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Sex differences in context-driven reinstatement of methamphetamine seeking is associated with distinct neuroadaptation in the dentate gyrus
Get PDFThe present study examined differences in operant responses in adult male and female rats during distinct phases of addiction. Males and females demonstrated escalation in methamphetamine (0.05 mg/kg, i.v.) intake with females showing enhanced latency to escalate, and bingeing. Following protracted abstinence, females show reduced responses during extinction, and have greater latency to extinguish compared with males, indicating reduced craving. Females demonstrated lower context-driven reinstatement compared to males, indicating that females have less motivational significance to the context associated with methamphetamine. Whole-cell patch-clamp recordings on dentate gyrus (DG) granule cell neurons (GCNs) were performed in acute brain slices from controls and methamphetamine experienced male and female rats and neuronal excitability were evaluated from GCNs. Reinstatement of methamphetamine seeking reduced spiking in males, and increased spiking in females compared to controls, demonstrating distinct neuroadaptations in intrinsic excitability of GCNs in males and females. Reduced excitability of GCNs in males were associated with enhanced levels of neural progenitor cells, expression of plasticity-related proteins including CaMKII and choline acetyltransferase in the DG. Enhanced excitability in females were associated with increased GluN2A/2B ratio, indicating changes in postsynaptic GluN subunit composition in the DG. Together, the present results indicate sexually dimorphic adaptive biochemical changes in excitatory neurotransmitter systems in the DG and highlight the importance of including sex as a biological variable in exploring neuroplasticity and neuroimmune changes that predict enhanced relapse to methamphetamine-seeking behaviors
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Inhibiting the Rebound Burst of Hippocampal Neural Progenitor Cells Decreases Reinstatement of Ethanol Seeking in Female Dependent Rats
Get PDFAlcohol use disorder (AUD) is a psychological disorder affecting millions worldwide, and current medications have limited efficacy. AUD studies in male rats have shown hippocampal neurobiological changes, such as proliferation of neural progenitor cells (NPCs) during withdrawal; however, the link between this phenomenon to increased risks of ethanol-seeking behavior is unclear. Furthermore, studies must be conducted in female rats to highlight sex differences in the neurobiology linked to AUD. Additionally, studies show that alcohol promotes neuroinflammation and disrupts blood-brain-barrier (BBB) integrity. In this study, we used a pharmacogenetic rat model (GFAP-TK rats), a CIE model of moderate to severe AUD (CIE), and fed Valcyte to inhibit NPC proliferation in understudied female rats to investigate neurobiological associations to AUD. CIE produced robust relapse to drinking behavior and reinstatement of ethanol seeking, and Valcyte diminished this effect. Hippocampal quantitative immunohistochemistry showed that Valcyte reduced Ki-67 cells compared to vehicle group, indicating ablation of NPC proliferation. 3-D structure analysis of Iba-1 microglia cells showed that CIE increased microglial activity in the hippocampus. Valcyte prevented this effect, suggesting suppression of neuroinflammatory response by inducing microglial inhibition. Reduced seeking by Valcyte was associated with normalization of plasma cytokines and chemokines, suggesting a role for NPCs in peripheral inflammation. Western blot analysis showed that CIE reduced Claudin-5 expression, and Valcyte rescued these protein levels, indicating increased BBB integrity. Altogether, blocking NPCs suppressed neuroinflammation factors, promoted BBB integrity, and decreased motivation to seek ethanol. These findings suggest a promising route to develop a new therapy for AUD
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Effect of Endostatin on Microglial Activation and Ethanol Seeking Behavior in Dependent Rats
Get PDFAlcohol use disorder, or AUD, is a brain disorder that affects millions of people with fewFDA approved effective treatments at reducing relapse, with none preventing relapse episodes.
Therefore, more research is necessary in order to find a targetable biochemical pathway for
therapeutics to treat AUD and prevent relapse in alcoholics. Recent data in rodent models of
moderate to severe AUD has demonstrated a correlation between higher levels of ethanol
seeking behavior and increased levels of PECAM-1 in the prefrontal cortex of the brain, a region
implicated in relapse to alcohol seeking. More notable is that increases in PECAM-1 is
associated with blood-brain barrier (BBB) leakage, suggesting endothelial cell damage in
relapsing alcoholics. Furthermore, published work from rodent studies and human postmortem
tissue analysis have indicated severe neuroimmune responses in the brain that is correlated with
increased relapse to drinking behavior. However, it remains unclear whether the activation state
of microglia, a major player in the neuroimmune response that occurs due to alcohol metabolism,
is associated with BBB leakage and endothelial cell damage in the prefrontal cortex. It was
hypothesized that endostatin, an angiogenic inhibitor, would inhibit PECAM-1 and thus decrease
BBB leakage. This was expected to reduce the peripheral neuroimmune response and prevent
alteration in microglial activation. In order to test this, the brain tissue of male rats with moderate
to severe AUD were analyzed. Specifically, quantitative immunohistochemistry and
stereological cell analysis were used to evaluate microglial activation which was positively
correlated to ethanol seeking behavior from the additional behavioral studies that were
performed. It was found that endostatin reduced PECAM-1 expression in the prefrontal cortex,
however, did not decrease ethanol seeking behavior of male rats. Additionally, microglial
activation remained similar to the rats that were not treated with endostatin. Therefore, it is
suggested that the peripheral immune response mediated by PECAM-1 is not directly linked to
ethanol seeking behavior and microglial activation in the prefrontal cortex in male rats. From
this, a new study investigating the internal neuroinflammation or immune response could help
determine the mechanism to decrease microglial activation and thus lower relapse. This would be
a significant advance in the effort to create an effective therapeutic for AUD
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The effects of abstinence from chronic ethanol administration on diffusion tensor imaging metrics and myelin-associated proteins in the medial prefrontal cortex
Get PDFPrevious studies have shown that ethanol dependence induced by repeating cycles of chronic intermittent ethanol vapor exposure (CIE) followed by protracted abstinence (CIE-PA) produces significant alterations in gliogenesis in the rodent medial prefrontal cortex (mPFC). Specifically, CIE-PA has been shown in previous studies to significantly dysregulate the process of myelinating oligodendrocytes in the mPFC by creating an unprecedented increase in premyelinating oligodendroglial progenitor cell (OPC) proliferation and survival, which has been associated with persistent elevated drinking behaviors during abstinence. In the current thesis, 63 male adult Wistar rats were subjected to seven weeks of CIE and were examined following 1 day(d), 7d, 21d, or 42d of abstinence. Neuroimaging, capable of detecting alterations to the myelination integrity of the mPFC in vivo, was performed in CIE-PA and age-matched non-vapor control rats, in parallel with conventional immunohistochemical methods to better characterize the physiological changes underlying any neuroimaging metric changes. This neuroimaging technique, called diffusion tensor imaging (DTI), successfully detected abstinence-related changes in the mPFC, specifically that CIE-PA produced transient increases in fractional anisotropy (FA) at the 7d abstinence time point compared to controls and other time points. Interestingly, this increase in FA, was associated temporally with increases in myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), 2’3’-cyclic-nucleotide 3’-phosphodiesterase (CNPase) that we observed also occurred at the 7d PA time point. Therefore, this study concluded that DTI is capable of detecting myelination related changes in the mPFC that result from CIE-PA
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Knockdown of Dopamine 1 Receptors in the Dorsal Striatum Increases Compulsive Drug Intake in Rats that Self-Administer Methamphetamine
Get PDFThe dorsal striatum is important for the development of drug addiction; However, the role of dorsal striatal dopamine D1 receptor (D1R) expressing medium-sized spiny neurons (MSNs) in regulating excessive methamphetamine (Meth) intake remains elusive. Here we seek to determine if reducing D1R expression in the dorsal striatum via RNA interference alters Meth self-administration. A viral vector-mediated approach was used to overexpress short hairpin RNA against D1Rs in the dorsal striatum. Dorsal striatal D1R knockdown increased active lever responses for Meth during a fixed-ratio (FR1) extended access paradigm compared to D1R-intact controls. Knocking down dorsal striatal D1Rs also produced a vertical and rightward shift in a self-administration dose-response paradigm. WB analysis revealed that dorsal striatal D1R knockdown (D1RshRNA) animals exhibit a reduction in D1R, PSD-95, and MAPK-1 expression in the dorsal striatum. Sucrose density gradient fractionation followed by semi-quantitative analysis of relative expression of proteins in subcellular membrane fractions demonstrated a redistribution of synaptic scaffolding proteins to membrane lipid raft (MLR) fractions in Meth-taking animals when compared to Meth-naïve animals. This redistribution occurred regardless of D1R knockdown. Our work indicates that reduced D1R expression-mediated enhancement of Meth addiction-like behaviors is associated with cellular adaptations that support dysfunctional dopamine signaling in the dorsal striatum
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Protracted Abstinence from Chronic Ethanol Experience alters Plasticity Related Proteins and Excitability of Pyramidal Neurons in the Rodent Medial Prefrontal Cortex
Get PDFThe rodent medial prefrontal cortex (mPFC) is a brain region that is homologous to the human PFC, a brain region within the limbic system that has been implicated in executive functions and in alcohol relapse. Previous studies in rodents have demonstrated that inducing ethanol dependence through chronic intermittent ethanol vapor exposure paradigms followed by forced protracted abstinence increases drinking during abstinence, and causes significant alterations in neuronal excitability and white matter/cerebrovascular composition in the mPFC. However, no studies have investigated the cellular and molecular changes associated with the neuroplastic adaptations that occur within this brain region during protracted abstinence. To investigate this, 69 adult male Wistar rats underwent seven weeks of chronic intermittent ethanol exposure and were then subject to either 1 day, 7 days, 21 days, or 42 days of protracted abstinence. Afterwards these animals were immediately euthanized and tissue from mPFC of their brains was collected for analysis via Western blotting or for analysis via ex vivo electrophysiology. We found that chronic intermittent ethanol experience followed by 21 days of protracted abstinence produced a significant decrease in levels of phosphorylated calcium calmodulin kinase II (CaMKII) along with a decrease in the ratio of glutamatergic subunits GluN2A/2B of the NMDA receptor. This reduction in CaMKII activity and the ratio of GluN2A/2B was associated with enhanced excitability of layer 2/3 pyramidal neurons in the mPFC. Together, the present results indicate adaptive biochemical changes in excitatory neurotransmitter systems in the mPFC and highlights the importance of exploring neuroplasticity changes that predict enhanced propensity to relapse alcohol-seeking behaviors
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Inhibition of the rebound burst of neural progenitor cells reduces ethanol seeking in female dependent rats: Regulation by neuroimmune and endothelial changes
Get PDFAlcohol use disorder (AUD) is highly prevalent in the United States with very few FDA approved treatments that do not completely reduce relapse to alcohol seeking. This is due to an incomplete understanding of the neurobiological substrates causing alcohol relapse. Previous studies in alcohol dependent male rats have shown cellular changes in the medial prefrontal cortex (mPFC), including increased oligodendrogenesis, heightened neuroimmune response, and decrease in blood-brain barrier (BBB) integrity associated with dependence induced drinking and relapse. Specifically during acute withdrawal, a proliferative burst of oligodendrocyte progenitor cells (OPCs) is prevalent in the mPFC which survive and mature into oligodendrocytes. Research focused on male rats have shown this dysfunctional production of OPCs is linked to a heightened neuroimmune response and endothelial disruption, but unclear if these changes are evident in females as well. Thus, it was investigated if these non-neuronal changes were also evident in dependent female rats. Like in males, quantitative immunohistochemistry depicted an increase in Ki-67 cells in the mPFC of females during acute withdrawal and concomitant increase in Olig2 cells during protracted abstinence. In addition, 3D structural analysis, Western blotting and ELISA detected a heightened neuroinflammatory response after relapse, with an increase in microglia soma area and cytokines levels including NF-kB, TNF-α, IL-13, and IL-10 in the mPFC. Furthermore, ELISA data showed an increase in VEGF levels indicating BBB dysfunction. The next question investigated was whether preventing this burst of OPCs in dependent female rats will decrease ethanol seeking behavior by regulating the neuroimmune responses and endothelial changes. To prevent the generation of Ki-67 cells, a pharmacogenetic approach was used, where transgenic GFAP-TK rats were orally fed a DNA synthesis inhibitor called Valcyte during maintenance through reinstatement. Valcyte treatment decreased ethanol seeking behavior during relapse and reinstatement, as well as enhanced latency to extinguish ethanol behaviors, showing decreased motivation to alcohol drinking. In addition, Valcyte prevented microglia activation in the mPFC and normalized levels of TNF-α, IL-4, and IL-6 in the plasma. However, Valcyte did not prevent endothelial damage in the mPFC, indicated by an increase in Ve-Cadherin and decrease in Claudin-5 expression. In conclusion, preventing the proliferative burst of Ki-67 cells in the mPFC during acute withdrawal decreased ethanol seeking behavior and prevented some aspects of the immune response seen during relapse and reinstatement of ethanol seeking behaviors
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Endostatin reduces relapse to ethanol seeking in dependent rats: Regulation by PECAM-1 and oligodendrocytes
Get PDFAlcohol use disorder (AUD) produces a variety of mental damage; AUD is a serious public health issue. Current FDA approved medications to treat AUD are partially effective, warranting the need for better therapies. Animal models of AUD are currently used to discover new therapies due to their robust face and predictive validity. Previous studies suggested that rats with AUD had higher ethanol seeking behavior during abstinence that correlated with enhanced expression of platelet endothelial cell adhesion molecules (PECAM-1; angiogenesis marker), increased number of oligodendrocyte progenitor cells (OPCs) and altered neuronal activation in medial prefrontal cortex (mPFC). However, it is unclear whether PECAM-1 directly promotes ethanol seeking behavior by reducing neuronal activity and increasing proliferating OPCs. Thus, to investigate the role of PECAM-1 in alcohol-dependent rats, we used endostatin, a broad-spectrum angiogenesis/PECAM-1 inhibitor. Results from behavior studies showed that in alcohol-dependent female rats, endostatin significantly reduced alcohol seeking during relapse. Postmortem tissue analysis using quantitative immunohistochemistry in the mPFC demonstrated that reduced seeking correlated with reduced PECAM-1, OPCs and neuronal activation. Western blotting analysis revealed that endostatin enhanced the activity of calcium calmodulin dependent kinase II (CaMKII), which could assist with normalizing the altered neuronal activity during abstinence. In conclusion, our analyses confirmed that the angiogenesis of PECAM-1 is responsible for enhancing ethanol seeking behavior by altering neuronal activation and oligodendrogenesis in female rat mPFC. Our research provided a possible mechanism to inhibit ethanol seeking and prevent the glial and neuronal damage caused by increased accumulation of PECAM-1 from alcohol drinking
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Neuroadaptations in the medial prefrontal cortex (mPFC) predict memory deficits dependent on the mPFC in alcohol dependent rats
Get PDFChronic intermittent ethanol vapor exposure (CIE) produces dependence, alters the structure of pyramidal neurons, and decreases oligodendroglial progenitors in the medial prefrontal cortex (mPFC). The effect of early abstinence from CIE on these neuroadaptations are unclear and unknown and were investigated. Adult male Wistar rats were exposed to CIE for 7 weeks, after which they underwent 3 days of abstinence. On the 4th day, CIE abstinent and age matched CIE naïve controls experienced trace fear conditioning (TFC) to determine the effect of early abstinence on neuroadaptations in the mPFC and retrieval of emotional memories, and the interactions between TFC and abstinence-induced neuroadaptations in the mPFC. To assess neuroadaptations, mPFC tissue was processed for Western blotting, immunohistochemistry and Golgi-Cox staining. CIE abstinent animals froze less during retrieval, indicating deficits in memory functions dependent on the mPFC. Abstinence enhanced dendritic complexity of layer 2/3 mPFC pyramidal neurons, as did trace fear conditioning. However, trace fear conditioning combined with CIE had no additive effect. Additionally, abstinence altered plasticity-related proteins in mPFC (increased total NR2A expression and decreased PSD-95) and TFC did not affect these adaptations. However, TFC produced profound alterations in oligodendroglial proteins (increases in transcription factor Olig2 and myelin basic protein (MBP)) and abstinence abolished these effects. Our findings indicate that neuroadaptations in the mPFC persist into early abstinence in CIE animals, and these deficits are associated with reduced memory functions dependent on the PF
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Endostatin reduces relapse to ethanol seeking in dependent rats: Indirect regulation by microglia
Get PDFAlcohol use disorder (AUD) affects millions of people around the world and kills many, therefore, it is considered a serious public health issue. AUD is an economic burden, causes brain damage, and causes dysfunction of heart, liver, lungs, and kidney. Currently there are a few FDA approved medications and mutual help groups to combat AUD, but none are successful in preventing relapse over long-term. Therefore, there is need for novel therapeutics to treat AUD. Previous studies suggested that in animal models of AUD, animals with moderate to severe AUD had higher ethanol seeking behavior during abstinence that correlated with enhanced expression of platelet endothelial cell adhesion molecule (PECAM-1, a marker for angiogenesis), increased blood brain barrier leakage, and altered neuronal activation in medial prefrontal cortex (mPFC), a brain region implicated in relapse to ethanol seeking behaviors. However, it was unclear how microglia, a cell type known to regulate neuroimmune responses was altered during abstinence. Observations from our lab show that endostatin reduces the expression of PECAM-1, prevents gliosis that occurs during abstinence, and decreased ethanol seeking behavior during relapse. Postmortem tissue analysis using quantitative immunohistochemistry in the mPFC demonstrated that ethanol seeking correlated with microglial activation in the mPFC and endostatin reduced ethanol seeking and concurrently reduced microglial activation. Microglial deactivation was quantified using stereological methods and 3D structural analysis by investigating changes in soma to tip distance, cell soma area, total dendritic length, and number of intersections relative to distance from soma in microglial cells labeled with Iba-1. Phenotypic analysis revealed that the number of intersections relative to the distance from the soma recovered to normal levels after treatment with endostatin in alcohol dependent rats; which indicates a deactivation of microglia. Our research showed that endostatin can indirectly deactivate microglia and alter the neuronal response. Our research provides a possible mechanism of action to limit ethanol seeking behavior by indirectly deactivating microglia in the mPFC.
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