Hilar Interneurons Research Articles

Spatial mRNA expression patterns of orexin receptors in the dorsal hippocampus.

Orexins are wake-promoting neuropeptides that originate from hypothalamic neurons projecting to widespread brain areas throughout the central nervous system. They modulate various physiological functions via their orexin 1 (OXR1) and 2 (OXR2) receptors, including sleep-wake rhythm but also cognitive functions such as memory formation. Here, we provide a detailed analysis of OXR1 and OXR2 mRNA expression profiles in the dorsal hippocampus as a key region for memory formation, using RNAscope multiplex in situ hybridization. Interconnected subareas relevant for cognition and memory such as the medial prefrontal cortex and the nucleus reuniens of the thalamus were assessed as well. Both receptor types display distinct profiles, with the highest percentage of OXR1 mRNA-positive cells in the hilus of the dentate gyrus. Here, the content of OXR1 mRNA per cell was slightly modulated at selected time points over a 12h light/ 12 dark light phase. Using RNAScope and quantitative polymerase chain reaction approaches, we began to address a cell-type specific expression of OXR1 in hilar GABAergic interneurons. The distinct expression profiles of both receptor subtypes within hippocampal subareas and circuits provide an interesting basis for future interventional studies on orexin receptor function in spatial and contextual memory.

Circuit-based intervention corrects excessive dentate gyrus output in the fragile X mouse model.

Abnormal cellular and circuit excitability is believed to drive many core phenotypes in fragile X syndrome (FXS). The dentate gyrus is a brain area performing critical computations essential for learning and memory. However, little is known about dentate circuit defects and their mechanisms in FXS. Understanding dentate circuit dysfunction in FXS has been complicated by the presence of two types of excitatory neurons, the granule cells and mossy cells. Here we report that loss of FMRP markedly decreased excitability of dentate mossy cells, a change opposite to all other known excitability defects in excitatory neurons in FXS. This mossy cell hypo-excitability is caused by increased Kv7 function in Fmr1 knockout (KO) mice. By reducing the excitatory drive onto local hilar interneurons, hypo-excitability of mossy cells results in increased excitation/inhibition ratio in granule cells and thus paradoxically leads to excessive dentate output. Circuit-wide inhibition of Kv7 channels in Fmr1 KO mice increases inhibitory drive onto granule cells and normalizes the dentate output in response to physiologically relevant theta-gamma coupling stimulation. Our study suggests that circuit-based interventions may provide a promising strategy in this disorder to bypass irreconcilable excitability defects in different cell types and restore their pathophysiological consequences at the circuit level.

Circuit-based intervention corrects excessive dentate gyrus output in the fragile X mouse model

Abnormal cellular and circuit excitability is believed to drive many core phenotypes in fragile X syndrome (FXS). The dentate gyrus is a brain area performing critical computations essential for learning and memory. However, little is known about dentate circuit defects and their mechanisms in FXS. Understanding dentate circuit dysfunction in FXS has been complicated by the presence of two types of excitatory neurons, the granule cells and mossy cells. Here we report that loss of FMRP markedly decreased excitability of dentate mossy cells, a change opposite to all other known excitability defects in excitatory neurons in FXS. This mossy cell hypo-excitability is caused by increased Kv7 function in Fmr1 knockout (KO) mice. By reducing the excitatory drive onto local hilar interneurons, hypo-excitability of mossy cells results in increased excitation/inhibition ratio in granule cells and thus paradoxically leads to excessive dentate output. Circuit-wide inhibition of Kv7 channels in Fmr1 KO mice increases inhibitory drive onto granule cells and normalizes the dentate output in response to physiologically relevant theta–gamma coupling stimulation. Our study suggests that circuit-based interventions may provide a promising strategy in this disorder to bypass irreconcilable excitability defects in different cell types and restore their pathophysiological consequences at the circuit level.

Exposure to alpha-glycosyl isoquercitrin from gestation to adulthood increases synaptic densities of glutamatergic and GABAergic inputs in the hippocampal dentate gyrus in rats

Exposure to alpha-glycosyl isoquercitrin (AGIQ) from gestation to adulthood has been shown to enhance synaptic plasticity in the hippocampal dentate gyrus (DG) and facilitate fear memory extinction in rats. Here, we investigated the effect of exposure to 0.5 % AGIQ in the diet from gestational day 6 to postnatal day 77 in rats on excitatory and inhibitory inputs in the DG. AGIQ increased VGLUT2 immunoreactivity in the granule cell layer and PSD95 immunoreactivity in the whole structure. AGIQ also increased VGAT immunoreactivity in all DG laminae as well as the number of hilar GABAergic interneurons expressing NMDAR2D in subpopulations expressing CB1R, parvalbumin or somatostatin. The GABAergic input was dominant to the glutamatergic input. These results suggest that AGIQ increases synaptic densities of both excitatory and inhibitory inputs in the DG. AGIQ may upregulate NMDAR2D in diverse interneuron subpopulations, thereby enhancing structural plasticity in granule cells, which might facilitate fear memory extinction.

Mild hypoxia-induced structural and functional changes of the hippocampal network.

Hypoxia causes structural and functional changes in several brain regions, including the oxygen-concentration-sensitive hippocampus. We investigated the consequences of mild short-term hypoxia on rat hippocampus in vivo. The hypoxic group was treated with 16% O2 for 1 h, and the control group with 21% O2. Using a combination of Gallyas silver impregnation histochemistry revealing damaged neurons and interneuron-specific immunohistochemistry, we found that somatostatin-expressing inhibitory neurons in the hilus were injured. We used 32-channel silicon probe arrays to record network oscillations and unit activity from the hippocampal layers under anaesthesia. There were no changes in the frequency power of slow, theta, beta, or gamma bands, but we found a significant increase in the frequency of slow oscillation (2.1-2.2 Hz) at 16% O2 compared to 21% O2. In the hilus region, the firing frequency of unidentified interneurons decreased. In the CA3 region, the firing frequency of some unidentified interneurons decreased while the activity of other interneurons increased. The activity of pyramidal cells increased both in the CA1 and CA3 regions. In addition, the regularity of CA1, CA3 pyramidal cells' and CA3 type II and hilar interneuron activity has significantly changed in hypoxic conditions. In summary, a low O2 environment caused profound changes in the state of hippocampal excitatory and inhibitory neurons and network activity, indicating potential changes in information processing caused by mild short-term hypoxia.

Genetic Ablation of Dentate Hilar Somatostatin-Positive GABAergic Interneurons is Sufficient to Induce Cognitive Impairment.

Aging is often associated with a decline in cognitive function. A reduction in the number of somatostatin-positive (SOM+) interneurons in the dentate gyrus (DG) has been described in cognitively impaired but not in unimpaired aged rodents. However, it remains unclear whether the reduction in SOM + interneurons in the DG hilus is causal for age-related cognitive dysfunction. We hypothesized that hilar SOM+ interneurons play an essential role in maintaining cognitive function and that a reduction in the number of hilar SOM + interneurons might be sufficient to induce cognitive dysfunction. Hilar SOM+ interneurons were ablated by expressing a diphtheria toxin transgene specifically in these interneurons, which resulted in a reduction in the number of SOM+ /GAD-67+ neurons and dendritic spine density in the DG. C-fos and Iba-1 immunostainings were increased in DG and CA3, but not CA1, and BDNF protein expression in the hippocampus was decreased. Behavioral testing showed a reduced recognition index in the novel object recognition test, decreased alternations in the Y maze test, and longer latencies and path lengths in the learning and reversal learning phases of the Morris water maze. Our results show that partial genetic ablation of SOM+ hilar interneurons is sufficient to increase activity in DG and CA3, as has been described to occur with aging and to induce an impairment of learning and memory functions. Thus, partial ablation of hilar SOM + interneurons may be a significant contributing factor to age-related cognitive dysfunction. These mice may also be useful as a cellularly defined model of hippocampal aging.

Natural antioxidant formula ameliorates lipopolysaccharide-induced impairment of hippocampal neurogenesis and contextual fear memory through suppression of neuroinflammation in rats

This study investigated the ameliorating effects of a natural antioxidant formula (NAF) consisting of Ginkgo biloba leaf extract, docosahexaenoic acid/eicosapentaenoic acid, ferulic acid, flaxseed oil, vitamin E, and vitamin B12 on a lipopolysaccharide (LPS)-induced cognitive dysfunction model in rats. Six-week-old rats received a diet containing 0.5% (w/w) NAF for 38 days from Day 1, and LPS (1 mg/kg body weight) was administered intraperitoneally once daily on Days 8 and 10. On Day 11, LPS alone increased interleukin-1β and tumor necrosis factor-α in the hippocampus and cerebral cortex and the numbers of M1-type microglia/macrophages and GFAP+ reactive astrocytes in the hilus of the hippocampal dentate gyrus. NAF treatment decreased brain proinflammatory cytokine levels and increased the number of M2-type microglia/macrophages. During Days 34–38, LPS alone impaired fear memory acquisition and the extinction learning process, and NAF facilitated fear extinction learning. On Day 38, LPS alone decreased the number of type-3 neural progenitor cells in the hippocampal neurogenic niche, and NAF restored the number of type-3 neural progenitor cells and increased the numbers of both immature granule cells in the neurogenic niche and reelin+ hilar interneurons. Thus, NAF exhibited anti-inflammatory effects and ameliorated LPS-induced adverse effects on hippocampal neurogenesis and fear memory learning, possibly through amplification of reelin signaling by hilar interneurons. These results suggest that neuroinflammation is a key factor in the development of LPS-induced impairment of fear memory learning, and supplementation with NAF in the present study helped to prevent hippocampal neurogenesis and disruptive neurobehaviors caused by neuroinflammation.

Atypical Neurogenesis, Astrogliosis, and Excessive Hilar Interneuron Loss Are Associated with the Development of Post-Traumatic Epilepsy.

Traumatic brain injury (TBI) remains a significant risk factor for post-traumatic epilepsy (PTE). The pathophysiological mechanisms underlying the injury-induced epileptogenesis are under investigation. The dentate gyrus-a structure that is highly susceptible to injury-has been implicated in the evolution of seizure development. Utilizing the murine unilateral focal control cortical impact (CCI) injury, we evaluated seizure onset using 24/7 EEG video analysis at 2-4 months post-injury. Cellular changes in the dentate gyrus and hilus of the hippocampus were quantified by unbiased stereology and Imaris image analysis to evaluate Prox1-positive cell migration, astrocyte branching, and morphology, as well as neuronal loss at four months post-injury. Isolation of region-specific astrocytes and RNA-Seq were performed to determine differential gene expression in animals that developed post-traumatic epilepsy (PTE+) vs. those animals that did not (PTE-), which may be associated with epileptogenesis. CCI injury resulted in 37% PTE incidence, which increased with injury severity and hippocampal damage. Histological assessments uncovered a significant loss of hilar interneurons that coincided with aberrant migration of Prox1-positive granule cells and reduced astroglial branching in PTE+ compared to PTE- mice. We uniquely identified Cst3 as a PTE+-specific gene signature in astrocytes across all brain regions, which showed increased astroglial expression in the PTE+ hilus. These findings suggest that epileptogenesis may emerge following TBI due to distinct aberrant cellular remodeling events and key molecular changes in the dentate gyrus of the hippocampus.

Selective inhibition of somatostatin-positive dentate hilar interneurons induces age-related cellular changes and cognitive dysfunction.

The cellular basis of age-related impairments of hippocampal function is not fully understood. In order to evaluate the role of somatostatin-positive (Sst+) interneurons in the dentate gyrus (DG) hilus in this process, we chemogenetically inhibited Sst+ interneurons in the DG hilus. Chronic chemogenetic inhibition (CCI) of these neurons resulted in increased c-Fos staining in the DG hilus, a decrease in the percentage of GAD67- and of Sst-expressing interneurons in the DG, and increased microglial activation in DG, CA3, and CA1. Total dendritic length and spine density were reduced in DG and CA1, suggesting reduced dendritic complexity. Behaviorally, the recognition index in an object recognition task and the percentage of spontaneous alternations in the Y-maze were decreased, while in both initial and reversal learning in the Morris water maze, the latencies to find the hidden platform were increased, suggesting cognitive dysfunction. Our findings establish a causal role for a reduced function of Sst+ interneurons in the DG hilus for cognitive decline and suggest that this reduced function may contribute to age-related impairments of learning and memory. Furthermore, our CCI mice may represent a cellularly defined model of hippocampal aging.

Amelioration of lipopolysaccharides-induced impairment of fear memory acquisition by alpha-glycosyl isoquercitrin through suppression of neuroinflammation in rats.

This study investigated the role of neuroinflammation in a lipopolysaccharides (LPS)-induced cognitive dysfunction model in rats using an antioxidant, α-glycosyl isoquercitrin (AGIQ). Six-week-old rats were dietary treated with 0.5% (w/w) AGIQ for 38 days, and LPS at 1 mg/kg body weight was administered intraperitoneally once daily on Days 8 and 10. On Day 11, LPS alone increased or tended to increase interleukin-1β and tumor necrosis factor-α in the hippocampus and cerebral cortex. Immunohistochemically, LPS alone increased the number of Iba1+ and CD68+ microglia, and GFAP+ astrocytes in the hilus of the hippocampal dentate gyrus (DG). AGIQ treatment decreased or tended to decrease brain proinflammatory cytokine levels and the number of CD68+ microglia in the DG hilus. In the contextual fear conditioning test during Day 34 and Day 38, LPS alone impaired fear memory acquisition, and AGIQ tended to recover this impairment. On Day 38, LPS alone decreased the number of DCX+ cells in the neurogenic niche, and AGIQ increased the numbers of PCNA+ cells in the subgranular zone and CALB2+ hilar interneurons. Additionally, LPS alone decreased or tended to decrease the number of synaptic plasticity-related FOS+ and COX2+ granule cells and AGIQ recovered them. The results suggest that LPS administration induced acute neuroinflammation and subsequent impairment of fear memory acquisition caused by suppressed synaptic plasticity of newborn granule cells following disruptive neurogenesis. In contrast, AGIQ exhibited anti-inflammatory effects and ameliorated LPS-induced adverse effects. These results suggest that neuroinflammation is a key factor in the development of LPS-induced impairment of fear memory acquisition.

Progressive disruption of neurodevelopment by mid-gestation exposure to lipopolysaccharides and the ameliorating effect of continuous alpha-glycosyl isoquercitrin treatment.

We investigated the effect of lipopolysaccharide (LPS)-induced maternal immune activation used as a model for producing neurodevelopmental disorders on hippocampal neurogenesis and behaviors in rat offspring by exploring the antioxidant effects of alpha-glycosyl isoquercitrin (AGIQ). Pregnant Sprague-Dawley rats were intraperitoneally injected with LPS (50 μg/kg body weight) at gestational days 15 and 16. AGIQ was administered in the diet to dams at 0.5% (w/w) from gestational day 10 until weaning at postnatal day 21 and then to offspring until adulthood at postnatal day 77. During postnatal life, offspring of LPS-injected animals did not show neuroinflammation or oxidative stress in the brain. At weaning, LPS decreased the numbers of type-2b neural progenitor cells (NPCs) and PCNA+ proliferating cells in the subgranular zone, FOS-expressing granule cells, and GAD67+ hilar interneurons in the dentate gyrus. In adulthood, LPS decreased type-1 neural stem cells, type-2a NPCs, and GAD67+ hilar interneurons, and downregulated Dpysl3, Sst, Fos, Mapk1, Mapk3, Grin2a, Grin2b, Bdnf, and Ntrk2. In adults, LPS suppressed locomotor activity in the open field test and suppressed fear memory acquisition and fear extinction learning in the contextual fear conditioning test. These results indicate that mid-gestation LPS injections disrupt programming of normal neurodevelopment resulting in progressive suppression of hippocampal neurogenesis and synaptic plasticity of newborn granule cells by suppressing GABAergic and glutamatergic neurotransmitter signals and BDNF/TrkB signaling to result in adult-stage behavioral deficits. AGIQ ameliorated most aberrations in hippocampal neurogenesis and synaptic plasticity, as well as behavioral deficits. Effective amelioration by continuous AGIQ treatment starting before LPS injections may reflect both anti-inflammatory and anti-oxidative stress effects during gestation and neuroprotective effects of continuous exposure through adulthood.

Molecular and electrophysiological features of GABAergic neurons in the dentate gyrus reveal limited homology with cortical interneurons.

GABAergic interneurons tend to diversify into similar classes across telencephalic regions. However, it remains unclear whether the electrophysiological and molecular properties commonly used to define these classes are discriminant in the hilus of the dentate gyrus. Here, using patch-clamp combined with single cell RT-PCR, we compare the relevance of commonly used electrophysiological and molecular features for the clustering of GABAergic interneurons sampled from the mouse hilus and primary sensory cortex. While unsupervised clustering groups cortical interneurons into well-established classes, it fails to provide a convincing partition of hilar interneurons. Statistical analysis based on resampling indicates that hilar and cortical GABAergic interneurons share limited homology. While our results do not invalidate the use of classical molecular marker in the hilus, they indicate that classes of hilar interneurons defined by the expression of molecular markers do not exhibit strongly discriminating electrophysiological properties.

Acute Delta 9-tetrahydrocannabinol administration differentially alters the hippocampal opioid system in adult female and male rats.

Our prior studies demonstrated that the rat hippocampal opioid system can undergo sex-specific adaptations to external stimuli that can influence opioid-associated learning processes. This opioid system extensively overlaps with the cannabinoid system. Moreover, acute administration of Δ9 Tetrahydrocannabinoid (THC), the primary psychoactive constituent of cannabis, can alter cognitive behaviors that involve the hippocampus. Here, we use light and electron microscopic immunocytochemical methods to examine the effects of acute THC (5mg/kg, i.p., 1h) on mossy fiber Leu-Enkephalin (LEnk) levels and the distribution and phosphorylation levels of delta and mu opioid receptors (DORs and MORs, respectively) in CA3 pyramidal cells and parvalbumin dentate hilar interneurons of adult female and male Sprague-Dawley rats. In females with elevated estrogen states (proestrus/estrus stage), acute THC altered the opioid system so that it resembled that seen in vehicle-injected females with low estrogen states (diestrus) and males: (1) mossy fiber LEnk levels in CA2/3a decreased; (2) phosphorylated-DOR levels in CA2/3a pyramidal cells increased; and (3) phosphorylated-MOR levels increased in most CA3b laminae. In males, acute THC resulted in the internalization of MORs in parvalbumin-containing interneuron dendrites which would decrease disinhibition of granule cells. In both sexes, acute THC redistributed DORs to the near plasma membrane of CA3 pyramidal cell dendrites, however, the dendritic region varied with sex. Additionally, acute THC also resulted in a sex-specific redistribution of DORs within CA3 pyramidal cell dendrites which could differentially promote synaptic plasticity and/or opioid-associated learning processes in both females and males.

A locus coeruleus to dentate gyrus noradrenergic circuit modulates aversive contextual processing

Dysregulation in contextual processing is believed to affect several forms of psychopathology, such as post-traumatic stress disorder (PTSD). The dentate gyrus (DG), a subregion of the hippocampus, is thought to be an important brain region for disambiguating new experiences from prior experiences. Noradrenergic (NE) neurons in the locus coeruleus (LC) are more tonically active during stressful events and send dense projections to the DG, yet an understanding of their function in DG-dependent contextual discrimination has not been established. Here, we isolate a key function of the LC-NE-DG circuit in contextual aversive generalization using selective manipulations and invivo single-cell calcium imaging. We report that activation of LC-NE neurons and terminal activity results in contextual generalization. We found that these effects required β-adrenergic-mediated modulation of hilar interneurons to ultimately promote aversive generalization, suggesting that disruption of noradrenergic tone may serve as an important avenue for treating stress-induced disorders.

Decreased excitatory drive onto hilar neuronal nitric oxide synthase expressing interneurons in chronic models of epilepsy

Excitation-inhibition imbalance of GABAergic interneurons is predisposed to develop chronic temporal lobe epilepsy (TLE). We have previously shown that virtually every neuronal nitric oxide synthase (nNOS)-positive cell is a GABAergic inhibitory interneuron in the denate gyrus. The present study was designed to quantify the number of nNOS-containing hilar interneurons using stereology in pilocapine- and kainic acid (KA)-exposed transgenic adult mice that expressed GFP under the nNOS promoter. In addition, we studied the properties of miniature excitatory postsynaptic current (mEPSC) and paired-pulse response ratio (PPR) of evoked EPSC in nNOS interneurons using whole cell recording techniques. Results showed that there were fewer nNOS-immunoreactive interneurons of chronically epileptic animals. Importantly, patch-clamp recordings revealed reduction in mEPSC frequency, indicating diminished global excitatory input. In contrast, PPR of evoked EPSC following the granule cell layer stimulation was increased in epileptic animals suggesting reduced neurotransmitter release from granule cell input. In summary, we propose that impaired excitatory drive onto hippocampal nNOS interneurons may be implicated in the development of refractory epilepsy.

Stress-related memories disrupt sociability and associated patterning of hippocampal activity: a role of hilar oxytocin receptor-positive interneurons

In susceptible individuals, memories of stressful experiences can give rise to debilitating socio-affective symptoms. This occurs even when the ability to retrieve such memories is limited, as seen in patients suffering from traumatic amnesia. We therefore hypothesized that the encoding, rather than retrieval, mechanisms of stress-related memories underlie their impact on social and emotional behavior. To test this hypothesis, we used combinations of stress-enhanced and state-dependent fear conditioning, which engage different encoding mechanisms for the formation of stress-related memories. We found that the encoding of stress-enhanced state-dependent memories robustly and sex specifically impairs sociability in male mice and disrupts the asymmetry of dentate gyrus (DG)/CA3 activity accompanying social interactions. These deficits were restored by chemogenetic inactivation of oxytocin receptor-positive interneurons localized in the hilus (Oxtr-HI), and by inactivation of dorsohippocampal efferents to the caudal lateral septum. Together, our data suggest that disrupted patterning of dorsohippocampal DG/CA3 activity underlies stress-induced sociability deficits, and that Oxtr-HI can be a cellular target for improving these deficits.

Cyanidin prevents hippocampal cell death and promotes astrocytosis in kainic acid-induced neurodegeneration

Temporal lobe epilepsy (TLE) is one of the non-communicable diseases characterized by dentate granule cell dispersion (GCD) and the loss of the CA1 and CA3 neurons. This study investigated the effects of cyanidin on neurodegeneration after KA injection. Male Wistar rats were divided into a saline-injected group as a control, KA alone and cyanidin treated group at a dose of 10 mg/kg BW seven days before and after KA injection. The histomorphological analysis revealed that GCD was reduced in the ipsilateral hippocampus of cyanidin treated group when compared with KA alone (P<0.05). There was no neurodegeneration observed in the contralateral hippocampus. In contrast, neuronal degeneration was limited in the ipsilateral CA1 (P<0.01) and the hilar interneurons (P<0.001) of cyanidin treated group when compared with KA alone. However, there was no significant difference in the number of CA3 neurons of the ipsilateral between KA alone and cyanidin treated group. GFAP staining revealed a higher number of reactive astrocytes in the ipsilateral hilus (P<0.001) and molecular layer (P<0.05) of cyanidin treated group when compared with KA alone. Taken together, cyanidin could prevent neurodegeneration and promote astrocyte proliferation as well as mitigated GCD following KA-induced hippocampal injury.

Sex Differences in Neuroplasticity- and Stress-Related Gene Expression and Protein Levels in the Rat Hippocampus Following Oxycodone Conditioned Place Preference

Prescription opioid abuse is a serious public health issue. Recently, we showed that female and male Sprague–Dawley rats acquire conditioned place preference (CPP) to the mu opioid receptor agonist oxycodone. Anatomical analysis of the hippocampus from these rats unveiled sex differences in the opioid system in a way that would support excitation and opiate associative learning processes especially in females. In this study, we examined the expression and protein densities of opioid, plasticity, stress and related kinase and signaling molecules in the hippocampus of female and male rats following oxycodone CPP. Oxycodone CPP females have: a) increases in ARC (activity regulated cytoskeletal-associated protein)-immunoreactivity (ir) in CA3 pyramidal cells; b) decreases in Npy (neuropeptide Y) gene expression in the medial hippocampus but higher numbers of NPY-containing hilar interneurons compared to males; c) increases in Crhr2 (corticotropin releasing factor receptor 2) expression in CA2/3; d) increases in Akt1 (AKT serine/threonine kinase 1) expression in medial hippocampus; and e) decreases in phosphorylated MAPK (mitogen activated protein kinase)-ir in CA1 and dentate gyrus. Oxycodone CPP males have: a) increases in Bdnf (brain derived-neurotrophic factor) expression, which is known to be produced in granule cells, relative to females; b) elevated Mapk1 expression and pMAPK-ir in the dentate hilus which harbors newly generated granule cells; and c) increases in CRHR1-ir in CA3 pyramidal cell soma. These sex-specific changes in plasticity, stress and kinase markers in hippocampal circuitry parallel previously observed sex differences in the opioid system after oxycodone CPP.

Chronic immobilization stress primes the hippocampal opioid system for oxycodone-associated learning in female but not male rats.

In adult female, but not male, Sprague Dawley rats, chronic immobilization stress (CIS) increases mossy fiber (MF) Leu-Enkephalin levels and redistributes delta- and mu-opioid receptors (DORs and MORs) in hippocampal CA3 pyramidal cells and GABAergic interneurons to promote excitation and learning processes following subsequent opioid exposure. Here, we demonstrate that CIS females, but not males, acquire conditioned place preference (CPP) to oxycodone and that CIS "primes" the hippocampal opioid system in females for oxycodone-associated learning. In CA3b, oxycodone-injected (Oxy) CIS females relative to saline-injected (Sal) CIS females exhibited an increase in the cytoplasmic and total densities of DORs in pyramidal cell dendrites so that they were similar to Sal- and Oxy-CIS males. Consistent with our earlier studies, Sal- and Oxy-CIS females but not CIS males had elevated DOR densities in MF-CA3 dendritic spines, which we have previously shown are important for opioid-mediated long-term potentiation. In the dentate gyrus, Oxy-CIS females had more DOR-labeled interneurons than Sal-CIS females. Moreover, Sal- and Oxy-CIS females compared to both groups of CIS males had elevated levels of DORs and MORs in GABAergic interneuron dendrites, suggesting capacity for greater synthesis or storage of these receptors in circuits important for opioid-mediated disinhibition. However, more plasmalemmal MORs were on large parvalbumin-containing dendrites of Oxy-CIS males compared to Sal-CIS males, suggesting a limited ability for increased granule cell disinhibition. These results suggest that low levels of DORs in MF-CA3 synapses and hilar GABAergic interneurons may contribute to the attenuation of oxycodone CPP in males exposed to CIS.

Sex Differences in the Rat Hippocampal Opioid System After Oxycodone Conditioned Place Preference

Although opioid addiction has risen dramatically, the role of gender in addiction has been difficult to elucidate. We previously found sex-dependent differences in the hippocampal opioid system of Sprague–Dawley rats that may promote associative learning relevant to drug abuse. The present studies show that although female and male rats acquired conditioned place preference (CPP) to the mu-opioid receptor (MOR) agonist oxycodone (3 mg/kg, I.P.), hippocampal opioid circuits were differentially altered. In CA3, Leu-Enkephalin-containing mossy fibers had elevated levels in oxycodone CPP (Oxy) males comparable to those in females and sprouted in Oxy-females, suggesting different mechanisms for enhancing opioid sensitivity. Electron microscopy revealed that in Oxy-males delta opioid receptors (DORs) redistributed to mossy fiber-CA3 synapses in a manner resembling females that we previously showed is important for opioid-mediated long-term potentiation. Moreover, in Oxy-females DORs redistributed to CA3 pyramidal cell spines, suggesting the potential for enhanced plasticity processes. In Saline-injected (Sal) females, dentate hilar parvalbumin-containing basket interneuron dendrites had fewer MORs, however plasmalemmal and total MORs increased in Oxy-females. In dentate hilar GABAergic dendrites that contain neuropeptide Y, Sal-females compared to Sal-males had higher plasmalemmal DORs, and near-plasmalemmal DORs increased in Oxy-females. This redistribution of MORs and DORs within hilar interneurons in Oxy-females would potentially enhance disinhibition of granule cells via two different circuits. Together, these results indicate that oxycodone CPP induces sex-dependent redistributions of opioid receptors in hippocampal circuits in a manner facilitating opioid-associative learning processes and may help explain the increased susceptibility of females to opioid addiction acquisition and relapse.