**6. Neuroprotective effect of LC neurons on dopaminergic neurons**

Since the noradrenergic nervous system experiences a loss of function before the dopaminergic system in PD (Braak et al., 2003b, 2006) and the noradrenergic nervous system innervates dopaminergic neurons in the SN and VTA (shown above); the noradrenergic nervous system could then affect the stability of dopaminergic neurons. There is data to support the hypothesis of a neuroprotective effect of the noradrenergic system upon dopaminergic neurons in animal models. To determine if the LC noradrenergic nervous system exerts a neuroprotective effect on dopaminergic neurons, a dopaminergic neurotoxin needs to be administered.

### **6.1 Animal models of PD**

There are several different animal models of PD which have been described in detail elsewhere (Betarbet et al., 2002; Dauer & Przedborski, 2003; Jackson-Lewis & Przedborski, 2007; Luchtman et al., 2009) and are not the focus of this chapter. The classic PD symptoms induced by a variety of dopaminergic neurotoxins are reduced number of SN dopaminergic neurons and reduced amount of DA in the striatum. The most routinely used dopaminergic neurotoxins are 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6 hydroxydopamine (6OHDA). The choice of neurotoxin depends on the species of animal to be studied. MPTP is typically the neurotoxin used to reduce the number of dopaminergic neurons in the SN (and to a lesser degree the VTA neurons) in mice and monkeys. MPTP is

The midbrain dopaminergic neurons in the SN and VTA also receive direct innervation from LC noradrenergic neurons (Fritschy & Grzanna, 1990; Jones & Moore, 1977; Jones & Yang, 1985; Phillipson, 1979; Simon et al., 1979; Swanson & Hartman, 1975). The SN and VTA regions have detectable levels of NET binding, also indicating direct noradrenergic innervation and some of the innervation is from the LC (Szot, Personal communications). The SN and VTA also have β-, α1- and α2-AR binding sites (Szot, Personal communication; Boyajian et al., 1987; Hudson et al., 1992; Lee et al., 1998; Rainbow et al., 1984; Rosin et al., 1996; Scheinin et al., 1994; Strazielle et al., 1999). The LC directly innervates and modifies the activity of midbrain dopaminergic neurons; the loss of LC noradrenergic neurons results in altered activity of dopaminergic neurons (Collingridge et al., 1979; Grenhoff et al., 1993,

There have been several studies to indicate the ability of LC noradrenergic neurons to modulate dopamine-induced behavior, especially when LC noradrenergic neurons are reduced (Antelman & Caggiula, 1977; Archer & Fredriksson, 2006**;** Chopin et al., 1999; Grimbergen et al., 2009; Mavridis et al., 1991; Rommelfanger et al., 2007; Taylor et al., 2009; Villegier et al., 2003; Wang et al., 2010). In addition, reducing LC function alone by lesioning LC neurons can produce motor symptoms that are observed in PD (Grimbergen et al., 2009; Wang et al., 2010). In support of the LC lesion studies and the consequence of reduced NE content, DBH knockout mice, which do not synthesize NE, also exhibit PD motor symptoms with age (Rommelfanger et al., 2007). These data suggest the loss of LC noradrenergic

Since the noradrenergic nervous system experiences a loss of function before the dopaminergic system in PD (Braak et al., 2003b, 2006) and the noradrenergic nervous system innervates dopaminergic neurons in the SN and VTA (shown above); the noradrenergic nervous system could then affect the stability of dopaminergic neurons. There is data to support the hypothesis of a neuroprotective effect of the noradrenergic system upon dopaminergic neurons in animal models. To determine if the LC noradrenergic nervous system exerts a neuroprotective effect on dopaminergic neurons, a dopaminergic neurotoxin

There are several different animal models of PD which have been described in detail elsewhere (Betarbet et al., 2002; Dauer & Przedborski, 2003; Jackson-Lewis & Przedborski, 2007; Luchtman et al., 2009) and are not the focus of this chapter. The classic PD symptoms induced by a variety of dopaminergic neurotoxins are reduced number of SN dopaminergic neurons and reduced amount of DA in the striatum. The most routinely used dopaminergic neurotoxins are 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6 hydroxydopamine (6OHDA). The choice of neurotoxin depends on the species of animal to be studied. MPTP is typically the neurotoxin used to reduce the number of dopaminergic neurons in the SN (and to a lesser degree the VTA neurons) in mice and monkeys. MPTP is

1995; Grenhoff & Svensson, 1989, 1993; Guiard et al., 2008; Wang et al., 2010).

function observed in PD may contribute to the motor symptoms of PD.

**6. Neuroprotective effect of LC neurons on dopaminergic neurons** 

**5.2 LC innervation to midbrain dopaminergic neurons** 

**5.3 LC altered dopaminergic behavior** 

needs to be administered.

**6.1 Animal models of PD** 

administered peripherally and depending on the dose and the number of times it is administered, a mild to severe degree of dopaminergic neuronal loss can be observed (Betarbet et al., 2002; Dauer & Przedborski, 2003; Jackson-Lewis & Przedborski, 2007; Luchtman et al., 2009). However, MPTP is not effective in rats (Jackson-Lewis & Przedborski, 2007). To reduce the number of SN dopaminergic neurons in rats, 6OHDA is administered directly into the medial forebrain bundle using stereotaxic surgery. The effect of 6OHDA in the SN is rapid and appears permanent (Walsh et al., 2011).

#### **6.2 Enhanced LC function reduces dopaminergic neurotoxin-induced damage 6.2.1 Neuroprotective effect of enhanced noradrenergic function in tottering and NET transgenic mice**

The tottering mouse has a mutation that results in hyperinnervation of noradrenergic terminals and increased concentration of NE throughout most regions of the forebrain. Administration of MPTP to these mutant mice has less of an effect on dopaminergic terminals in the striatum (i.e., loss of DA level) as compared to wild-type mice (Kilbourne et al., 1998). Another transgenic mouse that has enhanced noradrenergic function is the NET knockout mouse. NET knockout mice do not express the transporter protein for NE, which are localized specifically to noradrenergic neurons and responsible for removing NE from the synapse, resulting in enhanced NE in the synapse. Administration of MPTP to NET knockout mice, again, results in reduced damage to dopaminergic terminals in the striatum and DA levels in the striatum as compared to wild-type mice (Rommelfanger et al., 2004). These studies indicate that an enhanced noradrenergic system can protect dopaminergic neurons in the SN from damage.

#### **6.2.2 Neuroprotective effect of AR agents**

Another means of increasing noradrenergic function is to administer noradrenergic agonists. Administration of NET inhibitors to increase synaptic NE levels results in reduced dopaminergic damage on SN terminals in the striatum and DA levels, resembling the effect observed in the NET knockout mouse (Rommelfanger et al., 2004). Peripheral administration of α2-AR agonists such as clonidine and detomidine also reduces MPTPinduced reduction in striatal DA levels, while administration of α2-AR antagonists enhances MPTP-induced damage in mice (Fornai et al., 1995a). However, when 6OHDA is used as the dopaminergic neurotoxin in rats, the peripheral administration of α2-AR antagonists reduces the loss of DA in the striatum (Srinivasan & Schmidt, 2004b, c), the opposite of what is observed in mice with MPTP. The ability of α2-AR agents to either enhance or reduce damage on dopaminergic neurons could be attributed to the different species (rats versus mice) or the neurotoxin (MPTP versus 6OHDA) used. Another possible reason for the conflicting data of α2-AR agents is the complexity of the α2-AR. The α2-AR receptor is composed of three different subtypes: α2A-, α2B-, and α2C-AR. α2A- and α2C-ARs are localized on dendrites and terminals of noradrenergic neurons where they act as presynaptic autoreceptors to regulate the release of NE ( L'Heureux et al., 1986; Van Gaalen et al., 1997; Kawahara et al., 1999), as well as postsynaptic receptors on dendrites and terminals of NE target cell that regulate the release of other neurotransmitters (heteroreceptors). α2A-ARs are the most abundant α2-AR subtype in the brain, comprising approximately 90% of all central α2-ARs (Bucheler et al., 2002). The highest density of α2C-AR is in the striatum, while α2B-ARs have a very limited expression in the brain (Nicholas et al., 1993; Zeng & Lynch, 1991).

The Noradrenergic System is a Major Component in Parkinson's Disease 257

et al., 2003; Robertson et al., 1993; Szot et al., 2010) and that DSP4 does not selectively affect LC noradrenergic terminals (Grzanna et al., 1989; Kask et al., 1997; Szot et al., 2010; Theron et al., 1993; Wolfman et al., 1994). Work in our laboratory indicated that DSP4 may affect noradrenergic terminals and specific ARs in many forebrain regions (not just regions innervated by the LC), but it does so without a loss of LC noradrenergic neurons (Szot et al., 2010). Despite the data that suggests DSP4 may not result in a loss of LC noradrenergic neurons, DSP4 does result in a temporary reduction in terminal NE levels in many forebrain

Administration of DSP4 prior to methamphetamine, MPTP or 6OHDA into the medial forebrain bundle will increase the damage these dopaminergic neurotoxins exert on SN dopaminergic neurons and DA levels in the striatum (Fornai et al., 1995b, 1996, 1997; Marien et al., 1993; Srinivasan and Schmidt, 2003, 2004a). The enhanced susceptibility of dopaminergic neurons to damage from these dopaminergic neurotoxins is observed at a time when DSP4 results in a significant reduction in terminal NE levels. Administration of DSP4 after the dopaminergic neurotoxin does not enhance the susceptibility of dopaminergic neurons to damage (Fornai et al., 1997). These data indicate that a reduction in NE levels in forebrain regions will increase the susceptibility of dopaminergic neurons to damage. However, administration of MPTP to DBH knockout mice does not result in enhanced loss of SN neurons or DA levels in the striatum as compared to wild-type mice (Rommelfanger and Weinshenker, 2007). Administration of DSP4 prior to the administration of MPTP in the DBH knockout mouse does result in enhanced damage to dopaminergic neurons and terminals as compared to DBH knockout mice administered

MPTP alone (Rommelfanger and Weinshenker, 2007).

**6.3.2 6OHDA enhances susceptibility of dopaminergic neurons to damage** 

There are few studies examining the direct administration of the neurotoxin 6OHDA directly into those LC, though the few that do demonstrate a reduction in the number of LC noradrenergic neurons (Bing et al., 1991; Mavridis et al., 1991). Our laboratory has begun to examine the specificity of direct LC 6OHDA administration on LC neurons and lateral tegmental neurons as well as neurons in the SN and VTA. We have observed that direct administration of 6OHDA into the LC specifically reduces LC noradrenergic neurons, while there is no effect on SN or VTA dopaminergic neurons or on lateral tegmental neurons (data

There are two studies that have examined the ability of LC 6OHDA administration to increase MPTP-induced damage to dopaminergic neurons. Bing et al., (1994) examined the ability of LC 6OHDA to enhance MPTP-induced damage in the mouse, while Mavridis et al., (1991) examined LC 6OHDA in monkeys. Both of these studies demonstrated an enhanced susceptibility of dopaminergic neurons to damage following LC neuronal loss. Preliminary work in my laboratory has begun to examine the effects of 6OHDA administration into the LC of mice on the susceptibility of dopaminergic neurons to MPTP. We have examined the effect of MPTP (24 mg/kg, ip, twice, 2hrs apart) administered 3 days after bilateral administration of 6OHDA into the LC on LC, SN and VTA neurons. MPTP alone did not affect LC noradrenergic neurons, but MPTP alone significantly reduced DAergic neurons in the SN (28% reduced) with no effect on VTA neurons (7% reduced). However, the *combination* of 6OHDA + MPTP resulted in a further reduction in SN neurons (71% reduced) and a significant reduction in VTA neurons (54% reduced) (data not shown).

regions.

not shown).

Clonidine and detomidine do not discriminate between the different subtypes, so it is unclear how these agonists affect noradrenergic function: reduced NE release and function by acting on presynaptic autoreceptors or enhancing NEs action at postsynaptic receptors. The same complexity exists for the effects of α2-AR antagonists.
