Mu Opioid Receptor Modulation of Dopamine Neurons in the Periaqueductal Gray/Dorsal Raphe: A Role in Regulation of Pain

The periaqueductal gray (PAG) is a brain region involved in nociception modulation, and an important relay center for the descending nociceptive pathway through the rostral ventral lateral medulla. Given the dense expression of mu opioid receptors and the role of dopamine in pain, the recently characterized dopamine neurons in the ventral PAG (vPAG)/dorsal raphe (DR) region are a potentially critical site for the antinociceptive actions of opioids. The objectives of this study were to (1) evaluate synaptic modulation of the vPAG/DR dopamine neurons by mu opioid receptors and to (2) dissect the anatomy and neurochemistry of these neurons, in order to assess the downstream loci and functions of their activation. Using a mouse line that expresses eGFP under control of the tyrosine hydroxylase (TH) promoter, we found that mu opioid receptor activation led to a decrease in inhibitory inputs onto the vPAG/DR dopamine neurons. Furthermore, combining immunohistochemistry, optogenetics, electrophysiology, and fast-scan cyclic voltammetry in a TH-cre mouse line, we demonstrated that these neurons also express the vesicular glutamate type 2 transporter and co-release dopamine and glutamate in a major downstream projection structure—the bed nucleus of the stria terminalis. Finally, activation of TH-positive neurons in the vPAG/DR using Gq designer receptors exclusively activated by designer drugs displayed a supraspinal, but not spinal, antinociceptive effect. These results indicate that vPAG/DR dopamine neurons likely play a key role in opiate antinociception, potentially via the activation of downstream structures through dopamine and glutamate release

Disruption of Glucagon-Like Peptide 1 Signaling in Sim1 Neurons Reduces Physiological and Behavioral Reactivity to Acute and Chronic Stress

Organismal stress initiates a tightly orchestrated set of responses involving complex physiological and neurocognitive systems. Here, we present evidence for glucagon-like peptide 1 (GLP-1)-mediated paraventricular hypothalamic circuit coordinating the global stress response. The GLP-1 receptor (Glp1r) in mice was knocked down in neurons expressing single-minded 1, a transcription factor abundantly expressed in the paraventricular nucleus (PVN) of the hypothalamus. Mice with single-minded 1-mediated Glp1r knockdown had reduced hypothalamic-pituitary-adrenal axis responses to both acute and chronic stress and were protected against weight loss associated with chronic stress. In addition, regional Glp1r knockdown attenuated stress-induced cardiovascular responses accompanied by decreased sympathetic drive to the heart. Finally, Glp1r knockdown reduced anxiety-like behavior, implicating PVN GLP-1 signaling in behavioral stress reactivity. Collectively, these findings support a circuit whereby brainstem GLP-1 activates PVN signaling to mount an appropriate whole-organism response to stress. These results raise the possibility that dysfunction of this system may contribute to stress-related pathologies, and thereby provide a novel target for intervention

Impact of Corticosterone Treatment on Spontaneous Seizure Frequency and Epileptiform Activity in Mice with Chronic Epilepsy

Stress is the most commonly reported precipitating factor for seizures in patients with epilepsy. Despite compelling anecdotal evidence for stress-induced seizures, animal models of the phenomena are sparse and possible mechanisms are unclear. Here, we tested the hypothesis that increased levels of the stress-associated hormone corticosterone ( CORT) would increase epileptiform activity and spontaneous seizure frequency in mice rendered epileptic following pilocarpine-induced status epilepticus. We monitored video-EEG activity in pilocarpine-treated mice 24/7 for a period of four or more weeks, during which animals were serially treated with CORT or vehicle. CORT increased the frequency and duration of epileptiform events within the first 24 hours of treatment, and this effect persisted for up to two weeks following termination of CORT injections. Interestingly, vehicle injection produced a transient spike in CORT levels - presumably due to the stress of injection - and a modest but significant increase in epileptiform activity. Neither CORT nor vehicle treatment significantly altered seizure frequency; although a small subset of animals did appear responsive. Taken together, our findings indicate that treatment of epileptic animals with exogenous CORT designed to mimic chronic stress can induce a persistent increase in interictal epileptiform activity

Individual seizure timelines of epileptic animals receiving CORT first.

<p>Periods of vehicle and CORT treatment are highlighted in blue and red, respectively, while baseline periods are left white. Bars denote the number of seizures on a given day. No striking correlations between treatment and seizure incidence were observed for these animals.</p

Plasma corticosterone levels measured in a group of non-epileptic (control) mice.

<p>CORT levels were determined following vehicle (blue) and CORT (red) injection. Levels were measured just prior to injection (baseline), thirty minutes (30 m) after injection and two hours after injection. At 30 minutes, both vehicle and CORT injection elevated CORT levels significantly. At 120 minutes, CORT levels began to come down in the case of the vehicle injection, though still significantly higher than baseline levels. Following CORT injection, levels continued to increase beyond 30 minutes. ? ? ?, p<0.001 vs. baseline for both vehicle and CORT; ***, p<0.001 vs. all other groups and time points.</p

Schematic and graphs illustrating the experimental approach (A) and results (B–E) for animals treated with vehicle followed by CORT.

<p><b>A:</b> Experimental protocol for the vehicle-first treatment regimen. Video-EEG was monitored continuously throughout the entire regimen, with each period corresponding to 1–2 weeks. Twenty-four hour EEG segments quantified for epileptiform activity are denoted by arrows. The color of each part of the timeline corresponds to the bar color in the graphs shown below. <b>B:</b> CORT treatment significantly increased the number of epileptiform events relative to all other periods. <b>C:</b> CORT treatment significantly increased the total duration of epileptiform events relative to all other periods. Vehicle treatment also significantly increased the duration of events relative to the 1^st baseline period. <b>D:</b> No significant effects of vehicle or CORT treatment on seizure frequency were found. <b>E:</b> Mean seizure duration was statistically equivalent among treatment groups and baseline periods. Bar graphs show means ± SEM. *, p<0.05.</p

EEG recordings from epileptic animals.

<p><b>A:</b> Baseline EEG activity. <b>B:</b> A typical seizure event in an epileptic animal. This seizure was associated with behavioral rearing and loss of postural control (falling). <b>C:</b> An example of epileptiform activity recorded from an epileptic animal. The EEG segments shown below B and C are expansions of the periods marked by the solid bars. Animals were typically motionless or exhibited myoclonic jerks during epileptiform events.</p

Individual seizure timelines of epileptic animals receiving vehicle first.

<p>Periods of vehicle and CORT treatment are highlighted in blue and red, respectively, while baseline periods are left white. Bars denote the number of seizures on a given day. One animal exhibited seizures only during CORT treatment (E), while 2 animals (A & C) exhibited seizures primarily during periods of vehicle and CORT treatment. The remaining four animals showed no compelling correlation between seizure incidence and treatment, and overall there was no significant effect of treatment on seizure frequency.</p

Schematic and graphs illustrating the experimental approach (A) and results (B–E) for animals treated with CORT followed by vehicle.

<p><b>A:</b> Experimental protocol for the CORT-first treatment regimen. Video-EEG was monitored continuously throughout the entire regimen, with each period corresponding to 1–2 weeks. Twenty-four hour EEG segments quantified for epileptiform activity are denoted by arrows. The color of each part of the timeline corresponds to the bar color in the graphs shown below. <b>B:</b> CORT treatment significantly increased the number of epileptiform events relative to the preceding baseline period (1^st base). The number of epileptiform events was also increased during subsequent periods (2^nd baseline, vehicle) relative to the first baseline. <b>C:</b> CORT treatment significantly increased the total duration of epileptiform events relative to the preceding baseline period. The total duration of epileptiform events was also increased during subsequent periods relative to the first baseline. **, p<0.01 vs. 1^st baseline; ***, p<0.001 vs. 1^st baseline. <b>D:</b> No significant effects of vehicle or CORT treatment on seizure frequency were found. <b>E:</b> Mean seizure duration was statistically equivalent among treatment groups and baseline periods. Bar graphs show means ± SEM.</p