Shawn Gouty

Differential protection against MPTP or methamphetamine toxicity in dopamine neurons by deletion of ppN/OFQ expression

Nociceptin (N/OFQ) is an endogenous neuropeptide that plays a role in the behavioral deficits associated with Parkinsons disease (PD). The purpose of the present study was to characterize the protective effects of prepro (pp)N/OFQ gene deletion against two dopamine toxins, MPTP and methamphetamine (METH). Results demonstrate that ppN/OFQ gene deletion attenuates the loss of both the number of tyrosine hydroxylase (TH)‐positive neurons in the substantia nigra pars compacta (SNpc) and loss of TH and vesicular monoamine transporter‐2 (VMAT) immunoreactivity in the caudate putamen (CPu) of MPTP‐treated mice. This protection was unaffected by age or gender, although, when loss of TH exceeded 90% in 5–6 month‐old mice, the protective effect was greatly diminished. In contrast, METH administration preferentially damaged dopaminergic terminals in the CPu with little effect on dopamine neurons in the SNpc, an effect not reversed by ppN/OFQ gene deletion. To determine if N/OFQ and MPP+ act directly and synergistically on dopamine neurons, differentiated SH‐SY5Y cells were incubated with N/OFQ and/or MPP+. N/OFQ did not increase MPP+‐mediated cell loss, suggesting an indirect action of N/OFQ. These studies demonstrate that inhibition of the endogenous N/OFQ system may represent a new therapeutic target for prevention of neuronal loss associated with PD.

Ca2+/calmodulin-dependent protein kinase II in the rat cranial sensory ganglia

Immunohistochemistry for Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was performed on the rat cranial sensory ganglia. More than one half of neurons was immunoreactive for the enzyme in the trigeminal (60%), jugular (70%), petrosal (55%) and nodose ganglia (63%). These neurons were mainly small to medium-sized. The co-expression study demonstrated that one half of CaMKII-immunoreactive (ir) neurons was also immunoreactive for calcitonin gene-related peptide (CGRP) or the vanilloid receptor subtype 1 (VR1) in the trigeminal, jugular and petrosal ganglia. In the nodose ganglion, CaMKII-ir neurons were mostly devoid of CGRP-immunoreactivity (ir) (8.2%) whereas the co-expression with VR1-ir was common among such neurons (72%). In the facial skin, nasal mucosa and palate, the epithelium and taste bud were innervated by CaMKII-ir nerve fibers. In addition, the retrograde tracing study demonstrated that 39.6% and 44.8% of trigeminal neurons which were retrogradely traced with fluorogold from the facial skin and nasal mucosa exhibited CaMKII-ir. Forty-six percent of petrosal neurons which innervated the soft palate were immunoreactive for the enzyme.

Mass spectrometry imaging of rat brain lipid profile changes over time following traumatic brain injury.

BACKGROUND Mild traumatic brain injury (TBI) is a common public health issue that may contribute to chronic degenerative disorders. Membrane lipids play a key role in tissue responses to injury, both as cell signals and as components of membrane structure and cell signaling. This study demonstrates the ability of high resolution mass spectrometry imaging (MSI) to assess sequences of responses of lipid species in a rat controlled cortical impact model for concussion. NEW METHOD A matrix of implanted silver nanoparticles was implanted superficially in brain sections for matrix-assisted laser desorption (MALDI) imaging of 50μm diameter microdomains across unfixed cryostat sections of rat brain. Ion-mobility time-of-flight MS was used to analyze and map changes over time in brain lipid composition in a rats after Controlled Cortical Impact (CCI) TBI. RESULTS Brain MS images showed changes in sphingolipids near the CCI site, including increased ceramides and decreased sphingomyelins, accompanied by changes in glycerophospholipids and cholesterol derivatives. The kinetics differed for each lipid class; for example ceramides increased as early as 1 day after the injury whereas other lipids changes occurred between 3 and 7 days post injury. COMPARISON WITH EXISTING METHOD(S) Silver nanoparticles MALDI matrix is a sensitive new tool for revealing previously undetectable cellular injury response and remodeling in neural, glial and vascular structure of the brain. CONCLUSIONS Lipid biochemical and structural changes after TBI could help highlighting molecules that can be used to determine the severity of such injuries as well as to evaluate the efficacy of potential treatments.

α-Lipoic acid treatment prevents the diabetes-induced attenuation of the afferent limb of the baroreceptor reflex in rats

Autonomic neuropathies, common complications of prolonged diabetes, may result from diabetes-induced increased oxidative stress. Recently, we found that the afferent component of the baroreceptor reflex is attenuated in streptozotocin-induced diabetic rats. This study sought to determine the influence of the anti-oxidant, alpha-lipoic acid on the diabetes-induced deficits of the afferent limb of the baroreceptor reflex and on plasma malondialdehyde (a measure of lipid peroxidation). The number of c-Fos-ir neurons in the nucleus tractus solitarius in response to phenylephrine-induced baroreceptor activation was used as an index of the integrity of the afferent limb of the baroreceptor reflex. Groups of streptozotocin-induced diabetic and non-diabetic control rats, maintained from 8 to 16 weeks, were treated with alpha-lipoic acid (100 mg kg(-1) IP, 5x/week), or vehicle for the last 4 weeks prior to the experimental procedure. Vehicle-treated diabetic rats had elevated plasma malondialdehyde levels when compared to non-diabetic rats. alpha-Lipoic acid-treated diabetic rats had plasma malondialdehyde levels similar to those seen in non-diabetic rats and less than those of vehicle-treated diabetic rats at both the 8- and 16-week time points.alpha-Lipoic acid treatment did not affect the baseline (absence of baroreceptor activation) presence of c-Fos-ir in the nucleus tractus solitarius. In response to phenylephrine and regardless of treatment, the diabetic and control rats displayed increases in blood pressure and reflex bradycardia. As previously reported, phenylephrine-induced baroreceptor activation resulted in significantly fewer c-Fos-ir neurons in the nucleus tractus solitarius (commissural and caudal subpostremal regions) of diabetic rats when compared to non-diabetic rats at both 8- and 16-week time points. Four weeks of alpha-lipoic acid treatment reversed the diabetes-induced decrement in the numbers of c-Fos-ir neurons in the nucleus tractus solitarius in response to baroreceptor activation. alpha-Lipoic acid-treated diabetic rats showed the same phenylephrine-induced c-Fos response in the nucleus tractus solitarius as those of alpha-lipoic-acid- and vehicle-treated control rats at both 8- and 16-week time points. These data suggest that diabetes-induced oxidative stress plays a role in diabetes-induced baroreceptor dysfunction and that the alpha-lipoic acid may have a beneficial effect in treatment of diabetic autonomic neuropathy.

Attenuation of the afferent limb of the baroreceptor reflex in streptozotocin-induced diabetic rats.

Diabetic autonomic neuropathy is a common complication following prolonged diabetes. Alterations of cardiovascular reflexes contribute to the increased cardiovascular morbidity and mortality seen in diabetic patients. This study sought to better characterize these complications by investigating the afferent limb of the baroreceptor reflex in an experimental rat model of diabetes. Streptozotocin (STZ)-induced diabetic and euglycemic control rats were studied at 8- and 16-week time points after initiation of the experiment. Activation of the afferent limb of the baroreceptor reflex was assessed by measuring the numbers of c-Fos-immunoreactive (ir) neurons in the CNS site of termination of the baroreceptor afferent neurons, the nucleus of the solitary tract (NTS). Initial experiments established that baseline cardiovascular parameters and NTS expression of c-Fos-ir neurons were not different between diabetic and control rats at either time point. Phenylephrine (PE)-induced activation of baroreceptors resulted in a significant elevation in the numbers of c-Fos-ir neurons in the NTS of control rats. Although diabetic rats showed similar pressor responses to PE, the activation of c-Fos-ir neurons in the NTS of diabetic rats was significantly attenuated. At both 8 and 16 weeks, STZ-induced diabetic rats had significantly fewer c-Fos-ir neurons in the commissural NTS and in the caudal subpostrernal NTS when compared to the non-diabetic control animals receiving PE. These data suggest that STZ-induced diabetes, for a period of 8 and 16 weeks, results in reduced activity in the afferent baroreceptor input to the NTS, and are consistent with diabetes-induced damage to baroreceptor afferent nerves.

MPTP treatment increases expression of pre-pro-nociceptin/orphanin FQ mRNA in a subset of substantia nigra reticulata neurons.

Antagonists selectively inhibiting activation of the nociceptin/orphanin FQ (N/OFQ) receptor reduce motor symptoms in experimental models of Parkinsons disease, and genetic deletion of the ppN/OFQ gene offers partial protection of mid-brain dopamine neurons against the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP increased ppN/OFQ mRNA expression in the substantia nigra (SN). We have evaluated the temporal relationship of dopamine cell loss to increased ppN/OFQ mRNA expression in the substantia nigra after MPTP treatment, and characterized the cellular locations in which increased ppN/OFQ mRNA expression was observed after MPTP treatment. MPTP increased by about 5-fold the number of neurons expressing ppN/OFQ mRNA in the pars reticulata of SN (SNr) by 24 h after treatment and the elevation remained significant for at least 7 days. This period coincided with the timing of the loss of dopamine neurons from the pars compacta of substantia nigra (SNc) after MPTP. The increased expression of ppN/OFQ mRNA co-localized with a neuronal marker in the SNr. MPTP treatment resulted in a small increase in the numbers of neurons expressing ppN/OFQ in the SNc in mice from one mouse colony but the increase did not reach statistical significance in mice from another colony. No changes in ppN/OFQ-mRNA expression were observed in the ventral tegmental area (VTA), the caudate-putamen, the subthalamic nucleus, or in two other brains areas. These results demonstrate that increased N/OFQ expression in the SNr is closely associated with the MPTP-induced loss of dopamine neurons in the SNc in a widely used animal model of Parkinsons disease.

Histone Deacetylase Inhibition Rescues Maternal Deprivation-Induced GABAergic Metaplasticity through Restoration of AKAP Signaling

Adverse early-life experiences such as child neglect and abuse increase the risk of developing addiction and stress-related disorders through alterations in motivational systems including the mesolimbic dopamine (DA) pathway. Here we investigated whether a severe early-life stress (i.e., maternal deprivation, MD) promotes DA dysregulation through an epigenetic impairment of synaptic plasticity within ventral tegmental area (VTA) DA neurons. Using a single 24-hr episode of MD and whole-cell patch clamp recording in rat midbrain slices, we show that MD selectively induces long-term depression (LTD) and shifts spike timing-dependent plasticity (STDP) toward LTD at GABAergic synapses onto VTA DA neurons through epigenetic modifications of postsynaptic scaffolding A-kinase anchoring protein 79/150 (AKAP79/150) signaling. Histone deacetylase (HDAC) inhibition rescues GABAergic metaplasticity and normalizes AKAP signaling in MD animals. MD-induced reversible HDAC-mediated GABAergic dysfunction within the VTA may be a mechanistic link for increased propensity to mental health disorders following MD.

Morphine-induced synaptic plasticity in the VTA is reversed by HDAC inhibition

Dopamine (DA) dysfunction originating from the ventral tegmental area (VTA) occurs as a result of synaptic abnormalities following consumption of drugs of abuse and underlies behavioral plasticity associated with drug abuse. Drugs of abuse can cause changes in gene expression through epigenetic mechanisms in the brain that underlie some of the lasting neuroplasticity and behavior associated with addiction. Here we investigated the function of histone acetylation and histone deacetylase (HDAC)2 in the VTA in recovery of morphine-induced synaptic modifications following a single in vivo exposure to morphine. Using a combination of immunohistochemistry, Western blot, and whole cell patch-clamp recording in rat midbrain slices, we show that morphine increased HDAC2 activity in VTA DA neurons and reduced histone H3 acetylation at lysine 9 (Ac-H3K9) in the VTA 24 h after the injection. Morphine-induced synaptic changes at glutamatergic synapses involved endocannabinoid signaling to reduce GABAergic synaptic strength onto VTA DA neurons. Both plasticities were recovered by in vitro incubation of midbrain slices with a class I-specific HDAC inhibitor (HDACi), CI-994, through an increase in acetylation of histone H3K9. Interestingly, HDACi incubation also increased levels of Ac-H3K9 and triggered GABAergic and glutamatergic plasticities in DA neurons of saline-treated rats. Our results suggest that acute morphine-induced changes in VTA DA activity and synaptic transmission engage HDAC2 activity locally in the VTA to maintain synaptic modifications through histone hypoacetylation.

Mu opioid receptor activation enhances regulator of G protein signaling 4 association with the mu opioid receptor/G protein complex in a GTP‐dependent manner

The interaction of Regulator of G protein Signaling 4 (RGS4) with the rat mu opioid receptor (MOR)/G protein complex was investigated. Solubilized MOR from rat brain membranes was immunoprecipitated in the presence of RGS4 with antibodies against the N‐terminus of MOR (anti‐MOR10–70). Activation of MOR with [D‐Ala2, N‐Me‐Phe4, Gly5‐ol] enkephalin (DAMGO) during immunoprecipitation caused a 150% increase in Goα and a 50% increase in RGS4 in the pellet. When 10 μM GTP was included with DAMGO, there was an additional 72% increase in RGS4 co‐immunoprecipitating with MOR (p = 0.003). Guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) increased the amount of co‐precipitating RGS4 by 93% (compared to DAMGO alone, p = 0.008), and the inclusion of GTPγS caused the ratio of MOR to RGS4 to be 1 : 1 (31 fmoles : 28 fmoles, respectively). GTPγS also increased the association of endogenous RGS4 with MOR. In His6RGS4/Ni2+‐NTA agarose pull down experiments, 0.3 μM GTPγS tripled the binding of Goα to His6RGS4, whereas the addition of 100 μM GDP blocked this effect. Importantly, activation of solubilized MOR with DAMGO in the presence of 100 μM GDP and 0.3 μM GTPγS increased Goα binding to His6RGS4/Ni2+‐NTA agarose (p = 0.001).

Mass Spectrometric Imaging of Ceramide Biomarkers Tracks Therapeutic Response in Traumatic Brain Injury

Traumatic brain injury (TBI) is a serious public health problem and the leading cause of death in children and young adults. It also contributes to a substantial number of cases of permanent disability. As lipids make up over 50% of the brain mass and play a key role in both membrane structure and cell signaling, their profile is of particular interest. In this study, we show that advanced mass spectrometry imaging (MSI) has sufficient technical accuracy and reproducibility to demonstrate the anatomical distribution of 50 μm diameter microdomains that show changes in brain ceramide levels in a rat model of controlled cortical impact (CCI) 3 days post injury with and without treatment. Adult male Sprague-Dawley rats received one strike and were euthanized 3 days post trauma. Brain MS images showed increase in ceramides in CCI animals compared to control as well as significant reduction in ceramides in CCI treated animals, demonstrating therapeutic effect of a peptide agonist. The data also suggests the presence of diffuse changes outside of the injured area. These results shed light on the extent of biochemical and structural changes in the brain after traumatic brain injury and could help to evaluate the efficacy of treatments.