Whole transcriptome analysis to identify non-coding RNA regulators and hub genes in sperm of non-obstructive azoospermia by microarray, single-cell RNA sequencing, weighted gene co-expression network analysis, and mRNA-miRNA-lncRNA interaction analysis.

BACKGROUND: The issue of male fertility is becoming increasingly common due to genetic differences inherited over generations. Gene expression and evaluation of non-coding RNA (ncRNA), crucial for sperm development, are significant factors. This gene expression can affect sperm motility and, consequently, fertility. Understanding the intricate protein interactions that play essential roles in sperm differentiation and development is vital. This knowledge could lead to more effective treatments and interventions for male infertility. MATERIALS AND METHODS: Our research aim to identify new and key genes and ncRNA involved in non-obstructive azoospermia (NOA), improving genetic diagnosis and offering more accurate estimates for successful sperm extraction based on an individual's genotype. RESULTS: We analyzed the transcript of three NOA patients who tested negative for genetic sperm issues, employing comprehensive genome-wide analysis of approximately 50,000 transcript sequences using microarray technology. This compared gene expression profiles between NOA sperm and normal sperm. We found significant gene expression differences: 150 genes were up-regulated, and 78 genes were down-regulated, along with 24 ncRNAs up-regulated and 13 ncRNAs down-regulated compared to normal conditions. By cross-referencing our results with a single-cell genomics database, we identified overexpressed biological process terms in differentially expressed genes, such as "protein localization to endosomes" and "xenobiotic transport." Overrepresented molecular function terms in up-regulated genes included "voltage-gated calcium channel activity," "growth hormone-releasing hormone receptor activity," and "sialic acid transmembrane transporter activity." Analysis revealed nine hub genes associated with NOA sperm: RPL34, CYB5B, GOL6A6, LSM1, ARL4A, DHX57, STARD9, HSP90B1, and VPS36. CONCLUSIONS: These genes and their interacting proteins may play a role in the pathophysiology of germ cell abnormalities and infertility.

Adolescent morphine exposure changes the endogenous vlPAG opioid response to inflammatory pain in rats.

Adolescence is one of the most critical periods for brain development, and exposure to morphine during this period can have long-life effects on pain-related behaviors. The opioid system in the periaqueductal gray (PAG) is highly vulnerable to drug exposure. However, the impact of adolescent morphine exposure (AME) on the endogenous opioid system in the PAG is currently unknown. This study aims to investigate the long-lasting effects of AME on the endogenous opioid system and its involvement in altering nociceptive behaviors. Adolescent rats were given escalating doses of morphine (2.5-25 mg/kg, subcutaneous) or an equal volume of saline twice daily for 10 consecutive days (PND 31-40). After a 30-day washout period, adult rats underwent formalin tests following microinjection of morphine, naloxone, or saline into the ventrolateral PAG (vlPAG) region. The results indicated that morphine microinjection into the vlPAG of the adolescent morphine-treated group significantly reduced the nociceptive score. However, the analgesic response to morphine in this group was significantly lower compared to the saline-treated group during adolescence. Additionally, the nociceptive score significantly increased following naloxone but not saline microinjection into the vlPAG of the saline-treated group during adolescence, rather than the morphine-treated one. These findings indicate that AME has long-lasting effects on the endogenous opioid system in the vlPAG, which can consequently alter behaviors related to inflammatory pain in adulthood.

Unraveling the Significance of Nanog in the Generation of Embryonic Stem-like Cells from Spermatogonia Stem Cells: A Combined In Silico Analysis and In Vitro Experimental Approach.

Embryonic stem-like cells (ES-like cells) are promising for medical research and clinical applications. Traditional methods involve "Yamanaka" transcription (OSKM) to derive these cells from somatic cells in vitro. Recently, a novel approach has emerged, obtaining ES-like cells from spermatogonia stem cells (SSCs) in a time-related process without adding artificial additives to cell cultures, like transcription factors or small molecules such as pten or p53 inhibitors. This study aims to investigate the role of the Nanog in the conversion of SSCs to pluripotent stem cells through both in silico analysis and in vitro experiments. We used bioinformatic methods and microarray data to find significant genes connected to this derivation path, to construct PPI networks, using enrichment analysis, and to construct miRNA-lncRNA networks, as well as in vitro experiments, immunostaining, and Fluidigm qPCR analysis to connect the dots of Nanog significance. We concluded that Nanog is one of the most crucial differentially expressed genes during SSC conversion, collaborating with critical regulators such as Sox2, Dazl, Pou5f1, Dnmt3, and Cdh1. This intricate protein network positions Nanog as a pivotal factor in pathway enrichment for generating ES-like cells, including Wnt signaling, focal adhesion, and PI3K-Akt-mTOR signaling. Nanog expression is presumed to play a vital role in deriving ES-like cells from SSCs in vitro. Finding its pivotal role in this path illuminates future research and clinical applications.

The synthesis of broccoli sprout extract-loaded silk fibroin nanoparticles as efficient drug delivery vehicles: development and characterization.

Targeted drug delivery of biological molecules using the development of biocompatible, non-toxic and biodegradable nanocarriers can be a promising method for cancer therapy. In this study, silk fibroin protein nanoparticles (SFPNPs) were synthesized as a targeted delivery system for sulforaphane-rich broccoli sprout extract (BSE). The BSE-loaded SFPNPs were conjugated with polyethylene glycol and folic acid, and then their physicochemical properties were characterized via UV-Vis, XRD, FTIR, DLS, FE-SEM and EDX analyses. In vitro, the release profile, antioxidant and anticancer activities of NPs were also studied. The FE-SEM and DLS analyses indicated stable NPs with an average size of 88.5 nm and high zeta potential (-32 mV). The sulforaphane release profile from NPs was pH-dependent, with the maximum release value (70%) observed in simulated intestinal fluid (pH = 7.4). Encapsulation of BSE also decreased the release rate of sulforaphane from the capsules compared to free BSE. In vitro cytotoxicity of BSE and NPs on breast cancer cell lines (MCF-7) was concentration-dependent, and the IC50 for BSE and NPs were 54 and 210 µg ml-1, respectively. Moreover, the NPs demonstrated no appreciable cytotoxicity in normal mouse fibroblast (L929) cell lines. These results indicated that biocompatible NPs synthesized as controlled and long-term targeted drug delivery systems can be a potential candidate for breast cancer therapy.

Functions and mechanism of noncoding RNA in regulation and differentiation of male mammalian reproduction.

Noncoding RNAs (ncRNAs) are active regulators of a wide range of biological and physiological processes, including the majority of mammalian reproductive events. Knowledge of the biological activities of ncRNAs in the context of mammalian reproduction will allow for a more comprehensive and comparative understanding of male sterility and fertility. In this review, we describe recent advances in ncRNA-mediated control of mammalian reproduction and emphasize the importance of ncRNAs in several aspects of mammalian reproduction, such as germ cell biogenesis and reproductive organ activity. Furthermore, we focus on gene expression regulatory feedback loops including hormones and ncRNA expression to better understand germ cell commitment and reproductive organ function. Finally, this study shows the role of ncRNAs in male reproductive failure and provides suggestions for further research.

Acute adolescent morphine exposure improves dark avoidance memory and enhances long-term potentiation of ventral hippocampal CA1 during adulthood in rats.

Adolescence represents a distinctive vulnerable period when exposure to stressful situations including opioid exposure can entail lasting effects on brain and can change neural mechanisms involved in memory formation for drug-associated cues, possibly increasing vulnerability of adolescents to addiction. Herein, the effects of acute adolescent morphine exposure (AAME, two injections of 2.5 mg/kg SC morphine on PND 31) were therefore investigated 6 weeks later (adulthood) on avoidance memory and hippocampal long-term potentiation (LTP) at Schaffer collateral-CA1 synapses in transvers slices from the ventral hippocampus in adult male rats using field recordings technique. Animal body weight was measured from PND 31 throughout PND 40 and also in four time points with 1 week intervals from adolescence to adulthood (PNDs 48, 55, 62 and 69) to evaluate the effect of AAME on the weight gain. We showed that there were no effects on body weight, anxiety-like behaviour and locomotor activity, even until adulthood. There was an improved dark avoidance memory during adulthood. Finally, AAME had no effects on baseline synaptic responses and resulted in a decrease in the mean values of the field excitatory postsynaptic potential slopes required to evoke the half-maximal population spike amplitude and an enhancement of LTP magnitude (%) in the ventral CA1 during adulthood. Briefly, our results suggest long-lasting effects of acute adolescent morphine exposure on the ventral hippocampus, which begin the enhancing of synaptic plasticity and the improving of emotional memory in adulthood.

Advanced isolation, expansion and characterization research study on pig testicular cells during differentiation and proliferation.

Spermatogenesis is the complex process of sperm production to transmit paternal genetic information to the subsequent generation. This process is determined by the collaboration of several germ and somatic cells, most importantly spermatogonia stem cells and Sertoli cells. To characterize germ and somatic cells in the tubule seminiferous contort in pig and consequently has an impact on the analysis of pig fertility. Germ cells were extracted from pig testis by enzymatic digestion before being expanded on Sandos inbred mice (SIM) embryo-derived thioguanine and ouabain resistant fibroblasts (STO) feeder layer supplemented with FGF, EGF, and GDNF. Immunohistochemistry (IHC) and immunocytochemistry (ICC) analysis for Sox9, Vimentin, and PLZF markers were performed to examine the generated colonies of pig testicular cells. Electron microscopy was also utilized to analyze the morphological features of the extracted pig germ cells. IHC analysis revealed that Sox9 and Vimentin were expressed in the basal compartment of the seminiferous tubules. Moreover, ICC results showed that the cells have low expression of PLZF while expressing Vimentin. The heterogeneity of the in vitro cultured cells was detected via morphological analysis by the electron microscope. In this experimental study, we tried to reveal exclusive information which obviously could be helpful for future success in the achievement of proper therapies against infertility and sterility as an important global issue.

Immunohistochemistry and immunocytochemistry analysis of PLZF and VASA in mice testis during spermatogenesis.

Spermatogonial stem cells (SSCs) are the basis of male spermatogenesis and fertility. SSCs are distinguished by their ability to self-renew and differentiate into spermatozoa throughout the male reproductive life and pass genetic information to the next generation. Immunohistochemistry (IHC), immunocytochemistry (ICC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze the expression of PLZF and VASA in mice testis tissue. In this experimental study, whereas undifferentiated spermatogonial cells sharply expressed PLZF, other types of germ cells located in the seminiferous tubule were negative for this marker. Conversely, the germ cells near the basal membrane of the seminiferous tubule showed VASA expression, whereas the undifferentiated germ cells located on the basal membrane were negative. The ICC analysis indicated higher expression of PLZF in the isolated undifferentiated cells compared with differentiated germ cells. Fluidigm real-time RT-PCR results demonstrated a significant expression (P < 0.05) of VASA in the SSCs compared with differentiated cells and also showed expression of PLZF in undifferentiated spermatogonia. These results clearly proved the role of PLZF as a specific marker for SSCs, and can be beneficial for advanced research on in vitro differentiation of SSCs to functional sperms.

Biogenic synthesized copper oxide nanoparticles by Bacillus subtilis: Investigating antibacterial activity on the mexAB-oprM efflux pump genes and cytotoxic effect on MCF-7 cells.

One of the main characteristics of Pseudomonas aeruginosa is remarkable intrinsic antibiotic resistance which is associated with production of ß-lactamases and the expression of inducible efflux pumps. Nanoparticles (NPs) are a novel option for coping with this resistant bacteria. Hence, the aim of present study was production of CuO NPs via Bacillus subtilis and applied them to deal with resistant bacteria. For this purpose, first NPs were synthesized and were analyzed with different standard techniques containing scanning electron microscope, Fourier-transform infrared spectroscopy, and X-ray powder diffraction. Microdilution Broth Method and real-time polymerase chain reaction were used to antibacterial properties of the CuO NPs and expression of mexAB-oprM in clinical samples of P. aeruginosa, respectively. The cytotoxic effect of CuO NPs was also evaluated on MCF7 as a breast cancer cell line. Finally, the data were analyzed by one-way analysis of variance and Tukey's tests. The size of CuO NPs was in the range of 17-26 nm and showed antibacterial effect at <1000 µg/mL concentrations. Our evidence noted that the antibacterial effects of the CuO NPs occurred through the downregulation of mexAB-oprM and upregulation of mexR. The interesting point was that CuO NPs had an inhibitory effect on MCF7 cell lines with the optimal inhibition concentration at IC50 = 25.73 µg/mL. Therefore, CuO NPs can be considered as a promising medical candidate in the pharmaceutical industry.

A review of protein-protein interaction and signaling pathway of Vimentin in cell regulation, morphology and cell differentiation in normal cells.

The Vimentin intermediate filament (VIF) is an essential cytoskeleton component. It shows dynamically changing expression patterns throughout various phases of the differentiation process, suggesting that the protein is physiologically important. Vimentin's essential functions have recently been clear, so Vimentin-deficient of animals was described as a change of morphology and signaling pathway. Recent research has discovered many vital roles for Vimentin that were previously unknown. VIF emerges as an organizer of many essential proteins involved in movement and cell signaling. The highly dynamic and complicated phosphorylation of VIF seems to be a regulator mechanism for various activities. Changes in IF expression patterns are often linked with cancer progression, especially those leading to enhanced invasion and cellular migration. This review will discuss the function of Vimentin intermediate filaments in normal cell physiology, cell adhesion structures, cell shape, and signaling pathways. The genes interaction and gene network linked with Vimentin will be discussed in more studies. However, research aimed at understanding the function of Vimentin in different signaling cascades and gene interactions might offer novel methods for creating therapeutic medicines. Enrichr GEO datasets used gene ontology (GO) and pathway enrichment analyses. STRING online was used to predict the functional connections of proteins-proteins, followed by Cytoscape analysis to find the master genes. Cytoscape and STRING research revealed that eight genes, Fas, Casp8, Casp6, Fadd, Ripk1, Des, Tnnc2, and Tnnt3, were required for protein-protein interactions with Vimentin genes involved in cell differentiation.

Prenatal exposure to morphine enhances excitability in locus coeruleus neurons.

Opioid abuse during pregnancy may have noteworthy effects on the child's behavioral, emotional and cognitive progression. In this study, we assessed the effect of prenatal exposure to morphine on electrophysiological features of locus coeruleus (LC) noradrenergic neurons which is involved in modulating cognitive performance. Pregnant dams were randomly divided into two groups, that is a prenatal saline treated and prenatal morphine-treated group. To this end, on gestational days 11-18, either morphine or saline (twice daily, s.c.) was administered to pregnant dams. Whole-cell patch-clamp recordings were conducted on LC neurons of male offspring. The evoked firing rate, instantaneous frequency and action potentials half-width, and also input resistance of LC neurons significantly increased in the prenatal morphine group compared to the saline group. Moreover, action potentials decay slope, after hyperpolarization amplitude, rheobase current, and first spike latency were diminished in LC neurons following prenatal exposure to morphine. In addition, resting membrane potential, rise slope, and amplitude of action potentials were not changed by prenatal morphine exposure. Together, the current findings show a significant enhancement in excitability of the LC neurons following prenatal morphine exposure, which may affect the release of norepinephrine to other brain regions and/or cognitive performances of the offspring.

Microarray and in silico analysis of DNA repair genes between human testis of patients with nonobstructive azoospermia and normal cells.

DNA repair processes are critical to maintaining genomic integrity. As a result, dysregulation of repair genes is likely to be linked with health implications, such as an increased prevalence of infertility and an accelerated rate of aging. We evaluated all the DNA repair genes (322 genes) by microarray. This study has provided insight into the connection between DNA repair genes, including RAD23B, OBFC2A, PMS1, UBE2V1, ERCC5, SMUG1, RFC4, PMS2L5, MMS19, SHFM1, INO80, PMS2L1, CHEK2, TRIP13, and POLD4. The microarray analysis of six human cases with different nonobstructive azoospermia revealed that RAD23B, OBFC2A, PMS1, UBE2V1, ERCC5, SMUG1, RFC4, PMS2L5, MMS19, SHFM1, and INO80 were upregulated, and expression of PMS2L1, CHEK2, TRIP13, and POLD4 was downregulated versus the normal case. For this purpose, Enrich Shiny GO, STRING, and Cytoscape online evaluation was applied to predict proteins' functional and molecular interactions and then performed to recognize the master pathways. Functional enrichment analysis revealed that the biological process (BP) terms "base-excision repair, AP site formation," "nucleotide-excision repair, DNA gap filling," and "nucleotide-excision repair, preincision complex assembly" was significantly overexpressed in upregulated differentially expressed genes (DEGs). BP analysis of downregulated DEGs highlighted "histone phosphorylation," "DNA damage response, detection DNA response," "mitotic cell cycle checkpoint signaling," and "double-strand break repair." Overrepresented molecular function (MF) terms in upregulated DEGs included "Oxidized base lesion DNA N-glycosylase activity," "uracil DNA N-glycosylase activity," "bubble DNA binding" and "DNA clamp loader activity." Interestingly, MF investigation of downregulated DEGs showed overexpression in "heterotrimeric G-protein complex," "5'-deoxyribose-5-phosphate lyase activity," "minor groove of adenine-thymine-rich DNA binding," and "histone kinase activity." Our findings suggest that these genes and their interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.

Development of anxiety-like behaviors during adolescence: Persistent effects of adolescent morphine exposure in male rats.

Epidemiological studies show the prevalence of opioid use, misuse and abuse in adolescents, which imposes social and economic accountability worldwide. Chronic opioid exposure, especially in adolescents, may have lasting effects on emotional behaviors that persist into adulthood. The current experiments were therefore designed to study the effects of sustained opioid exposure during adolescence on anxiety-like behaviors. Adolescent male Wistar rats underwent increasing doses of morphine for 10 days (PNDs 31-40). After that the open field test (OFT) and elevated plus maze (EPM) test were performed over a 4-week postmorphine treatment from adolescence to adulthood. Moreover, the weight of the animals was measured at these time points. We found that chronic adolescent morphine exposure reduces the weight gain during the period of morphine treatment and 4 weeks after that. It had no significant effect on the locomotor activity in the animals. Moreover, anxiolytic-like behavior was observed in the rats exposed to morphine during adolescence evaluated by OFT and EPM test. Thus, long-term exposure to morphine during adolescence has the profound potential of altering the anxiety-like behavior profile in the period from adolescence to adulthood. The maturation of the nervous system can be affected by drug abuse during the developmental window of adolescence and these effects may lead to behaviorally stable alterations.

Review of Sertoli cell dysfunction caused by COVID-19 that could affect male fertility.

Spermatogenesis is a complex and elaborate differentiation process and is vital for male fertility. Sertoli cells play a major role in fertility and induce spermatogenesis by protecting, nourishing, and supporting germ cells. It has been speculated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could directly affect the male reproductive system, and therefore heredity and fertility. The similarity of SARS-CoV-2 to SARS-CoV could confirm this hypothesis because both viruses use angiotensin-converting enzyme (ACE2) as the receptor to enter human cells. ACE2 is expressed by Sertoli cells and other testicular cells, therefore COVID-19 has the potential to impair fertility by destroying Sertoli cells. This hypothesis should be evaluated and confirmed by monitoring fertility in patients with COVID-19.

Whole Exome Sequencing and In Silico Analysis of Human Sertoli in Patients with Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA) is a serious cause of male infertility. The Sertoli cell responds to androgens and takes on roles supporting spermatogenesis, which may cause infertility. This work aims to enhance the genetic diagnosis of NOA via the discovery of new and hub genes implicated in human NOA and to better assess the odds of successful sperm extraction according to the individual's genotype. Whole exome sequencing (WES) was done on three NOA patients to find key genes involved in NOA. We evaluated genome-wide transcripts (about 50,000 transcripts) by microarray between the Sertoli of non-obstructive azoospermia and normal cells. The microarray analysis of three human cases with different non-obstructive azoospermia revealed that 32 genes were upregulated, and the expressions of 113 genes were downregulated versus the normal case. For this purpose, Enrich Shiny GO, STRING, and Cytoscape online evaluations were applied to predict the functional and molecular interactions of proteins and then recognize the master pathways. The functional enrichment analysis demonstrated that the biological process (BP) terms "inositol lipid-mediated signaling", "positive regulation of transcription by RNA polymerase II", and "positive regulation of DNA-templated transcription" significantly changed in upregulated differentially expressed genes (DEGs). The BP investigation of downregulated DEGs highlighted "mitotic cytokinesis", "regulation of protein-containing complex assembly", "cytoskeleton-dependent cytokinesis", and the "peptide metabolic process". Overrepresented molecular function (MF) terms in upregulated DEGs included "ubiquitin-specific protease binding", "protease binding", "phosphatidylinositol trisphosphate phosphatase activity", and "clathrin light chain binding". Interestingly, the MF analysis of the downregulated DEGs revealed overexpression in "ATPase inhibitor activity", "glutathione transferase activity", and "ATPase regulator activity". Our findings suggest that these genes and their interacting hub proteins could help determine the pathophysiologies of germ cell abnormalities and infertility.

Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice.

BACKGROUND: Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. METHODS: In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology-immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer. RESULTS: ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P < 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. CONCLUSIONS: In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

Spermatogonia (SSCs) in the testis transmit genetic information to the next generation. Successful SSC transplantation into infertile men is an advanced therapeutic application in reproductive biology research. In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology­immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. IMH analysis showed different expression levels of Zbtb16 in both populations. Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene and the down-regulated expression of DAZL, VASA, and Zbtb16 during SSCs differentiation of (P < 0.05). Flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. We performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm. Data analysis confirmed that zbtb16 is expressed in the undifferentiated germ cells located on the basal membrane of seminiferous tubules and SSCs in vitro. Also, spermatogenesis was resumed, and fertility improved after transplantation of undifferentiated cells into busulfan-treated mice; thus, improvements in vitro SSCs transplantation, isolation and culture would be helpful in future clinical treatments to solve the reproductive problems of families influenced by infertility.

Maternal deprivation induces persistent adaptations in putative dopamine neurons in rat ventral tegmental area: in vivo electrophysiological study.

Early life aversive experiences can trigger persistent deficits in neuronal signaling within the mesolimbic pathway, most notably in the dopamine (DA) neurons of the ventral tegmental area (VTA). The identity of such cellular mechanisms currently appears as an important issue. To address this concern, we investigated whether early life maternal deprivation (MD) would affect the electrical activity of VTA DA neurons, via in vivo extracellular single-unit recording. Male Wistar rats were deprived of their dams for 3 h per day from postnatal days (PND) 1-14. Thereafter, the adult animals (PND 70-80) were tested for the discharge activity of putative VTA DA neurons. The VTA DA neurons displayed a decrease in firing rate and an increase in the variability of baseline discharge activity in deprived animals. MD also caused a decrease in burst firing of VTA DA neurons compared to control subjects. In summary, early life MD induces a hypoactive VTA DA system, which may contribute to lifespan psychopathologies.

PKC inhibitor reversed the suppressive effect of orexin-A on IPSCs of locus coeruleus neurons in naloxone-induced morphine withdrawal.

The locus coeruleus (LC) as a target of addictive drugs receives a dense projection of orexinergic fibres from the lateral hypothalamus (LH) and is accordingly a candidate site for the expression of the somatic aspects of morphine withdrawal. Recently it has been shown that the inhibitory synaptic currents of LC neurons decrease partly through orexin type 1 receptors in the context of naloxone-induced morphine withdrawal; however, its cellular mechanism remains unclear. In this study, whole-cell patch clamp recordings of LC neurons in brainstem slices were used to investigate the impact of protein kinase C (PKC) on GABAergic inhibitory post-synaptic currents (IPSCs) in the context of naloxone-induced morphine withdrawal. Male Wistar rats (P14-P21) received morphine (20 mg/kg, i.p.) daily for 7 consecutive days to induce morphine dependency. Our results showed that the application of PKC inhibitor (Go 6983; 1 µM) alone did not decrease the probability of GABA release in the LC neurons of the morphine-treated rats in the presence of naloxone. Although, Go 6983 reversed the reduction of the amplitude of evoked IPSCs (eIPSCs) and spontaneous IPSCs (sIPSCs) frequency induced by orexin-A but did not change the sIPSCs amplitude. These results indicate that the suppressive effect of orexin-A on IPSCs is probably reversed by PKC inhibitor in the LC neurons of morphine-treated rats in the context of naloxone withdrawal.

Adolescent morphine exposure increases nociceptive behaviors in rat model of formalin test.

The number of adolescents who use illicit drugs has increased dramatically. Adolescence is a critical period for brain development and maturation. The importance of the study of pain perception and the possible mechanisms involved is crystal clear. Up until now, there has been no evidence regarding the long-term effect of adolescence morphine administration on pain perception. The objective of the present study was to investigate long-lasting effect of adolescent morphine exposure on pain perception as well as analgesic response to a single dose of morphine injection. Adolescent and adult rats received morphine or saline, and then after 30 days of washout period, formalin test was performed. To evaluate morphine analgesia, in a separate group of animals, formalin test was performed after injection of a single dose of morphine during adulthood. The results demonstrated that the adolescent rats treated by morphine exhibited higher pain-related behaviors compared to the control group, while the same results were not observed in adult rats that had been treated by morphine. Moreover, there was no significant difference in analgesic response to a single dose of morphine between two experimental groups. This study demonstrates enduring effect of morphine exposure during adolescence on pain perception.

Exposure to opiates in male adolescent rats alters pain perception in the male offspring.

During the past decades, the use/misuse of opioids has increased dramatically among adolescent population. It is now well acknowledged that various morphological and physiological changes occur in the brain during adolescence. During this critical period, brain development and maturation could be affected by several factors including stress, drug abuse, nutritional status, etc. Although studies on transgenerational effects of substances such as alcohol, nicotine, and cocaine have focused on both paternal and maternal drug exposure, most reports on transgenerational effects of morphine are restricted to maternal exposure. Thus, in this study, we aimed to investigate the transgenerational effect of paternal morphine exposure during adolescence on pain perception and antinociceptive effect of morphine in rat offspring. Male rats received escalating doses of morphine for 10 days during postnatal days 31-40. Twenty days after the last morphine injection, male rats were mated with intact female rats, and then behavioral tests were conducted on the male offspring on postnatal day 60. Pain perception and morphine antinociception were evaluated using the formalin test. Our results demonstrated that morphine-sired and saline-sired animals differed in the interphase and phase 2 of the formalin test. These findings indicate a significant transgenerational effect of paternal morphine exposure on pain-related behaviors in rat offspring.