One of the main goals of our study was to see if increased excitation following rotenone intoxication causes dopaminergic cell death. To acquire supporting evidence, in vivo extracellular examination of SNc dopaminergic neurons is an appropriate way. Increased cholinergic input to dopaminergic neurons has been found to be a probable cause for dopaminergic cell death (Fonck C et al. 2003).
In vivo electrophysiological study was done to investigate the neuronal activity of the SN in reponse to HFS of hippocampus in a rat model of PD. To characterize the neuronal activity of the SNc neurons, in vivo extracellular recording was performed and the signals from 125 neurons were successfully collected. These neurons had a high firing rate and irregular firing pattern. Out of 125 neurons detected in the PD rats, there are 45 (R group), 40 (S group), and 40 (Curcumin) neurons.
Figure 1 shows three distinct firing patterns. When we compared the firing properties of the neuronal subtypes in the R group to the Curcumin, we discovered that the firing rates in Curcumin-treated neuronal populations are higher than in the R and S groups. Following that, we looked into the R and S groups. In comparison to the control group (S), the pathology group (R) was suppressed in the low-frequency. When the electrophysiological data was analyzed together (Fig. 1), it was discovered that the firing rate of the Curcumin group of neurons increased in response to HFS (100 Hz). We used subcutaneous dosing (2.5 mg/kg/day) to administer rotenone daily for up to 5 weeks. In the SN, there was a dose-dependent decrease in evoked neural activity and a decrease in firing strength. In the rotenone group, we found more expressed TP PTP responses than in the Curcumin group (Fig. 1). The pathological changes have been shown to have a significant impact on glutamatergic neurons. When compared to the non-lesioned side, glutamatergic neurons on the lesioned side had a distinct difference in cell appearance or quantification (Moran, R. J. et al. 2011). Glutamate-mediated excitotoxicity is thought to play a significant role in neuronal death during degenerative processes, and there is evidence that, under certain conditions, mitochondrial dysfunction may sensitize neurons to glutamate NMDA receptor-mediated excitotoxicity (Calabresi P et al. 2001). We also suggest that the mechanism of rotenone's action on the SN is excitatory. Marsden et al. (2007) demonstrated in rat slices that activation of NMDA receptors, which induces excitatory long-term depression (LTD) via AMPA endocytosis, increases the expression of GABAARs on the dendritic surface of hippocampal neurons (Marsden KC et al). One of the possible explanations for the cell-type-specific vulnerability induced by rotenone in the basal ganglia is the dopamine (DA) dependence of the rotenone-induced neurodegeneration. In fact, it is possible that the high endogenous DA levels present in both the pars compacta of the substantia nigra and the striatum render these structures selectively prone to toxicity induced by mitochondrial complex inhibition. Reduced dopamine levels in striatum and hippocampus were found in rotenone models (Ulusoy GK et al. 2011). Dopamine neurons arising from the ventral tegmental area and SNc contribute during the formation of rewarded behaviors (Bayer HM et al. 2005). Substantia nigra pars compacta (SNc) dopamine neurons are autonomously active; that is, they generate action potentials at a clock-like 2–4 Hz in the absence of synaptic input (Surmeier DJ et al. 2005). In this respect, they are much like cardiac pacemakers. Juvenile dopamine-containing neurons in the SNc use sodium influx as the pacemaking mechanisms common to neurons not affected in PD, but the sodium mechanism remains latent in adulthood (Chan CS et al. 2007). Instead, the autonomous activity is generated by Ca2 + influx. The SNc dopamine neurons rely on L-type Ca(v)1.3 Ca2 + channels (Surmeier DJ 2007). DA neurons fire phasic bursts in response to unpredicted rewards, and their phasic firing begins to track neutral stimuli that predict those rewards. This firing characteristic of DA neurons suggests that they are highly effective at pairing neutral stimuli to unconditioned stimuli, and this property provided evidence that DA signals are a neural substrate of reward prediction (Montague PR et al. 2004). Current Parkinson's disease treatments aim to increase dopamine levels in the striatum in order to alleviate the associated motor deficits. These include dopamine precursors (levodopa), dopamine agonists (pramipexole, ropinirole), and MAO-B inhibitors (selegiline, rasagiline) (Senek M et al. 2014). However, these approaches do not provide a long-term solution because their efficacy diminishes as dopaminergic neurodegeneration progresses. The unsatisfactory effects of traditional antiparkinsonian drugs prompted the search for novel alternatives.
In the view of the hippocampal neural appearance (Fig. 2A), the relatively large cells in this region were observed to be fusiform, medium-small sized cells were round or oval with thinner dendrites. Hippocampal neurons became round or oval, with small soma following 5 weeks Rotenone intoxication.
The results obtained in our study showed signs of neuronal damage at the level of the SNc and Hippocampus in Rotenone-group animals with an observable reduction in the number neurons (Figs. 2, 3). In physiological conditions, the Nissl bodies are big and abundant, showing that the function of neuronal protein synthesis are strong; on the other hand, when nerve cells are damaged, the number of Nissl bodies will be reduced or even disappear. Intraneural Nissl bodies of the SNc and hippocampus are lightly stained and appeared to be sparsely arranged in the Rotenone model group. However, deeper stained Nissl bodies with higher density in SNc and hippocampus neurons were found in the Curcumin group. Previous studies have revealed that the natural phenolic compound curcumin can reduce inflammation and oxidation, which makes it a potential therapeutic agent for neurodegenerative diseases. In this study, we investigated whether curcumin protects against rotenone induced dopaminergic neurotoxicity. Recent studies suggest that Curcumin can protect DA neurons from degeneration in experimental PD (Yu S et al. 2010). Besides, memory performance was also recognized to be greatly increased (Darbinyan L.V.et al. 2017, Song S et al. 2016). Another research recently found that pre-treatment with curcumin accompanied by PQ exposure to PINK1 siRNA cells showed increased mitochondrial membrane capacity and reduced apoptosis (Merwe C et al. 2017). Therefore, in PD treatment, curcumin provides strong promise. Over 70% of SNc input is GABAergic, including afferents from the rostromedial tegmental nucleus (Hong S et al. 2011), striatal patches (Fujiyama F et al. 2011) and SNr (Tepper JM et al. 1995). It also receives glutamatergic afferents from diverse subcortical structures including STN and PPN (Morikawa H et al. 2011). Further, local somatodendritic dopamine release influences both SNc and SNr . GABAergic cells fire more frequently, and evoke greater membrane depolarization (Kajikawa, Y. et al. 2011). The analysis of spike waveform duration was used to classify SNc neurons. GABAergic inputs effectively modulate the firing pattern of dopaminergic neurons in vivo. Local GABA(A) receptor blockade causes dopaminergic neurons to switch to a burst firing pattern, regardless of the original firing pattern. This is accompanied by a slight increase in the rate of spontaneous firing. GABAergic inputs from axonal collaterals of pars reticulata neurons appear to be a particular source of GABA tone for dopaminergic neurons (Tepper JM et al. 2007). Curcumol has been determined to allosterically modulate GABA receptors in a manner distinct from benzodiazepines (Liu, YM. et al. 2017). It promotes GABA-activated current in hippocampal neurons and cell lines expressing endogenous and recombinant GABAARs33, respectively (Ding, J. et al. 2014). In our experiment, after 35 days of curcumin administration, the activity of SNc neurons with depressive type TD responses tended to increase in response to HFS. Curcumin significantly increased the expression of TD and TD PTD responses in the SNc in response to hippocampal HFS compared to the control and Rotenone groups. GABAergic neuron loss was observed in a rat model of Parkinson's disease, which was also thought to be a feature of PD patients (Pienaar I. et al. 2013). It was shown that both acute blockade of D1/5 DAergic receptors (D1/5R) and chronic DA depletion abolish nigral LTD, and that activating D1/5R restores LTD expression in DA-depleted slices. Thus, impairments in DA-dependent adaptations of STN-SNr synapses observed in experimental parkinsonism (Dopamine-Dependent Long-Term Depression at Subthalamo-Nigral Synapses Is Lost in Experimental Parkinsonism (Julien Pierre Dupuis et al. 2013). SNc neurons appeared to have stronger inhibitory response to stimulation frequency and were silenced at 100 Hz in most cases. Higher prevalence of GABA synapses in SNr likely explains why SNr neurons exhibited a greater inhibitory response to electrical stimulation and following Curcumin treatment. We have previously showed that rotenone is a critical player in the host of cellular and synaptic changes (activity-dependent synaptic plasticity) induced in hippocampus by dopamine depletion (Darbinyan L.V. et al. 2017) and Curcumin protects hippocampal neurons against rotenone-induced cell death (Darbinyan L.V. et al. 2017). The above electrophysiological data show that curcumin protected PD animals against Rotenone injury, which histologically might be explained by activation of GABA neurons and signaling pathways. It was also determined that Curcumin significantly attenuated rotenone-induced dopaminergic neuronal oxidative stress-induced injury in the substantia nigra region of rats via the activation of the protein kinase B/nuclear factor erythroid 2-related factor 2 signaling pathway (Cui Q et al. 2016). Curcumin showed a neuroprotective effect against 6-OHDA-induced hippocampus neurons in rats, and the underlying mechanism promoted hippocampal tissue regeneration via activating the brain derived neurotrophic factor/Tropomyosin receptor kinase B pathway (Yang J et al. 2014).
Here, we show that Curcumin administered through the i.p. route provide substantial protection to SN DA neurons in a rotenone rat model of PD. Our study provides evidence for the therapy of Parkinson's disease as well as the underlying mechanism of curcumin's neuroprotective activity. Given the intricacy of molecular and neurological systems, more research is needed to pinpoint the precise process.