Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time¹. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)^2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning^4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs^6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode³. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings⁸. We provide details of our experimental setup and present representative recording traces for each of these techniques.     

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.     

Effect of antigen binding affinity and effector function on the pharmacokinetics and pharmacodynamics of anti-IgE monoclonal antibodies

Modulating the binding affinities to IgE or changing the FcγR binding properties of anti-IgE antibodies offers an opportunity to enhance the therapeutic potential of anti-IgE antibodies, but the influence of increased affinity to IgE or reduced Fc effector function on the pharmacological properties of anti-IgE therapies remains unclear. Our studies were designed to characterize the pharmacokinetics, pharmacodynamics and immune-complex distribution of two high-affinity anti-IgE monoclonal antibodies, high-affinity anti-IgE antibody (HAE) 1 and 2, in mice and monkeys. HAE1, also known as PRO98498, is structurally similar to omalizumab (Xolair®), a humanized anti-IgE IgG1 marketed for the treatment of asthma, but differs by 9 amino acid changes in the complementarity-determining region resulting in a 23-fold improvement in affinity. HAE2 is similar to HAE1, but its Fc region was altered to reduce binding to Fcγ receptors. As expected given the decreased binding to Fcγ receptors, systemic exposure to pre-formed HAE2:IgE complexes in mice was greater (six-fold) and distribution to the liver lower (four-fold) compared with HAE1:IgE complexes. In monkeys, systemic exposure to HAE1 was similar to that previously observed for omalizumab in this species, but required comparatively lower serum drug concentrations to suppress free IgE levels. HAE2 treatment resulted in greater exposure and greater increase of total IgE, relative to HAE1, because of decreased clearance of HAE2:IgE complexes. Overall, these data suggest that increased binding affinity to IgE may provide a more effective therapeutic for asthma patients, and that retaining FcγR binding of the anti-IgE antibody is important for elimination of anti-IgE:IgE complexes.     

A novel pattern recognition protein of the Chinese oak silkmoth,Antheraea pernyi,is involved in the pro-PO activating system

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein. [BMB Reports 2013; 46(7): 358-363]     

TcLr45凝集素类受体蛋白激酶基因克隆与序列分析

在小麦抗叶锈近等基因系TcLr45中扩增出一条凝集素类受体蛋白激酶基因片段。以该序列为靶序列进行3’和5’RCAE-PCR,获得该基因cDNA全长片段为2347bp,该序列具有完整的开放阅读框,共编码730个氨基酸,命名为LecRK-LR45。经软件分析, LecRK-LR45具有典型的凝集素类受体蛋白激酶结构,属于L类型。聚类分析结果表明,LecRK-LR45与山羊草、小麦（Lr34）中的凝集素类受体蛋白激酶基因具有较近的亲缘关系,与玉米、拟南芥的亲缘关系较远。该基因的克隆为研究小麦中凝集素类受体蛋白激酶的功能奠定基础。     

Sensorimotor gating and spatial learning in α7‐nicotinic receptor knockout mice

The role of acetylcholine and specific nicotinic receptors in sensorimotor gating and higher cognitive function has been controversial. Here, we used a commercially available mouse with a null mutation in the Chrna7^tm1Bay gene [α7‐nicotinic acetylcholine receptor (nAChR) knockout (KO) mouse] in order to assess the role of the α7‐nAChR in sensorimotor gating and spatial learning. We examined prepulse inhibition (PPI) of startle and nicotine‐induced enhancement of PPI. We also tested short‐ and long‐term habituation of the startle response as well as of locomotor behaviour in order to differentiate the role of this receptor in the habituation of evoked behaviour (startle) vs. motivated behaviour (locomotion). To address higher cognition, mice were also tested in a spatial learning task. Our results showed a mild but consistent PPI deficit in α7‐nAChR KO mice. Furthermore, they did not show nicotine‐induced enhancement of startle or PPI. Short‐ and long‐term habituation was normal in KO mice for both types of behaviours, evoked or motivated, and they also showed normal learning and memory in the Barnes maze. Thorough analysis of the behavioural data indicated a slightly higher degree of anxiety in α7‐nAChR KO mice; however, this could only be partially confirmed in an elevated plus maze test. In summary, our data suggest that α7‐nAChRs play a minor role in PPI, but seem to mediate nicotine‐induced PPI enhancement. We found no evidence to suggest that they are important for habituation or spatial learning .     

A study of the possible association between adenosine A2A receptor gene polymorphisms and attention‐deficit hyperactivity disorder traits

The adenosine A_2A receptor (ADORA2A) is linked to the dopamine neurotransmitter system and is also implicated in the regulation of alertness, suggesting a potential association with attention‐deficit hyperactivity disorder (ADHD) traits. Furthermore, animal studies suggest that the ADORA2A may influence ADHD‐like behavior. For that reason, the ADORA2A gene emerges as a promising candidate for studying the etiology of ADHD traits. The aim of this study was to examine the relationship between ADORA2A gene polymorphisms and ADHD traits in a large population‐based sample. This study was based on the Child and Adolescent Twin Study in Sweden (CATSS), and included 1747 twins. Attention‐deficit hyperactivity disorder traits were assessed through parental reports, and samples of DNA were collected. Associations between six single nucleotide polymorphisms (SNPs) and ADHD traits were examined, and results suggested a nominal association between ADHD traits and three of these SNPs: rs3761422, rs5751876 and rs35320474. For one of the SNPs, rs35320474, results remained significant after correction for multiple comparisons. These results indicate the possibility that the ADORA2A gene may be involved in ADHD traits. However, more studies replicating the present results are warranted before this association can be confirmed .     

Genetic variation in dopamine‐related gene expression influences motor skill learning in mice

Several neurodevelopmental disorders with a strong genetic basis, including attention‐deficit/hyperactivity disorder, autism spectrum disorders and developmental coordination disorder, involve deficits in fine motor skills. This phenotype may depend on heritable variation in components of the dopamine (DA) system, which is known to play a critical role in motor skill learning. In this study, we took advantage of two inbred strains of mice (BALB/c and C57BL/6) that differ markedly in the number of midbrain DA neurons in order to investigate the influence of such naturally occurring genetic variation on the acquisition and performance of fine motor skills. Gene expression analysis of midbrain, frontal cortex and striatum showed significant differences in the expression of presynaptic and postsynaptic dopaminergic (DAergic) markers (e.g. tyrosine hydroxylase, DA transporter, DA D4 receptor, DA D5 receptor and DARPP‐32) between these two strains. BALB/c mice had lower learning rate and performance scores in a complex skilled reaching task when compared with C57BL/6 mice. A negative correlation was found between the motor learning rate and level of DARPP‐32 mRNA expression in the frontal cortex contralateral to the trained forelimb. The rate of motor learning was also negatively correlated with the levels of DARPP‐32 and DA D1 receptor mRNAs in the striatum. Our results suggest that genetically driven variation in frontostriatal DAergic neurotransmission is a major contributor to individual differences in motor skill learning. Moreover, these findings implicate the D1R/cAMP/DARPP‐32 signaling pathway in those neurodevelopmental disorders that are associated with fine motor skill deficits.