The efficient introduction of genetic material into quiescent nerve cells is important in the study of brain function and for gene therapy of neurological disorders. A replication-deficient adenoviral vector that contained a reporter gene encoding beta-galactosidase infected rat nerve cells in vitro and in vivo. beta-Galactosidase was expressed in almost all sympathetic neurons and astrocytes in culture. After stereotactic inoculations into the rat hippocampus and the substantia nigra, beta-galactosidase activity was detected for 2 months. Infected cells were identified as microglial cells, astrocytes, or neurons with anatomical, morphological, and immunohistochemical criteria. No obvious cytopathic effect was observed.

A cDNA clone encoding the complete sequence of an active rat choline acetyltransferase (ChoAcTase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been isolated. Analysis of the deduced amino acid sequence reveals 85% and 31% identity with the porcine and Drosophila melanogaster enzymes, respectively. To further elucidate the molecular basis of neurotransmitter-related phenotypic plasticity, the expression of ChoAcTase mRNA was compared with that of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], in neurons from superior cervical ganglia grown in the following conditions: 1) normal medium, 2) high K+ medium, and 3) normal medium supplemented with 50% muscle-conditioned medium (CM). TH mRNA was expressed in all three media; its level rose in high K+ and decreased strikingly in the presence of CM. ChoAcTase mRNA could be visualized in CM, but fell to undetectable levels in normal and high K+ media. These results suggest that translational or post-translational mechanisms do not play a major role for the modulation of neurotransmitter-associated phenotype.

(3, 4). The analysis, in molecular terms, of the mechanisms underlying this plasticity requires the study of the genes encoding these two enzymes.We have identified (5-7) cDNA clones corresponding to rat and human tyrosine hydroxylases. Here, we describe the isolation of a cDNA clone-pChAT-1-that encodes an active porcine ChoAcTase enzyme. The nucleotide and complete amino acid sequence is reported. ¶ Some structural characteristics of porcine ChoAcTase are discussed, and the sequence is compared with that of Drosophila melanogaster reported by Itoh et al. (8). MATERIALS AND METHODSConstruction of a Randomly Primed cDNA Library in the XgtlO Vector. Total RNA from porcine ventral spinal cord was extracted as described by Lomedico and Saunders (9). Poly(A)+ RNA was purified by oligo(dT)-cellulose chromatography. Random DNA sequences 20-50 nucleotides in length were prepared by sonication and DNase I digestion of calf thymus DNA (10) and used as primers for cDNA synthesis. First-strand cDNA was synthesized from 2.5 ,g of ventral spinal cord poly(A)+ RNA with 30-fold excess of random primer. The second-strand synthesis and following steps were carried out using standard procedures (11, 12). The longest cDNAs [_500 base pairs (bp)] were selected on a 5-20o (wt/vol) sucrose gradient and ligated to the XgtlO vector. The amplified library contained =1.2 x 106 independent recombinant phages.Oligonucleotide Screening. The N-terminal sequence of porcine brain ChoAcTase was determined as described (13). A mixture of oligodeoxynucleotides, each containing eight different chains of 29 nucleotides, was prepared with a Biosearch DNA synthesizer model 8600 by the phosphoramidite method and purified by PAGE. The probes were end-labeled to a minimal specific activity of 8 x 108 cpm/,ug. About 106 recombinant phages were plated at 50,000-70,000 plaques per 13-cm plate, and duplicate filters were prepared. Filters were hybridized at 35°C with oligonucleotides in 6x SSC (lx SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7.0), 5x Denhardt's solution (lx Denhardt's solution = 0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin), 10% (wt/vol) dextran sulfate, 0.05% sodium pyrophosphate, herring sperm DNA at 0.1 mg/ml, and Escherichia coli tRNA at 0.1 mg/ml. Filters were then washed at 35°C, 40°C, and 45°C in 6x SSC containing 0.05% NaDodSO4. Positive clones were isolated after three successive rounds of screening. Phage DNA was prepared as Abbreviations: ChoAcTase, choline acetyltransferase; NVP, 4-(1-naphthylvinyl)pyridine. §To whom reprint requests should be addressed.

Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently been appreciated. Group 2 ILCs (ILC2s) are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in the expansion of ILC2s for maximal induction of type 2 immunity.