**6.2.3 Summary**

These studies indicate that increased noradrenergic function, either through genetic manipulation or pharmacologically, results in less damage to dopaminergic neurons by MPTP, indicating a neuroprotective effect of the noradrenergic nervous system on dopaminergic neurons. The major concern with these studies is that the enhanced noradrenergic function is not limited exclusively to the LC noradrenergic system. There is also the added complexity of the localization of the different noradrenergic receptors, preversus post-synaptically, and the differences in expression of AR between rats and mice (Szot, 2006) which may contribute to the conflicting results of α2-AR agonists. Future studies could examine the ability of other AR agonists that act postsynaptically to modulate susceptibility of dopaminergic neurons to damage or determine if pharmacological agents produce a neuroprotective effect after direct administration into a specific brain region such as the striatum or SN.

#### **6.3 Reduced LC function enhances susceptibility of dopaminergic neurons to damage**

An easier approach in determining if the LC noradrenergic nervous system can affect the susceptibility of dopaminergic neurons to damage is to reduce the number of LC noradrenergic neurons and innervation to forebrain regions. This approach specifically targets the LC noradrenergic neurons, mimicking what is observed in PD. LC noradrenergic neuronal loss has been shown to occur with administration of 6OHDA directly into the LC and peripheral administration of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4).

#### **6.3.1 DSP4 enhances susceptibility of dopaminergic neurons to damage**

DSP4 has been considered a LC selective noradrenergic neurotoxin since 1980s. The determination that DSP4 was a selective LC neurotoxin is based on documented changes in terminal noradrenergic fibers in regions innervated mainly by the LC. The appeal of DSP4 as a noradrenergic neurotoxin over other methods (like 6OHDA) is that DSP4 can produce this central affect by peripheral administration. DSP4 produces a rapid, though transient, reduction in terminal NE concentrations in the frontal cortex, hippocampus and cerebellum (Grzanna et al., 1989; Harro et al., 1999a, b; Hughes & Stanford, 1996, 1998; Jonsson et al., 1981; Kask et al., 1997; Ross, 1976; Szot et al., 2011; Theron et al., 1993; Wolfman et al., 1994). As indicated earlier, these regions receive sole innervation from the LC (Aston-Jones et al., 1995; Jones & Moore, 1977; Loughlin et al., 1986a, b; Mason & Fibiger, 1970; Moore & Bloom, 1979; Olsen & Fuxe, 1971; Ungerstedt, 1971; Waterhouse et al., 1983) so the original hypothesis was that DSP4 affected only LC neurons. NET binding sites are also reduced in specific forebrain regions, indicating a loss of innervation from the LC (Cheetham et al., 1996; Szot et al., 2010). The hypothesis that DSP4 was a neurotoxin was supported by TH-IR which demonstrated a gradual loss of LC noradrenergic neurons after by DSP4 administration (Grzanna et al., 1989). Since these initial descriptions of DSP4 as a noradrenergic neurotoxin, there have been other publications suggesting that DSP4 does not result in a loss of LC noradrenergic neurons (Booze et al., 1988; Lyon et al., 1983; Matsukawa

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.

These studies indicate that increased noradrenergic function, either through genetic manipulation or pharmacologically, results in less damage to dopaminergic neurons by MPTP, indicating a neuroprotective effect of the noradrenergic nervous system on dopaminergic neurons. The major concern with these studies is that the enhanced noradrenergic function is not limited exclusively to the LC noradrenergic system. There is also the added complexity of the localization of the different noradrenergic receptors, preversus post-synaptically, and the differences in expression of AR between rats and mice (Szot, 2006) which may contribute to the conflicting results of α2-AR agonists. Future studies could examine the ability of other AR agonists that act postsynaptically to modulate susceptibility of dopaminergic neurons to damage or determine if pharmacological agents produce a neuroprotective effect after direct administration into a specific brain region such

**6.3 Reduced LC function enhances susceptibility of dopaminergic neurons to damage**  An easier approach in determining if the LC noradrenergic nervous system can affect the susceptibility of dopaminergic neurons to damage is to reduce the number of LC noradrenergic neurons and innervation to forebrain regions. This approach specifically targets the LC noradrenergic neurons, mimicking what is observed in PD. LC noradrenergic neuronal loss has been shown to occur with administration of 6OHDA directly into the LC and peripheral administration of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4).

DSP4 has been considered a LC selective noradrenergic neurotoxin since 1980s. The determination that DSP4 was a selective LC neurotoxin is based on documented changes in terminal noradrenergic fibers in regions innervated mainly by the LC. The appeal of DSP4 as a noradrenergic neurotoxin over other methods (like 6OHDA) is that DSP4 can produce this central affect by peripheral administration. DSP4 produces a rapid, though transient, reduction in terminal NE concentrations in the frontal cortex, hippocampus and cerebellum (Grzanna et al., 1989; Harro et al., 1999a, b; Hughes & Stanford, 1996, 1998; Jonsson et al., 1981; Kask et al., 1997; Ross, 1976; Szot et al., 2011; Theron et al., 1993; Wolfman et al., 1994). As indicated earlier, these regions receive sole innervation from the LC (Aston-Jones et al., 1995; Jones & Moore, 1977; Loughlin et al., 1986a, b; Mason & Fibiger, 1970; Moore & Bloom, 1979; Olsen & Fuxe, 1971; Ungerstedt, 1971; Waterhouse et al., 1983) so the original hypothesis was that DSP4 affected only LC neurons. NET binding sites are also reduced in specific forebrain regions, indicating a loss of innervation from the LC (Cheetham et al., 1996; Szot et al., 2010). The hypothesis that DSP4 was a neurotoxin was supported by TH-IR which demonstrated a gradual loss of LC noradrenergic neurons after by DSP4 administration (Grzanna et al., 1989). Since these initial descriptions of DSP4 as a noradrenergic neurotoxin, there have been other publications suggesting that DSP4 does not result in a loss of LC noradrenergic neurons (Booze et al., 1988; Lyon et al., 1983; Matsukawa

**6.3.1 DSP4 enhances susceptibility of dopaminergic neurons to damage** 

The same complexity exists for the effects of α2-AR antagonists.

**6.2.3 Summary** 

as the striatum or SN.

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 regions.

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 not shown).

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).

The Noradrenergic System is a Major Component in Parkinson's Disease 259

neurons (Figure 2). What is known is that α2-AR agonists can facilitate movement by acting through the striatum (Hill and Brotchie, 1999). Therefore, α2-AR antagonists have been studied as a potential adjunct therapy to reduce L-DOPA-induced dyskinesia. α2-AR antagonists, yohimbine, idazoxan and fipamezole reduce L-DOPA dyskinesia in the MPTP and 6OHDA PD animal models and in Parkinsonian patients. These α2-AR antagonists reduce L-DOPA-induced side effects without affecting the anti-parkinsonian effect of L-DOPA (Brotchie, 2005; Buck et al., 2010; Colosimo and Craus, 2003; Chopin et al., 1986; Gomez-Mancilla and Bedard, 1993; Grondin et al., 2000; Henry et al., 1999; Lundblad et al., 2002; Rascol et al., 2001; Savola et al., 2003). However, the anti-dyskinetic effect of α2-AR antagonists may be limited to just L-DOPA; idazoxan did not reduce apomorphine-induced dyskinesia (Fox et al., 2001). The α2-ARs are not the only receptor capable of modulating L-DOPA-induced dyskinesia. Because β-ARs are found in large concentrations in the striatum (see above), studies are now beginning to determine if β-AR antagonists, such as propranolol, can reduce L-DOPA-induced dyskinesia. These studies indicate that propranolol also reduces L-DOPA-induced dyskinesia (Goshima et al., 1991; Reisine et al., 1982; Carpentier et al., 1996; Dekundy et al., 2007). α1-AR antagonist also reduces L-DOPAinduced dyskinesia; however, this receptor subtype has not been investigated to the same

The dopaminergic nervous system has been the major focus of research in PD for many years, and rightly so because it is the loss of dopaminergic neurons and function that is responsible for the appearance of the motor symptoms that define PD. However, the noradrenergic nervous system appears to play an important role in the progression of PD and in the therapy of PD. LC noradrenergic neurons are reduced early in the progression of PD, even before dopaminergic neurons. Animal models suggest that the loss of LC noradrenergic neurons will enhance the susceptibility of dopaminergic neurons to damage. Understanding the relationship of LC neuronal loss to enhanced dopaminergic susceptibility to damage introduces a time window in which pharmacological intervention could *prevent* the loss of dopaminergic neurons and the appearance of motor symptoms. But until this relationship is examined, the noradrenergic nervous system can be used to reduce Dyskinesia, the severe side effects of chronic L-DOPA therapy. These data indicate the importance of examining neurotransmitter systems other than DA and determining the

This work was supported by the Department of Veterans Affairs Research and Development Services Northwest Network Mental Illness Research, Education and Clinical Center at the Seattle Puget Sound Health Care Center. The authors thank Dr. Charles Wilkinson and Carl

Ader, J.P., Room, P. & Postema, F. (1980). Bilaterally diverging axon collaterals and

coutralateral projections from rat locus coeruleus neurons, demonstrated by

degree as α2- and β-AR antagonists (Buck and Ferbeger, 2010).

role of transmitters, such as NE, in affecting PD.

Sikkema for CSF catecholamine analysis.

**9. Acknowledgement** 

**10. References** 

**8. Conclusion** 

These data indicate that administration of the noradrenergic neurotoxin 6OHDA can increase the susceptibility of dopaminergic neurons to damage.

#### **6.4 Summary**

These data suggest that enhanced noradrenergic function can protect dopaminergic neurons from damage, while reducing noradrenergic function enhances the susceptibility of dopaminergic neurons to damage. The number of studies examining this important question are few, but the data indicates the LC noradrenergic nervous system can modulate the viability of dopaminergic neurons to damage. It is unknown why dopaminergic neurons in PD are reduced. The loss of dopaminergic neurons and the appearance of PD symptoms occur midway in the progression of the disorder, after the loss of LC noradrenergic neurons. Therefore, determining factors that can maintain the stability of dopaminergic neurons in the early stages of the disorder is imperative in preventing the loss of dopaminergic neurons and the onset of PD symptoms. Since LC neuronal loss occurs early in the progression of PD and is a prominent neuropathologic change in PD, an animal model examining the neuroprotective effect of the noradrenergic nervous system on dopaminergic neurons must exhibit reduced LC noradrenergic neuronal number, so DSP4 may not be the best animal model of LC neuronal loss. Future work will determine if there is a particular time following the loss of LC noradrenergic neurons that renders the dopaminergic neurons more susceptible to damage, or it may be that following administration of MPTP, animals with reduced LC function do not recover from the damage induced by MPTP.
