Organization and neural connections of the lateral complex in the brain of the desert locust.

The lateral complexes (LXs) are bilaterally paired neuropils in the insect brain that mediate communication between the central complex (CX), a brain center controlling spatial orientation, various sensory processing areas, and thoracic motor centers that execute locomotion. The LX of the desert locust consists of the lateral accessory lobe (LAL), and the medial and lateral bulb. We have analyzed the anatomical organization and the neuronal connections of the LX in the locust, to provide a basis for future functional studies. Reanalyzing the morphology of neurons connecting the CX and the LX revealed likely feedback loops in the sky compass network of the CX via connections in the gall of the LAL and a newly identified neuropil termed ovoid body. In addition, we characterized 16 different types of neuron that connect the LAL with other areas in the brain. Eight types of neuron provide information flow between both LALs, five types are LAL input neurons, and three types are LAL output neurons. Among these are neurons providing input from sensory brain areas such as the lobula and antennal neuropils. Brain regions most often targeted by LAL neurons are the posterior slope, the wedge, and the crepine. Two descending neurons with dendrites in the LAL were identified. Our data support and complement existing knowledge about how the LAL is embedded in the neuronal network involved in processing of sensory information and generation of appropriate behavioral output for goal-directed locomotion.

A multi-scale brain map derived from whole-brain volumetric reconstructions.

Animal nervous system organization is crucial for all body functions and its disruption can lead to severe cognitive and behavioural impairment1. This organization relies on features across scales-from the localization of synapses at the nanoscale, through neurons, which possess intricate neuronal morphologies that underpin circuit organization, to stereotyped connections between different regions of the brain2. The sheer complexity of this organ means that the feat of reconstructing and modelling the structure of a complete nervous system that is integrated across all of these scales has yet to be achieved. Here we present a complete structure-function model of the main neuropil in the nematode Caenorhabditis elegans-the nerve ring-which we derive by integrating the volumetric reconstructions from two animals with corresponding3 synaptic and gap-junctional connectomes. Whereas previously the nerve ring was considered to be a densely packed tract of neural processes, we uncover internal organization and show how local neighbourhoods spatially constrain and support the synaptic connectome. We find that the C. elegans connectome is not invariant, but that a precisely wired core circuit is embedded in a background of variable connectivity, and identify a candidate reference connectome for the core circuit. Using this reference, we propose a modular network architecture of the C. elegans brain that supports sensory computation and integration, sensorimotor convergence and brain-wide coordination. These findings reveal scalable and robust features of brain organization that may be universal across phyla.

Structural and developmental principles of neuropil assembly in C. elegans.

Neuropil is a fundamental form of tissue organization within the brain1, in which densely packed neurons synaptically interconnect into precise circuit architecture2,3. However, the structural and developmental principles that govern this nanoscale precision remain largely unknown4,5. Here we use an iterative data coarse-graining algorithm termed 'diffusion condensation'6 to identify nested circuit structures within the Caenorhabditis elegans neuropil, which is known as the nerve ring. We show that the nerve ring neuropil is largely organized into four strata that are composed of related behavioural circuits. The stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create neural structures that cage the strata within the nerve ring. We use high resolution light-sheet microscopy7,8 coupled with lineage-tracing and cell-tracking algorithms9,10 to resolve the developmental sequence and reveal principles of cell position, migration and outgrowth that guide stratified neuropil organization. Our results uncover conserved structural design principles that underlie the architecture and function of the nerve ring neuropil, and reveal a temporal progression of outgrowth-based on pioneer neurons-that guides the hierarchical development of the layered neuropil. Our findings provide a systematic blueprint for using structural and developmental approaches to understand neuropil organization within the brain.

Shore crabs reveal novel evolutionary attributes of the mushroom body.

Neural organization of mushroom bodies is largely consistent across insects, whereas the ancestral ground pattern diverges broadly across crustacean lineages resulting in successive loss of columns and the acquisition of domed centers retaining ancestral Hebbian-like networks and aminergic connections. We demonstrate here a major departure from this evolutionary trend in Brachyura, the most recent malacostracan lineage. In the shore crab Hemigrapsus nudus, instead of occupying the rostral surface of the lateral protocerebrum, mushroom body calyces are buried deep within it with their columns extending outwards to an expansive system of gyri on the brain's surface. The organization amongst mushroom body neurons reaches extreme elaboration throughout its constituent neuropils. The calyces, columns, and especially the gyri show DC0 immunoreactivity, an indicator of extensive circuits involved in learning and memory.

Outsourcing a visual neuropil - The central visual system of the median eyes of Galeodes granti Pocock, 1903 (Arachnida: Solifugae).

Only a few studies have examined the central visual system of Solifugae until now. To get new insights suitable for phylogenetic analysis we studied the R-cell (or retinula cell) projections and visual neuropils of Galeodes granti using various methods. G. granti possesses large median eyes and rudimentary lateral eyes. In this study, only the R-cells and neuropils of the median eyes were successfully stained. The R-cells terminate in two distinct visual neuropils. The first neuropil is located externally to the protocerebrum directly below the retina, the second neuropil lies in the cell body rind of the protocerebrum, and immediately adjacent is the arcuate body. This layout of the median eye visual system differs from Arachnopulmonata (Scorpiones + Tetrapulmonata). However, there are several similarities with Opiliones. In both, (1) the R-cells are connected to a first and second visual neuropil and not to any other region of the brain, (2) the first neuropil is not embedded in the cell body rind of the protocerebrum, it is rather external to the protocerebrum, (3) the second visual neuropil is embedded in the cell body rind, and (4) the second neuropil abuts the arcuate body. These findings may provide important new characters for the discussion on arachnid phylogeny.

Structure of the pecten neuropil pathway and its innervation by bimodal peg afferents in two scorpion species.

The pectines of scorpions are comb-like structures, located ventrally behind the fourth walking legs and consisting of variable numbers of teeth, or pegs, which contain thousands of bimodal peg sensillae. The associated neuropils are situated ventrally in the synganglion, extending between the second and fourth walking leg neuromeres. While the general morphology is consistent among scorpions, taxon-specific differences in pecten and neuropil structure remain elusive but are crucial for a better understanding of chemosensory processing. We analysed two scorpion species (Mesobuthus eupeus and Heterometrus petersii) regarding their pecten neuropil anatomy and the respective peg afferent innervation with anterograde and lipophilic tracing experiments, combined with immunohistochemistry and confocal laser-scanning microscopy. The pecten neuropils consisted of three subcompartments: a posterior pecten neuropil, an anterior pecten neuropil and a hitherto unknown accessory pecten neuropil. These subregions exhibited taxon-specific variations with regard to compartmentalisation and structure. Most notable were structural differences in the anterior pecten neuropils that ranged from ovoid shape and strong fragmentation in Heterometrus petersii to elongated shape with little compartmentalisation in Mesobuthus eupeus. Labelling the afferents of distinct pegs revealed a topographic organisation of the bimodal projections along a medio-lateral axis. At the same time, all subregions along the posterior-anterior axis were innervated by a single peg's afferents. The somatotopic projection pattern of bimodal sensillae appears to be common among arachnids, including scorpions. This includes the structure and organisation of the respective neuropils and the somatotopic projection patterns of chemosensory afferents. Nonetheless, the scorpion pecten pathway exhibits unique features, e.g. glomerular compartmentalisation superimposed on somatotopy, that are assumed to allow high resolution of substrate-borne chemical gradients.

The brain of Cataglyphis ants: Neuronal organization and visual projections.

Cataglyphis ants are known for their outstanding navigational abilities. They return to their inconspicuous nest after far-reaching foraging trips using path integration, and whenever available, learn and memorize visual features of panoramic sceneries. To achieve this, the ants combine directional visual information from celestial cues and panoramic scenes with distance information from an intrinsic odometer. The largely vision-based navigation in Cataglyphis requires sophisticated neuronal networks to process the broad repertoire of visual stimuli. Although Cataglyphis ants have been subjected to many neuroethological studies, little is known about the general neuronal organization of their central brain and the visual pathways beyond major circuits. Here, we provide a comprehensive, three-dimensional neuronal map of synapse-rich neuropils in the brain of Cataglyphis nodus including major connecting fiber systems. In addition, we examined neuronal tracts underlying the processing of visual information in more detail. This study revealed a total of 33 brain neuropils and 30 neuronal fiber tracts including six distinct tracts between the optic lobes and the cerebrum. We also discuss the importance of comparative studies on insect brain architecture for a profound understanding of neuronal networks and their function.

Multiple lineages enable robust development of the neuropil-glia architecture in adult Drosophila.

Neural remodeling is essential for the development of a functional nervous system and has been extensively studied in the metamorphosis of Drosophila Despite the crucial roles of glial cells in brain functions, including learning and behavior, little is known of how adult glial cells develop in the context of neural remodeling. Here, we show that the architecture of neuropil-glia in the adult Drosophila brain, which is composed of astrocyte-like glia (ALG) and ensheathing glia (EG), robustly develops from two different populations in the larva: the larval EG and glial cell missing-positive (gcm+ ) cells. Whereas gcm+ cells proliferate and generate adult ALG and EG, larval EG dedifferentiate, proliferate and redifferentiate into the same glial subtypes. Each glial lineage occupies a certain brain area complementary to the other, and together they form the adult neuropil-glia architecture. Both lineages require the FGF receptor Heartless to proliferate, and the homeoprotein Prospero to differentiate into ALG. Lineage-specific inhibition of gliogenesis revealed that each lineage compensates for deficiency in the proliferation of the other. Together, the lineages ensure the robust development of adult neuropil-glia, thereby ensuring a functional brain.

Visual pathways in the brain of the jumping spider Marpissa muscosa.

Some animals have evolved task differentiation among their eyes. A particular example is spiders, where most species have eight eyes, of which two (the principal eyes) are used for object discrimination, whereas the other three pairs (secondary eyes) detect movement. In the ctenid spider Cupiennius salei, these two eye types correspond to two visual pathways in the brain. Each eye is associated with its own first- and second-order visual neuropil. The second-order neuropils of the principal eyes are connected to the arcuate body, whereas the second-order neuropils of the secondary eyes are linked to the mushroom body. We explored the principal- and secondary eye visual pathways of the jumping spider Marpissa muscosa, in which size and visual fields of the two eye types are considerably different. We found that the connectivity of the principal eye pathway is the same as in C. salei, while there are differences in the secondary eye pathways. In M. muscosa, all secondary eyes are connected to their own first-order visual neuropils. The first-order visual neuropils of the anterior lateral and posterior lateral eyes are connected with a second-order visual neuropil each and an additional shared one (L2). In the posterior median eyes, the axons of their first-order visual neuropils project directly to the arcuate body, suggesting that the posterior median eyes do not detect movement. The L2 might function as an upstream integration center enabling faster movement decisions.

The brain of a nocturnal migratory insect, the Australian Bogong moth.

Every year, millions of Australian Bogong moths (Agrotis infusa) complete an astonishing journey: In Spring, they migrate over 1,000 km from their breeding grounds to the alpine regions of the Snowy Mountains, where they endure the hot summer in the cool climate of alpine caves. In autumn, the moths return to their breeding grounds, where they mate, lay eggs and die. These moths can use visual cues in combination with the geomagnetic field to guide their flight, but how these cues are processed and integrated into the brain to drive migratory behavior is unknown. To generate an access point for functional studies, we provide a detailed description of the Bogong moth's brain. Based on immunohistochemical stainings against synapsin and serotonin (5HT), we describe the overall layout as well as the fine structure of all major neuropils, including the regions that have previously been implicated in compass-based navigation. The resulting average brain atlas consists of 3D reconstructions of 25 separate neuropils, comprising the most detailed account of a moth brain to date. Our results show that the Bogong moth brain follows the typical lepidopteran ground pattern, with no major specializations that can be attributed to their spectacular migratory lifestyle. These findings suggest that migratory behavior does not require widespread modifications of brain structure, but might be achievable via small adjustments of neural circuitry in key brain areas. Locating these subtle changes will be a challenging task for the future, for which our study provides an essential anatomical framework.

The brain and the corresponding sense organs in calanoid copepods - Evidence of vestiges of compound eyes.

Copepoda is one of the crustacean taxa with still unresolved phylogenetic relationships within Tetraconata. Recent phylogenomic studies place them close to Malacostraca and Cirripedia. Little is known about the morphological details of the copepod nervous system, and the available data are sometimes contradictory. We investigated several representatives of the subgroup Calanoida using immunohistochemical labeling against alpha-tubulin and various neuroactive substances, combining this with confocal laser scanning analysis and 3D reconstruction. Our results show that the studied copepods exhibit only a single anterior protocerebral neuropil which is connected to the nerves of two protocerebral sense organs: the frontal filament organ and a photoreceptor known as the Gicklhorn's organ. We suggest, on the basis of its position and the innervation it provides, that Gicklhorn's organ is homologous to the compound eye in arthropods. With regard to the frontal filament organ, we reveal detailed innervation to the lateral protocerebrum and the appearance of spherical bodies that stain intensely against alpha tubulin. A potential homology of these bodies to the onion bodies in malacostacan crustaceans and in Mystacocarida is suggested. The nauplius eye in all the examined calanoids shows the same basic pattern of innervation with the middle cup sending its neurites into the median nerve, while the axons of the lateral cups proceed into both the median and the lateral nerves. The early development of the axonal scaffold of the nauplius eye neuropil from the proximal parts of the nauplius eye nerves follows the same pattern as in other crustaceans. In our view, this specific innervation pattern is a further feature supporting the homology of the nauplius eye in crustaceans.

The astrocyte network in the ventral nerve cord neuropil of the Drosophila third-instar larva.

Understanding neuronal function at the local and circuit level requires understanding astrocyte function. We have provided a detailed analysis of astrocyte morphology and territory in the Drosophila third-instar ventral nerve cord where there already exists considerable understanding of the neuronal network. Astrocyte shape varies more than previously reported; many have bilaterally symmetrical partners, many have a high percentage of their arborization in adjacent segments, and many have branches that follow structural features. Taken together, our data are consistent with, but not fully explained by, a model of a developmental growth process dominated by competitive or repulsive interactions between astrocytes. Our data suggest that the model should also include cell-autonomous aspects, as well as the use of structural features for growth. Variation in location of arborization territory for identified astrocytes was great enough that a standardized scheme of neuropil division among the six astrocytes that populate each hemi-segment is not possible at the third instar. The arborizations of the astrocytes can extend across neuronal functional domains. The ventral astrocyte in particular, whose territory can extend well into the proprioceptive region of the neuropil, has no obvious branching pattern that correlates with domains of particular sensory modalities, suggesting that the astrocyte would respond to neuronal activity in any of the sensory modalities, perhaps integrating across them. This study sets the stage for future studies that will generate a robust, functionally oriented connectome that includes both partners in neuronal circuits-the neurons and the glial cells, providing the foundation necessary for studies to elucidate neuron-glia interactions in this neuropil.

Slit neuronal secretion coordinates optic lobe morphogenesis in Drosophila.

The complexity of the nervous system requires the coordination of multiple cellular processes during development. Among them, we find boundary formation, axon guidance, cell migration and cell segregation. Understanding how different cell populations such as glial cells, developing neurons and neural stem cells contribute to the formation of boundaries and morphogenesis in the nervous system is a critical question in neurobiology. Slit is an evolutionary conserved protein essential for the development of the nervous system. For signaling, Slit has to bind to its cognate receptor Robo, a single-pass transmembrane protein. Although the Slit/Robo signaling pathway is well known for its involvement in axon guidance, it has also been associated to boundary formation in the Drosophila visual system. In the optic lobe, Slit is expressed in glial cells, positioned at the boundaries between developing neuropils, and in neurons of the medulla ganglia. Although it has been assumed that glial cells provide Slit to the system, the contribution of the neuronal expression has not been tested. Here, we show that, contrary to what was previously thought, Slit protein provided by medulla neurons is also required for boundary formation and morphogenesis of the optic lobe. Furthermore, tissue specific rescue using modified versions of Slit demonstrates that this protein acts at long range and does not require processing by extracellular proteases. Our data shed new light on our understanding of the cellular mechanisms involved in Slit function in the fly visual system morphogenesis.

Candidates for photic entrainment pathways to the circadian clock via optic lobe neuropils in the Madeira cockroach.

The compound eye of cockroaches is obligatory for entrainment of the Madeira cockroach's circadian clock, but the cellular nature of its entrainment pathways is enigmatic. Employing multiple-label immunocytochemistry, histochemistry, and backfills, we searched for photic entrainment pathways to the accessory medulla (AME), the circadian clock of the Madeira cockroach. We wanted to know whether photoreceptor terminals could directly contact pigment-dispersing factor-immunoreactive (PDF-ir) circadian pacemaker neurons with somata in the lamina (PDFLAs) or somata next to the AME (PDFMEs). Short green-sensitive photoreceptor neurons of the compound eye terminated in lamina layers LA1 and LA2, adjacent to PDFLAs and PDFMEs that branched in LA3. Long UV-sensitive compound eye photoreceptor neurons terminated in medulla layer ME2 without direct contact to ipsilateral PDFMEs that arborized in ME4. Multiple neuropeptide-ir interneurons branched in ME4, connecting the AME to ME2. Before, extraocular photoreceptors of the lamina organ were suggested to send terminals to accessory laminae. There, they overlapped with PDFLAs that mostly colocalized PDF, FMRFamide, and 5-HT immunoreactivities, and with terminals of ipsi- and contralateral PDFMEs. We hypothesize that during the day cholinergic activation of the largest PDFME via lamina organ photoreceptors maintains PDF release orchestrating phases of sleep-wake cycles. As ipsilateral PDFMEs express excitatory and contralateral PDFMEs inhibitory PDF autoreceptors, diurnal PDF release keeps both PDF-dependent clock circuits in antiphase. Future experiments will test whether ipsilateral PDFMEs are sleep-promoting morning cells, while contralateral PDFMEs are activity-promoting evening cells, maintaining stable antiphase via the largest PDFME entrained by extraocular photoreceptors of the lamina organ.

The reniform body: An integrative lateral protocerebral neuropil complex of Eumalacostraca identified in Stomatopoda and Brachyura.

Mantis shrimps (Stomatopoda) possess in common with other crustaceans, and with Hexapoda, specific neuroanatomical attributes of the protocerebrum, the most anterior part of the arthropod brain. These attributes include assemblages of interconnected centers called the central body complex and in the lateral protocerebra, situated in the eyestalks, paired mushroom bodies. The phenotypic homologues of these centers across Panarthropoda support the view that ancestral integrative circuits crucial to action selection and memory have persisted since the early Cambrian or late Ediacaran. However, the discovery of another prominent integrative neuropil in the stomatopod lateral protocerebrum raises the question whether it is unique to Stomatopoda or at least most developed in this lineage, which may have originated in the upper Ordovician or early Devonian. Here, we describe the neuroanatomical structure of this center, called the reniform body. Using confocal microscopy and classical silver staining, we demonstrate that the reniform body receives inputs from multiple sources, including the optic lobe's lobula. Although the mushroom body also receives projections from the lobula, it is entirely distinct from the reniform body, albeit connected to it by discrete tracts. We discuss the implications of their coexistence in Stomatopoda, the occurrence of the reniform body in another eumalacostracan lineage and what this may mean for our understanding of brain functionality in Pancrustacea.

Neuroarchitecture of the central complex of the desert locust: Tangential neurons.

The central complex (CX) comprises a group of midline neuropils in the insect brain, consisting of the protocerebral bridge (PB), the upper (CBU) and lower division (CBL) of the central body and a pair of globular noduli. It receives prominent input from the visual system and plays a major role in spatial orientation of the animals. Vertical slices and horizontal layers of the CX are formed by columnar, tangential, and pontine neurons. While pontine and columnar neurons have been analyzed in detail, especially in the fruit fly and desert locust, understanding of the organization of tangential cells is still rudimentary. As a basis for future functional studies, we have studied the morphologies of tangential neurons of the CX of the desert locust Schistocerca gregaria. Intracellular dye injections revealed 43 different types of tangential neuron, 8 of the PB, 5 of the CBL, 24 of the CBU, 2 of the noduli, and 4 innervating multiple substructures. Cell bodies of these neurons were located in 11 different clusters in the cell body rind. Judging from the presence of fine versus beaded terminals, the vast majority of these neurons provide input into the CX, especially from the lateral complex (LX), the superior protocerebrum, the posterior slope, and other surrounding brain areas, but not directly from the mushroom bodies. Connections are largely subunit- and partly layer-specific. No direct connections were found between the CBU and the CBL. Instead, both subdivisions are connected in parallel with the PB and distinct layers of the noduli.

Semaphorin 2b Regulates Sleep-Circuit Formation in the Drosophila Central Brain.

The fan-shaped body (FB) neuropil in the Drosophila brain central complex (CX) controls a variety of adult behaviors, including navigation and sleep. How neuronal processes are organized into precise layers and columns in the FB and how alterations in FB neural-circuit wiring affect animal behaviors are unknown. We report here that secreted semaphorin 2b (Sema-2b) acts through its transmembrane receptor Plexin B (PlexB) to locally attract neural processes to specific FB laminae. Aberrant Sema-2b/PlexB signaling leads to select disruptions in neural lamination, and these disruptions result in the formation of ectopic inhibitory connections between subsets of FB neurons. These structural alternations and connectivity defects are associated with changes in fly sleep and arousal, emphasizing the importance of lamination-mediated neural wiring in a central brain region critical for normal sleep behavior.

The Jewel Wasp Standard Brain: Average shape atlas and morphology of the female Nasonia vitripennis brain.

Nasonia, a genus of parasitoid wasps, is a promising model system in the study of developmental and evolutionary genetics, as well as complex traits such as learning. Of these "jewel wasps", the species Nasonia vitripennis is widely spread and widely studied. To accelerate neuroscientific research in this model species, fundamental knowledge of its nervous system is needed. To this end, we present an average standard brain of recently eclosed naïve female N. vitripennis wasps obtained by the iterative shape averaging method. This "Jewel Wasp Standard Brain" includes the optic lobe (excluding the lamina), the anterior optic tubercle, the antennal lobe, the lateral horn, the mushroom body, the central complex, and the remaining unclassified neuropils in the central brain. Furthermore, we briefly describe these well-defined neuropils and their subregions in the N. vitripennis brain. A volumetric analysis of these neuropils is discussed in the context of brains of other insect species. The Jewel Wasp Standard Brain will provide a framework to integrate and consolidate the results of future neurobiological studies in N. vitripennis. In addition, the volumetric analysis provides a baseline for future work on age- and experience-dependent brain plasticity.

The visual system of Thelyphonida (whip scorpions): Support for Arachnopulmonata.

Only a few studies have examined the central visual system of Thelyphonida (whip scorpions) until now. To obtain new insights suitable for phylogenetic analysis we studied the axonal trajectories and neuropil architecture of the central visual systems of two whip scorpion species (Mastigoproctus giganteus and Typopeltis dalyi) with different neuroanatomical techniques (Cobalt fills, Wigglesworth stains, and µCT). The central visual system of whip scorpion comprises one pair of median eyes and one pair of lateral eye triplets. The R-cells (or retinula cells) of both eye types each terminate in a first and a second visual neuropil. Furthermore, a few R-cell fibres from the median eyes leave the second median eye visual neuropil and terminate in the second and the first lateral eye neuropil. This means R-cell terminals from the lateral eyes and the median eyes overlap here. Additionally, the arcuate body and mushroom bodies are described. A detailed comparison of our findings with previously studied chelicerate central visual systems seems to support a monophyly of Arachnopulmonata, i.e. a clade comprising Tetrapulmonata (Thelyphonida, Schizomida, Amblypygi, and Araneae) and Scorpions. Furthermore, the architecture of the central visual systems hints at a close evolutionary relationship of Arachnopulmonata and Xiphosura.

Benchmarking miniaturized microscopy against two-photon calcium imaging using single-cell orientation tuning in mouse visual cortex.

Miniaturized microscopes are lightweight imaging devices that allow optical recordings from neurons in freely moving animals over the course of weeks. Despite their ubiquitous use, individual neuronal responses measured with these microscopes have not been directly compared to those obtained with established in vivo imaging techniques such as bench-top two-photon microscopes. To achieve this, we performed calcium imaging in mouse primary visual cortex while presenting animals with drifting gratings. We identified the same neurons in image stacks acquired with both microscopy methods and quantified orientation tuning of individual neurons. The response amplitude and signal-to-noise ratio of calcium transients recorded upon visual stimulation were highly correlated between both microscopy methods, although influenced by neuropil contamination in miniaturized microscopy. Tuning properties, calculated for individual orientation tuned neurons, were strongly correlated between imaging techniques. Thus, neuronal tuning features measured with a miniaturized microscope are quantitatively similar to those obtained with a two-photon microscope.