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Prescription opioids are powerful pain-controlling medications that have both benefits and potentially serious risks. Morphine is one of the preferred analgesics that are widely used to treat chronic pain. However, chronic morphine exposure has been found to cause both functional and structural changes in several brain regions, including the medial prefrontal cortex (mPFC), ventral tegmental area (VTA), and hippocampus (HPC), which lead to addictive behavior. Caveolin-1 (Cav-1), a scaffolding protein of membrane lipid rafts (MLRs), has been shown to organize GPCRs and multiple synaptic signaling proteins within the MLRs to regulate synaptic signaling and neuroplasticity. Previously, we showed that in vitro morphine treatment significantly elevates Cav-1 expression and causes neuroplasticity changes. In this study, we confirmed that chronic morphine exposure can significantly increase Cav-1 expression (P < 0.05) and microtubule-associated protein (MAP-2)-positive neuronal dendritic growth in the hippocampus. Moreover, the rewarding effect and dendritic growth in the HPC induced by chronic morphine exposure were significantly inhibited by hippocampal Cav-1 knockdown. Together, these data suggest that Cav-1 in the hippocampus plays an essential role in the neuroplasticity changes that underlie morphine addiction behaviors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Long-term repeated application of morphine induces plasticity changes in sensitive neurons, which represents the neurobiological basis of morphine addiction. Caveolin-1 (Cav-1) is an important scaffolding protein that plays a key role in the regulation of neurostructural plasticity. However, in-depth studies focused on the role of Cav-1 in morphine-induced changes in neurostructural plasticity are lacking. The present study explored the effects of Cav-1 on the expression levels of 2 markers of neurite outgrowth, growth association protein 43 (GAP-43) and microtubule-associated protein 2 (MAP-2), during the process of morphine-induced changes in the structural plasticity. A primary mouse cerebral cortical neuron culture was established, and Cav-1 knockdown was achieved. The results showed that morphine at a concentration of 10.0μmol/L had no adverse effect on neuronal viability, but enhanced the Cav-1 and GAP-43 levels and induced the outgrowth of MAP-2-labeled neurites. The qRT-PCR data showed that 10.0μmol/L of morphine increased the Cav-1 mRNA level and promoted Cav-1 expression at the transcriptional level. Moreover, Cav-1 knockdown inhibited the morphine-induced upregulation of GAP-43 expression and the prolongation of MAP-2-labeled neurites. In conclusion, morphine was capable of inducing changes in the structural plasticity of primary cultured mouse cortical neurons, and Cav-1 played an important role in this process. Inhibition of Cav-1 expression reduced the morphine-induced increase in the neuronal growth markers GAP-43 and MAP-2. Thus, Cav-1 may be a potential molecular target for inhibiting morphine-induced neuroplasticity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We previously reported that novel protein kinase C (nPKC) epsilon and N-methyl-d-aspartic acid (NMDA) receptors participated in morphine preconditioning (MP)-induced neuroprotection. In this study, we used Western blot analysis, 2,3,5-triphenyltetrazolium chloride (TTC) staining and lactate dehydrogenase (LDH) leakage assay to determine the involvement of conventional PKC isoforms (cPKC) in MP-induced neuroprotection against oxygen-glucose deprivation (OGD). Hippocampus slices (400-microm thickness) from healthy male BALB/c mice exposed to OGD for 5-45 min to mimic mild, moderate and severe ischemia in the presence of MP pretreatment. We found that OGD-induced damage in neuronal cell survival rate and LDH leakage could be improved by MP pretreatment (3 microM) within 20 min of OGD, which was abolished by concomitant incubation with non-selective opioid receptor antagonist naloxone (Nal, 50 microM). The results of Western blot analysis showed that only cPKCgamma membrane translocation, not alpha, betaI and betaII, increased under the condition of OGD 10 min and 2h reperfusion (OGD/2h), and this increment of cPKCgamma membrane translocation was inhibited by MP pretreatment. To further elucidate the role of cPKCgamma in MP-induced neuroprotection, we found that cPKCgamma membrane translocation inhibitor, Go6983 (6 nM) did not affect MP-induced neuroprotection while Go6983 alone exhibited a significant inhibition on OGD-induced increment in LDH leakage and decrease in cell survival rate. These phenomena were defined by the results that Go6983 could restore OGD-induced cPKCgamma membrane translocation, but had no further effect on MP-induced inhibition of cPKCgamma membrane translocation. These results demonstrated that MP can reduce OGD-induced neuronal injuries, and the down-regulation of cPKCgamma membrane translocation might be involved in the neuroprotection.

Elevated introcular pressure (IOP)-induced retinal neuron ischemic death includes an early phase of necrosis and prolonged phase of apoptosis. We used this ischemic model to observe the changes of sortilin and p75(NTR) protein expressions in rat retina. The results of Western blot analysis showed the expression of p75(NTR) at the band of 75 (mature form), 60 (non-glycosylated pieces) and 50 kDa (ectodomain shedding pieces), and the expression of sortilin at the 95 and 90 kDa (the mature form). The protein expressions of p75(NTR) (60 and 50 kDa pieces) and sortilin (90 kDa) increased significantly (p < 0.05) at days 3, 5 and 7 after retinal ischemia. This effect was also confirmed by immunofluorescence staining. Sortilin was primarily present in cell membrane of the ganglion cells layer (GCL) and large ganglion cell bodies by immunofluorescence labeling. There was little expression of p75(NTR) in the normal retina, while expression increased extensively in GCL, inner plexiform layer (IPL) and inner nuclear layer (INL) after retinal ischemia. p75(NTR) was shown to co-localize with neurofilament in the axons of neuronal cells by double-labeling. These results suggested that the protein expressions of 60 and 50 kDa forms of p75(NTR), and the 90 kDa mature form of sortilin increased in ischemia-induced retinal neuron of rats.

Accumulated studies have suggested that mitogen-activated protein kinase (MAPK) play a pivotal role in the development of cerebral hypoxic preconditioning (HPC). By using our "auto-hypoxia"-induced HPC mouse model, we have reported increased phosphorylation level of p38 MAPK, and decreased phosphorylation and protein expression levels of extracellular signal regulated kinases 1/2 (ERK1/2) in the brain of HPC mice. In the current study, we investigated the involvement of c-Jun N-terminal kinase (JNK) in the brain of HPC mice. By using Western blot analysis, we found that the phosphorylation levels of JNK at Thr183 and Tyr185 sites (phospho-Thr183/Tyr185 JNK), but not its protein expression, increased significantly (p<0.05, n=6 for each group) both in the hippocampus and frontal cortex of early (H1-H4) and delayed (H5 and H6) HPC mice than that of the normoxic group (H0, n=6). Similarly, enhanced phospho-Thr183/Tyr185 JNK was also observed by immunostaining in the hippocampus and frontal cortex of mice following series of hypoxic exposures (H3 and H6). In addition, we found that phospho-Thr183/Tyr185 JNK predominantly co-localized with a neuron-specific protein, neurogranin, in both the hippocampus and frontal cortex of HPC mice (H3) by using double-labeled immunofluorescence. These results suggest that the increased neuron-specific phosphorylation of JNK at Thr183/Tyr185, not protein expression, might be involved in the development of cerebral HPC of mice.

Nitric oxide (NO) is an important gas mediator in the signal transduction cascade regulating osmotic function in the hypothalamo-neurohypophysial system. We previously found that increased nitric oxide synthase (NOS) activity in the supraoptic nuclei (SON) and neural lobe following osmotic stimulation and NO could regulate the expression of Ca(2+)-activated K(+) channel (BK channels) protein in the magnocellular system during dehydration. The aim of the current study is to examine the role of NO in the regulation of nitrosocysteine and BK channel protein in the magnocellular system in dehydrated animals. Using Western blot analysis and quantitative immunofluorescent staining study, we found that water deprivation in rats significantly enhanced the expression of nitrosocysteine protein in SON and neural lobes. Immunohistochemistry study indicated that dehydration significantly increased the profiles of SON neurons co-expressing nitrosocysteine with BK-channel protein. Intracerebroventricular administration of L-NAME (an inhibitor of NO synthase) significantly reduced the neuronal profiles of nitrosocysteine, as well as their co-expression with BK-channel in SON of dehydrated rats. However, treatment of sodium nitroprusside (a donor of NO) increased this co-expression. Our results indicate that NO signaling cascade may control the expression of BK channels through the regulation of nitrosocysteine in SON and neural lobe of rats during osmotic regulation.

Studies have demonstrated that optic nerve transection results in apoptotic cell death of retinal ganglion cells (RGCs) and neurons within lateral geniculate nucleus (LGN). Heat shock protein (Hsp) 70 was reported to be involved in protecting cells from injury under various pathological conditions in vivo and in vitro. To determine the involvement of Hsp70 in protecting neurons within LGN against damage or loss induced by optic nerve injuries, we observed the changes in protein expression and distribution of Hsp70 in LGN at days 1, 3, 7, 14 and 28 after unilateral optic nerve transection in the left eye of Sprague-Dawley rats by using Western blot analysis and immunohistochemical staining. We found that the levels of Hsp70 protein expression increased significantly (p < 0.05, n = 6 for each group) in both right and left LGN of rats following left optic nerve transection 1-7 days. The maximum of Hsp70 expression reached at day 3. However, Hsp70 protein expression levels in both right and left LGN returned to control levels at 14 and 28 days after left optic nerve lesion. In addition, the increased Hsp70 expression, which mainly localized in the intergeniculate leaflet of LGN, was also observed by immunostaining in right LGN at the end of day 3 after the lesion. These results suggest that increased expression of Hsp70 may be involved in protecting neurons within LGN against damage or loss induced by left optic nerve transection at early stage.

Nitric oxide (NO) is a gas molecule to signal neurotransmission in the hypothalamo-neurohypophysial system during osmotic regulation. We previously reported that osmotic stimulation increased nitric oxide synthase (NOS) activity in the supraoptic nuclei (SON) and neural lobe. The aim of this study is to define the role of NO in the regulation of Ca(2+)-activated K(+) channels (BK channels) expression in the magnocellular system following dehydration. We used Western blot analysis and quantitative immunocytochemistry to conduct the experiment in rats. In the immunoblot study, we found that water deprivation significantly increased the expression of BK channels in the SON and neural lobes. Dehydration also enhanced the profiles of neurons expressing vasopressin and oxytocin significantly. In about 70% of these neurons, BK channels were co-localized in the same neuron, and their expression increased significantly during dehydration. We further examined the effects of intracerebroventricular administration of sodium nitroprusside (a donor of NO) and L-NAME (an inhibitor of NO synthase) on expression of BK channels in the SON. We found that compared to animals treated with the donor of NO, there were significant decreases in the expression of BK proteins in animals receiving L-NAME. These results suggest that NO may enhance the expression of BK channels in the supraoptic nuclei and neural lobe of rats following dehydration.

Accumulated reports have suggested that activation of protein kinase C (PKC) isoforms may involve the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the neuronal response to hypoxic stimuli. We have previously demonstrated that the membrane translocation or activation of conventional PKC (cPKC) betaII, gamma and novel PKC (nPKC) epsilon are increased in the early phase of cerebral hypoxic preconditioning in mice. However, the role of ERK1/2 in the development of cerebral hypoxic preconditioning is unclear. In the current study, we used Western blot analysis to investigate the effects of repetitive hypoxic exposure (H0-H6, n=6 for each group) on the levels of phosphorylation and protein expression of ERK1/2 in the frontal cortex and the whole hippocampus of mice. We found that the levels of phosphorylated ERK1/2, not protein expression of ERK1/2, decreased significantly in both cortex and hippocampus of the early hypoxic preconditioned mice (H1-H4), when compared to that of the normoxic group (p<0.05). In addition, a significant decrease (p<0.05) in the ERK1/2 protein expression, not the phosphorylated form of ERK1/2, was found both in the frontal cortex and hippocampus of mice followed hypoxia with previous hypoxia (H5 and H6). These results suggest that the decreased phosphorylation and downregulation of protein expression of ERK1/2 might be involved in the development of hypoxic preconditioning.

Neurogranin/RC3 (Ng/rodent cortex-enriched mRNA clone #3), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low Ca(2+) levels. Neurotransmitters triggering influx calcium induce neurogranin phosphorylation by PKC in physiological or pathophysiological conditions. Phosphorylated Ng reduces the affinity of Ng to bind CaM, which may affect the activities of calmodulin-dependent downstream enzymes, such as nitric oxide synthase (NOS), CaM-dependent protein kinase II (CaMKII) and adenylate cyclase (AC). These protein enzymes have been reported to play key roles in the development of ischemic/hypoxic preconditioning (I/HPC). We previously demonstrated that activation of cPKCbetaII and gamma isoforms may be involved in the early phase of cerebral hypoxic preconditioning. However, as a substrate of PKC, the role of Ng in the onset of cerebral hypoxic preconditioning is unknown. In this study, we examined the effects of repetitive hypoxic exposure on the status of Ng phosphorylation in the cortex and hippocampus of mice. Using Western blot analysis, we found that the levels of Ng phosphorylation in the cortex and hippocampus of the hypoxic group of mice increased significantly from that of the normoxic group (p<0.05). These results suggest that neurogranin protein may be involved in the development of cerebral hypoxic preconditioning.

Previous studies have shown that the level of total conventional protein kinase C (cPKC) membrane translocation (activation) was increased in the brain of hypoxic preconditioned mice. In order to find out which isoform of cPKC may participate in the development of cerebral hypoxic preconditioning (HPC), we used Western bolt and immunohistochemistry to observe the effects of repetitive hypoxic exposure (H1-H6, n = 6 for each group) on the level of cPKC isoform-specific protein expression and its membrane translocation in the cortex and hippocampus of mice. We found that the levels of cPKC betaII and gamma membrane translocation were increased significantly (p < 0.05 versus normoxic H0 group, n = 6) in response to repetitive hypoxic exposure (H1-H4) at an early phase of hypoxic preconditioning, but no significant changes of cPKC alpha and betaI membrane translocation were found during cPKC alpha, betaI, betaII and gamma protein expression both in hippocampus and cortex. In addition, an extensive subcellular redistribution of cPKC betaII and gamma was detected by immunohistochemistry staining in the cortex after repetitive hypoxic exposures (H3). However, a significant decrease in the expression of cPKC gamma protein (p < 0.05 versus H0 group) was found only in the cortex of delayed hypoxic preconditioned mice (H5-H6). These results suggest that the activation of cPKC betaII and gamma may be involved in the early phase of cerebral hypoxic preconditioning and the changes in cPKC gamma protein expression may participate in the development of the late phase of cerebral hypoxic preconditioning as well as selective vulnerability to hypoxia both in cortex and hippocampus.