Fluorescent Tract Tracing Research Articles

Cannabinoid CB2 receptors are expressed in glutamate neurons in the red nucleus and functionally modulate motor behavior in mice

Cannabinoids produce a number of central nervous system effects via the CB2 receptor (CB2R), including analgesia, antianxiety, anti-reward, hypoactivity and attenuation of opioid-induced respiratory depression. However, the cellular distributions of the CB2Rs in the brain remain unclear. We have reported that CB2Rs are expressed in midbrain dopamine (DA) neurons and functionally regulate DA-mediated behavior(s). Unexpectedly, high densities of CB2-like signaling were also found in a neighboring motor structure – the red nucleus (RN) of the midbrain. In the present study, we systematically explored CB2R expression and function in the RN. Immunohistochemistry and in situ hybridization assays showed high densities of CB2R-immunostaining and mRNA signal in RN magnocellular glutamate neurons in wildtype and CB1-knockout, but not CB2-knockout, mice. Ex vivo electrophysiological recordings in midbrain slices demonstrated that CB2R activation by JWH133 dose-dependently inhibited firing rates of RN magnocellular neurons in wildtype, but not CB2-knockout, mice, while having no effect on RN GABA neurons in transgenic GAD67-GFP reporter mice, suggesting CB2-mediated effects on glutamatergic neurons. In addition, microinjection of JWH133 into the RN produced robust ipsilateral rotations in wildtype, but not CB2-knockout mice, which was blocked by pretreatment with either a CB2 or DA D1 or D2 receptor antagonist, suggesting a DA-dependent effect. Finally, fluorescent tract tracing revealed glutamatergic projections from the RN to multiple brain areas including the ventral tegmental area, thalamus, and cerebellum. These findings suggest that CB2Rs in RN glutamate neurons functionally modulate motor activity, and therefore, constitute a new target in cannabis-based medication development for motor disorders.

Viral labeling of neurons synaptically connected to nucleus accumbens somatostatin interneurons.

The nucleus accumbens, a key brain reward region, receives synaptic inputs from a range of forebrain and brainstem regions. Many of these projections have been established using electrophysiology or fluorescent tract tracing. However, more recently developed viral tracing techniques have allowed for fluorescent labeling of synaptic afferents in a cell type-specific manner. Since the NAc is comprised of multiple cell types, these methods have enabled the delineation of the cell type-specific connectivity of principal medium spiny neurons in the region. The synaptic connectivity of somatostatin interneurons, which account for <5% of the neurons in the region, has been inferred from electrophysiological and immunohistochemical data, but has not yet been visualized using modern viral tracing techniques. Here, we use the pseudorabies virus (PRV)-Introvert-GFP virus, an alphaherpes virus previously shown to label synaptic afferents in a cell type-specific manner, to label first order afferents to NAc somatostatin interneurons. While we find GFP(+) labeling in several well established projections to the NAc, we also observe that several known projections to NAc did not contain GFP(+) cells, suggesting they do not innervate somatostatin interneurons in the region. A subset of the GFP(+) afferents are c-FOS(+) following acute administration of cocaine, showing that NAc somatostatin interneurons are innervated by some cells that respond to rewarding stimuli. These results provide a foundation for future studies aimed toward elucidating the cell type-specific connectivity of the NAc, and identify specific circuits that warrant future functional characterization.

Pre and postganglionic innervation of rat adrenal gland by fluorescent tract tracer – Fast blue

IntroductionTo search for pre and postganglionic neurons innervating the adrenal gland by injecting retrograde tract tracer fast blue in the adrenal medulla. MethodsThe motor innervation of rat adrenal gland was studied by a fluorescent tract tracer fast blue. 5 μl of 2% aqueous suspension of fast blue was injected into left adrenal gland. After a survival period of 4–5 days, spinal cord, sympathetic ganglia, suprarenal ganglion, coeliac ganglion and left adrenal gland were dissected out and 15 μm thick plastic sections (JB4 Polysciences) were examined under a fluorescent microscope. ResultsRetrogradely labeled preganglionic neurons were observed in the ipsilateral intermediolateral column of spinal cord from T3 to L2 spinal segments with maximum concentration of labeled neurons from T6 to T11. The labeled neurons were multipolar, spherical or fusiform in shape with transverse diameter 10–20 μm and vertical diameter varying from 12 to 30 μm. Postganglionic labeled neurons were also observed in the left suprarenal ganglion and left sympathetic ganglia (T5 –L2) with maximum concentration from T6 to L1. Labeled neurons varied from 12 to 30 μm in diameter and were randomly distributed throughout the ganglion. DiscussionThe preganglionic neurons from T3 to L2 spinal segments and postganglionic nerve fibers from ipsilateral sympathetic ganglia (T5 –L2) and suprarenal ganglion supplying the adrenal gland might be responsible for the hormone release by regulating blood flow and also by directly innervating the parenchymal cells.

Distribution of BDNF and trkB mRNA in the otic region of 3.5 and 4.5 day chick embryos as revealed with a combination of in situ hybridization and tract tracing.

We have used a recently developed technique which combines fluorescent tract tracing and in situ hybridization to study co-localization of neurotrophin mRNA and neurotrophin receptor mRNA expression simultaneously with the pattern of innervation in the developing chick ear. Efferent and afferent fibersfrom the VII/VIIIth cranial nerves were retrogradely and anterogradely filled with Dextran amines conjugated to Texas red and the brain stem was incubated for 2 hours in tissue culture medium. The tissue was subsequently fixed, sectioned frozen, mounted and subjected to in situ hybridization analysis using probes for brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor, trkB. The results show that afferent and efferent fibers to the ear innervate areas of the developing otocyst which express BDNF mRNA. We also found that neurons in the stato-acoustic ganglion express high levels of trkB mRNA whereas the subset of facial motor neurons that is efferent to the ear only had no or very low levels of trkB mRNA. From our results we conclude that chicken otic efferent fibers preferentially project to areas with BDNF mRNA expression. The very low levels of trkB mRNA in these motor neurons compared to afferent neurons innervating the same region suggest that other factors, perhaps co-expressed with BDNF, may support efferents. A possible involvement of afferents in guiding efferents to specific areas of the ear is suggested.

Cyto- and chemoarchitecture of the hypothalamic paraventricular nucleus in the C57BL/6J male mouse: a study of immunostaining and multiple fluorescent tract tracing.

The paraventricular nucleus of the hypothalamus (PVH) plays a critical role in the regulation of autonomic, neuroendocrine, and behavioral activities. This understanding has come from extensive characterization of the PVH in rats, and for this mammalian species we now have a robust model of basic PVH neuroanatomy and function. However, in mice, whose use as a model research animal has burgeoned with the increasing sophistication of tools for genetic manipulation, a comparable level of PVH characterization has not been achieved. To address this, we employed a variety of fluorescent tract tracing and immunostaining techniques in several different combinations to determine the neuronal connections and cyto- and chemoarchitecture of the PVH in the commonly used C57BL/6J male mouse. Our findings reveal a distinct organization in the mouse PVH that is substantially different from the PVH of male rats. The differences are particularly evident with respect to the spatial relations of two principal neuroendocrine divisions (magnocellular and parvicellular) and three descending preautonomic populations in the PVH. We discuss these data in relation to what is known about PVH function and provide the work as a resource for further studies of the neuronal architecture and function of the mouse PVH.

Cyto‐ and chemoarchitecture of the hypothalamic paraventricular nucleus in the C57BL/6J male mouse: A study of immunostaining and multiple fluorescent tract tracing

AbstractA composite confocal photomicrograph showing the organization an topographic relations of specific neuronal populations in the hypothalamic paraventricular nucleus (PVH) of a C57BL/6malemouse. One population of PVH neurons (presumptive neuroendocrine) was identified by immunocytochemical detection of the retrograde tracer Fluorogold (FG; green) following its injection in to the tail vein; a second population of PVH neurons (presumptive preautonomic) was back‐labeled following upper thoracic level spinal cord injection of the retrograde tracer Fast Blue (FB; blue). Subsequently, distinct populations of oxytocin (OXY; red)‐ and vasopressin (VAS; magenta)‐ expressing neurons were identified immunocytochemically. At this PVH level neurons labeled only with FG (green) include numerous presumptive parvicellular neuroendocrine neurons. None of the FB (blue) back‐labeled neurons were immunopositive for OXY or VAS at this PVH level; however all of the OXY‐ and VAS immunopositive neurons were back‐labeled with FG, although the labeling intensity varied. Thus, OXY‐expressing FG labeled neurons appear red/yellow, whereas VAS‐expressing FG labeled neurons appear magenta/white. The Journal of Comparative Neurology, Volume 520, Number 1, pages 6–33.

Serotonergic and Catecholaminergic Interactions with Co‐Localised Dopamine‐Melatonin Neurones in the Hypothalamus of the Female Turkey

Serotonin and catecholamines (dopamine, norepinephrine, epinephrine) have important roles as neurotransmitters in avian reproduction, but their anatomical relationship to the neuroendocrine circuitry that regulates reproduction is poorly understood. Our previous studies have shown that co-localised dopamine-melatonin (DA-MEL) neurones in the avian premammillary nucleus (PMM) are active during periods of photoresponsiveness and, therefore, are potentially photosensitive neurones. Because serotonergic and catecholaminergic neurotransmitters are important regulators of reproductive function in the female turkey, we hypothesised that the serotonergic/catecholaminergic neurones within the brainstem might interact with PMM DA-MEL neurones and constitute an important circuit for reproductive function. To examine this possible interaction, the retrograde fluorescent tract tracer, 1,1'dioctadecyl-3,3,3'3'-tetramethyleindocarbocyanine perchlorate (DiI) was injected into the PMM, and combined with serotonin, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and phenyl N-methyltransferse (PNMT) immunocytochemistry to reveal neuroanatomical connections. Changes in the activities of serotonergic, dopaminergic, adrenergic and noradrenergic neuronal systems projecting to the PMM were measured at different reproductive states with in situ hybridisation (ISH) techniques, using tryptophan hydroxylase 2 (TPH2) and TH mRNA expression, respectively. Cells labelled with DiI were found in anatomically discrete areas in or near the hypothalamus and the brainstem. Double immunocytochemistry confirmed that there were serotonin, DBH and PNMT fibres in close apposition to DA-MEL neurones. TPH2 mRNA expression in serotonin neurones was found in several nuclei, and its most abundant mRNA expression was seen in the nucleus Locus ceruleus of laying and incubating hens. TH mRNA expression levels in the six catecholaminegic areas labelled with DiI was measured across the different reproductive states. In the nucleus tractus solitarius (adrenergic), the highest level of TH mRNA expression was found in photorefractory hens and the lowest level in incubating hens. These observed patterns of serotonin/catecholamine neuronal distribution and their variable interactions with PMM DA-MEL neurones during different reproductive states may offer a significant neuroanatomical basis for understanding the control of avian reproductive seasonality.

Structural, Functional and Developmental Convergence of the Insect Mushroom Bodies with Higher Brain Centers of Vertebrates

Convergence of higher processing centers has been proposed for insects and vertebrates, but the extent of these similarities remains controversial. The present study demonstrates that one higher brain center of insects, the mushroom bodies, displays a number of similarities with mammalian higher brain centers that are arguably the products of adaptation to common behavioral ecologies, despite their deeply divergent origins. Quantitative neuroanatomy, immunohistochemistry, fluorescent tract tracing and BrdU labeling are employed to investigate the relationships among behavioral ecology and mushroom body size, sensory input and mode of development in one taxon, the scarab beetles (Coleoptera: Scarabaeidae). Comparisons are extended to a taxon in which similar mushroom body architectures have arisen independently, the cockroaches (Dictyoptera), and to published accounts of vertebrate brain evolution. This study demonstrates that evolutionary increases in higher brain center size and intrinsic neuron number are associated with flexibility in food acquisition behaviors in both vertebrates and insects. These evolutionarily expanded higher brain centers are divided into novel structural subcompartments that acquire novel processing functions. Increased numbers of neurons comprising enlarged higher brain centers are generated by expanded neural precursor pools, and the time for development of these brain centers is protracted. Taken together, these findings extend our understanding of how evolutionarily constrained neural substrates might converge under shared adaptive landscapes, even after 600 million years of divergence, and even at the level of higher brain centers that generate complex behaviors.

Thematic review series: Adipocyte Biology. Sympathetic and sensory innervation of white adipose tissue

During our study of the reversal of seasonal obesity in Siberian hamsters, we found an interaction between receptors for the pineal hormone melatonin and the sympathetic nervous system (SNS) outflow from brain to white adipose tissue (WAT). This ultimately led us and others to conclude that the SNS innervation of WAT is the primary initiator of lipid mobilization in these as well as other animals, including humans. There is strong neurochemical (norepinephrine turnover), neuroanatomical (viral tract tracing), and functional (sympathetic denervation-induced blockade of lipolysis) evidence for the role of the SNS in lipid mobilization. Recent findings suggest the presence of WAT sensory innervation based on strong neuroanatomical (viral tract tracing, immunohistochemical markers of sensory nerves) and suggestive functional (capsaicin sensory denervation-induced WAT growth) evidence, the latter implying a role in conveying adiposity information to the brain. By contrast, parasympathetic nervous system innervation of WAT is characterized by largely negative neuroanatomical evidence (viral tract tracing, immunohistochemical and biochemical markers of parasympathetic nerves). Functional evidence (intraneural stimulation and in situ microdialysis) for the role of the SNS innervation in lipid mobilization in human WAT is convincing, with some controversy regarding the level of sympathetic nerve activity in human obesity.

Sensory innervation of the suprarenal gland in the albino rat: a fluorescent tract tracer study.

The afferent innervation of the suprarenal gland was studied by using a fluorescent tract tracer in the adult albino rat. The left suprarenal gland was injected slowly with 5 microl of 2% aqueous suspension of Fast blue. After a survival period of 4-5 days, the dorsal root ganglia were dissected out and 15-microm-thick plastic (JB 4) sections were examined under the fluorescent microscope. The labelled neurons were seen from the third thoracic to second lumbar dorsal root ganglia, ipsilateral to the site of injection with maximum concentration from T6 to T11. These primary sensory neurons were round to oval in shape, varied from 7 microm to 40 microm in size, and were distributed randomly in the dorsal root ganglia. The labelling of the primary sensory neurons in the dorsal root ganglia confirms the presence of sensory nerve endings in the suprarenal gland that may be responsible for the vascular distension and hormonal release.

Catecholaminergic innervation of white adipose tissue in Siberian hamsters.

When Siberian hamsters are transferred from long summerlike days (LDs) to short winterlike days (SDs) they decrease their body weight, primarily as body fat. These SD-induced decreases in lipid stores are not uniform. Internally located white adipose tissue (WAT) pads are depleted preferentially of lipid, whereas the more externally located subcutaneous WAT pads are relatively spared. These data suggest a possible differential sympathetic neural control over catecholamine-induced lipolysis and that lipolytic rates are greater for internal vs. external WAT pads. Moreover, if these differential rates of lipolysis are due to differential sympathetic nervous system (SNS) drives on the pads, then fat pad-specific catecholaminergic innervation may exist. Therefore, we tested whether inguinal WAT (IWAT; an external pad) and epididymal WAT (EWAT; an internal pad) were innervated differentially. In addition, we tested whether norepinephrine (NE) turnover (TO) reflected the presumed greater SNS drive on EWAT vs. IWAT after SD exposure. Injections of fluorescent tract tracers [Fluoro-Gold or indocarbocyanine perchlorate (DiI)] demonstrated projections from the SNS ganglia T13-L3 to both fat pads. Retrograde labeling revealed a relatively separate pattern of distribution of labeled neurons in the ganglia projecting to each pad. In vivo anterograde transport of DiI resulted in labeling in both IWAT and EWAT that included staining around individual adipocytes and occasionally retrogradely labeled cells. The proportionately greater decrease in EWAT compared with IWAT mass after 5 wk of SD exposure was reflected in greater EWAT NE TO than found in their LD counterparts for this pad.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental study of GnRH neuronal projections to the medial basal hypothalamus of the male Djungarian hamster.

The present study in the male Djungarian hamster determined the neuroanatomical distribution and morphology of gonadotropin-releasing hormone (GnRH) neurons which innervate the medial basal hypothalamus during sexual maturation. Prepubertal, peripubertal, and postpubertal males were perfused, brains were removed, and crystals of the fluorescent tract tracer, DiI, were implanted directly into the median eminence of the brain. Eight weeks later, brains were sectioned and processed for GnRH immunofluorescence. At all ages, GnRH cell bodies were bipolar or unipolar; both subtypes were labeled with DiI in proportion to their respective numbers in each brain region. GnRH perikarya were distributed in a diffuse ventromedial continuum from the septum through the anterior hypothalamus. In prepubertal males, DiI was present in the majority of GnRH neurons (54% of total) that were located in brain regions rostral to and including the medial preoptic area. In lateral and caudal brain areas, fewer GnRH perikarya contained DiI (28% of total or less). With sexual maturation, fewer GnRH somata were labeled with DiI in areas rostral to the hypothalamus. The data suggest that bipolar and unipolar GnRH neurons in the forebrain, rostral to the preoptic area, are major contributors to the GnRH innervation of the median eminence in the male Djungarian hamster. With the onset of puberty, the finding that decreasing numbers of GnRH perikarya directly project to the medial basal hypothalamus suggests that fewer GnRH neurons constitute the final common pathway that controls gonadotropin secretion.

Multicolor Fluorescence Microscopy Using the Laser-Scanning Confocal Microscope

Neurons and glia can be labeled using fluorescence immunocytochemistry, fluorescent tract-tracers, and a variety of other techniques that use fluorescence. Combining these methods to label tissue with two or more fluorescent probes can increase the power of an experimental design. The present paper examines the issues related to the use of laser-scanning confocal microscopy to image combinations of fluorescent labels and recommends specific combinations of lasers, filters, and fluorophores.