**1. Introduction**

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The motor symptoms of Parkinson's disease (PD) are caused by degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc) and the resulting depletion of DA signaling in their target structure, the dorsal striatum (the nigrostriatal pathway). PD motor symptoms are successfully alleviated by systemic administration of a blood-brain barrier permeable DA precursor (levodopa) or DA receptor agonists (Olanow et al. 2001), however these treatments are marred by side-effects in some patients (Wood 2010), and increasingly unreliable response and shortened duration of effect coupled with the emergence of dyskinesias in most patients (Stocchi et al. 2010, Stocchi & Marconi 2010). These problems probably arise from loss of physiological storage, release and reuptake of nigrostriatal DA and ensuing down-stream changes in post-synaptic signaling, and from increased DA signaling in structures outside the nigrostriatal pathway, especially when D2 agonists are used. Targeted (nigrostriatal) reconstruction of physiological DA signaling, aimed at restoring nigrostriatal DA transmission to at least the level at disease outset, ought to alleviate PD motor symptoms without side-effects. However, attempts to achieve this by replacing SNc DA cells through transplantation or endogenous repair are often hampered by poor acquisition and maintenance of the DA phenotype in the microenvironment of the adult SNc (Brundin et al. 2000, Courtois et al. 2010, Torres et al. 2005, Bauer et al. 2000).

There is evidence that expression of tyrosine hydroxylase (TH, the rate-limiting enzyme in DA synthesis) by adult SNc and midbrain neurons is pliable. TH is down-regulated in cells that survive exposure to neurotoxins [6-hydroxy-dopamine (6-OHDA) or 1-methyl-4 phenyl-1,2,3,6-tetrahydropyridine (MPTP)], and can be up-regulated again, presumably in these same cells, by glial-derived neurotrophic factor (GDNF) (Bjorklund *et al.* 1997, Bowenkamp *et al.* 1996, Gash *et al.* 1996, Sauer & Oertel 1994). There is also a degree of spontaneous recovery in the number of TH immunoreactive (TH+) SNc cells following 6- OHDA, which occurs coincidentally with a decrease in the number of SNc cells that are not immunoreactive against TH (TH-) (Stanic *et al.* 2003), implying acquisition of DA phenotype by extant cells. The DA phenotype of SNc neurons also appears to be regulated in an activity-dependent way. Reduced SNc TH expression following striatal infarct is blocked by

Activity-Dependent Regulation of the Dopamine

**2.3 Osmotic minipump infusions** 

procedure.

**2.4 Behavior** 

Phenotype in the Adult Substantia Nigra: Prospects for Treating Parkinson's Disease 279

was sutured and antiseptic applied, an anti-inflammatory [3mg/kg Meloxicam (Metacam®),

Some weeks following the 6-OHDA lesion, when SNc TH+ cell loss was maximal, a cannula [ALZET® (Cupertino, CA, USA) or PlasticsOne® (Roanoke, VA, USA)] was implanted into the midbrain or striatum on the same side as the lesion, through which a drug (or vehicle) was infused continuously via an osmotic pump (ALZET® model #1002 in mice & model #2006 in rats) for a period of time (see Results section for specific timings). To prime the pumps prior to implantation they were filled with drug (or vehicle) and immersed in 37oC sterile saline overnight. The next day animals were anaesthetized and prepared as described above. The cannula was implanted at the following stereotaxic coordinates: (1) For SNc infusions in mice 3.0mm posterior to Bregma, 1.5mm lateral to the midline, 4.0mm deep; (2) For SNc infusions in rats 3.7mm anterior to Lambda, 1.9mm lateral to the midline, 7.8mm deep; (3) For striatal infusions in rats 0.5mm anterior to Bregma, 3.0mm lateral, 3.5mm deep. The cannula was glued to the skull with dental cement and the attached pump was placed

In rat experiments the period of drug delivery was extended beyond the lifetime (6 weeks) of the first implanted pump by replacing it with a new filled pump. This was done by anaesthetizing the rat with 1-2% isofluorane in air, making a small incision in the skin overlying the old pump, clamping the vinyl tube closed, removing the old pump (and flow moderator), replacing it with a new primed pump (and new flow moderator), releasing the clamp and suturing the skin. The implanted cannula remained undisturbed throughout this

The behavioral studies described below were performed on cohorts of animals typically comprising 12 animals, 6 treated with drug and 6 with vehicle. Each behavioral test was performed at the same time of day within each cohort and each animal was studied concurrently. If availability of equipment prevented this, animals were studied consecutively in a different pseudo-randomized order and equipment was thoroughly

Animals were injected intraperitonealy with 5mg/kg amphetamine and immediately placed into a cylindrical chamber (17cm diameter by 17cm high for mice & 31cm diameter by 31cm high for rats). In some rat experiments rotational behavior was also assessed for 20mins. prior to amphetamine. The behavior of each animal in its chamber was recorded on videotape for at least 1hour following amphetamine. On days where amphetamine was

Rotational behavior was measured off-line using Ethovision® XT animal tracking software (Noldus Information Technology, Wageningen, Netherlands). One rotation was defined as a cumulative 360o change in heading direction of the animal's centre of mass, with no time

cleaned with 80% ethanol after each animal to remove any distracting odors.

**2.4.1 Rotational response to amphetamine** 

administered, no other behavioral tests were performed.

s.c.] was administered, and the animal was left to recover in a warmed cage.

in a subcutaneous 'pocket' created in the interscapular region.

intraventricular administration of a GABAA receptor agonist (Soriano *et al.* 1997, Yamada *et al.* 1996); SNc TH mRNA is elevated followed systemic administration of nicotine or α-7 nicotinic acetylcholine receptor agonists (Serova & Sabban 2002); and we recently reported that the number of SNc TH+ cells in adult mice can be increased or decreased by direct brain infusions of drugs targeting SNc neuronal activity (Aumann *et al.* 2011, Aumann *et al.* 2008). These data are consistent with the possibility that the DA phenotype of adult SNc neurons can be gained or lost in an activity-dependent way.

Understanding how the DA phenotype of neurons in the adult SNc is regulated will highlight strategies and molecular (drug) targets to facilitate DA phenotype acquisition and maintenance by either transplanted or endogenous cells. The aims of the experiments described in this report were to determine whether infusions of drugs that increase the number of SNc DA (TH+) cells in normal mice: (1) increase the number of these cells in a 6- OHDA rodent model of PD; and (2) restore motor deficits.
