Injections Of Fluorescent Tracers Research Articles

Topography of visual and somatosensory inputs to the pontine nuclei in zebra finches (Taeniopygia guttata).

Birds have a comprehensive network of sensorimotor projections extending from the forebrain and midbrain to the cerebellum via the pontine nuclei, but the organization of these circuits in the pons is not thoroughly described. Inputs to the pontine nuclei include two retinorecipient areas, nucleus lentiformis mesencephali (LM) and nucleus of the basal optic root (nBOR), which are important structures for analyzing optic flow. Other crucial regions for visuomotor control include the retinorecipient ventral lateral geniculate nucleus (GLv), and optic tectum (TeO). These visual areas, together with the somatosensory area of the anterior (rostral) Wulst, which is homologous to the primary somatosensory cortex in mammals, project to the medial and lateral pontine nuclei (PM, PL). In this study, we used injections of fluorescent tracers to study the organization of these visual and somatosensory inputs to the pontine nuclei in zebra finches. We found a topographic organization of inputs to PM and PL. The PM has a lateral subdivision that predominantly receives projections from the ipsilateral anterior Wulst. The medial PM receives bands of inputs from the ipsilateral GLv and the nucleus laminaris precommisulis, located medial to LM. We also found that the lateral PL receives a strong ipsilateral projection from TeO, while the medial PL and region between the PM and PL receive less prominent projections from nBOR, bilaterally. We discuss these results in the context of the organization of pontine inputs to the cerebellum and possible functional implications of diverse somato-motor and visuomotor inputs and parcellation in the pontine nuclei.

Yuanzhi powder facilitated Aβ clearance in APP/PS1 mice: Target to the drainage of glymphatic system and meningeal lymphatic vessels

Ethnopharmacological relevanceYuanzhi Powder (YZP) is a classical Chinese medicine prescription, which is suitable for the treatment of dementia by “dispelling phlegm and opening orifice”. The therapeutic efficacy of YZP on Alzheimer's disease (AD) has been previously reported in our work. However, it remains unclear whether the neuroprotective effect of YZP is linked to β-amyloid(Aβ) clearance through cerebral lymphatic drainage. Aim of the studyThe aim was to determine the protective efficacy of YZP against AD and investigate the potential mechanism for eliminating excessive Aβ deposition. Materials and methodsAPP/PS1 mice were divided into four groups of 8 mice each: APP/PS1 group, DONE group, L-YZP group, and H-YZP group. Additionally, 8 wild-type littermates were assigned to the control group (WT group). After 8 weeks of consecutive intragastric administration, behavioral tests, including the open field test, novel object recognition test and Morris Water Maze test, were employed to assess the cognitive abilities of all groups of mice. Nissl staining, immunohistochemistry, and western blotting were utilized to evaluate clearance of excessive Aβ deposition and pathological changes. Furthermore, immunofluorescence was applied to visualize the drainage of the cerebral lymphatic system after fluorescent tracer injection in the cisterna magna. ResultsThe administration of YZP significantly attenuated cognitive deficits, cleared excessive Aβ deposition, and improved pathological damage in APP/PS1 mice. Furthermore, YZP effectively enhanced glymphatic system drainage by restoring AQP4 polarization and inhibiting reactive astrogliosis. Additionally, YZP facilitated the drainage of meningeal lymphatic vessels (MLVs) by augmenting their diameter and coverage. Lastly, YZP promoted the elimination of Aβ from the brain to deep cervical lymph nodes. ConclusionsThe administration of YZP may ameliorate the cognitive deficits and pathological damage in APP/PS1 mice by effectively clearing excessive Aβ deposition. The underlying mechanisms potentially involve Aβ clearance through the cerebral lymphatic system, which includes the glymphatic system and MLVs.

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium.

Endocytosis allows cells to internalise a wide range of molecules from their environment and to maintain their plasma membrane composition. It is vital during development and for maintenance of tissue homeostasis. The ability to visualise endocytosis in vivo requires suitable assays to monitor the process. Here, we describe imaging-based assays to visualise endocytosis in the neuroepithelium of living zebrafish embryos. Injection of fluorescent tracers into the brain ventricles followed by live imaging was used to study fluid-phase or receptor-mediated endocytosis, for which we used receptor-associated protein (RAP, encoded by Lrpap1) as a ligand for low-density lipoprotein receptor-related protein (LRP) receptors. Using dual-colour imaging combined with expression of endocytic markers, it is possible to track the progression of endocytosed tracers and to monitor trafficking dynamics. Using these assays, we reveal a role for the Lowe syndrome protein Ocrl in endocytic trafficking within the neuroepithelium. We also found that the RAP-binding receptor Lrp2 (encoded by lrp2a) appears to contribute only partially to neuroepithelial RAP endocytosis. Altogether, our results provide a basis to track endocytosis within the neuroepithelium in vivo and support a role for Ocrl in this process. This article has an associated First Person interview with the first author of the paper.

Pretecto‐ and ponto‐cerebellar pathways to the pigeon oculomotor cerebellum follow a zonal organization

Both birds and mammals have relatively large forebrains and cerebella. In mammals, there are extensive sensory-motor projections to the cerebellum through the pontine nuclei originating from several parts of the cerebral cortex. Similar forebrain-to-cerebellum pathways exist in birds, but the organization of this circuitry has not been studied extensively. Birds have two nuclei at the base of the brainstem that are thought to be homologous to the pontine nuclei of mammals, the medial and lateral pontine nuclei (PM, PL). Additionally, birds are unique in that they have a pretectal nucleus called the medial spiriform nucleus (SpM) that, like the pontine nuclei, also receives projections from the forebrain and projects to the oculomotor cerebellum (OCb; folia VI to VIII). The OCb also receives input from the pretectal nucleus lentiformis mesencephali (LM), which analyzes visual optic flow information resulting from self-movement. In this study, we used single or double injections of fluorescent tracers to study the organization of these inputs from PM, PL, SpM and LM to the OCb in pigeons. We found that these inputs follow a zonal organization. The most medial zone in the OCb, zone A1, receives bilateral inputs from the lateral SpM, PL and LM. Zones A2 and C receive a bilateral projection from the medial SpM, and a mostly contralateral projection from PM and LM. We discuss how the pathway to zone A1 processes mainly visuo-motor information to spinal premotor areas, whereas the pathways to zone A2/C processes somato-motor and visuo-motor information and may have a feedback/modulatory role.

Inducing sterile pyramidal neuronal death in mice to model distinct aspects of gray matter encephalitis

Up to one person in a population of 10,000 is diagnosed once in lifetime with an encephalitis, in 50–70% of unknown origin. Recognized causes amount to 20–50% viral infections. Approximately one third of affected subjects develops moderate and severe subsequent damage. Several neurotropic viruses can directly infect pyramidal neurons and induce neuronal death in cortex and hippocampus. The resulting encephalitic syndromes are frequently associated with cognitive deterioration and dementia, but involve numerous parallel and downstream cellular and molecular events that make the interpretation of direct consequences of sudden pyramidal neuronal loss difficult. This, however, would be pivotal for understanding how neuroinflammatory processes initiate the development of neurodegeneration, and thus for targeted prophylactic and therapeutic interventions. Here we utilized adult male NexCreERT2xRosa26-eGFP-DTA (= ‘DTA’) mice for the induction of a sterile encephalitis by diphtheria toxin-mediated ablation of cortical and hippocampal pyramidal neurons which also recruits immune cells into gray matter. We report multifaceted aftereffects of this defined process, including the expected pathology of classical hippocampal behaviors, evaluated in Morris water maze, but also of (pre)frontal circuit function, assessed by prepulse inhibition. Importantly, we modelled in encephalitis mice novel translationally relevant sequelae, namely altered social interaction/cognition, accompanied by compromised thermoreaction to social stimuli as convenient readout of parallel autonomic nervous system (dys)function. High resolution magnetic resonance imaging disclosed distinct abnormalities in brain dimensions, including cortical and hippocampal layering, as well as of cerebral blood flow and volume. Fluorescent tracer injection, immunohistochemistry and brain flow cytometry revealed persistent blood–brain-barrier perturbance and chronic brain inflammation. Surprisingly, blood flow cytometry showed no abnormalities in circulating major immune cell subsets and plasma high-mobility group box 1 (HMGB1) as proinflammatory marker remained unchanged. The present experimental work, analyzing multidimensional outcomes of direct pyramidal neuronal loss, will open new avenues for urgently needed encephalitis research.

Brain Glymphatic/Lymphatic Imaging by MRI and PET.

Since glymphatic was proposed and meningeal lymphatic was discovered, MRI and even PET were introduced to investigate brain parenchymal interstitial fluid (ISF), cerebrospinal fluid (CSF), and lymphatic outflow in rodents and humans. Previous findings by ex vivo fluorescent microscopic, and in vivo two-photon imaging in rodents were reproduced using intrathecal contrast (gadobutrol and the similar)-enhanced MRI in rodents and further in humans. On dynamic MRI of meningeal lymphatics, in contrast to rodents, humans use mainly dorsal meningeal lymphatic pathways of ISF-CSF-lymphatic efflux. In mice, ISF-CSF exchange was examined thoroughly using an intra-cistern injection of fluorescent tracers during sleep, aging, and neurodegeneration yielding many details. CSF to lymphatic efflux is across arachnoid barrier cells over the dorsal dura in rodents and in humans. Meningeal lymphatic efflux to cervical lymph nodes and systemic circulation is also well-delineated especially in humans onintrathecal contrast MRI. Sleep- or anesthesia-related changes of glymphatic-lymphatic flow and the coupling of ISF-CSF-lymphatic drainage are major confounders ininterpreting brain glymphatic/lymphatic outflow in rodents. PET imaging in humans should be interpreted based on human anatomy and physiology, different in some aspects, using MRI recently. Based on the summary in this review, we propose non-invasive and longer-term intrathecal SPECT/PET or MRI studies to unravel the roles of brain glymphatic/lymphatic in diseases.

Quantitative Determination of Glymphatic Flow Using Spectrophotofluorometry.

Following intrathecal injection of fluorescent tracers, ex vivo imaging of brain vibratome slices has been widely used to study the glymphatic system in the rodent brain. Tracer penetration into the brain is usually quantified by image-processing, even though this approach requires much time and manual operation. Here, we illustrate a simple protocol for the quantitative determination of glymphatic activity using spectrophotofluorometry. At specific time-points following intracisternal or intrastriatal injection of fluorescent tracers, certain brain regions and the spinal cord were harvested and tracers were extracted from the tissue. The intensity of tracers was analyzed spectrophotometrically and their concentrations were quantified from standard curves. Using this approach, the regional and dynamic delivery of subarachnoid CSF tracers into the brain parenchyma was assessed, and the clearance of tracers from the brain was also determined. Furthermore, the impairment of glymphatic influx in the brains of old mice was confirmed using our approach. Our method is more accurate and efficient than the imaging approach in terms of the quantitativedetermination of glymphatic activity, and this will be useful in preclinical studies.

Characterization of multi-cellular dynamics of angiogenesis and vascular remodelling by intravital imaging of the wounded mouse cornea

Establishment of the functional blood vasculature involves extensive cellular rearrangement controlled by growth factors, chemokines and flow-mediated shear forces. To record these highly dynamic processes in mammalians has been technically demanding. Here we apply confocal and wide field time-lapse in vivo microscopy to characterize the remodelling vasculature of the wounded mouse cornea. Using mouse lines with constitutive or inducible endogenous fluorescent reporters, in combination with tracer injections and mosaic genetic recombination, we follow processes of sprouting angiogenesis, sprout fusion, vessel expansion and pruning in vivo, at subcellular resolution. We describe the migratory behaviour of endothelial cells of perfused vessels, in relation to blood flow directionality and vessel identity. Live-imaging following intravascular injection of fluorescent tracers, allowed for recording of VEGFA-induced permeability. Altogether, live-imaging of the remodelling vasculature of inflamed corneas of mice carrying endogenous fluorescent reporters and conditional alleles, constitutes a powerful platform for investigation of cellular behaviour and vessel function.

Seeing through porous media: An experimental study for unveiling interstitial flows

AbstractWe describe a novel inexpensive method, utilizing particle image velocimetry (PIV) and refractive index‐matching (RIM) for visualizing and quantifying the flow field within bio‐amended porous media. To date, this technique has been limited to idealized particles, whose refractive index does not match that of fresh water and thus requires specialized and often toxic or hazardous fluids. Here, we use irregularly shaped grains made of hydrogel as the solid matrix and water as the fluid. The advantage of using water is that it provides, for the first time, the opportunity to study both hydraulic and biological processes, which typically occur in soils and streambeds. By using RIM coupled with PIV (RIM‐PIV), we measured the interstitial flow field within a cell packed with granular material consisting of hydrogel grains in a size range of 1–8 mm, both in the presence and in the absence of Sinorhizobium meliloti bacteria (strain Rm8530). We also performed experiments with fluorescent tracer (fluorescein) and fluorescent microbes (Shewanella GPF MR‐1) to test the capability of visualizing solute transport and microbial movements. Results showed that the RIM‐PIV can measure the flow field for both biofilm‐free and biofilm‐covered hydrogel grains. The fluorescent tracer injection showed the ability to visualize both physical (concave surfaces and eddies) and biological (biofilms) transient storage zones, whereas the fluorescent microbe treatment showed the ability to track microbial movements within fluids. We conclude that the proposed methodology is a promising tool to visualize and quantify biofilm attachment, growth, and detachment in a system closer to natural conditions than a 2D flow cell experiment.

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls.

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM. The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

Online LI-rTMS during a Visual Learning Task: Differential Impacts on Visual Circuit and Behavioral Plasticity in Adult Ephrin-A2A5-/- Mice.

Repetitive transcranial magnetic stimulation (rTMS) induces plasticity in normal and abnormal neural circuitries, an effect that may be influenced by intrinsic brain activity during treatment. Here, we study potential synergistic effects between low-intensity rTMS (LI-rTMS) and concurrent neural activity in promoting circuit reorganization and enhancing visual behavior. We used ephrin-A2A5–/– mice, which are known to possess visuotopic mapping errors that are ameliorated by LI-rTMS, and assessed the impact of stimulation when mice were engaged in a visual learning task. A detachable coil was affixed to each mouse, and animals underwent 2 wk of 10-min daily training in a two-choice visual discrimination task with concurrent LI-rTMS or sham stimulation. No-task controls (+LI-rTMS/sham) were placed in the task arena without visual task training. At the end of the experiment, visuomotor tracking behavior was assessed, and corticotectal and geniculocortical pathway organization was mapped by injections of fluorescent tracers into the primary visual cortex. Consistent with previous results, LI-rTMS alone improved geniculocortical and corticotectal topography, but combining LI-rTMS with the visual learning task prevented beneficial corticotectal reorganization and had no additional effect on geniculocortical topography or visuomotor tracking performance. Unexpectedly, there was a significant increase in the total number of trials completed by task + LI-rTMS mice in the visual learning task. Comparison with wild-type mice revealed that ephrin-A2A5–/– mice had reduced accuracy and response rates, suggesting a goal-directed behavioral deficit, which was improved by LI-rTMS. Our results suggest that concurrent brain activity during behavior interacts with LI-rTMS, altering behavior and different visual circuits in an abnormal system.

Laboratory Scale Investigation of Dispersion Effects on Saltwater Movement due to Cutoff Wall Installation

In the numerical investigation of saltwater transport in coastal aquifers, we need to correctly evaluate the hydrodynamic dispersion in the flow field. In this study, we focused on the role of dispersivity in the removal process of residual saltwater in a laboratory scale cutoff wall experiment. From a pulse-type fluorescent tracer injection experiment in a saturated porous media of glass beads with a mean diameter of 0.088 cm, the estimated longitudinal and transverse dispersivities were found to be 0.07 cm and 0.0025 cm, respectively. Numerical analysis of the saltwater intrusion and subsequent removal after cutoff wall installation using SEAWAT and the generated dispersivity ratio (αL/αT) of 28 reproduces well the measured salt concentration changes with time. Whereas, if a dispersivity ratio of 10 is used in the numerical simulation, transverse dispersion in the saltwater and freshwater mixing zone becomes large and the residual saltwater is removed faster than the laboratory experiment. Inversely, if 100 was used, the residual saltwater removal time took longer. The transverse dispersion is a key parameter in the mechanical dispersion of saltwater in the mixing zone after cutoff wall installation.

In Vivo Analysis of the Neurovascular Niche in the Developing Xenopus Brain.

The neurovascular niche is a specialized microenvironment formed by the interactions between neural progenitor cells (NPCs) and the vasculature. While it is thought to regulate adult neurogenesis by signaling through vascular-derived soluble cues or contacted-mediated cues, less is known about the neurovascular niche during development. In Xenopus laevis tadpole brain, NPCs line the ventricle and extend radial processes tipped with endfeet to the vascularized pial surface. Using in vivo labeling and time-lapse imaging in tadpoles, we find that intracardial injection of fluorescent tracers rapidly labels Sox2/3-expressing NPCs and that vascular-circulating molecules are endocytosed by NPC endfeet. Confocal imaging indicates that about half of the endfeet appear to appose the vasculature, and time-lapse analysis of NPC proliferation and endfeet-vascular interactions suggest that proliferative activity does not correlate with stable vascular apposition. Together, these findings characterize the neurovascular niche in the developing brain and suggest that, while signaling to NPCs may occur through vascular-derived soluble cues, stable contact between NPC endfeet and the vasculature is not required for developmental neurogenesis.

Abstract WMP82: Exosomes Derived From Cerebral Endothelial Cells of Young Adult Rats Reduced Cognitive Deficits in Aged Diabetic Rats

Introduction: Diabetes mellitus (DM) is associated with cognitive decline and dementia in the elderly. The glymphatic system mediates clearance of the interstitial solutes in the brain by exchange of cerebrospinal and interstitial fluid (CSF and ISF). We recently demonstrated that DM in aged rat induces impairment of the glymphatic system and cognitive decline. Exosomes, membrane vesicles, mediate intercellular communication by transferring their cargo into recipient cells. The present study investigated whether cerebral endothelial exosomes (CEE) ameliorate glymphatic system impairment and improve cognitive function in aged DM rats. Methods and Results: DM was induced in male Wistar rats (13 months, n=48) by injection of nicotinamide and streptozotocin. Two months after DM, rats were treated with CEE (1x10 11 exosomes/rat, IV) twice a week for 4 weeks. Age matched DM and non-DM rats were used as controls. CEE were harvested from the cultured cerebral endothelial cells of health young adult rats. Exchanges of CSF and ISF were measured by intracisternal injection of fluorescent tracer, Texas Red-dextran (TR, 3kD). Confocal microscopic analysis of brain slices revealed a progressive slowdown of ISF clearance in the hippocampi, assessed by retention of TR starting at 2.5 fold at 2M (13±5 vs 5±3% of area) and increasing to 4 fold at 4M (21±4 vs 5±2%) of DM. Paravascular amyloid β (Aβ) accumulation was only detected at 4M of DM. The CEE treatment significantly (p<0.05) reduced TR retention (10±4%) at 4M of DM and also decreased Aβ accumulation (2±1 vs 6±2/mm 2 ) and parenchymal fibrin deposition (9±5 vs 23±5/mm 2 ) compared to untreated DM rats. Moreover, the CEE treatment significantly improved hippocampal related learning and memory measured by the Morris Water Maze and odor-based novelty recognition for olfactory memory, without altering the glucose level. In vitro, cerebral endothelial cells isolated from 2M DM rats exhibited substantial dysfunction as measured by capillary-like tube formation and cell migration, whereas incubation with the CEE substantially reversed endothelial dysfunction. Conclusions: The CEE treatment reduces DM-induced glymphatic and cerebral endothelial dysfunctions, leading to improvement of cognitive function in aged DM rats.

Coronary stem development in wild-type and Tbx1 null mouse hearts.

Coronary artery (CA) stems connect the ventricular coronary tree with the aorta. Defects in proximal CA patterning are a cause of sudden cardiac death. In mice lacking Tbx1, common arterial trunk is associated with an abnormal trajectory of the proximal left CA. Here we investigate CA stem development in wild-type and Tbx1 null embryos. Genetic lineage tracing reveals that limited outgrowth of aortic endothelium contributes to proximal CA stems. Immunohistochemistry and fluorescent tracer injections identify a periarterial vascular plexus present at the onset of CA stem development. Transplantation experiments in avian embryos indicate that the periarterial plexus originates in mesenchyme distal to the outflow tract. Tbx1 is required for the patterning but not timing of CA stem development and a Tbx1 reporter allele is expressed in myocardium adjacent to the left but not right CA stem. This expression domain is maintained in Sema3c(-/-) hearts with a common arterial trunk and leftward positioned CA. Ectopic myocardial differentiation is observed on the left side of the Tbx1(-/-) common arterial trunk. A periarterial plexus bridges limited outgrowth of the aortic endothelium with the ventricular plexus during CA stem development. Molecular differences associated with left and right CA stems provide new insights into the etiology of CA patterning defects.

Arrested development of the dorsal column following neonatal spinal cord injury in the opossum, Monodelphis domestica.

Developmental studies of spinal cord injury in which regrowth of axons occurs across the site of transection rarely distinguish between the recovery of motor-controlling pathways and that of ascending axons carrying sensory information. We describe the morphological changes that occur in the dorsal column (DC) of the grey short-tailed opossum, Monodelphis domestica, following spinal cord injury at two early developmental ages. The spinal cords of opossums that had had their mid-thoracic spinal cords completely transected at postnatal day 7 (P7) or P28 were analysed. Profiles of neurofilament immunoreactivity in transected cords showing DC development were differentially affected by the injury compared with the rest of the cord and cytoarchitecture was modified in an age- and site-dependent manner. The ability of DC neurites to grow across the site of transection was confirmed by injection of fluorescent tracer below the injury. P7 transected cords showed labelling in the DC above the site of original transection indicating that neurites of this sensory tract were able to span the injury. No growth of any neuronal processes was seen after P28 transection. Thus, DC is affected by spinal injury in a differential manner depending on the age at which the transection occurs. This age-differential response, together with other facets of remodelling that occur after neonatal spinal injury, might explain the locomotor adaptations and recovery observed in these animals.

The peculiar visual brain of the split-eyed whirligig beetle Dineutus sublineatus

Event Abstract Back to Event The peculiar visual brain of the split-eyed whirligig beetle Dineutus sublineatus Chan Lin1, 2* and Nicholas J. Strausfeld2, 3 1 University of Arizona, Graduate Interdisciplinary Program in Entomology & Insect Science, United States 2 University of Arizona, Center for Insect Science, United States 3 University of Arizona, Department of Neuroscience, United States Whirligig beetles (Coleoptera: Gyrinidae) are aquatic insects living on the water surface. They are equipped with two separate pairs of compound eyes, an upper pair viewing above the water and a lower pair viewing downwards beneath the water line. Little is known about how their optic lobes serve the split eyes. Using reduced silver-staining, immunolabeling and fluorescent tracer injections, we found that their elaborate optic lobes are split into two halves: separated upper and lower laminas; upper and lower medullas; and a bilobed partially split lobula. Visual relay neurons from the upper medulla send axons to the central brain where they provide the exclusive afferent supply to both ipsilateral and contralateral calyces of the paired mushroom bodies. The whirligig beetle is the only insect species so far found to be so equipped [1]. Lobula plate neuropil, which in other insects plays a major role in mediating stabilized flight, is found only in conjunction with the lower lobe of the lobula. Investigations of the larval visual system reveal a precious lobula plate that is supplied by three larval laminas serving the three dorsally located stemmata (single-lens larval eyes), which are adjacent to the developing upper compound eye [2]. Because the whirligig larvae are subaquatic ambush predators, it is theorized that the precocious development of a lobula plate may be employed for detecting movement by potential prey items, as has been reported previously from observations of larval tiger beetles (Cicindela) that are also ambush predators [3, 4]. In D. sublineatus, the precocious lobula plate appears to degenerate during pupal metamorphosis and, as a result, the adult whirligig beetle lacks the upper lobula plate. Other adult optic neuropils, including the lamina, medulla and lobula, begin developing alongside the larval optic neuropils as early as the 3rd instar larval stage. How this early development of adult neuropils contributes to the split optic lobes and how a new lower lobula plate is developed during pupal metamorphosis are presently under investigation. The unique organization and development scheme of the whirligig beetle’s optic lobes suggest distinct functional differences between the upper and the lower compound eyes. Acknowledgements The study was supported by the University of Arizona Regents’ Fund, the Air Force Office of Scientific Research (AFOSR FA95501010299) (to NJS), and stipends from the Taiwan Government Scholarship for Study Abroad and the University of Arizona’s Center for Insect Science (to CL).

Zebrin-Immunopositive and -Immunonegative Stripe Pairs Represent Functional Units in the Pigeon Vestibulocerebellum

The cerebellum is a site of complex sensorimotor integration and contains up to 80% of the neurons in the brain, yet comparatively little is known about the organization of sensorimotor systems within the cerebellum. It is known that afferent projections and Purkinje cell (PC) response properties are organized into sagittal "zones" in the cerebellum. Moreover, the isoenzyme aldolase C [also known as zebrin II (ZII)] is heterogeneously expressed in cerebellar PCs such that there are sagittal stripes of PCs with high expression (ZII+) interdigitated with stripes of little or no expression (ZII-). In the present study, we show how the ZII stripes in folium IXcd of the vestibulocerebellum in pigeons are related to response properties of PCs. IXcd consists of seven pairs of ZII+/- stripes denoted P1+/- (medial) to P7+/- (lateral). Electrophysiological studies have shown that vestibulocerebellar PCs respond to particular patterns of optic flow resulting from self-motion in three-dimensional space. In our study, we recorded optic flow preferences from PCs in IXcd, marked recording locations with injections of fluorescent tracer, and subsequently immunoreacted coronal sections for ZII. We found that the PCs within a ZII+/- stripe pair all responded best to the same pattern of optic flow. That is, a ZII+/- stripe pair forms a functional unit in the cerebellum. This is the first demonstration that the function of PCs is associated with ZII stripes across the mediolateral extent of an entire folium.

Contrasting Patterns of Cortical Input to Architectural Subdivisions of the Area 8 Complex: A Retrograde Tracing Study in Marmoset Monkeys

Contemporary studies recognize 3 distinct cytoarchitectural and functional areas within the Brodmann area 8 complex, in the caudal prefrontal cortex: 8b, 8aD, and 8aV. Here, we report on the quantitative characteristics of the cortical projections to these areas, using injections of fluorescent tracers in marmoset monkeys. Area 8b was distinct from both 8aD and 8aV due to its connections with medial prefrontal, anterior cingulate, superior temporal polysensory, and ventral midline/retrosplenial areas. In contrast, areas 8aD and 8aV received the bulk of the projections from posterior parietal cortex and dorsal midline areas. In the frontal lobe, area 8aV received projections primarily from ventrolateral areas, while both 8aD and 8b received dense inputs from areas on the dorsolateral surface. Whereas area 8aD received the most significant auditory projections, these were relatively sparse, in comparison with those previously reported in macaques. Finally, area 8aV was distinct from both 8aD and 8b by virtue of its widespread input from the extrastriate visual areas. These results are compatible with a homologous organization of the prefrontal cortex in New and Old World monkeys, and suggest significant parallels between the present pathways, revealed by tract-tracing, and networks revealed by functional connectivity analysis in Old World monkeys and humans.