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Acquisition of a behavioral task is influenced by many factors. The relative timing of stimuli is such a factor and is especially relevant for tasks relying on short-term memory, like working memory paradigms, because of the constant evolution and decay of neuronal activity evoked by stimuli. Here, we assess two aspects of stimulus timing on the acquisition of an olfactory delayed nonmatch-to-sample (DNMS) task. We demonstrate that head-fixed male mice learn to perform the task more quickly when the initial training uses a shorter sample-test odor delay without detectable loss of generalizability. Unexpectedly, we observed a slower task acquisition when the odor–reward interval was shorter. The effect of early reward timing was accompanied by a shortening of reaction times and more frequent sporadic licking. Analysis of this result using a drift-diffusion model indicated that a primary consequence of early reward delivery is a lowered threshold to act, or a lower decision bound. Because an accurate performance with a lower decision bound requires greater discriminability in the sensory representations, this may underlie the slower learning rate with early reward arrival. Together, our results reflect the possible effects of stimulus timing on stimulus encoding and its consequence on the acquisition of a complex task.SIGNIFICANCE STATEMENTThis study describes how head-fixed mice acquire a working memory task (olfactory delayed nonmatch-to-sample task). We simplified and optimized the stimulus timing, allowing robust and efficient training of head-fixed mice. Unexpectedly, we found that early reward timing leads to slower learning. Analysis of this data using a computational model (drift-diffusion model) revealed that the reward timing affects the behavioral threshold, or how quickly animals respond to a stimulus. But, to still be accurate with early reaction times, the sensory representation needs to become even more refined. This may explain the slower learning rate with early reward timing.

In sensory systems of the brain, mechanisms exist to extract distinct features from stimuli to generate a variety of behavioural repertoires. These often correspond to different cell types at various stages in sensory processing. In the mammalian olfactory system, complex information processing starts in the olfactory bulb, whose output is conveyed by mitral and tufted cells (MCs and TCs). Despite many differences between them, and despite the crucial position they occupy in the information hierarchy, Cre-driver lines that distinguish them do not yet exist. Here, we sought to identify genes that are differentially expressed between MCs and TCs of the mouse, with an ultimate goal to generate a cell-type specific Cre-driver line, starting from a transcriptome analysis using a large and publicly available single-cell RNA-seq dataset (Zeisel et al., 2018). Many genes were differentially expressed, but only a few showed consistent expressions in MCs and at the specificity required. After further validating these putative markers using in-situ hybridization, two genes, namely Pkib and Lbdh2, remained as promising candidates. Using CRISPR/Cas9-mediated gene editing, we generated Cre-driver lines and analysed the resulting recombination patterns. This indicated that our new inducible Cre-driver line, Lbhd2-CreERT2, can be used to genetically label MCs in a tamoxifen dose-dependent manner, both in male and female mice, as assessed by soma locations, projection patterns and sensory-evoked responses in vivo. Hence this is a promising tool for investigating cell-type specific contributions to olfactory processing and demonstrates the power of publicly accessible data in accelerating science. SIGNIFICANCE STATEMENT: In the brain, distinct cell types play unique roles. It is therefore important to have tools for studying unique cell types specifically. For the sense of smell in mammals, information is processed first by circuits of the olfactory bulb, where two types of cells, mitral cells and tufted cells, output different information. We generated a transgenic mouse line that enables mitral cells to be specifically labelled or manipulated. This was achieved by looking for genes that are specific to mitral cells using a large and public gene expression dataset, and creating a transgenic mouse using the gene editing technique, CRISPR/Cas9. This will allow scientists to better investigate parallel information processing underlying the sense of smell.

Knowing what factors affect the acquisition of a behavioural task is central to understanding the mechanisms of learning and memory. It also has practical implications, as animal behavioural experiments used to probe cognitive functions often require long training durations. Delayed Match (or Non-Match)-to-Sample (DMS/DNMS) tasks are relatively complex tasks used to study working memory and sensory perception, but their use in the mouse remains hampered by the lengthy training involved. In this study, we assessed two aspects of stimulus timing on the acquisition of an olfactory DNMS task: how the sample-test odour delay durations and the reward timing affect the acquisition rate. We demonstrate that head-fixed mice learn to perform an olfactory DNMS task more quickly when the initial training uses a shorter sample-test odour delay without detectable loss of generalisability. Unexpectedly, we observed a slower task acquisition when the odour-reward interval was shorter. This effect was accompanied by a shortening of reaction times and more frequent sporadic licking. Analysis of this result using a drift-diffusion model indicated that a primary consequence of early reward delivery is a lower decision bound. Since an accurate performance with a lower decision bound requires greater discriminability in the sensory representations, this may underlie the slower learning rate with early reward arrival. Together, our results reflect the possible effects of stimulus timing on stimulus encoding and its consequence on the acquisition of a complex task.

Sensory systems are often tasked to analyse complex signals from the environment, separating relevant from irrelevant parts. This process of decomposing signals is challenging when a mixture of signals does not equal the sum of its parts, leading to an unpredictable corruption of signal patterns. In olfaction, nonlinear summation is prevalent at various stages of sensory processing. Here, we investigate how the olfactory system deals with binary mixtures of odours under different brain states, using two-photon imaging of olfactory bulb (OB) output neurons. Unlike previous studies using anaesthetised animals, we found that mixture summation is more linear in the early phase of evoked responses in awake, head-fixed mice performing an odour detection task, due to dampened responses. Despite this, and responses being more variable, decoding analyses indicated that the data from behaving mice was well discriminable. Curiously, the time course of decoding accuracy did not correlate strictly with the linearity of summation. Further, a comparison with naïve mice indicated that learning to accurately perform the mixture detection task is not accompanied by more linear mixture summation. Finally, using a simulation, we demonstrate that, while saturating sublinearity tends to degrade the discriminability, the extent of the impairment may depend on other factors, including pattern decorrelation. Altogether, our results demonstrate that the mixture representation in the primary olfactory area is state-dependent, but the analytical perception may not strictly correlate with linearity in summation.

Volatile chemicals in the environment provide ethologically important information to many animals. However, how animals learn to use this information is only beginning to be understood. This review highlights recent experimental advances elucidating olfactory learning in rodents, ranging from adaptations to the environment to task-dependent refinement and multisensory associations. The broad range of phenomena, mechanisms, and brain areas involved demonstrate the complex and multifaceted nature of olfactory learning.

In each sensory system of the brain, mechanisms exist to extract distinct features from stimuli to generate a variety of behavioural repertoires. These often correspond to different cell types at some stage in sensory processing. In the mammalian olfactory system, complex information processing starts in the olfactory bulb, whose output is conveyed by mitral and tufted cells (MCs and TCs). Despite many differences between them, and despite the crucial position they occupy in the information hierarchy, little is known how these two types of projection neurons differ at the mRNA level. Here, we sought to identify genes that are differentially expressed between MCs and TCs, with an ultimate goal to generate a cell-type specific Cre-driver line, starting from a transcriptome analysis using a large and publicly available single-cell RNA-seq dataset (Zeisel et al., 2018). Despite many genes showing differential expressions, we identified only a few that were abundantly and consistently expressed only in MCs. After further validating these putative markers usingin-situhybridization, two genes, namelyPkibandLbdh2, remained as promising candidates. Using CRISPR/Cas9-mediated gene editing, we generated Cre-driver lines and analysed the resulting recombination patterns. This analysis indicated that our new inducible Cre-driver line,Lbhd2-CreERT2, can be used to genetically label MCs in a tamoxifen dose-dependent manner, as assessed by soma locations, projection patterns and sensory-evoked responses. Hence this line is a promising tool for future investigations of cell-type specific contributions to olfactory processing and demonstrates the power of publicly accessible data in accelerating science.

Odours are transported in turbulent plumes, which result in rapid concentration fluctuations1,2 that contain rich information about the olfactory scenery, such as the composition and location of an odour source2–4. However, it is unclear whether the mammalian olfactory system can use the underlying temporal structure to extract information about the environment. Here we show that ten-millisecond odour pulse patterns produce distinct responses in olfactory receptor neurons. In operant conditioning experiments, mice discriminated temporal correlations of rapidly fluctuating odours at frequencies of up to 40 Hz. In imaging and electrophysiological recordings, such correlation information could be readily extracted from the activity of mitral and tufted cells—the output neurons of the olfactory bulb. Furthermore, temporal correlation of odour concentrations5 reliably predicted whether odorants emerged from the same or different sources in naturalistic environments with complex airflow. Experiments in which mice were trained on such tasks and probed using synthetic correlated stimuli at different frequencies suggest that mice can use the temporal structure of odours to extract information about space. Thus, the mammalian olfactory system has access to unexpectedly fast temporal features in odour stimuli. This endows animals with the capacity to overcome key behavioural challenges such as odour source separation5, figure–ground segregation6 and odour localization7 by extracting information about space from temporal odour dynamics. Fast temporal dynamics of the olfactory environment can be perceived by mice and used to perform scene segmentation.

A development of a biocompatible, optical stimulation device capable of adhering to the brain surface and activating spatially separated brain regions is necessary for in vivo optogenetic applications. In this study, a hollow structure for isolating the microLED epitaxial layer was fabricated using the anisotropic KOH wet-etching method. Using a thermal release sheet, a method to transfer microLEDs onto a biocompatible parylene film was established without rotation or misalignment of the microLEDs while retaining their characteristics. Accordingly, a flexible microLED array film was fabricated, which adhered to the surface of the brain of a mouse and exhibited blue emission.

Odours are transported in turbulent plumes resulting locally in highly fluctuating odour concentration (Celani et al., 2014; Murlis et al., 1992; Mylne and Mason, 1991; Shraiman and Siggia, 2000). Yet, whether mammals can make use of the ensuing temporal structure (Celani et al., 2014; Crimaldi and Koseff, 2001; Murlis et al., 1992; Mylne and Mason, 1991; Schmuker et al., 2016; Vickers, 2000) to extract information about the olfactory environment remains unknown. Here, we use dual-energy photoionisation recording with >300 Hz bandwidth to simultaneously determine odour concentrations of two odours in air. We show that temporal correlation of odour concentrations reliably predicts whether odorants emerge from the same or different sources in normal turbulent environments outside and in laboratory conditions. To replicate natural odour dynamics in a reproducible manner we developed a multichannel odour delivery device allowing presentation of several odours with 10 ms temporal resolution. Integrating this device in an automated operant conditioning system we demonstrate that mice can reliably discriminate the correlation structure of odours at frequencies of up to 40 Hz. Consistent with this finding, output neurons in the olfactory bulb show segregated responses depending on the correlation of odour stimuli with populations of 10s of neurons sufficient to reach behavioural performance. Our work thus demonstrates that mammals can perceive temporal structure in odour stimuli at surprisingly fast timescales. This in turn might be useful for key behavioural challenges (Jacobs, 2012) such as odour source separation (Hopfield, 1991), figure-ground separation (Rokni et al., 2014) or odour localisation (Vergassola et al., 2007; Vickers, 2000).

Adapting neural representation to rapidly changing behavioural demands is a key challenge for the nervous system. Here, we demonstrate that the output of the primary olfactory area, the mouse olfactory bulb, is already a target of dynamic and reproducible modulation. The modulation depends on the stimulus tuning of a given neuron, making olfactory responses more discriminable through selective amplification in a demand-specific way.

The raphe nuclei provide serotonergic innervation widely in the brain, thought to mediate a variety of neuromodulatory effects. The mammalian olfactory bulb (OB) is a prominent recipient of serotonergic fibers, particularly in the glomerular layer (GL), where they are thought to gate incoming signals from the olfactory nerve. The dorsal raphe nucleus (DRN) and the median raphe nucleus (MRN) are known to densely innervate the OB. The majority of such projections are thought to terminate in the GL, but this has not been explicitly tested. We sought to investigate this using recombinant adeno-associated viruses (rAAV)-mediated expression of green fluorescent protein (GFP)-synaptophysin targeted specifically to neurons of the DRN or the MRN. With DRN injections, labeled fibers were found mostly in the granule cell layer (GCL), not the GL. Conversely, dense labeling in the GL was observed with MRN injections, suggesting that the source of GL innervation is the MRN, not the DRN, as previously thought. The two raphe nuclei thus give dual innervation within the OB, with distinct innervation patterns. J. Comp. Neurol. 523:805–813, 2015. © 2015 Wiley Periodicals, Inc.

Summary The olfactory bulb (OB) is the first site of synaptic odor information processing, yet a wealth of contextual and learned information has been described in its activity. To investigate the mechanistic basis of contextual modulation, we use whole-cell recordings to measure odor responses across rapid learning episodes in identified mitral/tufted cells (MTCs). Across these learning episodes, diverse response changes occur already during the first sniff cycle. Motivated mice develop active sniffing strategies across learning that robustly correspond to the odor response changes, resulting in enhanced odor representation. Evoking fast sniffing in different behavioral states demonstrates that response changes during active sampling exceed those predicted from feedforward input alone. Finally, response changes are highly correlated in tufted cells, but not mitral cells, indicating there are cell-type-specific effects on odor representation during active sampling. Altogether, we show that active sampling is strongly associated with enhanced OB responsiveness on rapid timescales., Highlights • During rapid olfactory learning, responses of mitral and tufted cells change overtly • These changes reliably track the development of active sniffing • This enhances odor detectability and discriminability in a cell-type-specific way • Active sniffing impacts odor responses through both bottom-up and top-down mechanisms, Using whole-cell recordings in the olfactory bulb of mice across a range of behavioral states, Jordan et al. show that odor responses are dynamically enhanced during learning and task engagement, tightly mirroring alterations in active sniffing.

Spike timing-based representations of sensory information depend on embedded dynamical frameworks within neural structures that establish the rules of local computation and interareal communication. Here, we investigated the dynamical properties of mouse olfactory bulb circuitry. Neurochemical activation or optogenetic stimulation of sensory afferents evoked persistent (minutes) gamma oscillations in the local field potential. These oscillations arose from slower, GABA(A) receptor-independent intracolumnar oscillators coupled by GABA(A)-ergic synapses into a faster, broadly coherent network oscillation. Consistent with the theoretical properties of coupled-oscillator networks, the spatial extent of zero-phase coherence was bounded in slices by the reduced density of lateral interactions. The intact in vivo network, however, exhibits long-range lateral interactions theoretically sufficient to enable zero-phase coherence across the complete network. These coupled-oscillator dynamics thereby establish a common clock, robust to biological heterogeneities, that is capable of supporting gamma-band phase coding across the spiking output of olfactory bulb principal neurons.

WOS: 000415310800007 PubMed ID: 29144972 Science is ideally suited to connect people from different cultures and thereby foster mutual understanding. To promote international life science collaboration, we have launched "The Science Bridge'' initiative. Our current project focuses on partnership between Western and Middle Eastern neuroscience communities. Medical Research Council [MC_UP_1202/5]

Circuits in the brain possess the ability to orchestrate activities on different timescales, but the manner in which distinct circuits interact to sculpt diverse rhythms remains unresolved. The olfactory bulb is a classic example of a place in which slow theta and fast gamma rhythms coexist. Furthermore, inhibitory interneurons that are generally implicated in rhythm generation are segregated into distinct layers, neatly separating local and global motifs. We combined intracellular recordings in vivo with circuit-specific optogenetic interference to examine the contribution of inhibition to rhythmic activity in the mouse olfactory bulb. We found that the two inhibitory circuits controlled rhythms on distinct timescales: local, glomerular networks coordinated theta activity, regulating baseline and odor-evoked inhibition, whereas granule cells orchestrated gamma synchrony and spike timing. Notably, granule cells did not contribute to baseline rhythms or sniff-coupled odor-evoked inhibition. Thus, activities on theta and gamma timescales are controlled by separate, dissociable inhibitory networks in the olfactory bulb., Deutsche Forschungsgemeinschaft (DFG-SPP1392), Max Planck Society for the Advancement of Science, Alexander von Humboldt-Stiftung, Germany. Federal Ministry of Education and Research (US-German collaboration computational neuroscience), Medical Research Council (Great Britain) (MC_UP_1202/5), University of Tubingen (ExcellenzCluster Cell Networks)

Perturbations in neural circuits can provide mechanistic understanding of the neural correlates of behavior. In M71 transgenic mice with a “monoclonal nose”, glomerular input patterns in the olfactory bulb are massively perturbed and olfactory behaviors are altered. To gain insights into how olfactory circuits can process such degraded inputs we characterized odor-evoked responses of olfactory bulb mitral cells and interneurons. Surprisingly, calcium imaging experiments reveal that mitral cell responses in M71 transgenic mice are largely normal, highlighting a remarkable capacity of olfactory circuits to normalize sensory input. In vivo whole cell recordings suggest that feedforward inhibition from olfactory bulb periglomerular cells can mediate this signal normalization. Together, our results identify inhibitory circuits in the olfactory bulb as a mechanistic basis for many of the behavioral phenotypes of mice with a “monoclonal nose” and highlight how substantially degraded odor input can be transformed to yield meaningful olfactory bulb output.

How wakefulness shapes neural activity is a topic of intense discussion. In the awake olfactory bulb, high activity with weak sensory-evoked responses has been reported in mitral/tufted cells (M/TCs). Using blind whole-cell recordings, we found 33% of M/TCs to be 'silent', yet still show strong sensory responses, with weak or inhibitory responses in 'active' neurons. Thus, a previously missed M/TC subpopulation can exert powerful influence over the olfactory bulb.

Background and purpose: Specific and selective inhibitors for mGlu1 receptors are presently inadequate. A new generation of non-competitive mGlu1 antagonists with low nanomolar potencies is emerging. We evaluated two new compounds, YM-298198 and JNJ16259685, for effectiveness, potency and specificity for the first time in a brain slice preparation. Experimental approach: Patch-clamp recording of Purkinje neurones in cerebellar slices were obtained. The slow mGlu1mediated EPSP was used to establish a concentration-response curve. Fast excitatory synaptic inputs were tested for nonspecific effects. Key results: YM-298198 and JNJ16259685 inhibited the synaptic activation of mGlu1 in a concentration-dependent manner (IC50 values of 24 nM and 19 nM, respectively). The antagonists were slow to inhibit and to reverse on washout, probably due to their lipophilic nature. There were no non-specific effects on fast AMPA receptor-mediated synaptic transmission in the cerebellum. Conclusions and implications: These compounds are more than a thousand-fold more potent than previously available compounds. Their selectivity and specificity will be very useful for studying the role of mGlu1 receptors both in vitro and in vivo. British Journal of Pharmacology (2007) 151, 870–876; doi:10.1038/sj.bjp.0707286; published online 14 May 2007

CPCCOEt (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) is frequently used to test for the involvement of mGlu1 receptors. Using whole-cell voltage recording from Purkinje cells in slices of rat cerebellum we find that CPCCOEt, at concentrations used to block mGlu1 receptors, causes an enhancement of the climbing fibre response. Application of alternative antagonists with activity at mGlu1 neither mimicked nor occluded the effects of CPCCOEt. Receptor antagonists demonstrated that this non-mGlu1 action of CPCCOEt was not mediated by other mGlu receptors or GABAB receptors. Voltage-clamped climbing fibre EPSCs are unaffected by CPCCOEt whilst application of a glutamate transport blocker did not occlude the CPCCOEt effect. This suggests that a postsynaptic voltage-dependent component of the complex climbing fibre response is the target. We have found no evidence for the involvement of the hyperpolarisation-activated current, Ih, and calcium-activated conductances. Voltage-gated sodium, calcium and potassium channels are possible targets with inhibition of a potassium channel the most likely. Awareness of this non-mGlu-mediated effect of CPCCOEt is likely to be important for the correct interpretation of its actions.

SummaryLocal inhibitory circuits are thought to shape neuronal information processing in the central nervous system, but it remains unclear how specific properties of inhibitory neuronal interactions translate into behavioral performance. In the olfactory bulb, inhibition of mitral/tufted cells via granule cells may contribute to odor discrimination behavior by refining neuronal representations of odors. Here we show that selective deletion of the AMPA receptor subunit GluA2 in granule cells boosted synaptic Ca2+ influx, increasing inhibition of mitral cells. On a behavioral level, discrimination of similar odor mixtures was accelerated while leaving learning and memory unaffected. In contrast, selective removal of NMDA receptors in granule cells slowed discrimination of similar odors. Our results demonstrate that inhibition of mitral cells controlled by granule cell glutamate receptors results in fast and accurate discrimination of similar odors. Thus, spatiotemporally defined molecular perturbations of olfactory bulb granule cells directly link stimulus similarity, neuronal processing time, and discrimination behavior to synaptic inhibition.

Making choices is a fundamental aspect of human life. For over a century experimental economists have characterized the decisions people make based on the concept of a utility function. This function increases with increasing desirability of the outcome, and people are assumed to make decisions so as to maximize utility. When utility depends on several variables, indifference curves arise that represent outcomes with identical utility that are therefore equally desirable. Whereas in economics utility is studied in terms of goods and services, the sensorimotor system may also have utility functions defining the desirability of various outcomes. Here, we investigate the indifference curves when subjects experience forces of varying magnitude and duration. Using a two-alternative forced-choice paradigm, in which subjects chose between different magnitude–duration profiles, we inferred the indifference curves and the utility function. Such a utility function defines, for example, whether subjects prefer to lift a 4-kg weight for 30 s or a 1-kg weight for a minute. The measured utility function depends nonlinearly on the force magnitude and duration and was remarkably conserved across subjects. This suggests that the utility function, a central concept in economics, may be applicable to the study of sensorimotor control., Economists use the concept of a utility function, which increases with increasing desirability of the outcome, to characterize human decision making. This concept is shown here to apply to the control of movement

Several forms of learning, including classical conditioning of the eyeblink, depend upon the cerebellum. In examining mechanisms of eyeblink conditioning in rabbits, reversible inactivations of the control circuitry have begun to dissociate aspects of cerebellar cortical and nuclear function in memory consolidation. It was previously shown that post-training cerebellar cortical, but not nuclear, inactivations with the GABA(A) agonist muscimol prevented consolidation but these findings left open the question as to how final memory storage was partitioned across cortical and nuclear levels. Memory consolidation might be essentially cortical and directly disturbed by actions of the muscimol, or it might be nuclear, and sensitive to the raised excitability of the nuclear neurons following the loss of cortical inhibition. To resolve this question, we simultaneously inactivated cerebellar cortical lobule HVI and the anterior interpositus nucleus of rabbits during the post-training period, so protecting the nuclei from disinhibitory effects of cortical inactivation. Consolidation was impaired by these simultaneous inactivations. Because direct application of muscimol to the nuclei alone has no impact upon consolidation, we can conclude that post-training, consolidation processes and memory storage for eyeblink conditioning have critical cerebellar cortical components. The findings are consistent with a recent model that suggests the distribution of learning-related plasticity across cortical and nuclear levels is task-dependent. There can be transfer to nuclear or brainstem levels for control of high-frequency responses but learning with lower frequency response components, such as in eyeblink conditioning, remains mainly dependent upon cortical memory storage.