Enhanced green fluorescent protein (GFP) irreversibly loses not only fluorescence but also antigenicity recognized with conventional anti-GFP antibodies by heat denaturation. This hinders combinatory applications of the GFP immunodetection technique with heat-requiring procedures, such as in situ hybridization histochemistry, antigen retrieval, and Western blot. Here we produced new rabbit and guinea pig antibodies against heat-denatured GFP. The polyclonal antibodies affinity-purified with the antigen column detected a single band corresponding to the molecular size of GFP in Western blot analysis, with mouse brain expressing GFP from the GAD67 locus. By immunofluorescence labeling, the new antibodies detected GFP molecules in heat (≥70C)-treated sections but not in untreated sections of the mouse brain. When the sections were incubated at ≥37C with in situ hybridization buffer containing 50% formamide, a denaturing reagent, the sections lost immunoreactivity with the conventional anti-GFP antibodies but acquired immunoreactivity with the new antibodies to heat-denatured GFP. Finally, GFP immunofluorescence was successfully visualized with the new antibodies in sections of the GFP-expressing mice labeled by fluorescence in situ hybridization histochemistry against GAD67 mRNA. Thus, the antibodies produced in this study may provide an opportunity to combine GFP immunodetection with procedures requiring heat treatment. This manuscript contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.
J. Neurochem. (2010) 113 , 1555–1564. Abstract Enkephalin (ENK) has been implicated in nociceptive transmission in the spinal cord while its functional role is not clear because of difficulties in ideally visualizing ENKergic neurons. We thus developed preproenkephalin–green fluorescent protein transgenic mice to better identify ENKergic neurons. Real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) together with immunohistochemistry and in situ hybridization were first employed to confirm the successful transgenic manipulation and its application in showing spinal ENKergic neurons. The proportions of ENKergic neurons in the spinal cord laminae I, II, III and IV–VI among dorsal horn neurons were 15.8 ± 3.1%, 39.5 ± 3.3%, 11.8 ± 1.9% and 10.7 ± 2.1%, respectively. Double labeling with other molecules was then performed to further clarify the neurochemical properties of spinal ENKergic neurons. GABA was found to exist in 42.9 ± 2.8% of ENKergic neurons that were mainly located in lamina I–III. The proportions of parvalbumin‐, calretinin‐, calbindin‐ and neuronal nitric oxide synthase‐positive cells among the ENKergic neurons were 5.2 ± 0.7%, 42.6 ± 2.3%, 25.8 ± 2.2% and 11.1 ± 1.6%, respectively. Compared with previously findings obtained with ENK antibody labeling, this line of newly generated mice can be a reliable tool for the study of specific spinal ENKergic neuronal population.
Abstract Whether or not the striosome compartment of the neostriatum contained preproenkephalin (PPE)‐expressing neurons remained unresolved. To address this question by developing a sensitive detection method, we generated transgenic mice expressing enhanced green fluorescent protein (GFP) under the specific transcriptional control of the PPE gene. Eight transgenic lines were established, and three of them showed GFP expression which was distributed in agreement with the reported localization of PPE mRNA in the central nervous system. Furthermore, in the matrix compartment of the neostriatum of the three lines, intense GFP immunoreactivity was densely distributed in the neuronal cell bodies and neuropil, and matrix neurons displayed > 94% co‐localization for GFP and PPE immunoreactivities. In sharp contrast, GFP immunoreactivity was very weak in the striosome compartment, which was characterized by intense immunoreactivity for mu‐opioid receptors (MOR). Although neostriatal neurons were divided into GFP‐immunopositive and ‐negative groups in both the striosome and matrix compartments, GFP immunoreactivity of cell bodies was much weaker (∼1/5) in GFP‐positive striosomal neurons than in GFP‐positive matrix neurons. A similar reciprocal organization of PPE and MOR expression was also suggested in the ventral striatum, because GFP immunoreactivity was weaker in intensely MOR‐immunopositive regions than in the surrounding MOR‐negative regions. As PPE‐derived peptides are endogenous ligands for MOR in the neostriatum and few axon collaterals of matrix neurons enter the striosome compartment, the present results raised the question of the target of those peptides produced abundantly by matrix neurons.
Abstract Pathway-selective gene delivery would be critical for future gene therapy against neuropsychiatric disorders, traumatic neuronal injuries, or neurodegenerative diseases, because the impaired functions depend on neural circuits affected by the insults. Pathway-selective gene delivery can be achieved by double viral vector techniques, which combine an injection of a retrograde transport viral vector into the projection area of the target neurons and that of an anterograde viral vector into their somas. In this study, we tested the efficiency of gene delivery with different combinations of viral vectors to the pathway extending from the ventral tegmental area (VTA) to the cortical motor regions in rats, considered to be critical in the promotion of motor recovery from neural injuries. It was found that retrograde recombinant adeno-associated virus 2-retro (rAAV2reto) combined with anterograde AAVDJ (type2/type4/type5/type8/type9/avian/bovine/caprine chimera) exhibited the highest transduction efficiency in the short term (3–6 weeks) but high toxicity in the long term (3 months). In contrast, the same rAAV2reto combined with anterograde AAV5 displayed moderate transduction efficiency in the short term but low toxicity in the long term. These data suggest that the combination of anterograde AAV5 and retrograde rAAV2retro is suitable for safe and efficient gene delivery to the VTA-cortical pathway.
To understand the connectome of the axonal arborizations of dopaminergic midbrain neurons, we investigated the anterograde spread of highly sensitive viral tracers injected into the ventral tegmental area (VTA) and adjacent areas in 3 macaques. In 2 monkeys, injections were centered on the lateral VTA with some spread into the substantia nigra, while in one animal the injection targeted the medial VTA with partial spread into the ventro-medial thalamus. Double-labeling with antibodies against transduced fluorescent proteins (FPs) and tyrosine hydroxylase indicated that substantial portions of transduced midbrain neurons were dopaminergic. Interestingly, cortical terminals were found either homogeneously in molecular layer I, or more heterogeneously, sometimes forming patches, in the deeper laminae II-VI. In the animals with injections in lateral VTA, terminals were most dense in somatomotor cortex and the striatum. In contrast, when the medial VTA was transduced, dense terminals were found in dorsal prefrontal and temporal cortices, while projections to striatum were sparse. In all monkeys, orbitofrontal and occipito-parietal cortex received strong and weak innervation, respectively. Thus, the dopaminergic ventral midbrain sends heterogeneous projections throughout the brain. Furthermore, our results suggest the existence of subgroups in meso-dopaminergic neurons depending on their location in the primate ventral midbrain.
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