Scott W. Rogers

Neurotoxins Distinguish Between Different Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations

Neuronal and muscle nicotinic acetylcholine receptor subunit combinations expressed in Xenopus oocytes were tested for sensitivity to various neurotoxins. Extensive blockade of the α3β2 neuronal subunit combination was achieved by 10 nM neuronal bungarotoxin. Partial blockade of the α4β2 neuronal and α1β1γδ muscle subunit combinations was caused by 1,000 nM neuronal bungarotoxin. The α2β2 neuronal subunit combination was insensitive to 1,000 nM neuronal bungarotoxin. Nearly complete blockade of all neuronal subunit combinations resulted from incubation with 2 nM neosurugatoxin, whereas 200 nM neosurugatoxin was required for partial blockade of the α1β1γδ muscle subunit combination. The α2β2 and α3β2 neuronal subunit combinations were partially blocked by 10,000 nM lophotoxin analog‐1, whereas complete blockade of the α4β2 neuronal and α1β1γδ muscle subunit combinations resulted from incubation with this concentration of lophotoxin analog‐1. The α1β1γδ muscle subunit combination was blocked by the α‐conotoxins G1A and M1 at concentrations of 100 nM. All of the neuronal subunit combinations were insensitive to 10,000 nM of both α‐conotoxins. Thus, neosurugatoxin and the α‐conotoxins distinguish between muscle and neuronal subunit combinations, whereas neuronal bungarotoxin and lophotoxin analog‐1 distinguish between different neuronal subunit combinations on the basis of differing α subunits.

Mouse strain-specific nicotinic acetylcholine receptor expression by inhibitory interneurons and astrocytes in the dorsal hippocampus.

The response by individuals to nicotine is likely to reflect the interaction of this compound with target nAChRs. However, resolving how different genetic backgrounds contribute to unique mouse strain‐specific responses to this compound remains an important and unresolved issue. To examine this question in detail, expression of the nicotine acetylcholine receptor (nAChR) subunits α3, α4, α5, α7, β2, and β4 was measured in the dorsal hippocampus using immunohistochemistry in mouse strains or lines BALB/c, C3H/J, C57BL/6, CBA/J, DBA/2, Long Sleep (LS), Short Sleep (SS), and CF1. The nAChRs in all mice colocalized with glutamic acid decarboxylase (GAD)‐positive interneurons that were subclassified into at least four groups based on nAChR subunit heterogeneity. A notable difference between mouse strains was the expression of nAChRs by astrocyte subpopulations in CA1 subregions whose numbers vary inversely with nAChR‐immunostained neurons. This novel relationship also correlated with published parameters of strain sensitivity to nicotine. Attempts to identify the origin of this significant difference in nAChR expression among strains included comparison of the entire nAChRα4 gene sequence. Although multiple polymorphisms were identified, including two that changed nAChRα4 amino acid coding, none of these clearly correlate with strain‐related differences in cell type‐specific nAChR expression. These findings suggest that mouse strain‐specific behavioral and physiological responses to nicotine are likely to be a reflection of a complex interplay between genetic factors that shape differences in expression and cellular architecture of this modulatory neurotransmitter system in the mammalian nervous system. J. Comp. Neurol. 468:334–346, 2004.

Neuronal and astrocyte expression of nicotinic receptor subunit β4 in the adult mouse brain

Neuronal nicotinic acetylcholine receptor (nAChR) expression and function are customized in different brain regions through assembling receptors from closely related but genetically distinct subunits. Immunohistochemical analysis of one of these subunits, nAChRβ4, in the mouse brain suggests an extensive and potentially diverse role for this subunit in both excitatory and inhibitory neurotransmission. Prominent immunostaining included: 1) the medial habenula, efferents composing the fasciculus retroflexus, and the interpeduncular nucleus; 2) nuclei and ascending tracts of the auditory system inclusive of the medial geniculate; 3) the sensory cortex barrel field and cell bodies of the ventral thalamic nucleus; 4) olfactory‐associated structures and the piriform cortex; and 5) sensory and motor trigeminal nuclei. In the hippocampus, nAChRβ4 staining was limited to dendrites and soma of a subset of glutamic acid dehydrogenase‐positive neurons. In C57BL/6 mice, but to a lesser extent in C3H/J, CBA/J, or CF1 mice, a subpopulation of astrocytes in the hippocampal CA1 region prominently expressed nAChRβ4 (and nAChRα4). Collectively, these results suggest that the unique functional and pharmacological properties exerted by nAChRβ4 on nAChR function could modify and specialize the development of strain‐specific sensory and hippocampal‐related characteristics of nicotine sensitivity including the development of tolerance. J. Comp. Neurol. 468:322–333, 2004.

The nicotinic receptor genes.

Summary: The causative factor(s) of Alzheimers disease (AD) are presently unknown. However, it has been shown that the number as well as the fraction of high‐ to low‐affinity nicotine binding sites is altered in patients suffering from this disease. This finding, along with the identification of seven genes which code for nicotinic receptors expressed in the mammalian brain, has led to the idea that one nicotinic receptor subtype may be specifically altered in AD. The present article reviews how, through a molecular genetic approach, a family of genes coding for nicotinic acetylcholine receptor subtypes was uncovered. Also discussed is the use of in situ hybridization to determine the distribution of expression of the mRNA encoding for each receptor subtype and the patch clamp technique to characterize their biophysical properties. Determination of the promoters of these genes, as well as the properties of the expressed receptor subtypes, may make it possible to design new specific nicotinic receptor subtype drugs that will treat not only the symptoms of AD but the progression of the disease process as well.

Nicotinic Receptor Alpha7 Expression Identifies a Novel Hematopoietic Progenitor Lineage

How inflammatory responses are mechanistically modulated by nicotinic acetylcholine receptors (nAChR), especially by receptors composed of alpha7 (α7) subunits, is poorly defined. This includes a precise definition of cells that express α7 and how these impact on innate inflammatory responses. To this aim we used mice generated through homologous recombination that express an Ires-Cre-recombinase bi-cistronic extension of the endogenous α7 gene that when crossed with a reporter mouse expressing Rosa26-LoxP (yellow fluorescent protein (YFP)) marks in the offspring those cells of the α7 cell lineage (α7lin+). In the adult, on average 20–25 percent of the total CD45+ myeloid and lymphoid cells of the bone marrow (BM), blood, spleen, lymph nodes, and Peyers patches are α7lin+, although variability between litter mates in this value is observed. This hematopoietic α7lin+ subpopulation is also found in Sca1+cKit+ BM cells suggesting the α7 lineage is established early during hematopoiesis and the ratio remains stable in the individual thereafter as measured for at least 18 months. Both α7lin+ and α7lin– BM cells can reconstitute the immune system of naïve irradiated recipient mice and the α7lin+:α7lin– beginning ratio is stable in the recipient after reconstitution. Functionally the α7lin+:α7lin– lineages differ in response to LPS challenge. Most notable is the response to LPS as demonstrated by an enhanced production of IL-12/23(p40) by the α7lin+ cells. These studies demonstrate that α7lin+ identifies a novel subpopulation of bone marrow cells that include hematopoietic progenitor cells that can re-populate an animal’s inflammatory/immune system. These findings suggest that α7 exhibits a pleiotropic role in the hematopoietic system that includes both the direct modulation of pro-inflammatory cell composition and later in the adult the role of modulating pro-inflammatory responses that would impact upon an individual’s lifelong response to inflammation and infection.

Identification of cultured cells expressing ligand-gated cationic channels

We have identified cultured cells that express ligand-gated cation channels using a simple method which may also be applied to the screening of chemical agents for their use as agonists or antagonists. This assay is based upon the observation that many ligand-gated cation channels are permeable to lithium and agonists induce the flux of lithium into the cells which contain them. Since the accumulation of intracellular lithium can alter the cell cycle, the measurement of [3H]thymidine ([3H]thy) incorporation should reflect this occurrence. This expectation was realized using the PC12 cell line which expresses neuronal-like nicotinic acetylcholine receptor (nAChR). When cholinergic agonists are applied to PC12 cells in the presence of lithium-containing buffer and cells are subsequently pulsed with [3H]thy, the radiolabel incorporation into these cells relative to controls is reduced. If cholinergic antagonists are included or if the concentration of agonist either rapidly desensitizes receptors or is insufficient to induce channel opening, the reduction in [3H]thy incorporation is not observed. This method also provides a rapid way to screen cultured cell lines for those that express ligand-gated cation channels. This assay offers the potential to be automated for the low cost screening of drugs which act upon ligand-gated ion channels.

The Glutamate Receptors: Genes, Structure and Expression

Many plausible theories of learning, pattern recognition and memory depend upon changes in the efficiency of chemical synapses (Cajal 1911; Hebb 1949; Hopfield 1982; Kandel and Schwartz 1982). It seems unlikely that these theories will be testable until the structure, function and regulation of receptor and ion channel molecules are understood in detail.

Lung epithelial response to cigarette smoke and modulation by the nicotinic alpha 7 receptor

Cigarette smoking (CS) is a principal contributor to a spectrum of devastating lung diseases whose occurrence and severity may vary between individuals and not appear for decades after prolonged use. One explanation for the variability and delay in disease onset is that nicotine, the addictive component of CS, acts through the ionotropic nicotinic acetylcholine receptor (nAChR) alpha7 (α7) to modulate anti-inflammatory protection. In this study we measured the impact α7 signaling has on the mouse distal lung response to side-stream CS exposure for mice of the control genotype (α7G) and those in which the α7-receptor signaling mechanisms are restricted by point mutation (α7E260A:G). Flow cytometry results show that after CS there is an increase in a subset of CD11c (CD11chi) alveolar macrophages (AMs) and histology reveals an increase in these cells within the alveolar space in both genotypes although the α7E260A:G AMs tend to accumulate into large aggregates rather than more widely distributed solitary cells common to the α7G lung after CS. Changes to lung morphology with CS in both genotypes included increased tissue cavitation due to alveolar expansion and bronchial epithelium dysplasia in part associated with altered club cell morphology. RNA-Seq analysis revealed changes in epithelium gene expression after CS are largely independent of the α7-genotype. However, the α7E260A:G genotype did reveal some unique variations to transcript expression of gene sets associated with immune responsiveness and macrophage recruitment, hypoxia, genes encoding mitochondrial respiration complex I and extracellular fibrillary matrix proteins (including alterations to fibrotic deposits in the α7G proximal airway bronchioles after CS). These results suggest α7 has a central role in modulating the response to chronic CS that could include altering susceptibility to associated lung diseases including fibrosis and cancer.