Gergely Zachar

The plover neurotranscriptome assembly: transcriptomic analysis in an ecological model species without a reference genome

We assembled a de novo transcriptome of short‐read Illumina RNA‐Seq data generated from telencephalon and diencephalon tissue samples from the Kentish plover, Charadrius alexandrinus. This is a species of considerable interest in behavioural ecology for its highly variable mating system and parental behaviour, but it lacks genomic resources and is evolutionarily distant from the few available avian draft genome sequences. We assembled and identified over 21 000 transcript contigs with significant expression in our samples, showing high homology to exonic sequences in avian draft genomes. From these, we identified >31 000 high‐quality SNPs and > 2500 simple sequence repeats (SSRs). We also analysed expression patterns in our data to identify potential candidate genes related to differences in male and female behaviour, identifying over 200 nonoverlapping putative autosomal transcripts that show significant expression differences between males and females. Gene ontology analysis revealed that female‐biased transcripts were significantly enriched for cerebral functions related to learning, cognition and memory, and male‐biased transcripts were mostly enriched for terms related to neural function such as neuron projection and synapses. This data set provides one of the first de novo transcriptome assemblies from non‐normalized short‐read next‐generation data and outlines an effective strategy for measuring sequence and expression variability simultaneously without the aid of a reference genome.

Chiral analysis of amino acid neurotransmitters and neuromodulators in mouse brain by CE-LIF.

Chiral CE method has been developed for quantitative determination of d‐amino acid modulators of NMDA glutamate receptor; d‐serine and d‐aspartate along with l‐glutamate and l‐aspartate in biological samples. These ligands are suggested to be involved in regulation of NMDA receptor related brain functions, such as neurogenesis, neuronal plasticity, and memory formation. For sensitive determination of the amino acids LIF detection was chosen, and a fluorogenic reagent, 7‐fluoro‐4‐nitro‐2,1,3‐benzoxadiazole was used for derivatization. An amino‐modified β‐CD, 6‐monodeoxy‐6‐mono(3‐hydroxy)propylamino‐β‐CD (HPA‐β‐CD) was applied as chiral selector. Determinations were accomplished in a polyacrylamide coated capillary and reverse polarity was used for the analysis of the negatively charged analytes. The method was optimized and validated; 6 mM HPA‐β‐CD in 50 mM HEPES buffer, pH 7 was appropriate to achieve baseline separation of the analytes. The limit of quantification with acceptable accuracy is 0.05 μM for both d‐amino acids. The method was used for the determination of d‐aspartate and d‐serine content in various brain regions of adult mice.

Afferent connections of septal nuclei of the domestic chick (Gallus domesticus): A retrograde pathway tracing study

The afferents to the septum of the domestic chicken were studied using retrograde tracers, rhodamine conjugated latex bead or Fast Blue, placed in different septal subregions. The results were verified by anterograde tracer injections deposited to selected areas. The main telencephalic afferents to the septum arise ipsilaterally from the hippocampal formation, dorsolateral corticoid area, piriform cortex, amygdaloid pallium, and the ventral pallidum. Contralateral afferents originate from the lateral septum and the amygdaloid pallium. A massive bilateral projection arises from the lateral hypothalamus. Other hypothalamic afferents arise from the periventricular, paraventricular and anterior medial nuclei, and the premammillary and mammillary areas. The dorsal thalamic nuclei (dorsal medial anterior and posterior) and the reticular dorsal nuclei also contribute septal afferents. Brainstem afferents arise bilaterally from the ventral tegmental area, substantia nigra, central gray, A8, locus coeruleus, ventral subcoeruleus nucleus, and raphe nuclei. The main terminal fields for septal afferents lie in the lateral septal nucleus and the belt of medial septal nucleus. The core of the latter is invaded mainly by fibers from the brainstem, presumably belonging to the ascending activating system. The septal afferents of the chicken are largely similar to those of other avian and nonavian species. The most prominent differences with previous pigeon data were found in the subregional selectivity of the hippocampal formation, dorsolateral corticoid area, mammillary nuclei, some dorsal thalamic nuclei, substantia nigra, and subcoeruleus nuclei in their projections to defined septal nuclei. J. Comp. Neurol. 511:109–150, 2008.

Postnatal changes in the distribution and density of neuronal nuclei and doublecortin antigens in domestic chicks (Gallus domesticus)

To understand better the rate of neurogenesis and the distribution of new neurons in posthatch domestic chicks, we describe and compare the expression of the neuronal nuclei protein (NeuN, a.k.a. Fox‐3) and doublecortin antigens in the whole brain of chicks 2 days, 8 days, and 14 weeks posthatch. In the forebrain ventricular and paraventricular zones, the density of bromodeoxyuridine‐, NeuN‐, and doublecortin‐labeled cells was compared between chicks 24 hours and 7 days after an injection of bromodeoxyuridine (2 and 8 days posthatch, respectively). The distribution of NeuN‐labeled neurons was similar to Nissl‐stained tissue, with the exception of some areas where neurons did not express NeuN: cerebellar Purkinje cells and olfactory bulb mitral cells. The ventral tegmental area of 2‐day‐old chicks was also faintly labeled. The distribution of doublecortin was similar at all timepoints, with doublecortin‐labeled profiles located throughout all forebrain areas as well as in the cerebellar granule cell layer. However, doublecortin labeling was not detectable in any midbrain or brainstem areas. Our data indicate that a significant number of new neurons is still formed in the telencephalon of posthatch domestic chicks, whereas subtelencephalic areas (except for the cerebellum) finish their neuronal expansion before hatching. Most newly formed cells in chicks leave the paraventricular zone after hatching, but a pool of neurons stays in the vicinity of the ventricular zone and matures in situ within 7 days. Proliferating cells often migrate laterally along forebrain laminae into still‐developing brain areas. J. Comp. Neurol., 2012.

The organisation of the basal ganglia in the domestic chick (Gallus domesticus): Anatomical localisation of DARPP-32 in relation to glutamate

Subpallial structures are highly conserved across the different vertebrate species. They are instrumental in the neural processing relevant to adaptive learning, decision making, motivation and behavioural strategies. Of the striatal regions, our attention has been focussed on the medial and ventral striatum (MSt), now parcellated into subregions, and also including the nucleus accumbens (Ac). Similar to mammals, the avian Ac and MSt receive glutamatergic input from the pallium and dopaminergic input from the substantia nigra and ventral tegmental area. Coincidence between glutamatergic and dopaminergic synaptic activities in the ventral/medial striatum, including the Ac, is required for memory to be formed for a given pairing of stimulus and a hedonic quality or behavioural salience. The underlying mechanism involves the activation of NMDA and dopaminergic receptors, as well as the phosphorylation of dopamine-cAMP-regulated phosphoprotein (DARPP-32). Using quantitative electron microscopy of chick specimens double-labelled against glutamate and DARPP-32 we observed direct synaptic connections between glutamate immunoreactive axon terminals and DARPP-32 labelled dendrites in the MSt and also in the posterolateral telencephalon (nidopallium caudolaterale, a prefrontal cortex equivalent region) and the hippocampus. Glutamate immunoreactive axons synapsed with both DARPP-32 immunoreactive (DARPP-32+) and DARPP-32 negative (DARPP-32-) dendrites, forming asymmetrical junctions, in all brain regions observed. The existence of direct synaptic contacts between excitatory amino acid containing axon terminals and DARPP-32 containing dopaminoceptive neurons of the chicken MSt underlines the functional homology with mammalian striatal systems.

Chondroitin sulphate proteoglycan-based perineuronal net establishment is largely activity-independent in chick visual system☆

Extracellular matrix components consisting of large aggregating chondroitin sulphate proteoglycans accumulate around neuronal perikarya to establish perineuronal nets. These perineuronal nets surround subpopulations of neurons in many vertebrates including man. In chickens, perineuronal nets show very fast matrix maturation after hatching which is probably due to the rapid establishment of neuronal morphology and immediate functional and behavioural performance of the animals. In mammals, maturation of extracellular matrix including perineuronal nets largely depends upon specific afferent activation. The present study shows that extracellular matrix maturation in mesencephalic, diencephalic and telencephalic visual centers of chicks tectofugal system is not principally determined by light activation. Perineuronal nets show an equally developed phenotypic character on monocularly light deprived animals in all investigated brain regions. Results suggest that establishment of extracellular matrix and perineuronal nets are largely activity-independent in the investigated precocial bird.

Abundance and location of DARPP-32 in striato-tegmental circuits of domestic chicks.

The striatum is reciprocally connected to the brainstem dopaminergic nuclei and receives a strong dopaminergic input. In the present study the spatial relation between the dopaminergic and dopaminoceptive structures of the avian medial striatum (formerly: lobus parolfactorius) was observed by confocal laser scanning microscope in the domestic chick (Gallus domesticus). We also analysed the connections in the area ventralis tegmentalis and the substantia nigra. To label the dopaminergic structures, anti-tyrosine hydroxylase was used and DARPP-32 (dopamine and cAMP regulated phosphoprotein) was a marker of dopaminoceptive elements. The tyrosine hydroxylase positive fibres formed baskets of juxtapositions around the DARPP-32 containing cells of the medial striatum. However, such baskets were also observed to juxtapose DARPP-32 immunonegative cells. In the tegmentum, DARPP-32 was observed in axons descending from the telencephalon via the ansa lenticularis. These varicose fibers innervated the ventral tegmental area and substantia nigra and were often juxtaposed to dopaminergic neurons and dendrites. Approximately 40% of the striatal projection neurons targeting the ventral tegmentum, and 60% of striatal projection neurons targeting the nigra were immunoreactive to DARPP-32, as revealed by retrograde pathway tracing with Fast Blue. Endogenous dopamine may exert a retrograde synaptic effect on the afferent striato-tegmental fibers, apart from the reported extrasynaptic action. The abundance of juxtapositions observed in the avian brainstem and medial striatum corroborates the possibility of reciprocal striato-tegmental circuits, relevant to the reinforcement of behaviour.

Comparison of quantitative performance of three fluorescence labels in CE/LIF analysis of aspartate and glutamate in brain microdialysate.

Three different fluorescent tags have been compared for the quantitative analysis of aspartate and glutamate in brain microdialysate samples. Separation conditions have been optimized to achieve short analysis time using reversed polarity separation in coated capillary. Method validation has revealed similar quantification limit of 0.1 μM of analytes using either of the labels, although LOD values were different: 7.8–9.8 nM for 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole, 3.5 nM for fluorescein‐5‐isothiocyanate and 1.3–1.5 nM for carboxyfluorescein succinimidyl ester derivatives. The almost two orders of magnitude difference between LOD and LOQ values is likely due to the unreliable derivatization reaction at low sample concentration. Based on the superior stability, FITC derivatization was used for the analysis of biological samples. The applicability of the method has been demonstrated by analyzing basal and potassium evoked amino acid concentrations in individual brain microdialysate samples.

Determination of stanozolol and 3′-hydroxystanozolol in rat hair, urine and serum using liquid chromatography tandem mass spectrometry

BackgroundAnabolic androgenic steroids, such as stanozolol, are typically misused by athletes during preparation for competition. Out-of-competition testing presents a unique challenge in the current anti-doping detection system owing to logistic reasons. Analysing hair for the presence of a prohibited drug offers a feasible solution for covering the wider window in out-of-competition testing. To assist in vivo studies aiming to establish a relationship between drug levels detected in hair, urine and blood, sensitive methods for the determination of stanozolol and its major metabolite 3′-hydroxystanozolol were developed in pigmented hair, urine and serum, using brown Norway rats as a model system and liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsFor method development, spiked drug free rat hair, blood and urine samples were used. The newly developed method was then applied to hair, urine and serum samples from five brown Norway rats after treatment (intraperitoneal) with stanozolol for six consecutive days at 5.0 mg/kg/day. The assay for each matrix was linear within the quantification range with determination coefficient (r2) values above 0.995. The respective assay was capable of detecting 0.125 pg/mg stanozolol and 0.25 pg/mg 3′-hydroxystanozolol with 50 mg hair; 0.063 ng/mL stanozolol and 0.125 ng/mL 3′-hydroxystanozolol with 100 μL of urine or serum. The accuracy, precision and extraction recoveries of the assays were satisfactory for the detection of both compounds in all three matrices. The average concentrations of stanozolol and 3′-hydroxystanozolol, were as follows: hair = 70.18 ± 22.32 pg/mg and 13.01 ± 3.43 pg/mg; urine = 4.34 ± 6.54 ng/mL and 9.39 ± 7.42 ng/mL; serum = 7.75 ± 3.58 ng/mL and 7.16 ± 1.97 ng/mL, respectively.ConclusionsThe developed methods are sensitive, specific and reproducible for the determination of stanozolol and 3′-hydroxystanozolol in rat hair, urine and serum. These methods can be used for in vivo studies further investigating stanozolol metabolism, but also could be extended for doping testing. Owing to the complementary nature of these tests, with urine and serum giving information on recent drug use and hair providing retrospective information on habitual use, it is suggested that blood or urine tests could accompany hair analysis and thus avoid false doping results.

Context-dependent prey avoidance in chicks persists following complete telencephalectomy.

Young naive domestic chicks readily attack green insects and avoid insects painted red but show no discrimination of the same colours when applied to fruit-like objects, a discrimination that has been depicted as context-dependent preference. To study the neural representation of such preference we performed bilateral telencephalectomy on 1-day-old domestic chicks and tested them on an unlearned prey discrimination paradigm. Here we show that following complete decerebration, young domestic chicks preferentially peck at red fruit versus red insects and tend to choose green insects over green fruit indistinguishably from unoperated chicks. The present study provides the first direct evidence that sophisticated context-dependent, unlearned colour preference is processed by subtelencephalic areas of an amniote species.