Associated degeneration of ventral tegmental area dopaminergic neurons in the rat nigrostriatal lactacystin model of parkinsonism and their neuroprotection by valproate

Abstract

Parkinson's disease (PD) manifests clinically as bradykinesia, rigidity, and development of a resting tremor, primarily due to degeneration of dopaminergic nigrostriatal pathways in the brain. Intranigral administration of the irreversible ubiquitin proteasome system inhibitor, lactacystin, has been used extensively to model nigrostriatal degeneration in rats, and study the effects of candidate neuroprotective agents on the integrity of the dopaminergic nigrostriatal system. Recently however, adjacent extra-nigral brain regions such as the ventral tegmental area (VTA) have been noted to also become affected in this model, yet their integrity in studies of candidate neuroprotective agents in the model have largely been overlooked. Here we quantify the extent and distribution of dopaminergic degeneration in the VTA of rats intranigrally lesioned with lactacystin, and quantify the extent of VTA dopaminergic neuroprotection after systemic treatment with an epigenetic therapeutic agent, valproate, shown previously to protect dopaminergic SNpc neurons in this model. We found that unilateral intranigral administration of lactacystin resulted in a 53.81% and 31.72% interhemispheric loss of dopaminergic SNpc and VTA neurons, respectively. Daily systemic treatment of lactacystin lesioned rats with valproate however resulted in dose-dependant neuroprotection of VTA neurons. Our findings demonstrate that not only is the VTA also affected in the intranigral lactacystin rat model of PD, but that this extra-nigral brain region is substrate for neuroprotection by valproate, an agent shown previously to induce neuroprotection and neurorestoration of SNpc dopaminergic neurons in this model. Our results therefore suggest that valproate is a candidate for extra-nigral as well as intra-nigral neuroprotection.

Keywords: Dopamine; Histone deacetylase inhibitor; Lactacystin; Parkinson’s disease; Substantia Nigra pars compacta; Valproate; Ventral tegmental area.

Fig. 1

Animal study design. *Lacta(+) Week 1, Lacta(+)VPA(−), Lacta(+)VPA(+) and Lacta(+)VPA(++) groups were intranigrally injected with lactacystin. Control groups (Lacta(−)VPA(−) and Lacta(−)VPA(++)) remained surgically naïve.

Fig. 2

Lactacystin induced loss of TH+ and Nissl+ cell loss in the VTA. (A) Representative image of TH immunohistochemistry within the brain showing proximity and discrimination of the SNpc from the VTA. Mean percentage interhemispheric (ipsilateral lesioned vs. contralateral non-lesioned hemisphere) loss of (B) TH+ cells and (C) Nissl+ cells in the SNpc and VTA of lactacystin (10 μg) lesioned animals, 1 and 5 weeks post-lesion. Distribution of (D) TH+ and (E) Nissl+ cell loss at each rostrocaudal level of the VTA in intranigral lactacystin lesioned animals, 5 weeks post-lesion. Each level is defined by its approximate distance caudal to bregma. Dotted line represents the level of the intranigral injection of lactacystin. Data presented as mean ± SEM. Statistical significance indicated with asterisks: *p < 0.05, **p < 0.01. n = 6/7 per group. Scale bar equal to 500 μm. Abbreviations: iSNpc, ipsilateral Substantia Nigra pars compacta; cSNpc, contralateral Substantia Nigra pars compacta; iVTA, ipsilateral ventral tegmental area; cVTA, contralateral central ventral tegmental area.

Fig. 3

Micrographs demonstrating loss and protection of TH+ cells in the VTA. Representative example images of the TH and Nissl stained VTA in each treatment group: (A) Lacta(+) Week1; (B) Lacta(−)VPA(−); (C) Lacta(−)VPA(++); (D) Lacta(+)VPA(−); (E) Lacta(+)VPA(+); (F) Lacta(+)VPA(++). All sections are approximately −5.8 mm caudal to bregma. Scale bar equal to 500 μm.

Fig. 4

Valproate treatment causes dose-dependant protection of dopaminergic neurons in the VTA in lactacystin lesioned animals. Stereological estimated (A) TH+ and (B) Nissl+ neuron numbers in the VTA of rats suggest a dose-dependant neuroprotective/restorative effect of valproate in the intranigral lactacystin (10 μg) rat model of Parkinson’s disease. This is exemplified by the percentage interhemispheric loss of TH+ (C) and Nissl+ (D) neurons calculated between hemispheres of the VTA. Data presented as mean ± SEM. Statistical significance indicated with asterisks: *p < 0.05; **p < 0.01, ***p < 0.001. n = 6/7 per group.