α4 nicotinic receptors on GABAergic neurons mediate a cholinergic analgesic circuit in the substantia nigra pars reticulata.

Nicotinic acetylcholine receptors (nAChRs) regulate pain pathways with various outcomes depending on receptor subtypes, neuron types, and locations. But it remains unknown whether α4ß2 nAChRs abundantly expressed in the substantia nigra pars reticulata (SNr) have potential to mitigate hyperalgesia in pain states. We observed that injection of nAChR antagonists into the SNr reduced pain thresholds in naïve mice, whereas injection of nAChR agonists into the SNr relieved hyperalgesia in mice, subjected to capsaicin injection into the lower hind leg, spinal nerve injury, chronic constriction injury, or chronic nicotine exposure. The analgesic effects of nAChR agonists were mimicked by optogenetic stimulation of cholinergic inputs from the pedunculopontine nucleus (PPN) to the SNr, but attenuated upon downregulation of α4 nAChRs on SNr GABAergic neurons and injection of dihydro-ß-erythroidine into the SNr. Chronic nicotine-induced hyperalgesia depended on α4 nAChRs in SNr GABAergic neurons and was associated with the reduction of ACh release in the SNr. Either activation of α4 nAChRs in the SNr or optogenetic stimulation of the PPN-SNr cholinergic projection mitigated chronic nicotine-induced hyperalgesia. Interestingly, mechanical stimulation-induced ACh release was significantly attenuated in mice subjected to either capsaicin injection into the lower hind leg or SNI. These results suggest that α4 nAChRs on GABAergic neurons mediate a cholinergic analgesic circuit in the SNr, and these receptors may be effective therapeutic targets to relieve hyperalgesia in acute and chronic pain, and chronic nicotine exposure.

Reversal of hyperactive subthalamic circuits differentially mitigates pain hypersensitivity phenotypes in parkinsonian mice.

Although pain is a prevalent nonmotor symptom in Parkinson's disease (PD), it is undertreated, in part because of our limited understanding of the underlying mechanisms. Considering that the basal ganglia are implicated in pain sensation, and that their synaptic outputs are controlled by the subthalamic nucleus (STN), we hypothesized that the STN might play a critical role in parkinsonian pain hypersensitivity. To test this hypothesis, we established a unilateral parkinsonian mouse model with moderate lesions of dopaminergic neurons in the substantia nigra. The mice displayed pain hypersensitivity and neuronal hyperactivity in the ipsilesional STN and in central pain-processing nuclei. Optogenetic inhibition of STN neurons reversed pain hypersensitivity phenotypes in parkinsonian mice, while hyperactivity in the STN was sufficient to induce pain hypersensitivity in control mice. We further demonstrated that the STN differentially regulates thermal and mechanical pain thresholds through its projections to the substantia nigra pars reticulata (SNr) and the internal segment of the globus pallidus (GPi)/ventral pallidum (VP), respectively. Interestingly, optogenetic inhibition of STN-GPi/STN-VP and STN-SNr projections differentially elevated mechanical and thermal pain thresholds in parkinsonian mice. In summary, our results support the hypothesis that the STN and its divergent projections play critical roles in modulating pain processing under both physiological and parkinsonian conditions, and suggest that inhibition of individual STN projections may be a therapeutic strategy to relieve distinct pain phenotypes in PD.

Differential modulation of subthalamic projection neurons by short-term and long-term electrical stimulation in physiological and parkinsonian conditions.

The subthalamic nucleus (STN) is one of the best targets for therapeutic deep brain stimulation (DBS) to control motor symptoms in Parkinson's disease. However, the precise circuitry underlying the effects of STN-DBS remains unclear. To understand how electrical stimulation affects STN projection neurons, we used a retrograde viral vector (AAV-retro-hSyn-eGFP) to label STN neurons projecting to the substantia nigra pars reticulata (SNr) (STN-SNr neurons) or the globus pallidus interna (GPi) (STN-GPi neurons) in mice, and performed whole-cell patch-clamp recordings from these projection neurons in ex vivo brain slices. We found that STN-SNr neurons exhibited stronger responses to depolarizing stimulation than STN-GPi neurons. In most STN-SNr and STN-GPi neurons, inhibitory synaptic inputs predominated over excitatory inputs and electrical stimulation at 20-130 Hz inhibited these neurons in the short term; its longer-term effects varied. 6-OHDA lesion of the nigrostriatal dopaminergic pathway significantly reduced inhibitory synaptic inputs in STN-GPi neurons, but did not change synaptic inputs in STN-SNr neurons; it enhanced short-term electrical-stimulation-induced inhibition in STN-SNr neurons but reversed the effect of short-term electrical stimulation on the firing rate in STN-GPi neurons from inhibitory to excitatory; in both STN-SNr and STN-GPi neurons, it increased the inhibition but attenuated the enhancement of firing rate induced by long-term electrical stimulation. Our results suggest that STN-SNr and STN-GPi neurons differ in their synaptic inputs, their responses to electrical stimulation, and their modification under parkinsonian conditions; STN-GPi neurons may play important roles in both the pathophysiology and therapeutic treatment of Parkinson's disease.

D2 receptor activation relieves pain hypersensitivity by inhibiting superficial dorsal horn neurons in parkinsonian mice.

Chronic pain is a common and undertreated nonmotor symptom in Parkinson's disease (PD). Although chronic pain is improved by L-dopa in some PD patients, the underlying mechanisms remain unclear. In this study, we established PD mice by unilateral microinjection of 6-OHDA in the medial forebrain bundle to investigate the contribution of spinal cord dopamine receptors to parkinsonian pain hypersensitivity. The von Frey filament tests and thermal pain tests revealed that these PD mice displayed decreased nociceptive thresholds in both hindpaws; intrathecal injection of L-dopa or apomorphine significantly increased the mechanical and thermal nociceptive thresholds, and the analgesic effect was mimicked by ropinirole (a D2 receptor agonist), but not SKF38393 (a D1/D5 receptor agonist), and blocked by sulpiride (a D2 receptor antagonist), but not SKF83566 (a D1/D5 receptor antagonist). Whole-cell recordings in lumber spinal cord slices showed that superficial dorsal horn (SDH) neurons in PD mice exhibited hyperexcitability, including more depolarized resting membrane potentials and more action potentials evoked by depolarizing current steps, which were mitigated by ropinirole. Furthermore, ropinirole inhibited the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in SDH neurons more strongly in PD mice than in control mice. However, sulpiride caused less disinhibition of sEPSCs in PD mice than in control mice. Taken together, our data reveal that pain hypersensitivity in PD mice is associated with hyperexcitability of SDH neurons, and both events are reversed by activation of spinal D2 receptors. Therefore, spinal D2 receptors can be promising therapeutic targets for the treatment of PD pain.

A nigra-subthalamic circuit is involved in acute and chronic pain states.

ABSTRACT: The basal ganglia modulate somatosensory pain pathways, but it is unclear whether a common circuit exists to mitigate hyperalgesia in pain states induced by peripheral nociceptive stimuli. As a key output nucleus of the basal ganglia, the substantia nigra pars reticulata (SNr) may be a candidate for this role. To test this possibility, we optogenetically modulated SNr GABAergic neurons and examined pain thresholds in freely behaving male mice in inflammatory and neuropathic pain states as well as comorbid depression in chronic pain. We observed that stimulation of either SNr GABAergic neurons or their projections to the subthalamic nucleus (STN) significantly alleviated nociceptive responses in all pain states on the contralateral side and comorbid depression in chronic pain, and that this analgesic effect was eliminated when SNr-STN GABAergic projection was blocked. However, SNr modulation did not affect baseline pain thresholds. We also found that SNr-STN GABAergic projection was attenuated in pain states, resulting in disinhibition of STN neurons. Thus, impairment of the SNr-STN GABAergic circuit may be a common pathophysiology for the maintenance of hyperalgesia in both inflammatory and neuropathic pain states and the comorbid depression in chronic pain; compensating this circuit has potential to effectively treat pain related conditions.

Internal States Influence the Representation and Modulation of Food Intake by Subthalamic Neurons.

Deep brain stimulation of the subthalamic nucleus (STN) is an effective therapy for motor deficits in Parkinson's disease (PD), but commonly causes weight gain in late-phase PD patients probably by increasing feeding motivation. It is unclear how STN neurons represent and modulate feeding behavior in different internal states. In the present study, we found that feeding caused a robust activation of STN neurons in mice (GCaMP6 signal increased by 48.4% ± 7.2%, n = 9, P = 0.0003), and the extent varied with the size, valence, and palatability of food, but not with the repetition of feeding. Interestingly, energy deprivation increased the spontaneous firing rate (8.5 ± 1.5 Hz, n = 17, versus 4.7 ± 0.7 Hz, n = 18, P = 0.03) and the depolarization-induced spikes in STN neurons, as well as enhanced the STN responses to feeding. Optogenetic experiments revealed that stimulation and inhibition of STN neurons respectively reduced (by 11% ± 6%, n = 6, P = 0.02) and enhanced (by 36% ± 15%, n = 7, P = 0.03) food intake only in the dark phase. In conclusion, our results support the hypothesis that STN neurons are activated by feeding behavior, depending on energy homeostatic status and the palatability of food, and modulation of these neurons is sufficient to regulate food intake.

Galantamine reversed early postoperative cognitive deficit via alleviating inflammation and enhancing synaptic transmission in mouse hippocampus.

Postoperative cognitive dysfunction (POCD) is commonly seen in patients undergoing major surgeries and may persist. Although neuroinflammation is one of the important contributors to the development of POCD, the mechanisms underlying POCD remain unclear. We performed stabilized tibial fracture operation in male mice. In comparison with sham mice (anesthesia only), the surgery mice exhibited cognitive deficits in a fear conditioning paradigm at postsurgery day 3-7, and increased numbers of microglia and elevated levels of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) without change of anti-inflammatory cytokines (IL-4 and IL-10) in the hippocampus. Electrophysiological recordings from CA1 hippocampal neurons revealed that POCD mice exhibited impairment in AMPA receptor-mediated evoked excitatory postsynaptic currents (eEPSCs) without alteration in the rectification property of AMPA receptors. Interestingly, daily intraperitoneal administration of galantamine, an inhibitor of acetylcholinesterase, reversed cognitive dysfunction in surgery mice and attenuated accumulation of microglia and protein levels of IL-1ß, IL-6 and TNF-α in the hippocampus. Additionally, galantamine potentiated AMPA receptor-mediated eEPSCs in the hippocampus more prominent in surgery mice than in sham mice. Therefore, enhancement of cholinergic tone in the hippocampus might be a therapeutic strategy for early POCD in terms of suppression of inflammation and normalization of excitatory synaptic transmission.

Determination of Simvastatin in human plasma by liquid chromatography-mass spectrometry.

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for the determination of simvastatin (I) has been developed. After extraction by ethyl acetate, using lovastatin (II) as internal standard, solutes are separated on a C(18) column with a mobile phase consisting of methanol-water (9:1). Detection is performed on an atmospheric pressure ionization single quadruple mass spectrometer equipped with an ESI interface and operates in positive ionization mode. Simvastatin quantification was realized by computing peak area ratio (I/II) of the extracts analyzed in SIM mode (m/z: 441 and m/z: 427 for I and II, respectively) and comparing them with calibration curve (r=0.9997). Accuracy and precision for the assay were determined by calculating the intra-batch and inter-batch variation at three concentrations 0.1, 5.0, 10.0 ng/ml; the intra batch relative standard deviation (RSD) was less than 10% and ranged from 1.8 to 8.5%, respectively; the inter-batch RSD was less than 20% and ranged from 4.1 to 16.5%. The limit of detection was 0.05 ng/ml.