Association of postganglionic sympathetic neurons with primary afferents in sympathetic-sensory co-cultures

Functional coupling between sympathetic postganglionic neurons and sensory neurons is thought to play an essential role in the pathogenesis of certain chronic pain syndromes following peripheral tissue and nerve injury. The mechanism(s) underlying this interaction are enigmatic. The relative anatomical inaccessibility of sympathetic and sensory neurons in vivo complicates study of their interrelationships. We have developed a system for long-term co-culturing of explants of sympathetic chain ganglia and dorsal root ganglia from newborn rats. Co-cultures were labelled for tyrosine hydroxylase-like immunoreactivity and studied at the light and electron microscopic levels. Explanted ganglia of both types survived well in co-culture. They maintained their tissue type-specific histological properties, including neuronal and glial morphology, and characteristic glial–neuronal associations. Moreover, neurons maintained their characteristic neurochemical identity, at least to the extent that sympathetic neurons continued to express tyrosine hydroxylase and dorsal root ganglion neurons did not. Sympathetic neurons emitted numerous outgrowing processes (axons) some of which came into association with sensory neurons in the explanted dorsal root ganglia. Some apparently specific sympathetic-sensory contacts were observed, suggesting that a functional interaction may develop between sympathetic axons and sensory neurons in vitro.     

Pyridoxal phosphatase: cytochemical localization in GERL and other organelles of rat neurons.

A phosphatase, hydrolyzing pyridoxal-5-phosphate (P5P), a physiologically active component of the vitamin B6 complex and an essential co-enzyme in the synthesis of neurotransmitters, has been localized cytochemically in the perikarya of neurons in the peripheral, autonomic and central nervous systems of the rat. Neurons in dorsal root ganglia, sympathetic ganglia and ventral horn of spinal cord were studied by light and electron microscopy, while Purkinje cells, neurons in the dentate nucleus of the cerebellum, thalamus, and hypothalamus were studied by light microscopy only. Optimal conditions for demonstrating this activity in aldehyde-fixed tissue were determined with dorsal root ganglia. At the optimal pH of 5.0, neurons in these ganglia and in all other neurons studied show pyridoxal-5-phosphatase (P5Pase) activity in GERL. Small neurons in dorsal root ganglia also display enzyme activity in the endoplasmic reticulum (ER); activities in GERL and ER are also appreciably high at neutral pH. Small and large neurons in these ganglia, and neurons of sympathetic ganglia, show variable P5Pase activity in the Golgi apparatus. These localizations differ from the usual sites of both acid phosphatase and alkaline phosphatase activities. The P5Pase activity, demonstrated cytochemically, is a new acid hydrolase activity in GERL.     

Galanin-immunoreactive nerves in the female rat paracervical ganglion and uterine cervix: distribution and reaction to capsaicin

Summary The presence and distribution of galanin-immunoreactivity was examined in the uterine cervix and paracervical autonomic ganglia of the female rat. Some animals were treated with capsaicin to determine if galanin-immunoreactivity was present in small-diameter primary afferent nerves. Other animals were treated with the noradrenergic neurotoxin 6-hydroxydopamine to ascertain if galanin-immunoreactivity was present in sympathetic noradrenergic nerves. Galanin-immunoreactive nerve fibers were sparse in the cervical myometrium and vasculature, but numerous in the paracervical ganglion where they appeared to innervate principal neurons. Immunoreactivity was also present in dorsal root ganglia, dorsal horn of spinal cord, and inferior mesenteric ganglia. Capsaicin treatment resulted in a marked reduction of galanin-immunoreactivity in the spinal cord dorsal horn, but not in the dorsal root ganglia, paracervical ganglia, or cervix (although there was a substantial reduction of substance P-, neurokinin A-, and calcitonin gene-related peptide-immunoreactivity in the dorsal horn, dorsal root ganglia, and uterine cervix). 6-Hydroxydopamine treatment did not cause any appreciable change in the galanin-immunoreactivity in any tissues. We conclude that galanin-like immunoreactivity is expressed in nerve fibers innervating the paracervical ganglia and uterine cervix of the female rat. This immunoreactivity is probably present in afferent nerves and could play a role in neuroendocrine reflexes and in reproductive function.     

Expression of the unique NCAM VASE exon is independently regulated in distinct tissues during development

During development of the rat central nervous system, neural cell adhesion molecule (NCAM) mRNAs containing in the extracellular domain a 30-bp alternative exon, here named VASE, replace RNAs that lack this exon. The presence of this alternative exon between previously described exons 7 and 8 changes the predicted loop structure of the derived polypeptide from one resembling an immunoglobulin constant region domain to one resembling an immunoglobulin variable domain. This change could have significant effects on NCAM polypeptide function and cell-cell interaction. In this report we test multiple rat tissues for the presence of additional alternative exons at this position and also examine the regulation of splicing of the previously described exon. To sensitively examine alternative splicing, polymerase chain reactions (PCRs) with primers flanking the exon 7/exon 8 alternative splicing site were performed. Four categories of RNA samples were tested for new exons: whole brain from embryonic day 11 to adult, specific brain regions dissected from adult brain, clonal lines of neural cells in vitro, and muscle cells and tissues cultured in vitro and obtained by dissection. Within the limits of the PCR methodology, no evidence for any alternative exon other than the previously identified VASE was obtained. The regulation of expression of this exon was found to be complex and tissue specific. Expression of the 30-bp exon in the heart and nervous system was found to be regulated independently; a significant proportion of embryonic day 15 heart NCAM mRNAs contain VASE while only a very small amount of day 15 nervous system mRNAs contain VASE. Some adult central nervous system regions, notably the olfactory bulb and the peripheral nervous system structures adrenal gland and dorsal root ganglia, express NCAM which contains very little VASE. VASE is undetectable in NCAM PCR products from the olfactory epithelium. Other nervous system regions express significant quantities of NCAM both with and without VASE. Clonal cell lines in culture generally expressed very little VASE. These results indicate that a single alternative exon, VASE, is found in NCAM immunoglobulin-like loop 4 and that distinct tissues and nervous system regions regulate expression of VASE independently both during development and in adult animals.     

Molecular characterization of the family of choline transporter-like proteins and their splice variants

We show here that the choline transporter-like (CTL) family is more extensive than initially described with five genes in humans and complex alternative splicing. In adult rat tissues, CTL2-4 mRNAs are mainly detected in peripheral tissues, while CTL1 is widely expressed throughout the nervous system. During rat post-natal development, CTL1 is expressed in several subpopulations of neurones and in the white matter, where its spatio-temporal distribution profile recalls that of myelin basic protein, an oligodendrocyte marker. We identified two major rat splice variants of CTL1 (CTL1a and CTL1b) differing in their carboxy-terminal tails with both able to increase choline transport after transfection in neuroblastoma cells. In the developing brain, CTL1a is expressed in both neurones and oligodendroglial cells, whereas CTL1b is restricted to oligodendroglial cells. These findings suggest specific roles for CTL1 splice variants in both neuronal and oligodendrocyte physiology.     

Nucleotide sequence of the bovine cyclic-AMP responsive DNA binding protein (CREB2) cDNA

The bovine cyclic AMP responsive binding protein cDNA (CREB2) was isolated from a lambda-gt11 cDNA expression library using a 32P labelled oligonucleotide corresponding to the 21 bp enhancer sequence present in the BLV LTR. The deduced amino acid sequence revealed that CREB2 contains a leucine zipper structure (residue 295 to 316), a basic amino acid domain (residue 268 to 291) and several potential phosphorylation sites.     

Chimeric proteins composed of Jun and CREB define domains required for interaction with the human T-cell leukemia virus type 1 Tax protein.

The regulation of human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat gene expression is dependent on three cis-acting elements known as 21-bp repeats and the transactivator protein Tax. Mutagenesis has demonstrated that sequences in each of the 21-bp repeats can be divided into three domains designated A, B, and C. Tax stimulates the binding of CREB to the B domain, which is essential for Tax activation of HTLV-1 gene expression. In this study, we demonstrate that Tax will stimulate the binding of CREB to the HTLV-1 21-bp repeats but does not stimulate CREB binding to the consensus cyclic AMP response element (CRE) element found in the somatostatin promoter. However, Tax stimulates CREB binding to a consensus CRE in the context of the 21-bp repeats, indicating the importance of these sequences in stimulating CREB binding. To determine the mechanism by which Tax stimulates CREB binding and determine potential interactions between Tax and CREB, we used the mammalian two-hybrid system in conjunction with in vitro binding and gel retardation assays. Two-hybrid analysis indicated that mutations in either the basic or leucine zipper region of CREB prevented interactions with Tax. Since several studies have demonstrated that Tax will also stimulate the binding of a variety of different basic region-leucine zipper proteins to their cognate binding sites, we assayed whether chimeric proteins composed of portions of CREB and another basic region-leucine zipper protein, Jun, could be used to map domains required for interactions with Tax. These studies were possible because we did not detect in vivo or in vitro interactions between Tax and Jun. The amino acid sequence of the CREB basic region and a portion of its leucine zipper were required for both in vivo and in vitro interactions with Tax and increased binding of CREB to the 21-bp repeats in response to Tax. These studies define the domains in CREB required for both in vivo and in vitro interactions by the HTLV-1 Tax protein.     

Profiling the phospho-status of the BKCa channel alpha subunit in rat brain reveals unexpected patterns and complexity

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BK(Ca)) channels are tetramers of alpha subunits (BKalpha) either alone or together with beta subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca(2+). The cytoplasmic C terminus of BKalpha is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanoflow liquid chromatography tandem mass spectrometry on BK(Ca) channels affinity-purified from rat brain to analyze in vivo BKalpha phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho- and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BKalpha differentially modulates the voltage- and Ca(2+)-dependence of channel activation. These results demonstrate that the pore-forming subunit of BK(Ca) channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BKalpha structure and function.