Neural transcriptome of constitutional Pten dysfunction in mice and its relevance to human idiopathic autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with a clear, but heterogeneous, genetic component. Germline mutations in the tumor suppressor Pten are a well-established risk factor for ASD with macrocephaly, and conditional Pten mouse models have impaired social behavior and brain development. Some mutations observed in patients disrupt the normally balanced nuclear-cytoplasmic localization of the Pten protein, and we developed the Pten(m3m4) model to study the effects of a cytoplasm-predominant Pten. In this model, germline mislocalization of Pten causes inappropriate social behavior with intact learning and memory, a profile reminiscent of high-functioning ASD. These animals also exhibit histological evidence of neuroinflammation and expansion of glial populations by 6 weeks of age. We hypothesized that the neural transcriptome of this model would be altered in a manner that could inform human idiopathic ASD, a constitutional condition. Using total RNA sequencing, we found progressive disruption of neural gene expression in Pten(m3m4) mice from 2-6 weeks of age, involving both immune and synaptic pathways. These alterations include downregulation of many highly coexpressed human ASD-susceptibility genes. Comparison with a human cortical development coexpression network revealed that genes disrupted in Pten(m3m4) mice were enriched in the same areas as those of human ASD. Although Pten-related ASD is relatively uncommon, our observations suggest that the Pten(m3m4) model recapitulates multiple molecular features of human ASD, and that Pten operates far upstream of common pathways within ASD pathogenesis.

Innervation of tracheal epithelium and smooth muscle by neurons in airway ganglia.

The neurochemical profiles of neurons in ferret tracheal ganglia has been characterized, but their projections to smooth muscle and epithelium in ferret trachea has not been examined. The purpose of this study is to determine the location of cell bodies that project VIP-, SP-, and NPY-containing fibers to the ferret tracheal smooth muscle and epithelium. Segments of ferret trachea were cultured for 0, 1, 3, or 7 days, some in the presence of 3 microm capsaicin. VIP, SP, or NPY nerve fiber density was measured using morphometric procedures. A retrograde tracer, rhodamine-labeled microspheres, identified neurons projecting to the epithelium. The density of SP fibers in the epithelium was reduced after culture, but VIP innervation was not different. In tracheal smooth muscle, the density of VIP- and SP-IR fibers was not different during the culture period, but NPY fiber density was reduced at all culture times. Capsaicin treatment did not affect nerve fiber density in the tracheal smooth muscle but produced a significant reduction in the density of epithelial VIP- and SP-IR nerve fibers after 1 day. Rhodamine-labeled microspheres were identified in VIP-containing nerve cell bodies of the ferret tracheal plexus. VIP innervation to the airway epithelium in ferret originates both from cell bodies in airway ganglia and cell bodies in sensory ganglia. The pathway from airway ganglia suggest the existence of a local reflex mechanisms initiated by epithelial irritation.

Neurochemical characterization of intrinsic neurons in ferret tracheal plexus.

Although neuroanatomical and neurophysiological features of neurons in the ferret trachea have been studied, the neural mediators associated with this plexus have not been completely characterized. The purpose of this study was to examine the occurrence of choline acetyltransferase (ChAT), nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), and substance P(SP) in the intrinsic neurons of this plexus. The distribution of double- and triple-labeled neurons was quantified in cryostat sections and in whole mounted specimens to evaluate the neurochemical profiles. About 85% of the nerve cell bodies with ChAT immunoreactivity (ChAT-IR) were located in ganglia of the longitudinal trunks or the closely associated bridge ganglia. Approximately 15% of ChAT-positive neurons were in ganglia of the superficial muscular plexus. Conversely, VIP-IR neurons were most frequent in the superficial muscular plexus (>75%) and, <10% were observed in the longitudinal trunks or bridge neurons. Most NOS- and SP-IR neurons were also located in the superficial muscular plexus. The following distribution of neurochemical profiles was determined for neurons of the superficial muscular plexus: 11% only NOS, 20% only VIP, 5% only SP, 67% NOS and VIP, and 40% VIP and SP. NOS, VIP, and SP were frequently localized in the same nerve cell body. The occurrence of nerve terminals containing only SP located around the borders of individual NOS/VIP/SP-containing neurons suggests possible sensory innervation to the airway neurons. The results demonstrate that: (1) most cholinergic nerves do not contain VIP, NOS, or SP; (2) cholinergic neurons are predominantly located in the longitudinal trunk ganglia; (3) VIP, NOS, and SP are predominantly located in the superficial muscular plexus ganglia; and (4) nerve terminals containing exclusively SP, suggesting possible sensory origin, are closely associated with some neurons in the plexus.

Distribution of vasoactive intestinal peptide- and substance P-containing nerves originating from neurons of airway ganglia in cat bronchi.

This study examined the possibility that vasoactive intestinal peptide (VIP)- and substance P (SP)-containing nerve fibers in bronchial smooth muscle, glands, epithelium, and blood vessels originate from neurons of airway ganglia. Explants of airway walls were maintained in culture with the expectation that nerve fibers from neurons of airway ganglia would remain viable, whereas fibers originating from neurons not present in the airway walls would degenerate. Airways were dissected and placed into culture dishes containing CMRL 1066 medium for 3, 5, and 7 days. In controls (noncultured), VIP- and SP-like immunoreactivity was observed in nerve fibers associated with bronchial smooth muscle, glands, and blood vessel walls and in nerve cell bodies of airway ganglia. Nerve fibers containing SP were also observed within the bronchial epithelium. After 3, 5, and 7 days in culture, VIP- and SP-containing fibers were identified in all of the same locations except in the airway epithelium where SP-containing fibers could not be demonstrated. VIP and SP were frequently colocalized in the same nerve fibers of bronchial smooth muscle and glands in controls and cultured airways. There were no statistically significant differences in nerve fiber density for either VIP- or SP-containing fibers in bronchial smooth muscle between controlled and cultured airways. VIP concentrations in cultured airways were significantly less than in controls. The results suggest that a large proportion of VIP- and SP-containing nerve fibers supplying bronchial smooth muscle, glands, and blood vessels in the airways originate from neurons of airway ganglia.

Origin and colocalization of CGRP- and SP-reactive nerves in cat airway epithelium.

A combination of neuroanatomic techniques was used to examine the origin and neuropeptide content of nerve fibers in the airway epithelium of adult cats. By the use of immunocytochemical methods, the peptides substance P (SP) and calcitonin gene-related peptide (CGRP) were colocalized in airway epithelial nerve fibers. Two days after wheat germ agglutinin (WGA) was injected into the nodose ganglion, fibers containing WGA immunoreactivity (IR) were detected in the airway epithelium. SP-like immunoreactivity (LI) and CGRP-LI were demonstrated separately in the WGA-IR fibers, establishing their origin from nerve cell bodies of nodose ganglion. Vagal transection inferior to the nodose ganglion reduced the number of SP- and CGRP-IR fibers by greater than 90% in ipsilateral airways. In contralateral airways, SP-IR fibers were substantially reduced, whereas the effect on CGRP-IR fibers was not statistically significant. Vagotomy superior to the nodose ganglion did not alter the density of peptide-IR fibers. The results prove that SP- and CGRP-IR nerve fibers of cat airway epithelium originate from nerve cell bodies in the nodose ganglion and that SP- and CGRP-like peptides may be stored together in some nerve fibers of the airway epithelium.