Localization of substance P-like and enkephalin-like immunoreactivity within preganglionic terminals of the avian ciliary ganglion: light and electron microscopy

The avian ciliary ganglion receives its only recognized input from the nucleus of Edinger-Westphal. This is known to be a cholinergic input. In the present study, using fluorescein isothiocyanate and peroxidase- antiperoxidase immunohistochemical methods, substance P-like and enkephalin-like immunoreactivity has been found within preganglionic terminals of the avian ciliary ganglion. The ciliary ganglion is known to consist of two distinct cell populations: small choroid cells that project to the smooth muscle coat of the choroid and large ciliary neurons that send axons to both the iris and the ciliary body. Preganglionic terminals on choroid cells consist of small boutonal endings, whereas ciliary neurons receive a calyx-like cap ending around the hilus of the cell. Substance P-like and enkephalin-like immunoreactivity was localized to preganglionic axons and to both boutonal and calyx-like terminations upon cells of the ciliary ganglion. Electron microscopic studies of both substance P-like and enkephalin-like immunoreactive terminals revealed small clear core vesicles (approximately 58 nm in diameter) and two sizes of dense core vesicles (approximately 85 and approximately 119 nm in diameter). Immunoreactive staining was observed only in the smaller dense core vesicles. The unlabeled clear core vesicles were clustered at synaptic release sites, while the immunoreactive and larger unlabeled dense core vesicles usually were not near these synaptic specializations. These observations strongly imply that neuropeptides co-occur with acetylcholine in preganglionic axons of the ciliary ganglion

Neurotransmitter organization of the nucleus of Edinger-Westphal and its projection to the avian ciliary ganglion

Two morphologically distinct types of preganglionic endings are observed in the avian ciliary ganglion: boutonal and cap-like. Boutonal endings synapse on ciliary ganglion neurons (called choroidal neurons) innervating choroidal blood vessels, while cap-like endings synapse on ciliary ganglion neurons (called ciliary neurons) controlling the lens and pupil. Some of both types of preganglionic endings contain the neuropeptides substance P (SP) and/or leucine-enkephalin (LENK). Although both types of preganglionic terminals are also known to be cholinergic, there has been no direct evidence that SP and LENK are found in cholinergic endings in the ciliary ganglion. The present studies in pigeons, which involved the use of single- and double-label immunohistochemical techniques, were undertaken to examine this issue, as well as to (1) determine the relative percentages of the boutonal and cap-like endings that contain SP, LENK, or both SP and LENK; and (2) determine if the two different types of terminals in the ciliary ganglion arise from different subdivisions of the nucleus of Edinger-Westphal (EW). Single- and double-label immunohistochemical studies revealed that all neurons of EW, regardless of whether they contained immunohistochemically detectible amounts of SP or LENK, are cholinergic. In the medial subdivision of EW (EWM), which was found to contain approximately 700 neurons, 20.2% of these neurons were observed to contain both SP and LENK, while 11.6% were observed to contain SP only and 10.7% were observed to contain LENK only. In contrast, in lateral EW (EWL), which was found to contain approximately 500 neurons, 16.2% of the neurons were observed to contain both SP and LENK, while 19.2% of the neurons were observed to contain SP only and 12.6% were observed to contain LENK only. Retrograde-labeling studies involving horseradish peroxidase injections into the ciliary ganglion revealed that EW was the sole source of input to the ciliary ganglion and all, or nearly all, neurons in EW innervate the ciliary ganglion. Immunohistochemical labeling of the ciliary ganglion neurons with an antiserum against choline acetyltransferase revealed that approximately 900 choroidal neurons and approximately 600 ciliary neurons are present in the ganglion, all of which receive cholinergic preganglionic endings. Of the choroidal neurons, 94% receive butonal terminals containing both SP and LENK, while only 2% receive SP+ only boutonal endings and 2% receive LENK+ only butonal endings. Of the ciliary neurons, 25% receive cap-like endings containing both SP and LENK, 30% receive cap-like endings containing only SP and 3% receive cap-like endings containing only LENK. Total unilateral lesions of EW resulted in the loss of all SP+ or LENK+ terminals in the ipsilateral ganglion. Subtotal EW lesions that spared either part of EWM or part of EWL revealed that boutonal endings arise from EWM neurons and cap-like endings from EWL neurons. The present results suggest that the choroidal neurons, which regulate choroidal blood flow, may be relatively uniform in their functional properties since they nearly all receive boutonal endings from EWM that co-contain SP, LENK, and acetylcholine. In contrast, the ciliary neurons, which receive their preganglionic input from EWL, may consist of at least three major functionally distinct subgroups: (1) those receiving SP/LENK/acetylcholine-containing cap-like endings; (2) those receiving SP/acetylcholine-containing cap-like endings; and (3) those receiving acetylcholine-containing cap-like endings. The functional diversity of ciliary neurons may in part be related to the fact that some ciliary neurons innervate the iris and others the ciliary body

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.Comment: 41 page

Motion processing with wide-field neurons in the retino-tecto-rotundal pathway

The retino-tecto-rotundal pathway is the main visual pathway in non-mammalian vertebrates and has been found to be highly involved in visual processing. Despite the extensive receptive fields of tectal and rotundal wide-field neurons, pattern discrimination tasks suggest a system with high spatial resolution. In this paper, we address the problem of how global processing performed by motion-sensitive wide-field neurons can be brought into agreement with the concept of a local analysis of visual stimuli. As a solution to this problem, we propose a firing-rate model of the retino-tecto-rotundal pathway which describes how spatiotemporal information can be organized and retained by tectal and rotundal wide-field neurons while processing Fourier-based motion in absence of periodic receptive-field structures. The model incorporates anatomical and electrophysiological experimental data on tectal and rotundal neurons, and the basic response characteristics of tectal and rotundal neurons to moving stimuli are captured by the model cells. We show that local velocity estimates may be derived from rotundal-cell responses via superposition in a subsequent processing step. Experimentally testable predictions which are both specific and characteristic to the model are provided. Thus, a conclusive explanation can be given of how the retino-tecto-rotundal pathway enables the animal to detect and localize moving objects or to estimate its self-motion parameters

Subdivisions of the Auditory Midbrain (N. Mesencephalicus Lateralis, pars dorsalis) in Zebra Finches Using Calcium-Binding Protein Immunocytochemistry

The midbrain nucleus mesencephalicus lateralis pars dorsalis (MLd) is thought to be the avian homologue of the central nucleus of the mammalian inferior colliculus. As such, it is a major relay in the ascending auditory pathway of all birds and in songbirds mediates the auditory feedback necessary for the learning and maintenance of song. To clarify the organization of MLd, we applied three calcium binding protein antibodies to tissue sections from the brains of adult male and female zebra finches. The staining patterns resulting from the application of parvalbumin, calbindin and calretinin antibodies differed from each other and in different parts of the nucleus. Parvalbumin-like immunoreactivity was distributed throughout the whole nucleus, as defined by the totality of the terminations of brainstem auditory afferents; in other words parvalbumin-like immunoreactivity defines the boundaries of MLd. Staining patterns of parvalbumin, calbindin and calretinin defined two regions of MLd: inner (MLd.I) and outer (MLd.O). MLd.O largely surrounds MLd.I and is distinct from the surrounding intercollicular nucleus. Unlike the case in some non-songbirds, however, the two MLd regions do not correspond to the terminal zones of the projections of the brainstem auditory nuclei angularis and laminaris, which have been found to overlap substantially throughout the nucleus in zebra finches

Age and Diet Affect Gene Expression Profiles in Canine Liver Tissue

BACKGROUND: The liver plays a central role in nutrient and xenobiotic metabolism, but its functionality declines with age. Senior dogs suffer from many of the chronic hepatic diseases as elderly humans, with age-related alterations in liver function influenced by diet. However, a large-scale molecular analysis of the liver tissue as affected by age and diet has not been reported in dogs. METHODOLOGY/PRINCIPAL FINDINGS: Liver tissue samples were collected from six senior (12-year old) and six young adult (1-year old) female beagles fed an animal protein-based diet (APB) or a plant protein-based diet (PPB) for 12 months. Total RNA in the liver tissue was extracted and hybridized to Affymetrix GeneChip® Canine Genome Arrays. Using a 2.0-fold cutoff and false discovery rate <0.10, our results indicated that expression of 234 genes was altered by age, while 137 genes were differentially expressed by diet. Based on functional classification, genes affected by age and/or diet were involved in cellular development, nutrient metabolism, and signal transduction. In general, gene expression suggested that senior dogs had an increased risk of the progression of liver disease and dysfunction, as observed in aged humans and rodents. In particular for aged liver, genes related to inflammation, oxidative stress, and glycolysis were up-regulated, whereas genes related to regeneration, xenobiotic metabolism, and cholesterol trafficking were down-regulated. Diet-associated changes in gene expression were more common in young adult dogs (33 genes) as compared to senior dogs (3 genes). CONCLUSION: Our results provide molecular insight pertaining to the aged canine liver and its predisposition to disease and abnormalities. Therefore, our data may aid in future research pertaining to age-associated alterations in hepatic function or identification of potential targets for nutritional management as a means to decrease incidence of age-dependent liver dysfunction

Fluorescent Labeling of Newborn Dentate Granule Cells in GAD67-GFP Transgenic Mice: A Genetic Tool for the Study of Adult Neurogenesis

Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP+ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP+ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber) terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells

When Are New Hippocampal Neurons, Born in the Adult Brain, Integrated into the Network That Processes Spatial Information?

Adult-born neurons in the dentate gyrus (DG) functionally integrate into the behaviorally relevant hippocampal networks, showing a specific Arc-expression response to spatial exploration when mature. However, it is not clear when, during the 4- to 6-week interval that is critical for survival and maturation of these neurons, this specific response develops. Therefore, we characterized Arc expression after spatial exploration or cage control conditions in adult-born neurons from rats that were injected with BrdU on one day and were sacrificed 1, 7, 15, 30, and 45 days post-BrdU injection (PBI). Triple immunostaining for NeuN, Arc, and BrdU was analyzed through the different DG layers. Arc protein expression in BrdU-positive cells was observed from day 1 to day 15 PBI but was not related to behavioral stimulation. The specific Arc-expression response to spatial exploration was observed from day 30 and 45 in about 5% of the BrdU-positive cell population. Most of the BrdU-positive neurons expressing Arc in response to spatial exploration (∼90%) were located in DG layer 1, and no Arc expression was observed in cells located in the subgranular zone (SGZ). Using the current data and that obtained previously, we propose a mathematical model suggesting that new neurons are unlikely to respond to exploration by expressing Arc after they are 301 days old, and also that in a 7-month-old rat the majority (60%) of the neurons that respond to exploration must have been born during adulthood; thus, suggesting that adult neurogenesis in the DG is highly relevant for spatial information processing

Caveats of chronic exogenous corticosterone treatments in adolescent rats and effects on anxiety-like and depressive behavior and hypothalamic-pituitary-adrenal (HPA) axis function

<p>Abstract</p> <p>Background</p> <p>Administration of exogenous corticosterone is an effective preclinical model of depression, but its use has involved primarily adult rodents. Using two different procedures of administration drawn from the literature, we explored the possibility of exogenous corticosterone models in adolescence, a time of heightened risk for mood disorders in humans.</p> <p>Methods</p> <p>In experiment 1, rats were injected with 40 mg/kg corticosterone or vehicle from postnatal days 30 to 45 and compared with no injection controls on behavior in the elevated plus maze (EPM) and the forced swim test (FST). Experiment 2 consisted of three treatments administered to rats from postnatal days 30 to 45 or as adults (days 70 to 85): either corticosterone (400 μg/ml) administered in the drinking water along with 2.5% ethanol, 2.5% ethanol or water only. In addition to testing on EPM, blood samples after the FST were obtained to measure plasma corticosterone. Analysis of variance (ANOVA) and alpha level of <it>P </it>< 0.05 were used to determine statistical significance.</p> <p>Results</p> <p>In experiment 1, corticosterone treatment of adolescent rats increased anxiety in the EPM and decreased immobility in the FST compared to no injection control rats. However, vehicle injected rats were similar to corticosterone injected rats, suggesting that adolescent rats may be highly vulnerable to stress of injection. In experiment 2, the intake of treated water, and thus doses delivered, differed for adolescents and adults, but there were no effects of treatment on behavior in the EPM or FST. Rats that had ingested corticosterone had reduced corticosterone release after the FST. Ethanol vehicle also affected corticosterone release compared to those ingesting water only, but differently for adolescents than for adults.</p> <p>Conclusions</p> <p>The results indicate that several challenges must be overcome before the exogenous corticosterone model can be used effectively in adolescents.</p