Extensive characterization of Tupaia belangeri neuropeptidome using an integrated mass spectrometric approach

Neuropeptidomics is used to characterize endogenous peptides in the brain of tree shrews (Tupaia belangeri). Tree shrews are small animals similar to rodents in size but close relatives of primates, and are excellent models for brain research. Currently, tree shrews have no complete proteome information available on which direct database search can be allowed for neuropeptide identification. To increase the capability in the identification of neuropeptides in tree shrews, we developed an integrated mass spectrometry (MS)-based approach that combines methods including data-dependent, directed, and targeted liquid chromatography (LC)-Fourier transform (FT)-tandem MS (MS/MS) analysis, database construction, de novo sequencing, precursor protein search, and homology analysis. Using this integrated approach, we identified 107 endogenous peptides that have sequences identical or similar to those from other mammalian species. High accuracy MS and tandem MS information, with BLAST analysis and chromatographic characteristics were used to confirm the sequences of all the identified peptides. Interestingly, further sequence homology analysis demonstrated that tree shrew peptides have a significantly higher degree of homology to equivalent sequences in humans than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Our results provide the first extensive characterization of the peptidome in tree shrews, which now permits characterization of their function in nervous and endocrine system. As the approach developed fully used the conservative properties of neuropeptides in evolution and the advantage of high accuracy MS, it can be portable for identification of neuropeptides in other species for which the fully sequenced genomes or proteomes are not available.     

BOLD temporal dynamics of rat superior colliculus and lateral geniculate nucleus following short duration visual stimulation

Background

The superior colliculus (SC) and lateral geniculate nucleus (LGN) are important subcortical structures for vision. Much of our understanding of vision was obtained using invasive and small field of view (FOV) techniques. In this study, we use non-invasive, large FOV blood oxygenation level-dependent (BOLD) fMRI to measure the SC and LGN''s response temporal dynamics following short duration (1 s) visual stimulation.

Methodology/Principal Findings

Experiments are performed at 7 tesla on Sprague Dawley rats stimulated in one eye with flashing light. Gradient-echo and spin-echo sequences are used to provide complementary information. An anatomical image is acquired from one rat after injection of monocrystalline iron oxide nanoparticles (MION), a blood vessel contrast agent. BOLD responses are concentrated in the contralateral SC and LGN. The SC BOLD signal measured with gradient-echo rises to 50% of maximum amplitude (PEAK) 0.2±0.2 s before the LGN signal (p<0.05). The LGN signal returns to 50% of PEAK 1.4±1.2 s before the SC signal (p<0.05). These results indicate the SC signal rises faster than the LGN signal but settles slower. Spin-echo results support these findings. The post-MION image shows the SC and LGN lie beneath large blood vessels. This subcortical vasculature is similar to that in the cortex, which also lies beneath large vessels. The LGN lies closer to the large vessels than much of the SC.

Conclusions/Significance

The differences in response timing between SC and LGN are very similar to those between deep and shallow cortical layers following electrical stimulation, which are related to depth-dependent blood vessel dilation rates. This combined with the similarities in vasculature between subcortex and cortex suggest the SC and LGN timing differences are also related to depth-dependent dilation rates. This study shows for the first time that BOLD responses in the rat SC and LGN following short duration visual stimulation are temporally different.     

Integrate-and-fire vs Poisson models of LGN input to V1 cortex: noisier inputs reduce orientation selectivity

One of the reasons the visual cortex has attracted the interest of computational neuroscience is that it has well-defined inputs. The lateral geniculate nucleus (LGN) of the thalamus is the source of visual signals to the primary visual cortex (V1). Most large-scale cortical network models approximate the spike trains of LGN neurons as simple Poisson point processes. However, many studies have shown that neurons in the early visual pathway are capable of spiking with high temporal precision and their discharges are not Poisson-like. To gain an understanding of how response variability in the LGN influences the behavior of V1, we study response properties of model V1 neurons that receive purely feedforward inputs from LGN cells modeled either as noisy leaky integrate-and-fire (NLIF) neurons or as inhomogeneous Poisson processes. We first demonstrate that the NLIF model is capable of reproducing many experimentally observed statistical properties of LGN neurons. Then we show that a V1 model in which the LGN input to a V1 neuron is modeled as a group of NLIF neurons produces higher orientation selectivity than the one with Poisson LGN input. The second result implies that statistical characteristics of LGN spike trains are important for V1’s function. We conclude that physiologically motivated models of V1 need to include more realistic LGN spike trains that are less noisy than inhomogeneous Poisson processes.     

Saccades differentially modulate human LGN and V1 responses in the presence and absence of visual stimulation

Saccades occur several times each second in normal human vision. The visual image moves across the retina at high velocity during a saccade, yet no blurring of the visual scene is perceived . Active suppression of visual input may account for this perceptual continuity, but the neural mechanisms underlying such saccadic suppression remain unclear. We used functional MRI to specifically examine responses in the lateral geniculate nucleus (LGN) and primary visual cortex (V1) during saccades. Activity in both V1 and LGN was strongly modulated by saccades. Furthermore, this modulation depended on whether visual stimulation was present or absent. In complete darkness, saccades led to reliable signal increases in V1 and LGN, whereas in the presence of visual stimulation, saccades led to suppression of visually evoked responses. These findings represent unequivocal evidence for saccadic suppression in human LGN and retinotopically defined V1 and are consistent with the earliest site of saccadic suppression lying at or before V1.     

A visual thalamocortical slice

We describe a thalamocortical slice preparation in which connectivity between the mouse lateral geniculate nucleus (LGN) and primary visual cortex (V1) is preserved. Through DiI injections in fixed brains we traced and created a three-dimensional model of the mouse visual pathways. From this computer model we designed a slice preparation that contains a projection from LGN to V1. We prepared brain slices with these predicted coordinates and demonstrated anatomical LGN-V1 connectivity in these slices after LGN tracer injections. We also revealed functional LGN-V1 connectivity by stimulating LGN electrically and detecting responses in layer 4 of V1 using calcium imaging, field potential recordings and whole-cell recordings. We also identified layer-4 neurons that receive direct thalamocortical input. Finally, we compared cortical activity after LGN stimulation with spontaneous cortical activity and found significant overlap of the spatiotemporal dynamics generated by both types of events.     

Repetitive and Retinotopically Restricted Activation of the Dorsal Lateral Geniculate Nucleus with Optogenetics

Optogenetics allows the control of cellular activity using focused delivery of light pulses. In neuroscience, optogenetic protocols have been shown to efficiently inhibit or stimulate neuronal activity with a high temporal resolution. Among the technical challenges associated with the use of optogenetics, one is the ability to target a spatially specific population of neurons in a given brain structure. To address this issue, we developed a side-illuminating optical fiber capable of delivering light to specific sites in a target nucleus with added flexibility through rotation and translation of the fiber and by varying the output light power. The designed optical fiber was tested in vivo in visual structures of ChR2-expressing transgenic mice. To assess the spatial extent of neuronal activity modulation, we took advantage of the hallmark of the visual system: its retinotopic organization. Indeed, the relative position of ganglion cells in the retina is transposed in the cellular topography of both the dorsal lateral geniculate nucleus (LGN) in the thalamus and the primary visual cortex (V1). The optical fiber was inserted in the LGN and by rotating it with a motor, it was possible to sequentially activate different neuronal populations within this structure. The activation of V1 neurons by LGN projections was recorded using intrinsic optical imaging. Increasing light intensity (from 1.4 to 8.9 mW/mm²) led to increasing activation surfaces in V1. Optogenetic stimulation of the LGN at different translational and rotational positions was associated with different activation maps in V1. The position and/or orientation of the fiber inevitably varied across experiments, thus limiting the capacity to pool data. With the optogenetic design presented here, we demonstrate for the first time a transitory and spatially-concise activation of a deep neuronal structure. The optogenetic design presented here thus opens a promising avenue for studying the function of deep brain structures.     

Optokinetic visual detection in the rat visual centres. A [14C]-2-deoxy-D-glucose study

Following forty-five min of binocular optokinetic stimulation (OKS) the autoradiographic maps of [14C]-2-deoxy-D-glucose (2DG) assumption of Long-Evans brain reveal clearly different patterns of optical density within visual centres. The most superficial layers of superior colliculus (SC) and a pretectal area including the nucleus of the optic tract (NOT) appear symmetrically, strongly darker than other visual structures such as lateral geniculate nucleus (LGN) and visual cortex (VC). Whereas the lack of metabolic increase at LGN and VC levels entirely confirms the non-involvement of the geniculo-cortical path in mediating the optomotor response following OKS in Rodents, it is postulated that the symmetrical increase of 2DG uptake even upon unidirectional OKS found even at pretectal level may represent a commissural transfer of visual information between homologous pretectal areas like the nuclei of the optic tract.     

猫外侧膝状体神经元HRP示踪结合生长抑素的免疫组织化学研究

本文采用HRP逆行示踪的方法,在猫视皮质的17区多点微量注射30％HRP,逆行标记外侧膝状体核（LGN）至视皮质的中继神经元,继用免疫金银法作生长抑素（SS）免疫组织化学,试图双标记LGN的中断神经元。结果显示：光镜下HRP标记细胞与SS是性细胞清晰可辨,HRP标记细胞内为较粗的棕色颗粒,分布于胞浆和树突基部;而SS免疫阳性细胞内的颗粒为银染黑色颗粒;HRP和SS双标记神经元内,上述两种颗粒共存,警备LGN的A、A1和C板层均有SS免疫阳性凶的分布;HRP标记细胞分布于A和A1板层;双标记神经元位于A和A1板层;C板层未见。本文结合以前的研究认为,SS在LGN至视皮质传导通路中的作用,可能与视觉信息的传递和调制有关。     

γ节律振荡机制在视觉研究中的新进展

γ节律振荡是大脑皮质中常见的,频率在30～80 Hz之间的神经振荡模式,在初级视觉通道中能观察到多种起源的γ节律振荡.在小鼠、猫与猴V1的视觉诱发的γ节律振荡主要起源于L2/3和L4B,并对刺激参数敏感.猫与小鼠初级视觉通道(视网膜、LGN与V1)中观察到起源于视网膜由亮度诱发的高频γ节律振荡;在猴LGN却没有观察到γ节律振荡,而在V1上记录到亮度诱发的γ活动.γ节律振荡的产生与抑制性中间神经元网络有重要的关系,其中抑制性中间神经元中PV细胞被认为与自发γ节律振荡的产生相关. SOM细胞的参与对低频γ节律振荡(20～40 Hz)的产生起到关键作用;而光栅诱发的高频γ节律振荡(65～80 Hz)主要与PV细胞有关.动物在不同生理状态、发育阶段与脑疾病状态下光栅诱发的γ节律振荡存在较大差异,反映大脑对视觉信息加工的变化.     

Predictive Feedback Can Account for Biphasic Responses in the Lateral Geniculate Nucleus

Biphasic neural response properties, where the optimal stimulus for driving a neural response changes from one stimulus pattern to the opposite stimulus pattern over short periods of time, have been described in several visual areas, including lateral geniculate nucleus (LGN), primary visual cortex (V1), and middle temporal area (MT). We describe a hierarchical model of predictive coding and simulations that capture these temporal variations in neuronal response properties. We focus on the LGN-V1 circuit and find that after training on natural images the model exhibits the brain's LGN-V1 connectivity structure, in which the structure of V1 receptive fields is linked to the spatial alignment and properties of center-surround cells in the LGN. In addition, the spatio-temporal response profile of LGN model neurons is biphasic in structure, resembling the biphasic response structure of neurons in cat LGN. Moreover, the model displays a specific pattern of influence of feedback, where LGN receptive fields that are aligned over a simple cell receptive field zone of the same polarity decrease their responses while neurons of opposite polarity increase their responses with feedback. This phase-reversed pattern of influence was recently observed in neurophysiology. These results corroborate the idea that predictive feedback is a general coding strategy in the brain.     

Isolation of [Pro2,Met13]somatostatin-14 and somatostatin-14 from the frog brain reveals the existence of a somatostatin gene family in a tetrapod.

Two somatostatin-related peptides were isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the most abundant component was identical to that of mammalian somatostatin-14. The primary structure of the second component, present in approximately 5% of the abundance of somatostatin-14, was established as Ala-Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Met-Cys. This sequence shows two substitutions (Pro for Gly2 and Met for Ser13) compared with mammalian somatostatin-14. The data provide evidence for a somatostatin gene family in tetrapods as well as in teleost fish.     

Structural, functional, and evolutionary characterization of novel members of the allatostatin receptor family from insects

By using degenerate primers based on known mammalian somatostatin receptors and the recently identified Drosophila allatostatin receptors (AlstR), we have cloned a novel receptor for the neuropeptide, allatostatin, from the cockroach Periplaneta americana. The receptor exhibits about 60% amino acid identity in the transmembrane regions when compared to the two known AlstRs from Drosophila melanogaster. In addition, two cDNA fragments were obtained from the stick insect Carausius morosus, one of which is similar to Drosophila AlstR, whereas the other is more similar to mammalian somatostatin receptors. Functional expression in Xenopus oocytes shows that the Periplaneta-AlstR exhibits high affinity to endogenous cockroach allatostatin peptides. Studies with synthetic peptides demonstrate that agonistic activity is mediated by the conserved C-terminal pentapeptide YXFGL-amide; in this sequence, amidation of the C-terminus is obligatory to maintain affinity. Thus, our studies provide a molecular basis for understanding the widespread biological activities of the allatostatin peptides.     

The Expression Patterns of MHC Class I Molecules in the Developmental Human Visual System

It has been considered that healthy neurons in central nervous system (CNS) do not express major histocompatibility complex (MHC) class I molecules. However, recent studies clearly demonstrated the expression of functional MHC class I in the mammalian embryonic, neonatal and adult brain. Until now, it is still unknown whether MHC I molecules are expressed in the development of human brain. We collected nine human brain tissues from fetuses aged from 21 to 31 gestational weeks (GW), one newborn of postnatal 55 days and one adult. The expression of MHC class I molecules was detected during the development of visual system in human brain by immunohistochemistry and immunofluorescence. MHC class I proteins were located at lateral geniculate nucleus (LGN) and the expression was gradually increased from 21 GW to 31 GW and reached high levels at 30–31 GW when fine-scale refinement phase was mediated by neural electric activity. However, there was no expression of MHC class I molecules in the visual cortical cortex during all the developmental stages examined. We also concluded that MHC class I molecules were mainly expressed in neurons but not in astrocytes at LGN. In the developing visual system, the expression of β2M protein on neurons was not found in our study.     

Proteomic characteristics of the liver and skeletal muscle in the Chinese tree shrew (Tupaia belangeri chinensis)

Valid animal models are useful for studying the pathophysiology of specific disorders, such as neural disease, diabetes and cancer. Previous molecular phylogeny studies indicate that the tree shrew is in the same order as (or a close sister to) primates, and thus may be an ideal model in which to study human disease. In this study, the proteome of liver and muscle tissue in tree the shrew was identified by combining peptide fractionation and LC-MS/MS identification. In total, 2146 proteins were detected, including 1759 proteins in liver samples and 885 proteins in skeletal muscle samples from the tree shrew. Further sub-source analysis revealed that nearly half of the identified proteins (846 proteins and 418 proteins) were derived from human database. In this study, we are the first to describe the characteristics of the proteome from the liver and skeletal muscle of the tree shrew. Phylogenetic tree analysis based on these proteomic data showed that the tree shrew is closer to primates (human) than to glires (the mouse and rat).     

Peptides and neurotransmission in the central nervous system

Radioimmunoassays of brain extracts have shown that several peptides occur in high concentrations in the CNS. The releasing-factor peptides TRF, LRF, somatostatin, CRF and GRF have the highest concentration in the hypothalamic extracts. High levels of somatostatin, CCK octapeptide, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) are found in cortical extracts. Substance P, CCK, NPY, and enkephalins are present in high concentrations in basal ganglia and mesolimbic areas. Pharmacological doses of these peptides result in several behavioural and vegetative effects. Immunocytochemical studies show that the CNS peptides are localised in neurones and in synaptic vesicles. In vitro studies with brain tissues show that peptides are capable of modifying the ongoing classical neurotransmission. In depressive patients several neuropeptides (CCK, CRF and NPY) have been shown to have low CSF levels. Patients dying of senile dementia have low cortical levels of somatostatin, CRF and substance P. In schizophrenic patients CCK peptides have shown to improve some symptoms. At present the therapeutic potentials of peptides are poorly known. More studies are required to understand their role in neurotransmission and related pathological states.     

Recent advances in mammalian RFamide peptides: the discovery and functional analyses of PrRP, RFRPs and QRFP

Since the first discovery of a peptide with RFamide structure at its C-terminus (i.e., an RFamide peptide) from an invertebrate in 1977, numerous studies on RFamide peptides have been conducted, and a variety have been identified in various phyla throughout the animal kingdom. The first reported mammalian RFamide peptides were neuropeptide FF (NPFF) and neuropeptide AF (NPAF) in 1985. However, for many years after this, no new novel RFamide peptides were identified in mammals. A breakthrough in discovering mammalian RFamide peptides was made possible by reverse pharmacology on the basis of orphan G protein-coupled receptor (GPCR) research. The first report of an RFamide peptide identified from orphan GPCR research was prolactin (PRL)-releasing peptide (PrRP) in 1998. To date, a total of five RFamide peptide genes have been discovered in mammals. Orphan GPCR research has contributed considerably to the identification of these peptides and their receptor genes. This paper examines these mammalian RFamide peptides focusing especially on PrRP, RFamide-related peptides (RFRPs) and, the most recently identified, pyroglutamylated RFamide peptide (QRFP), the discovery of all of which the authors were at least partly involved in. We review here the strategies employed for the identification of these peptides and examine their characteristics, tissue distribution, receptors and functions.     

Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus)

Summary Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities. These results are consistent with the hypothesis that peptides having significant sequence homology with human neuropeptide Y and porcine neuropeptide Y are present in the nerve fibers that permeate the islet.     

Influence of the motor cortex on lateral geniculate responses evoked in the cat by contralateral superior colliculus stimulation

It is shown that in nembutal anesthetized cats, a single stimulation of motor cortex (MC) causes a response in lateral geniculate nucleus (LGN). The development of this response had a conditioning effect on the LGN response evoked by stimulation of the contralateral superior colliculus (SC), markedly inhibiting it. The degree of this inhibition depended on the time interval between the cortical conditioning stimulation and the tectal test stimulation. A single conditioning MC stimulation did not noticeably change the LGN responses evoked by a light stimulus, but markedly inhibited visual responses from deep SC layers (those regions which on stimulation gave rise to LGN responses). From the results, it is suggested that the MC monitors the execution of tectal influences on LGN function at the tectal level rather than the geniculate level, and it is precisely by this means that it regulates saccadic suppression of LGN function, in the realization of which, as presumed earlier, the SC takes part.A. I. Karaev Institute of Physiology, Azerbaijan Academy of Sciences, Baku. Translated from Neirofiziologiya, Vol. 24, No. 4, July–August 1992.     

Complementary and dynamic type II cadherin expression associated with development of the primate visual system

The middle temporal visual area (MT, also known as V5) is a visual association area that is particularly evolved in the primate brain. The MT receives input from the primary visual area (V1), constitutes part of the dorsal visual pathway, and plays an essential role in processing motion. Connections between the MT and V1 in the primate brain are formed after birth, and are related to the maturation of visual system. However, it remains to be determined what molecular mechanisms control the formation and maturation of the visual system. Cadherins are transmembrane proteins, originally isolated as cell adhesion molecules, which have multiple roles in synapse formation and function. To investigate potential involvement of cadherins in development of the primate visual system, we examined type II cadherin expression (cadherin‐6, ‐8, ‐12) in cortical and thalamic visual areas of pre‐ and postnatal brains of the common marmoset (Callithrix jacchus). In the prenatal brain, cadherin‐6 was dominantly expressed in the pulvino‐MT pathway whereas cadherin‐8 was dominant in the lateral geniculate nucleus (LGN)‐V1 pathway. During postnatal development, there was a downregulation of cadherin‐6 and upregulation of cadherin‐8 expression in the MT. The timing of this cadherin exchange preceded the development of V1‐MT connections. Our results suggest the possibility that changes in cadherin expression are involved in the development of the primate visual system, and that a switch in cadherin expression may be a general mechanism to control neural plasticity of highly cognitive abilities.     

Evolutionary origin and divergence of PQRFamide peptides and LPXRFamide peptides in the RFamide peptide family. Insights from novel lamprey RFamide peptides

Among the RFamide peptide groups, PQRFamide peptides, such as neuropeptide FF (NPFF) and neuropeptide AF (NPAF), share a common C-terminal Pro-Gln-Arg-Phe-NH(2) motif. LPXRFamide (X = L or Q) peptides, such as gonadotropin-inhibitory hormone (GnIH), frog growth hormone-releasing peptide (fGRP), goldfish LPXRFamide peptide and mammalian RFamide-related peptides (RFRPs), also share a C-terminal Leu-Pro-Leu/Gln-Arg-Phe-NH(2) motif. Such a similar C-terminal structure suggests that these two groups may have diverged from a common ancestral gene. In this study, we sought to clarify the evolutionary origin and divergence of these two groups, by identifying novel RFamide peptides from the brain of sea lamprey, one of only two extant groups of the oldest lineage of vertebrates, Agnatha. A novel lamprey RFamide peptide was identified by immunoaffinity purification using the antiserum against LPXRFamide peptide. The lamprey RFamide peptide did not contain a C-terminal LPXRFamide motif, but had the sequence SWGAPAEKFWMRAMPQRFamide (lamprey PQRFa). A cDNA of the precursor encoded one lamprey PQRFa and two related peptides. These related peptides, which also had the C-terminal PQRFamide motif, were further identified as mature endogenous ligands. Phylogenetic analysis revealed that lamprey PQRFamide peptide precursor belongs to the PQRFamide peptide group. In situ hybridization demonstrated that lamprey PQRFamide peptide mRNA is expressed in the regions predicted to be involved in neuroendocrine and behavioral functions. This is the first demonstration of the presence of RFamide peptides in the agnathan brain. Lamprey PQRFamide peptides are considered to have retained the most ancestral features of PQRFamide peptides.