Ruth E. Siegel

Evidence for neurotransmitter plasticity in vivo. II: Immunocytochemical studies of rat sweat gland innervation during development

Previous studies of the cholinergic sympathetic innervation of rat sweat glands provide evidence for a change in neurotransmitter phenotype from noradrenergic to cholinergic during development. To define further the developmental history of cholinergic sympathetic neurons, we have used immunocytochemical techniques to examine developing and mature sweat gland innervation for the presence of the catecholamine synthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) and for two neuropeptides present in the mature cholinergic innervation, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP). In 7-day old animals, intensely TH- and DBH-immunoreactive axons were closely associated with the forming glands. The intensity of both the TH and DBH immunofluorescence decreased as the glands and their innervation developed. Neither TH-IR nor DBH-IR disappeared entirely; faint immunoreactivity for both enzymes was reproducibly detected in mature animals. In contrast to noradrenergic properties, the expression of peptide immunoreactivities appeared relatively late. No VIP-IR or CGRP-IR was detectable in the sweat gland innervation at 4 or 7 days. In some glands VIP-IR first appeared in axons at 10 days, and was evident in all glands by 14 days. CGRP-IR was detectable only after 14 days. In addition to VIP-IR and CGRP-IR, we examined the sweat gland innervation for several neuropeptides which have been described in noradrenergic sympathetic neurons including neuropeptide Y, somatostatin, substance P, and leu- and met-enkephalin; these peptides were not evident in either developing or mature sweat gland axons. Our observations provide further evidence for the early expression and subsequent modulation of noradrenergic properties in a population of cholinergic sympathetic neurons in vivo. In addition, the asynchronous appearance during development of the two neuropeptide immunoreactivities raises the possibility that the expression of peptide phenotypes may be controlled independently.

Neurotrophic action of VIP on spinal cord cultures

Vasoactive intestinal peptide-like immunofluorescence was observed in 3-5% of the neurons in 4 week old dissociated cultures from fetal mouse spinal cord and dorsal root ganglion. Radioimmunoassay indicated that VIP was spontaneously released into the culture medium. This release was inhibited by tetrodotoxin (TTX). Previous studies indicated that 50-60% of the spinal cord neurons die between days 7 and 21 in vitro. Blockade of electrical activity with TTX during days 8-15 resulted in a 30% decrease in the number of neurons as compared to control cultures. Addition of VIP (0.1 nM) to TTX-treated cultures prevented neuronal cell death. When VIP alone was added to the cultures, no significant difference in the number of neurons from controls was observed. The possibility that VIP influences cholinergic neurons was tested by measuring choline acetyltransferase (CAT) activity at various periods during development. A 48 hour treatment with 0.1 nM VIP increased CAT activity by 50%. The CAT stimulation was observed only during a period in development when naturally occurring neuronal cell death was taking place. The increases in CAT activity were dose dependent within a range of 10(-12) M to 10(-10) M VIP; however, higher concentrations of VIP attenuated the increases in enzyme activity.

Detection of preprocholecystokinin and preproenkephalin A mRNAs in rat brain by hybridization histochemistry using complementary RNA probes.

The distributions of mRNAs encoding preprocholecystokinin and preproenkephalin A in the rat brain were examined by in situ hybridization histochemistry. Complementary RNA probes 0.5-1.0 kilobase in length were synthesized in vitro with 35S-ribonucleotides using SP6 polymerase and a transcription vector containing the SP6 promotor. Following hybridization to fixed tissue sections, signals were detected by autoradiography after relatively short exposure times. These studies demonstrate that neuropeptide mRNAs can be detected within specific cells in the brain and that the patterns of hybridization match the localization of the peptides visualized by immunohistochemistry. Minimum numbers of mRNA copies encoding the peptides were determined by quantitative autoradiography.

The mRNAs encoding GABAA/benzodiazepine receptor subunits are localized in different cell populations of the bovine cerebellum.

The expression of the mRNAs encoding the alpha and beta subunits of the GABAA/benzodiazepine receptor was examined in the bovine cerebellum by in situ hybridization histochemistry. The alpha subunit mRNA, which encodes the benzodiazepine binding site, was localized in all Purkinje and granule cells and in some cells of the molecular layer. The distribution of the beta subunit mRNA, which encodes the GABA binding site, only partially overlapped with that of the alpha subunit mRNA. While cells in the granule cell layer expressed the beta subunit mRNA, no message could be detected in other cell populations. These findings suggest that the subunit composition of the GABAA/benzodiazepine receptor is heterogeneous and that additional, as yet unidentified, beta subunits exist.

A novel γ subunit of the GABAA receptor identified using the polymerase chain reaction

We have utilized a polymerase chain reaction (PCR) strategy to identify a novel subunit, γ3, of the GABAA receptor. The γ3 cDNA encodes a mature protein of 450 amino acids that contains structural features typically conserved among subunits of the GABAA receptor family. The γ3 subunit shares approximately 66% sequence identity with the γ2 subunit but only 38% and 29% with α1 and β1 subunits, respectively. Localization of the γ3 mRNA indicates that it is widely distributed throughout the mouse brain in a pattern similar to that observed for mRNAs encoding the γ2 subunits.

Expression of the GABAA receptor delta subunit is selectively modulated by depolarization in cultured rat cerebellar granule neurons.

The levels of several GABAA receptor subunit mRNAs increase as cerebellar granule neurons migrate to their adult positions and receive excitatory mossy fiber input. Despite the temporal similarity of these increases in transcript expression in vivo, studies in cultured granule neurons demonstrated that the subunit mRNAs are differentially regulated. To address the possibility that neuronal activity regulates transcript expression, GABAA receptor subunit mRNA levels were assessed in cultured granule neurons grown in chemically defined, serum-free medium containing either nondepolarizing (5 mM) or depolarizing (25 mm) KCl concentrations. Whereas the δ subunit mRNA was almost undetectable in cultures maintained in nondepolarizing medium, an eightfold increase occurred between days 2 and 4 in cultures grown in depolarizing medium. Furthermore, δ subunit transcript expression was reduced by 76 ± 6% when neurons in depolarizing medium were switched into nondepolarizing medium. The importance of depolarization in the initiation and maintenance of subunit transcript expression in granule neurons was selective for the GABAA receptor δ subunit. These changes in transcript expression involved calcium entry through L-type calcium channels. Nifedipine treatment (1 μm) both reduced intracellular calcium and decreased δ subunit mRNA expression by 79 ± 4%. Furthermore, inhibition of Ca2+/calmodulin-dependent protein kinases (CaM kinases) by KN-62 (1 μm) also reduced δ subunit transcript expression. These studies demonstrate that KCl-induced depolarization, a condition that mimics the effects of neuronal activity, selectively modulates GABAA receptor δ subunit mRNA expression through a pathway involving calcium entry and activation of a CaM kinase.

Differential Regulation of GABAA Receptor Subunit mRNAs in Rat Cerebellar Granule Neurons: Importance of Environmental Cues

Abstract: Levels of the GABAA receptor α1‐, α6‐, β2‐, β3‐, γ2‐, and δ‐subunit mRNAs in cerebellar granule neurons rise concurrently during the second week of postnatal ontogeny. Previous studies in culture have suggested that extrinsic signals control these increases, but little is known about the nature of the regulatory cues. To determine when granule neurons become competent to express these six subunit mRNAs in mature patterns and to gain insight into their regulation, reverse transcriptase‐PCR was used to examine transcript expression in cultured granule neurons prepared at 2‐day intervals from postnatal days 2 through 10. Although only one pattern of expression was observed in vivo, three patterns were detected in culture. First, the levels of the α1‐ and α6‐subunit mRNAs were constant in cultures prepared at all ages. Second, the levels of the β2‐, β3‐, and γ2‐subunit mRNAs were constant in cultures prepared at postnatal days 2–6 but increased in those prepared at days 8–10. Third, the δ‐subunit mRNA level increased over time in culture regardless of cerebellar age at plating. Moreover, only δ‐subunit transcript expression was modulated by cell density. These findings indicate that the subunit transcripts are differentially regulated by multiple environmental cues.

Neuregulin induces GABAA receptor β2 subunit expression in cultured rat cerebellar granule neurons by activating multiple signaling pathways

The GABAA receptor β subunit is required to confer sensitivity to γ‐aminobutyric acid (GABA), the major inhibitory neurotransmitter in the CNS. In previous studies we demonstrated that the growth and differentiation factor neuregulin 1 (NRG1) selectively induced expression of the β2 subunit mRNA and encoded protein in rat cerebellar granule neurons in culture. In the present report we examine the signaling pathways that mediate this effect. These studies demonstrate that the effects of NRG1 on β2 subunit polypeptide expression require activation of the ErbB4 receptor tyrosine kinase; its effects are inhibited by pharmacological blockade of ErbB4 phosphorylation or reduction of receptor level with an antisense oligodeoxynucleotide. The NRG1‐induced activation of ErbB4 stimulates the mitogen‐activated protein kinase (MAPK), phosphatidylinositol 3‐kinase (PI3K) and cyclin‐dependent kinase‐5 (cdk5) pathways. Pharmacological blockade of any of these pathways inhibits increased β2 subunit expression, demonstrating that all three pathways are required to mediate the effects of NRG1 on GABAA receptor subunit expression in cerebellar granule neurons. These studies provide novel information concerning the actions of NRG1 on GABAA receptor expression in the CNS.

Elevated potassium stimulates enkephalin biosynthesis in bovine chromaffin cells

Exposure of bovine adrenal medullary cells in culture to a depolarizing concentration of potassium (50 mM), causes a rapid rise in both cellular and secreted Met-enkephalin peptide. The induction of peptide is preceded by the appearance of a nuclear preproenkephalin transcript and subsequent increases in cytoplasmic preproenkephalin mRNA. These data suggest that the depolarizing medium acts by enhancing enkephalin gene transcription. Potassium stimulation of Met-enkephalin biosynthesis requires the presence of extracellular Ca2+ and is not observed in either low Ca2+ medium or in the presence of D600, a Ca2+ channel blocker. As similar depolarizing stimuli inhibit enkephalin biosynthesis in the rat adrenal gland, these findings suggest that the regulation of enkephalin peptide content in neuroendocrine cells is highly species specific.

Immature granule neurons from cerebella of different ages exhibit distinct developmental potentials.

Cultured cerebellar granule neurons exhibit different developmental potentials in culture dependent on cerebellar age at plating. In cultures prepared at postnatal days (P)2-6, when all granule neurons reside in the external germinal layer (EGL) in vivo, levels of the GABA(A) receptor beta2 and gamma2 subunit mRNAs are constant. In contrast, in cultures prepared at P8-10, when neurons have begun to migrate into the internal granule cell layer (IGL), the mRNAs increase several-fold in a pattern mimicking that found in vivo. To determine the relationship between neuronal differentiation in culture and potential to express GABA(A) receptor beta2 and gamma2 subunit transcripts in the mature pattern, neuronal maturity in P6 and P10 cultures was compared. Bromodeoxyuridine labeling studies demonstrated that P10 as well as P6 cultures contained neurons only from the EGL. Moreover, the maturation of cultured P10 and P6 neurons appeared virtually identical. Cells dissociated at both ages expressed mRNAs encoding the EGL markers MATH-1 and TAG-1. The MATH-1 transcript disappeared from cultures maintained 4 days when expression of the GABA(A) receptor alpha6 subunit, a marker of mature cells, was initiated. Thus, although cultured P6 and P10 granule neurons exhibit the same maturation markers, P10 neurons presumably have been modulated by environmental cues specifying increases in GABA(A) receptor beta2 and gamma2 subunit expression. This possibility is supported by the finding that extracts of dissociated P10 cells but not P6 cells induce increases in GABA(A) receptor subunit expression in P6 cultures.