Janelle M.P. Pakan

Small molecule phosphorescent probes for O2 imaging in 3D tissue models

Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O2 sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present several new cell-penetrating phosphorescent conjugates of a Pt(ii)-tetrakis(pentafluorophenyl)porphine (PtPFPP) dye produced by click-modification with thiols, and perform their evaluation as O2 imaging probes for 3D tissue models. The hydrophilic glucose (Pt-Glc) and galactose (Pt-Gal) conjugates demonstrated minimal aggregation and self-quenching in aqueous media, and efficient in-depth staining of different cell types and multi-cellular aggregates at working concentrations ≤10 μM. The Pt-Glc probe was applied in high-resolution phosphorescence lifetime based O2 imaging (PLIM) in multi-cellular spheroids of cancer cells (PC12), primary neural cells (neurospheres) and slices of brain tissue, where it showed good analytical performance, minimal effects on cell viability and appropriate responses to O2 with phosphorescence lifetimes changing from 20 μs in air-saturated to 57 μs under deoxygenated conditions. In contrast, mono- and tetra-substituted oligoarginine conjugates of PtPFPP showed marked aggregation and unstable photophysical properties precluding their use as O2 sensing probes.

Purkinje Cell Compartmentation as Revealed by Zebrin II Expression in the Cerebellar Cortex of Pigeons (Columba livia)

Purkinje cells in the cerebellum express the antigen zebrin II (aldolase C) in many vertebrates. In mammals, zebrin is expressed in a parasagittal fashion, with alternating immunopositive and immunonegative stripes. Whether a similar pattern is expressed in birds is unknown. Here we present the first investigation into zebrin II expression in a bird: the adult pigeon (Columba livia). Western blotting of pigeon cerebellar homogenates reveals a single polypeptide with an apparent molecular weight of 36 kDa that is indistinguishable from zebrin II in the mouse. Zebrin II expression in the pigeon cerebellum is prominent in Purkinje cells, including their dendrites, somata, axons, and axon terminals. Parasagittal stripes were apparent with bands of Purkinje cells that strongly expressed zebrin II (+ve) alternating with bands that expressed zebrin II weakly or not at all (−ve). The stripes were most prominent in folium IXcd, where there were seven +ve/−ve stripes, bilaterally. In folia VI–IXab, several thin stripes were observed spanning the mediolateral extent of the folia, including three pairs of +ve/−ve stripes that extended across the lateral surface of the cerebellum. In folium VI the zebrin II expression in Purkinje cells was stronger overall, resulting in less apparent stripes. In folia II–V, four distinct +ve/−ve stripes were apparent. Finally, in folia I (lingula) and X (nodulus) all Purkinje cells strongly expressed zebrin II. These data are compared with studies of zebrin II expression in other species, as well as physiological and neuroanatomical studies that address the parasagittal organization of the pigeon cerebellum. J. Comp. Neurol. 501:619–630, 2007.

Behavioral-state modulation of inhibition is context-dependent and cell type specific in mouse visual cortex

Cortical responses to sensory stimuli are modulated by behavioral state. In the primary visual cortex (V1), visual responses of pyramidal neurons increase during locomotion. This response gain was suggested to be mediated through inhibitory neurons, resulting in the disinhibition of pyramidal neurons. Using in vivo two-photon calcium imaging in layers 2/3 and 4 in mouse V1, we reveal that locomotion increases the activity of vasoactive intestinal peptide (VIP), somatostatin (SST) and parvalbumin (PV)-positive interneurons during visual stimulation, challenging the disinhibition model. In darkness, while most VIP and PV neurons remained locomotion responsive, SST and excitatory neurons were largely non-responsive. Context-dependent locomotion responses were found in each cell type, with the highest proportion among SST neurons. These findings establish that modulation of neuronal activity by locomotion is context-dependent and contest the generality of a disinhibitory circuit for gain control of sensory responses by behavioral state. DOI: http://dx.doi.org/10.7554/eLife.14985.001

The Optic Tectum of Birds: Mapping Our Way to Understanding Visual Processing

Over the past few decades there has been a massive amount of research on the geniculo-striate visual system in primates. However, studies of the avian visual system have provided a rich source of data contributing to our understanding of visual processing. In this paper we review the connectivity and function of the optic tectum (homolog of the superior colliculus) in birds. We highlight the retinotopic projections that the optic tectum has with the isthmal nuclei, and the functional topographic projections that the optic tectum has with the nucleus rotundus and entopallium (homologs of the pulvinar and extrastriate cortex, respectively) where retinotopy has been sacrificed. This work has been critical in our understanding of basic visual processes including attention, parallel processing, and the binding problem.

Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O2 levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O2 imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O2 in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O2 levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O2 in 3D tissue models.

Compartmentation of the cerebellar cortex of hummingbirds (Aves: Trochilidae) revealed by the expression of zebrin II and phospholipase Cβ4

The parasagittal organization of the mammalian cerebellar cortex into zones has been well characterized by immunohistochemical, hodological and physiological studies in recent years. The pattern of these parasagittal bands across the cerebellum is highly conserved across mammals, but whether a similar conservation of immunohistochemically defined parasagittal bands occurs within birds has remained uncertain. Here, we examine the compartmentation of the cerebellar cortex of a group of birds with unique cerebellar morphology-hummingbirds (Trochilidae). Immunohistochemical techniques were used to characterize the expression of zebrin II (aldolase C) and phospholipase C beta 4 (PLC beta 4) in the cerebellar cortex of two hummingbird species. A series of zebrin II immunopositive/immunonegative parasagittal stripes was apparent across most folia representing three major transverse zones: an anterior zone with a central stripe flanked by three lateral stripes on either side; a central zone of high/low immunopositive stripes; and a posterior zone with a central stripe flanked by four to six lateral stripes on either side. In addition, both folia I and X were uniformly immunopositive. The pattern of PLC beta 4 immunoreactivity was largely complementary-PLC beta 4 positive stripes were zebrin II negative and vice versa. The similarity of zebrin II expression between the hummingbirds and the pigeon indicates that the neurochemical compartmentation of the cerebellar cortex in birds is highly conserved, but species differences in the number and width of stripes do occur.

Organization of Visual Mossy Fiber Projections and Zebrin Expression in the Pigeon Vestibulocerebellum

Extensive research has revealed a fundamental organization of the cerebellum consisting of functional parasagittal zones. This compartmentalization has been well documented with respect to physiology, biochemical markers, and climbing fiber afferents. Less is known about the organization of mossy fiber afferents in general, and more specifically in relation to molecular markers such as zebrin. Zebrin is expressed by Purkinje cells that are distributed as a parasagittal array of immunopositive and immunonegative stripes. We examined the concordance of zebrin expression with visual mossy fiber afferents in the vestibulocerebellum (folium IXcd) of pigeons. Visual afferents project directly to folium IXcd as mossy fibers and indirectly as climbing fibers via the inferior olive. These projections arise from two retinal recipient nuclei: the lentiformis mesencephali (LM) and the nucleus of the basal optic root (nBOR). Although it has been shown that these two nuclei project to folium IXcd, the detailed organization of these projections has not been reported. We injected anterograde tracers into LM and nBOR to investigate the organization of mossy fiber terminals and subsequently related this organization to the zebrin antigenic map. We found a parasagittal organization of mossy fiber terminals in folium IXcd and observed a consistent relationship between mossy fiber organization and zebrin stripes: parasagittal clusters of mossy fiber terminals were concentrated in zebrin‐immunopositive regions. We also describe the topography of projections from LM and nBOR to the inferior olive and relate these results to previous studies on the organization of climbing fibers and zebrin expression. J. Comp. Neurol. 518:175–198, 2010.

Two optic flow pathways from the pretectal nucleus lentiformis mesencephali to the cerebellum in pigeons (Columba livia)

Neurons in the pretectal nucleus lentiformis mesencephali (LM) are involved in the analysis of optic flow. LM provides mossy fiber inputs to folia VI–VIII of the posterior cerebellum and IXcd of the vestibulocerebellum. Previous research has shown that the vestibulocerebellum is involved in visual‐vestibular integration supporting gaze stabilization. The function of folia VI–VIII in pigeons is not well understood; however, these folia receive input from a tectopontine system, which is likely involved with analyzing local motion as opposed to optic flow. We sought to determine whether the mossy fiber input from LM to IXcd differs from that to VI–VIII. Fluorescent retrograde tracers were injected into these folia, and the pattern of labeling in LM was observed. Large multipolar neurons were labeled throughout the rostrocaudal extent of LM. There was a clear mediolateral difference: 74.3% of LM neurons projecting to IXcd were located in the lateral subnucleus of LM (LMl), whereas 73.8% of LM neurons projecting to VI–VIII were found in medial LM (LMm). This suggests that the subnuclei of LM have differing roles. In particular, the LMl‐IXcd pathway is involved in generating the optokinetic response. We suggest that the pathway from LMm to VI–VIII is integrating optic flow and local motion to support various oculomotor and visuomotor behaviors, including obstacle avoidance during locomotion. J. Comp. Neurol. 499:732–744, 2006.

Organization of the cerebellum: correlating zebrin immunochemistry with optic flow zones in the pigeon flocculus.

The cerebellar cortex has a fundamental parasagittal organization that is apparent in the physiological response properties of Purkinje cells (PCs) and the expression of several molecular markers such as zebrin II (ZII). ZII is heterogeneously expressed in PCs such that there are sagittal stripes of high expression [ZII immunopositive (ZII+)] interdigitated with stripes of little or no expression [ZII immunonegative (ZII-)]. Several studies in rodents have suggested that climbing fiber (CF) afferents from an individual subnucleus in the inferior olive project to either ZII+ or ZII- stripes but not both. In this report, we show that this is not the case in the pigeon flocculus. The flocculus (the lateral half of folia IXcd and X) receives visual-optokinetic information and is important for generating compensatory eye movements to facilitate gaze stabilization. Previous electrophysiological studies from our lab have shown that the pigeon flocculus consists of four parasagittal zones: 0, 1, 2, and 3. PC complex spike activity (CSA), which reflects CF input, in zones 0 and 2 responds best to rotational optokinetic stimuli about the vertical axis (VA zones), whereas CSA in zones 1 and 3 responds best to rotational optokinetic stimuli about the horizontal axis (HA zones). In addition, folium IXcd consists of seven pairs of ZII+/- stripes. Here, we recorded CSA of floccular PCs to optokinetic stimuli, marked recording locations, and subsequently visualized ZII expression in the flocculus. VA neurons were localized to the P4+/- and P6+/- stripes and HA neurons were localized to the P5+/- and P7- stripes. This is the first study showing that a series of adjacent ZII+/- stripes are tied to specific physiological functions as measured in the responses of PCs to natural stimulation. Moreover, this study shows that the functional zone in the pigeon flocculus spans a ZII+/- stripe pair, which is contrary to the scheme proposed from rodent research.

Radial glial cells: key organisers in CNS development.

Radial glia are elongated bipolar cells present in the CNS during development. Our understanding of the unique roles these cells play has significantly expanded in the last decade. Historically, radial glial cells were primarily thought to provide an architectural framework for neuronal migration. Recent research reveals that radial glia play a more dynamic and integrated role in the development of the brain and spinal cord. They represent a major progenitor pool during early development and can give rise to a small population of multipotent cells in neurogenic niches of the adult CNS. Radial glial cells are a heterogeneous population, with divergent and often poorly understood roles across different brain and spinal cord regions during development; this heterogeneity extends to specialised adult subtypes, such as tanycytes, Müller glial cells and Bergman glial cells which possess morphological similarities to radial glial but play distinct functional roles in the CNS.