Addiction Research

Introduction
Parkinson's disease (PD), originally described in 1817 by James Parkinson, is currently regarded as the most common degenerative disorder of the ageing brain after the Alzheimer's dementia. It is characterized by muscle rigidity, tetrad of tremor at rest, postural instability, bradykinesia and in extreme cases akinesia [1]. The symptoms are the results of decreased stimulation of the motor cortex by the basal ganglia, normally caused by the insufficient formation and action of dopamine, which is produced in the dopaminergic neurons of the substantia nigra pars compacta (SNpc) of brain.

PD patients are characterized by systemic mitochondrial dysfunction, marked by inhibition of complex I of the mitochondrial electron transport chain. To model the systemic defect in complex I reported in PD, researchers have used rotenone exposure. Rotenone is a commonly used pesticide and potent, specific inhibitor of mitochondrial complex I. Rotenone because of its lipophilic nature, crosses biological membranes easily and independent of transporters. As a result, systemic rotenone exposure inhibits complex I uniformly throughout brain [2]. Unilateral infusion of rotenone reproduces neurochemical and neuropathological features of hemi-Parkinsonism in rats and indicates an active involvement of oxidative stress in rotenone induced nigrostriatal neurodegeneration [3].

Dopamine (DA), one of the major neurotransmitter in central nervous system is involved in the control of motor and cognitive functions [4]. Dopamine supplied as medication cannot cross the blood-brain barrier [5]. Targeting neurotransmitter systems beyond the dopamine system is an interesting approach, both for the motor and nonmotor problems of PD [6]. Non-dopaminergic neurotransmission is also affected in PD. The dysfunction of nondopaminergic systems explains the principal non-dopaminergic symptoms, such as ‘axial’ signs and cognitive impairment. The non-dopaminergic neurotransmitters affected in PD are noradrenaline (norepinephrine), serotonin (5-hydroxytryptamine; 5-HT), glutamate, gamma-aminobutyric acid (GABA), acetylcholine and neuropeptides [7]. Dysfunction of these systems can lead to some of the motor symptoms of the disease and provide targets for pharmacological interventions to treat these symptoms. Accordingly, alterations in serotonergic function accounts for behavioural disturbances commonly observed during PD. Recent advances in understanding the role of 5-HT in Parkinsonism and the generation of side-effects of dopamine replacement therapy (e.g. wearing-off and levodopa-induced dyskinesia) have identified 5-HT1A, 5-HT1B and 5-HT2C receptors as potential therapeutic targets in Parkinson's disease [8].

Although Parkinson's disease is characterized primarily by loss of nigrostriatal dopaminergic neurons, there is a concomitant loss of norepinephrine neurons in the locus coeruleus (LC). Norepinephrine is important for regulating the activity of dopamine neurons. The dopamine neurons and the norepinephrine neurons function in concert. As the dopamine neurons start dying, the norepinephrine neurons compensate by signalling the surviving dopamine cells to dramatically increase their activity and the output of dopamine. Results of Rommelfanger et al. [9] indicate that having a normal complement of dopamine neurons is not enough for normal motor function but that norepinephrine also needs to be present to ensure proper dopamine release. Rommelfanger and Weinshenker [10] examined the evidence that NE is neuroprotective and that LC degeneration sensitizes DA neurons to damage. In patients with Parkinson's disease, the cerebellar cortical norepinephrine levels were significantly below normal [11]. Altered norepinephrine metabolism contributes to some aspects of intellectual dysfunction in PD [12-14]. Restoring NE can have important therapeutic potential in PD.

In the present work, the effects of 5-HT, DA and NE supplementation intranigrally to substantia nigra as treatment individually on rotenone induced Hemi-Parkinson’s disease in rats were analyzed. Dopaminergic D1 and D2 binding parameters investigated its role in the regulation of dopamine receptor subtypes in the cerebral cortex of the experimental rats. Real-Time PCR work was done to confirm the binding parameters.

Experimental and Methods
Animals
Experiments were carried out on adult male Wistar rats of 250- 300g body weight purchased from Kerala Agricultural University, Mannuthy. They were housed in separate cages under 12 hrs light and 12 hrs dark periods and were maintained on standard food pellets and water ad libitum. Adequate measures were also taken to minimize pain and discomfort of the animals. All animal care and procedures were taken in accordance with the Institutional, National Institute of Health guidelines and CPCSEA guidelines.

Chemicals
Biochemicals
Rotenone, dopamine, 5-hydroxy tryptamine, norepinephrine, ascorbic acid, pargyline, calcium chloride, sulpiride, SCH 23390, amphetamine and apomorphine were purchased from Sigma Chemical Co., St. Louis, MI, USA. All other reagents were of analytical grade purchased locally.

Radiochemicals
[3H] SCH 23390 (Sp. activity 83Ci/mmol), [3 H]YM-09151-2((Sp. activity 85Ci/mmol) and [3 H] Dopamine (Sp. activity 45Ci/mmol) were purchased from NEN Life Sciences Products, Inc. Boston, USA.

Molecular Biology Chemicals
Tri-reagent kit was purchased from Sigma Chemical Co., St. Louis, MI, USA. ABI PRISM High Capacity cDNA Archive kit, Primers and Taqman probes for Real-Time PCR - Dopamine D1 (Rn_02043440), Dopamine D2 (Rn_00561126) endogenous control (β-actin) were purchased from Applied Biosystems, Foster City, CA, USA.

Experimental Design
The experimental rats were divided into the following groups i) Control ii) Rotenone infused (Rotenone) iii) Rotenone infused supplemented with DA (Rotenone + DA) iv) Rotenone infused supplemented with 5-HT (Rotenone + 5-HT) v) Rotenone infused supplemented with NE (Rotenone + NE). Each group consisted of 6-8 animals.

Unilateral intranigral infusion of Rotenone
Rats were anesthetized with urethane (100 mg/100g body weight. i.p.). The animal was placed in the flat skull position on a cotton bed on a stereotaxic frame (BenchmarkTM, USA) with incisor bar fixed at 3.5 mm below the interaural line. Rotenone dissolved in DMSO: PEG (1:1) was infused 1μl into the right SNpc at a flow rate of 0.2μl/min. After stopping the infusion of the toxin, the probe was kept in the same position for a further 5 min for complete diffusion of the drug and then slowly retracted. The stereotaxic coordinates for SNpc were: lateral (L) =+0.20 cm, antero-posterior (AP, from the bregma point) =−0.53 cm and dorsi-ventral (DV) = +0.75 cm. The stereotaxic co-ordinates were calculated for the dopaminergic neuronal cell body region, SNpc following the “Rat Brain Atlas” [15]. All the groups except Control group were infused with Rotenone and in control animals, 1 μl of the vehicle (DMSO: PEG (1:1)) was infused into the right SNpc. Proper postoperative care was provided till the animals recovered completely.

Rotational Behavior
Amphetamine-induced rotational behavior was assessed as described earlier [16,17]. Rats were tested on the 14th day after intranigral injection of Rotenone. After acclimatization for at least 10 min, amphetamine (5 mg/kg, i.p.) was administered. Animals that had completed a 360â?¦ circle towards the intact (contralateral) side and the lesioned (ipsilateral) side were counted for 240 min continuously and recorded separately. Rats were tested on the 16th day following intranigral injection of Rotenone. After acclimatization in the experimental cage for at least 10 min, apomorphine (1 mg/kg, s.c.) was administered. Animals that had completed a 360â?¦ circle towards the intact (contralateral) and the lesioned (ipsilateral) sides were counted for 60 min continuously and recorded separately. Animals showing more than 350 contralateral rotations during the initial 1 hr were separated on the 16th day. (Animals that showed no significant contralateral rotations were excluded from the study).

Treatment
Animals in group iii, iv, v were anaesthetized with urethane (100 mg/100g body weight. i.p) on the 18th day, and Stereotaxic single dose of 1µl of DA (10µg/µl), 5-HT (10µg/µl) and NE (10µg/µl) was infused into the right SNpc at a flow rate of 0.2 μl/min into the respective groups. The stereotaxic co-ordinates are: lateral (L) =+0.20 cm, antero-posterior (AP) =−0.53 cm and dorsi-ventral (DV) = +0.75 cm from the bregma point. After stopping the infusion, the probe was kept in the same position for a further 5 min for complete diffusion of the drug and then slowly retracted.

Tissue Preparation
All the control and experimental rats were sacrificed on the 25th day by decapitation. The brain regions and body parts were dissected out quickly over ice [18] and the tissues were stored at -700C for various experiments. All animal care and procedures were in accordance with Institutional and National Institute of Health guidelines.

Dopamine Receptor Binding Studies Using [3 H]Radioligands In the Brain Regions of Control and Experimental Rats
Dopamine receptor binding studies using [3H] Dopamine
DA receptor assay was done using [3H]DA according to Madras et al. Cerebral cortex were homogenised in a polytron homogeniser with 20 volumes of cold 50mM Tris-HCl buffer, along with 1mM EDTA, 0.01%ascorbic acid, 4mM MgCl2 , 1.5mM CaCl2 , pH. 7.4 and centrifuged at 38,000 x g for 30min at 4°C. The pellet was washed twice by rehomogenization and centrifuged twice at 38,000 x g for 30min at 4°C. This was resuspended in appropriate volume of the buffer containing the above mentioned composition.

Binding assays were done using different concentrations i.e., 0.25nM-1.5nM of [3 H]DA in 50mM Tris-HCl buffer, along with 1mM EDTA, 0.01% ascorbic acid, 1mM MgCl2 , 2mM CaCl2 , 120mM NaCl, 5mM KCl, pH.7.4 in a total incubation volume of 250µl containing 200-300µg of proteins. Specific binding was determined using 100µM unlabelled dopamine. Tubes were incubated at 25°C for 60 min. and filtered rapidly through GF/B filters (Whatman). The filters were washed quickly by three successive washing with 5.0ml of ice cold 50mM Tris buffer, pH 7.4. Bound radioactivity was counted with cocktail-T in a Wallac 1409 liquid scintillation counter.

Dopamine D1 Receptor Binding Studies Using [3H]SCH 23390
Dopamine D1 receptor binding assay using [3 H]SCH 23390 in the cerebral cortex were done [19]. The tissues were weighed and homogenized in 10 volumes of ice cold 50mM Tris-HCl buffer, along with 1mM EDTA, 4mM MgCl2 , 1.5mM CaCl2 , 5mM KCl, pH 7.4. The homogenate was centrifuged at 40,000 x g for 30min. The pellet was washed and centrifuged with 50 volumes of the buffer at 40,000 x g for 30min. This was suspended in appropriate volume of the buffer containing the above mentioned composition.

Binding assays were done using different concentrations i.e., 0.5 - 5.0nM of [3 H]SCH 23390 in 50mM Tris-HCl buffer (pH 7.4), along with 1mM EDTA, 4mM MgCl2 , 1.5 mM CaCl2 , 5mM KCl with 12µM pargyline and 0.1% ascorbic acid in a total incubation volume of 250µl containing 150-200µg protein. Specific binding was determined using 50µM unlabelled SCH 23390. Competition studies were carried out with 1.0nM [3 H]SCH 23390 in each tube with unlabelled ligand concentrations varying from 10-9-10-4 M of SCH 23390.

Tubes were incubated at 25°C for 60 min. and filtered rapidly through GF/B filters. The filters were washed quickly by three successive washing with 5.0ml of ice cold 50mM Tris buffer, (pH 7.4). Bound radioactivity was counted with cocktail-T in a Wallac 1409 liquid scintillation counter.

Dopamine D2 receptor binding studies using [3H]YM-09151-2
Dopamine D2 receptor binding assay was done according to the modified procedure of Madras et al., Unis et al. [20,21]. The dissected Cerebral Cortex was weighed and homogenised in 10 volumes of ice cold 50mM Tris-HCl buffer, pH.7.4 along with 1mM EDTA, 5mM MgCl2 , 1.5mM CaCl2 , 120mM NaCl and 5mM KCl. The homogenate was centrifuged at 40,000xg for 30 min. The pellet was washed and centrifuged with 50 volumes of the buffer at 40,000xg for 30 min. This was suspended in appropriate volume of the buffer containing the above mentioned composition.

Binding assays were done using different concentrations i.e., 0.1 - 2.0nM of [3 H]YM-09151-2 in 50mM Tris-HCl buffer, pH 7.4, along with 1mM EDTA, 5mM MgCl2 , 1.5mM CaCl2 , 120mM NaCl, 5mM KCl, 10µM pargyline and 0.1% ascorbic acid in a total incubation volume of 250µl containing 150-200µg of protein. Specific binding was determined using 5.0 µM unlabelled sulpiride.

Tubes were incubated at 25°C for 60 min. and filtered rapidly through GF/B filters (Whatman). The filters were washed quickly by three successive washing with 5.0 ml of ice cold 50mM Tris buffer, pH 7.4. Bound radioactivity was counted with cocktail-T in a Wallac 1409 liquid scintillation counter.

Protein Determination
Protein was measured according to Lowry et al. [22] using bovine serum albumin as standard. The intensity of the purple blue colour formed was proportional to the amount of protein which was read in Spectrophotometer at 660nm.

Analysis of the Receptor Binding Data
Linear Regression Analysis for Scatchard Plots
The data was analysed by Scatchard et al. [23]. The specific binding was determined by subtracting non-specific binding from the total. The binding parameters, maximal binding (Bmax) and equilibrium dissociation constant (Kd ), were derived by linear regression analysis by plotting the specific binding of the radioligand on X-axis and bound/free on Y-axis. The maximal binding is a measure of the total number of receptors present in the tissue and the equilibrium dissociation constant is the measure of the affinity of the receptors for the radioligand. The Kd is inversely related to receptor affinity.

Gene Expression Studies of Dopamine Receptor Subtypes in Cerebral Cortex of Control and Experimental Rats Preparation of RNA
RNA was isolated from the different brain regions of control and experimental rats using the Tri reagent from Sigma Chemical Co., St. Louis, MI, USA.

Isolation of RNA
Tissue (25-50mg) homogenates were made in 0.5ml Tri Reagent and was centrifuged at 12,000 x g for 10 minutes at 4°C. The clear supernatant was transferred to a fresh tube and it was allowed to stand at room temperature for 5min. 100µl of chloroform was added to it, mixed vigorously for 15sec and allowed to stand at room temperature for 15min. The tubes were then centrifuged at 12,000 x g for 15min at 4°C. Three distinct phases appear after centrifugation. The bottom red organic phase contained protein, interphase contained DNA and a colourless upper aqueous phase contained RNA. The upper aqueous phase was transferred to a fresh tube and 250μl of isopropanol was added and the tubes were allowed to stand at room temperature for 10min. The tubes were centrifuged at 12,000 x g for 10min at 4°C. RNA precipitate forms a pellet on the sides and bottom of the tube. The supernatants were removed and the RNA pellet was washed with 500µl of 75% ethanol, vortexed and centrifuged at 12,000 x g for 5 min at 4°C. The pellets were semi dried and dissolved in minimum volume of DEPC-treated water. 2μl of RNA was made up to 1ml and absorbance was measured at 260nm and 280nm. For pure RNA preparation the ratio of absorbance at 260/280 was 1.7. The concentration of RNA was calculated as one absorbance 260 = 42µg.