Transient expression of somatostatin peptide is a widespread feature of developing sensory and sympathetic neurons in the embryonic rat

Previous studies from this and other laboratories demonstrated that many embryonic sensory ganglion cells in the rat transiently express the catecholamine synthesizing enzyme tyrosine hydroxylase (TH), a trait not expressed by most mature sensory neurons. We, therefore, sought to determine whether transient expression was uniquely associated with catecholaminergic traits, or, alternatively, whether embryonic ganglion cells transiently expressed peptidergic properties as well. Of the four peptides examined {somatostatin [somatotropin release inhibiting factor] (SRIF), galanin (Gal), calcitonin gene-related peptide (CGRP), and substance P (SP)}, only SRIF was found to be transiently expressed during early stages of sensory gangliogenesis. Surprisingly, SRIF immunoreactivity was observed in virtually all cranial and spinal sensory ganglion cells on embryonic day (E) 12.5. In addition to perikaryal labeling, intense SRIF immunoreactivity was also observed in the central and peripheral processes of E12.5 sensory neurons, suggesting the peptide may be released from nerve endings. The time course of SRIF appearance in cranial ganglion cells paralleled that previously described for TH, and double labeling studies revealed extensive co-localization of these two phenotypes. By E16.5, however, the number of neurons expressing SRIF had diminished markedly, indicating that SRIF is only transiently expressed by most sensory neurons during early stages of ganglion development. An unexpected finding was that transient expression of SRIF is also a prominent feature of sympathetic ganglion cells; however, the temporal pattern of staining in the sympathetic and sensory ganglia differed substantially. Whereas virtually no SRIF staining was observed in E12.5 sympathetics, the vast majority of cells in the E16.5 superior cervical ganglion (SCG) were labeled. This contrasted sharply with the adult SCG, in which only low levels of SRIF expression were found. These findings demonstrate that SRIF peptide is transiently expressed at high levels in peripheral sensory and sympathetic neurons during embryogenesis. The time course and widespread distribution of SRIF expression indicates that the peptide may play a role in early stages of ganglion cell growth and development. Moreover, these data, in conjunction with previous studies demonstrating SRIF immunoreactivity in developing central neurons, suggest that transient expression of this peptide is a common property of diverse neuronal cell types. © 1992 John Wiley & Sons, Inc.     

Developmental regulation of tyrosine hydroxylase expression in primary sensory neurons of the rat

The regulation of transmitter phenotype in primary sensory neurons remains poorly understood. However, recent studies of catecholaminergic (CA) sensory neurons suggest that expression of this particular phenotype may be related to innervation of specific peripheral tissues. In the glossopharyngeal petrosal ganglion (PG) of adult rats, for example, the vast majority of CA sensory neurons innervate a single target, the carotid body. The present study was undertaken, therefore, to begin investigating factors that underlie CA differentiation in sensory neurons, using the rat PG as a model system. Immunocytochemical, biochemical, and morphometric methods were used to investigate the normal time course of CA development in the PG in vivo, employing tyrosine hydroxylase (TH) as a phenotypic marker. These studies revealed two temporally distinct waves of TH expression during embryogenesis. TH immunoreactivity was initially detectable on Embryonic Day (E) 11.5; the number of stained cells increased markedly by E12.5 and then fell off sharply to near 0 by E15.5. Simultaneous immunostaining for TH and neurofilament proteins revealed a high proportion of double-labeled perikarya on E12.5, indicating that the transiently TH-positive cells are neurons. A second, sustained phase of TH expression began on E16.5, and by Postnatal Day 1 adult numbers of TH-containing ganglion cells were present. Western blot analysis demonstrated that TH levels per cell rose 3.5-fold in the perinatal period, indicating that maturation of this particular catecholaminergic trait in PG sensory neurons is highly regulated around birth. Morphometric techniques were used to define the relationship between neurons that transiently exhibit TH immunoreactivity early in gangliogenesis and those that maintain enzyme expression in the mature PG. These studies revealed separate and distinct growth curves for the early and late TH cells, respectively, demonstrating that the appearance, disappearance, and reappearance of immunoreactive cells reflects the differentiation of two separate populations of PG neurons. Moreover, these data indicate that TH expression in the population of CA cells that persists in the mature PG begins around E16.5. This is after peripheral target innervation has begun, raising the possibility that neuron-target interactions regulate biochemical differentiation of these CA sensory neurons.     

Regulation of tyrosine hydroxylase mRNA in catecholaminergic cells of embryonic rat: analysis by in situ hybridization

In situ hybridization was used to examine the appearance of mRNA specific for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine (CA) biosynthesis, in neural crest derivatives of the rat embryo. These derivatives include sympathetic ganglia and transient catecholaminergic cells of embryonic intestine. Messenger RNA is first detected in sympathetic ganglia at E11.5, the age corresponding to the initial immunocytochemical expression of TH protein. In older embryos increased accumulation of TH-specific mRNA in sympathetic ganglia parallels the increase in TH immunoreactivity. By contrast, mRNA for TH is difficult to detect in embryonic intestines at E11.5 but is found instead in cells clustered at the dorsal boundaries of the pharynx and foregut. Cells expressing TH mRNA are infrequently found in embryonic intestines at any age, even though TH protein is immunohistochemically apparent. Treatment of pregnant rats with doses of reserpine, known to increase circulating levels of glucocorticoid hormones and prolong the expression of TH protein in embryonic gut cells, dramatically but transiently increases the number of gut cells at E12.5 with detectable TH mRNA. After E13.5 TH mRNA is undetectable even in reserpine-treated guts. Reserpine treatment also increases the labeling density in sympathetic ganglia. Taken together, these data are consistent with the hypothesis that the microenvironment of the embryonic intestine affects gene expression directly to alter phenotype. Moreover, although reserpine administration briefly increases TH mRNA levels, the effect is short-lived and does not alter neurotransmitter phenotypic conversion.     

Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic polypeptide, distantly related to transforming growth factor-beta (TGF- beta), originally isolated by virtue of its ability to induce dopamine uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic neurons, and more recently shown to be a potent neurotrophic factor for motorneurons. The biological activities and distribution of this molecule outside the central nervous system are presently unknown. We report here on the mRNA expression, biological activities and initial receptor binding characterization of GDNF and a shorter spliced variant termed GDNF beta in different organs and peripheral neurons of the developing rat. Both GDNF mRNA forms were found to be most highly expressed in developing skin, whisker pad, kidney, stomach and testis. Lower expression was also detected in developing skeletal muscle, ovary, lung, and adrenal gland. Developing spinal cord, superior cervical ganglion (SCG) and dorsal root ganglion (DRG) also expressed low levels of GDNF mRNA. Two days after nerve transection, GDNF mRNA levels increased dramatically in the sciatic nerve. Overall, GDNF mRNA expression was significantly higher in peripheral organs than in neuronal tissues. Expression of either GDNF mRNA isoform in insect cells resulted in the production of indistinguishable mature GDNF polypeptides. Purified recombinant GDNF promoted neurite outgrowth and survival of embryonic chick sympathetic neurons. GDNF produced robust bundle-like, fasciculated outgrowth from chick sympathetic ganglion explants. Although GDNF displayed only low activity on survival of newborn rat SCG neurons, this protein was found to increase the expression of vasoactive intestinal peptide and preprotachykinin-A mRNAs in cultured SCG neurons. GDNF also promoted survival of about half of the neurons in embryonic chick nodose ganglion and a small subpopulation of embryonic sensory neurons in chick dorsal root and rat trigeminal ganglia. Embryonic chick sympathetic neurons expressed receptors for GDNF with Kd 1-5 x 10(-9) M, as measured by saturation and displacement binding assays. Our findings indicate GDNF is a new neurotrophic factor for developing peripheral neurons and suggest possible non-neuronal roles for GDNF in the developing reproductive system.     

Neurofilament immunoreactivity and acetylcholinesterase activity in the developing sympathetic tissues of the rat.

In this study, the ontogenetic appearance of three neuronal markers, tyrosine hydroxylase (TH), neurofilament (NF) proteins and acetylcholinesterase (AChE), have been compared in the neural tube and derivatives of the neural crest with special consideration on developing rat sympathetic tissues. The tree markers appeared for the first time on embryonic day E 12.5. At this age, NF immunoreactivity was located in the cells on the ventro- and dorsolateral edges of the neural tube, i.e., in the regions where the cells had reached the postmitotic stage. In addition, on day E 12.5, NF-immunoreactive fibers were located in the dorsal and ventral roots and the spinal and sympathetic ganglia. This suggests rapid extension of neurites. In contrast to NF, AChE first appeared on day E 12.5 in cell somata of spinal and sympathetic ganglia and only after that in axons. Thus, it can be considered as a marker of differentiating neuronal cell bodies. In the developing sympathoadrenal cells, TH is expressed before NF and AChE. However, the migrating TH immunoreactive sympathetic cells are constantly followed by NF immunoreactive fibers, suggesting that sympathetic tissues may receive innervation from preganglionic axons at the very beginning of their ontogeny. During the later development, all sympathetic tissues contain two major cell groups: 1) one with a moderate TH immunoreactivity, NF immunoreactivity and AChE activity and 2) the other with an intense TH immunoreactivity but lacking NF immunoreactivity or AChE activity. The former includes principal neurons, neuron-like cells of the paraganglia and noradrenaline cells of the adrenal medullae, and the latter includes ganglionic small intensely fluorescent (SIF) cells, paraganglionic cells and medullary adrenaline cells.     

Neurofilament immunoreactivity and acetylcholinesterase activity in the developing sympathetic tissues of the rat

Summary In this study, the ontogenetic appearance of three neuronal markers, tyrosine hydroxylase (TH), neurofilament (NF) proteins and acetylcholinesterase (AChE), have been compared in the neural tube and derivatives of the neural crest with special consideration on developing rat sympathetic tissues. The tree markers appeared for the first time on embryonic day E 12.5. At this age, NF immunoreactivity was located in the cells on the ventro- and dorsolateral edges of the neural tube, i.e., in the regions where the cells had reached the postmitotic stage. In addition, on day E 12.5, NF-immunoreactive fibers were located in the dorsal and ventral roots and the spinal and sympathetic ganglia. This suggests rapid extension of neurites. In contrast to NF, AChE first appeared on day E 12.5 in cell somata of spinal and sympathetic ganglia ond only after that in axons. Thus, it can be considered as a marker of differentiating neuronal cell bodies. In the developing sympathoadrenal cells, TH is expressed before NF and AChE. However, the migrating TH immunoreactive sympathetic cells are constantly followed by NF immunoreactive fibers, suggesting that sympathetic tissues may receive innervation from preganglionic axons at the very beginning of their ontogeny. During the later development, all sympathetic tissues contain two major cell groups: 1) one with a moderate TH immunoreactivity, NF immunoreactivity and AChE activity and 2) the other with an intense TH immunoreactivity but lacking NF immunoreactivity or AChE activity. The former includes principal neurons, neuron-like cells of the paraganglia and noradrenaline cells of the adrenal medullae, and the latter includes ganglionic small intensely fluorescent (SIF) cells, paraganglionic cells and medullary adrenaline cells.     

Proliferation and differentiation of embryonic chick sympathetic neurons: effects of ciliary neurotrophic factor

At early developmental stages (embryonic day 7, E7), chick paravertebral sympathetic ganglia contain a cell population that divides in culture while expressing various neuronal properties. In an attempt to identify factors that control neuronal proliferation, we found that ciliary neurotrophic factor (CNTF) specifically inhibits the proliferation of those cells expressing neuronal markers. In addition, CNTF affects the differentiation of sympathetic ganglion cells by inducing the expression of vasoactive intestinal peptide immunoreactivity (VIP-IR). After 1 day in culture, tyrosine hydroxylase immunoreactivity (TH-IR) was expressed by about 86% of the cells whereas VIP-IR was virtually absent. In the presence of CNTF, 50%-60% of the cells expressed VIP-IR after 4 days in culture; however, none of the cells expressed VIP-IR in the absence of CNTF. These results, and the demonstration of cells that express both VIP and TH-IR, indicate that VIP is induced in cells that initially express tyrosine hydroxylase. The findings suggest a potential role for CNTF as a factor affecting the proliferation and differentiation of developing sympathetic neurons.     

Differentiation of noradrenergic traits in the principal neurons and small intensely fluorescent cells of the parasympathetic sphenopalatine ganglion of the rat

Catecholamine synthetic enzymes are found in many cranial parasympathetic principal neurons, and in the small intensely fluorescent (SIF) cells that populate parasympathetic as well as sympathetic ganglia. While there is evidence that the acquisition of noradrenergic properties in sympathetic neuron precursors depends on factors that these cells encounter in the trunk environment, the mechanisms that direct the development of noradrenergic traits in cranial parasympathetic neurons and SIF cells are not understood. The present study examines the time course of appearance of tyrosine hydroxylase (TH) immunoreactivity in the principal neurons and SIF cells of the rat sphenopalatine ganglion. We show that the sphenopalatine ganglion of normal adult rats contains both a small population of TH-immunoreactive principal neurons and many SIF cells. The TH-immunoreactive principal neurons do not synthesize or store detectable catecholamines, even though the majority of sphenopalatine ganglion neurons do contain 1-amino acid decarboxylase catalytic activity. Sphenopalatine ganglion principal neurons do not accumulate detectable levels of exogenous catecholamines. This observation suggests that they lack a high affinity norepinephrine uptake system. In contrast to what has been observed previously for sympathetic neurons, the appearance of TH immunoreactivity in sphenopalatine neurons is not temporally correlated with the cessation of neural crest cell migration. The first TH-immunoreactive neurons do not appear in the sphenopalatine ganglion until Embryonic Day 16.5, 2 days after the ganglion has condensed and process outgrowth has begun. The number of sphenopalatine neurons that express TH immunoreactivity increases dramatically between Embryonic Day 18.5 and Postnatal Day 1, but then decreases. In fact, the percentage of sphenopalatine neurons that express TH immunoreactivity is almost fivefold higher in newborn than in adult rats. SIF cells cannot be definitively identified in the sphenopalatine ganglion until after Embryonic Day 18.5. The time course of appearance of TH immunoreactivity in sphenopalatine ganglion cells raises the possibility that TH expression is stimulated in these cells by factors encountered either at their condensation site or at their target, such as glucocorticoids or nerve growth factor. The relatively late appearance of SIF cells in the sphenopalatine ganglion argues against the hypothesis that SIF cells are the precursors of all autonomic neurons.     

Distribution and ontogeny of SP, CGRP, SOM, and VIP in chick sensory and sympathetic ganglia

The distribution and ontogeny of four neuropeptides in developing chick lumbosacral sensory and sympathetic ganglia were studied using immunohistochemical techniques. Antibodies to two of these peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), stained small neurons in the medial part of the dorsal root ganglia from embryonic Day 5 and Day 10, respectively, whereas neurons in the lateral part of the ganglia were negative; this distribution persisted throughout development. Both sets of neurons apparently send fibers to the dorsal horn of the spinal cord: SP to laminae I and II, and CGRP to lamina I, suggesting that the SP- and CGRP-positive sensory neurons are nociceptive or thermoreceptive. This correlation between the presence of SP or CGRP in a neuron and a particular functional modality thus provides evidence for a functional distinction between the mediodorsal and ventrolateral zones that are apparent during the development of chick dorsal root ganglia. Moreover, this study suggests that the type of neuron that develops within the dorsal root ganglion correlates with its position within the ganglion. In contrast to SP and CGRP, somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) immunoreactivities were not seen in the lumbosacral sensory ganglia at any stage during development. However, both were present in sympathetic ganglia: SOM from embryonic Day 4.5 and VIP from embryonic Day 10. VIP immunoreactivity persisted throughout development in a large number of sympathetic neurons, but the number of cells with SOM immunoreactivity decreased from embryonic Day 10 onward. SOM therefore appears to be present only transiently in most chick lumbosacral sympathetic cells.     

Developmental Changes in Expression of a 60-kDa Somatostatin Receptor Immunoreactivity in the Rat Brain

Abstract: The neuropeptide somatostatin (SRIF) exerts several important physiological actions in the adult CNS through interactions with membrane-bound receptors. SRIF expression is developmentally regulated and this regulation is most apparent in the cerebellum, where SRIF immunoreactivity is expressed at early postnatal ages and then disappears toward adulthood. The transitory nature of SRIF expression at a time of major changes in cerebellum suggests that this peptide may have a role in cerebellar development. To further investigate the role of the SRIF transmitter system during development, we have examined the levels of expression of SRIF receptors in the developing rat brain by immunoblotting using antiserum selective for a 60-kDa brain SRIF receptor. In whole rat brain, SRIF receptor immunoreactivity first appears at embryonic day 13 (E13), is elevated at E16. increases at birth, peaks at early postnatal ages, and then gradually declines with age. No apparent changes in size of the receptor occur with age. No consistent changes in levels of SRIF receptor immunoreactivity are detected from early postnatal ages to adulthood in the hippocampus, cerebral cortex, and striatum, but levels gradually decline in the hypothalamus. In contrast, SRIF receptor immunoreactivity is expressed transiently in cerebellum. SRIF receptor immunoreactivity is detectable in cerebellum at E16, increases in levels at birth, is apparent from postnatal day 3 to postnatal day 8, and then disappears. The transitory nature of SRIF receptor expression in cerebellum is unique and parallels the expression of SRIF immunoreactivity in this brain region. These findings support the hypothesis that SRIF has a role in cerebellar development.     

A monoclonal anti-glycoconjugate antibody defines a stage and position-dependent gradient in the developing sympathoadrenal system

Summary The expression of complex carbohydrate antigens was analysed in developing sympathoadrenal cells of the rat using monoclonal antibodies that react with unique carbohydrate structures. CC1 and CC4 are monoclonal antibodies that react specifically with -N-acetylgalactosamine and -galactose/-fucose moieties, respectively. CC1-reactive glycoconjugates are expressed in embryonic superior cervical ganglion (SCG) cells as early as embryonic day 15 (E15). CC4 is expressed in the SCG only for a brief period starting at E18 and then disappearing at P5. During their transient period of expression, CC1 antigens are expressed uniformly throughout the SCG at E15–17, but are then restricted to the rostral portion of the SCG from E18 to P4. CC4 is also concentrated in the rostral portion of the SCG between E21 and P4. In the adrenal medulla, CC1 and CC4 antigens display a post-natal onset of expression commencing approximately at P14 and continue to be expressed on a subset of cells which contain tyrosine hydroxylase (TH). The expression of CC1, however, is restricted to phenylethanolamine-N-methyltransferase-(PNMT)-negative chromaffin cells, whereas CC4 is not. CC1 and CC4-expressing cells appear to be scattered throughout the adrenal medulla without any particular topographic orientation. These findings suggest that the CC1 monoclonal antibody defines a stage-specific differentiation antigen in the sympathoadrenal lineage. Additionally, the CC1 antigen may confer important positional information in the embryonic SCG by distinguishing rostral from caudal neuronal cell bodies.     

Developmental expression of the calcium‐activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium

Calcium‐activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium‐activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 657–675, 2014     

Developmental expression of galanin-like immunoreactivity by members of the avian sympathoadrenal cell lineage

The developmental coexpression of galanin-like immunoreactivity with the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) was studied in the avian embryo sympathoadrenal system using double-labeling immunocytochemistry. Galanin-like immunoreactivity is expressed by various catecholaminergic cell populations, namely sympathoblasts, chromaffin and small intensely fluorescent (SIF) cells, but not by principal neurons of the paravertebral sympathetic ganglia. Both galanin and somatostatin immunoreactivities are coexpressed in the adrenal and sympathetic ganglion primordia by the neural precursors, but the subsequent expression pattern of both peptides differs. Our results support the hypothesis that early sympathoblasts express a large repertoire of neuroactive substances and that the expression of these becomes restricted during further development as the sympathoblasts become principal neurons.     

NPY-, galanin-, VIP/PHI-, CGRP- and substance P-immunoreactive neuronal subpopulations in cat autonomic and sensory ganglia and their projections

Summary The neuronal subpopulations in the cat stellate, lower lumbar and sacral sympathetic ganglia were studied with regard to the cellular distribution of immunoreactivity to tyrosine hydroxylase (TH), acetylcholinesterase (AChE) and various neuronal peptides. Coexistence of neuropeptide Y (NPY)- and galanin (GAL)-like immunoreactivity (LI) was found in a high proportion of the neuronal cell bodies; these cells also contained immunoreactivity to TH, confirming their presumably noradrenergic nature. Some TH- and GAL-immunoreactive principal ganglion cells lacked NPY-LI. Two populations (scattered and clustered) of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-positive cell bodies were found in the sympathetic ganglia studied. The scattered VIP/PHI neurons also contained AChE-LI, calcitonin gene-related peptide (CGRP)-and, following culture, substance P (SP)-LI. The clustered type only contained AChE-LI. In the submandibular and sphenopalatine ganglia, neurons were AChE- and VIP/ PHI-immunoreactive but lacked CGRP- and SP-LI. Many GAL- and occasional TH-positive neurons were found in these ganglia. In the spinal ganglia, single NPY-immunoreactive sensory neuronal cells were observed, in addition to CGRP- and SP-positive neurons. The present results show that there are at least two populations of sympathetic cholinergic neurons in the cat. Retrograde tracing experiments indicate that the scattered type of cholinergic neurons contains four vasodilator peptides (VIP, PHI, CGRP, SP) and provides an important input to sweat glands, whereas the clustered type (containing VIP and PHI) mainly innervates blood vessels in muscles.