Different classes of input and output neurons reveal new features in microglomeruli of the adult Drosophila mushroom body calyx

To investigate how sensory information is processed, transformed, and stored within an olfactory system, we examined the anatomy of the input region, the calyx, of the mushroom bodies of Drosophila melanogaster. These paired structures are important for various behaviors, including olfactory learning and memory. Cells in the input neuropil, the calyx, are organized into an array of microglomeruli each comprising the large synaptic bouton of a projection neuron (PN) from the antennal lobe surrounded by tiny postsynaptic neurites from intrinsic Kenyon cells. Extrinsic neurons of the mushroom body also contribute to the organization of microglomeruli. We employed a combination of genetic reporters to identify single cells in the Drosophila calyx by light microscopy and compared these with cell shapes, synapses, and circuits derived from serial-section electron microscopy. We identified three morphological types of PN boutons, unilobed, clustered, and elongated; defined three ultrastructural types, with clear- or dense-core vesicles and those with a dark cytoplasm having both; reconstructed diverse dendritic specializations of Kenyon cells; and identified Kenyon cell presynaptic sites upon extrinsic neurons. We also report new features of calyx synaptic organization, in particular extensive serial synapses that link calycal extrinsic neurons into a local network, and the numerical proportions of synaptic contacts between calycal neurons. All PN bouton types had more ribbon than nonribbon synapses, dark boutons particularly so, and ribbon synapses were larger and with more postsynaptic elements (2-14) than nonribbon (1-10). The numbers of elements were in direct proportion to presynaptic membrane area. Extrinsic neurons exclusively had ribbon synapses.     

Synaptic connections of cholinergic antennal lobe relay neurons innervating the lateral horn neuropile in the brain of Drosophila melanogaster

Presumed cholinergic projection neurons (PNs) in the brain of the fruit fly Drosophila melanogaster, immunoreactive to choline acetyltransferase (ChAT), convey olfactory information between the primary sensory antennal lobe neuropile and the mushroom body calyces, and finally terminate in the lateral horn (LH) neuropile. The texture and synaptic connections of ChAT PNs in the LH and, comparatively, in the smaller mushroom body calyces were investigated by immuno light and electron microscopy. The ChAT PN fibers of the massive inner antennocerebral tract (iACT) extend into all portions of the LH, distributing in a nonrandom fashion. Immunoreactive boutons accumulate in the lateral margins of the LH, whereas the more proximal LH exhibits less intense immunolabeling. Boutons with divergent presynaptic sites, unlabeled as well as ChAT-immunoreactive, appear to be the preponderant mode of synaptic input throughout the LH. Synapses of ChAT-labeled fibers appear predominantly as divergent synaptic boutons (diameters 1-3 microm), connected to unlabeled postsynaptic profiles, or alternatively as a minority of tiny postsynaptic spines (diameters 0.05-0.5 microm) among unlabeled profiles. Together these spines encircle unidentified presynaptic boutons of interneurons which occupy large areas of the LH. Thus, synaptic circuits in the LH differ profoundly from those of the PNs in the mushroom body calyx, where ChAT spines have not been encountered. Synaptic contacts between LH ChAT elements were not observed. The synaptic LH neuropile may serve as an output area for terminals of the ChAT PNs, their presynaptic boutons providing input to noncholinergic relay neurons. The significance of the postsynaptic neurites of the ChAT PNs is discussed; either local or other interneurons might connect the ChAT PNs within the LH, or PNs might receive inputs arising from outside the LH.     

Age-related plasticity in the synaptic ultrastructure of neurons in the mushroom body calyx of the adult honeybee Apis mellifera

The mushroom bodies are high-order sensory integration centers in the insect brain. In the honeybee, their main sensory input regions are large, doubled calyces with modality-specific, distinct sensory neuropil regions. We investigated adult structural plasticity of input synapses in the microglomeruli of the olfactory lip and visual collar. Synapsin-immunolabeled whole-mount brains reveal that during the natural transition from nursing to foraging, a significant volume increase in the calycal subdivisions is accompanied by a decreased packing density of boutons from input projection neurons. To investigate the associated ultrastructural changes at pre- and postsynaptic sites of individual microglomeruli, we employed serial-section electron microscopy. In general, the membrane surface area of olfactory and visual projection neuron boutons increased significantly between 1-day-old bees and foragers. Both types of boutons formed ribbon and non-ribbon synapses. The percentage of ribbon synapses per bouton was significantly increased in the forager. At each presynaptic site the numbers of postsynaptic partners-mostly Kenyon cell dendrites-likewise increased. Ribbon as well as non-ribbon synapses formed mainly dyads in the 1-day-old bee, and triads in the forager. In the visual collar, outgrowing Kenyon cell dendrites form about 140 contacts upon a projection neuron bouton in the forager compared with only about 95 in the 1-day-old bee, resulting in an increased divergence ratio between the two stages. This difference suggests that synaptic changes in calycal microcircuits of the mushroom body during periods of altered sensory activity and experience promote behavioral plasticity underlying polyethism and social organization in honeybee colonies.     

Synaptic organization of the mushroom body calyx in Drosophila melanogaster

The calyx neuropil of the mushroom body in adult Drosophila melanogaster contains three major neuronal elements: extrinsic projection neurons, presumed cholinergic, immunoreactive to choline acetyltransferase (ChAT-ir) and vesicular acetylcholine transporter (VAChT-ir) antisera; presumed gamma-aminobutyric acid (GABA)ergic extrinsic neurons with GABA-like immunoreactivity; and local intrinsic Kenyon cells. The projection neurons connecting the calyx with the antennal lobe via the antennocerebral tract are the only source of cholinergic elements in the calyces. Their terminals establish an array of large boutons 2-7 microm in diameter throughout all calycal subdivisions. The GABA-ir extrinsic neurons, different in origin, form a network of fine fibers and boutons codistributed in all calycal regions with the cholinergic terminals and with tiny profiles, mainly Kenyon cell dendrites. We have investigated the synaptic circuits of these three neuron types using preembedding immuno-electron microscopy. All ChAT/VAChT-ir boutons form divergent synapses upon multitudinous surrounding Kenyon cell dendrites. GABA-ir elements also regularly contribute divergent synaptic input onto these dendrites, as well as occasional inputs to boutons of projection neurons. The same synaptic microcircuits involving these three neuron types are repeatedly established in glomeruli in all calycal regions. Each glomerulus comprises a large cholinergic bouton at its core, encircled by tiny vesicle-free Kenyon cell dendrites as well as by a number of GABAergic terminals. A single dendritic profile may thereby receive synaptic input from both cholinergic and GABAergic elements in close vicinity at presynaptic sites with T-bars typical of fly synapses. ChAT-ir boutons regularly have large extensions of the active zones. Thus, Kenyon cells may receive major excitatory input from cholinergic boutons and considerable postsynaptic inhibition from GABAergic terminals, as well as, more rarely, presynaptic inhibitory signaling. The calycal glomeruli of Drosophila are compared with the cerebellar glomeruli of vertebrates. The cholinergic boutons are the largest identified cholinergic synapses in the Drosophila brain and an eligible prospect for studying the genetic regulation of excitatory presynaptic function.     

F-actin at identified synapses in the mushroom body neuropil of the insect brain

The distribution of f-actin stained by fluorescent phalloidin was investigated in the brain of several insect species, with a special focus on the mushroom body. For localizing f-actin in identified neurons and at synapses, additional staining with fluorescent dextrans and anti-synapsin I immunostaining was employed. Intense f-actin staining was consistently found in synaptic complexes of the mushroom body calyces (calycal microglomeruli [MG]). These MG contain a central core of presynaptic boutons, predominantly belonging to deutocerebral cholinergic excitatory projection neurons, which are surrounded by a shell of numerous Kenyon cell (KC) dendritic tips. In the cricket Gryllus bimaculatus, high-resolution confocal laser scanning imaging revealed colocalization of f-actin with KC dendritic spine parts within MG. Although presynaptic boutons appear to be mainly devoid of f-actin-phalloidin fluorescence, there appears to be an accumulation of f-actin in KC dendritic spines synaptically contacting the boutons. Electron microscopy of boutons and dextran-stained KC dendrites revealed their pre- and postsynaptic sites, with KCs being strictly postsynaptic elements. Their subsynaptic membrane appositions are considered to be associated with f-actin. Focal accumulation of f-actin in the dendritic tips of KCs was found to be a general feature of MG, with either spheroidal or indented boutons of different sizes, as encountered in the mushroom bodies of the cricket, honey bee, ant, and fruit fly. The structural similarities of calycal MG and f-actin accumulation in KC dendrites with cerebellar microglomeruli are considered comparatively. The accumulation of f-actin in KC dendrites is discussed in view of mushroom body plasticity and its potential role in learning and memory formation.     

Fine structural organization of the hemiellipsoid body of the land hermit crab, Coenobita clypeatus

Electron microscopical observations of the hemiellipsoid bodies of the land hermit crab Coenobita clypeatus resolve microglomerular synaptic complexes that are comparable to those observed in the calyces of insect mushroom bodies and which characterize olfactory inputs onto intrinsic neurons. In an adult hermit crab, intrinsic neurons and one class of efferent neurons originate from neuronal somata of globuli cells covering the hemiellipsoid bodies. Counts of their nucleoli show that about 120,000 globuli cells supply each hemiellipsoid body in an adult hermit crab. This number is comparable to the number of globuli cells supplying mushroom bodies of certain insects, such as honey bees and cockroaches. Counts of axons in tracts leading from the olfactory lobes to the hemiellipsoid bodies resolve 20,000 afferent axons, however, an order of magnitude greater than known for any insect. These afferent axons provide numerous swollen varicosities, each presynaptic to many small profiles, and thus comparable to the microglomeruli that characterize insect mushroom body calyces. Also, common to mushroom bodies and hemiellipsoid bodies are arrangements of intrinsic neurons, afferent neurons containing dense core vesicles, and systems of serial synaptic complexes that relate to postsynaptic profiles of efferent neurons. Together, the ultrastructural organization of the hemiellipsoid bodies of C. clypeatus supports the proposition that this center may share a common origin with the insect mushroom body despite obvious divergent evolution of overall shape.     

GABA-immunoreactive neurons in the mushroom bodies of the honeybee: an electron microscopic study

Synaptic contacts of gamma-aminobutyric acid (GABA) -immunoreactive neurons in honeybee mushroom bodies were studied by using electron microscopic immunocytochemistry. In the lip region of the calyx neuropil, GABA-immunoreactive profiles formed synapses onto both small postsynaptic profiles (76%) and large immunonegative boutons (4%), which were likely to belong to the intrinsic and extrinsic mushroom body neurons, respectively. Three morphologic types of the large immunonegative boutons were distinguished: "light," "dark," and "dense core"; all of them received synaptic inputs from the GABA-immunoreactive profiles. A significant proportion of the synapses formed by the GABA-immunoreactive neurons in the lip region (20%) were input synapses from immunonegative neurons. Analysis of thin serial sections showed that the output and input synapses formed microcircuits in which both large immunonegative boutons and small postsynaptic profiles were involved. We interpret these findings to show that negative feedforward and feedback loops exist within the microcircuits of the lip region.     

A novel type of microglomerular synaptic complex in the polarization vision pathway of the locust brain

The lateral accessory lobes (LALs) are prominent integration centers in the insect brain. In the desert locust Schistocerca gregaria, they are connected with the anterior optic tubercles (AOTus), with the central complex, and with the ventral nerve cord. Two subcompartments of the LALs, the lateral triangle and the median olive, are easily recognized by their prominent granular texture. Both areas are part of the polarization vision pathway in the locust brain; they receive input from projection neurons of the AOTu and are the site of presumed dendritic arborizations of tangential neurons of the lower division of the central body. Both types of neuron are sensitive to polarized light and most likely play a role in sky compass navigation of the locust. We show here that neurons from the AOTu and tangential neurons of the central body form large microglomerular contacts in the median olive and lateral triangle. Presynaptic elements from the AOTu end in small numbers of large cup-shaped terminals. These cups enclose many small gamma-aminobutyric acid (GABA)-immunoreactive (-ir) profiles from tangential neurons of the lower division of the central body. Each cup-shaped profile makes numerous (>150) dyadic output synapses with the small postsynaptic GABA-ir profiles. No synaptic connections were found between the small core profiles. The microglomerular organization of the median olive and lateral triangle is unlike that of any other synaptic microglomeruli reported for the insect brain. It might provide precise spike timing information possibly used to extract spatial information by comparison of binocular inputs in the central complex.     

Polyneuronal innervation of spiny stellate neurons in cat visual cortex

Our hypothesis was that spiny stellate neurons in layer 4 of cat visual cortex receive polyneuronal innervation. We characterised the synapses of four likely sources of innervation by three simple criteria: the type of synapse, the target (spine, dendritic shaft), and the area of the presynaptic bouton. The layer 6 pyramids had the smallest boutons and formed asymmetric synapses mainly with the dendritic shaft. The thalamic afferents had the largest boutons and formed asymmetric synapses mainly with spines. The spiny stellates had medium-sized boutons and formed asymmetric synapses mainly with spines. We used these to make a “template” to match against the boutons forming synapses with the spiny stellate dendrite. Of the asymmetric synapses, 45% could have come from layer 6 pyramidal neurons, 28% from spiny stellate neurons, and 6% from thalamic afferents. The remaining 21% of asymmetric synapses could not be accounted for without assuming some additional selectivity of the presynaptic axons. Additional asymmetric synapses may come from a variety of sources, including other cortical neurons and subcortical nuclei such as the claustrum. Of the symmetric synapses, 84% could have been provided by clutch cells, which form large boutons. The remainder, formed by small boutons, probably come from other smooth neurons in layer 4, e.g., neurogliaform and bitufted neurons. Our analysis supports the hypothesis that the spiny stellate receives polyneuronal innervation, perhaps from all the sources of boutons in layer 4. Although layer 4 is the major recipient of thalamic afferents, our results show that they form only a few percent of the synapses of layer 4 spiny stellate neurons.     

Synaptic connectivity of urinary bladder afferents in the rat superficial dorsal horn and spinal parasympathetic nucleus

That visceral sensory afferents are functionally distinct from their somatic analogues has been known for a long time but the detailed knowledge of their synaptic connections and neurotransmitters at the first relay nucleus in the spinal cord has been limited. To provide information on these topics, we investigated the synapses and neurotransmitters of identified afferents from the urinary bladder to the superficial laminae of the rat spinal dorsal horn (DH) and the spinal parasympathetic nucleus (SPN) by tracing with horseradish peroxidase, quantitative electron microscopical analysis, and immunogold staining for GABA and glycine. In the DH, most bladder afferent boutons formed synapses with 1–2 postsynaptic dendrites, whereas in the SPN, close to a half of them formed synapses with 3–8 postsynaptic dendrites. The number of postsynaptic dendrites and dendritic spines per bladder afferent bouton, both measures of synaptic divergence and of potential for synaptic plasticity at a single bouton level, were significantly higher in the SPN than in the DH. Bladder afferent boutons frequently received inhibitory axoaxonic synapses from presynaptic endings in the DH but rarely in the SPN. The presynaptic endings were GABA- and/or glycine-immunopositive. The bouton volume, mitochondrial volume, and active zone area, all determinants of synaptic strength, of the bladder afferent boutons were positively correlated with the number of postsynaptic dendrites. These findings suggest that visceral sensory information conveyed via the urinary bladder afferents is processed differently in the DH than in the SPN, and differently from the way somatosensory information is processed in the spinal cord.     

Reciprocal synapses between inner hair cell spines and afferent dendrites in the organ of corti of the mouse

We provide, for the first time, ultrastructural evidence for the differentiation of reciprocal synapses between afferent dendrites of spiral ganglion neurons and inner hair cells. Cochlear synaptogenesis of inner hair cells in the mouse occurs in two phases: before and after the onset of hearing at 9-10 postnatal (PN) days. In the first phase, inner hair cells acquire afferent innervation (1-5 PN). Reciprocal synapses form around 9-10 PN on spinous processes emitted by inner hair cells into the dendritic terminals, predominantly in conjunction with ribbon afferent synapses. During the second phase, which lasts up to 14 PN, synaptogenesis is led by the olivocochlear fibers of the lateral bundle, which induce the formation of compound and spinous synapses. The afferent dendrites themselves also develop recurrent presynaptic spines or form mounds of synaptic vesicles apposed directly across inner hair cell ribbon synapses. Thus, in the adult 2-month mouse, afferent dendrites of spiral ganglion neurons are not only postsynaptic but also presynaptic to inner hair cells, providing a synaptic loop for an immediate feedback response. Reciprocal synapses, together with triadic, converging, and serial synapses, are an integral part of the afferent ribbon synapse complex. We define the neuronal circuitry of the inner hair cell and propose that these minicircuits form synaptic trains that provide the neurological basis for local cochlear encoding of the initial acoustic signals.     

Ultrastructure and synaptic differences of the boutons of the projection neurons between the lip and collar regions of the mushroom bodies in the ant, Cataglyphis albicans

The mushroom bodies of insects are viewed as key neuropils for sensory integration and perhaps learning and memory. In Hymenoptera, particularly ants, the calyx of the mushroom bodies is divided into two main regions, the lip and the collar. Although most ants are highly dependent on olfaction and have enlarged calyces comprised mostly of lip, some ant groups are also highly visual and have well-developed collars. The desert ant Cataglyphis albicans, known for its navigational abilities, shifts from the dark olfactory demanding nest interior to the visually demanding desert environment, and unlike many other ants their mushroom bodies are comprised of both a well-developed lip and collar. In this study, using electron microscope serial-sectioning and 3D-reconstructions, we show that axonal processes that innervate the lip and collar are inherently different in structure and synaptic connectivity. The boutons of the lip are larger, with more synaptic vesicles and larger synapses than the collar, while boutons of the collar have more postsynaptic partners per synapse. Our morphological findings suggest that the signals originating from olfactory projection neurons that innervate the lip appear stronger and more likely to propagate than signals that innervate the collar, while the signals entering the collar appear relatively weaker and are further integrated between more postsynaptic partners. We discuss the differences of the signaling properties between the lip and collar projection neurons and suggest that the greater postsynaptic integration in the collar is presumably for spatial processing for visual navigation in Cataglyphis.     

Synapse replacement in the striatum of the adult rat following unilateral cortex ablation

Defining the selective pattern of synapse replacement that occurs in different areas of the damaged brain is essential for predicting the limits of functional compensation that can be achieved after various types of brain injury. Here we describe the time course of dendritic reorganization, spine loss and recovery, and synapse replacement in the striatum following a unilateral cortex ablation. We found that the time course for the transient loss and recovery of dendritic spines on medium spiny I (MSI) neurons, the primary postsynaptic target for corticostriatal axons, paralleled the time course for the removal of degenerating axon terminals from the neuropil and the formation of new synapses on MSI neurons. Reinnervation of the deafferented striatum occurred chiefly by axon terminals that formed asymmetric synapses with dendritic spines of MSI neurons, and the mean density of asymmetric synapses recovered to 86% of the sham-operated rat value by 30 days postlesion. In addition, the synaptic circuitry of the reconstructed striatum was characterized by an increase in the number of multiple synaptic boutons (MSBs), i.e., presynaptic axon terminals that make contact with more than one dendritic spine. Whether the postsynaptic contacts of MSBs are formed with the dendritic spines of the same or a different parent dendrite in the striatum is unknown. Nevertheless, these data suggest that the formation of MSBs is an essential part of the compensatory response to the loss of input from the ipsilateral cortex following the aspiration lesion and may serve to modulate activity-dependent adaptive changes in the reconstructed striatum that can lead to functional recovery.     

Presynaptic Ultrastructural Plasticity Along CA3→CA1 Axons During Long‐Term Potentiation in Mature Hippocampus

In area CA1 of the mature hippocampus, synaptogenesis occurs within 30 minutes after the induction of long‐term potentiation (LTP); however, by 2 hours many small dendritic spines are lost, and those remaining have larger synapses. Little is known, however, about associated changes in presynaptic vesicles and axonal boutons. Axons in CA1 stratum radiatum were evaluated with 3D reconstructions from serial section electron microscopy at 30 minutes and 2 hours after induction of LTP by theta‐burst stimulation (TBS). The frequency of axonal boutons with a single postsynaptic partner was decreased by 33% at 2 hours, corresponding perfectly to the 33% loss specifically of small dendritic spines (head diameters <0.45 μm). Docked vesicles were reduced at 30 minutes and then returned to control levels by 2 hours following induction of LTP. By 2 hours there were fewer small synaptic vesicles overall in the presynaptic vesicle pool. Clathrin‐mediated endocytosis was used as a marker of local activity, and axonal boutons containing clathrin‐coated pits showed a more pronounced decrease in presynaptic vesicles at both 30 minutes and 2 hours after induction of LTP relative to control values. Putative transport packets, identified as a cluster of less than 10 axonal vesicles occurring between synaptic boutons, were stable at 30 minutes but markedly reduced by 2 hours after the induction of LTP. APV blocked these effects, suggesting that the loss of axonal boutons and presynaptic vesicles was dependent on N‐methyl‐D‐aspartic acid (NMDA) receptor activation during LTP. These findings show that specific presynaptic ultrastructural changes complement postsynaptic ultrastructural plasticity during LTP. J. Comp. Neurol. 521:3898–3912, 2013. © 2013 Wiley Periodicals, Inc.     

Environment- and age-dependent plasticity of synaptic complexes in the mushroom bodies of honeybee queens

Diversity in behavior plays a crucial role for the division of labor in insect societies. Social insects such as honeybees provide excellent model systems to investigate neuronal principles underlying behavioral plasticity. The two female castes, queens and workers, differ substantially in anatomy, physiology, aging and behavior. The different phenotypes are induced by environmental factors rather than genetic differences. Here we investigated environment- and age-dependent effects on the synaptic organization within higher order neuropils of the honeybee brain. Synaptic complexes (microglomeruli) in sensory-input regions of the mushroom bodies, prominent higher sensory integration centers, were analyzed quantitatively using fluorescent markers and confocal microscopy. Pre- and postsynaptic compartments of individual microglomeruli were labeled by anti-synapsin immunolabeling and f-actin detection with phalloidin in dendritic spines of mushroom-body intrinsic neurons. The results demonstrate that in queens the numbers of microglomeruli in the olfactory and visual input regions of the mushroom-body calyx are significantly lower than in workers. In queens raised in incubators, microglomeruli were affected by differences in pupal rearing temperature within the range of naturally occurring temperatures (32-36 degrees C). The highest numbers of microglomeruli developed at a lower temperature compared to workers (33.5 vs. 34.5 degrees C). We found a striking adult plasticity of microglomeruli numbers throughout the extended life-span of queens. Whereas microglomeruli in the olfactory lip increased with age ( approximately 55%), microglomeruli in the visual collar significantly decreased ( approximately 35%). We propose that developmental and adult plasticity of the synaptic circuitry in the mushroom-body calyx might underlie caste- and age-specific adaptations in behavior.     

Compound synapses within the GABAergic innervation of the auditory inner hair cells in the adolescent mouse

Ultrastructural investigation of the γ-aminobutyric acid (GABA) component of the inner spiral bundle in adolescent mice revealed a pathway of glutamic acid decarboxylase (GAD)-positive and -negative fibers and vesiculated endings that contact inner hair cells and their afferents through a complex of axosomatic and axodendritic synapses. Ultrastructural details were investigated by using conventional electron microscopy. Several synaptic arrangements were observed: Main axosomatic synapses form between vesiculated endings and individual or adjoining inner hair cells (interreceptor synapses). Spinous synapses form on long, spinelike processes that protrude from inner hair cells to reach distant efferent endings. The efferent endings associate with inner hair cells and their synaptic afferents through compound synapses—serial, “converging,” and triadic—otherwise characteristic of sensory relay nuclei. Serial synapses form by the sequential presynaptic alignment of the efferent→receptor→afferent components. Converging synapses result from the simultaneous apposition of a receptor ribbon synapse and a presynaptic efferent terminal on a recipient afferent dendrite. Triadic synapses comprise a vesiculated efferent ending in contact with an inner hair cell and with its synaptic afferent. Additionally, efferent endings may form simple axodendritic and axoaxonal synapses with GAD-negative vesiculated endings. The combination of different synaptic arrangements leads to short chains of compound synapses. It is assumed that these synaptic patterns seen in the adolescent mouse represent adult synaptology. The patterns of synaptic connectivity suggest an integrative role for the GABA/GAD lateral efferent system, and imply its involvement in the pre- and postsynaptic modulation of auditory signals. J. Comp. Neurol. 377:423–442, 1997. © 1997 Wiley-Liss, Inc.     

Synaptic circuitry of identified neurons in the antennal lobe of Drosophila melanogaster

In Drosophila melanogaster olfactory sensory neurons (OSNs) establish synapses with projection neurons (PNs) and local interneurons within antennal lobe (AL) glomeruli. Substantial knowledge regarding this circuitry has been obtained by functional studies, whereas ultrastructural evidence of synaptic contacts is scarce. To fill this gap, we studied serial sections of three glomeruli using electron microscopy. Ectopic expression of a membrane‐bound peroxidase allowed us to map synaptic sites along PN dendrites. Our data prove for the first time that each of the three major types of AL neurons is both pre‐ and postsynaptic to the other two types, as previously indicated by functional studies. PN dendrites carry a large proportion of output synapses, with approximately one output per every three input synapses. Detailed reconstructions of PN dendrites showed that these synapses are distributed unevenly, with input and output sites partially segregated along a proximal–distal gradient and the thinnest branches carrying solely input synapses. Moreover, our data indicate synapse clustering, as we found evidence of dendritic tiling of PN dendrites. PN output synapses exhibited T‐shaped presynaptic densities, mostly arranged as tetrads. In contrast, output synapses from putative OSNs showed elongated presynaptic densities in which the T‐bar platform was supported by several pedestals and contacted as many as 20 postsynaptic profiles. We also discovered synaptic contacts between the putative OSNs. The average synaptic density in the glomerular neuropil was about two synapses/µm³. These results are discussed with regard to current models of olfactory glomerular microcircuits across species. J. Comp. Neurol. 524:1920–1956, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.     

Neuropeptide Y-immunoreactive terminals form axo-axonic synaptic arrangements in the substantia gelatinosa (lamina II) of the cat spinal dorsal horn

The ultrastructural organization of nerve terminals containing neuropeptide Y-immunoreactivity was studied in the substantia gelatinosa of the cat spinal dorsal horn. Seventy immunoreactive boutons were examined through serial sections and 67 of them were found to form between one and five synaptic junctions with dendrites (59.5% of synapses), somata (3% of synapses) and other axon terminals (37.5% of synapses). The postsynaptic axon terminals were often the central boutons of glomeruli. These findings suggest that neuropeptide Y regulates spinal sensory transmission through both a postsynaptic action upon dorsal horn neurons and a presynaptic action upon primary afferent terminals.     

Neuronal organization of the hemiellipsoid body of the land hermit crab, Coenobita clypeatus: correspondence with the mushroom body ground pattern

Malacostracan crustaceans and dicondylic insects possess large second-order olfactory neuropils called, respectively, hemiellipsoid bodies and mushroom bodies. Because these centers look very different in the two groups of arthropods, it has been debated whether these second-order sensory neuropils are homologous or whether they have evolved independently. Here we describe the results of neuroanatomical observations and experiments that resolve the neuronal organization of the hemiellipsoid body in the terrestrial Caribbean hermit crab, Coenobita clypeatus, and compare this organization with the mushroom body of an insect, the cockroach Periplaneta americana. Comparisons of the morphology, ultrastructure, and immunoreactivity of the hemiellipsoid body of C. clypeatus and the mushroom body of the cockroach P. americana reveal in both a layered motif provided by rectilinear arrangements of extrinsic and intrinsic neurons as well as a microglomerular organization. Furthermore, antibodies raised against DC0, the major catalytic subunit of protein kinase A, specifically label both the crustacean hemiellipsoid bodies and insect mushroom bodies. In crustaceans lacking eyestalks, where the entire brain is contained within the head, this antibody selectively labels hemiellipsoid bodies, the superior part of which approximates a mushroom body's calyx in having large numbers of microglomeruli. We propose that these multiple correspondences indicate homology of the crustacean hemiellipsoid body and insect mushroom body and discuss the implications of this with respect to the phylogenetic history of arthropods. We conclude that crustaceans, insects, and other groups of arthropods share an ancestral neuronal ground pattern that is specific to their second-order olfactory centers.