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Wide-field calcium imaging is often used to measure brain dynamics in behaving mice. With a large field of view and a high sampling rate, wide-field imaging can monitor activity from several distant cortical areas simultaneously, revealing cortical interactions. Interpretation of wide-field images is complicated, however, by the absorption of light by hemoglobin, which can substantially affect the measured fluorescence. One approach to separating hemodynamics and calcium signals is to use multiwavelength backscatter recordings to measure light absorption by hemoglobin. Following this approach, we develop a spatially detailed regression-based method to estimate hemodynamics. This Spatial Model is based on a linear form of the Beer–Lambert relationship but is fit at every pixel in the image and does not rely on the estimation of physical parameters. In awake mice of three transgenic lines, the Spatial Model offers improved separation of hemodynamics and changes in GCaMP fluorescence. The improvement is pronounced near blood vessels and, in contrast with the Beer–Lambert equations, can remove vascular artifacts along the sagittal midline and in general permits more accurate fluorescence-based determination of neuronal activity across the cortex. NEW & NOTEWORTHY This paper addresses a well-known and strong source of contamination in wide-field calcium-imaging data: hemodynamics. To guide researchers toward the best method to separate calcium signals from hemodynamics, we compare the performance of several methods in three commonly used mouse lines and present a novel regression model that outperforms the other techniques we consider.

Scientists have long conjectured that the neocortex learns the structure of the environment in a predictive, hierarchical manner. According to this conjecture, expected, predictable features are differentiated from unexpected ones by comparing bottom-up and top-down streams of information. It is theorized that the neocortex then changes the representation of incoming stimuli, guided by differences in the responses to expected and unexpected events. In line with this conjecture, different responses to expected and unexpected sensory features have been observed in spiking and somatic calcium events. However, it remains unknown whether these unexpected event signals occur in the distal apical dendrites where many top-down signals are received, and whether these signals govern subsequent changes in the brain’s stimulus representations. Here, we show that both somata and distal apical dendrites of cortical pyramidal neurons exhibit distinct unexpected event signals that systematically change over days. These findings were obtained by tracking the responses of individual somata and dendritic branches of layer 2/3 and layer 5 pyramidal neurons over multiple days in primary visual cortex of awake, behaving mice using two-photon calcium imaging. Many neurons in both layers 2/3 and 5 showed large differences between their responses to expected and unexpected events. Interestingly, these responses evolved in opposite directions in the somata and distal apical dendrites. These differences between the somata and distal apical dendrites may be important for hierarchical computation, given that these two compartments tend to receive bottom-up and top-down information, respectively.

3-photon excitation permits fluorescence microscopy through the intact skull. We demonstrate 3-photon fluorescence imaging of neuronal structure and calcium activity in a chronic transcranial window preparation up to 300 μm into brain below the intact skull.

Visual perception and behavior are mediated by cortical areas that have been distinguished using architectonic and retinotopic criteria. We employed fluorescence imaging and GCaMP6 reporter mice to generate retinotopic maps, revealing additional regions of retinotopic organization that extend into barrel and retrosplenial cortices. Aligning retinotopic maps to architectonic borders, we found a mismatch in border location, indicating that architectonic borders are not aligned with the retinotopic transition at the vertical meridian. We also assessed the representation of visual space within each region, finding that four visual areas bordering V1 (LM, P, PM and RL) display complementary representations, with overlap primarily at the central hemifield. Our results extend our understanding of the organization of mouse cortex to include up to 16 distinct retinotopically organized regions. DOI: http://dx.doi.org/10.7554/eLife.18372.001, eLife digest Our eyes send information about the world around us to a region on the surface of the brain called the visual cortex, which is made up of a series of interconnected areas. Researchers have studied the anatomy and activity of these areas to generate maps that show how these areas are arranged. For example, architectonic borders are drawn where there are abrupt changes in the density of cells, or the degree to which they are stained by certain chemicals. Maps based on architectonics and those based on brain activity are thought to exhibit matching borders between visual areas. Zhuang et al. used animaging approach to produce detailed maps of the visual cortex of mice. The approach uses a fluorescent protein called GCaMP6 to indicate levels of activity in the brain while the mice were exposed to visual cues. Furthermore, Zhuang et al. added a second step to this approach to reveal the architectonic borders of the areas in the visual cortex. This made it possible to compare the locations of activity-based and anatomical borders in a single mouse. Zhuang et al. found that maps of the visual cortex based on architectonics do not completely match those based on activity. These findings help reconcile the differences between maps of mouse visual cortex produced by other studies. It is not clear whether a similar mismatch in architectonic and activity-based border locations exists in other animals, such as primates. DOI: http://dx.doi.org/10.7554/eLife.18372.002

3-photon excitation permits fluorescence microscopy through the intact skull, but is highly sensitive to aberration. We estimate aberration encountered in focusing 1250 nm light through a glass-on-acrylic window for chronic transcranial in vivo imaging.

Modern neuroscience has demonstrated how the adult brain has the ability to profoundly remodel its neurons in response to changes in external stimuli or internal states. However, adult brain plasticity, although possible throughout life, remains restricted mostly to subcellular levels rather than affecting the entire cell. New neurons are continuously generated in only a few areas of the adult brain—the olfactory bulb and the dentate gyrus—where they integrate into already functioning circuitry. In these regions, adult neurogenesis adds another dimension of plasticity that either complements or is redundant to the classical molecular and cellular mechanisms of plasticity. This review extracts clues regarding the contribution of adult-born neurons to the different circuits of the olfactory bulb and specifically how new neurons participate in existing computations and enable new computational functions.

Most organisms use their olfactory system to detect and analyze chemical cues from the external world to guide essential behaviors. From worms to vertebrates, chemicals are detected by odorant receptors expressed by olfactory sensory neurons, which in vertebrates send an axon to the primary processing center called the olfactory bulb (OB). Within the OB, sensory neurons form excitatory synapses with projection neurons and with inhibitory interneurons. Thus, because of complex synaptic interactions, the output of a given projection neuron is determined not only by the sensory input, but also by the activity of local inhibitory interneurons that are regenerated throughout life in the process of adult neurogenesis. Herein, we discuss how it is optimized and why.

International audience; In the mammalian brain, the anatomical structure of neural circuits changes little during adulthood. As a result, adult learning and memory are thought to result from specific changes in synaptic strength. A possible exception is the olfactory bulb (OB), where activity guides interneuron turnover throughout adulthood. These adult-born granule cell (GC) interneurons form new GABAergic synapses that have little synaptic strength plasticity. In the face of persistent neuronal and synaptic turnover, how does the OB balance flexibility, as is required for adapting to changing sensory environments, with perceptual stability? Here we show that high dendritic spine turnover is a universal feature of GCs, regardless of their developmental origin and age. We find matching dynamics among postsynaptic sites on the principal neurons receiving the new synaptic inputs. We further demonstrate in silico that this coordinated structural plasticity is consistent with stable, yet flexible, decorrelated sensory representations. Together, our study reveals that persistent, coordinated synaptic structural plasticity between interneurons and principal neurons is a major mode of functional plasticity in the OB.

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

We explored the structural basis of voltage sensing in the HCN1 hyperpolarization-activated cyclic nucleotide-gated cation channel by examining the relative orientation of the voltage sensor and pore domains. The opening of channels engineered to contain single cysteine residues at the extracellular ends of the voltage-sensing S4 (V246C) and pore-forming S5 (C303) domains is inhibited by formation of disulfide or cysteine:Cd(2+) bonds. As Cd(2+) coordination is promoted by depolarization, the S4-S5 interaction occurs preferentially in the closed state. The failure of oxidation to catalyze dimer formation, as assayed by Western blotting, indicates the V246C:C303 interaction occurs within a subunit. Intriguingly, a similar interaction has been observed in depolarization-activated Shaker voltage-dependent potassium (Kv) channels at depolarized potentials but such an intrasubunit interaction is inconsistent with the X-ray crystal structure of Kv1.2, wherein S4 approaches S5 of an adjacent subunit. These findings suggest channels of opposite voltage-sensing polarity adopt a conserved S4-S5 orientation in the depolarized state that is distinct from that trapped upon crystallization.

Sensory perception emerges from the confluence of sensory inputs that encode the composition of external environment and top-down feedback that conveys information from higher brain centers. In olfaction, sensory input activity is initially processed in the olfactory bulb (OB), serving as the first central relay before being transferred to the olfactory cortex. In addition, the OB receives dense connectivity from feedback projections, so the OB has the capacity to implement a wide array of sensory neuronal computation. However, little is known about the impact and the regulation of this cortical feedback. Here, we describe a novel mechanism to gate glutamatergic feedback selectively from the anterior olfactory cortex (AOC) to the OB. Combining in vitro and in vivo electrophysiological recordings, optogenetics, and fiber-photometry-based calcium imaging applied to wild-type and conditional transgenic mice, we explore the functional consequences of circuit-specific GABA type-B receptor (GABA(B)R) manipulation. We found that activation of presynaptic GABA(B)Rs specifically depresses synaptic transmission from the AOC to OB inhibitory interneurons, but spares direct excitation to principal neurons. As a consequence, feedforward inhibition of spontaneous and odor-evoked activity of principal neurons is diminished. We also show that tunable cortico-bulbar feedback is critical for generating beta, but not gamma, OB oscillations. Together, these results show that GABA(B)Rs on cortico-bulbar afferents gate excitatory transmission in a target-specific manner and thus shape how the OB integrates sensory inputs and top-down information. SIGNIFICANCE STATEMENT The olfactory bulb (OB) receives top-down inputs from the olfactory cortex that produce direct excitation and feedforward inhibition onto mitral and tufted cells, the principal neurons. The functional role of this feedback and the mechanisms regulating the balance of feedback excitation and inhibition remain unknown. We found that GABA(B) receptors are expressed in cortico-bulbar axons that synapse on granule cells and receptor activation reduces the feedforward inhibition of spontaneous and odor-driven mitral and tufted cells' firing activity. In contrast, direct excitatory inputs to these principal neurons remain unchanged. This study demonstrates that activation of GABA(B) receptors biases the excitation/inhibition balance provided by cortical inputs to the OB, leading to profound effects on early stages of sensory information processing.

The study of cholinergic signaling in the mammalian CNS has been greatly facilitated by the advent of mouse lines that permit the expression of reporter proteins, such as opsins, in cholinergic neurons. However, the expression of opsins could potentially perturb the physiology of opsin-expressing cholinergic neurons or mouse behavior. Indeed, the published literature includes examples of cellular and behavioral perturbations in preparations designed to drive expression of opsins in cholinergic neurons. Here we investigate expression of opsins, cellular physiology of cholinergic neurons and behavior in two mouse lines, in which channelrhodopsin-2 (ChR2) and archaerhodopsin (Arch) are expressed in cholinergic neurons using the Cre-lox system. The two mouse lines were generated by crossing ChAT-Cre mice with Cre-dependent reporter lines Ai32(ChR2-YFP) and Ai35(Arch-GFP). In most mice from these crosses, we observed expression of ChR2 and Arch in only cholinergic neurons in the basal forebrain and in other putative cholinergic neurons in the forebrain. In small numbers of mice, off-target expression occurred, in which fluorescence did not appear limited to cholinergic neurons. Whole-cell recordings from fluorescently-labeled basal forebrain neurons revealed that both proteins were functional, driving depolarization (ChR2) or hyperpolarization (Arch) upon illumination, with little effect on passive membrane properties, spiking pattern or spike waveform. Finally, performance on a behavioral discrimination task was comparable to that of wild-type mice. Our results indicate that ChAT-Cre x reporter line crosses provide a simple, effective resource for driving indicator and opsin expression in cholinergic neurons with few adverse consequences and are therefore an valuable resource for studying the cholinergic system.

Contrast enhancement in sensory systems often relies on spatial filters implemented by lateral inhibition. However, in this issue of Neuron , Fantana et al. provide evidence that lateral inhibition in the olfactory bulb selectively acts between sparse populations of principal neurons without regard to spatial relations.

Neuronal regeneration occurs naturally in a few restricted mammalian brain regions, but its functional significance remains debated. Here we search for unique features in the synaptic outputs made by adult-born granule cell interneurons in the mouse olfactory bulb using optogenetic targeting of specific neuronal ages. We find that adult-born interneurons are resistant to presynaptic GABAB-mediated depression of GABA release compared with interneurons born just after birth that exhibit strong GABABneuromodulation. Correlated with this functional change, we found altered localization of the GABABR1 protein within adult-born granule cells. These results suggest that adult neurogenesis produces a population of functionally unique GABAergic synapses in the olfactory bulb.

Optogenetic techniques are used widely to perturb and interrogate neural circuits in behaving animals, but illumination can have additional effects, such as the activation of endogenous opsins in the retina. We found that illumination, delivered deep into the brain via an optical fiber, evoked a behavioral artifact in mice performing a visually guided discrimination task. Compared with blue (473 nm) and yellow (589 nm) illumination, red (640 nm) illumination evoked a greater behavioral artifact and more activity in the retina, the latter measured with electrical recordings. In the mouse, the sensitivity of retinal opsins declines steeply with wavelength across the visible spectrum, but propagation of light through brain tissue increases with wavelength. Our results suggest that poor retinal sensitivity to red light was overcome by relatively robust propagation of red light through brain tissue and stronger illumination of the retina by red than by blue or yellow light. Light adaptation of the retina, via an external source of illumination, suppressed retinal activation and the behavioral artifact without otherwise impacting behavioral performance. In summary, long wavelength optogenetic stimuli are particularly prone to evoke behavioral artifacts via activation of retinal opsins in the mouse, but light adaptation of the retina can provide a simple and effective mitigation of the artifact.

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations(1,2). With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channel rhodopsin-2 (ChR2) allows researchers to activate neurons with light(3,4). By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Objective Conventional anticonvulsants reduce neuronal excitability through effects on ion channels and synaptic function. Anticonvulsant mechanisms of the ketogenic diet remain incompletely understood. Because carbohydrates are restricted in patients on the ketogenic diet, we evaluated the effects of limiting carbohydrate availability by reducing glycolysis using the glycolytic inhibitor 2-deoxy-D-glucose (2DG) in experimental models of seizures and epilepsy. Methods Acute anticonvulsant actions of 2DG were assessed in vitro in rat hippocampal slices perfused with 7.5mM [K+]o, 4-aminopyridine, or bicuculline, and in vivo against seizures evoked by 6Hz stimulation in mice, audiogenic stimulation in Fring's mice, and maximal electroshock and subcutaneous pentylenetetrazol (Metrazol) in rats. Chronic antiepileptic effects of 2DG were evaluated in rats kindled from olfactory bulb or perforant path. Results 2DG (10mM) reduced interictal epileptiform bursts induced by 7.5mM [K+]o, 4-aminopyridine, and bicuculline, and electrographic seizures induced by high [K+]o in CA3 of hippocampus. 2DG reduced seizures evoked by 6Hz stimulation in mice (effective dose [ED]50 = 79.7mg/kg) and audiogenic stimulation in Fring's mice (ED50 = 206.4mg/kg). 2DG exerted chronic antiepileptic action by increasing afterdischarge thresholds in perforant path (but not olfactory bulb) kindling and caused a twofold slowing in progression of kindled seizures at both stimulation sites. 2DG did not protect against maximal electroshock or Metrazol seizures. Interpretation The glycolytic inhibitor 2DG exerts acute anticonvulsant and chronic antiepileptic actions, and has a novel pattern of effectiveness in preclinical screening models. These results identify metabolic regulation as a potential therapeutic target for seizure suppression and modification of epileptogenesis. Ann Neurol 2009;65:435–448.

SRp38 is an atypical SR protein splicing regulator. To define the functions of SRp38 in vivo, we generated SRp38 null mice. The majority of homozygous mutants survived only until E15.5 and displayed multiple cardiac defects. Evaluation of gene expression profiles in the SRp38(-/-) embryonic heart revealed a defect in processing of the pre-mRNA encoding cardiac triadin, a protein that functions in regulation of Ca(2+) release from the sarcoplasmic reticulum during excitation-contraction coupling. This defect resulted in significantly reduced levels of triadin, as well as those of the interacting protein calsequestrin 2. Purified SRp38 was shown to bind specifically to the regulated exon and to modulate triadin splicing in vitro. Extending these results, isolated SRp38(-/-) embryonic cardiomyocytes displayed defects in Ca(2+) handling compared with wild-type controls. Taken together, our results demonstrate that SRp38 regulates cardiac-specific alternative splicing of triadin pre-mRNA and, reflecting this, is essential for proper Ca(2+) handling during embryonic heart development.

The prion protein PrP(C) is infamous for its role in disease, but its normal physiological function remains unknown. Here we found a previously unknown behavioral phenotype of Prnp(-/-) mice in an odor-guided task. This phenotype was manifest in three Prnp knockout lines on different genetic backgrounds, which provides strong evidence that the phenotype is caused by a lack of PrP(C) rather than by other genetic factors. Prnp(-/-) mice also showed altered behavior in a second olfactory task, suggesting that the phenotype is olfactory specific. Furthermore, PrP(C) deficiency affected oscillatory activity in the deep layers of the main olfactory bulb, as well as dendrodendritic synaptic transmission between olfactory bulb granule and mitral cells. Notably, both the behavioral and electrophysiological alterations found in Prnp(-/-) mice were rescued by transgenic neuronal-specific expression of PrP(C). These data suggest that PrP(C) is important in the normal processing of sensory information by the olfactory system.

The piriform (or primary olfactory) cortex is a trilaminar structure that is the first cortical destination of olfactory information, receiving monosynaptic input from the olfactory bulb. Here, we show that the main input layer of the piriform cortex, layer II, is dominated by two classes of principal neurons, superficial pyramidal (SP) and semilunar (SL) cells, with strikingly different properties. Action potentials in SP cells are followed by a Ni(2+)-sensitive afterdepolarization that promotes burst firing, whereas SL cells fire nonbursting action potentials that are followed by a powerful afterhyperpolarization. Synaptic inputs from the olfactory bulb onto SP cells exhibit prominent paired-pulse facilitation, which is attributable to residual presynaptic Ca(2+) and a low probability of neurotransmitter release. In contrast, the same inputs onto SL cells do not facilitate. These distinctive synaptic and firing properties cause SP and SL cells to respond differently to in vivo-like bursts of afferent stimulation: SP cells tend to fire bursts of output action potentials at a higher frequency than the input, whereas SL cells tend to fire at a lower frequency than the input. When connected together in the canonical circuit of the piriform cortex, SP and SL cells transform the pattern of synaptic inputs they receive from the olfactory bulb, dispersing the firing rate and latency of output action potentials to an extent that depends on the strength of the input. Thus, the presence of two types of principal cells in layer II of the piriform cortex may underlie coding strategies used for the representation of odors.

What we perceive in the olfactory world is different from what odor molecules are in the air. With hundreds or even thousands of aerosolized molecules present in any single environment, our brain must be able to simplify incoming information so that our perceptual categories, like rose, musk, or

SummaryCa2+/calmodulin-mediated negative feedback is a prototypical regulatory mechanism for Ca2+-permeable ion channels. In olfactory sensory neurons (OSNs), such regulation on the cyclic nucleotide-gated (CNG) channel is considered a major mechanism of OSN adaptation. To determine the role of Ca2+/calmodulin desensitization of the olfactory CNG channel, we introduced a mutation in the channel subunit CNGB1b in mice that rendered the channel resistant to fast desensitization by Ca2+/calmodulin. Contrary to expectations, mutant OSNs showed normal receptor current adaptation to repeated stimulation. Rather, they displayed slower response termination and, consequently, reduced ability to transmit olfactory information to the olfactory bulb. They also displayed reduced response decline during sustained odorant exposure. These results suggest that Ca2+/calmodulin-mediated CNG channel fast desensitization is less important in regulating the sensitivity to recurring stimulation than previously thought and instead functions primarily to terminate OSN responses.

Transgenic mouse lines are invaluable tools for neuroscience but as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically-encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, though rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.