Acetylcholine Neurotransmitter Receptor Densities in the Striatum of Hemiparkinsonian Rats Following Botulinum Neurotoxin-A Injection

Abstract

Cholinergic neurotransmission has a pivotal function in the caudate-putamen, and is highly associated with the pathophysiology of Parkinson's disease. Here, we investigated long-term changes in the densities of the muscarinic receptor subtypes M₁, M₂, M₃ (mAchRs) and the nicotinic receptor subtype α₄β₂ (nAchRs) in the striatum of the 6-OHDA-induced hemiparkinsonian (hemi-PD) rat model using quantitative in vitro receptor autoradiography. Hemi-PD rats exhibited an ipsilateral decrease in striatal mAchR densities between 6 and 16%. Moreover, a massive and constant decrease in striatal nAchR density by 57% was found. A second goal of the study was to disclose receptor-related mechanisms for the positive motor effect of intrastriatally injected Botulinum neurotoxin-A (BoNT-A) in hemi-PD rats in the apomorphine rotation test. Therefore, the effect of intrastriatally injected BoNT-A in control and hemi-PD rats on mAchR and nAchR densities was analyzed and compared to control animals or vehicle-injected hemi-PD rats. BoNT-A administration slightly reduced interhemispheric differences of mAchR and nAchR densities in hemi-PD rats. Importantly, the BoNT-A effect on striatal nAchRs significantly correlated with behavioral testing after apomorphine application. This study gives novel insights of 6-OHDA-induced effects on striatal mAchR and nAchR densities, and partly explains the therapeutic effect of BoNT-A in hemi-PD rats on a cellular level.

Keywords: Botulinum neurotoxin-A; Parkinson's disease; acetylcholine; basal ganglia; hemiparkinsonian rat model; receptors.

Figure 1

(A,B) Contrast-enhanced color-coded images showing the regional distribution of M₁ receptor density labeled with [³H]pirenzepine in a control rat (A,B) after 6-OHDA lesion (group L6W1M) in a control rat (A) and after 6-OHDA lesion (group L6W1M) (B). (C) Mean receptor density (fmol/mg protein; averaged over all post-lesion survival times) in the left/contralateral (black column) and right/ipsilateral hemispheres (gray column) of the 5 groups: C, control; L, 6-OHDA lesion; B, BoNT-A only; LB, L + BoNT-A; LV. L + vehicle. All data is expressed as means ± SD. (D) Scatter plots and regression analyses of the right-left differences of M₁ receptor density for all 18 groups and time points (five groups with different survival times); see Table 1 for explanation of abbreviations. Significant regressions are labeled by a continuous line.

Figure 2

(A,B) Contrast-enhanced color-coded images showing the regional distribution of M₂ receptor density labeled with the agonist [³H]oxotremorine-M in a control rat (A,B) after 6-OHDA lesion (group L6W1M) in a control rat (A) and after 6-OHDA lesion (group L6W1M) (B). (C) Mean receptor density (fmol/mg protein; averaged over all post-lesion survival times) in the left/contralateral (black column) and right/ipsilateral hemispheres (gray column) of the 5 groups (see Figure 1). All data is expressed as means ± SD. (D) Scatter plots and regression analyses of the right-left differences of M₂ receptor density for all 18 groups and time points (five groups with different survival times); see Table 1 for explanation of abbreviations. Significant regressions are labeled by a continuous line.

Figure 3

(A,B) Contrast-enhanced color-coded images showing the regional distribution of M₂ and M₄ receptor densities receptor density labeled with the antagonist [³H]AF-DX 384 in a control rat (A,B) after 6-OHDA lesion (group L6W6M) in a control rat (A) and after 6-OHDA lesion (group L6W6M) (B). (C) Mean receptor density (fmol/mg protein; averaged over all post-lesion survival times) in the left/contralateral (black column) and right/ipsilateral hemispheres (gray column) of the five groups (see Figure 1). All data is expressed as means ± SD. Asterisks mark significant differences to the respective side of controls (^*p < 0.05, ^***p < 0.001). Rhombs signs highlight interhemispheric significance within each group (^###p < 0.001). (D) Scatter plots and regression analyses of the right-left differences of M₂ receptor density for all 18 groups and time points (five groups with different survival times); see Table 1 for explanation of abbreviations. Significant regressions are labeled by a continuous line.

Figure 4

(A,B) Contrast-enhanced color-coded images showing the regional distribution of M₃ receptor density labeled with [³H]4-DAMP in a control rat (A,B) after 6-OHDA lesion (group L6W1M) in a control rat (A) and after 6-OHDA lesion (group L6W1M) (B). (C) Mean receptor density (fmol/mg protein; averaged over all post-lesion survival times) in the left/contralateral (black column) and right/ipsilateral hemispheres (gray column) of the five groups (see Figure 1). All data is expressed as means ± SD. Asterisks mark significant differences to the respective side of controls (^***p < 0.001). (D) Scatter plots and regression analyses of the right-left differences of M₂ receptor density for all 18 groups and time points (five groups with different survival times); see Table 1 for explanation of abbreviations. Significant regressions are labeled by a continuous line.

Figure 5

(A,B) Contrast-enhanced color-coded images showing the regional distribution of nicotinic α₄β₂ receptor density labeled with [³H]epibatidine in a control rat (A,B) after 6-OHDA lesion (group L6W3M) in a control rat (A) and after 6-OHDA lesion (group L6W3M) (B). (C) Mean receptor density (fmol/mg protein; averaged over all post-lesion survival times) in the left/contralateral (black column) and right/ipsilateral hemispheres (gray column) of the five groups (see Figure 1). All data is expressed as means ± SD. Asterisks mark significant differences to the respective side of controls (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). Rhombs signs highlight interhemispheric significance within each group (^###p < 0.001). (D) Scatter plots and regression analyses of the right-left differences of M₂ receptor density for all 18 groups and time points (five groups with different survival times); see Table 1 for explanation of abbreviations. Significant regressions are labeled by a continuous line. (E) Relationship between interhemispheric differences in the α₄β₂ receptor densities of groups (LB and LV) and rotational behavior (anti-clockwise: +, clockwise: –).