Studies have shown that following opiate withdrawal, the spontaneous discharge rate of locus coeruleus (LC) neurons remarkably increases. Combination of intrinsic mechanisms with extrinsic excitatory modulations mediates the withdrawal-induced hyperactivity of LC neurons. The nucleus paragigantocellularis (PGi) provides the main excitatory inputs to LC and plays a pivotal role in opiate withdrawal. In the present study the direct facilitatory role of PGi on opiate withdrawal-induced hyperactivity of LC neurons was investigated using a newly developed brain slice, containing both LC and PGi. HRP retrograde neuronal tracing was used to verify the existence of both LC and PGi neurons in the developed slice. The spontaneous discharge rate (SDR), resting membrane potential (RMP) and spontaneous excitatory post-synaptic currents (sEPSCs) were recorded in LC neurons using whole cell patch clamp recording. Results showed that the net SDR and the net RMP of LC neurons in slices containing both LC and PGi neurons are significantly higher than slices lacking intact (uncut) PGi inputs. Also, the frequency of sEPSCs in those LC neurons receiving PGi inputs significantly increased compared to the slices containing no intact PGi inputs. Altogether, our results propose that increase in PGi-mediated excitatory transmission might facilitate the opiate withdrawal-induced hyperactivity of LC neurons.

Opioids have always been used in clinical practice for pain management. However, development of tolerance to their effects following long term administration, seriously restricts further clinical use of these drugs. In this regard, μ-opioid receptor (MOR) desensitization, as an initial step in development of opioid tolerance, is of particular significance. Previous studies support the involvement of orexinergic system in development of opioid tolerance. Locus coeruleus (LC) nucleus has been shown to modulate pain and development of tolerance. Opioid receptors (particularly μ) are densely expressed within the LC. Moreover, it receives widespread orexinergic inputs and orexin type 1 receptors (OX1Rs) are also highly expressed in this brain region. In the present study, the effect of orexin-A (OXA) on met-enkephalin (ME)-induced MOR desensitization was investigated in locus coeruleus neurons of male Wistar rats (2-3weeks of age). ME (30μM), as a potent MOR agonist, was applied for 10min and the outward K+ current was recorded using whole cell patch clamp recording. The percentage of decrease in ME-induced K+ current was considered as the degree of MOR desensitization. Results indicated that OXA (100nM) enhances ME-induced MOR desensitization via affecting OX1Rs in rat locus coeruleus neurons and this effect is mediated by a protein kinase C dependent mechanism within the LC. The activity of orexinergic system might potentiate the signaling pathways underlying opioid-induced receptor desensitization.

Tobacco smoking is recognized as a life-threatening risk factor worldwide. Initiation of smoking primarily occurs during adolescence which is a critical developmental phase characterized by specific neurobehavioral alterations. The effect of adolescent nicotine exposure on vulnerability to opioid addiction has not been previously addressed. Furthermore, lateral paragigantocellularis (LPGi) is a key modulator of opiate effects. In this study we investigated the effect of adolescent nicotine treatment on development of morphine tolerance and dependence as well as LPGi neuronal responses to morphine during adulthood. Male Wistar rats received subcutaneous injections of either nicotine or saline during adolescence and then development of morphine tolerance and dependence was assessed during adulthood by tail-flick and withdrawal tests, respectively. In vivo single-unit recording was performed to examine the LPGi neuronal activities. Results indicated that adolescent nicotine exposure significantly facilitates the development of tolerance to analgesic effect of morphine and increases the expression of morphine withdrawal signs in adulthood. Also, it was observed that following adolescent nicotine treatment, the extent of morphine-induced excitation is attenuated in LPGi neurons of adult rats. Moreover, the onset of morphine-induced inhibition was increased in these animals. Neither the baseline, nor the regularity of firing was affected in our observations. It could be concluded that nicotine challenge during adolescence may enhance the future vulnerability to opioid addiction through induction of persistent neuroadaptations in LPGi neurons.

Various intrinsic and extrinsic factors can increase the spontaneous discharge rate of locus coeruleus (LC) neurons. Among the extrinsic ones, orexinergic neurons of the lateral hypothalamus (LH) send widespread projections to LC region. Accumulating evidence support the involvement of glutamate in mediating the excitatory effect of orexin-A on LC neurons. In addition, both orexinergic and glutamatergic systems have been shown to play critical roles in molecular changes underlying the development of morphine dependence. The present study was designed to investigate the interaction between orexin-A and glutamate in modulating the firing rate of LC neurons. Regarding the role of both orexinergic and glutamatergic systems in morphine dependence, this effect was also investigated in morphine dependent rats. For this purpose, spontaneous discharge rate of LC neurons was recorded using the whole-cell patch clamp recording method in presence of orexin-A, glutamate or orexin-A plus glutamate in acutely prepared brain slices. Results indicated that superfusion of either orexin-A or glutamate enhances the firing rate of LC neurons in both dependent and non-dependent rats. However, co-application of orexin-A and glutamate elicited a significant synergism in enhancement of firing rate only in morphine dependent animals. In conclusion, it seems that development of morphine dependence promotes adaptations in locus coeruleus neurons that potentiate the orexin-glutamate interaction.

The mechanisms involved in the anti-seizure effects of low-frequency stimulation (LFS) have not been completely determined. However, Gi-protein-coupled receptors, including D2-like receptors, may have a role in mediating these effects. In the present study, the role of D2-like receptors in LFS' anti-seizure action was investigated. Rats were kindled with semi-rapid (6 stimulations per day), electrical stimulation of the hippocampal CA1 area. In LFS-treated groups, subjects received four trials of LFS at 5 min, 6 h, 24 h, and 30 h following the last kindling stimulation. Each LFS set occurred at 5 min intervals, and consisted of 4 trains. Each train contained 200, 0/1 ms long, monophasic square wave pulses at 1 Hz. Haloperidol (D2-like receptors antagonist, 2 µm) and/or bromocriptine (D2-like receptors agonist 2 µg/µlit) were microinjected into the lateral ventricle immediately after the last kindling, before applying LFS. Obtained results showed that applying LFS in fully-kindled subjects led to a depotentiation-like decrease in kindling-induced potentiation and reduced the amplitude and rise slope of excitatory and inhibitory post-synaptic currents in whole-cell recordings from CA1 pyramidal neurons. In addition, LFS restored the kindling-induced, spatial learning and memory impairments in the Barnes maze test. A D2-like receptor antagonist inhibited these effects of LFS, while a D2-like receptor agonist mimicked these effects. In conclusion, a depotentiation-like mechanism may be involved in restoring LFS' effects on learning and memory, and synaptic plasticity. These effects depend on D2-like receptors activity.

Adipose-derived stem cells (ADSCs), with suitable and easy access, are multipotential cells that have the ability for differentiation into other mesodermal and transdifferentiate into neural phenotype cells. In this study, Lithium chloride (LiCl) was used for in vitro transdifferentiation of rat ADSCs into neuron-like cells (NLCs).

Chronic pain is recognized as an important problem in communities. The locus coeruleus (LC) with extensive ascending and descending projections has a critical role in modulating pain. Some studies indicate how the locus coeruleus-noradrenaline system can remain more active after nociceptive stimulation. In the present study, we examined whether formalin-induced inflammatory pain may affect the electrophysiological properties of LC neurons after 24 h. Inflammatory pain was induced by a subcutaneous injection of 2% formalin (10 μL) into the hind paw of 2-3 week-old male Wistar rats. After 24 h, horizontal slices of brain stem containing the locus coeruleus were prepared and whole-cell patch-clamp recordings were carried out on LC neurons. Findings revealed that LC neurons from formalin injected rats had a significant enhancement in firing rate, half-width and instantaneous frequency of action potentials, but their resting membrane potential, input resistance and afterhyperpolarization amplitude almost remained unchanged. In addition, action potential peak amplitude, maximum rise slope, maximum decay slope, first spike latency and rheobase current significantly decreased in LC neurons obtained from formalin-treated rats. Here, for the first time, we demonstrate that inflammatory pain after 24 h induces hyperexcitability in LC neurons, which in turn may result in changes in noradrenaline release and pain processing.

The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen-/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers.

The properties of self-renewal and division in spermatogonial stem cells (SSCs) support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and selfrenewal of germline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system.

Vimentin is a type of intermediate filament (IF) and one of the first filaments expressed in spermatogenesis. Vimentin plays numerous roles, consisting of the determination of cell shape, differentiation, cell motility, the maintenance of cell junctions, intracellular trafficking, and assisting in keeping normal differentiating germ cell morphology. This study investigated the vimentin expression in two populations of undifferentiated and differentiated spermatogonia. We examined vimentin expression in vivo and in vitro by immunocytochemistry (ICC), immunohistochemistry (IMH), and Fluidigm real-time polymerase chain reaction. IMH data showed that the high vimentin expression was localized in the middle of seminiferous tubules, and low expression was in the basal membrane. ICC analysis of the colonies by isolated differentiated spermatogonia indicated the positive expression for the vimentin antibody, but vimentin's expression level in the undifferentiated population was negative under in vitro conditions. Fluidigm real-time PCR analysis showed significant vimentin expression in differentiated spermatogonia compared to undifferentiated spermatogonia (p < 0.05). Our results showed that vimentin is upregulated in the differentiation stages of spermatogenesis, proving that vimentin is an intermediate filament with crucial roles in the differentiation stages of testicular germ cells. These results support the advanced investigations of the spermatogenic process, both in vitro and in vivo.

Orexin neuropeptides are implicated in the expression of morphine dependence. Locus coeruleus (LC) nucleus is an important brain area involving in the development of withdrawal signs of morphine and contains high expression of orexin type 1 receptors (OX1Rs). Despite extensive considerations, effects of immediate inhibition of OX1Rs by a single dose administration of SB-334867 prior to the naloxone-induced activation of LC neurons remains unknown. Therefore, we examined the direct effects of OX1Rs acute blockade on the neuronal activity of the morphine-dependent rats which underwent naloxone administration. Adult male rats underwent subcutaneous administration of 10 mg/kg morphine (two times/day) for a ten-day period. On the last day of experiment, intra-cerebroventricular administration of 10 μg/μl antagonist of OX1Rs, SB-334867, was performed just before intra-peritoneal injection of 2 mg/kg naloxone. Thereafter, in vivo extracellular single unit recording was employed to evaluate the electrical activity of LC neuronal cells. The outcomes demonstrated that morphine tolerance developed following ten-day of injection. Then, naloxone administration causes hyperactivity of LC neuronal cells, whereas a single dose administration of SB-334867 prior to naloxone prevented the enhanced activity of neurons upon morphine withdrawal. Our findings indicate that increased response of LC neuronal cells to applied naloxone could be prevented by the acute inhibition of the OX1Rs just before the naloxone treatment.

Current estimates indicate that opioid use and misuse are a rising epidemic, which presents a substantial socioeconomic burden around the world. Chronic opioid consumption, specifically during the critical period of adolescence, can lead to enduring effects not only in individuals but also in future generations. Utilizing rodent model, we have previously reported the impacts of paternal exposure to chronic morphine during adolescence on neurobehavioral features in progenies. Currently, the potential transgenerational effects of paternal morphine exposure during adolescence on anxiety-like behavior and short-term memory remains unknown. Male Wistar rats were exposed to increasing doses of morphine for ten days in adolescence (PND 30-39). Thereafter, following a 30-days drug-free period, the treated male rats mated with naïve females. The anxiety-like behavior and short-term memory performance were assessed in adult male and female offspring (PND 60) using open field and Y-maze tests. Both male and female progenies of morphine-treated sires revealed a significant reduction in the movement velocity compared to progenies of saline-treated sires as measured by open field test. Morphine-sired male but not female offspring also showed a non-significant large decreasing effect on time spent in the center and frequency of entries to the center of open field box. Moreover, a significant reduction in the number of entries and percent of time spent in the novel arm was observed in male and female morphine-sired offspring, as measured using Y-maze test. Growth outcomes also did not demonstrate any difference in the number of dam's fertility, pups birth, and death between morphine-sired and saline-sired groups in both sexes. Collectively, paternal exposure to morphine during adolescence induces sex-specific and selective disturbances in short-term memory while anxiety-like behavior was slightly disturbed.

Investigations have shown that the circadian rhythm can affect the mechanisms associated with drug dependence. In this regard, we sought to assess the negative consequence of morphine withdrawal syndrome on conditioned place aversion (CPA) and lateral paragigantocellularis (LPGi) neuronal activity in morphine-dependent rats during light (8:00-12:00) and dark (20:00-24:00) cycles. Male Wistar rats (250-300 g) were received 10 mg/kg morphine or its vehicle (Saline, 2 mL/kg/12 h, s.c.) in 13 consecutive days for behavioral assessment tests. Then, naloxone-induced conditioned place aversion and physical signs of withdrawal syndrome were evaluated during light and dark cycles. In contrast to the behavioral part, we performed in vivo extracellular single-unit recording for investigating the neural response of LPGi to naloxone in morphine-dependent rats on day 10 of morphine/saline exposure. Results showed that naloxone induced conditioned place aversion in both light and dark cycles, but the CPA score during the light cycle was larger. Moreover, the intensity of physical signs of morphine withdrawal syndrome was more severe during the light cycle (rest phase) compare to the dark one. In electrophysiological experiments, results indicated that naloxone evoked both excitatory and inhibitory responses in LPGi neurons and the incremental effect of naloxone on LPGi activity was stronger in the light cycle. Also, the neurons with the excitatory response exhibited higher baseline activity in the dark cycle, but the neurons with the inhibitory response showed higher baseline activity in the light cycle. Interestingly, the baseline firing rate of neurons recorded in the light cycle was significantly different in response (excitatory/inhibitory) -dependent manner. We concluded that naloxone-induced changes in LPGi cellular activity and behaviors of morphine-dependent rats can be affected by circadian rhythm and the internal clock.

The clinical use of opioids is restricted by its deleterious impacts on respiratory system. Gaining a better understanding of an individual's susceptibility to adverse opioid effects is important to recognize patients at risk. Ancestral drug addiction has been shown to be associated with alterations in drug responsiveness in the progenies. In the current study, we sought to evaluate the effects of preconception paternal morphine consumption on respiratory parameters in response to acute morphine in male offspring during adulthood, using plethysmography technique. Male Wistar rats administered 10 days of increasing doses of morphine in the period of adolescence. Thereafter, following a 30-day abstinence time, adult males copulated with naïve females. The adult male offspring were examined for breathing response to morphine. Our results indicated that sires who introduce chronic morphine during adolescence leads to increase irregularity of respiratory pattern and asynchronization between inter-breath interval (IBI) and respiratory volume (RV) time series in male offspring. These findings provide evidence that chronic morphine use by parents even before pregnancy can affect respiratory pattern and response to morphine in the offspring.

Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male's life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis.

Adolescence is a gradual period of transition from childhood to adulthood. It is considered as a sensitive developmental time point that long lasting changes occur in the brain. The present study examined adolescent chronic escalating morphine administration on morphine tolerance and dependence in adulthood. Adolescent male Wistar rats (30days old) were administered increasing doses of morphine (2.5 to 25mg/kg, s.c.) every 12h, for 10days. Control rats received saline according to the same protocol. Thereafter, during adulthood (65-75days old), tolerance to antinociceptive effect of morphine was induced by subcutaneous injection of 3mg/kg morphine, once a day for 7days. Morphine analgesia was measured in the animals by tail flick test every two days, 10min before and 30min after morphine administration. Also, in another test, adult rats were administered morphine (10mg/kg, s.c.) twice a day for 9days to become morphine dependent. On day 10, naloxone (2mg/kg, i.p.) was injected 2h after morphine administration. Somatic signs of morphine withdrawal were then recorded in a clear Plexiglas test chamber for 25min. Results showed that adolescent morphine treatment significantly facilitates the development of tolerance to the analgesic effect of morphine and increases morphine withdrawal signs (grooming, head tremor, sniffing, scratching and teeth chattering) in adulthood compared to the saline group. Facilitation of morphine tolerance and potentiation of withdrawal signs suggests that chronic escalating morphine treatment during adolescence causes long-lasting effects on development of morphine tolerance and dependence in adulthood.

During the cultivation of spermatogonial stem cells (SSCs) and their conversion into embryonic stem-like (ES-like) cells, transitional ES-like colonies and epiblast-like cells were observable. In the present experimental study, we aimed to analyze the efficiency of the multipotency or pluripotency potential of ES-like cells, transitional colonies and epiblast-like cells.

Sox2 (SRY box2) is an essential transcription factor that plays a vital role in spermatogenesis and regulates the genes in this process. Sox2 is important for pluripotency, self-renewal, and even spermatogonial stem cell differentiation. This gene is found in pluripotent and specialized cells, and it is involved in their biological activities.

Peripheral nerve injury (PNI) is believed to cause maladaptive changes at synaptic level, leading to neuropathic pain which is difficult to treat with common analgesic drugs. Noradrenergic locus coeruleus (LC) neurons have a crucial role in neuropathic pain modulation. In this study we examined whether chronic constriction injury (CCI) could affect glutamatergic synaptic transmission in LC neurons. CCI was performed on P10 to P12 Sprague Dawley pups. Seven days after CCI, horizontal slices of brainstem (300μm thick) were prepared and whole-cell patch clamp recording was performed. Evoked and spontaneous excitatory postsynaptic currents (eEPSC and sEPSC) were recorded from LC neurons at a holding potential of -70mV, in the presence of bicuculline (20μM). The sEPSCs recorded from LC neurons of neuropathic rats showed a significant increase in amplitude, but not in frequency. The eEPSC amplitude in neurons of rats under gone CCI was significantly increased compared to the control group (P<0.05). The paired pulse ratio (PPR) elicited with different inter-stimulus intervals (50-250ms) did not show any difference between neurons of CCI and control pups. This study shows that PNI increases excitatory synaptic transmission in LC neurons 7days after chronic constriction injury. The observed synaptic potentiation is mainly due to postsynaptic mechanisms.

Orexin neuropeptides are involved in opiate-induced physical dependence and expression of withdrawal signs following drug abstinence. Orexin type-1 receptors (OXR1) are expressed throughout the rostroventrolateral medulla (RVLM), particularly in the lateral paragigantocellularis (LPGi) nucleus. The present study examined whether blocking OXR1 in LPGi region could affect the development of morphine dependence and so behavioral manifestations induced by morphine withdrawal in rats. Male Wistar rats weighing 250-300 g were used. In order to induce drug dependence, morphine was injected subcutaneously (s.c.) (6, 16, 26, 36, 46, 56, and 66 mg/kg, 2 ml/kg) at an interval of 24 h for 7 days. Animals were divided into experimental groups in which the orexin type-1 receptor antagonist, SB-334867 (0.2 μl, 3 mM), or its vehicle were injected into the LPGi nucleus for 7 days before each morphine injection. On day 8, naloxone (2.5 mg/kg, i.p.) was administered and morphine withdrawal behaviors were monitored for 25 min. Our results indicated that the inhibition of OXR1 in LPGi nucleus significantly reduces the development of morphine dependence and behavioral signs elicited by the administration of naloxone in morphine-dependent rats.