Identification of vagal afferent nerve endings in the mouse colon and their spatial relationship with enterochromaffin cells.

Understanding how the gut communicates with the brain, via sensory nerves, is of significant interest to medical science. Enteroendocrine cells (EEC) that line the mucosa of the gastrointestinal tract release neurochemicals, including the largest quantity of 5-hydroxytryptamine (5-HT). How the release of substances, like 5-HT, from enterochromaffin (EC) cells activates vagal afferent nerve endings is unresolved. We performed anterograde labelling from nodose ganglia in vivo and identified vagal afferent axons and nerve endings in the mucosa of whole-mount full-length preparations of mouse colon. We then determined the spatial relationship between mucosal-projecting vagal afferent nerve endings and EC cells in situ using 3D imaging. The mean distances between vagal afferent nerve endings in the mucosa, or nearest varicosities along vagal afferent axon branches, and the nearest EC cell were 29.6 ± 19.2 µm (n = 107, N = 6) and 25.7 ± 15.2 µm (n = 119, N = 6), respectively. No vagal afferent endings made close contacts with EC cells. The distances between EC cells and vagal afferent endings are many hundreds of times greater than known distances between pre- and post-synaptic membranes (typically 10-20 nm) that underlie synaptic transmission in vertebrates. The absence of any close physical contacts between 5-HT-containing EC cells and vagal afferent nerve endings in the mucosa leads to the inescapable conclusion that the mechanism by which 5-HT release from ECs in the colonic mucosa occurs in a paracrine fashion, to activate vagal afferents.

Multiple Posterior Insula Projections to the Brainstem Descending Pain Modulatory System.

The insular cortex is an important hub for sensory and emotional integration. It is one of the areas consistently found activated during pain. While the insular's connections to the limbic system might play a role in the aversive and emotional component of pain, its connections to the descending pain system might be involved in pain intensity coding. Here, we used anterograde tracing with viral expression of mCherry fluorescent protein, to examine the connectivity of insular axons to different brainstem nuclei involved in the descending modulation of pain in detail. We found extensive connections to the main areas of descending pain control, namely, the periaqueductal gray (PAG) and the raphe magnus (RMg). In addition, we also identified an extensive insular connection to the parabrachial nucleus (PBN). Although not as extensive, we found a consistent axonal input from the insula to different noradrenergic nuclei, the locus coeruleus (LC), the subcoereuleus (SubCD) and the A5 nucleus. These connections emphasize a prominent relation of the insula with the descending pain modulatory system, which reveals an important role of the insula in pain processing through descending pathways.

The gut-brain axis: spatial relationship between spinal afferent nerves and 5-HT-containing enterochromaffin cells in mucosa of mouse colon.

Cross talk between the gastrointestinal tract and brain is of significant relevance for human health and disease. However, our understanding of how the gut and brain communicate has been limited by a lack of techniques to identify the precise spatial relationship between extrinsic nerve endings and their proximity to specific cell types that line the inner surface of the gastrointestinal tract. We used an in vivo anterograde tracing technique, previously developed in our laboratory, to selectively label single spinal afferent axons and their nerve endings in mouse colonic mucosa. The closest three-dimensional distances between spinal afferent nerve endings and axonal varicosities to enterochromaffin (EC) cells, which contain serotonin (5-hydroxytryptamine; 5-HT), were then measured. The mean distances (± standard deviation) between any varicosity along a spinal afferent axon or its nerve ending, and the nearest EC cell, were 5.7 ± 6.0 µm (median: 3.6 µm) and 26.9 ± 18.6 µm (median: 24.1 µm), respectively. Randomization of the spatial location of EC cells revealed similar results to this actual data. These distances are ∼200-1,000 times greater than those between pre- and postsynaptic membranes (15-25 nm) that underlie synaptic transmission in the vertebrate nervous system. Our findings suggest that colonic 5-HT-containing EC cells release substances to activate centrally projecting spinal afferent nerves likely via diffusion, as such signaling is unlikely to occur with the spatial fidelity of a synapse.NEW & NOTEWORTHY We show an absence of close physical contact between spinal afferent nerves and 5-HT-containing EC cells in mouse colonic mucosa. Similar relative distances were observed between randomized EC cells and spinal afferents compared with actual data. This spatial relationship suggests that substances released from colonic 5-HT-containing EC cells are unlikely to act via synaptic transmission to neighboring spinal afferents that relay sensory information from the gut lumen to the brain.

Reconsidering the Border between the Visual and Posterior Parietal Cortex of Mice.

The posterior parietal cortex (PPC) contributes to multisensory and sensory-motor integration, as well as spatial navigation. Based on primate studies, the PPC is composed of several subdivisions with differing connection patterns, including areas that exhibit retinotopy. In mice the composition of the PPC is still under debate. We propose a revised anatomical delineation in which we classify the higher order visual areas rostrolateral area (RL), anteromedial area (AM), and Medio-Medial-Anterior cortex (MMA) as subregions of the mouse PPC. Retrograde and anterograde tracing revealed connectivity, characteristic for primate PPC, with sensory, retrosplenial, orbitofrontal, cingulate and motor cortex, as well as with several thalamic nuclei and the superior colliculus in the mouse. Regarding cortical input, RL receives major input from the somatosensory barrel field, while AM receives more input from the trunk, whereas MMA receives strong inputs from retrosplenial, cingulate, and orbitofrontal cortices. These input differences suggest that each posterior PPC subregion may have a distinct function. Summarized, we put forward a refined cortical map, including a mouse PPC that contains at least 6 subregions, RL, AM, MMA and PtP, MPta, LPta/A. These anatomical results set the stage for a more detailed understanding about the role that the PPC and its subdivisions play in multisensory integration-based behavior in mice.

Changes of motor corticobulbar projections following different lesion types affecting the central nervous system in adult macaque monkeys.

Functional recovery from central nervous system injury is likely to be partly due to a rearrangement of neural circuits. In this context, the corticobulbar (corticoreticular) motor projections onto different nuclei of the ponto-medullary reticular formation (PMRF) were investigated in 13 adult macaque monkeys after either, primary motor cortex injury (MCI) in the hand area, or spinal cord injury (SCI) or Parkinson's disease-like lesions of the nigro-striatal dopaminergic system (PD). A subgroup of animals in both MCI and SCI groups was treated with neurite growth promoting anti-Nogo-A antibodies, whereas all PD animals were treated with autologous neural cell ecosystems (ANCE). The anterograde tracer BDA was injected either in the premotor cortex (PM) or in the primary motor cortex (M1) to label and quantify corticobulbar axonal boutons terminaux and en passant in PMRF. As compared to intact animals, after MCI the density of corticobulbar projections from PM was strongly reduced but maintained their laterality dominance (ipsilateral), both in the presence or absence of anti-Nogo-A antibody treatment. In contrast, the density of corticobulbar projections from M1 was increased following opposite hemi-section of the cervical cord (at C7 level) and anti-Nogo-A antibody treatment, with maintenance of contralateral laterality bias. In PD monkeys, the density of corticobulbar projections from PM was strongly reduced, as well as that from M1, but to a lesser extent. In conclusion, the densities of corticobulbar projections from PM or M1 were affected in a variable manner, depending on the type of lesion/pathology and the treatment aimed to enhance functional recovery.

EMX1 regulates NRP1-mediated wiring of the mouse anterior cingulate cortex.

Transcription factors act during cortical development as master regulatory genes that specify cortical arealization and cellular identities. Although numerous transcription factors have been identified as being crucial for cortical development, little is known about their downstream targets and how they mediate the emergence of specific neuronal connections via selective axon guidance. The EMX transcription factors are essential for early patterning of the cerebral cortex, but whether EMX1 mediates interhemispheric connectivity by controlling corpus callosum formation remains unclear. Here, we demonstrate that in mice on the C57Bl/6 background EMX1 plays an essential role in the midline crossing of an axonal subpopulation of the corpus callosum derived from the anterior cingulate cortex. In the absence of EMX1, cingulate axons display reduced expression of the axon guidance receptor NRP1 and form aberrant axonal bundles within the rostral corpus callosum. EMX1 also functions as a transcriptional activator of Nrp1 expression in vitro, and overexpression of this protein in Emx1 knockout mice rescues the midline-crossing phenotype. These findings reveal a novel role for the EMX1 transcription factor in establishing cortical connectivity by regulating the interhemispheric wiring of a subpopulation of neurons within the mouse anterior cingulate cortex.

Parahippocampal and retrosplenial connections of rat posterior parietal cortex.

The posterior parietal cortex has been implicated in spatial functions, including navigation. The hippocampal and parahippocampal region and the retrosplenial cortex are crucially involved in navigational processes and connections between the parahippocampal/retrosplenial domain and the posterior parietal cortex have been described. However, an integrated account of the organization of these connections is lacking. Here, we investigated parahippocampal connections of each posterior parietal subdivision and the neighboring secondary visual cortex using conventional retrograde and anterograde tracers as well as transsynaptic retrograde tracing with a modified rabies virus. The results show that posterior parietal as well as secondary visual cortex entertain overall sparse connections with the parahippocampal region but not with the hippocampal formation. The medial and lateral dorsal subdivisions of posterior parietal cortex receive sparse input from deep layers of all parahippocampal areas. Conversely, all posterior parietal subdivisions project moderately to dorsal presubiculum, whereas rostral perirhinal cortex, postrhinal cortex, caudal entorhinal cortex and parasubiculum all receive sparse posterior parietal input. This indicated that the presubiculum might be a major liaison between parietal and parahippocampal domains. In view of the close association of the presubiculum with the retrosplenial cortex, we included the latter in our analysis. Our data indicate that posterior parietal cortex is moderately connected with the retrosplenial cortex, particularly with rostral area 30. The relative sparseness of the connectivity with the parahippocampal and retrosplenial domains suggests that posterior parietal cortex is only a modest actor in forming spatial representations underlying navigation and spatial memory in parahippocampal and retrosplenial cortex. © 2017 Wiley Periodicals, Inc.

Corticobulbar projections from distinct motor cortical areas to the reticular formation in macaque monkeys.

Corticospinal and corticobulbar descending pathways act in parallel with brainstem systems, such as the reticulospinal tract, to ensure the control of voluntary movements via direct or indirect influences onto spinal motoneurons. The aim of this study was to investigate the corticobulbar projections from distinct motor cortical areas onto different nuclei of the reticular formation. Seven adult macaque monkeys were analysed for the location of corticobulbar axonal boutons, and one monkey for reticulospinal neurons' location. The anterograde tracer BDA was injected in the premotor cortex (PM), in the primary motor cortex (M1) or in the supplementary motor area (SMA), in 3, 3 and 1 monkeys respectively. BDA anterograde labelling of corticobulbar axons were analysed on brainstem histological sections and overlapped with adjacent Nissl-stained sections for cytoarchitecture. One adult monkey was analysed for retrograde CB tracer injected in C5-C8 hemispinal cord to visualise reticulospinal neurons. The corticobulbar axons formed bilateral terminal fields with boutons terminaux and en passant, which were quantified in various nuclei belonging to the Ponto-Medullary Reticular Formation (PMRF). The corticobulbar projections from both PM and SMA tended to end mainly ipsilaterally in PMRF, but contralaterally when originating from M1. Furthermore, the corticobulbar projection was less dense when originating from M1 than from non-primary motor areas (PM, SMA). The main nuclei of bouton terminals corresponded to the regions where reticulospinal neurons were located with CB retrograde tracing. In conclusion, the corticobulbar projection differs according to the motor cortical area of origin in density and laterality.

Ipsilateral corticotectal projections from the primary, premotor and supplementary motor cortical areas in adult macaque monkeys: a quantitative anterograde tracing study.

The corticotectal projection from cortical motor areas is one of several descending pathways involved in the indirect control of spinal motoneurons. In non-human primates, previous studies reported that cortical projections to the superior colliculus (SC) originated from the premotor cortex (PM) and the primary motor cortex, whereas no projection originated from the supplementary motor area (SMA). The aim of the present study was to investigate and compare the properties of corticotectal projections originating from these three cortical motor areas in intact adult macaques (n = 9). The anterograde tracer biotinylated dextran amine was injected into one of these cortical areas in each animal. Individual axonal boutons, both en passant and terminaux, were charted and counted in the different layers of the ipsilateral SC. The data confirmed the presence of strong corticotectal projections from the PM. A new observation was that strong corticotectal projections were also found to originate from the SMA (its proper division). The corticotectal projection from the primary motor cortex was quantitatively less strong than that from either the premotor or SMAs. The corticotectal projection from each motor area was directed mainly to the deep layer of the SC, although its intermediate layer was also a consistent target of fairly dense terminations. The strong corticotectal projections from non-primary motor areas are in position to influence the preparation and planning of voluntary movements.

Synaptic contact between median preoptic neurons and subfornical organ neurons projecting to the paraventricular hypothalamic nucleus.

It is known that the median preoptic nucleus (POMe) sends dense projections to the subfornical organ (SFO). However, the functional significance of these projections have not been well discussed. In this electron microscopic study, we investigated the presence of synapses between POMe-derived axon terminals and SFO neurons that project to the paraventricular hypothalamic nucleus (PVN). After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex, into the PVN, many labeled neurons were found in the SFO. In contrast, after injection of an anterograde tracer, biotinylated dextran amine, in the POMe, abundant labeled axon varicosities were observed in the SFO. Using electron microscopy, synapses were identified between retrogradely labeled dendrites and cell bodies, and anterogradely labeled axon terminals, indicating that POMe neurons innervate SFO neurons projecting to the PVN. The possibility that POMe neurons play multiple roles in the neuronal circuit responsible for vasopressin release and/or cardiovascular regulation is also discussed.

Efferent Axonal Projections of the Habenular Complex in the Fire-Bellied Toad Bombina orientalis.

The habenular complex and its associated axonal pathways are often thought of as phylogenetically conserved features of the brain among vertebrates despite the fact that detailed studies of this brain region are limited to a few species. Here, the gross morphology and axonal projection pattern of the habenular complex of an anuran amphibian, the fire-bellied toad Bombina orientalis, was studied to allow comparison with the situation in other vertebrates. Axonal pathways were traced using biocytin applications in dissected brain preparations. The results show that the rostral part of the left dorsal nucleus is enlarged in this species, while the rostral ventral nucleus and caudal parts do not show left-right size differences. Biocytin applications revealed widespread axonal projections of the habenular complex to the posterior tuberculum/dorsal hypothalamic region, ventral tegmentum, interpeduncular nucleus (IPN), and raphe median. Additionally, axons targeting the lateral hypothalamus originated from the ventral habenular nuclei. The results also suggest an asymmetrical pattern of projection to the IPN in the rostral part of the habenular complex, where the left habenula preferentially targeted the dorsal IPN while the right habenula preferentially targeted the ventral IPN. The caudal habenular nuclei showed no asymmetry of projections as both sides targeted the ventral IPN. Comparison of the habenular complex axonal connectivity across vertebrates argues against strong phylogenetic conservation of the axonal projection patterns of different habenular nuclei.

Mechanisms underlying the gut-brain communication: How enterochromaffin (EC) cells activate vagal afferent nerve endings in the small intestine.

How the gastrointestinal tract communicates with the brain, via sensory nerves, is of significant interest for our understanding of human health and disease. Enterochromaffin (EC) cells in the gut mucosa release a variety of neurochemicals, including the largest quantity of 5-hydroxytryptamine (5-HT) in the body. How 5-HT and other substances released from EC cells activate sensory nerve endings in the gut wall remains a major unresolved mystery. We used in vivo anterograde tracing from nodose ganglia to determine the spatial relationship between 5-HT synthesizing and peptide-YY (PYY)-synthesizing EC cells and their proximity to vagal afferent nerve endings that project to the mucosa of mouse small intestine. The shortest mean distances between single 5-HT- and PYY-synthesizing EC cells and the nearest vagal afferent nerve endings in the mucosa were 33.1 ± 14.4 µm (n = 56; N = 6) and 70.3 ± 32.3 µm (n = 16; N = 6). No morphological evidence was found to suggest that 5-HT- or PYY-containing EC cells form close morphological associations with vagal afferents endings, or varicose axons of passage. The large distances between EC cells and vagal afferent endings are many hundreds of times greater than those known to underlie synaptic transmission in the nervous system (typically 10-15 nm). Taken together, the findings lead to the inescapable conclusion that communication between 5-HT-containing EC cells and vagal afferent nerve endings in the mucosa of the mouse small intestinal occurs in a paracrine fashion, via diffusion. New and Noteworthy None of the findings here are consistent with a view that close physical contacts occur between 5-HT-containing EC cells and vagal afferent nerve endings in mouse small intestine. Rather, the findings suggest that gut-brain communication between EC cells and vagal afferent endings occurs via passive diffusion. The morphological data presented do not support the view that EC cells are physically close enough to vagal afferent endings to communicate via fast synaptic transmission.

The discovery of a new type of innervation in lymphoid organs.

It is well known that the main forms of innervation are synapses and free nerve endings, while other forms of innervation have not been reported. Here, we explore a new way of innervating lymphoid organs. Male Sprague-Dawley rats were used for studying the innervation of sympathetic nerve fibers in lymph nodes by means of anterograde tracking, immunoelectron microscopy, three-dimension reconstruction analysis, and immunofluorescence labeling. The results showed that the Fluoro-Ruby labeled nerve endings targeted only a group of cells in the lymph nodes and entered the cells through the plasma membrane. The electron microscopy showed that the biotinylated glucan amine reaction elements were distributed in the cytoplasm, and most of the biotinylated glucan amine active elements were concentrated on the microtubule and microfilament walls. Birbeck particles with rod-shaped and/or tennis racket like structures can be seen in the labeled cells at high magnification, and Birbeck particles contain biotinylated glucan amine-reactive elements. The immunofluoresence results showed that the Fluoro-Ruby-labeled nerve innervating cells expressed CD207 and CD1a protein. This result confirmed that the labeled cells were Langerhans cells. Our findings suggested that Langerhans cells might serve as a "bridge cell" for neuroimmune cross-talking in lymph organs, which play an important role in transmitting signals of the nervous system to immune system. This study also opened up a new way for further study of immune regulation mechanism.

Hippocampal-medial entorhinal circuit is differently organized along the dorsoventral axis in rodents.

The general understanding of hippocampal circuits is that the hippocampus and the entorhinal cortex (EC) are topographically connected through parallel identical circuits along the dorsoventral axis. Our anterograde tracing and in vitro electrophysiology data, however, show a markedly different dorsoventral organization of the hippocampal projection to the medial EC (MEC). While dorsal hippocampal projections are confined to the dorsal MEC, ventral hippocampal projections innervate both dorsal and ventral MEC. Further, whereas the dorsal hippocampus preferentially targets layer Vb (LVb) neurons, the ventral hippocampus mainly targets cells in layer Va (LVa). This connectivity scheme differs from hippocampal projections to the lateral EC, which are topographically organized along the dorsoventral axis. As LVa neurons project to telencephalic structures, our findings indicate that the ventral hippocampus regulates LVa-mediated entorhinal-neocortical output from both dorsal and ventral MEC. Overall, the marked dorsoventral differences in hippocampal-entorhinal connectivity impose important constraints on signal flow in hippocampal-neocortical circuits.

Regional and cell-type-specific afferent and efferent projections of the mouse claustrum.

The claustrum (CLA) is a conspicuous subcortical structure interconnected with cortical and subcortical regions. Its regional anatomy and cell-type-specific connections in the mouse remain not fully determined. Using multimodal reference datasets, we confirmed the delineation of the mouse CLA as a single group of neurons embedded in the agranular insular cortex. We quantitatively investigated brain-wide inputs and outputs of CLA using bulk anterograde and retrograde viral tracing data and single neuron tracing data. We found that the prefrontal module has more cell types projecting to the CLA than other cortical modules, with layer 5 IT neurons predominating. We found nine morphological types of CLA principal neurons that topographically innervate functionally linked cortical targets, preferentially the midline cortical areas, secondary motor area, and entorhinal area. Together, this study provides a detailed wiring diagram of the cell-type-specific connections of the mouse CLA, laying a foundation for studying its functions at the cellular level.

BoutonNet: an automatic method to detect anterogradely labeled presynaptic boutons in brain tissue sections.

Neurons emit axons, which form synapses, the fundamental unit of the nervous system. Neuroscientists use genetic anterograde tracing methods to label the synaptic output of specific neuronal subpopulations, but the resulting data sets are too large for manual analysis, and current automated methods have significant limitations in cost and quality. In this paper, we describe a pipeline optimized to identify anterogradely labeled presynaptic boutons in brain tissue sections. Our histologic pipeline labels boutons with high sensitivity and low background. To automatically detect labeled boutons in slide-scanned tissue sections, we developed BoutonNet. This detector uses a two-step approach: an intensity-based method proposes possible boutons, which are checked by a neural network-based confirmation step. BoutonNet was compared to expert annotation on a separate validation data set and achieved a result within human inter-rater variance. This open-source technique will allow quantitative analysis of the fundamental unit of the brain on a whole-brain scale.

Failure of AAV retrograde tracer transduction in hypothalamic projections to the periaqueductal gray matter.

Connectomics is an important field of neuroscience that examines how neurons are connected and form functional circuits that underly the brain's functions. Conventional tracers based on dye have led to great advances in mapping these connections, and now, neurotropic viruses are contributing to connectomics. In this work, two retrograde adeno-associated virus failed to transduce in projections from hypothalamic neurons to periaqueductal gray matter (PAG) but worked well in cortical connections to PAG. One of this virus also marked a substantial amount of PAG efferent projections, therefore working as an anterograde tracer. We also used hydroxystilbamidine (FluoroGold™) as a gold standard in retrograde tracing for comparison with the projections shown by the retrograde virus. As determined in past works, FluoroGold™ shows connections from the hypothalamus and cortex to the PAG. Also, an anterograde AAV was compared with one of the retrograde AAV, which showed a similar pattern of axonal projections and terminal fields. Hence, although neurotropic viruses are revolutionizing connectomics and other areas, their mechanism, neurotropism, and cell invasion need to be addressed. Their use is a great challenge and requires further studies to clarify their interaction with the nervous system's cells.

Whole-brain monosynaptic inputs and outputs of glutamatergic neurons of the vestibular nuclei complex in mice.

Vestibular nuclei complex (VN) glutamatergic neurons play a critical role in the multisensory and multimodal processing. The dysfunction of VN leads to a series of vestibular concurrent symptoms, such as disequilibrium, spatial disorientation, autonomic disorders and even emotion disorders. However, the reciprocal neural connectivity in the whole brain of VN glutamatergic neurons was incompletely understood. Here, we employed a cell-type-specific, cre-dependent, modified virus vector to retrogradely and anterogradely trace VN glutamatergic neurons in the VGLUT2-IRES-Cre mouse line. We identified and analyzed statistically the afferents and efferents of VN glutamatergic neurons in the whole brain, and also reconstructed monosynaptic inputs distribution of VN glutamatergic neurons at the three-dimensional level with the combination of a fluorescence micro-optical sectioning tomography system (fMOST). We found that VN glutamatergic neurons primarily received afferents from 57 nuclei and send efferents to 59 nuclei in the whole brain, intensively located in the brainstem and cerebellum. Projections from nuclei in the cerebellum targeting VN glutamatergic neurons mainly performed the balance control - the principal function of the vestibular system. In addition, VN glutamatergic neurons sent projections to oculomotor nucleus, trochlear nucleus and abducens nucleus dominating the eye movement. Except for the maintenance of balance, VN glutamatergic neurons were also directly connected with other functional regions, such as sleep-wake state (locus coeruleus, dorsal raphe nucleus, and laterodorsal tegmental nucleus, gigantocellular reticular nucleus, lateral paragigantocellular nucleus, periaqueductal gray, subcoeruleus nucleus, parvicellular reticular nucleus, paramedian raphe nucleus), and emotional regulation (locus coeruleus and dorsal raphe nucleus). Hence, this study revealed a comprehensive whole-brain neural connectivity of VN glutamatergic neurons and provided with a neuroanatomic foundation to further study on central vestibular circuits.

Connections of the mouse subfornical region of the lateral hypothalamus (LHsf).

The lateral hypothalamus is a major integrative hub with a complex architecture characterized by intricate and overlapping cellular populations expressing a large variety of neuro-mediators. In rats, the subfornical lateral hypothalamus (LHsf) was identified as a discrete area with very specific outputs, receiving a strong input from the nucleus incertus, and involved in defensive and foraging behaviors. We identified in the mouse lateral hypothalamus a discrete subfornical region where a conspicuous cluster of neurons express the mu opioid receptor. We thus examined the inputs and outputs of this LHsf region in mice using retrograde tracing with the cholera toxin B subunit and anterograde tracing with biotin dextran amine, respectively. We identified a connectivity profile largely similar, although not identical, to what has been described in rats. Indeed, the mouse LHsf has strong reciprocal connections with the lateral septum, the ventromedial hypothalamic nucleus and the dorsal pre-mammillary nucleus, in addition to a dense output to the lateral habenula. However, the light input from the nucleus incertus and the moderate bidirectional connectivity with nucleus accumbens are specific to the mouse LHsf. A preliminary neurochemical study showed that LHsf neurons expressing mu opioid receptors also co-express calcitonin gene-related peptide or somatostatin and that the reciprocal connection between the LHsf and the lateral septum may be functionally modulated by enkephalins acting on mu opioid receptors. These results suggest that the mouse LHsf may be hodologically and functionally comparable to its rat counterpart, but more atypical connections also suggest a role in consummatory behaviors.

Influence of Lysine and TRITC Conjugation on the Size and Structure of Dextran Nanoconjugates with Potential for Biomolecule Delivery to Neurons.

As a potent nonviral system for biomolecular delivery to neurons via their axons, we have studied molecular characteristics of lysinated fluorescent dextran nanoconjugates with degrees of conjugation of 0.54-15.2 mol lysine and 0.25-7.27 mol tetramethyl rhodamine isothiocyanate (TRITC) per mol dextran. We studied the influence of conjugation with lysine and TRITC on the size and structure of different molecular weight dextrans and their mobility within axons. Dynamic light scattering (DLS) and small-angle neutron scattering (SANS) experiments revealed significant differences in the size and structure of unmodified and modified dextrans. Unexpectedly, lower-molecular-weight conjugated dextrans exhibited higher molecular volumes, which we propose is due to fewer intramolecular interactions than in higher-molecular-weight conjugated dextrans. Assessment of retrograde and anterograde movement of lysine- and TRITC-conjugated dextrans in axons in the lumbar spinal cord of chicken embryos showed that lower-molecular-weight dextrans translocate more efficiently than higher-molecular-weight dextrans, despite having larger molecular volumes. This comparative characterization of different molecular weight dextrans will help define optimal features for intracellular delivery.