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We reconstructed the intrinsic axons of 32 neurons in the guinea pig inferior colliculus (IC) following juxtacellular labeling. Biocytin was injected into cells in vivo, after first analyzing physiological response properties. Based on axonal morphology there were two classes of neuron: (1) laminar cells (14/32, 44%) with an intrinsic axon and flattened dendrites confined to a single fibrodendritic lamina and (2) translaminar cells (18/32, 56%) with axons that terminated in two or more laminae in the central nucleus (ICc) or the surrounding cortex. There was also one small, low-frequency cell with bushy-like dendrites that was very sensitive to interaural timing differences. The translaminar cells were subdivided into three groups of cells with: (a) stellate dendrites that crossed at least two laminae (8/32, 25%); (b) flattened dendrites confined to one lamina and that had mainly en passant axonal swellings (7/32, 22%) and (c) short, flattened dendrites and axons with distinctive clusters of large terminal boutons in the ICc (3/32, 9%). These terminal clusters were similar to those of cortical basket cells. The 14 laminar cells all had sustained responses apart from one offset response. Almost half the non-basket type translaminar cells (7/15) had onset responses while the others had sustained responses. The basket cells were the only ones to have short-latency (7–9 ms), chopper responses and this distinctive temporal response should allow them to be studied in more detail in future. This is the first description of basket cells in the auditory brainstem, but more work is required to confirm their neurotransmitter and precise post-synaptic targets.

Recent experimental observations have shown evidence of an unexpected sudden drop-off in the dense core vesicles (DCVs) content at the ends of certain types of axon endings. This article seeks to determine whether these observations may be explained without modifying the parameters characterizing the ability of distal en passant boutons to capture and accumulate DCVs. We developed a mathematical model that is based on the conservation of captured and transiting DCVs in boutons. The model consists of 77 ordinary differential equations and is solved using a standard Matlab solver. We hypothesize that the drop in DCV content in distal boutons is due to an insufficient supply of anterogradely moving DCVs coming from the soma. As anterogradely moving DCVs are captured (and eventually destroyed) in more proximal boutons on their way to the end of the terminal, the fluxes of anterogradely moving DCVs between the boutons become increasingly smaller, and the most distal boutons are left without DCVs. We tested this hypothesis by modifying the flux of DCVs entering the terminal and found that the number of most distal boutons left unfilled increases if the DCV flux entering the terminal is decreased. The number of anterogradely moving DCVs in the axon can be increased either by the release of a portion of captured DCVs into the anterograde component or by an increase of the anterograde DCV flux into the terminal. This increase could lead to having enough anterogradely moving DCVs such that they could reach the most distal bouton and then turn around by changing molecular motors that propel them. The model suggests that this could result in an increased concentration of resident DCVs in distal boutons beginning with bouton 2 (the most distal is bouton 1). This is because in distal boutons, DCVs have a larger chance to be captured from the transiting state as they pass the boutons moving anterogradely and then again as they pass the same boutons moving retrogradely.

Recent experimental observations have shown evidence of an unexpected sudden drop-off in the dense core vesicles (DCVs) content at the ends of certain types of axon endings. This paper seeks to determine whether these observations may be explained without modifying the parameters characterizing the ability of distalen passantboutons to capture and accumulate DCVs. We developed a mathematical model that is based on the conservation of captured and transiting DCVs in boutons. The model consists of 77 ordinary differential equations and is solved using a standard Matlab solver. We hypothesize that the drop in DCV content in distal boutons is due to an insufficient supply of anterogradely moving DCVs coming from the soma. As anterogradely moving DCVs are captured (and eventually destroyed) in more proximal boutons on their way to the end of the terminal, the fluxes of anterogradely moving DCVs between the boutons become increasingly smaller, and the most distal boutons are left without DCVs. We tested this hypothesis by modifying the flux of DCVs entering the terminal and found that the number of most distal boutons left unfilled increases if the DCV flux entering the terminal is decreased. The number of anterogradely moving DCVs in the axon can be increased either by the release of a portion of captured DCVs into the anterograde component or by an increase of the anterograde DCV flux into the terminal. This increase could lead to having enough anterogradely moving DCVs such that they could reach the most distal bouton and then turn around by changing molecular motors that propel them. The model suggests that this could result in an increased concentration of resident DCVs in distal boutons beginning with bouton 2 (the most distal is bouton 1). This is because in distal boutons, DCVs have a larger chance to be captured from the transiting state as they pass the boutons moving anterogradely and then again as they pass the same boutons moving retrogradely.

Summary Analyses of microtubule (MT) plus end dynamics at glutamatergic en passant boutons can be carried out in cultured primary neurons isolated from mouse or rat embryos or ex vivo in acute slices isolated from mice that had been electroporated in utero. Here, we describe a protocol for setting up and analyzing live image recordings of primary neurons and acute hippocampal slices expressing tagged versions of the MT plus end binding protein EB3 and the presynaptic vesicle markers vGlut1 or VAMP2. For complete information on the use and execution of this protocol, please refer to Qu et al. (2019)., Graphical abstract, Highlights • Analysis of MT plus end dynamics at excitatory boutons in cultured neurons • Analysis of MT plus end dynamics at excitatory boutons in acute hippocampal slices • Pharmacological stimulation of MT nucleation at boutons in cultured neurons, Analyses of microtubule (MT) plus end dynamics at glutamatergic en passant boutons can be carried out in cultured primary neurons isolated from mouse or rat embryos or ex vivo in acute slices isolated from mice that had been electroporated in utero. Here, we describe a protocol for setting up and analyzing live image recordings of primary neurons and acute hippocampal slices expressing tagged versions of the MT plus end binding protein EB3 and the presynaptic vesicle markers vGlut1 or VAMP2.

Neuronal cultures are widely used in neuroscience research. However, the randomness of circuits in conventional cultures prevents accurate in vitro modeling of cortical development and of the pathogenesis of neurological and psychiatric disorders. A basic feature of cortical circuits that is not captured in standard cultures of dissociated cortical cells is directional connectivity. In this work, a polydimethylsiloxane (PDMS)-based device that achieves directional connectivity between micro 3D cultures is demonstrated. The device consists of through-holes for micro three-dimensional (μ3D) clusters of cortical cells connected by microtrenches for axon and dendrite guidance. The design of the trenches relies in part on the concept of axonal edge guidance, as well as on the novel concept of specific dendrite targeting. This replicates dominant excitatory connectivity in the cortex, enables the guidance of the axon after it forms a synapse in passing (an “en passant” synapse), and ensures that directional selectivity is preserved over the lifetime of the culture. The directionality of connections was verified morphologically and functionally. Connections were dependent on glutamatergic synapses. The design of this device has the potential to serve as a building block for the reconstruction of more complex cortical circuits in vitro.

Control of microtubule (MT) nucleation and dynamics is critical for neuronal function. Whether MT nucleation is regulated at presynaptic boutons and influences overall presynaptic activity remains unknown. By visualizing MT plus end dynamics at individual excitatory en passant boutons in axons of cultured hippocampal neurons and in hippocampal slices expressing EB3-EGFP and vGlut1-Cherry, we found that dynamic MTs preferentially grow from presynaptic boutons, show biased directionality being almost always oriented towards the distal tip of the axon, and can be induced by neuronal activity. Silencing of γ-tubulin expression reduced presynaptic MT nucleation and depletion of either HAUS1 or HAUS7-augmin subunits increased the percentage of retrograde comets initiated at boutons, indicating that γ-tubulin and augmin are required for activity-dependent de novo nucleation of uniformly distally-oriented dynamic MTs. We analyzed the dynamics of a wide range of axonal organelles as well as synaptic vesicles (SVs) relative to vGlut1(+) stable presynaptic boutons in a time window during which MT nucleation at boutons is promoted upon induction of neuronal activity, and found that γ-tubulin-dependent presynaptic MT nucleation controls bidirectional (SV) interbouton transport, and regulates evoked SV exocytosis. Hence, en passant boutons act as hotspots for activity-dependent de novo MT nucleation, which controls neurotransmission by providing dynamic tracks for bidirectional delivery of SVs between sites of neurotransmitter release.

In neurons, neuropeptides are synthesized in the soma and are then transported along the axon in dense core vesicles (DCVs). DCVs are captured in varicosities located along the axon terminal called en passant boutons, which are active terminal sites that accumulate and release neurotransmitters. Recently developed experimental techniques allow for the estimation of the age of DCVs in various locations in the axon terminal. Accurate simulation of the mean age of DCVs in boutons requires the development of a model that would account for resident, transiting-anterograde, and transiting-retrograde DCV populations. In this paper, such a model is developed. The model is applied to simulating DCV transport in Drosophila type II motoneurons. The model simulates DCV transport and capture in the axon terminals and makes it possible to predict the age density distribution of DCVs in en passant boutons as well as DCV's mean age in boutons. The predicted prevalence of older organelles in distal boutons may explain the "dying back" pattern of axonal degeneration observed in dopaminergic neurons in Parkinson's disease. The predicted difference of two hours between the age of older DCVs residing in distal boutons and the age of younger DCVs residing in proximal boutons is consistent with an approximate estimate of age difference deduced from experimental observations. The age density of resident DCVs is found to be bimodal, which is because DCVs are captured from two transiting states: the anterograde transiting state that contains younger DCVs and the retrograde transiting state that contains older DCVs., Final, accepted manuscript version

The intracellular transport system in neurons is specialized to an extraordinary degree, enabling the delivery of critical cargo to sites in axons or dendrites that are far removed from the cell center. Vesicles formed in the cell body are actively transported by kinesin motors along axonal microtubules to presynaptic sites that can be located over a meter away. Both growth factors and degradative vesicles carrying aged organelles or aggregated proteins take the opposite route, driven by dynein motors. Distance is not the only challenge; precise delivery of cargos to sites of need must also be accomplished. For example, localized delivery of presynaptic components to hundreds of thousands of ‘en passant’ synapses distributed along the length of a single axon in some neuronal subtypes provides a layer of complexity that must be successfully navigated to maintain synaptic transmission. Here, we review recent advances in the field of axonal transport with a focus on conceptual developments, and highlight our growing quantitative understanding of neuronal trafficking and its role in maintaining the synaptic function that underlies higher cognitive processes such as learning and memory.

Although the endoplasmic reticulum (ER) is present throughout axons, and IP3 and ryanodine receptors are widely expressed in nerve terminals, whether Ca2+ release from presynaptic stores contributes to action potential (AP)-evoked Ca2+ transients remains controversial. We investigated the release of Ca2+ from ER stores in boutons en passant of neocortical layer 5 pyramidal neurons. A hallmark of these stores is that they spontaneously release Ca2+ at a low frequency. Using a high-affinity Ca2+ indicator, we documented and characterised such spontaneous Ca2+ transients (sCaTs), which occurred at a rate of ~0.2 per min and raised the intracellular Ca2+ concentration ([Ca2+]i) by ~2 µM in the absence of exogenous buffers. Caffeine increased the average frequency of sCaTs by 90%, without affecting their amplitude and decay kinetics. Therefore, presynaptic ryanodine receptors were likely involved. To determine if presynaptic ER stores contribute to intracellular Ca2+ accumulation during repetitive stimulation, we measured [Ca2+]i during 2 s long trains of APs evoked at 10-50 Hz. We found that for frequencies

Addition and removal of presynaptic terminals reconfigures neuronal circuits of the mammalian neocortex, but little is known about how this presynaptic structural plasticity is controlled. Since mitochondria can regulate presynaptic function, we investigated whether the presence of axonal mitochondria relates to structural plasticity of presynaptic boutons in mouse neocortex. We found that the overall density of axonal mitochondria did not appear to influence loss and gain of boutons. However, positioning of mitochondria at individual presynaptic sites did relate to increased stability of those boutons. In line with this, synaptic localisation of mitochondria increased as boutons aged and showed differing patterns of localisation aten passantandterminauxboutons. These results suggest that mitochondria accumulate locally at boutons over time to increase bouton stability.

SUMMARY In principle, one way to define the functional role of a neuron would be to identify all the synaptic input it receives and all synaptic output it confers onto its targets. With serial electron microscopy we annotated all the input synapses (862) and output synapses (626) associated with one inhibitory interneuron in the visual thalamus of the mouse. This neuron’s neurites covered a broad swath of lateral geniculate nucleus and spanned multiple functionally distinct regions. Every one of its neurites formed synapses onto hundreds of thalamocortical cells of several different types. All but one small neurite also had dendrite-like properties and received input from retinal ganglion cell axons. Pre- and postsynaptic associations with other inhibitory interneurons were also distributed throughout the interneuron’s territory. We observed a diverse array of local synaptic motifs and three fundamentally different types of inhibitory neurites. Many thalamocortical cells were innervated weakly by this interneuron by single en passant shaft synapses. But a subset of the interneuron’s thalamocortical cell targets received multiple synaptic inputs from targeted inhibitory neurites that climbed along the thalamocortical cell’s dendrite with an assemblage of fasciculated retinal ganglion cell axons. Because of the diverse range of synaptic relationships exhibited by this one neuron, this cell defies a single functional label and seems rather to be using extremely local synaptic processing to participate in many different functions.

Not only the amplitude but also the time course of a presynaptic Ca(2+) transient determine multiple aspects of synaptic transmission. In small bouton-type synapses, the mechanisms underlying the Ca(2+) decay kinetics have not been fully investigated. Here, factors that shape an action-potential-evoked Ca(2+) transient were quantitatively studied in synaptic boutons of neocortical layer 5 pyramidal neurons. Ca(2+) transients were measured with different concentrations of fluorescent Ca(2+) indicators and analyzed based on a single-compartment model. We found a small endogenous Ca(2+)-binding ratio (7 ± 2) and a high activity of Ca(2+) transporters (0.64 ± 0.03 ms(−1)), both of which enable rapid clearance of Ca(2+) from the boutons. However, contrary to predictions of the single-compartment model, the decay time course of the measured Ca(2+) transients was biexponential and became prolonged during repetitive stimulation. Measurements of [Ca(2+)](i) along the adjoining axon, together with an experimentally constrained model, showed that the initial fast decay of the Ca(2+) transients predominantly arose from the diffusion of Ca(2+) from the boutons into the axon. Therefore, for small boutons en passant, factors like terminal volume, axon diameter, and the concentration of mobile Ca(2+)-binding molecules are critical determinants of Ca(2+) dynamics and thus Ca(2+)-dependent processes, including short-term synaptic plasticity.

Neuromodulators play a critical role in sensorimotor processing via various actions, including pre- and postsynaptic signal modulation and direct modulation of signal encoding in peripheral dendrites. Here, we present a new mechanism that allows state-dependent modulation of signal encoding in sensory dendrites by neuromodulatory projection neurons. We studied the impact of antidromic action potentials (APs) on stimulus encoding using the anterior gastric receptor (AGR) neuron in the heavily modulated crustacean stomatogastric ganglion (STG). We found that ectopic AP initiation in AGR’s axon trunk is under direct neuromodulatory control by the inferior ventricular (IV) neurons, a pair of descending projection neurons. IV neuron activation elicited a long-lasting decrease in AGR ectopic activity. This modulation was specific to the site of AP initiation and could be mimicked by focal application of the IV neuron co-transmitter histamine. IV neuron actions were diminished after blocking H2receptors in AGR’s axon trunk, suggesting a direct axonal modulation. This local modulation did not affect the propagation dynamics of en passant APs. However, decreases in ectopic AP frequency prolonged sensory bursts elicited distantly near AGR’s dendrites. This frequency-dependent effect was mediated via the reduction of antidromic APs, and the diminishment of backpropagation into the sensory dendrites. Computational models suggest that invading antidromic APs interact with local ionic conductances, the rate constants of which determine the sign and strength of the frequency-dependent change in sensory sensitivity. Antidromic APs therefore provide descending projection neurons with a means to influence sensory encoding without affecting AP propagation or stimulus transduction.

The ultrastructure of motoneurons and synapses in the somatic zone of the oculomotor nerve nuclei was studied in C57 Black/6 mice. Axodendritic and axosomatic synapses were characterized by round densities (round subjunctional densities) located in the postsynaptic part of the synapse some distance from the postsynaptic density. The synaptic cleft close to subjunctional densities was widened to up to 30 μm. Other parts of the cleft in the same synapse could be narrowed almost twofold. Axosomatic and axodendritic spiny synapses were found. Motoneuron bodies and dendrites bore synapses of the en passant type. Gap junctions were not seen.

The goal of this paper is to use mathematical modeling to investigate the fate of dense core vesicles (DCVs) captured in en passant boutons located in nerve terminals. One possibility is that all DCVs captured in boutons are destroyed, another possibility is that captured DCVs can escape and reenter the pool of transiting DCVs that move through the boutons, and a third possibility is that some DCVs are destroyed in boutons, while some reenter the transiting pool. We developed a model by applying the conservation of DCVs in various compartments composing the terminal, to predict different scenarios that emerge from the above assumptions about the fate of DCVs captured in boutons. We simulated DCV transport in type Ib and type III terminals. The simulations demonstrate that, if no DCV destruction in boutons is assumed and all captured DCVs reenter the transiting pool, the DCV fluxes evolve to a uniform circulation in a type Ib terminal at steady-state and the DCV flux remains constant from bouton to bouton. Because at steady-state the amount of captured DCVs is equal to the amount of DCVs that reenter the transiting pool, no decay of DCV fluxes occurs. In a type III terminal at steady-state, the anterograde DCV fluxes decay from bouton to bouton, while retrograde fluxes increase. This is explained by a larger capture efficiency of anterogradely moving DCVs than of retrogradely moving DCVs in type III boutons, while the captured DCVs that reenter the transiting pool are assumed to be equally split between anterogradely and retrogradely moving components. At steady-state, the physiologically reasonable assumption of no DCV destruction in boutons results in the same number of DCVs entering and leaving a nerve terminal. Because published experimental results indicate no DCV circulation in type III terminals, modeling results suggest that DCV transport in these type III terminals may not be at steady-state. To better understand the kinetics of DCV capture and release, future experiments in type III terminals at different times after DCV release (molting) may be proposed.

Organelles such as neuropeptide-containing dense-core vesicles (DCVs) and mitochondria travel down axons to supply synaptic boutons. DCV distribution among en passant boutons in small axonal arbors is mediated by circulation with bidirectional capture. However, it is not known how organelles are distributed in extensive arbors associated with volume transmission and neuromodulation by monoamines and neuropeptides and mammalian dopamine neuron vulnerability. Therefore, we studied presynaptic organelle distribution in Drosophila octopamine neurons that innervate ∼20 muscles with ∼1500 boutons. Unlike in smaller arbors, distal boutons in these arbors contain fewer DCVs and mitochondria, although active zones are present. Absence of vesicle circulation is evident by proximal nascent DCV delivery, limited impact of retrograde transport and older distal DCVs. Traffic studies show that DCV axonal transport and synaptic capture are not scaled for extensive innervation, thus limiting distal delivery. Activity-induced synaptic endocytosis and synaptic neuropeptide release are also reduced distally. We propose that limits in organelle transport and synaptic capture compromise distal synapse maintenance and function in extensive axonal arbors, thereby affecting development, plasticity and vulnerability to neurodegenerative disease.

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB’s α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall. DOI: http://dx.doi.org/10.7554/eLife.26975.001

N-methyl-D-aspartate receptors (NMDARs) are present in both pyramidal neurons and interneurons of the hippocampus. These receptors play an important role in the adult structural plasticity of excitatory neurons, but their impact on the remodeling of interneurons is unknown. Among hippocampal interneurons, somatostatin-expressing cells located in the stratum oriens are of special interest because of their functional importance and structural characteristics: they display dendritic spines, which change density in response to different stimuli. In order to understand the role of NMDARs on the structural plasticity of these interneurons, we have injected acutely MK-801, an NMDAR antagonist, to adult mice which constitutively express enhanced green fluorescent protein (EGFP) in these cells. We have behaviorally tested the animals, confirming effects of the drug on locomotion and anxiety-related behaviors. NMDARs were expressed in the somata and dendritic spines of somatostatin-expressing interneurons. Twenty-four hours after the injection, the density of spines did not vary, but we found a significant increase in the density of their en passant boutons (EPB). We have also used entorhino-hippocampal organotypic cultures to study these interneurons in real-time. There was a rapid decrease in the apparition rate of spines after MK-801 administration, which persisted for 24 h and returned to basal levels afterwards. A similar reversible decrease was detected in spine density. Our results show that both spines and axons of interneurons can undergo remodeling and highlight NMDARs as regulators of this plasticity. These results are specially relevant given the importance of all these players on hippocampal physiology and the etiopathology of certain psychiatric disorders.

Dense core vesicles (DCVs) are neuronal organelles that transport diverse cargos important for neuron growth and function, such as neuropeptides, monoamines, and neurotrophic factors. Dysregulation of DCV transport is linked to numerous disorders. Neuropeptides, one of the key components transported by DCVs, are synthesized in the neuron soma. They are then packaged in DCVs and transported toward nerve terminals. Published experimental results suggest that DCVs circulate in nerve terminals, undergoing repeated anterograde and retrograde transport cycles. Some DCVs are captured as they travel through en passant boutons, which are sequential varicose sites in axon terminals. Other DCVs that were not captured while passing the boutons switch direction when they leave the terminal and re-enter circulation. The chapter reviews recent modeling approaches to simulating DCV transport in axon terminals. In particular, differences in modeling DCV transport in terminals with type Ib and type III boutons are discussed. The effects of varying the rate of DCV synthesis in the soma are investigated. The ability of the model to predict the trends observed in experiments is also discussed. Testable predictions made by the model are examined.

This paper is motivated by a recent experimental research by Levitan and colleagues (Wong MY, Zhou C, Shakiryanova D, Lloyd TE, Deitcher DL, Levitan ES. Neuropeptide delivery to synapses by long-range vesicle circulation and sporadic capture. Cell 2012; 148(5): 1029-1038), which discovered and explained the circulation of dense core vesicles (DCVs) between the neuron soma and nerve terminals. We developed a compartmental mechanistic model to simulate this circulation. The model includes five compartments, the axonal compartment and four en passant boutons, which are small axonal varicosities that are present in many axon terminals. By postulating expressions for DCV fluxes between the compartments and for the rates of DCV capture in the boutons and utilizing conservation of DCVs, ODEs modeling concentrations in each of the compartments were developed. The equations were then solved numerically. The obtained results provide insight into how DCV circulation develops, what the circulation is at steady state, and how it may be affected by defects in retrograde transport.

We used biotinylated dextran amine (BDA) to anterogradely label individual axons projecting from primary somatosensory cortex (S1) to four different cortical areas in rats. A major goal was to determine whether axon terminals in these target areas shared morphometric similarities based on the shape of individual terminal arbors and the density of two bouton types: en passant (Bp) and terminaux (Bt). Evidence from tridimensional reconstructions of isolated axon terminal fragments (n=111) did support a degree of morphological heterogeneity establishing two broad groups of axon terminals. Morphological parameters associated with the complexity of terminal arbors and the proportion of beaded Bp vs stalked Bt were found to differ significantly in these two groups following a discriminant function statistical analysis across axon fragments. Interestingly, both groups occurred in all four target areas, possibly consistent with a commonality of presynaptic processing of tactile information. These findings lay the ground for additional work aiming to investigate synaptic function at the single bouton level and see how this might be associated with emerging properties in postsynaptic targets.

The claustrum is a subcortical structure reciprocally connected with most areas of neocortex. This strategic location suggests an integrative role of the claustrum across different sensory modalities. However, our knowledge of the synaptic relationship between the neocortex and the claustrum is basic. In this study, we address this question through a structural investigation of the claustral projection to the ipsilateral primary visual cortex of the cat. Light microscopic reconstructions of axons from the entire thickness of cortex showed a very sparse innervation of the entire cortical depth, with most synaptic boutons in layers 2/3 and 6. Axons bearing numerous boutonsterminauxand boutonsen passantbranched in these laminae. The sparse innervation did not seem to be compensated by particularly large synapses, given that the postsynaptic densities in the superficial layers are of comparable sizes (0.1 μm2) to other cortical synapses. All claustral synapses were asymmetric and in most cases targeted spines (87% in layer 4, 94% in layers 2/3 and 97% in layer 6). The pattern of innervation together with the known physiology of this projection suggests that the claustrum has a modulatory effect on visual cortex.

The primary visual cortex (V1) and V2 together form approximately 24% of the total neocortex of the macaque monkey and have each other as their major partners. The major target of the V1 projection to V2 is layer 4, where it forms clusters of boutons, which form asymmetric (excitatory) synapses mainly with dendritic spines (75%). The remainder form synapses with dendritic shafts. The synapses found on spines were often more complex, perforated postsynaptic densities than those found on dendritic shafts. The reciprocal projection from V2 to V1 targeted layers 1, 2/3, and 5 and was formed of axons of different morphologies. One axon type, originating from superficial layer pyramidal cells, had a morphology resembling those of local pyramidal cell collaterals. These axons arborized in layers 1, 2/3, and 5 of V1. Another type of axon, arborizing in layer 1, was slender (0.3 microm), unbranched, unmyelinated, and uniformly covered with boutons terminaux and formed asymmetric synapses mainly with slender spines. Yet a third type of axon also confined to layer 1, was thick (>1 microm), branched, heavily myelinated, and formed separate small clusters of large ( approximately 1 microm) en passant multisynaptic boutons that formed asymmetric synapses mainly with large flat spines. These data show the existence of a reciprocal excitatory loop between V1 and V2 that is formed by different axonal types, each with preferred layers of termination.

After being synthesized in the soma, neuropeptides are packaged in dense core vesicles (DCVs) and transported toward nerve terminals. It is known, from published experimental results, that in terminals with type Ib boutons DCVs circulate in the terminal, undergoing repeated anterograde and retrograde transport, while in type III terminals DCVs do not circulate in the terminal. Our goal here is to investigate whether the increased DCV production in the soma can lead to the appearance of DCV circulation in type III boutons. For this purpose we developed a mathematical model that simulates DCV transport in various terminals. Our model reproduces some important experimental results, such as those concerning DCV circulation in type Ib and type III terminals. We used the developed model to make testable predictions. The model predicts that an increased DCV production rate in the soma leads to increased DCV circulation in type Ib boutons and to the appearance of DCV circulation in type III boutons. The model also predicts that there are different stages in the development of DCV circulation in the terminals after they were depleted of DCVs due to neuropeptide release.Copyright © 2015 by ASME

The medial nucleus of the amygdala (Me) is a component of the neural circuit for the interpretation of multimodal sensory stimuli and the elaboration of emotions and social behaviors in primates. We studied the presence, distribution, diverse shape, and connectivity of dendritic spines in the human Me of adult postmortem men. Data were obtained from the five types of multipolar neurons found in the Me using an adapted Golgi method and light microscopy, the carbocyanine DiI fluorescent dye and confocal microscopy, and transmission electron microscopy. Three-dimensional reconstruction of spines showed a continuum of shapes and sizes, with the spines either lying isolated or forming clusters. These dendritic spines were classified as stubby/wide, thin, mushroom-like, ramified or with an atypical morphology including intermediate shapes, double spines, and thorny excrescences. Pleomorphic spines were found from proximal to distal dendritic branches suggesting potential differences for synaptic processing, strength, and plasticity in the Me neurons. Furthermore, the human Me has large and thin spines with a gemmule appearance, spinules, and filopodium. The ultrastructural data showed dendritic spines forming monosynaptic or multisynaptic contacts at the spine head and neck, and with asymmetric or symmetric characteristics. Additional findings included en passant, reciprocal, and serial synapses in the Me. Complex long-necked thin spines were observed in this subcortical area. These new data reveal the diversity of the dendritic spines in the human Me likely involved with the integration and processing of local synaptic inputs and with functional implications in physiological and various neuropathological conditions.

This review describes the spiny stellate cells (SCC) which constitute a unique category of cortical interneurons and represent a major component of intracolumnar, lateral and collosal excitation of neocortical pyramidal and nonpyramidal neurons. SCC axons establish contacts with apical dendrites of pyramidal neurons forming cartridge-type synapses en passant with them. SCC establish recurrent connections with inhibitory interneurons and other SCC and also form contacts on themselves through autapses. SCC subtypes are identified, their structure, neurochemical specialization and spatial organization in human and animal neocortex are described. On the basis of the results of author's own investigations and literature data, critical analysis of up-to-date concepts of the histophysiology of recurrent excitatoty and inhibitory neurocirculation in the cortical module, is presented.

While recent studies of synaptic stability in adult cerebral cortex have focused on dendrites, how much axons change is unknown. We have used advances in axon labeling by viruses and in vivo two−photon microscopy to investigate axon branching and bouton dynamics in primary visual cortex (V1) of adult Macaque monkeys. A nonreplicative adeno−associated virus bearing the gene for enhanced green fluorescent protein (AAV.EGFP) provided persistent labeling of axons, and a custom−designed two−photon microscope enabled repeated imaging of the intact brain over several weeks. We found that large−scale branching patterns were stable but that a subset of small branches associated with terminaux boutons, as well as a subset of en passant boutons, appeared and disappeared every week. Bouton losses and gains were both not, vert, similar7% of the total population per week, with no net change in the overall density. These results suggest ongoing processes of synaptogenesis and elimination in adult V1

The membrane potential changes following action potentials in thin unmyelinated cortical axons with en passant boutons may be important for synaptic release and conduction abilities of such axons. In the lack of intra-axonal recording techniques we have used extracellular excitability testing as an indirect measure of the after-potentials. We recorded from individual CA3 soma in hippocampal slices and activated the axon with a range of stimulus intensities. When conditioning and test stimuli were given to the same site the excitability changes were partly masked by local effects of the stimulating electrode at intervals 1 Hz, a condition that activates a hyperpolarization-activated current (Ih) in these axons, the decay was faster than at lower stimulation rates. This effect was reduced by the Ih blocker ZD7288. These data suggest that the decay time course of the action potential-induced hyperexcitability is determined by the membrane time constant.

Neuropeptides are synthesized in the neuron soma; they are packaged in dense core vesicles (DCVs) which undergo axonal transport toward nerve terminals. Published experimental results suggest that in terminals with type Ib boutons DCVs circulate in the terminal, undergoing repeated anterograde and retrograde transport, while in type III terminals DCVs do not circulate in the terminal. In this paper we developed a mathematical model that allowed us to investigate the effects of an increased rate of DCV production in the soma. We demonstrated that our model reproduces some important experimental results, in particular those concerning DCV circulation in type Ib and type III terminals. Our model makes testable predictions. Probably the most important prediction concerns the effect of an increased DCV production rate in the soma, which we anticipate leads to increased DCV circulation in type Ib boutons and to the appearance of DCV circulation in type III boutons. Other predictions concern various stages of development of DCV circulation in the terminals after they were depleted of DCVs due to neuropeptide release.

Our confocal three-dimensional analyses revealed substantial differences in the innervation to vibrissal follicle-sinus complexes (FSCs) in the rat and cat. This is the first study using anti-protein gene product 9.5 (PGP9.5) immunolabeling and confocal microscopy on thick sections to examine systematically the terminal arborizations of the various FSC endings and to compare them between two species, the rat and the cat, that have similar-appearing FSCs but different exploratory behaviors, such as existence or absence of whisking. At least eight distinct endings were clearly discriminated three dimensionally in this study: 1) Merkel endings at the rete ridge collar, 2) circumferentially oriented lanceolate endings, 3) Merkel endings at the level of the ring sinus, 4) longitudinally oriented lanceolate endings, 5) club-like ringwulst endings, 6) reticular endings, 7) spiny endings, and 8) encapsulated endings. Of particular contrast, each nerve fiber that innervates Merkel cells at the level of the ring sinus in the rat usually terminates as a single, relatively small cluster of endings, whereas in the cat they terminate en passant as several large clusters of endings. Also, individual arbors of reticular endings in the rat ramify parallel to the vibrissae and distribute over wide, overlapping territories, whereas those in the cat ramify perpendicular and terminate in tightly circumscribed territories. Otherwise, the inner conical body of rat FSCs contains en passant, circumferentially oriented lanceolate endings that are lacking in the cat, whereas the cavernous sinus of the cat has en passant corpuscular endings that are lacking in the rat. Surprisingly, the one type of innervation that is the most similar in both species is a major set of simple, club-like endings, located at the attachment of the ringwulst, that had not previously been recognized as a morphologically unique type of innervation. Although the basic structure of the FSCs is similar in the rat and cat, the numerous differences in innervation suggest that these species would have different tactile capabilities and perceptions possibly related to their different vibrissa-related exploratory behaviors.

We developed a mathematical model for simulating neuropeptide transport inside dense core vesicles (DCVs) in axon terminals containing en passant boutons. The motivation for this research is a recent experimental study by Levitan and colleagues (Bulgari et al., 2014) which described DCV transport in nerve terminals of type Ib and type III as well as in nerve terminals of type Ib with the transcription factor DIMM. The goal of our modeling is validating the proposition put forward by Levitan and colleagues that the dramatic difference in DCV number in type Ib and type III terminals can be explained by the difference in DCV capture in type Ib and type III boutons rather than by differences in DCV anterograde transport and half-life of resident DCVs. The developed model provides a tool for studying the dynamics of DCV transport in various types of nerve terminals. The model is also an important step in gaining a better mechanistic understanding of transport processes in axons and identifying directions for the development of new models in this area.

The dendritic morphology in neocortical grafts was studied with three-dimensional confocal laser scanning microscopy after microinjection of Lucifer Yellow into individual cells. The grafts had been implanted into infarct cavities in the neocortex of hypertensive rats 46 weeks earlier. The carbocyanine dye method was used to identify afferent (host to transplant) and efferent (transplant to host) connections. Pyramidal, nonpyramidal and glial cells were present in the transplants. Some dendrites had an almost normal appearance, but abnormalities (atypical orientation of apical, basal or oblique apical dendrites) were observed. Some bi-apical pyramidal neurons and pyramidal neurons with obliquely oriented apical dendrites were also observed. Carbocyanine dye-labelled fibres of different diameter formed a dense network in the transplant, enabling the border between transplant and host tissue to be clearly recognized. No labelled fibres were observed to enter the host brain. Fibres with "boutons en passant" and no preferential orientation were noted. It is proposed that Lucifer Yellow microinjection may be a useful method in studies aimed at improving graft morphology. Failure to demonstrate host to transplant connections with the carbocyanine dye method was contrary to earlier studies in which tracers were applied in vivo. A combined use of in-vivo and post-mortem tracer techniques is needed to establish the reason for the discrepancy.

Axon collaterals were identified in 21 of 24 preganglionic neurons in the lateral band of the sacral parasympathetic nucleus of the cat. Following the intracellular injection of HRP or neurobiotin the axons from 20 of these neurons were followed and 53 primary axon collaterals were found to originate from unmyelinated segments and from nodes of Ranvier. Detailed mapping done in the five best labeled cells showed bilateral axon collaterals distributions up to 25,000 μm in length with 950 varicosities and unilateral distributions up to 12,561 μm with 491 varicosities. The axon collaterals appeared to be unmyelinated, which was confirmed at EM, and were small in diameter (average 0.3 μm). Varicosities were located mostly in laminae I, V, VII, VIII and X and in the lateral funiculi. Most varicosities were not in contact with visible structures but some were seen in close apposition to Nissl stained somata and proximal dendrites. Varicosities had average minor diameters of 1.3 μm and major diameters of 2.3 μm. Most were boutons en passant while 10–20% were boutons termineaux. EM revealed axodendritic and axoaxonic synapses formed by varicosities and by the axons between varicosities. It is estimated that the most extensive of these axon collaterals systems may contact over 200 spinal neurons in multiple locations. These data lead to the conclusion that sacral preganglionic neurons have multiple functions within the spinal cord in addition to serving their target organ. As most preganglionic neurons in this location innervate the urinary bladder, it is possible that bladder preganglionic neurons have multiple functions.

The normal maturation of the auditory space map in the deeper layers of the ferret superior colliculus (SC) depends on signals provided by the superficial visual layers, but it is unknown where or how these signals influence the developing auditory responses. Here we report that tracer injections in the superficial layers label axons with en passant and terminal boutons, both in the deeper layers of the SC and in their primary source of auditory input, the nucleus of the brachium of the inferior colliculus (nBIC). Electron microscopy confirmed that biocytin-labelled SC axons form axodendritic synapses on nBIC neurons. Injections of biotinylated dextran amine in the nBIC resulted in anterograde labelling in the deeper layers of the SC, as well as retrogradely labelled superficial and deep SC neurons, whose distribution varied systematically with the rostrocaudal placement of the injection sites in the nBIC. Topographical order in the projection from the SC to the ipsilateral nBIC was confirmed using fluorescent microspheres. We demonstrated the existence of functional SC-nBIC connections by making whole-cell current-clamp recordings from young ferret slices. Both monosynaptic and polysynaptic EPSPs were generated by electrical stimulation of either the superficial or deep SC layers. In addition to unimodal auditory units, both visual and bimodal visual‐auditory units were recorded in the nBIC in vivo and their incidence was higher in juvenile ferrets than in adults. The SC-nBIC circuit provides a potential means by which visual and other sensory or premotor signals may be delivered to the nBIC to calibrate the representation of auditory space.

Formation of terminal synapses at sites such as the neuromuscular junction involves transformation of the motile growth cone into the nonmotile synaptic terminal. However, transformation does not need to be the mechanism when a neurite forms multiple widely spaced synaptic varicosities along a target in an en passant configuration. Synaptic varicosities could form here by specialization of the neurite after the growth cone has advanced past the site. We examined this issue by using cocultures of identified sensory (SN) and motor (L7) neurons from Aplysia. Living SNs were labeled with fluorescent dye and their neurites were observed at high resolution every few minutes growing along the axon of L7, allowing a fine-grained analysis of the behavior of the growth cone at the sites of synapse formation. All varicosities whose formation was observed indeed developed from the growth cone. Sensory varicosities were shown by electron microscopy to contain features characteristic of active zones for transmitter release within a day of their formation on the motor axon. Growth cone advance slowed or stopped transiently during varicosity formation, but the motile activity of the peripheral region of the growth cone (veils and filopodia) was maintained. These results suggest that target "stop signals" involved in the formation of synapses, at least of the en passant variety, may be of a different type from the growth inhibitory molecules, such as the collapsins, which guide axons to their targets.

Local collateral projections from the medial superior olivary nucleus in the gerbil auditory brainstem were examined to study the possible communication of this nucleus with periolivary cell groups. The projections were investigated using intracellular and extracellular labeling with Biocytin in the medial superior olive (MSO) in brainstem tissue slices. Collateral axons were found to branch from the main axons of the central cells of the MSO as the latter passed through a dorsally neighboring periolivary nucleus, the superior paraolivary nucleus (SPN), toward the ipsilateral inferior colliculus (IC), their traditionally accepted target. Bouton-like endings and en passant varicosities of these collaterals appeared to contact the somata and proximal dendrites of cells within the SPN. Furthermore, close observation revealed that these collaterals terminate on at least two types of SPN cells. Intracellular labeling of the collateral axons of the MSO neurons combined with retrograde prelabeling of their target cells, however, revealed that the collaterals selectively contact the cells of the SPN that project to the ipsilateral IC. A link between the MSO and SPN has not been reported previously. This connection is of interest since SPN cells themselves project either to the cochlear nuclei (CN) or the IC. The MSO-SPN projection identified here raises the possibility that the latter may serve as an ancillary channel to convey MSO information to the IC.

Transmission electron micrographs from serial sections were obtained from the neural pole of outer hair cells (OHCs) in the Japanese macaque (Macaca fuscata) and reconstructions of nerve terminals were made using computer software. Data are based on observations of six cells in the basal turn, eight in the middle turn and four in the apex. In general, the number of afferent (type II) terminals on each OHC increased from base to apex, and for a given turn, the numbers appeared unrelated to OHC row. On the other hand, the number of efferent terminals was greater in the middle turn than in other areas, and the number decreased from row 1 to row 3. Reciprocal synapses increased in frequency from the upper basal turn apicalward. The total number of terminals synapsing on an individual OHC increased from base to apex by nearly 100%. Three-dimensional reconstructions showed that nerve fibers terminating on basal and middle turn OHCs ascended directly from sub-OHC regions to synapse on the subnuclear regions of the OHC. In contrast, apical turn fibers ran horizontally at the level of the subnuclear region and the terminals appeared as en passant swellings along a single fiber. Although physiological data are wanting for the macaque, the anatomical findings suggest that functional differences may exist along the length of the cochlea.

Xenopus tadpole motoneurons make cholinergic synapses within the spinal cord. This excitation changes with longitudinal position and contributes to the excitation that controls motor activity and its longitudinal spread during swimming. To explore the anatomic constraints on this excitation, backfilling has been used to examine the anatomy and distribution of the whole population of spinal motoneurons, to define the extent of their central axons and to find where they make synapses. Motoneuron features show considerable variation but do not allow their separation into primary and secondary. Most motoneurons have descending central axons and it is likely that central synapses are made from these axons as longitudinal dendritic extent is very limited. Motoneuron density reaches a broad plateau over the mid-trunk region at 12–13 per 100 μm. Soma size does not change with longitudinal position, but the dorsoventral extent of the dendrites decreases caudally, whereas the central axon length increases. Motoneuron distribution data were used to estimate the longitudinal distribution of central motoneuron axons. This has a broad plateau at 12–14 per 100 μm over much of the trunk and only decreases significantly caudal to the anus. This distribution correlates with cholinergic excitation during swimming. Transmission electron microscopy of motoneurons backfilled with horseradish peroxidase was used to show that central motoneuron axons make en passant synapses with motoneuron dendrites and the dendrites of other unstained neurons. By using measures of synapse frequency and total dendrite length, trunk motoneurons are estimated to each receive 100–200 synapses. J. Comp. Neurol. 411:472–486, 1999. © 1999 Wiley-Liss, Inc.

A temporal dispersion windowis the time required for a volley of action potentials on presynaptic axons to cross the dendritic arbor of a postsynaptic neuron. The volley produces a series of unitary postsynaptic potentials (PSPs) on the postsynaptic neuron. Temporal dispersion is, thus, one factor that can influence the integration of unitary PSPs and the production of action potentials in cortical neurons. Temporal dispersion windows for neurons in the visual cortex of the freshwater turtle, Pseudemys scripta, were estimated by characterizing geniculate afferents and the morphology of neurons in the visual cortex. Horseradish peroxidase injections in the thalamus revealed thin and unmyelinated terminal arbors that run horizontally from lateral to medial across the cortex, forming en passant synapses across the dendrites of cortical neurons. Axons with two calibers were seen, one with diameters between 0.5 and 2.0 "m, and a second with diameters below the resolution limit of the light microscope. The conduction velocity of geniculate afferents in the cortex was measured at 0.18 m/sec§0.04 using the latency of extracellular field potentials evoked by electrical stimulation of the lateral forebrain bundle. The positions and dendritic arbors were characterized in Golgi preparations. Seven morphologically distinct neuron types were positioned to intersect the geniculate afferents in Golgi preparations. The spatial overlap between the dendritic arbors of these cells and the geniculate afferents varied from 128 to 850 "m. Temporal dispersion windows for the seven cell types ranged from 0.7 to 4.7 msec, estimated using a geniculate fiber conduction velocity of 0.18 m/sec. Estimated conduction velocities of 0.04 m/sec for small-caliber fibers produce temporal dispersion windows of 3.2 to 21.3 m/sec.

The fast electrosensory pathway (FEP) of gymnotiform fish is mediated by tuberous electroreceptor organs innervated by ganglion cells that synapse with spherical cells of the electrosensory lateral line lobe (ELL). Spherical cells project to the magnocellular mesencephalic nucleus. The electrosensory environment was represented somatotopically within ELL. The mandibular (MN) and the supraorbital (SON) nerves projected to rostral ELL (occupying 19-28% and 4-10%, respectively), and the posterior branch of the anterior lateral line nerve (pALLN) projected to caudal ELL (occupying 56-64%). Labeling with horseradish peroxidase or biotinylated dextran-amine demonstrated three kinds of synaptic endings coupling primary afferents to spherical cells: multiple synaptic knobs, medium-sized calyxes, and very large calyxes. Multiple synaptic knobs arose from MN and SON primary afferents and were found in a narrow rostral area covering the centrolateral (CLS) and lateral (LS) segments of ELL. Medium and large calyxes, proceeding from the same nerves, predominated in the remaining parts of the three segments of ELL containing spherical cells. Calyx-type endings were also found in the LS-occupying regions in which the pALLNs projected. Calyx-type endings formed gap junctions but also contained vesicles and showed submembrane specializations typical of chemical synapses. The postsynaptic spherical cells were linked by dendrosomatic gap junctions and were also contacted by unlabeled en passant synaptic boutons, whose fine structure suggested chemical transmission. Electrophysiological studies indicated that spherical cell responsiveness diminished after electrosensory stimulation. This apparently inhibitory phenomenon may be subserved by the unlabeled synaptic boutons, which possibly originate from interneurons that have yet to be identified.

In the motor, somatosensory, and auditory systems of rodents and cats, the corticothalamic connection is composed of a main projection formed by small endings and a minor projection terminating with giant endings. To establish whether the corticothalamic projection originating from motor cortical areas in primates exhibits the same duality, the anterograde tracer biotinylated dextran amine was injected in eight macaque monkeys in the primary motor (M1; n = 3), the supplementary motor (SMA; n = 3) and the dorsal premotor (PMd; n = 2) cortical areas to label corticothalamic axons. The corticothalamic projection originating from these three motor cortical areas was characterized by the presence of axon terminals constituting the same two types of endings, observed both as boutons en passant and terminaux. The population of small endings exhibited a mean cross-sectional maximum diameter of 0.95 microm (S.D. = 0.23), a range of diameters not overlapping that of giant endings (mean diameter = 3.46 microm, S.D. = 0.74 microm). Topographically, the giant endings originating from M1 were located in the same thalamic nucleus (ventroposterolateral nucleus, oral part) in which the small endings were found. In contrast, the giant endings originating from SMA and PMd were located in a thalamic nucleus (mediodorsal nucleus) distinct from the main termination zone formed by small endings. Along the rostrocaudal axis, the giant endings were distributed in a restricted zone, irrespective of the origin of the projection (M1, SMA, PMd). The dual morphology of corticothalamic endings, previously found in rodents and cats, is present in the motor system of subhuman primates for both primary and nonprimary motor cortical areas.

We have examined the morphology of afferent endings that originate in three distinct cell groups and terminate in the lateral posterior nucleus of the thalamus (LP). Retino-LP projections were sparse, occurred throughout the nucleus, and could be classified into 1) simple en passant varicosities and terminal swellings found on poorly branched fibers in all LP subdivisions, 2) string-like configurations of varicosities detected largely in the medial subdivision of the LP, and 3) terminals resembling retinogeniculate endings occurring mainly in the rostral part of the superficial subdivision of the LP adjacent to the dorsal nucleus of the lateral geniculate body. Cortico-LP terminals fell into three classes: 1) single varicosities decorating the tips of short appendages on fine preterminal and terminal axons; 2) tiny, round varicosities studding the axon shaft; and 3) boutons of variable shape visible on medium-caliber corticothalamic fibers. Tecto-LP terminals exhibited a large variation in morphology and density. Those found most commonly could be classified into two groups: 1) individual swellings and 2) terminal clusters arranged in a tubular configuration that enclosed a central channel, most likely occupied by the dendrite of a postsynaptic neuron. An unusual tecto-LP terminal consisted of an ovoid swelling (up to 20 microns in the long axis) from which emerged several long, thin extensions and was seen at the tips of large-diameter axons. These results show that, despite having overlapping projection zones, each set of afferents that projects to the LP elaborates terminal specializations that are structurally distinct from others projecting to the same target area.

We investigated the spatial patterns of synaptic profiles in en passant synapses between the premotor axon of a peripherally synapsing interneuron (PAPSI) and a set of individually identifiable motoneuron axons in Drosophila melanogaster. These synaptic profiles are distributed as the axons travel parallel to each other in a bundle; the synapses begin as the axons leave the thoracic ganglion and continue peripherally for 45-65 microm. We found that the number of synaptic profiles per micron length of the motoneuron axons was greatest close to the ganglion; the cumulative distribution of profiles could be fitted to curves of the form f(x) = alpha(1 - e(-beta x)), where x = the distance from the thoracic ganglion, and alpha and beta are constants. The distribution of synaptic profiles was also examined in a mutant strain, Passover (Pas), known to affect connectivity in a pathway that includes the PAPSI. The synaptic profiles between the PAPSI and the motoneuron axons appeared ultrastructurally unremarkable in Pas. Also, the total number of synaptic profiles between the PAPSI and the motoneuron axons did not differ between Pas and wild type flies. However, the distribution of synaptic profiles among the individual motoneuron axons did differ significantly from wild type flies, as did the area of contiguity between the motoneuron axons and the PAPSI, which was much greater in Pas than in wild type flies.

The distribution and morphology of axons projecting from the medial superior olivary nucleus to the dorsal nucleus of the lateral lemniscus were studied in the adult cat. Injections of Phaseolus vulgaris-leucoagglutinin, biocytin, or dextran-rhodamine in the medial superior olivary nucleus labeled axons that ascended in the lateral lemniscus. Before entering the inferior colliculus, collateral branches of these labeled axons ended in the dorsal nucleus of the lateral lemniscus in thin, horizontal bands forming laminae that extended throughout the rostral-caudal length of the dorsal nucleus of the lateral lemniscus. A dorsal-ventral topography was apparent in the position of the lamina with respect to the injection site, but no relation between the rostral-caudal location of labeled endings and the injection site was observed. There was a divergent pattern of connections within the horizontal laminae rather than a point-to-point organization. The terminal branches of the collateral axons exhibited round or oval boutons en passant and terminaux. Individual arbors reconstructed from serial sections distributed varicosities in circumscribed domains that were only a subcomponent of the area of the afferent laminae in which they were distributed. The spatial relationships of axonal domains of several axons labeled from a single injection in the medial superior olivary nucleus suggest a mosaic pattern in the laminar connections with the dorsal nucleus of the lateral lemniscus.

The lobus parolfactorius (LPO) has been implicated in memory formation associated with passive avoidance training of young posthatch domestic chicks. The anatomical circuitry underlying memory formation in the chick is likely to involve the intermediate medial hyperstriatum ventrale–archistriatum–LPO arc. In the present work, we attempted to combine an ultrastructural characterisation of archistriatal afferent terminals in LPO with a description of the synaptic structure of LPO, in particular those elements that are immunoreactive to glutamate and GABA. Ventral archistriatal regions of 7-day-old domestic chicks were iontophoretically injected with Phaseolus vulgaris leucoagglutinin and the anterograde transport of the tracer was detected in the LPO. Selected samples from these birds, and also from other day-old chicks, were resin-embedded and reacted for l -glutamate or GABA, using the postembedding immunocytochemical method. Glutamate was abundant in the neuropil of LPO and typically seen in axodendritic or axospinous terminals with asymmetrical junctions, often multiple or perforated postsynaptic appositions. Conversely, GABA was often present in aspinous dendrites, probably representing GABAergic local circuit neurons or (putative striatonigral) projection neurons. Archistriatal efferents terminating in LPO formed small en passant or terminal varicosities, with infrequent asymmetrical axospinous synapses. Glutamate was not detected in these boutons. The findings imply that the functional state of LPO, based on powerful glutamatergic excitation, may be modified by a non-glutamatergic archistriatal input.

Corticopulvinar axons were anterogradely labeled by Phaseolus vulgaris-leucoagglutinin injections in the occipitotemporal cortex of the macaque to determine quantitative parameters of divergence and convergence, arbor size and shape, and distribution of terminal specializations. Forty individual axons were analyzed by serial section reconstruction and divided into two major groups. The majority of axons have numerous (typically 500-1,000) small, spinous endings (boutons terminaux). These axons have terminal fields that are beam-like or elongated (E, corresponding to classical type 1) and highly divergent (1.0-3.0 mm). These frequently innervate several of the traditionally designated pulvinar subdivisions; namely inferior pulvinar (PI) and the ventral part of interal pulvinar (PL); medial pulvinar (PM) and dorsal PL, and (one axon) PM, dorsal PL, and PI. Some axons, however (R or round, corresponding to classical type 2), have a small number (typically 70-160) of primarily large, beaded endings (boutons en passant), which concentrate in sharply delimited, round arbors (diameters 100-125 microns). R axons appear to be larger caliber than E axons (1.0-1.5 microns vs. 0.5-1.0 micron, respectively). These differences in phenotype are probably associated with distinct types of projection neurons. In visual areas, corticopulvinar terminations are reported to originate from pyramidal cell subpopulations in layer 5. Indirect evidence, presented here, suggests that the more numerous medium-sized neurons give rise to E axons, and the sparser giant pyramids give rise to R corticopulvinar axons. If this is correct, corticopulvinar connectivity may be involved in multiple transformations. Spatially, axons of giant neurons (with basal dendrites that collect intracortically from a disc-like area, about 1.0 mm in diameter) converge onto a small number of pulvinar neurons. Axons of medium neurons (with basal dendrites that occupy a small intracortical disc, about 0.3 mm in diameter) diverge over 1.0-3.0 mm in the pulvinar and may form many contacts. Giant neurons, although numerically few in relation to medium pyramids (1 or 2: 50?), are likely to have distinctive membrane properties (functionally equivalent to bursting neurons?). Their larger boutons and axon caliber may be associated with a faster transmission that compensates for their small numbers. In primates, the E and R duality does not characterize cortical projections to the caudate, lateral geniculate nucleus, pons, or superior colliculus and thus may be essentially linked to pulvinar-specific processes.

Thalamocortical projections originating from the parafascicular nucleus were reinvestigated using biocytin or biotylinated dextran amine as anterograde tracers in the rat. After stereotaxic injection of the marker in the lateral part of the parafascicular nucleus, labelled ascending fibres were observed running ipsilaterally to the frontal motor and anterior cingulate areas. Labelled fibres gave rise in layer VI to a plexus of thin ramifications ending in layer V, where sparse boutons en passant and terminaux were seen in close apposition to pyramidal cells. Few retrogradely labelled pyramidal somata, contacted by labelled varicosities, were also observed. Electron microscopy demonstrated the synaptic nature of the labelled contacts, displaying asymmetrical junctions and a round vesicular content. The direct loop parafascicular-motor cortex-parafascicular may be of great functional significance in motor control.

The European medicinal leech, Hirudo verbana, is a complex predator with a substantial repertoire of behaviors, most of which can be observed in the isolated nerve cord as “fictive” motor patterns. Each of the 21 midbody segmental ganglia of ~400 neurons is highly iterated and controls an individual body segment. Propagating behaviors (such as swimming, crawling, and shortening) rely on inter-ganglionic communications routed through connective nerves. However, for some observed behaviors, it is not known if the entire behavioral circuit is contained within each individual ganglion or if input from distal ganglia (> 2 segments away) plays a role in the resulting motor action and timing. Specifically in whole-body shortening (WBS), motor output is generated with left-right symmetry and propagates extremely rapidly towards the posterior of the animal, resulting in intense and rapid contraction of the body wall. Using en passant recordings (in which an extracellular electrode records all signals passing a specific location in the connective nerves) with extracellular motor neuron recordings, we have characterized the onset and latencies of WBS as a function of stimulation site. Our results indicate that WBS is primarily initiated locally and radiates outward with a fixed rapid propagation velocity. A possible explanation for the speed of WBS is the recruitment of the fast-conducting S cell network, which is known to conduct signals quickly enough to match the speed of WBS. However, our experiments reveal that lesioning Favre’s nerve to disrupt the S cell network results in a fixed delay past the lesion and no change to the propagation velocity. This suggests that pathways traveling through the lateral connectives may be re-recruiting the S cell network past the lesion. In order to identify the source of these neural signals, we use a novel application of the smoothed nonlinear energy operator to detect spikes and then class them by energy amplitude. The hierarchically classed spikes can then be removed from the data in an iterative process. This preprocessing improves our ability to separate sources in our highly overconstrained system. After separating our sources, superparamagnetic clustering is used to objectively sort spike sources. By measuring the variation in the timing of the sorted spike trains as a function of stimulus location and lesion presence, we hope to localize sources to specific ganglia. We will also use the observed patterns and timings to statistically characterize WBS for future experiments.