**3.1 CNS pathophysiology of 5-HT system in PD**

#### **3.1.1 Neuroanatomical evidence in PD patients**

Even though degeneration of DA neurons in the SNpc remains the best identified neuropathological hallmark in PD, there exists increasing evidence suggesting PD-related pathology in the principle 5-HT cell bodies, the raphe nuclei and other regions innervated by raphe neurons (Braak et al., 2003).

Multiple studies have reported the presence of LB in the caudal group of raphe nuclei, like the raphe magnus and raphe pallidus, in early PD, sometimes occurring even before the onset of motor symptoms (Braak et al., 2003; Del Tredici et al., 2002; Parkkinen et al., 2008). It is interesting to note that these caudal raphe nuclei contain 5-HT neurons associated with functions like pain perception, and gastrointestinal motility that are manifest as early symptoms in PD patients prior to motor complaints (Chaudhuri and Schapira, 2009). The rostral raphe nuclei consisting of dorsal and medial raphe nuclei are equally affected in PD and according to Braak staging, are affected before the SNpc but after the caudal raphe nuclei (Braak et al., 2003).

Despite reports of raphe LB formation, evidence for the degeneration of 5-HT neurons in the rostral raphe nuclei is variable; post-mortem analysis of PD brains by Paulus and Jellinger (1991) revealed a profound loss of 5-HT neurons, however, other studies have not (Halliday et al., 1990; Mann and Yates, 1983). Several studies have employed transcranial sonography to study the midbrain raphe nuclei. This work has revealed abnormal pathology in the form of hypoechogenicity or an absence of sonographic signals in PD vs. control subjects. Interestingly, PD patients in one study also suffered from higher incidence of depression, reflecting a direct relationship between raphe nuclei loss and PD-related depression (Becker et al., 1997; Berg and Gaenslen, 2010; Walter et al., 2007b). MRI imaging studies carried out in depressed PD patients have also demonstrated a loss of homogeneity in the midbrain raphe consistent with neuronal compromise and/or cell loss (Berg et al., 1999).

PD-related pathology of the 5-HT system is not limited to the cell bodies of the raphe nuclei. Convincing evidence exists for damaged 5-HT projections and terminals as well. For example, post-mortem studies in PD patients have described significant loss of 5-HT markers, such as brain 5-HT concentrations. In cortical and the basal ganglia regions 5-HT content has been reported to be reduced by as much as 50% compared to controls (Birkmayer and Birkmayer, 1987). Kish and colleagues (2008) investigated the integrity of the forebrain 5-HT system. In contrast to DA loss, which was preferential to the putamen, 5- HT loss was more prominent in the caudate for all 5-HT markers including 5-HT (-66%), the 5-HT metabolite 5-HIAA (-42%), 5-HT transporter (SERT), (-56%) and the rate limiting enzyme in 5-HT synthesis tryptophan hydroxylase (-59%). These corroborated ante-mortem

Pathophysiology of Non-Dopaminergic Monoamine

**3.2 Non-motor symptoms** 

et al., 1991; Huot et al., 2010a).

al., 2000; 2004).

**3.2.1 5-HT loss and non-motor symptoms** 

potential targets for therapeutic strategies in the treatment of PD.

neuroanatomical and functional alterations of the 5-HT system.

Systems in Parkinson's Disease: Implications for Mood Dysfunction 533

well as other regions (Fox and Brotchie, 2000; Huot et al., 2010c; Radja et al., 1993). It is important to note that these changes may not be direct evidence of 5-HT neuropathology but definitely provide an insight into neuroplasticity of the 5-HT system that may unravel

An indirect marker for 5-HT alterations in PD is the assessment of responses to 5-HT challenge tests. Of these, the most common is the endocrine response to the 5-HT releasing agent, Fenfluramine. In normal subjects Fenfluramine produces robust increases in prolactin and corticosterone levels. However, in PD patients it was found that this endocrine response was impaired (Kostic et al., 1996; Volpi et al., 1997). Such effects may also correlate with non-motor symptoms since PD patients suffering from depression also displayed blunted prolactin responses in comparison to non-depressed PD patients (Kostic et al., 1996). Collectively these findings provide substantial evidence for neurochemical,

As previously mentioned depression and anxiety are some of the most common nonmotor symptoms in PD and are even associated with an elevated risk towards the development of PD (Leentjens et al., 2003; Schuurman et al., 2002; Shiba et al., 2000). The underlying pathophysiological mechanisms remain to be completely understood; however, it is well established that 5-HT dysfunction plays an important role in several mood-disorders in non-PD patients (Michelsen et al., 2008). Depression not only reduces the quality of life for PD patients but has a negative effect on caregivers as well (Schrag et

During the progression of PD it has been observed that brain regions, like rostral raphe, thalamus and cortex, that mediate mood disturbances in PD are severely affected by the presence of Lewy bodies (Braak and Del Tredici, 2008). Currently, most evidence linking abnormal serotonergic neurotransmission to mood disturbances in PD is corroborative but points to a role for 5-HT pathology. For example, depressed PD patients display reduced brainstem raphe echogenicity, in comparison to non-depressed PD patients (Walter et al., 2007a). Post-mortem comparisons of neuronal density in the dorsal raphe nucleus between depressed and non-depressed PD patients found lower neuronal density in depressed PD patients (Paulus and Jellinger, 1991). In vivo studies measuring cerebrospinal fluid levels found lower levels of 5-HIAA in depressed PD patients indicating reduced 5-HT metabolism (Mayeux et al., 1986). Imaging studies have been less conclusive and have found either no change in SERT uptake (Kim et al., 2003) or reported elevated 5-HT receptor binding in depressed PD patients when compared to non-depressed PD patients (Boileau et al., 2008). Interestingly, acute tryptophan depletion in a small group of PD patients did not produce depression or anxiety in these patients (Leentjens et al., 2006). Another major nonmotor symptom affecting PD patients is the development of psychosis that may lead to development of paranoid delusions in some PD patients (Ravina et al., 2007). The underlying cause remains to be elucidated and some investigators have postulated that there may be a serotonergic involvement. 5-HT2 receptors, responsible for hallucinations and psychosis, are relatively intact or may even be upregulated in the cortex of PD patients suffering from psychosis compared to PD patients free from any psychotic disorder (Cheng

observations in PD patients that examined levels of 5-HIAA in cerebrospinal fluid and have found significant reductions when compared to control patients. Interestingly, the deficits in cerebrospinal fluid 5-HIAA levels were more pronounced in PD patients with depression in comparison to non-depressed PD patients, again supporting a relationship between decreased 5-HT function and depression in PD (Mayeux et al., 1984; Mayeux et al., 1986).

Development of additional imaging technologies, like PET and SPECT, has facilitated the measurement of SERT and thus the evaluation of the integrity of the 5-HT terminal (Meyer et al., 2007). In vivo SPECT studies, using non-specific ligands for SERT, found decreased binding in the cortex and hypothalamus of PD patients (Berding et al., 2003a; Berding et al., 2003b). However, these findings have been contradicted by studies that did not find any changes in the mid-brain but rather reduction in the thalamic nuclei of PD patients (Caretti et al., 2008; Kim et al., 2003; Roselli et al., 2010). Decreased SERT binding has been observed by use of PET imaging using more specific ligands. Under these circumstances reduced SERT was observed in the striatum, frontal cortex, caudate nucleus, putamen and the mid-brain raphe region of patients with PD (Albin et al., 2008; Guttman et al., 2007; Kerenyi et al., 2003). SERT binding is also labile, changing as PD progresses. For example, in the early stages of PD, SERT binding has been shown to be reduced in only in the striatum, thalamus and cingulate cortex. In later symptomatic stages of PD these alterations appear to extend to the prefrontal cortex and the raphe nuclei (Haapaniemi et al., 2001; Politis et al., 2010). Such findings suggest that a progressive reduction in SERT binding may serve as good a bio-marker for the diagnosis and development of treatment strategies for PD patients.

In addition to neuronal integrity, 5-HT receptors are also affected in PD. Modification of pre- and post-synaptic 5-HT receptors has been observed in various animal and human studies of PD. While it is not clear whether these compensatory changes are due to lost 5-HT input, DA innervation, or DA replacement, it is established that dopaminergic tone regulates the expression of several 5-HT receptors. 5-HT1A receptor binding is not consistently affected in the 6-OHDA model of PD; however, studies in MPTP-treated macaques suggest increases in striatal and cortical binding (Frechilla et al., 2001; Huot et al., 2010b). 5-HT1B receptor binding is significantly increased in the striatum (54%) and the globus pallidus (33%). Intranigral lesions have also been reported to increase 5-HT4 receptor density in the caudate and the globus pallidus (Di Matteo et al., 2008). Studies using in situ hybridization and autoradiographic radioligand binding have revealed few changes in 5- HT1A and 5-HT2B receptor binding (Numan et al., 1995; Zhang et al., 2008); however, 5-HT2A receptors have been shown to increase in the striatum (Zhang et al., 2008). The possibility exists that striatal 5-HT2A and 5-HT2C receptor are differentially regulated in 6-OHDAlesioned animals and the changes observed in these receptors could be a reflection of the compensatory changes in the PD-afflicted brain. Some of the changes in 5-HT receptor binding are reversible after treatment with l-DOPA, Zhang and colleagues (2008) reported a reversal of increased striatal 5-HT2A receptor mRNA in a 6-OHDA rodent model of PD after l-DOPA treatment. Interestingly, l-DOPA did not alter the changes in striatal 5-HT2C receptor mRNA levels. It appears that changes in regulation of the 5-HT2A receptor are dependent on striatal DA levels and the 5-HT2C loss could be due to nigrostriatal loss, thus reflecting a difference in regulation between the two receptor sub-types. The 5-HT receptor changes seen in PD patients are partly similar to changes in the experimental PD models. Similar increases were seen in the density of 5-HT2A and 5-HT2C receptor in the striatum as well as other regions (Fox and Brotchie, 2000; Huot et al., 2010c; Radja et al., 1993). It is important to note that these changes may not be direct evidence of 5-HT neuropathology but definitely provide an insight into neuroplasticity of the 5-HT system that may unravel potential targets for therapeutic strategies in the treatment of PD.

An indirect marker for 5-HT alterations in PD is the assessment of responses to 5-HT challenge tests. Of these, the most common is the endocrine response to the 5-HT releasing agent, Fenfluramine. In normal subjects Fenfluramine produces robust increases in prolactin and corticosterone levels. However, in PD patients it was found that this endocrine response was impaired (Kostic et al., 1996; Volpi et al., 1997). Such effects may also correlate with non-motor symptoms since PD patients suffering from depression also displayed blunted prolactin responses in comparison to non-depressed PD patients (Kostic et al., 1996). Collectively these findings provide substantial evidence for neurochemical, neuroanatomical and functional alterations of the 5-HT system.
