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**Chapter 5**

**Provisional chapter**

**Regulation of Dendritogenesis in Sympathetic Neurons**

In postganglionic sympathetic neurons, the size of the dendritic arbor determines presynaptic convergence, which correlates with tonic activity, and aberrant dendritic morphology is associated with disease. There is, therefore, great interest in understanding how dendritic morphology is regulated in these neurons. Early studies established a role for target-derived nerve growth factor (NGF) in regulating the size of the dendritic arbor of sympathetic neurons *in vivo*. However, *in vitro* studies revealed that even in the presence of optimal concentrations of NGF, rat sympathetic neurons cultured in the absence of serum or non-neuronal cells survive and elaborate extensive axonal arbors, but fail to form dendrites. Subsequently, it was discovered that bone morphogenetic proteins (BMPs) trigger cultured sympathetic neurons to extend a dendritic arbor comparable to that of their *in vivo* counterparts. The goals of this chapter are to: (i) summarize these early experiments; (ii) discuss evidence substantiating a role for BMPs in glial-induced dendritic growth *in vitro* and regulation of dendritic growth *in vivo*; (iii) review what is known about the molecular mechanisms by which NGF, BMPs and other factors influence dendritic arborization of sympathetic neurons; and (iv) identify key data gaps in

understanding of how dendrites are regulated in sympathetic neurons.

**Keywords:** afferent input, BMPs, dendrites, neuronal polarity, NGF, p75, reactive oxygen species (ROS), Rit, Smad, STAT, sympathetic neurons, target-derived factors

Differences in dendritic morphology between neurons are a striking feature of the vertebrate nervous system with important functional implications. The shape of dendrites influences the propagation and integration of postsynaptic potentials [1], and determines presynaptic

**Regulation of Dendritogenesis in Sympathetic** 

© 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.80480

Vidya Chandrasekaran and Pamela J. Lein

Vidya Chandrasekaran and Pamela J. Lein

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.80480

**Abstract**

**1. Introduction**

**Neurons**

#### **Regulation of Dendritogenesis in Sympathetic Neurons Regulation of Dendritogenesis in Sympathetic Neurons**

DOI: 10.5772/intechopen.80480

Vidya Chandrasekaran and Pamela J. Lein Vidya Chandrasekaran and Pamela J. Lein

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.80480

#### **Abstract**

In postganglionic sympathetic neurons, the size of the dendritic arbor determines presynaptic convergence, which correlates with tonic activity, and aberrant dendritic morphology is associated with disease. There is, therefore, great interest in understanding how dendritic morphology is regulated in these neurons. Early studies established a role for target-derived nerve growth factor (NGF) in regulating the size of the dendritic arbor of sympathetic neurons *in vivo*. However, *in vitro* studies revealed that even in the presence of optimal concentrations of NGF, rat sympathetic neurons cultured in the absence of serum or non-neuronal cells survive and elaborate extensive axonal arbors, but fail to form dendrites. Subsequently, it was discovered that bone morphogenetic proteins (BMPs) trigger cultured sympathetic neurons to extend a dendritic arbor comparable to that of their *in vivo* counterparts. The goals of this chapter are to: (i) summarize these early experiments; (ii) discuss evidence substantiating a role for BMPs in glial-induced dendritic growth *in vitro* and regulation of dendritic growth *in vivo*; (iii) review what is known about the molecular mechanisms by which NGF, BMPs and other factors influence dendritic arborization of sympathetic neurons; and (iv) identify key data gaps in understanding of how dendrites are regulated in sympathetic neurons.

**Keywords:** afferent input, BMPs, dendrites, neuronal polarity, NGF, p75, reactive oxygen species (ROS), Rit, Smad, STAT, sympathetic neurons, target-derived factors

#### **1. Introduction**

Differences in dendritic morphology between neurons are a striking feature of the vertebrate nervous system with important functional implications. The shape of dendrites influences the propagation and integration of postsynaptic potentials [1], and determines presynaptic

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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

convergence [2, 3]. These observations coupled with evidence that aberrant dendritic structure is strongly associated with neurologic disease [4, 5] have generated significant interest in understanding how dendrites are regulated.

required for dendritic growth in sympathetic neurons. When grown in low-density cultures in the absence of serum or non-neuronal cells, but in the presence of optimal concentrations

Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 93

*In vitro* studies have further revealed that the initiation and maintenance of dendritic growth in sympathetic neurons is regulated by trophic interactions. Addition of serum to NGFcontaining medium stimulates sympathetic neurons to form dendrites, although under these culture conditions, the dendritic arbor is significantly less complex than is observed *in vivo* [31]. In contrast, co-culture with ganglionic glial cells causes these neurons to form a dendritic arbor comparable to that of their *in vivo* counterparts [32]. Subsequently, it was discovered that the addition of bone morphogenetic proteins (BMPs) to the culture medium similarly triggers sympathetic neurons to form a complex dendritic arbor [29, 33]. Multiple BMP family members have been shown to stimulate dendritic growth in cultured sympathetic neurons, including BMPs 2, 4, 5, 6, 7 and 60A; however, this activity appears to be restricted to the dpp and 60A BMP subfamilies since BMP-3 and other members of the TGFβ superfamily, including TGFβ1, TGFβ2, TGFβ3, activin A, inhibin, and GDNF, have no effect on dendritic growth

The trophic actions of BMPs are specific to dendritic growth in that BMPs do not support cell survival, nor do they enhance axonal growth in cultured sympathetic neurons [29]. Consistent with observations of dendritic growth in sympathetic neurons *in vivo*, the dendrite-promoting activity of BMP-7 is independent of synaptic or electrical activity [35], but is modulated by NGF [29, 36]. Importantly, the dendritic arbor induced by BMPs in cultured sympathetic neurons is comparable to that of their *in vivo* counterparts with respect to not only size and complexity, but also accumulation and post-translational modification of dendrite-specific cytoskeletal and membrane proteins, exclusion of axonal proteins, transport of select mRNA, and formation of synaptic contacts of the appropriate polarity [29, 34, 35]. These observations indicate that BMPs selectively induce the execution of a developmental program in sympathetic neurons that controls both quantitative and qualitative aspects of dendritic growth.

These observations suggest that BMPs mediate the effects of ganglionic glia and target tissues on dendritic growth in sympathetic neurons. Immunocytochemical and *in situ* hybridization studies indicate that the spatiotemporal expression of BMPs 5, −6, and −7 in rat SCG is consistent with a role in the initial stages of dendritogenesis [33, 37]. *In vitro*, both SCG glia and neurons express BMP mRNA and protein when grown in the absence or presence of each other [37]. However, co-culture of sympathetic neurons with ganglionic glia markedly increases BMP protein coincident with a significant decrease in levels of the soluble BMP antagonists, follistatin and noggin [37]. Functional assays indicate that glial-induced dendritic growth is significantly reduced by BMP-7 antibodies and completely blocked by exogenous noggin and follistatin [37]. Collectively, these data suggest a model in which glia influence the rapid perinatal expansion of the dendritic arbor in sympathetic neurons by increasing BMP activity via modulation of the balance between BMPs and their antagonists. Whether this model holds

The question of whether BMPs also contribute to target effects on dendritic growth has yet to be addressed experimentally. Sympathetic targets, including the eye, heart, lung, kidney, and

of NGF, sympathetic neurons form an axon but no dendrites [29, 30].

in cultured sympathetic neurons [29, 33, 34].

true *in vivo* has yet to be tested.

Postganglionic sympathetic neurons are a well-characterized model for studying dendrite development and plasticity [6]. The dendritic arbor of these neurons is relatively complex with an average of two to six primary dendrites, depending on the animal species, and multiple orders of branching [7]. In postganglionic sympathetic neurons, the size of the dendritic arbor correlates with not only the number and pattern of synaptic inputs [3, 8], but also tonic activity [8, 9]. As is true of central neurons, aberrant morphology of sympathetic neuron dendrites is associated with disease. For example, dendritic hypertropy of sympathetic neurons in stellate and superior cervical ganglia (SCG) is observed in the spontaneously hypertensive rat [10, 11], and is thought to contribute to the pathogenesis of hypertension in this model [11]. Therapeutic intervention with statins not only decreases sympathetic activity and normalizes blood pressure in the spontaneously hypertensive rat [12], but also decreases dendritic arborization of both stellate and SCG neurons [13].

In this chapter, we will review what is known about the molecular and cellular mechanisms that regulate dendrites in postganglionic sympathetic neurons, and identify key data gaps.

#### **2. Early studies of dendritic growth in sympathetic neurons**

The majority of dendritic growth in postganglionic sympathetic neurons occurs during the postnatal period; however, dendrites continue to grow into adulthood [14–16], and *in situ* imaging of mature sympathetic neurons has demonstrated that their dendritic arbors continually grow and retract throughout life [17, 18]. In the rat, neonatal deafferentation has negligible effect on dendritic growth in SCG neurons throughout the first month of life [19–21], indicating that dendritic growth in sympathetic neurons does not require afferent input. In contrast, target tissues strongly influence dendritic growth in these neurons. Experimentally reducing target size causes dendritic arbors of SCG neurons to be smaller than normal; conversely, increasing the target size significantly increases the size of the dendritic arbor [16]. The influence of target is further illustrated by observations that sympathetic neurons within the same ganglion that project to different targets exhibit varying dendritic morphologies [18, 19].

The effect of target tissues on dendritic growth in sympathetic neurons is mediated, at least in part, by nerve growth factor (NGF) [22–25]. Separation of neurons from target tissues by axonal ligation causes dendritic atrophy in the few neurons that survive, and this effect is attenuated by systemic administration of NGF [26, 27]. However, exogenous NGF reverses axotomy-induced dendritic retraction by <50%, even though cell survival is completely rescued [27], indicating that additional target-derived factors are needed to fully account for the effects of target on dendritic growth. Consistent with this conclusion, the dendritic complexity of axotomized sympathetic neurons recovers to control levels upon ganglion cell reinnervation of the periphery [28]. *In vitro* studies further suggest that factors in addition to NGF are required for dendritic growth in sympathetic neurons. When grown in low-density cultures in the absence of serum or non-neuronal cells, but in the presence of optimal concentrations of NGF, sympathetic neurons form an axon but no dendrites [29, 30].

convergence [2, 3]. These observations coupled with evidence that aberrant dendritic structure is strongly associated with neurologic disease [4, 5] have generated significant interest in

Postganglionic sympathetic neurons are a well-characterized model for studying dendrite development and plasticity [6]. The dendritic arbor of these neurons is relatively complex with an average of two to six primary dendrites, depending on the animal species, and multiple orders of branching [7]. In postganglionic sympathetic neurons, the size of the dendritic arbor correlates with not only the number and pattern of synaptic inputs [3, 8], but also tonic activity [8, 9]. As is true of central neurons, aberrant morphology of sympathetic neuron dendrites is associated with disease. For example, dendritic hypertropy of sympathetic neurons in stellate and superior cervical ganglia (SCG) is observed in the spontaneously hypertensive rat [10, 11], and is thought to contribute to the pathogenesis of hypertension in this model [11]. Therapeutic intervention with statins not only decreases sympathetic activity and normalizes blood pressure in the spontaneously hypertensive rat [12], but also decreases dendritic

In this chapter, we will review what is known about the molecular and cellular mechanisms that regulate dendrites in postganglionic sympathetic neurons, and identify key data gaps.

The majority of dendritic growth in postganglionic sympathetic neurons occurs during the postnatal period; however, dendrites continue to grow into adulthood [14–16], and *in situ* imaging of mature sympathetic neurons has demonstrated that their dendritic arbors continually grow and retract throughout life [17, 18]. In the rat, neonatal deafferentation has negligible effect on dendritic growth in SCG neurons throughout the first month of life [19–21], indicating that dendritic growth in sympathetic neurons does not require afferent input. In contrast, target tissues strongly influence dendritic growth in these neurons. Experimentally reducing target size causes dendritic arbors of SCG neurons to be smaller than normal; conversely, increasing the target size significantly increases the size of the dendritic arbor [16]. The influence of target is further illustrated by observations that sympathetic neurons within the same ganglion that project to different targets exhibit varying

The effect of target tissues on dendritic growth in sympathetic neurons is mediated, at least in part, by nerve growth factor (NGF) [22–25]. Separation of neurons from target tissues by axonal ligation causes dendritic atrophy in the few neurons that survive, and this effect is attenuated by systemic administration of NGF [26, 27]. However, exogenous NGF reverses axotomy-induced dendritic retraction by <50%, even though cell survival is completely rescued [27], indicating that additional target-derived factors are needed to fully account for the effects of target on dendritic growth. Consistent with this conclusion, the dendritic complexity of axotomized sympathetic neurons recovers to control levels upon ganglion cell reinnervation of the periphery [28]. *In vitro* studies further suggest that factors in addition to NGF are

**2. Early studies of dendritic growth in sympathetic neurons**

understanding how dendrites are regulated.

92 Autonomic Nervous System

arborization of both stellate and SCG neurons [13].

dendritic morphologies [18, 19].

*In vitro* studies have further revealed that the initiation and maintenance of dendritic growth in sympathetic neurons is regulated by trophic interactions. Addition of serum to NGFcontaining medium stimulates sympathetic neurons to form dendrites, although under these culture conditions, the dendritic arbor is significantly less complex than is observed *in vivo* [31]. In contrast, co-culture with ganglionic glial cells causes these neurons to form a dendritic arbor comparable to that of their *in vivo* counterparts [32]. Subsequently, it was discovered that the addition of bone morphogenetic proteins (BMPs) to the culture medium similarly triggers sympathetic neurons to form a complex dendritic arbor [29, 33]. Multiple BMP family members have been shown to stimulate dendritic growth in cultured sympathetic neurons, including BMPs 2, 4, 5, 6, 7 and 60A; however, this activity appears to be restricted to the dpp and 60A BMP subfamilies since BMP-3 and other members of the TGFβ superfamily, including TGFβ1, TGFβ2, TGFβ3, activin A, inhibin, and GDNF, have no effect on dendritic growth in cultured sympathetic neurons [29, 33, 34].

The trophic actions of BMPs are specific to dendritic growth in that BMPs do not support cell survival, nor do they enhance axonal growth in cultured sympathetic neurons [29]. Consistent with observations of dendritic growth in sympathetic neurons *in vivo*, the dendrite-promoting activity of BMP-7 is independent of synaptic or electrical activity [35], but is modulated by NGF [29, 36]. Importantly, the dendritic arbor induced by BMPs in cultured sympathetic neurons is comparable to that of their *in vivo* counterparts with respect to not only size and complexity, but also accumulation and post-translational modification of dendrite-specific cytoskeletal and membrane proteins, exclusion of axonal proteins, transport of select mRNA, and formation of synaptic contacts of the appropriate polarity [29, 34, 35]. These observations indicate that BMPs selectively induce the execution of a developmental program in sympathetic neurons that controls both quantitative and qualitative aspects of dendritic growth.

These observations suggest that BMPs mediate the effects of ganglionic glia and target tissues on dendritic growth in sympathetic neurons. Immunocytochemical and *in situ* hybridization studies indicate that the spatiotemporal expression of BMPs 5, −6, and −7 in rat SCG is consistent with a role in the initial stages of dendritogenesis [33, 37]. *In vitro*, both SCG glia and neurons express BMP mRNA and protein when grown in the absence or presence of each other [37]. However, co-culture of sympathetic neurons with ganglionic glia markedly increases BMP protein coincident with a significant decrease in levels of the soluble BMP antagonists, follistatin and noggin [37]. Functional assays indicate that glial-induced dendritic growth is significantly reduced by BMP-7 antibodies and completely blocked by exogenous noggin and follistatin [37]. Collectively, these data suggest a model in which glia influence the rapid perinatal expansion of the dendritic arbor in sympathetic neurons by increasing BMP activity via modulation of the balance between BMPs and their antagonists. Whether this model holds true *in vivo* has yet to be tested.

The question of whether BMPs also contribute to target effects on dendritic growth has yet to be addressed experimentally. Sympathetic targets, including the eye, heart, lung, kidney, and 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 of the animal.

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

Regulation of Dendritogenesis in Sympathetic Neurons http://dx.doi.org/10.5772/intechopen.80480 95

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

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

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

[49], suggesting that Smad 4 may play a role in limiting dendritic growth *in vivo*.

for the initiation and early growth of dendrites in sympathetic neurons.

of BMP signaling in dendritogenesis *in vivo*.

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