**3. Signaling pathways that regulate dendritic growth in sympathetic neurons**

Research over the past few decades has provided insights into the signaling pathways and molecular mechanisms that control dendritic growth in sympathetic neurons. As discussed in the preceding section, BMPs and NGF play predominant roles in the initiation and maintenance of dendrites in these autonomic neurons. While the importance of these growth factors as regulators of dendritic growth in sympathetic neurons is well established, the downstream effectors that link BMP and NGF to increased dendritic growth are not fully understood. In this section, we will discuss the signaling pathways activated by these growth factors, the evidence implicating downstream effectors of BMPs and NGF in dendritic regulation, and the identification of factors that interact with these signaling pathways to alter their influence on the dendritic arborization of sympathetic neurons.

#### **3.1. BMP signaling**

BMPs mediate their cellular effects by binding to a heteromeric receptor complex of transmembrane serine/threonine kinas receptor subunits comprised of a type I receptor [BMP type I receptor A (BMPR1A), which is also known as activin receptor-like kinase**-**3 (ALK-3) or BMP type I receptor B (BMPRIB, also known as ALK-6)], and a type II receptor [BMP type II receptor (BMPRII), activin type II receptor (ActRII), or activin type IIB receptor (ActRIIB)] [41, 42]. Ligand binding causes type II receptors to phosphorylate type I receptors, which then phosphorylate Smads 1, 5, and/or 8, also known as receptor Smads (R-Smads). Phosphorylated R-Smads complex with Smad 4, triggering translocation of the Smad complex to the nucleus to regulate gene transcription [42, 43]. Studies in *Smad* knockout animals and *Smad* deficient cells suggest that BMPs can also signal through Smad-independent pathways via activation of mitogen-activated protein kinase (MAPK), c-jun amino terminal kinase (JNK), p38-MAPK, phosphoinositol-3-kinase (PI3K), LIM kinase 1 (LIMK1) or small GTPases [43–46].

BMP signaling pathways are active in sympathetic ganglia during developmental periods corresponding to the initiation, extension and maintenance of dendrites. Quantitative PCR, *in situ* hybridization, and immunocytochemistry studies have confirmed the presence of mRNA and protein for BMPRIA, BMPRIB, ActRII and BMPRII in neurons of mouse SCG starting as early as embryonic day 14, and persisting into postnatal development and even adulthood [47, 48]. A role for BMPRIA in regulating dendritic growth in sympathetic neurons *in vivo* was confirmed in a BMPRIA conditional knockout mouse generated by crossing a *Dbhi-Cre* mouse line to a *BmprIa* floxed line [49]. *Dbhi-Cre* is expressed in sympathetic ganglia around E10.5, thus, *BmprIa* expression is knocked out after neuronal specification, but before initiation of dendritic growth. The loss of BMPRIA expression in sympathetic ganglia significantly diminished but did not completely block dendritic growth *in vivo* [49]. At postnatal day 3, the number of primary dendrites and the size of the dendritic arbor were not significantly different in sympathetic neurons of SCG from *BmprIa* conditional knockout mice *vs*. SCG neurons from congenic wildtype controls. At later developmental times, however, dendritic arborization was significantly decreased in SCG neurons of *BmprIa* knockout vs. wildtype mice. These data suggest that, *in vivo*, BMP signaling is required for the maintenance of dendrites, but not for the initiation and early growth of dendrites in sympathetic neurons.

Several caveats of the *in vivo* study suggest that the lack of effect of *BmprIa* knockout on early stages of dendritic growth *in vivo* may not be discrepant with *in vitro* data showing that BMPs are necessary and sufficient for induction of dendritic growth in sympathetic neurons [29, 49]. First, while the authors confirmed that *BmprIa* mRNA was not generated in the SCG after embryonic day 11.5, they did not examine BMPRIA protein levels in the SCG of knockout animals. Thus, the possibility that BMPRIA protein was still present during early stages of dendritic growth cannot be ruled out. Moreover, recent biochemical studies have shown that BMPRs cycle between various cellular compartments, and the association of the receptors with endocytosis machinery is required for Smad-mediated signal transduction [50]. Thus, additional studies are needed to characterize BMPR turnover and changes in receptor localization in wildtype *vs*. *BMPR* knockouts. Another, perhaps more likely, explanation is that sympathetic neurons in the *BmprIa* knockout mice express other type I receptors, such as the activin receptors, that may be activated by BMPs to trigger dendritic growth at early developmental stages. Further studies targeting other BMPRs are warranted to fully elucidate the role of BMP signaling in dendritogenesis *in vivo*.

#### *3.1.1. Smad-dependent transcriptional regulation of dendritic growth*

blood vessels, express significant levels of BMPs during embryonic development, throughout the postnatal period, and into adulthood [38–40]. Thus, target tissues may be a source of BMPs to sympathetic neurons not only during initial expansion of the dendritic arbor, but also in the maintenance and remodeling of dendritic arbors that continues throughout the life

**3. Signaling pathways that regulate dendritic growth in sympathetic** 

the dendritic arborization of sympathetic neurons.

Research over the past few decades has provided insights into the signaling pathways and molecular mechanisms that control dendritic growth in sympathetic neurons. As discussed in the preceding section, BMPs and NGF play predominant roles in the initiation and maintenance of dendrites in these autonomic neurons. While the importance of these growth factors as regulators of dendritic growth in sympathetic neurons is well established, the downstream effectors that link BMP and NGF to increased dendritic growth are not fully understood. In this section, we will discuss the signaling pathways activated by these growth factors, the evidence implicating downstream effectors of BMPs and NGF in dendritic regulation, and the identification of factors that interact with these signaling pathways to alter their influence on

BMPs mediate their cellular effects by binding to a heteromeric receptor complex of transmembrane serine/threonine kinas receptor subunits comprised of a type I receptor [BMP type I receptor A (BMPR1A), which is also known as activin receptor-like kinase**-**3 (ALK-3) or BMP type I receptor B (BMPRIB, also known as ALK-6)], and a type II receptor [BMP type II receptor (BMPRII), activin type II receptor (ActRII), or activin type IIB receptor (ActRIIB)] [41, 42]. Ligand binding causes type II receptors to phosphorylate type I receptors, which then phosphorylate Smads 1, 5, and/or 8, also known as receptor Smads (R-Smads). Phosphorylated R-Smads complex with Smad 4, triggering translocation of the Smad complex to the nucleus to regulate gene transcription [42, 43]. Studies in *Smad* knockout animals and *Smad* deficient cells suggest that BMPs can also signal through Smad-independent pathways via activation of mitogen-activated protein kinase (MAPK), c-jun amino terminal kinase (JNK), p38-MAPK,

phosphoinositol-3-kinase (PI3K), LIM kinase 1 (LIMK1) or small GTPases [43–46].

BMP signaling pathways are active in sympathetic ganglia during developmental periods corresponding to the initiation, extension and maintenance of dendrites. Quantitative PCR, *in situ* hybridization, and immunocytochemistry studies have confirmed the presence of mRNA and protein for BMPRIA, BMPRIB, ActRII and BMPRII in neurons of mouse SCG starting as early as embryonic day 14, and persisting into postnatal development and even adulthood [47, 48]. A role for BMPRIA in regulating dendritic growth in sympathetic neurons *in vivo* was confirmed in a BMPRIA conditional knockout mouse generated by crossing a *Dbhi-Cre* mouse line to a *BmprIa* floxed line [49]. *Dbhi-Cre* is expressed in sympathetic ganglia around E10.5, thus, *BmprIa* expression is knocked out after neuronal specification, but before initiation of dendritic growth. The loss of BMPRIA expression in sympathetic ganglia significantly

of the animal.

94 Autonomic Nervous System

**neurons**

**3.1. BMP signaling**

Canonical BMP signaling involves the Smad family of transcription factors. Immunocytochemical analyses of primary rat SCG neurons have demonstrated that Smad 1/5/8 translocates to the nucleus within 20 minutes of exposure to BMP-7, with maximal nuclear translocation observed within 2 hours of adding BMP-7 to the culture medium. Transfection with a Smad1 dominant negative mutant, Smad1 (3SA), blocked BMP-7-induced dendritic growth in primary sympathetic neurons [51], indicating that Smad 1 activation is necessary for induction of dendritic growth by BMP-7. In contrast to the *BmprIa* conditional knockouts, conditional knockdown of *Smad4* in sympathetic neurons, generated by crossing *Dbhi-Cre* mice to *Smad4* floxed mice, increased dendritic length and the size of the dendritic arbor in SCG neurons [49], suggesting that Smad 4 may play a role in limiting dendritic growth *in vivo*.

A comprehensive analyses of Smad-dependent dendritic growth in sympathetic neurons provided evidence for early transcriptional regulation of dendritic growth downstream of BMP-7 [52]. In neuronal cell cultures from embryonic rat SCG, BMP-7-induced dendritic growth could be blocked by pharmacologic inhibition of transcription with actinomycin-D when the inhibitor was added within the first 24 hours of BMP-7 exposure but not when it was added after 48 hours of BMP-7 exposure. Microarray analyses identified over 250 genes that were differentially regulated by BMP-7 within the first 24 hours after adding BMP-7 to the culture medium. Of these, 56 mRNAs were altered within the first 6 hours and 185 mRNAs were differentially regulated at 24 hours after BMP exposure [52]. Many of the differentially regulated genes were linked to signaling pathways previously implicated in dendritogenesis in other neuronal cell types or neuronal morphogenesis and/or axonal guidance, such as BMP, Notch, integrin, Wnt, and NGF signaling molecules. However, the functional relevance of most of these genes to dendritic growth in sympathetic neurons has yet to be determined. Moreover, recent reports of limited correlation between transcriptome and proteome analysis in yeast, plants and mice [53, 54], suggest that in order to generate a more complete understanding of the molecular pathways that link BMPs to dendritic growth in sympathetic neurons, detailed proteome analyses are needed to complement the existing transcriptomic dataset.

in the dendritic actin cytoskeleton [68, 69]. p75NTR has been shown to interact with RhoA in the yeast two-hybrid system [70]. In cultured rat sympathetic neurons, exposure to BMP-7 increases the levels of the GTP-bound form of RhoA, but not GTP-Rac1 or GTP-Cdc42, as determined by a GTP pull down assay, and triggers RhoA translocation from the cytoplasm to the membrane [13]. The observation that BMP-7-induced dendritic growth in primary sympathetic neurons requires RhoA activation [13], suggests a model in which BMP-7 sequentially activates BMPRIA, Smad 1/5/8, p75NTR, and then RhoA to induce dendritic growth in

Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 97

The shape of the dendritic arbor of developing sympathetic neurons is determined by interactions between positive and negative regulators of dendritic growth. A number of signaling pathways have been shown to interact with BMP signaling to modulate the number of dendrites, total dendritic length and dendritic branching in sympathetic neurons. In this section, we will review known positive and negative regulators of Smad signaling that impact BMP-

*Proteasome-mediated signaling.* The ubiquitin-proteasome pathway is unique in that it is one of the few signaling pathways identified thus far whose interactions with BMP signaling enhance dendritic arborization in sympathetic neurons. In the nervous system, proteasomemediated protein degradation pathways are important for self-renewal of neurons, axonal growth, vesicle release, receptor turnover, signal transduction and synaptic plasticity [71– 73]. The ubiquitin-proteasome pathway has been implicated in regulating dendritic growth in the mammalian central nervous system, with multiple E3 ubiquitin ligases, including Anaphase promoting complex (APC), cullin RING-type E3 ubiquitin ligases (CUL) and neuronal precursor cell expressed and developmentally downregulated protein (Nedd4), determined to be necessary for the extension and maintenance of the dendritic arbor of central

Biochemical studies have shown that R-Smads interact with components of the proteasome complex, as well as enzymes and proteins involved in the ubiquitination-deubiquitination of proteins, in many systems, and the ability of Smads to regulate transcription is dependent on association with the proteasome complex in various cell types [77, 78]. Interactions between BMP signaling molecules and the proteasome pathway have been reported in perinatal rat sympathetic neuronal cultures prior to dendritic growth induction by BMP-7 [51]. In this study, interactions between Smad1 and multiple proteasome components were confirmed using a yeast two-hybrid assay, and pharmacologic inhibition of proteasome activity by lactacystin and ALLN (N-acetyl-Leu-Leu-norleucinal) selectively blocked BMP-7-induced dendritic growth in primary sympathetic neurons in the absence of any effect on axonal growth [51]. These proteasome inhibitors also suppressed Smad-mediated transcriptional regulation in a biochemical assay using a Tlx -luciferase construct transfected into P19 cells [51]. One caveat of this study is that although there was clearly a functional interaction between BMP signaling and the proteasome pathway in the context of dendritic growth, a biochemical

**3.2. Signaling pathways that interact with Smad signaling to modulate BMP-**

sympathetic neurons.

neurons [71, 74–76].

**induced dendritic growth**

induced dendritogenesis in sympathetic neurons.

*3.2.1. Positive regulators of Smad signaling that enhance dendritic growth*

One gene identified as being strongly upregulated by BMP-7 in primary sympathetic neurons, the gene encoding the p75 neurotrophin receptor (p75NTR) [52], has been evaluated for a role in BMP-induced dendritic growth. A member of the tumor necrosis factor (TNF) receptor family, p75NTR regulates diverse neurobiological processes, including axonal growth, synaptic plasticity, dendritic growth in central neurons, and neuronal cell death [55–61]. p75NTR binds diverse ligands to mediate its effects, including NGF, other neurotrophins, myelin-derived polypeptides, such as myelin-associated glycoprotein (MAG) or Nogo, and β-amyloid peptide [60, 62, 63]. In cultured embryonic rat SCG neurons, p75NTR mRNA and protein expression are significantly upregulated within 24 hours of exposure to BMP-7 [52, 64], and pharmacologic inhibition of signaling via BMPRI prevents induction of p75NTR protein expression in primary sympathetic neurons exposed to BMP-7 [64]. Functional studies revealed that BMP7 does not trigger dendritic growth in primary sympathetic neurons derived from SCG of p75NTR knockout mice; conversely, ligand-independent activation of p75NTR via overexpression of a p75NTR cDNA construct in p75NTR−/− neurons [65], phenocopies the dendrite-promoting effects of BMP-7 [64]. Morphometric analyses of SCG from wildtype *vs*. p75NTR knockout mice at 3, 6 and 12–16 weeks of age indicated that genetic deletion of p75NTR does not prevent dendritic growth, but does significantly stunt dendritic maturation in sympathetic neurons. These data support the hypotheses that p75NTR is involved in downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that p75NTR signaling positively modulates dendritic complexity in sympathetic neurons *in vivo*.

An outstanding question regarding p75NTR effects on dendritic growth in sympathetic neurons is the identity of ligand(s) and co-receptor(s) that p75NTR interacts with to mediate BMP-induced dendritic growth. Several lines of evidence argue against a direct interaction between p75NTR and the BMP receptor complex: (*i*) inhibiting transcription blocks both BMP-induced dendritic growth and upregulation of p75NTR mRNA [52]; (*ii*) pharmacologic blockade of BMPRI signaling blocks p75NTR induction [64]; and (*iii*) overexpressing p75NTR in cultured p75NTR−/− neurons induces dendritic growth even in the absence of BMP-7 [64]. NGF is a potential activating ligand, and NGF is required for BMP-induced dendritic growth [29, 36]. However, the observation that p75NTR interactions with the primary receptor for NGF, TrkA, serve to limit synapse formation in sympathetic neurons [66], argues against p75NTR-TrkA interactions enhancing dendritic development. Likewise, pro-neurotrophin activation of the p75NTR-sortilin complex serves to promote sympathetic axon degeneration and cell death [67], suggesting that this interaction is also unlikely to stimulate dendritic growth. The identity of the p75NTR ligand that mediates BMP-7-induced dendritic growth remains an outstanding question.

Similarly, the downstream effector molecule(s) that link p75NTR to increased dendritic arborization remain to be determined. Key candidates include the Rho GTPases. Rho GTPases function as central regulators of dendritic morphology, linking extracellular signals to changes in the dendritic actin cytoskeleton [68, 69]. p75NTR has been shown to interact with RhoA in the yeast two-hybrid system [70]. In cultured rat sympathetic neurons, exposure to BMP-7 increases the levels of the GTP-bound form of RhoA, but not GTP-Rac1 or GTP-Cdc42, as determined by a GTP pull down assay, and triggers RhoA translocation from the cytoplasm to the membrane [13]. The observation that BMP-7-induced dendritic growth in primary sympathetic neurons requires RhoA activation [13], suggests a model in which BMP-7 sequentially activates BMPRIA, Smad 1/5/8, p75NTR, and then RhoA to induce dendritic growth in sympathetic neurons.

#### **3.2. Signaling pathways that interact with Smad signaling to modulate BMPinduced dendritic growth**

neuronal cell types or neuronal morphogenesis and/or axonal guidance, such as BMP, Notch, integrin, Wnt, and NGF signaling molecules. However, the functional relevance of most of these genes to dendritic growth in sympathetic neurons has yet to be determined. Moreover, recent reports of limited correlation between transcriptome and proteome analysis in yeast, plants and mice [53, 54], suggest that in order to generate a more complete understanding of the molecular pathways that link BMPs to dendritic growth in sympathetic neurons, detailed

One gene identified as being strongly upregulated by BMP-7 in primary sympathetic neurons, the gene encoding the p75 neurotrophin receptor (p75NTR) [52], has been evaluated for a role in BMP-induced dendritic growth. A member of the tumor necrosis factor (TNF) receptor family, p75NTR regulates diverse neurobiological processes, including axonal growth, synaptic plasticity, dendritic growth in central neurons, and neuronal cell death [55–61]. p75NTR binds diverse ligands to mediate its effects, including NGF, other neurotrophins, myelin-derived polypeptides, such as myelin-associated glycoprotein (MAG) or Nogo, and β-amyloid peptide [60, 62, 63]. In cultured embryonic rat SCG neurons, p75NTR mRNA and protein expression are significantly upregulated within 24 hours of exposure to BMP-7 [52, 64], and pharmacologic inhibition of signaling via BMPRI prevents induction of p75NTR protein expression in primary sympathetic neurons exposed to BMP-7 [64]. Functional studies revealed that BMP7 does not trigger dendritic growth in primary sympathetic neurons derived from SCG of p75NTR knockout mice; conversely, ligand-independent activation of p75NTR via overexpression of a p75NTR cDNA construct in p75NTR−/− neurons [65], phenocopies the dendrite-promoting effects of BMP-7 [64]. Morphometric analyses of SCG from wildtype *vs*. p75NTR knockout mice at 3, 6 and 12–16 weeks of age indicated that genetic deletion of p75NTR does not prevent dendritic growth, but does significantly stunt dendritic maturation in sympathetic neurons. These data support the hypotheses that p75NTR is involved in downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that p75NTR signaling

proteome analyses are needed to complement the existing transcriptomic dataset.

96 Autonomic Nervous System

positively modulates dendritic complexity in sympathetic neurons *in vivo*.

mediates BMP-7-induced dendritic growth remains an outstanding question.

Similarly, the downstream effector molecule(s) that link p75NTR to increased dendritic arborization remain to be determined. Key candidates include the Rho GTPases. Rho GTPases function as central regulators of dendritic morphology, linking extracellular signals to changes

An outstanding question regarding p75NTR effects on dendritic growth in sympathetic neurons is the identity of ligand(s) and co-receptor(s) that p75NTR interacts with to mediate BMP-induced dendritic growth. Several lines of evidence argue against a direct interaction between p75NTR and the BMP receptor complex: (*i*) inhibiting transcription blocks both BMP-induced dendritic growth and upregulation of p75NTR mRNA [52]; (*ii*) pharmacologic blockade of BMPRI signaling blocks p75NTR induction [64]; and (*iii*) overexpressing p75NTR in cultured p75NTR−/− neurons induces dendritic growth even in the absence of BMP-7 [64]. NGF is a potential activating ligand, and NGF is required for BMP-induced dendritic growth [29, 36]. However, the observation that p75NTR interactions with the primary receptor for NGF, TrkA, serve to limit synapse formation in sympathetic neurons [66], argues against p75NTR-TrkA interactions enhancing dendritic development. Likewise, pro-neurotrophin activation of the p75NTR-sortilin complex serves to promote sympathetic axon degeneration and cell death [67], suggesting that this interaction is also unlikely to stimulate dendritic growth. The identity of the p75NTR ligand that The shape of the dendritic arbor of developing sympathetic neurons is determined by interactions between positive and negative regulators of dendritic growth. A number of signaling pathways have been shown to interact with BMP signaling to modulate the number of dendrites, total dendritic length and dendritic branching in sympathetic neurons. In this section, we will review known positive and negative regulators of Smad signaling that impact BMPinduced dendritogenesis in sympathetic neurons.

#### *3.2.1. Positive regulators of Smad signaling that enhance dendritic growth*

*Proteasome-mediated signaling.* The ubiquitin-proteasome pathway is unique in that it is one of the few signaling pathways identified thus far whose interactions with BMP signaling enhance dendritic arborization in sympathetic neurons. In the nervous system, proteasomemediated protein degradation pathways are important for self-renewal of neurons, axonal growth, vesicle release, receptor turnover, signal transduction and synaptic plasticity [71– 73]. The ubiquitin-proteasome pathway has been implicated in regulating dendritic growth in the mammalian central nervous system, with multiple E3 ubiquitin ligases, including Anaphase promoting complex (APC), cullin RING-type E3 ubiquitin ligases (CUL) and neuronal precursor cell expressed and developmentally downregulated protein (Nedd4), determined to be necessary for the extension and maintenance of the dendritic arbor of central neurons [71, 74–76].

Biochemical studies have shown that R-Smads interact with components of the proteasome complex, as well as enzymes and proteins involved in the ubiquitination-deubiquitination of proteins, in many systems, and the ability of Smads to regulate transcription is dependent on association with the proteasome complex in various cell types [77, 78]. Interactions between BMP signaling molecules and the proteasome pathway have been reported in perinatal rat sympathetic neuronal cultures prior to dendritic growth induction by BMP-7 [51]. In this study, interactions between Smad1 and multiple proteasome components were confirmed using a yeast two-hybrid assay, and pharmacologic inhibition of proteasome activity by lactacystin and ALLN (N-acetyl-Leu-Leu-norleucinal) selectively blocked BMP-7-induced dendritic growth in primary sympathetic neurons in the absence of any effect on axonal growth [51]. These proteasome inhibitors also suppressed Smad-mediated transcriptional regulation in a biochemical assay using a Tlx -luciferase construct transfected into P19 cells [51]. One caveat of this study is that although there was clearly a functional interaction between BMP signaling and the proteasome pathway in the context of dendritic growth, a biochemical interaction between Smads and proteasomes were not demonstrated in primary sympathetic neurons. Further studies are necessary to fully understand the genetic and biochemical interactions between Smads and ubiquitin-proteasome pathway during dendritogenesis in sympathetic neurons.

IFNγ-mediated dendritic retraction [92, 94]. These data provide evidence that neuropoietic cytokines negatively regulate dendritic growth in perinatal sympathetic neurons via activation of STAT proteins. However, many questions remain, including the mechanism by which nuclear translocation of STAT proteins limits the activity of SMAD proteins, and the down-

*Mitogen-activated protein kinase (MAPK) signaling.* MAPK family members, including extra-

ine–threonine kinases that function as downstream effectors of many extracellular signaling molecules, including NGF, epidermal growth factor (EGF), and fibroblast growth factor (FGF) [95, 96]. The activation of MAPK regulates gene expression to alter various cellular activities such as cell division, growth, survival and cell death [95, 96]. Previous studies in *Xenopus* embryos and cells such as radial glia, astrocytes, hippocampal neurons, and PC12 cells, have demonstrated interplay between Smad and MAPK signaling pathways, and shown that phosphorylation of the linker on the Smad protein by MAPK is necessary for coordinating the cellular effects of BMPs and other growth factors [43, 97–99]. Interplay between BMP and MAPK signaling pathways is also observed during dendritogenesis in rat and mouse sympathetic neurons. Pharmacologic inhibition of MAPK signaling with PD98059 or transfection of a dominant negative MEK1 or ERK2 mutant increases the number of dendrites and total dendritic arbor induced following BMP-7 exposure, whereas overexpression of MEK1 decreases BMP-7 induced dendritic growth [100]. Inhibition of MAPK signaling in primary sympathetic neurons also increases Smad nuclear accumulation following BMP-7 exposure, and FGF was identified as one of the ligands responsible for activating the ERK signaling pathway in these neurons [100]. These findings suggest that, in perinatal sympathetic neurons, FGF activates the MAPK pathway, which inhibits the nuclear accumulation of SMADs, thereby limiting

In addition to modulating BMP effects on dendritic arborization via activation of MAPK signaling, FGFs regulate neuronal differentiation via the integrative nuclear FGFR1 signaling (INFS) pathway [101]. FGFR1 is expressed in the nucleus of adult rat SCG neurons following axotomy [102]. Nuclear localization of FGFR1 is also increased in perinatal rat sympathetic neurons following exposure to BMP-7, and transfection of a mutant FGFR1 receptor inhibits FGFR1 nuclear localization and decreases the dendritic response to BMP-7 [103]. These data suggest that the INFS-mediated FGF signaling pathway functions downstream of BMP sig-

Rit GTPase, a member of the small GTPase family, has also been shown to activate ERK1/2 in primary sympathetic neurons, and the transfection of dominant negative (dn) Rit or constitutively active (ca) Rit were observed to increase or decrease BMP-induced dendritic growth, respectively [36]. Rit GTPase also negatively modulates dendritic growth as a downstream target of IFNγ signaling as demonstrated by inhibition of IFNγ-mediated dendritic retraction in primary sympathetic neurons transfected with dnRit constructs [104]. Addition of IFNγ to cultures of pheochromocytoma cells, which are often used as a model for sympathetic neurons, increased levels of GTP-Rit, and transfection of dnRit inhibited IFNγ-induced activation of p38 MAPK [104]. These observations suggest that a novel Rit-p38 MAP kinase signaling pathway functions in parallel with the canonical JAK–STAT signaling pathway to mediate IFNγ-induced dendritic retraction. Collectively, these studies provide evidence for crosstalk

naling to limit BMP-induced dendritogenesis in sympathetic neurons.


Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 99

stream targets of STAT and SMAD signaling during dendritogenesis.

cellular signal-regulated kinase (ERK), p38, and c-Jun NH2

BMP-induced dendritic growth.

*Signaling mediated by reactive oxygen species (ROS).* Under physiological conditions, ROS are generated by the mitochondrial electron transport chain or as a consequence of the activity of NADPH oxidase (NOX) [79–81]. While the deleterious effects of ROS at high concentrations are well documented, over the past decade, there is growing appreciation of the beneficial role of ROS at physiological concentrations [80–84], and of the importance of the NOX family of enzymes in regulating many aspects of neuronal development including neurogenesis, neurite outgrowth and synaptic plasticity [80, 83, 85]. NOX2 is expressed in neonatal rat SCG neuronal cell cultures [86], and exposure of cultured embryonic SCG neurons to BMP-7 increases the expression of NOX2 [87]. Pharmacologic inhibition or siRNA knockdown of NOX2 significantly decreases BMP-7-induced dendritic growth in primary sympathetic neurons, an effect that is also observed in cultures co-exposed to BMP-7 and any of three mechanistically and structurally distinct antioxidants that block ROS generation [87]. Antioxidants block BMP-induced dendritic growth downstream of Smad signaling since BMP-induced nuclear translocation of Smads is unaffected by antioxidant treatment [87].

Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth, and suggest that ROS-mediated signaling positively modulates dendritic complexity in sympathetic neurons. One caveat of this study, however, is that while BMP-7 was observed to increase NOX2 levels and oxygen consumption in sympathetic neurons, increased ROS levels were not detected in sympathetic neurons exposed to BMP-7. Likely, this reflects the fact that physiologic BMP signaling generates levels of ROS that are below the detection threshold for standard ROS detection assays. Further work is needed to determine whether these *in vitro* observations translate to a role for ROS in regulating dendritic growth in sympathetic ganglia *in vivo*.

#### *3.2.2. Negative regulators of SMAD signaling that influence dendritic growth*

*STAT signaling.* The interferons (IFN) and the neuropoietic cytokines, which include interleukins (IL), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF), signal through the activation and nuclear translocation of signal transducers and activators of transcription (STAT) proteins, which then regulate transcription of target genes [88]. Previous studies have shown that cytokines that signal through STATs *vs*. members of the TGFβ superfamily of signaling molecules tend to exert opposing effects on cellular function [89–91]. Similar antagonistic interactions between STAT-dependent cytokine signaling pathways and the BMP signaling pathways have been documented during dendritogenesis in perinatal sympathetic neurons. In primary perinatal sympathetic neurons, addition of LIF, CNTF or gamma-IFN (IFNγ) significantly decreased the number of primary dendrites and total dendritic length in cultures exposed to BMP-7, but had no effect on axonal growth or cell viability [92–94]. In addition, all three cytokines triggered retraction of preexisting dendrites [92, 94], suggesting a role for them in the dendritic retraction observed following neuronal injury. Addition of LIF, CNTF or IFNγ to primary sympathetic neurons resulted in nuclear translocation of STAT, and transfection of a dominant negative STAT1 construct blocked IFNγ-mediated dendritic retraction [92, 94]. These data provide evidence that neuropoietic cytokines negatively regulate dendritic growth in perinatal sympathetic neurons via activation of STAT proteins. However, many questions remain, including the mechanism by which nuclear translocation of STAT proteins limits the activity of SMAD proteins, and the downstream targets of STAT and SMAD signaling during dendritogenesis.

interaction between Smads and proteasomes were not demonstrated in primary sympathetic neurons. Further studies are necessary to fully understand the genetic and biochemical interactions between Smads and ubiquitin-proteasome pathway during dendritogenesis in sym-

*Signaling mediated by reactive oxygen species (ROS).* Under physiological conditions, ROS are generated by the mitochondrial electron transport chain or as a consequence of the activity of NADPH oxidase (NOX) [79–81]. While the deleterious effects of ROS at high concentrations are well documented, over the past decade, there is growing appreciation of the beneficial role of ROS at physiological concentrations [80–84], and of the importance of the NOX family of enzymes in regulating many aspects of neuronal development including neurogenesis, neurite outgrowth and synaptic plasticity [80, 83, 85]. NOX2 is expressed in neonatal rat SCG neuronal cell cultures [86], and exposure of cultured embryonic SCG neurons to BMP-7 increases the expression of NOX2 [87]. Pharmacologic inhibition or siRNA knockdown of NOX2 significantly decreases BMP-7-induced dendritic growth in primary sympathetic neurons, an effect that is also observed in cultures co-exposed to BMP-7 and any of three mechanistically and structurally distinct antioxidants that block ROS generation [87]. Antioxidants block BMP-induced dendritic growth downstream of Smad signaling since BMP-induced

Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth, and suggest that ROS-mediated signaling positively modulates dendritic complexity in sympathetic neurons. One caveat of this study, however, is that while BMP-7 was observed to increase NOX2 levels and oxygen consumption in sympathetic neurons, increased ROS levels were not detected in sympathetic neurons exposed to BMP-7. Likely, this reflects the fact that physiologic BMP signaling generates levels of ROS that are below the detection threshold for standard ROS detection assays. Further work is needed to determine whether these *in vitro* observations translate to a role for

*STAT signaling.* The interferons (IFN) and the neuropoietic cytokines, which include interleukins (IL), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF), signal through the activation and nuclear translocation of signal transducers and activators of transcription (STAT) proteins, which then regulate transcription of target genes [88]. Previous studies have shown that cytokines that signal through STATs *vs*. members of the TGFβ superfamily of signaling molecules tend to exert opposing effects on cellular function [89–91]. Similar antagonistic interactions between STAT-dependent cytokine signaling pathways and the BMP signaling pathways have been documented during dendritogenesis in perinatal sympathetic neurons. In primary perinatal sympathetic neurons, addition of LIF, CNTF or gamma-IFN (IFNγ) significantly decreased the number of primary dendrites and total dendritic length in cultures exposed to BMP-7, but had no effect on axonal growth or cell viability [92–94]. In addition, all three cytokines triggered retraction of preexisting dendrites [92, 94], suggesting a role for them in the dendritic retraction observed following neuronal injury. Addition of LIF, CNTF or IFNγ to primary sympathetic neurons resulted in nuclear translocation of STAT, and transfection of a dominant negative STAT1 construct blocked

nuclear translocation of Smads is unaffected by antioxidant treatment [87].

ROS in regulating dendritic growth in sympathetic ganglia *in vivo*.

*3.2.2. Negative regulators of SMAD signaling that influence dendritic growth*

pathetic neurons.

98 Autonomic Nervous System

*Mitogen-activated protein kinase (MAPK) signaling.* MAPK family members, including extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2 -terminal kinase (JNK), are serine–threonine kinases that function as downstream effectors of many extracellular signaling molecules, including NGF, epidermal growth factor (EGF), and fibroblast growth factor (FGF) [95, 96]. The activation of MAPK regulates gene expression to alter various cellular activities such as cell division, growth, survival and cell death [95, 96]. Previous studies in *Xenopus* embryos and cells such as radial glia, astrocytes, hippocampal neurons, and PC12 cells, have demonstrated interplay between Smad and MAPK signaling pathways, and shown that phosphorylation of the linker on the Smad protein by MAPK is necessary for coordinating the cellular effects of BMPs and other growth factors [43, 97–99]. Interplay between BMP and MAPK signaling pathways is also observed during dendritogenesis in rat and mouse sympathetic neurons. Pharmacologic inhibition of MAPK signaling with PD98059 or transfection of a dominant negative MEK1 or ERK2 mutant increases the number of dendrites and total dendritic arbor induced following BMP-7 exposure, whereas overexpression of MEK1 decreases BMP-7 induced dendritic growth [100]. Inhibition of MAPK signaling in primary sympathetic neurons also increases Smad nuclear accumulation following BMP-7 exposure, and FGF was identified as one of the ligands responsible for activating the ERK signaling pathway in these neurons [100]. These findings suggest that, in perinatal sympathetic neurons, FGF activates the MAPK pathway, which inhibits the nuclear accumulation of SMADs, thereby limiting BMP-induced dendritic growth.

In addition to modulating BMP effects on dendritic arborization via activation of MAPK signaling, FGFs regulate neuronal differentiation via the integrative nuclear FGFR1 signaling (INFS) pathway [101]. FGFR1 is expressed in the nucleus of adult rat SCG neurons following axotomy [102]. Nuclear localization of FGFR1 is also increased in perinatal rat sympathetic neurons following exposure to BMP-7, and transfection of a mutant FGFR1 receptor inhibits FGFR1 nuclear localization and decreases the dendritic response to BMP-7 [103]. These data suggest that the INFS-mediated FGF signaling pathway functions downstream of BMP signaling to limit BMP-induced dendritogenesis in sympathetic neurons.

Rit GTPase, a member of the small GTPase family, has also been shown to activate ERK1/2 in primary sympathetic neurons, and the transfection of dominant negative (dn) Rit or constitutively active (ca) Rit were observed to increase or decrease BMP-induced dendritic growth, respectively [36]. Rit GTPase also negatively modulates dendritic growth as a downstream target of IFNγ signaling as demonstrated by inhibition of IFNγ-mediated dendritic retraction in primary sympathetic neurons transfected with dnRit constructs [104]. Addition of IFNγ to cultures of pheochromocytoma cells, which are often used as a model for sympathetic neurons, increased levels of GTP-Rit, and transfection of dnRit inhibited IFNγ-induced activation of p38 MAPK [104]. These observations suggest that a novel Rit-p38 MAP kinase signaling pathway functions in parallel with the canonical JAK–STAT signaling pathway to mediate IFNγ-induced dendritic retraction. Collectively, these studies provide evidence for crosstalk between BMP signaling and MAPK signaling during dendritogenesis in sympathetic neurons, and suggest that in contrast to its effects in central neurons, MAPK signaling functions as a negative regulator of BMP-induced dendritic growth in sympathetic neurons.

not well characterized. Early growth response-3 (Egr3), a transcriptional regulator known to be induced by NGF via MAPK signaling, has been identified as a potential downstream regulator of NGF-induced dendritic growth [111]. Sympathetic neurons from a conditional *Egr3* knockout generated using *Dbhi-Cre* have fewer primary dendrites and shorter dendritic arbors compared to sympathetic neurons from congenic wildtype animals. In addition, *Egr3* knockout animals exhibit defects in axonal guidance, and in innervation of autonomic targets [111]. Although, Egr3 had previously been shown to not be required for NGF effects on neuronal survival [112], some neuronal cell death was observed in germline mutants for Egr3. It is, therefore, possible that the dendritic effects of knocking out *Egr3* are secondary to adverse

Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 101

Neuronal depolarization induced by electric field stimulation or the addition of potassium chloride to primary postnatal sympathetic neurons cultured was shown to trigger the formation of dendrites in the presence of NGF that retracted in the absence of neuronal activity [113]. Neuronal depolarization enhanced stability of microtubules and activated calcium calmodulin dependent kinase II (CaMKII) in dendrites. The latter was shown to be causally related to the effects of neuronal depolarization on dendritic growth: pharmacologic inhibition of CaMKII activity using KN62 or mAIP completely blocks activity-dependent dendritic

Signaling by integrin-linked kinase (ILK) and glycogen synthase kinase-3β (GSK-3β) have also been shown to be downstream effectors of activity-dependent dendritic growth in postnatal sympathetic neurons [114]. ILK and GSK-3β are serine threonine kinases that are downstream effectors of integrin and neurotrophin signaling [115]. ILK has been shown to phosphorylate and inactivate GSK-3β to regulate NGF-mediated axonal growth [116]. Increased phosphorylation of GSK-3β protein was observed in cultured postnatal rat SCG neurons in response to increased neuronal activity, and inhibition of ILK activity by QLT0254, as well as transfection of dominant negative ILK or siRNA for ILK, blocked activity-dependent dendritic growth in these neurons. Similarly, inhibition of GSK-3β activity using kenpaullone or genetic knockdown of GSK-3β expression increased the number of primary dendrites formed in response to potassium chloride, suggesting that GSK-3β inhibition is necessary for early stages of activity-

Interestingly, unlike BMP-induced dendritic growth, inhibition of ERK activity inhibited activity-dependent dendritic growth in postnatal sympathetic neurons *in vitro* [113, 114]. Pharmacologic inhibition of ERK by PD98059 blocked activity-dependent dendritic growth, inhibition of GSK-3β increased depolarization-dependent ERK activation, and inhibition of ERK reversed the enhanced dendritic growth observed with GSK-3β inhibition [113, 114]. The reasons for the opposing roles of ERK in BMP-induced vs. activity-dependent dendritic

The experimental evidence clearly implicate NGF, BMP and neuronal activity as positive regulators of dendritic growth in perinatal sympathetic neurons *in vitro*. However, the interaction between these three pathways and their relative contributions to the induction and

effects on neuronal cell viability or changes in target innervation.

growth in cultured sympathetic neurons [113].

dependent dendritic growth in sympathetic neurons.

growth remain unexplored.

**4. The path forward**

*Retinoic acid signaling.* In the developing nervous system, BMPs and retinoic acid work synergistically to regulate neural tube patterning and specification of neuronal identity [105–108]. Similarly, interplay between BMP and retinoic acid signaling have been documented during dendritogenesis in sympathetic neurons; however, their interaction in this latter context is antagonistic [106]. Genes required for synthesis of retinoic acid are expressed in cultured perinatal rat SCG neurons, and retinoids synthesized by SCG explants were able to activate transcriptional reporters in retinoic acid-responsive F9 cells [106]. The addition of retinoic acid, or specific retinoic acid receptor and retinoid X receptor agonists, to the medium of cultured sympathetic neurons inhibited BMP-induced dendritic growth without altering axonal growth or cell viability [106]. What is not known, however, is the point of convergence between these two pathways in the context of dendritic growth. Further studies are also needed to determine whether retinoic acid regulates the dendritic arborization of sympathetic neurons *in vivo*.

*Signaling by pituitary adenylate cyclase (PACAP) and vasoactive intestinal peptide (VIP).* PACAP is a member of the secretin/VIP family of peptides that regulates the development of cells within the sympathoadrenal lineage [109]. In sympathetic neurons, PACAP regulates cell survival, proliferation, and catecholamine secretion, and release of PACAP and VIP from preganglionic neurons stimulates depolarization of sympathetic neurons [109]. In cultured perinatal rat sympathetic neurons, PACAP38 and VIP decrease BMP-induced dendritic growth, as evidenced by a decrease in the percentage of cells with dendrites, number of dendrites per neuron, and size of the dendritic arbor [110]. Using receptor specific antagonists and antibodies against phosphorylated cyclic AMP response element binding (CREB) protein, the PACAP response was shown to be mediated by PAC1 receptor activation, which results in the nuclear accumulation of phospho-CREB. Inhibition of adenylate cyclase activity by SQ22526 overcomes the inhibitory effects of PACAP on dendritic growth, and agents that increase cAMP levels, such as forskolin, inhibit BMP-induced dendritic growth [110]. These data suggest that peptides released by preganglionic nerves modulate dendritic growth in postganglionic sympathetic neurons by a cAMP-dependent mechanism.

In summary, signaling by cytokines, growth factors, small molecules, and peptides, such as retinoic acid, PACAP and VIP, antagonize BMP signaling during dendritogenesis in sympathetic neurons. Most of the relevant data were collected from studies of primary perinatal sympathetic neurons cultured from rodent SCG. While the findings from this model have provided a glimpse into the complexity of the interactions that influence dendritic arborization of these neurons, further studies are required to understand the mechanisms by which these factors interact to regulate dendritic growth, how these pathways are spatially and temporally coordinated to influence dendritic arborization of sympathetic neurons *in vivo*, and to determine their relevance to the human condition.

#### **3.3. Other pathways that regulate dendritic growth in sympathetic neurons**

As described earlier, NGF is an important regulator of dendritic growth in sympathetic neurons. However, the molecular mechanisms by which NGF regulates dendritic growth are not well characterized. Early growth response-3 (Egr3), a transcriptional regulator known to be induced by NGF via MAPK signaling, has been identified as a potential downstream regulator of NGF-induced dendritic growth [111]. Sympathetic neurons from a conditional *Egr3* knockout generated using *Dbhi-Cre* have fewer primary dendrites and shorter dendritic arbors compared to sympathetic neurons from congenic wildtype animals. In addition, *Egr3* knockout animals exhibit defects in axonal guidance, and in innervation of autonomic targets [111]. Although, Egr3 had previously been shown to not be required for NGF effects on neuronal survival [112], some neuronal cell death was observed in germline mutants for Egr3. It is, therefore, possible that the dendritic effects of knocking out *Egr3* are secondary to adverse effects on neuronal cell viability or changes in target innervation.

Neuronal depolarization induced by electric field stimulation or the addition of potassium chloride to primary postnatal sympathetic neurons cultured was shown to trigger the formation of dendrites in the presence of NGF that retracted in the absence of neuronal activity [113]. Neuronal depolarization enhanced stability of microtubules and activated calcium calmodulin dependent kinase II (CaMKII) in dendrites. The latter was shown to be causally related to the effects of neuronal depolarization on dendritic growth: pharmacologic inhibition of CaMKII activity using KN62 or mAIP completely blocks activity-dependent dendritic growth in cultured sympathetic neurons [113].

Signaling by integrin-linked kinase (ILK) and glycogen synthase kinase-3β (GSK-3β) have also been shown to be downstream effectors of activity-dependent dendritic growth in postnatal sympathetic neurons [114]. ILK and GSK-3β are serine threonine kinases that are downstream effectors of integrin and neurotrophin signaling [115]. ILK has been shown to phosphorylate and inactivate GSK-3β to regulate NGF-mediated axonal growth [116]. Increased phosphorylation of GSK-3β protein was observed in cultured postnatal rat SCG neurons in response to increased neuronal activity, and inhibition of ILK activity by QLT0254, as well as transfection of dominant negative ILK or siRNA for ILK, blocked activity-dependent dendritic growth in these neurons. Similarly, inhibition of GSK-3β activity using kenpaullone or genetic knockdown of GSK-3β expression increased the number of primary dendrites formed in response to potassium chloride, suggesting that GSK-3β inhibition is necessary for early stages of activitydependent dendritic growth in sympathetic neurons.

Interestingly, unlike BMP-induced dendritic growth, inhibition of ERK activity inhibited activity-dependent dendritic growth in postnatal sympathetic neurons *in vitro* [113, 114]. Pharmacologic inhibition of ERK by PD98059 blocked activity-dependent dendritic growth, inhibition of GSK-3β increased depolarization-dependent ERK activation, and inhibition of ERK reversed the enhanced dendritic growth observed with GSK-3β inhibition [113, 114]. The reasons for the opposing roles of ERK in BMP-induced vs. activity-dependent dendritic growth remain unexplored.

### **4. The path forward**

between BMP signaling and MAPK signaling during dendritogenesis in sympathetic neurons, and suggest that in contrast to its effects in central neurons, MAPK signaling functions

*Retinoic acid signaling.* In the developing nervous system, BMPs and retinoic acid work synergistically to regulate neural tube patterning and specification of neuronal identity [105–108]. Similarly, interplay between BMP and retinoic acid signaling have been documented during dendritogenesis in sympathetic neurons; however, their interaction in this latter context is antagonistic [106]. Genes required for synthesis of retinoic acid are expressed in cultured perinatal rat SCG neurons, and retinoids synthesized by SCG explants were able to activate transcriptional reporters in retinoic acid-responsive F9 cells [106]. The addition of retinoic acid, or specific retinoic acid receptor and retinoid X receptor agonists, to the medium of cultured sympathetic neurons inhibited BMP-induced dendritic growth without altering axonal growth or cell viability [106]. What is not known, however, is the point of convergence between these two pathways in the context of dendritic growth. Further studies are also needed to determine whether retinoic acid regulates the dendritic arborization of sympathetic

*Signaling by pituitary adenylate cyclase (PACAP) and vasoactive intestinal peptide (VIP).* PACAP is a member of the secretin/VIP family of peptides that regulates the development of cells within the sympathoadrenal lineage [109]. In sympathetic neurons, PACAP regulates cell survival, proliferation, and catecholamine secretion, and release of PACAP and VIP from preganglionic neurons stimulates depolarization of sympathetic neurons [109]. In cultured perinatal rat sympathetic neurons, PACAP38 and VIP decrease BMP-induced dendritic growth, as evidenced by a decrease in the percentage of cells with dendrites, number of dendrites per neuron, and size of the dendritic arbor [110]. Using receptor specific antagonists and antibodies against phosphorylated cyclic AMP response element binding (CREB) protein, the PACAP

accumulation of phospho-CREB. Inhibition of adenylate cyclase activity by SQ22526 overcomes the inhibitory effects of PACAP on dendritic growth, and agents that increase cAMP levels, such as forskolin, inhibit BMP-induced dendritic growth [110]. These data suggest that peptides released by preganglionic nerves modulate dendritic growth in postganglionic

In summary, signaling by cytokines, growth factors, small molecules, and peptides, such as retinoic acid, PACAP and VIP, antagonize BMP signaling during dendritogenesis in sympathetic neurons. Most of the relevant data were collected from studies of primary perinatal sympathetic neurons cultured from rodent SCG. While the findings from this model have provided a glimpse into the complexity of the interactions that influence dendritic arborization of these neurons, further studies are required to understand the mechanisms by which these factors interact to regulate dendritic growth, how these pathways are spatially and temporally coordinated to influence dendritic arborization of sympathetic neurons *in vivo*, and to

As described earlier, NGF is an important regulator of dendritic growth in sympathetic neurons. However, the molecular mechanisms by which NGF regulates dendritic growth are

**3.3. Other pathways that regulate dendritic growth in sympathetic neurons**

receptor activation, which results in the nuclear

as a negative regulator of BMP-induced dendritic growth in sympathetic neurons.

neurons *in vivo*.

100 Autonomic Nervous System

response was shown to be mediated by PAC1

sympathetic neurons by a cAMP-dependent mechanism.

determine their relevance to the human condition.

The experimental evidence clearly implicate NGF, BMP and neuronal activity as positive regulators of dendritic growth in perinatal sympathetic neurons *in vitro*. However, the interaction between these three pathways and their relative contributions to the induction and maintenance of dendritic growth in sympathetic neurons *in vivo* are not fully understood. Multiple mechanistically and structural diverse signaling molecules have been implicated as negative regulators of dendritic growth *in vitro*. The spatial and temporal regulation of these pathways, how they interact with each other and with positive modulators of dendritic growth to shape dendritic arbors, and their functional significance *in vivo* remain outstanding questions. In addition, further studies are needed to understand the genes and proteins that are regulated downstream of each of these signaling pathways and to identify those that serve as hubs for interactions between multiple signaling pathways to regulate the final dendritic arbor of sympathetic neurons. Finally, a key data gap is the relevance of these observations in experimental models to the human condition.

[3] Purves D. Body and Brain: A Trophic Theory of Neural Connections. Cambridge, MA:

Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 103

[4] Kweon JH, Kim S, Lee SB. The cellular basis of dendrite pathology in neurodegenerative

[5] Copf T. Impairments in dendrite morphogenesis as etiology for neurodevelopmental disorders and implications for therapeutic treatments. Neuroscience and Biobehavioral

[6] Lein PJ, Fryer AD, Higgins D. Cell culture: Autonomic and enteric neurons. In: Squire LR, editor. Encyclopedia of Neuroscience. Oxford, UK: Academic Press; 2009. pp. 625-632

[7] Purves D, Lichtman JW. Geometrical differences among homologous neurons in mam-

[8] Ivanov A, Purves D. Ongoing electrical activity of superior cervical ganglion cells in mammals of different size. The Journal of Comparative Neurology. 1989;**284**(3):398-404

[9] De Castro F, Sanchez-Vives MV, Munoz-Martinez EJ, Gallego R. Effects of postganglionic nerve section on synaptic transmission in the superior cervical ganglion of the

[10] Peruzzi D, Hendley ED, Forehand CJ. Hypertrophy of stellate ganglion cells in hypertensive, but not hyperactive, rats. The American Journal of Physiology. 1991;**261**(4 Pt 2):

[11] Kondo M, Terada M, Shimizu D, Fujiwara T, Tabei R. Morphometric study of the superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage. Virchows Archiv. B, Cell Pathology Including Molecular Pathology.

[12] Kishi T, Hirooka Y, Mukai Y, Shimokawa H, Takeshita A. Atorvastatin causes depressor and sympatho-inhibitory effects with upregulation of nitric oxide synthases in strokeprone spontaneously hypertensive rats. Journal of Hypertension. 2003;**21**(2):379-386 [13] Kim WY, Gonsiorek EA, Barnhart C, Davare MA, Engebose AJ, Lauridsen H, Bruun D, Lesiak A, Wayman G, Bucelli R, Higgins D, Lein PJ. Statins decrease dendritic arborization in rat sympathetic neurons by blocking RhoA activation. Journal of Neurochemistry.

[14] Andrews TJ, Li D, Halliwell J, Cowen T. The effect of age on dendrites in the rat superior

[15] Snider WD. Rostrocaudal differences in dendritic growth and synaptogenesis in rat sympathetic chain ganglia. The Journal of Comparative Neurology. 1986;**244**(2):245-253.

[16] Voyvodic JT. Peripheral target regulation of dendritic geometry in the rat superior cervi-

cervical ganglion. Journal of Anatomy. 1994;**184**(Pt 1):111-117

cal ganglion. The Journal of Neuroscience. 1989;**9**(6):1997-2010

Reviews. 2016;**68**:946-978. DOI: 10.1016/j.neubiorev.2016.04.008

Harvard University Press; 1988

diseases. BMB Reports. 2017;**50**(1):5-11

mals. Science. 1985;**228**(4697):298-302

R979-R984

1990;**58**(5):371-376

2009;**108**(4):1057-1071

DOI: 10.1002/cne.902440210

guinea-pig. Neuroscience. 1995;**67**(3):689-695

### **Acknowledgements**

The authors acknowledge Dennis Higgins, PhD (University of Buffalo) who dedicated his research career to understanding the regulation of dendritic growth in sympathetic neurons, and whose enthusiasm for this topic, and scientific research in general, was the inspiration and motivation for much of the authors' own work in this field. This work was supported by the National Institutes of Health (grants R01 NS097808, R01 ES014901 and R21 NS45037).

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Vidya Chandrasekaran1 and Pamela J. Lein2 \*


#### **References**


[3] Purves D. Body and Brain: A Trophic Theory of Neural Connections. Cambridge, MA: Harvard University Press; 1988

maintenance of dendritic growth in sympathetic neurons *in vivo* are not fully understood. Multiple mechanistically and structural diverse signaling molecules have been implicated as negative regulators of dendritic growth *in vitro*. The spatial and temporal regulation of these pathways, how they interact with each other and with positive modulators of dendritic growth to shape dendritic arbors, and their functional significance *in vivo* remain outstanding questions. In addition, further studies are needed to understand the genes and proteins that are regulated downstream of each of these signaling pathways and to identify those that serve as hubs for interactions between multiple signaling pathways to regulate the final dendritic arbor of sympathetic neurons. Finally, a key data gap is the relevance of these observations in

The authors acknowledge Dennis Higgins, PhD (University of Buffalo) who dedicated his research career to understanding the regulation of dendritic growth in sympathetic neurons, and whose enthusiasm for this topic, and scientific research in general, was the inspiration and motivation for much of the authors' own work in this field. This work was supported by the National Institutes of Health (grants R01 NS097808, R01 ES014901 and R21 NS45037).

experimental models to the human condition.

**Acknowledgements**

102 Autonomic Nervous System

**Conflict of interest**

**Author details**

**References**

Vidya Chandrasekaran1

The authors declare no conflict of interest.

and Pamela J. Lein2

Journal of Experimental Biology. 1984;**112**:129-145

University Press; 1967. pp. 372-393

\*Address all correspondence to: pjlein@ucdavis.edu

\*

[1] Miller JP, Jacobs GA. Relationships between neuronal structure and function. The

[2] Purpura DP. Comparative physiology of dendrites. In: Quarton GC, Melnechuk T, Schmitt FO, editors. The Neurosciences: A Study Program. New York: Rockefeller

1 Department of Biology, Saint Mary's College of California, Moraga, CA, USA

2 Department of Molecular Biosciences, University of California, Davis, CA, USA


[17] Purves D, Hadley RD, Voyvodic JT. Dynamic changes in the dendritic geometry of individual neurons visualized over periods of up to three months in the superior cervical ganglion of living mice. The Journal of Neuroscience. 1986;**6**(4):1051-1060

[32] Tropea M, Johnson MI, Higgins D. Glial cells promote dendritic development in rat sym-

Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 105

[33] Beck HN, Drahushuk K, Jacoby DB, Higgins D, Lein PJ. Bone morphogenetic protein-5 (bmp-5) promotes dendritic growth in cultured sympathetic neurons. BMC

[34] Guo X, Rueger D, Higgins D. Osteogenic protein-1 and related bone morphogenetic proteins regulate dendritic growth and the expression of microtubule-associated pro-

[35] Lein P, Guo X, Hedges AM, Rueger D, Johnson M, Higgins D. The effects of extracellular matrix and osteogenic protein-1 on the morphological differentiation of rat sympathetic neurons. International Journal of Developmental Neuroscience. 1996;**14**(3):203-215

[36] Lein PJ, Guo X, Shi GX, Moholt-Siebert M, Bruun D, Andres DA. The novel GTPase Rit differentially regulates axonal and dendritic growth. The Journal of Neuroscience.

[37] Lein PJ, Beck HN, Chandrasekaran V, Gallagher PJ, Chen HL, Lin Y, Guo X, Kaplan PL, Tiedge H, Higgins D. Glia induce dendritic growth in cultured sympathetic neurons by modulating the balance between bone morphogenetic proteins (BMPs) and BMP antago-

[38] Lyons KM, Hogan BL, Robertson EJ. Colocalization of BMP 7 and BMP 2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development.

[39] Vukicevic S, Latin V, Chen P, Batorsky R, Reddi AH, Sampath TK. Localization of osteogenic protein-1 (bone morphogenetic protein-7) during human embryonic development: High affinity binding to basement membranes. Biochemical and Biophysical Research

[40] Wall NA, Blessing M, Wright CV, Hogan BL. Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6). The

[41] Katagiri T, Watabe T. Bone morphogenetic proteins. Cold Spring Harbor Perspectives in

[42] Massague J, Wotton D. Transcriptional control by the TGF-beta/Smad signaling system.

[43] Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta

[44] Lee-Hoeflich ST, Causing CG, Podkowa M, Zhao X, Wrana JL, Attisano L. Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendrito-

The EMBO Journal. 2000;**19**(8):1745-1754. DOI: 10.1093/emboj/19.8.1745

tein-2 in rat sympathetic neurons. Neuroscience Letters. 1998;**245**(3):131-134

2007;**27**(17):4725-4736. DOI: 10.1523/JNEUROSCI.5633-06.2007

nists. The Journal of Neuroscience. 2002;**22**(23):10377-10387

Mechanisms of Development. 1995;**50**(1):71-83

Communications. 1994;**198**(2):693-700

Journal of Cell Biology. 1993;**120**(2):493-502

Biology. 2016;**8**(6). DOI: 10.1101/cshperspect.a021899

family signalling. Nature. 2003;**425**(6958):577-584

genesis. The EMBO Journal. 2004;**23**(24):4792-4801

pathetic neurons in vitro. Glia. 1988;**1**(6):380-392

Neuroscience. 2001;**2**:12


[32] Tropea M, Johnson MI, Higgins D. Glial cells promote dendritic development in rat sympathetic neurons in vitro. Glia. 1988;**1**(6):380-392

[17] Purves D, Hadley RD, Voyvodic JT. Dynamic changes in the dendritic geometry of individual neurons visualized over periods of up to three months in the superior cervical

[18] Andrews TJ, Thrasivoulou C, Nesbit W, Cowen T. Target-specific differences in the dendritic morphology and neuropeptide content of neurons in the rat SCG during develop-

[19] Luebke JI, Wright LL. Characterization of superior cervical ganglion neurons that project to the submandibular glands, the eyes, and the pineal gland in rats. Brain Research.

[20] Smolen AJ, Beaston-Wimmer P. Dendritic development in the rat superior cervical gan-

[21] Voyvodic JT. Development and regulation of dendrites in the rat superior cervical gan-

[22] Goedert M, Otten U, Thoenen H. Biochemical effects of antibodies against nerve growth factor on developing and differentiated sympathetic ganglia. Brain Research.

[23] Purves D, Snider WD, Voyvodic JT. Trophic regulation of nerve cell morphology and innervation in the autonomic nervous system. Nature. 1988;**336**(6195):123-128

[24] Ruit KG, Osborne PA, Schmidt RE, Johnson EM Jr, Snider WD. Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse. The

[25] Ruit KG, Snider WD.Administration or deprivation of nerve growth factor during development permanently alters neuronal geometry. The Journal of Comparative Neurology.

[26] Purves D, Nja A. Effect of nerve growth factor on synaptic depression after axotomy.

[27] Nja A, Purves D. The effects of nerve growth factor and its antiserum on synapses in the superior cervical ganglion of the guinea-pig. The Journal of Physiology. 1978;**277**:55-75

[28] Yawo H. Changes in the dendritic geometry of mouse superior cervical ganglion cells following postganglionic axotomy. The Journal of Neuroscience. 1987;**7**(11):3703-3711 [29] Lein P, Johnson M, Guo X, Rueger D, Higgins D. Osteogenic protein-1 induces dendritic

[30] Bruckenstein DA, Higgins D. Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not

[31] Bruckenstein DA, Higgins D. Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. II. Serum promotes dendritic growth. Developmental

growth in rat sympathetic neurons. Neuron. 1995;**15**(3):597-605

Biology. 1988;**128**(2):337-348. DOI: 10.1016/0012-1606(88)90296-5

dendrites. Developmental Biology. 1988;**128**(2):324-336

ganglion of living mice. The Journal of Neuroscience. 1986;**6**(4):1051-1060

ment and aging. The Journal of Comparative Neurology. 1996;**368**(1):33-44

1992;**589**(1):1-14

104 Autonomic Nervous System

1978;**148**(1):264-268

1991;**314**(1):106-113

Nature. 1976;**260**(5551):535-536

glion. Brain Research. 1986;**394**(2):245-252

Journal of Neuroscience. 1990;**10**(7):2412-2419

glion. The Journal of Neuroscience. 1987;**7**(3):904-912


[45] Xu X, Han J, Ito Y, Bringas P Jr, Deng C, Chai Y. Ectodermal Smad4 and p38 MAPK are functionally redundant in mediating TGF-beta/BMP signaling during tooth and palate development. Developmental Cell. 2008;**15**(2):322-329. DOI: 10.1016/j.devcel.2008.06.004

[57] Kenchappa RS, Zampieri N, Chao MV, Barker PA, Teng HK, Hempstead BL, Carter BD. Ligand-dependent cleavage of the P75 neurotrophin receptor is necessary for NRIF nuclear translocation and apoptosis in sympathetic neurons. Neuron. 2006;**50**(2):219-

Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 107

[58] Roux PP, Barker PA. Neurotrophin signaling through the p75 neurotrophin receptor.

[59] Salama-Cohen P, Arevalo MA, Meier J, Grantyn R, Rodriguez-Tebar A. NGF controls dendrite development in hippocampal neurons by binding to p75NTR and modulating the cellular targets of Notch. Molecular Biology of the Cell. 2005;**16**(1):339-347. DOI:

[60] Shu YH, Lu XM, Wei JX, Xiao L, Wang YT. Update on the role of p75NTR in neurological disorders: A novel therapeutic target. Biomedicine & Pharmacotherapy. 2015;**76**:17-23.

[61] Underwood CK, Coulson EJ. The p75 neurotrophin receptor. The International Journal of Biochemistry & Cell Biology. 2008;**40**(9):1664-1668. DOI: 10.1016/j.biocel.2007.06.010

[62] Bengoechea TG, Chen Z, O'Leary DA, Masliah E, Lee KF. P75 reduces beta-amyloidinduced sympathetic innervation deficits in an Alzheimer's disease mouse model. Proceedings of the National Academy of Sciences of the United States of America.

[63] Wang KC, Kim JA, Sivasankaran R, Segal R, He Z. P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature. 2002;**420**(6911):74-78. DOI: 10.1038/

[64] Courter LA, Shaffo FC, Ghogha A, Parrish DJ, Lorentz CU, Habecker BA, Lein PJ. BMP7 induced dendritic growth in sympathetic neurons requires p75(NTR) signaling.

[65] Roux PP, Bhakar AL, Kennedy TE, Barker PA. The p75 neurotrophin receptor activates Akt (protein kinase B) through a phosphatidylinositol 3-kinase-dependent pathway. The Journal of Biological Chemistry. 2001;**276**(25):23097-23104. DOI: 10.1074/jbc.M011520200

[66] Sharma N, Deppmann CD, Harrington AW, St Hillaire C, Chen ZY, Lee FS, Ginty DD. Long-distance control of synapse assembly by target-derived NGF. Neuron.

[67] Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen MS, Jacobsen C, Kliemannel M, Schwarz E, Willnow TE, Hempstead BL, Petersen CM. Sortilin is essential for proNGFinduced neuronal cell death. Nature. 2004;**427**(6977):843-848. DOI: 10.1038/nature02319

[68] Redmond L, Ghosh A. The role of Notch and Rho GTPase signaling in the control of dendritic development. Current Opinion in Neurobiology. 2001;**11**(1):111-117

[69] Van Aelst L, Cline HT. Rho GTPases and activity-dependent dendrite development.

[70] Yamashita T, Tucker KL, Barde YA. Neurotrophin binding to the p75 receptor modulates

Developmental Neurobiology. 2016;**76**(9):1003-1013. DOI: 10.1002/dneu.22371

232. DOI: 10.1016/j.neuron.2006.03.011

10.1091/mbc.E04-05-0438

nature01176

DOI: 10.1016/j.biopha.2015.10.010

Progress in Neurobiology. 2002;**67**(3):203-233

2009;**106**(19):7870-7875. DOI: 10.1073/pnas.0901533106

2010;**67**(3):422-434. DOI: 10.1016/j.neuron.2010.07.018

Current Opinion in Neurobiology. 2004;**14**(3):297-304

rho activity and axonal outgrowth. Neuron. 1999;**24**(3):585-593


[57] Kenchappa RS, Zampieri N, Chao MV, Barker PA, Teng HK, Hempstead BL, Carter BD. Ligand-dependent cleavage of the P75 neurotrophin receptor is necessary for NRIF nuclear translocation and apoptosis in sympathetic neurons. Neuron. 2006;**50**(2):219- 232. DOI: 10.1016/j.neuron.2006.03.011

[45] Xu X, Han J, Ito Y, Bringas P Jr, Deng C, Chai Y. Ectodermal Smad4 and p38 MAPK are functionally redundant in mediating TGF-beta/BMP signaling during tooth and palate development. Developmental Cell. 2008;**15**(2):322-329. DOI: 10.1016/j.devcel.2008.06.004

[46] Yu L, Hebert MC, Zhang YE. TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGT-beta responses. The EMBO Journal. 2002;**21**(14):3749-3759.

[47] O'Keeffe GW, Gutierrez H, Howard L, Laurie CW, Osorio C, Gavalda N, Wyatt SL, Davies AM. Region-specific role of growth differentiation factor-5 in the establishment of sympathetic innervation. Neural Development. 2016;**11**:4. DOI: 10.1186/s13064-016-0060-3 [48] Zhang D, Mehler MF, Song Q, Kessler JA. Development of bone morphogenetic protein receptors in the nervous system and possible roles in regulating trkC expression. The

[49] Majdazari A, Stubbusch J, Muller CM, Hennchen M, Weber M, Deng CX, Mishina Y, Schutz G, Deller T, Rohrer H. Dendrite complexity of sympathetic neurons is controlled during postnatal development by BMP signaling. The Journal of Neuroscience.

[50] Ehrlich M. Endocytosis and trafficking of BMP receptors: Regulatory mechanisms for fine-tuning the signaling response in different cellular contexts. Cytokine & Growth

[51] Guo X, Lin Y, Horbinski C, Drahushuk KM, Kim IJ, Kaplan PL, Lein P, Wang T, Higgins D. Dendritic growth induced by BMP-7 requires Smad1 and proteasome activity. Journal

[52] Garred MM, Wang MM, Guo X, Harrington CA, Lein PJ. Transcriptional responses of cultured rat sympathetic neurons during BMP-7-induced dendritic growth. PLoS One.

[53] Ghazalpour A, Bennett B, Petyuk VA, Orozco L, Hagopian R, Mungrue IN, Farber CR, Sinsheimer J, Kang HM, Furlotte N, Park CC, Wen PZ, Brewer H, Weitz K, Camp DG 2nd, Pan C, Yordanova R, Neuhaus I, Tilford C, Siemers N, Gargalovic P, Eskin E, Kirchgessner T, Smith DJ, Smith RD, Lusis AJ. Comparative analysis of proteome and transcriptome variation in mouse. PLoS Genetics. 2011;**7**(6):e1001393. DOI: 10.1371/

[54] Nie L, Wu G, Culley DE, Scholten JC, Zhang W. Integrative analysis of transcriptomic and proteomic data: Challenges, solutions and applications. Critical Reviews in

[55] Dechant G, Barde YA. The neurotrophin receptor p75(NTR): Novel functions and implications for diseases of the nervous system. Nature Neuroscience. 2002;**5**(11):1131-1136.

[56] Horton A, Laramee G, Wyatt S, Shih A, Winslow J, Davies AM. NGF binding to p75 enhances the sensitivity of sensory and sympathetic neurons to NGF at different stages of development. Molecular and Cellular Neurosciences. 1997;**10**(3-4):162-172. DOI: 10.

Biotechnology. 2007;**27**(2):63-75. DOI: 10.1080/07388550701334212

2013;**33**(38):15132-15144. DOI: 10.1523/JNEUROSCI.4748-12.2013

Factor Reviews. 2016;**27**:35-42. DOI: 10.1016/j.cytogfr.2015.12.008

DOI: 10.1093/emboj/cdf366

106 Autonomic Nervous System

Journal of Neuroscience. 1998;**18**(9):3314-3326

of Neurobiology. 2001;**48**(2):120-130

journal.pgen.1001393

DOI: 10.1038/nn1102-1131

1006/mcne.1997.0650

2011;**6**(7):e21754. DOI: 10.1371/journal.pone.0021754


[71] Hamilton AM, Zito K. Breaking it down: The ubiquitin proteasome system in neuronal morphogenesis. Neural Plasticity. 2013;**2013**:196848. DOI: 10.1155/2013/196848

[83] Munnamalai V, Suter DM. Reactive oxygen species regulate F-actin dynamics in neuronal growth cones and neurite outgrowth. Journal of Neurochemistry. 2009;**108**(3):644-

Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 109

[84] Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of

[85] Massaad CA, Klann E. Reactive oxygen species in the regulation of synaptic plasticity and memory. Antioxidants & Redox Signaling. 2011;**14**(10):2013-2054. DOI: 10.1089/

[86] Cao X, Demel SL, Quinn MT, Galligan JJ, Kreulen D. Localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers. Autonomic

[87] Chandrasekaran V, Lea C, Sosa JC, Higgins D, Lein PJ. Reactive oxygen species are involved in bmp-induced dendritic growth in cultured rat sympathetic neurons. Molecular and Cellular Neurosciences. 2015;**67**:116-125. DOI: 10.1016/j.mcn.2015.06.007

[88] O'Shea JJ, Gadina M, Kanno Y. Cytokine signaling: Birth of a pathway. Journal of

[89] Iwamoto T, Oshima K, Seng T, Feng X, Oo ML, Hamaguchi M, Matsuda S. STAT and SMAD signaling in cancer. Histology and Histopathology. 2002;**17**(3):887-895. DOI:

[90] Tang LY, Heller M, Meng Z, Yu LR, Tang Y, Zhou M, Zhang YE. Transforming growth factor-beta (TGF-beta) directly activates the JAK1-STAT3 axis to induce hepatic fibrosis in coordination with the SMAD pathway. The Journal of Biological Chemistry.

[91] Ulloa L, Doody J, Massague J. Inhibition of transforming growth factor-beta/SMAD signalling by the interferon-gamma/STAT pathway. Nature. 1999;**397**(6721):710-713. DOI:

[92] Guo X, Chandrasekaran V, Lein P, Kaplan PL, Higgins D. Leukemia inhibitory factor and ciliary neurotrophic factor cause dendritic retraction in cultured rat sympathetic

[93] Guo X, Metzler-Northrup J, Lein P, Rueger D, Higgins D. Leukemia inhibitory factor and ciliary neurotrophic factor regulate dendritic growth in cultures of rat sympathetic

[94] Kim IJ, Beck HN, Lein PJ, Higgins D. Interferon gamma induces retrograde dendritic retraction and inhibits synapse formation. The Journal of Neuroscience.

[95] Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in human diseases. Biochimica et Biophysica Acta. 2010;**1802**(4):396-405. DOI: 10.1016/j.bbadis.2009.12.009

neurons. Brain Research. Developmental Brain Research. 1997;**104**(1-2):101-110

Neuroscience. 2009;**151**(2):90-97. DOI: 10.1016/j.autneu.2009.07.010

Immunology. 2011;**187**(11):5475-5478. DOI: 10.4049/jimmunol.1102913

2017;**292**(10):4302-4312. DOI: 10.1074/jbc.M116.773085

neurons. The Journal of Neuroscience. 1999;**19**(6):2113-2121

Biochemistry & Cell Biology. 2007;**39**(1):44-84. DOI: 10.1016/j.biocel.2006.07.001

661. DOI: 10.1111/j.1471-4159.2008.05787.x

ars.2010.3208

10.14670/HH-17.887

10.1038/17826

2002;**22**(11):4530-4539


[83] Munnamalai V, Suter DM. Reactive oxygen species regulate F-actin dynamics in neuronal growth cones and neurite outgrowth. Journal of Neurochemistry. 2009;**108**(3):644- 661. DOI: 10.1111/j.1471-4159.2008.05787.x

[71] Hamilton AM, Zito K. Breaking it down: The ubiquitin proteasome system in neuronal morphogenesis. Neural Plasticity. 2013;**2013**:196848. DOI: 10.1155/2013/196848

[72] Oddo S. The ubiquitin-proteasome system in Alzheimer's disease. Journal of Cellular and Molecular Medicine. 2008;**12**(2):363-373. DOI: 10.1111/j.1582-4934.2008.00276.x

[73] Sun L, Trausch-Azar JS, Ciechanover A, Schwartz AL. Ubiquitin-proteasome-mediated degradation, intracellular localization, and protein synthesis of Myod and Id1 during muscle differentiation. The Journal of Biological Chemistry. 2005;**280**(28):26448-26456.

[74] Kawabe H, Neeb A, Dimova K, Young SM Jr, Takeda M, Katsurabayashi S, Mitkovski M, Malakhova OA, Zhang DE, Umikawa M, Kariya K, Goebbels S, Nave KA, Rosenmund C, Jahn O, Rhee J, Brose N. Regulation of Rap2A by the ubiquitin ligase Nedd4-1 controls neurite development. Neuron. 2010;**65**(3):358-372. DOI: 10.1016/j.neuron.2010.01.007

[75] Kim AH, Puram SV, Bilimoria PM, Ikeuchi Y, Keough S, Wong M, Rowitch D, Bonni A. A centrosomal Cdc20-APC pathway controls dendrite morphogenesis in postmitotic

[76] Litterman N, Ikeuchi Y, Gallardo G, O'Connell BC, Sowa ME, Gygi SP, Harper JW, Bonni A. An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning. PLoS Biology. 2011;**9**(5):e1001060. DOI: 10.1371/

[77] Izzi L, Attisano L. Regulation of the TGFbeta signalling pathway by ubiquitin-mediated

[78] Zhu H, Kavsak P, Abdollah S, Wrana JL, Thomsen GH. A smad ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature. 1999;**400**(6745):687-

[79] Brennan AM, Suh SW, Won SJ, Narasimhan P, Kauppinen TM, Lee H, Edling Y, Chan PH, Swanson RA. NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation. Nature Neuroscience. 2009;**12**(7):857-863. DOI: 10.1038/

[80] Kennedy KA, Sandiford SD, Skerjanc IS, Li SS. Reactive oxygen species and the neuronal fate. Cellular and Molecular Life Sciences. 2012;**69**(2):215-221. DOI: 10.1007/s00018-

[81] Kowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE. Mitochondria and reactive oxygen species. Free Radical Biology & Medicine. 2009;**47**(4):333-343. DOI: 10.1016/j.

[82] Colavitti R, Pani G, Bedogni B, Anzevino R, Borrello S, Waltenberger J, Galeotti T. Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR. The Journal of Biological Chemistry.

degradation. Oncogene. 2004;**23**(11):2071-2078. DOI: 10.1038/sj.onc.1207412

neurons. Cell. 2009;**136**(2):322-336. DOI: 10.1016/j.cell.2008.11.050

DOI: 10.1074/jbc.M500373200

108 Autonomic Nervous System

journal.pbio.1001060

693. DOI: 10.1038/23293

freeradbiomed.2009.05.004

2002;**277**(5):3101-3108. DOI: 10.1074/jbc.M107711200

nn.2334

011-0807-2


[96] Plotnikov A, Zehorai E, Procaccia S, Seger R. The MAPK cascades: Signaling components, nuclear roles and mechanisms of nuclear translocation. Biochimica et Biophysica Acta. 2011;**1813**(9):1619-1633. DOI: 10.1016/j.bbamcr.2010.12.012

[109] Ghzili H, Grumolato L, Thouennon E, Tanguy Y, Turquier V, Vaudry H, Anouar Y. Role of PACAP in the physiology and pathology of the sympathoadrenal system. Frontiers

Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 111

[110] Drahushuk K, Connell TD, Higgins D. Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide inhibit dendritic growth in cultured sympathetic

[111] Quach DH, Oliveira-Fernandes M, Gruner KA, Tourtellotte WG. A sympathetic neuron autonomous role for Egr3-mediated gene regulation in dendrite morphogenesis and target tissue innervation. The Journal of Neuroscience. 2013;**33**(10):4570-4583. DOI:

[112] Eldredge LC, Gao XM, Quach DH, Li L, Han X, Lomasney J, Tourtellotte WG.Abnormal sympathetic nervous system development and physiological dysautonomia in Egr3-

[114] Naska S, Park KJ, Hannigan GE, Dedhar S, Miller FD, Kaplan DR. An essential role for the integrin-linked kinase-glycogen synthase kinase-3 beta pathway during dendrite initiation and growth. The Journal of Neuroscience. 2006;**26**(51):13344-13356. DOI:

[115] Dedhar S, Williams B, Hannigan G. Integrin-linked kinase (ILK): A regulator of integrin

[116] Arevalo JC, Chao MV. Axonal growth: Where neurotrophins meet Wnts. Current Opinion in Cell Biology. 2005;**17**(2):112-115. DOI: 10.1016/j.ceb.2005.01.004

and growth-factor signalling. Trends in Cell Biology. 1999;**9**(8):319-323

deficient mice. Development. 2008;**135**(17):2949-2957. DOI: 10.1242/dev.023960 [113] Vaillant AR, Zanassi P, Walsh GS, Aumont A, Alonso A, Miller FD. Signaling mechanisms underlying reversible, activity-dependent dendrite formation. Neuron.

in Neuroendocrinology. 2008;**29**(1):128-141. DOI: 10.1016/j.yfrne.2007.10.001

neurons. The Journal of Neuroscience. 2002;**22**(15):6560-6569

10.1523/JNEUROSCI.5481-12.2013

10.1523/JNEUROSCI.4462-06.2006

2002;**34**(6):985-998


[109] Ghzili H, Grumolato L, Thouennon E, Tanguy Y, Turquier V, Vaudry H, Anouar Y. Role of PACAP in the physiology and pathology of the sympathoadrenal system. Frontiers in Neuroendocrinology. 2008;**29**(1):128-141. DOI: 10.1016/j.yfrne.2007.10.001

[96] Plotnikov A, Zehorai E, Procaccia S, Seger R. The MAPK cascades: Signaling components, nuclear roles and mechanisms of nuclear translocation. Biochimica et Biophysica

[97] Guo X, Wang XF. Signaling cross-talk between TGF-beta/BMP and other pathways. Cell

[98] Massague J. Integration of Smad and MAPK pathways: A link and a linker revisited.

[99] Pera EM, Ikeda A, Eivers E, De Robertis EM. Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. Genes & Development.

[100] Kim IJ, Drahushuk K, Kim W, Lein PJ, Andres DA, Higgins D. Extracellular signalregulated kinases regulate dendritic growth in rat sympathetic neurons. The Journal of

[101] Stachowiak MK, Fang X, Myers JM, Dunham SM, Berezney R, Maher PA, Stachowiak EK. Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forwardand-gate" signaling module that controls cell growth and differentiation. Journal of

[102] Klimaschewski L, Meisinger C, Grothe C. Localization and regulation of basic fibroblast growth factor (FGF-2) and FGF receptor-1 in rat superior cervical ganglion after

[103] Horbinski C, Stachowiak EK, Chandrasekaran V, Miuzukoshi E, Higgins D, Stachowiak MK. Bone morphogenetic protein-7 stimulates initial dendritic growth in sympathetic neurons through an intracellular fibroblast growth factor signaling pathway. Journal of

[104] Andres DA, Shi GX, Bruun D, Barnhart C, Lein PJ. Rit signaling contributes to interferon-gamma-induced dendritic retraction via p38 mitogen-activated protein kinase activation. Journal of Neurochemistry. 2008;**107**(5):1436-1447. DOI: 10.1111/j.1471-

[105] Altmann CR, Brivanlou AH. Neural patterning in the vertebrate embryo. International

[106] Chandrasekaran V, Zhai Y, Wagner M, Kaplan PL, Napoli JL, Higgins D. Retinoic acid regulates the morphological development of sympathetic neurons. Journal of

[107] Melton KR, Iulianella A, Trainor PA. Gene expression and regulation of hindbrain and

[108] Wilson L, Maden M. The mechanisms of dorsoventral patterning in the vertebrate neural tube. Developmental Biology. 2005;**282**(1):1-13. DOI: 10.1016/j.ydbio.2005.02.027

spinal cord development. Frontiers in Bioscience. 2004;**9**:117-138

Cellular Biochemistry. 2003;**90**(4):662-691. DOI: 10.1002/jcb.10606

axotomy. Journal of Neurobiology. 1999;**38**(4):499-506

Acta. 2011;**1813**(9):1619-1633. DOI: 10.1016/j.bbamcr.2010.12.012

Research. 2009;**19**(1):71-88. DOI: 10.1038/cr.2008.302

Genes & Development. 2003;**17**(24):2993-2997

2003;**17**(24):3023-3028. DOI: 10.1101/gad.1153603

Neuroscience. 2004;**20**(8):2013-2037

110 Autonomic Nervous System

Neurochemistry. 2002;**80**(1):54-63

Review of Cytology. 2001;**203**:447-482

Neurobiology. 2000;**42**(4):383-393

4159.2008.05708.x


**Chapter 6**

**Provisional chapter**

**A5 and A6 Noradrenergic Cell Groups: Implications for**

**A5 and A6 Noradrenergic Cell Groups: Implications for** 

Central pontine A5 and A6 noradrenergic cell groups are two of the main sources of noradrenaline release at the spinal cord, at the level of the superficial dorsal horn, the motoneuron pools of the ventral horn, lamina X and the thoracic and sacral intermediolateral cell columns. Noradrenergic ascending or descending pathways originating in the A5 or A6 noradrenergic cell groups are highly sensitive to stress and to other high-arousal states. These noradrenergic groups present extensive projections that play a key role in the modulation of all antinociceptive and autonomic responses elicited by painful or threatening situations. Depending on the locations of these projections, different possible roles for each noradrenergic cell groups are suggested. The A6 noradrenergic cell group might have the greatest effect on somatosensory transmission and the A5 group on sympathetic function. Consistent with this, stimulation of central noradrenergic pathways evokes an array of stresslike and antinociceptive effects, including changes in blood pressure, heart rate and respiratory rate. In addition, it also produces an increase in excitability, which leads to a high degree of arousal and a potentiation of cortical and subcortical mechanism generating the necessary cognitive, behavioral and autonomic responses to confront these physical or psychological situations.

**Keywords:** pontine noradrenergic cell groups, A5 region, locus coeruleus,

Noradrenergic (NA) central pathways located at the level of the brainstem were initially described by Dahlström and Fuxe in 1964 and contain several clusters or groups of neurons

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.79389

**Cardiorespiratory Control**

**Cardiorespiratory Control**

Manuel Víctor López-González, Marta González-García and Marc Stefan Dawid-Milner

Manuel Víctor López-González, Marta González-García and Marc Stefan Dawid-Milner

http://dx.doi.org/10.5772/intechopen.79389

cardiovascular control, analgesia

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **A5 and A6 Noradrenergic Cell Groups: Implications for Cardiorespiratory Control A5 and A6 Noradrenergic Cell Groups: Implications for Cardiorespiratory Control**

DOI: 10.5772/intechopen.79389

Manuel Víctor López-González, Marta González-García and Marc Stefan Dawid-Milner Manuel Víctor López-González, Marta González-García and Marc Stefan Dawid-Milner

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79389

#### **Abstract**

Central pontine A5 and A6 noradrenergic cell groups are two of the main sources of noradrenaline release at the spinal cord, at the level of the superficial dorsal horn, the motoneuron pools of the ventral horn, lamina X and the thoracic and sacral intermediolateral cell columns. Noradrenergic ascending or descending pathways originating in the A5 or A6 noradrenergic cell groups are highly sensitive to stress and to other high-arousal states. These noradrenergic groups present extensive projections that play a key role in the modulation of all antinociceptive and autonomic responses elicited by painful or threatening situations. Depending on the locations of these projections, different possible roles for each noradrenergic cell groups are suggested. The A6 noradrenergic cell group might have the greatest effect on somatosensory transmission and the A5 group on sympathetic function. Consistent with this, stimulation of central noradrenergic pathways evokes an array of stresslike and antinociceptive effects, including changes in blood pressure, heart rate and respiratory rate. In addition, it also produces an increase in excitability, which leads to a high degree of arousal and a potentiation of cortical and subcortical mechanism generating the necessary cognitive, behavioral and autonomic responses to confront these physical or psychological situations.

**Keywords:** pontine noradrenergic cell groups, A5 region, locus coeruleus, cardiovascular control, analgesia

#### **1. Introduction**

Noradrenergic (NA) central pathways located at the level of the brainstem were initially described by Dahlström and Fuxe in 1964 and contain several clusters or groups of neurons

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

classified from A1 to A7. These clusters extend rostrocaudally from the lateral pons to the caudal ventrolateral medulla. Afferent and efferent connections are sent and come from very different locations along the central nervous system (CNS) and are implicated in physiological and behavioral functions associated with a wide cascade of processes, such as homeostasis, arousal, memory, learning, autonomic and behavioral responses to stress and pain, among others [1, 2].

at thoracic levels. These descending projections of the NA cell groups are crucial in explaining their functional implications in central cardiorespiratory control and in other important

A5 and A6 Noradrenergic Cell Groups: Implications for Cardiorespiratory Control

http://dx.doi.org/10.5772/intechopen.79389

115

The projections from A6 cells use two main pathways: through the spinal cord in the ventral funiculi and through the dorsal surface of the dorsal horn. The A6 NA cell group supplies the highest concentration of synaptic endings at all levels. It includes all regions of the spinal gray matter, but it is especially dense at the level of the dorsal horn, although it has a small number of axons to the ventral horn and IML [5]. Extensive literature for this exists, not only at an anatomical level [6–12] but also with electrophysiological evidence [13, 14]. Intra and extracellular neuronal recording studies provide the assignment to caudal A6 NA neurons with a role in regulating the excitability of the cell bodies of somatic alpha motoneurons located within

It is well established that the spinally projecting axons of the A5 NA group mainly travel through the spinal cord within the lateral funiculi to end at the level of the IML cell column

**Figure 1.** Schematic diagram of a sagittal section of human brain in which the main pontine noradrenergic nuclei (A5 and A6) and their main efferent connections are positioned. (A5) A5 noradrenergic cell group. (A6) A6 noradrenergic cell group, Locus Coeruleus. (Amyg) Amygdala. (CVL) Caudal ventrolateral medulla. (DMH-PeF) Dorsomedial Hypothalamic nucleus and perifornical area. (Hyppoc) Hyppocampus. (IML) Intermedio lateral cell collum of the spinal cord. (KF) Kölliker-Fuse nucleus. (LH) Lateral Hypothalamus. (NTS) Nucleus Tractus Solitarii. (PAG) Periaqueductal

gray. (PBc) Parabrachial complex. (PVN) Paraventricular nucleus. (RVL) Rostral ventrolateral medulla.

autonomic functions involved in behavioral responses to stress or pain.

**2.1. Spinal projections from the A6 (locus coeruleus)**

the ventral horn of the spinal cord.

**2.2. Spinal projections from the A5**

NA neurons are characterized by the presence, within the synaptic terminal, of the cytoplasmatic enzymatic machinery, which is necessary to biosynthesize noradrenaline from the amino acid tyrosine through a precise and sequential enzymatic reaction. Tyrosine hydroxylase (TH) is the limiting enzyme. It transforms tyrosine into dihydroxyphenylalanine (L-DOPA), which is converted into dopamine by L-DOPA decarboxylase. Finally, dopamine is used as a substrate by dopamine-β-hydroxylase (DBH), which transforms dopamine into noradrenaline [3]. DBH immunodetection is specific for NA neurons and NA central demand [4]. Although once the noradrenaline is a precursor to adrenaline synthesis, the immunodetection of DBH is not restricted to noradrenergic neurons except in the cases where the referred group is isolated from adrenergic neurons (as A6 and A5). After its release into the synaptic cleft, noradrenaline can bind to the pre- or post-synaptic adrenergic receptors and activates intracellular signaling cascades depending on the specific function of the subtype of the adrenergic receptor activated (facilitatory or inhibitory receptors).

Briefly, in terms of the precise location of the different NA cell groups: the A1 NA cell group is found in the ventrolateral medulla; the A2, located close to the dorsal vagal complex, has an intimate relationship (as part of) with caudal NTS complex, starting in very caudal level of medulla until the open of fourth ventricle; A3 neurons are included within the medullary reticular formation, and neurons of the A4 cell group are situated in the surroundings of the fourth ventricle. The precise location of the most studied NA cell groups, the A5, A6 and A7, is the following: the A5 NA cell group is located in the ventrolateral pons; A6, which represents the locus coeruleus, is located in the lateral floor of the fourth ventricle and, finally, A7 is found in the lateral part of the pons. These last three groups of NA neurons represent the most important NA clusters with projections to the spinal cord [5, 6].

Early studies using retrograde transport of horseradish peroxidase combined with immunostaining for DBH or retrograde transport of anti-DBH antibodies demonstrated that the NA endings of the spinal cord arise from the A5, A6 and A7 cell groups in the pons [7]. The projections from the neurons located in the A5, A6 and A7 cell groups are found throughout the spinal cord, but the highest density of synaptic contacts is established at the level of the superficial dorsal horn, the motoneuron pools of the ventral horn, lamina X and the thoracic and sacral intermediolateral cell columns (IML) [5].

In this chapter, the main focus is centered on the main pontine NA cell groups, which project to the spinal cord (A5 and A6), and their implications for cardiorespiratory control.
