitation of all interneuron types.

Poster #10 Functional and structural characteristics of layer 2/3 neurons in adult mouse barrel cortex Barz C. S., Feldmeyer D. The accessibility for paired and multiple recordings and drug application make the barrel cortex slice a popular system for studying neuronal and circuit properties. For technical reasons, however, slice studies have mostly been performed in young rats and mice (typically up to 3 weeks of age). In contrast, neuronal properties in adult barrel cortex (> 6 weeks) have not been described comprehensively so far. In particular, detailed knowledge about the morphological and electrophysiological features of inhibitory neurons is lacking. The present study characterizes the structure and function of layer 2/3 excitatory and inhibitory neurons in adult mouse barrel cortex. Whole-cell patch clamp recordings with simultaneous biocytin fillings were performed in thalamocortical brain slices obtained from C57BL/6J mice aged 6-21 weeks. Post-hoc histological processing and 3D morphological reconstructions were carried out for detailed analyses of dendritic and axonal processes. Based on the morphological and electrophysiological cell properties, different subclasses of excitatory and inhibitory neurons were identified. The results provide an important basis for understanding the barrel cortex microcircuitry in adult mice, as well as changes during long-term skill learning and alterations in developmental disease models.

Poster #11 Characterization of non-fast spiking interneurons in layer 4 of rat barrel cortex Vishalini Sivarajan, Guanxiao Qi, Dirk Feldmeyer GABAergic interneurons (INs) play an important role in providing balanced cortical excitation, synchronized activity, and maintaining neuronal oscillatory networks. Abnormalities of these functions have been associated with several neurological and psychiatric disorders. Despite the low population of GABAergic interneurons (10-20%), they display a vast heterogeneity in their structure, function and neurochemical markers expression. Quantitative classification of GABAergic interneurons based on reliable and stable criteria is crucial to understand the different subtypes and their functions. In this study, we investigated the different subtypes of non-fast spiking (nFS) INs and their functional properties in layer 4 (L4) of rat barrel cortex using whole-cell patch-clamp recordings, and morphological 3D reconstructions. We performed quantitative morphological classification based on the positioning and orientation of axonal projection patterns through different layers and columns. We identified several distinct morphological subtypes of nFS interneurons, in particular, lateral projecting interneurons. This kind of projection pattern from one barrel to neighboring barrels is a novel finding, and we believe that this kind of interneurons could play an important role in lateral inhibition. Paired recordings of nFS L4 INs with other neuron types showed that nFS INs establish weak synaptic connections with a low release probability and small amplitude; the connections showed paired-pulse facilitation. This is markedly different from the fast spiking interneurons, which form strong synaptic connections that exhibit invariably paired-pulse depression. Our findings reveal that nFS INs are elements of the L4 micro-circuitry that exhibit very distinct connectivity patterns and functional roles in the neuronal network.

Poster #12 Bi-phasic modulation of cortical excitability Francois Pauzin, Veronika Gondzik, Desire Humanes-Valera, Patrik Krieger Tactile sensory information is processed in the primary somatosensory cortex, a structure organized in six different horizontal neuronal layers that are interconnected to each other by vertical axonal projections. Each of them can influence the response to sensory stimuli, but the specific role played by each layer in cortical processing is still not fully understood. Here we show using optogenetical tools that layer 6 in the primary somatosensory cortex of the mouse plays a crucial role in controlling the gain of whisker evoked activity in neurons of all cortical layers. This gain modulation results from the coordinated action of layer 6 projections to inhibitory neurons whose cell body resides in deep cortical layers and subcortical projections to the thalamus, the main gateway for tactile information to reach the cerebral cortex. This study thus establishes L6 as a major mediator of cortical gain modulation, enlightening the role of cortico-cortical and cortico-thalamic feedback in sensory processing.

Poster #13 Inter-columnar synaptic transmission in thalamo-recipient layers 4 and 6A of rat barrel cortex Guanxiao Qi and Dirk Feldmeyer In the primary somatosensory (barrel) cortex of rodents, layer 4 (L4) and (to a lesser degree) L6A are the main thalamorecipient layers. Within a barrel column excitatory and inhibitory neurons form local recurrent synaptic microcircuits. However, little is known about synaptic connections between cortical columns. The purpose of this study is a functional and structural characterization of monosynaptic connections be-tween L4 or L6A neurons located in two neighboring columns. Monosynaptic connections (n=24) between L4 neurons located in two neighboring barrels were obtained; the average inter-soma distance of pre- and postsynaptic neurons was 146 ± 46 µm. Of these L4 ‘intercolumnar’ connections 12 were excitatory-excitatory and 10 excitatory-inhibitory. The pre- and postsynaptic excitatory neurons were either spiny stellate cells (SSCs) or start pyramidal neurons. The postsynaptic inhibitory neurons were predominantly fast-spiking (FS) interneurons. In addition, two monosynaptic inhibitory connections from FS interneurons in one barrel to SSCs in the other barrel were recorded. Recordings from synaptically coupled L6A pyramidal neurons located in two neighboring columns were also obtained.; the average inter-soma distance of which was 226 ± 27 µm (n=5). For L6A excitatory connections, the presynaptic neurons were exclusively cortico-cortical (CC) cells and postsynaptic neurons are either CC cells or cortico-thalamic cells. Our data show that there is direct inter-columnar signaling via monosynaptic excitatory or inhibitory connections in both thalamorecipient layers 4 and 6A, which is like to play an important role in shaping receptive field properties of barrel cortical neurons.

Poster #14 Expression pattern and distribution of the forkhead-box protein P2 (FoxP2) in layer 6 of the somatosensory rat barrel cortex Manuel Marx, Werner Hucko and Dirk Feldmeyer In rodents, the transcription factor forkhead-box protein P2 (FoxP2) is highly and exclusively expressed in neocortical layer 6. However, little information about FoxP2 on the cellular level is available so far. Here, we examined layer 6A and layer 6B (L6A, L6B) neurons using whole-cell patch clamp single and paired recordings. In addition, neurons were simultaneously filled with biocytin and a fluorescent dye to allow a correlated analysis of FoxP2 expression using antibody labeling and neuronal morphology. FoxP2 is consistently expressed from late subplate stages (postnatal days P0-4) into adulthood (>P35). We did not find any age-dependent changes in the FoxP2 cell count. FoxP2 was only expressed in glutamatergic L6A and L6B neurons. All GABAergic L6A and L6B interneurons were found to be FoxP2(-). The expression of FoxP2 was also found to depend on the type of excitatory L6 neuron. In layer 6A, corticocortical (CC) pyramidal neurons were consistently FoxP2(-) while corticothalamic (CT) pyramidal neurons were FoxP2(+). In layer 6B, non-pyramidal excitatory neurons (i.e. multipolar, tangential, horizontal and inverted neurons) were generally FoxP2(-). In contrast, all L6B pyramidal neurons showed a clear FoxP2 expression, i.e. FoxP2(+). We identified three L6-L6 connection types between FoxP2(+)-FoxP2(+), FoxP2(+)-FoxP2(-) and FoxP2(-)-FoxP2(-) neurons. Moreover, FoxP2 is highly expressed in subcortical regions like the striatum and in thalamic nuclei. We hypothesize that FoxP2 is involved in corticocortical, corticothalamic and corticostriatal microcircuits and may play a key role in motor coordination.

Poster #15 Neural representation of multiwhisker tactile information in the secondary somatosensory cortex Matías A. Goldin*, Evan R. Harrell*, Daniel E. Shulz * contributed equally Tactile object discrimination in rodents is mediated by the vibrissal system and its related processing areas in the brain. Efficient discrimination relies on spatiotemporally correlated whisker deflections that provide the raw information that animals use to make inferences about an object. Here we studied the neuronal coding of these spatiotemporal correlations by applying complex, multi-vibrissal stimuli in a controlled fashion using a matrix of piezo-electric benders applied to the 24 caudal macrovibrissae of isoflurane-anesthetized rats [Jacob et al. 2010]. This stimulator has already facilitated a detailed characterization of the coding of inter-whisker correlation in the primary somatosensory cortex (SI) [Estebanez et al. 2012], and here we aim to extend this description to the secondary somatosensory cortex (SII). We recorded activity of multiple single units with up to 64 channel multi-site extracellular electrodes. Using a forward correlation approach, we found both single and multiwhisker receptive fields, which tended to be more elongated along whisker rows. We determined the response latencies and found that they are compatible with SII being driven directly by thalamic inputs. Our working hypothesis is that SII has a strong representation of large-scale, multi-whisker patterns of stimulation, since vibrissal somatotopy in SII is much less defined than in SI. This feature was studied with a reverse correlation approach providing white noise stimuli in a fully correlated (all identical) or uncorrelated (all different) fashion across 24 macrovibrissae. Preliminary results show that global inter-whisker correlation has a relatively stronger representation in SII than in SI.

Poster #16 Laminar and columnar refinement of sensory coding in developing mouse barrel cortex Alexander van der Bourg, Jenq-Wei Yang, Vicente Reyes-Puerta, Balazs Laurenczy, Martin Wieckhorst, Maik C. Stüttgen, Heiko J. Luhmann and Fritjof Helmchen Rodent rhythmic whisking behavior matures during a critical period two weeks after birth. The related adaptations of neocortical function remain poorly understood. Here, we characterized neuronal dynamics in mouse barrel cortex evoked by various spatiotemporal whisker stimulation patterns across all cortical layers before, during and after the onset of whisking behavior. By employing multi-electrode recordings and two-photon calcium imaging in anesthetized mice, we found layer-specific changes in stimulus-evoked activity from postnatal day P10 to P28, which decreases in layer 2/3 (L2/3) and L4 neurons but increases in L5 and L6. During the same time period, neuronal activity progressively spread further between neighboring barrel columns, especially for superficial layers. Repetitive stimulation of multiple whiskers showed distinct response profiles in the stimulated barrel columns depending on the direction and temporal separation of the stimuli. Calcium imaging of individual L2/3 neurons revealed that, in addition to progressive sparsification and decorrelation of neuronal activity, response selectivity to axial vs. lateral whisker movement emerged around the critical period. Our results demonstrate a layer-specific refinement of sensory responses to spatially and temporally distinct stimuli in developing mouse barrel cortex at the onset of exploratory behavior.

Poster #17 Anatomical implications for multisensory areas in ferret cortex Lianne Klaver, Chaira Serrarens, Umberto Olcese, Cyriel Pennartz & Conrado Bosman Multisensory integration (MI) refers to the combination of information from multiple senses to produce coherent perceptual experiences. Classically, MI has been studied in subcortical areas, yet little is known about the anatomical organization underlying MI responses in neocortex. To study multisensory integration in the cortex, we use domestic ferrets (Mustela putorius furo), which are presumed to have a large capacity available for specialized integration areas, due to cortical expansion. Previously identified integration areas in cats include the antero- and posteromedial lateral suprasylvian visual areas (AMLS and PMLS), located in the multisensory zone between the primary auditory and visual cortices. Furthermore, it has been shown in ferrets that multisensory areas are present outside these multisensory zones, for example in the anterior ectosylvian visual area (AEV) and in primary and secondary auditory areas of the ferret. Nevertheless, an accurate description of putative integration areas in multisensory zones is still missing. Here, we aimed to provide an anatomical overview of the connectivity between unisensory and multisensory integrative areas. We used BDA (biotinylated dextran amine), an anterograde tracer, to determine overlapping projections from primary auditory and visual cortices. We confirmed the presence of projections from primary visual and auditory cortex to AEV, primary and secondary unisensory areas. Furthermore, preliminary results reveal previously unidentified projections from primary visual and auditory cortex to several multisensory zones located between these unisensory areas in the ferret. We believe that these results will be relevant for a wide audience studying auditory, visual and audiovisual processing in the ferret.

Poster #18 Reduced feedforward inhibition within layer IV of the barrel cortex and altered sensory performance in serotonin transporter knockout rats. Stéphanie Miceli, Nael Nadif Kasri, Joep Joosten, Chao Huang, Lara Kepser, Rémi Proville, Martijn Selten, Fenneke van Eijs, Alireza Azarfar, Judith R. Homberg, Tansu Celikel, and Dirk Schubert High extracellular serotonin (5-HT) levels during development alter input into the primary somatosensory cortex (S1) of rodents by impairing neural transmission between the thalamus and cortical input layer IV (LIV). As one consequence rodent models of impaired 5-HT transporter (SERT) function show a disruption in their topological organization of the somatosensory system both on thalamocortical and intracortical level. Here we investigate in SERT-/- rats how elevated 5-HT affects the feed forward inhibition circuits, which control afferent sensory information in LIV of S1. We show that an increased exposure to 5-HT during early cortical development affects the function of inhibitory networks within S1 by resulting in fewer functional soma-targeting inhibitory synapses and a depolarized GABA reversal potential in S1 LIV. On the neural population level stimulation of LIV networks resulted in increased excitatory responses in the intracortical circuits. Consequently, we show that reduced efficiency of the feedforward inhibition circuit and increased excitability along the LIV to LII/III pathway may underlie an accelerated and more efficient sensory performance during tactile behavior. Our results imply that serotonergic signaling during development affects activity driven cortical network maturation and the processing of sensory information.

Poster #19 Contribution of Distinct Progenitor Lineages to Neuronal Diversity in Layer 4 of the Mouse Barrel Cortex Teresa Guillamon-Vivancos, Maria Medalla, William Tyler, Tarik Haydar and Jennifer Luebke The striking diversity of neurons in the mammalian neocortex is key for the specification of cortical areas, but how this diversity is achieved during development is still poorly understood. Radial Glial Cells (RGCs), the neural stem cells of the developing cortex, also generate intermediate progenitor cells (IPCs), which in turn are able to generate new neurons. Our overall hypothesis is that distinct IPCs are responsible for generation of neuronal diversity in the neocortex. Our group recently showed that neurons derived from different IPCs have distinct morphological and electrophysiological properties in layers 2/3 of the mouse frontal cortex. In this study we aimed to determine whether different IPCs contribute to neuronal diversity in layer 4 of the barrel cortex, where several morphological and physiological types of neurons have been described. Using a novel genetic fatemapping technique we were able to simultaneously label progenitors that express Tbr2 and progenitors that don’t in different colors, as well as the neurons that derive from them. Through in utero electroporation at embryonic day E13.5 we labeled neurons in layer 4 of the barrel cortex. We studied whether neurons derived from distinct IPCs distributed differently across the depth and the distinct compartments of the barrel field. We also used whole-cell patch clamp recordings to assess the electrophysiological properties of these neurons. During the recordings, neurons were filled and their morphology studied using high-resolution confocal microscopy. This study contributes to a better understanding of how different streams of neurogenesis contribute to adult neuronal diversity and cortical organization.

Poster #20 The afferent connectome of VIP expressing GABAergic interneurons in the mouse barrel cortex Georg Hafner, Robin J. Wagener, Julien Guy, Mirko Witte, Martin K. Schwarz, Karl-Klaus Conzelmann and Jochen F. Staiger The very diverse population of inhibitory interneurons plays a fundamental role in shaping cortical activity. Vasoactive intestinal polypeptide (VIP) expressing interneurons have received attention as major integrators of long-range input into the local cortical network. However, from which areas they receive projections has not been studied systematically. We visualized the brain-wide monosynaptic afferent inputs to VIP interneurons in the mouse barrel cortex using retrograde rabies virus tracing. This technique specifically labels presynaptic cells with enhanced green fluorescent protein. More than 90% of cells presynaptic to VIP neurons were found locally within the barrel cortex. Other reliably labeled cortical areas included ipsilateral primary somatosensory (outside the whisker representation) and secondary somatosensory, visual, auditory, motor and cingulate cortex as well as contralateral barrel cortex. Subcortical projections originated from several thalamic nuclei and the basal forebrain. In addition, we characterized the neurochemistry of presynaptic inputs within the barrel cortex. Using in-situ hybridization and immunohistochemistry, about 20% of inputs were characterized as inhibitory and subclassified into somatostatin and parvalbumin positive cells. In conclusion, our anatomical data suggest that VIP neurons are mostly driven by local excitatory inputs but have the capacity to integrate long-range inputs.

Poster #21 Local and thalamic origins of ongoing and sensory evoked cortical correlations. Keti Cohen-Kashi, Boaz Mohar, Akiva N. Rappaport and Ilan Lampl The contribution of local circuits versus remote inputs in the generation of synchronized activity in sensory cortices is poorly understood. In sensory cortices, ascending information from the thalamus targets cells in L4. However, the impact of these inputs is controversial. The majority of the synaptic contacts in L4 are between neighboring cortical cells. Yet, electrophysiological studies have suggested that thalamic inputs are powerful enough to evoke the observed subthreshold response of L4 cells, without requiring inputs from neighboring cortical cells. How then such a low number of thalamic inputs can have such a profound impact on cortical response? A widely accepted mechanism that explains the robust activation of L4 cells is thalamic synchrony, implying that thalamic inputs of neighboring L4 cells are highly correlated. To investigate the role of both inputs in generation of cortical synchronization we isolated the thalamic excitatory inputs of cortical cells by optogenetically silencing cortical firing. In anesthetized mice we measured the correlation between isolated thalamic synaptic inputs of simultaneously patched nearby L4 cells of the barrel cortex. In contrast to correlated activity of excitatory synaptic inputs in the intact cortex, isolated thalamic inputs exhibit asynchronous spontaneous and sensory evoked inputs. These results were further supported in awake mice, where we recorded the excitatory inputs of individual cortical cells simultaneously with the local field potential (LFP) in a nearby site. Our results therefore indicate that cortical synchronization emerges by intracortical coupling.

Poster #22 Olfactory bulb and piriform cortex exhibit vibrissa responses that depend on barrel cortex activity. Ana Parabucki, Ilan Lampl Crucial aspect of sensory perception is that it is often a result of processes in which multiple cues, coming from different modalities (such as visual, auditory, touch and olfactory) are integrated into a unified concept. This multisensory integration greatly improves the reaction time and detection of stimuli, hence enhancing the representation of the external environment by reducing uncertainties in sensory assessment. A large body of multisensory integration studies indicated that most cross-modal interactions occur at high stages of processing. However, specific tactile-auditory inhibitory interactions were reported in the brainstem. We found that in anaesthetized mice stimulation of the vibrissae evoked a robust local field potential (LFP) response and elevated firing rate in the first stage of olfactory processing – the olfactory bulb (OB). Moreover, paired recordings showed a strong response to whisker stimulation in the piriform cortex (PC), which precedes the onset of OB response. Pharmacological and optogenetical inactivation of the barrel cortex substantially reduced the OB responses. In awake animals whisker stimulation evoked no clear response in the OB unless the animal was exposed to aversive odors. Together these findings the existence of a novel form of multisensory integration in which top-down inputs from the primary sensory cortex of one modality (barrel cortex) evoke robust neuronal activity at the early stage of sensory processing in another modality (olfactory bulb).

Poster #23 Layer 6 contributes to sensory induced potentiation in somatosensory and motor cortex Desire Humanes-Valera, Patrik Krieger Cortico-cortical communication has an important role in sensory processing. To study this communication we have focused on the sensorimotor circuit of the rodent vibrissal system. Sensory inputs were elicited by moving the whiskers using electrical stimulation of the whisker pad. Using a 2 Hz stimulation protocol, we induced a potentiation of whisker evoked responses in the sensorimotor circuit. Our results show that in somatosensory cortex (S1) responses to whisker movements, in both granular and supragranular layers, increase after the potentiation protocol. Moreover, motor cortex (M1) responses also increased, but only in layer 5, which is a major input layer for S1 projections. Cortical projections originating in layer 6 in S1 have an important function controlling columnar modulation. To study the contribution to the observed potentiation we blocked a sub-population of L6 pyramidal cells. Silencing the output of these layer 6 cells blocked the potentiation in L2/3 in S1 and in L5 in M1. The potentiation can be induced by subcortical and cortical mechanisms. In our results, we observed that the L6 blockade interfered with the intracortically mediated potentiation, but not the direct thalamic potentiation of layer 4, thus suggesting that silencing L6 did not affect the subcortical mechanisms.

Poster #24 Vibrissa motor cortex activity suppresses contralateral whisker movement Christian L. Ebbesen, Guy Doron, Constanze Lenschow, and Michael Brecht Anatomical, stimulation and lesion data point to a role of vibrissa motor cortex in the control of whisker movement. Motor cortex is classically thought to play a key role in movement generation, but most studies have found only weak correlations between vibrissa motor cortex activity and whisking. The exact role of vibrissa motor cortex in motor control remains unknown. To address this question we recorded vibrissa motor cortex neurons during various forms of vibrissal touch, all of which were associated with increased movement and forward positioning of whiskers. Free whisking, palpation of objects and social touch all resulted in similar vibrissa motor cortex responses: (i) Population activity decreased. (ii) The vast majority (~80%) of significantly modulated single cells decreased their firing. (iii) Rate-decreasing cells were the most strongly modulated cells. To understand the cellular basis of this decrease of activity, we performed juxtacellular recordings, nanostimulation and in vivo whole-cell recordings in head-fixed animals. Social facial touch – a strongly engaging stimulus – resulted in decreased spiking, massively decreased cell excitability and a ~1.5 mV hyperpolarization in vibrissa motor cortex neurons. To assess how activation of vibrissa motor cortex impacts whisking we performed intracortical microstimulation, which led to whisker retraction, as if to abort vibrissal touch. Finally, we blocked vibrissa motor cortex. A variety of inactivation protocols resulted in increased contralateral whisker movements and contralateral whisker protraction, as if to engage in vibrissal touch. Surprisingly, our observations collectively point to movement suppression as a prime function of vibrissa motor cortex activity.

Poster #25 Air-Track: A real-world floating environment for active sensing in head-fixed mice Mostafa A. Nashaat, Hatem Oraby, York Winter, Robert N. S. Sachdev, Matthew E. Larkum Natural behavior occurs in multiple sensory and motor modalities. These multimodal behavioral interactions are embodied in the various iterations of the head-fixed rodent stepping on a treadmill or walking on an air ball while navigating a virtual reality, or discriminating between somatosensory stimuli. A goal of these new developments is to elicit natural behavior by mimicking a quasinatural environment and to study the effect of locomotion on cortical responses at the cellular level. Here we have developed an “Air-Track” where head-fixed mice navigate a real environment. The environment has walls in the shape of a small plus maze, and surfaces for the animal to discriminate with whiskers or both. The Air-Track system combines a virtual reality – the animal is moving an Air-Track, but is not physically moving itself— and a physical reality, where movement by the mouse positions physical discriminanda that mouse uses in making decisions. In a two alternative forced choice task, the animal moves the Air-Track back and forth and rotates the maze from lane to lane, performing contextual behavior. In the course of this behavior, whisker and paw movement can be tracked in a newly developed online method. The track design can be modified and used to study decision-making quantitatively in quasi-natural complex environments. With the Air-Track, we come closest to providing a real environment for eliciting the natural behavior of mice in closed spaces.

Poster #26 Multidimensional response correlations enhance population code reliability and allow prediction of single trial noise in mouse visual cortex Jorrit S. Montijn, Guido T. Meijer, Carien S. Lansink, Cyriel M.A. Pennartz Sensory neurons are often tuned to particular stimulus features, but their responses to repeated presentation of the same stimulus can vary greatly over subsequent trials. This presents a problem for understanding the functioning of the brain, as downstream neuronal populations ought to construct accurate stimulus representations, even upon singular exposure. To study how trial-by-trial fluctuations (i.e., noise) in neuronal activity influence cortical representations of sensory input, we performed chronic imaging of GCaMP6-expressing populations in mouse V1 and observed that higher-dimensional response correlations within local populations can be used to predict single-trial, single-neuron noise. These multidimensional correlations are structured such that variability in the response of single neurons is relatively harmless to the population representation of grating orientation and natural movies. We propose that multidimensional coding may represent a canonical principle of cortical circuits explaining why the apparent noisiness of neuronal responses is compatible with accurate neural representations of stimulus features.

Poster #27 Organization of Barrel Cortex Layer 5B subcortical projections Anton Sumser, Bert Sakmann and Alexander Groh Neurons in cortical layer 5B (L5B) connect the cortex to various subcortical areas. Possibly the best-studied L5B cortico-subcortical connection is formed between L5B neurons in barrel cortex (BC) and the posterior medial nucleus of the thalamus (POm). L5B neurons form sparse but powerful giant synapses which can drive target POm via giant EPSPs (>10mV). Much less is known about the organization of L5B giant boutons in other parts of the brain. A complete target map of L5B neurons in barrel cortex is missing and it is unclear if L5B cortico-subcortical pathways retain somatotopic, i.e. body map organization. By dual, small and non-overlapping anterograde viral tracer injections in deep layers of BC, we labeled cortical bouton fields in the whole brain. Subsequent large-scale confocal scanning microscopy and slice alignment enabled us to semi-automatically reconstruct the projection fields of two adjacent BC L5B areas for each experiment. In total we extracted and reconstructed the 3D location of approximately 3 million large profiles from seven such dual injection experiments. We found that L5B in BC targets 13 subcortical areas (7 in thalamus, 5 in brainstem, 1 in midbrain). Bouton numbers, density and projection volume varied greatly between nuclei. However, in all investigated areas we consistently found somatotopic segregation of the projections from different barrel columns in BC, albeit with varying precision. In conclusion, corticofugal L5B targets many subcortical structures via sparse but somatotopically organized pathways.

Poster #28 Correlation between Cortical State and Locus Coeruleus Activity: Implications for Sensory Coding in Rat Barrel Cortex Zeinab Fazlali, Yadollah Ranjbar-Slamloo, Mehdi Adibi and Ehsan Arabzadeh Locus Coeruleus (LC) neuromodulatory nucleus in the brainstem is known to modulate the activity of individual cells as well as global brain state. Here, we quantified the link between spontaneous LC activity, cortical state and sensory processing in the rat Barrel Cortex (BC). Under urethane anesthesia, we simultaneously recorded unit activity from LC and BC along with prefrontal EEG while presenting brief whisker deflections of various amplitudes. The ratio of low to high frequency components of EEG (referred to as the L/H ratio) identified the cortical state. We found that spontaneous activity of all recorded units in LC exhibited a negative correlation with the L/H ratio. Cross-correlation analysis revealed that changes in LC firing rate preceded changes in state: the cross-correlation function between LC firing profile and L/H ratio showed the strongest correlation at -1.2 s. We further quantified BC neuronal responses to whisker stimulation during the synchronized (high L/H ratio) and desynchronized (low L/H ratio) states. In the desynchronized state, BC neurons showed lower stimulus detection threshold, lower trial-to-trial variability, and shorter response latency. The most prominent change in BC response was observed during the late phase of evoked activity (100-400 ms post stimulus onset): the desynchronized state significantly increased the late response almost for every recorded BC unit. These findings provide evidence for the involvement of the LC norepinephrine neuromodulatory system, in desynchronization of cortical state and a consequent enhancement of sensory coding efficiency.

Poster #29 Somatosensory Processing and Plasticity Deficits in Mouse Barrel Cortex Project #1: Konrad Juczewski, Helen von Richthofen, Claudia Bagni, Tansu Celikel, Gilberto Fisone, Patrik Krieger Project #2: Stuart D. Greenhill, Konrad Juczewski, Annelies de Haan, Gillian Seaton, Kevin Fox, Neil R. Hardingham Touch is an important source of sensory information. Disturbances to the development of the somatosensory system have serious consequences for social behavior and may lead to many neurodevelopmental disorders. In our studies we used two different mouse models of disease: Fmr1 KO mouse with a knock out gene Fmr1 (a model of fragile X syndrome); and DISC1-cc transgenic mouse with transient disruption of signaling (DISC1 is a molecule implicated in psychiatric disorders). In Fmr1 KO mice we focused on analyzing somatosensory processing defects that lead to hypersensitivity to touch in Fragile X Syndrome (FXS) patients. We show neuronal mechanisms that appear to underlie hypersensitivity to somatosensory stimuli (whisker deflections) thus causing an altered behavior observed in Fmr1 KO mice. In addition we provide evidence showing how the processing and encoding of tactile information have been affected. In DISC1-cc mice we activated truncated protein DISC1-cc for a controlled period of time at different points during the early postnatal development. Development is shaped by sensory experience, especially during phases known as critical periods. Disruption of experience during a critical period normally produces neurons that lack specificity for sensory experience in adulthood. We found that transient disruption of DISC1 signaling during a critical period of development produced neurons that lack plasticity in adulthood. Adult plasticity deficits may be associated with cognitive deficits and the delayed onset of psychiatric symptoms in late adolescence. Using the whisker system as a model system we have obtained insight into potential disease mechanism causing human brain disorders.