Induction Of C-fos Protein Research Articles

Sodium Leak Channel in Glutamatergic Neurons of the Lateral Parabrachial Nucleus Modulates Inflammatory Pain in Mice.

Elevated excitability of glutamatergic neurons in the lateral parabrachial nucleus (PBL) is associated with the pathogenesis of inflammatory pain, but the underlying molecular mechanisms are not fully understood. Sodium leak channel (NALCN) is widely expressed in the central nervous system and regulates neuronal excitability. In this study, chemogenetic manipulation was used to explore the association between the activity of PBL glutamatergic neurons and pain thresholds. Complete Freund's adjuvant (CFA) was used to construct an inflammatory pain model in mice. Pain behaviour was tested using von Frey filaments and Hargreaves tests. Local field potential (LFP) was used to record the activity of PBL glutamatergic neurons. Gene knockdown techniques were used to investigate the role of NALCN in inflammatory pain. We further explored the downstream projections of PBL using cis-trans-synaptic tracer virus. The results showed that chemogenetic inhibition of PBL glutamatergic neurons increased pain thresholds in mice, whereas chemogenetic activation produced the opposite results. CFA plantar modelling increased the number of C-Fos protein and NALCN expression in PBL glutamatergic neurons. Knockdown of NALCN in PBL glutamatergic neurons alleviated CFA-induced pain. CFA injection induced C-Fos protein expression in central nucleus amygdala (CeA) neurons, which was suppressed by NALCN knockdown in PBL glutamatergic neurons. Therefore, elevated expression of NALCN in PBL glutamatergic neurons contributes to the development of inflammatory pain via PBL-CeA projections.

Lactoferrin Modulates Induction of Transcription Factor c-Fos in Neuronal Cultures

Lactoferrin (Lf) is a multifunctional protein from the transferrin family. Of particular interest is the ability of Lf to affect a wide range of neuronal processes by modulating the expression of genes involved in long-term neuroplasticity. The expression of the immediate early gene c-fos that is rapidly activated in response to external influences, and its product, transcription factor c-Fos, is widely used as a marker of long-term neuronal plasticity. The present study aims to examine the effect of human Lf on the induction of transcription factor c-Fos in the primary mouse neuronal cultures after stimulation and to determine the cellular localization of human Lf and its colocalization with induced c-Fos protein. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 7 of culturing, human Lf was added to the medium. After 24 h (day 8 in culture), c-Fos protein was induced in cells by triple application of 50 mM KCl. c-Fos content was analyzed using the immunofluorescent method 2 h after stimulation. Stimulation promoted exogenous Lf translocation into the nuclei of cultured neuronal cells, which correlated with increased induction of transcription factor c-Fos and was accompanied by nuclear colocalization of these proteins. These results attest to the potential of Lf as a modulator of neuronal processes and open up new prospects in studying the mechanisms of the regulatory effects of lactoferrin on cell function.

C-Fos expression response to olanzapine, amisulpride, aripiprazole, and quetiapine single administration in the rat forebrain: Effect of a mild stress preconditioning

Antipsychotics have been shown to stimulate different forebrain areas, whereas some of them are sensitive to stress. In the present study, effect of a single administration of olanzapine (OLA), amisulpride (AMI), aripiprazole (ARI), and quetiapine (QUE) on the activity of cells in the striatal dorsolateral (stDL) area, the periventricular zone (peVZ), the septal ventrolateral (seVL) nucleus, and the accumbens nucleus shell (shACC) and core (coACC) was investigated in male rats preconditioned with a mild stress complex (CMS) for 20 days. The objective of the study was to extend the anatomical-functional knowledge on the mechanism of selected antipsychotics with the goals: 1) to analyze the ability of the selected antipsychotics to induce c-Fos protein expression in the above mentioned forebrain structures and to map the pattern of their topography and 2) to find out whether longer-lasting mild stress preconditioning may modify the impact of the selected antipsychotics on the activity of cells in the forebrain areas in adult rats. Ten groups of rats were used. CMS complex contained five stressors: cage crowding, air-puff noising, wet bedding, predator stress, and forced swimming. AMI (20 mg/kg), OLA (5 mg/kg), QUE (15 mg/kg), and ARI (10 mg/kg/b.w.) were administered intraperitoneally and 90 min later the animals transcardially perfused by fixative. c-Fos was visualized by ABC complex. In unstressed animals, OLA and ARI elevated c-Fos expression in all areas studied, AMI and QUE in all areas except stDL, seVL and coACC, shACC FL-2 (shACC posterior level), respectively. CMS potentiated the effect of AMI in coACC, and QUE in shACC FL-2 and suppressed the effect of AMI in peVZ, and ARI in peVZ and seVL. The present data provide new insights into activity of cells in response to CMS challenge, which might be helpful in understanding the diverse clinical effects of atypical antipsychotics.

Previous Early-life Stress Modifies Acute Corticosterone-induced Synaptic Plasticity in the Medial Prefrontal Cortex of Adolescent Rats

Stress can either strengthen coping strategies or enhance the risk of depression and anxiety. Synaptic plasticity is one of the key brain functions that can be affected by stress. We have previously shown that early-life stress in the form of maternal separation (MS) impairs functional synaptic plasticity in the medial prefrontal cortex (mPFC), i.e., long-term potentiation (LTP), in adolescent rats. It has been postulated that a previous experience of prolonged stress can modify the response to a subsequent acute stress challenge and influence coping strategies. Therefore, in the present study, we examined how previous MS experience influenced acute stress-induced changes in the LTP and expression of genes and proteins engaged in synaptic plasticity in the mPFC of adolescent rats. To mimic acute stress, we applied acute injections of corticosterone (CORT) and its vehicle (VEH). In control rats, acute CORT injection enhanced LTP in the mPFC. In contrast, MS rats generally exhibited an impairment of LTP that was not further affected by CORT. Moreover, for many studied parameters, such as induction of cFos and Arc mRNA and protein and activation of BDNF, GDNF and NCAM mRNA, MS rats showed diminished, vague or absent responses to acute VEH/CORT compared with those of control rats. These results suggest that previous early-life stress experiences may induce adaptive plasticity within the mPFC, which influences the response to acute stress challenge and coping strategies in adolescents. Depending on the specific environmental context, this phenomenon may lead to either future vulnerability or future resilience to stress-related psychopathologies.

Activation of the thalamic parafascicular nucleus by electrical stimulation of the peripheral vestibular nerve in rats.

The parafascicular nucleus (PFN) of the thalamus is a primary structure in the feedback circuit of the basal ganglia-thalamo-cortical system, as well as in the neural circuit of the vestibulo-thalamo-striatal pathway. We investigated the characteristics of the functional connectivity between the peripheral vestibular system and the PFN in rats. A single electrical stimulation was applied to the horizontal semicircular canal nerve in the peripheral vestibular end-organs. This resulted in polysynaptic local field potentials (LFPs) in the PFN, which were composed of long-lasting multiple waves. The LFPs were prominently seen contralateral to the stimulation site. The PFN LFPs were suppressed by transient chemical de-afferentation of peripheral vestibular activity using a 5% lidocaine injection into the middle ear. The spontaneous firing rate of the single units increased after electrical stimulation to the horizontal canal nerve in a frequency-dependent manner. The induction of cFos protein was more prominent in the contralateral PFN than in the ipsilateral PFN following horizontal semicircular canal nerve stimulation. The functional vestibulo-parafascicular connection is a neural substrate for the transmission of vestibular sensory information to the basal ganglia.

Psychedelics Produce Complex And Heterogeneous Transcriptional Responses In Diverse Cortical Cell Types In The Brain Of Rats As Determined By New Flow Cytometric Methods Allowing For Sorting Of Distinct Cell And Neuronal Populations From Whole Brain

Psychedelics, also known as classic serotonin hallucinogens, are currently being investigated by several groups for their potential as therapeutic agents for a variety of psychiatric indications. Although the neural circuitry underlying the effects of psychedelics is being established by others, the role of specific cell types and populations within the brain that respond to these agents remain largely uninvestigated. We conducted a series of experiments examining the transcriptional effects of the psychedelic and selective 5‐HT2 receptor agonist, (R)‐DOI on a variety of cell types in the brain. Pre‐existing flow cytometry methods were initially used to sort and to examine the transcriptional effects of (R)‐DOI on activated and non‐activated neurons, as well as on specific non‐neuronal populations, including astrocytes, in the medial prefronal cortex (mPFC) and somatosensory cortex (SSC). QRTPCR analysis of mRNA extracted from sorted cortical cells indicated that a variety of cell types produce cfos and other immediate early genes (IEGs) in response to (R)‐DOI, including parvalbumin and somatostatin expressing interneurons, HTR2A expressing neurons, and astrocytes. The induction of cFos protein in a subset of somatostatin‐expressing and 5‐HT2A receptor‐expressing neurons was confirmed using immunohistochemistry. Additionally, a pronounced redistribution of 5‐HT2A receptors to the peri‐nuclear region of neurons in cortical neurons was observed following (R)‐DOI administration. To separate neurons by cell type, we developed a new methodology using flow cytometry. This involved developing methods that isolated cells with cytoplasm and extracellular membranes intact, rather than only nuclei, from whole brain. Pure populations of somatostatin and parvalbumin expressing interneurons were each isolated from whole cortex in the SSC and mPFC. Further separation of these particular cell types into activated and non‐activated populations using antibodies directed against cFos allowed us to analyze IEG induction within specific subpopulations of cortical cells. QRTPCR analyses of sorted neurons from distinct cell types revealed that different IEGs (such as egr2) are not induced uniformly in all activated (cFos+) cells by (R)‐DOI. Our results highlight the diversity of cells that are recruited by psychedelic drugs (e.g. excitatory glutamatergic neurons, GABA interneurons, astrocytes), and the differential transcriptional responses of these cells. Importantly, we demonstrate the utility of our new flow cytometric methods for investigating cell‐type‐specific transcriptional and translational effects of drug administration in the brain. Significantly, our new technique can be generally applied to study the effects of any manipulation (ie., drug‐induced, genetic, or environmental) within specific neural populations of whole brain.Support or Funding InformationLSU Health Science Center. Heffter Research Institute.

Stauntonia hexaphylla (Lardizabalaceae) leaf methanol extract inhibits osteoclastogenesis and bone resorption activity via proteasome-mediated degradation of c-Fos protein and suppression of NFATc1 expression.

BackgroundNatural plants, including common vegetables and fruits, have been recognized as essential sources for drug discovery and the development of new, safe, and economical medicaments. Stauntonia hexaphylla (Lardizabalaceae) is widely distributed in Korea, Japan, and China, and is a popular herbal supplement in Korean and Chinese folk medicine owing to its analgesic, sedative, and diuretic properties. However, the exact pharmacological effects of S. hexaphylla extract, particularly its effect on osteoclastogenesis, are not known.MethodsOsteoclast differentiation and function were identified with tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assay, and the underling mechanisms were determined by real-time RT-PCR and western blot analysis.ResultsS. hexaphylla was found to inhibit early-stage receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without cytotoxicity and bone-resorbing activity in mature osteoclasts in a dose-dependent manner. This S. hexaphylla-mediated blockade of osteoclastogenesis involved abrogation of the NF-κB, ERK, and c-Src-Btk-PLCγ2 calcium signal pathways. Interestingly, we found that S. hexaphylla down-regulated RANKL-associated c-Fos protein induction by suppressing its translation. Furthermore, ectopic overexpression of c-Fos and NFATc1 rescued the inhibition of osteoclast differentiation by S. hexaphylla. Furthermore, S. hexaphylla inhibited the c-Fos- and NFATc1-regulated expression of genes required for osteoclastogenesis, such as TRAP, OSCAR, β3-integrin, ATP6v0d2, and CtsK.ConclusionsThese findings suggest that S. hexaphylla might be useful for the development of new anti-osteoporosis agents.

Abstract 3501: Aspirin modulates oncogene expression in hct 116 colon cancer cells

Abstract Aspirin is a 100 year old Non-steroidal anti-inflammatory drug (NSAID). Beside its use a cardio-protective agent, it is also used for treatment of headache, fever, and inflammation. Many studies have shown that regular intake of aspirin for 5 years or more reduces the risk of colon, breast, prostate, lung and skin cancers. However, mechanism for such actions is not very well understood. Since transformation of a normal cell to cancer cell often involves mutation and activation of proto-oncogene, we initiated this research to understand if aspirin modulates the level of oncogene products. In this study we focused on three different oncogenes; c-FOS, c-JUN and c-RAF. c-FOS and c-JUN are transcription factors, they form a heterodimer called Activator protein (AP-1) transcription factor and regulate the expression of other genes. We observed that, in serum starved HCT-116 colon cancer cells Aspirin and Salicylic acid inhibited serum induced c-FOS protein expression, however, levels of c-JUN protein were not inhibited. In fact Aspirin and Salicylic acid increased c-JUN levels marginally over the serum induced levels at all time points examined. Exposure of cells to Aspirin and Salicylic acid inhibited c-RAF levels in a concentration dependent fashion. Inhibition was observed at physiologically achieved concentration of 250 -500 micro molar Salicylic acid suggesting that Aspirin may mediate its effects through the formation of Salicylic acid. We are currently investigating how inhibition of c-RAF levels by Aspirin and Salicylic acid affects downstream signaling molecules such as p42/p44 MAP kinases. Modulation of proto oncogenes by Aspirin may represent an important pathway by which Aspirin may mediate its anti-cancer effects. Citation Format: Rakesh Dachineni, Goqiang Ai, Jayarama B. Gunaje. Aspirin modulates oncogene expression in hct 116 colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3501. doi:10.1158/1538-7445.AM2014-3501

Nociception originating from the crural fascia in rats

Little is documented in the literature as to the function of muscle fascia in nociception and pain. The aim of this study was to examine the distribution of presumptive nociceptive nerve fibers, to characterize fascial thin-fiber sensory receptors, and to examine the spinal projection of nociceptive input from the rat crural fascia (CF). Nerve fibers labeled with specific antibodies to calcitonin gene-related peptide (CGRP) and peripherin were found to be densely distributed in the distal third of the CF. Thin-fiber receptors (Aδ- and C-fibers) responding to pinching stimuli to the CF with sharpened watchmaker’s forceps, identified in vivo with the teased fiber technique from the common peroneal nerve, exist in the CF. Forty-three percent of the mechano-responsive fascial C-fibers were polymodal receptors (nociceptors) responding to mechanical, chemical (bradykinin), and heat stimuli, whereas almost all Aδ-fibers were responsive only to mechanical stimuli. Repetitive pinching stimulus to the CF induced c-Fos protein expression in the middle to medial part of superficial layers ie, laminae I–II of the spinal dorsal horn at segments L2 to L4, peaking at L3. These results clearly demonstrate the following: 1) peptidergic and non-peptidergic axons of unmyelinated C-fibers with nerve terminals are distributed in the CF; 2) peripheral afferents responding to noxious stimuli exist in the fascia, and 3) nociceptive information from the CF is mainly processed in the spinal dorsal horn at the segments L2 to L4. These results together indicate that the “muscle fascia,” a tissue often overlooked in pain research, can be an important source of nociception under normal conditions.

An environment-dependent modulation of cortical neural response by forebrain cholinergic neurons in awake rat

The forebrain cholinergic neurons project to cortex, including the hippocampus and the cingulate cortex (Cg). However, the relative influence of these neurons on behavior-linked neural processing in the two cortical areas remains unclear. We have now examined the effect of destruction of the cholinergic neurons with microinjection of the immunotoxin 192 IgG-saporin into the medial septum on the induction of c-Fos protein, an index of neuronal synaptic excitation, in the two forebrain areas to varied episodic experiences. Separate groups of rats were (a) re-exposed to the laboratory where they had previously undergone a surgery for intraseptal microinjection or (b) exposed to a novel environment. Re-exposure evoked a differential increase in the number of c-Fos positive neurons in dorsal CA1 compared to novelty, while a robust increase was observed in the Cg selectively in the novel environment. Both the differential and the selective increases were strongly attenuated by the cholinergic destruction with intraseptal-immunotoxin. These findings suggest that the cholinergic modulation of the neural processing in the two forebrain areas varies partly in an environment-dependent fashion affecting CA1 neural activation on repeat exposure to an environment where they had a relatively complex aversive experience while favoring Cg neural activation more during novelty.

Inhomogeneous Rostrocaudal Activation of the Hippocampus in Mice after Exploration of a Novel Space

Hippocampal activity in C57BL/6 mice was studied during exploration of novel spaces of different levels of complexity and learning by neuromapping using the induction of c-Fos protein by nervous activity. c-Fos-immunopositive neurons were counted at eight levels along the whole of the rostrocaudal axis of the hippocampus. In experiment 1, mice were given six opportunities on a single day to find the entrance to the home cage through one of the arms of a modified eight-arm radial maze. Animals of the active control group were released into the home cage via an isolated arm. Mice tested in the eight-arm maze showed an inhomogeneous increase in c-Fos expression along the rostrocaudal axis: activation in the caudal part of fields CA1 and CA3 and the dentate fascia was significantly greater than that in the rostral part. In experiment 2, animals were tested in a novel open field. In this situation, the increase in expression in the caudal part of field CA1 was greater than that in the rostral part, though in field CA3 and the dentate fascia there was uniform activation over the whole rostrocaudal extent. Both experiments identified positive correlations between the density of c-Fos-positive neurons and measures of investigative activity. These data provide evidence that placing of animals in a novel space leads to inhomogeneous activation of the hippocampal field along its rostrocaudal axis.

Obese mice exhibit an altered behavioural and inflammatory response to lipopolysaccharide

SUMMARYObesity is associated with an increase in the prevalence and severity of infections. Genetic animal models of obesity (ob/ob and db/db mice) display altered centrally-mediated sickness behaviour in response to acute inflammatory stimuli such as lipopolysaccharide (LPS). However, the effect of diet-induced obesity (DIO) on the anorectic and febrile response to LPS in mice is unknown. This study therefore determined how DIO and ob/ob mice respond to a systemic inflammatory challenge. C57BL/6 DIO and ob/ob mice, and their respective controls, were given an intraperitoneal (i.p.) injection of LPS. Compared with controls, DIO and ob/ob mice exhibited an altered febrile response to LPS (100 μg/kg) over 8 hours. LPS caused a greater and more prolonged anorexic effect in DIO compared with control mice and, in ob/ob mice, LPS induced a reduction in food intake and body weight earlier than it did in controls. These effects of LPS in obese mice were also seen after a fixed dose of LPS (5 μg). LPS (100 μg/kg) induced Fos protein expression in several brain nuclei of control mice, with fewer Fos-positive cells observed in the brains of obese mice. An altered inflammatory response to LPS was also observed in obese mice compared with controls: changes in cytokine expression and release were detected in the plasma, spleen, liver and peritoneal macrophages in obese mice. In summary, DIO and ob/ob mice displayed an altered behavioural response and cytokine release to systemic inflammatory challenge. These findings could help explain why obese humans show increased sensitivity to infections.

Expression of Novel Opsins and Intrinsic Light Responses in the Mammalian Retinal Ganglion Cell Line RGC-5. Presence of OPN5 in the Rat Retina

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca2+mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca2+ levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca2+ mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.

Central Effects of Ghrelin, a Unique Peptide, on Appetite and Fluid/Water Drinking Behavior

Ghrelin is a stomach-derived peptide discovered as a ligand of the orphan G-protein coupled receptor. Ghrelin is now recognized as a major orexigenic neuropeptide. Immunohistochemical studies demonstrated that centrally administered ghrelin induced c-fos protein expression in many areas in the brain. Indeed, centrally administered ghrelin has various effects such as stimulating feeding, arousal, increasing gastric acid secretion, release of hormones from the pituitary, and inhibition of water intake. In particular, we recently showed that ghrelin was an antidipsogenic peptide with a simultaneous orexigenic effect. This may be of important, because most spontaneous daily water intake is temporally associated with feeding. Here, we summarise recent findings on the integration of central effects of ghrelin that regulate feeding, release hormones from the pituitary and inhibit fluid/water intake.

Regulation of Neuronal Activation by Alpha2A Adrenergic Receptor Agonist

Stress factors induce neuronal activation in brain areas that are related to anxiety and fear. High doses of caffeine induce neuronal activation with Ca2+ influx followed by expression of the immediate early gene c-fos. In the present study, we investigated c-Fos protein expression in stress-responsive brain areas induced by caffeine, as well as the role of alpha2A receptor in the regulation of neuronal activation. Immunohistochemical analysis showed that an acute effect of caffeine induced c-Fos protein expression in the hippocampus, the bed nucleus of stria terminalis (BNST), the lateral septum, the basolateral and central amygdala, the paraventricular hypothalamic nucleus (PVN), the locus coeruleus, and the lateral parabrachial nucleus (LPBN). However, c-Fos expression was attenuated after repeated treatment of caffeine, spaced 24 h apart, compared to a single acute effect. Alpha2A receptor activation with the agonist guanfacine attenuated the acute effect of caffeine in terms of c-Fos expression in neurons in the CA1-CA3 areas of hippocampus, the locus coeruleus and the LPBN as compared with effect of caffeine alone, whereas the number of c-Fos expressing neurons increased in the lateral septum, the dorsal BNST, the central amygdala, and the PVN, areas that are densely innervated by noradrenergic neurons. Guanfacine alone induced c-Fos protein expression in neurons in the central amygdala, the dorsal BNST, the PVN, the LPBN, and the caudal nucleus of the solitary tract. Guanfacine alone also induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in neurons expressing c-Fos in the dorsal BNST, the central amygdala, and the LPBN. These results suggest that alpha2A receptor activation modulates synaptic transmission in neuronal circuits that are correlated with stress in vivo.

Delayed c-Fos activation in human cells triggers XPF induction and an adaptive response to UVC-induced DNA damage and cytotoxicity

The oncoprotein c-Fos has been commonly found differently expressed in cancer cells. Our previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (UVC) light. Here, we demonstrate that in human diploid fibroblasts UV-triggered induction of c-Fos protein is a delayed and long-lasting event. Sustained upregulation of c-Fos goes along with transcriptional stimulation of the NER gene xpf, which harbors an AP-1 binding site in the promoter. Data gained on c-Fos knockdown and c-Fos overexpressing human cells provide evidence that c-Fos/AP-1 stimulates upregulation of XPF, thereby increasing the cellular repair capacity protecting from UVC-induced DNA damage. When these cells are pre-exposed to a low non-toxic UVC dose and challenged with a subsequent high dose of UVC irradiation, they show accelerated repair of UVC-induced DNA adducts and reduced cell kill. The data indicate a protective role of c-Fos induction by triggering an adaptive response pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-010-0546-9) contains supplementary material, which is available to authorized users.

The immediate early genes, c-fos, c-jun and AP-1, are early markers of platinum analogue toxicity in human proximal tubular cell primary cultures

These studies tested the hypothesis that c-fos, c-jun and AP-1 are early markers of platinum analogue-induced proximal tubule nephrotoxicity in primary rat proximal tubule (RPT) and human proximal tubule (HPT) cell cultures. The order of platinum analogue toxicity was cisplatin > transplatin > carboplatin in RPT and HPT cultures. Following a 2-h platinum analogue treatment, c-fos protein expression correlated with toxicity. Maximal c-fos protein levels were observed at 8-h (RPT) and 4-h (HPT) post-platinum analogue treatment. c-jun and AP-1 protein levels were maximal 4-h and 8-h, respectively, post cisplatin treatment in HPT cultures. In contrast, c-jun and AP-1 protein were not detected in RPT cultures. c-fos and c-jun mRNA levels were maximal at 60 and 120-min in RPT cell cultures, respectively, whilst c-fos and c-jun mRNA levels were maximal at 120-min in HPT cultures. Differences between HPT and RPT responses to cisplatin reveal inter-species differences associated with induction of c-fos and c-jun mRNA and protein, which in turn form the functional AP-1 complex prior to the onset of cellular toxicity. These studies highlight the utility of HPT cultures as an in vitro model system, and the potential of c-fos and c-jun as early markers of nephrotoxicity to screen therapeutic lead compounds.

Signal transduction and gene expression in cultured accessory olfactory bulb neurons

Glutamate and norepinephrine (NE) are believed to mediate the long-lasting synaptic plasticity in the accessory olfactory bulb (AOB) that underlies pheromone recognition memory. The mechanisms by which these neurotransmitters bring about the synaptic changes are not clearly understood. In order to study signals that mediate synaptic plasticity in the AOB, we used AOB neurons in primary culture as a model system. Because induction of pheromone memory requires coincident glutamatergic and noradrenergic input to the AOB, and requires new protein synthesis, we reasoned that glutamate and NE must induce gene expression in the AOB. We used a combination of agonists that stimulate α1 and α2 adrenergic receptors in combination with N-methyl- d-aspartic acid and tested expression of the immediate-early gene (IEG) c-Fos. We found that the glutamatergic and noradrenergic stimulation caused significant induction of c-Fos mRNA and protein. Induction of c-Fos was significantly reduced in the presence of inhibitors of protein kinase C, mitogen-activated protein kinase (MAPK) and phospholipase C. These results suggest that glutamate and NE induce gene expression in the AOB through a signaling pathway mediated by protein kinase C and MAPK.

Activation of 5-HT 2C receptors acutely induces Per1 gene expression in the rat SCN in vitro

The suprachiasmatic nucleus of the hypothalamus receives dense serotonergic projections from the raphe nuclei. This input may be involved in the transmission of photic information with activation of 5-HT receptors at night having a photo-mimetic effect. 5-HT 2C receptors have been implicated in this role, with 5-HT 2C receptor agonist administration at night resulting in the induction of cFOS protein, and a phase delay of melatonin metabolite excretion rhythms in a manner similar to light. To investigate the site of action of 5-HT 2C receptor agonists, rat SCN explants were isolated in culture, allowing exposure of agonists to denervated tissue. Any response could then be attributed to the actions of the 5-HT 2C receptor agonist on cells within the SCN, rather than at other distant sites or on pre-synaptic receptors. Rat SCN tissue was microdissected and prepared in tissue culture conditions. The acute effect of the 5-HT 2A/2C receptor agonist DOI and glutamate administration at various circadian times on c-fos and Per1 expressions was assessed. Glutamate induced the expression of c-fos at CT6, CT16 and CT22, but induced Per1 expression at CT16 only. DOI altered c-fos expression at all times examined with an increase at CT6 and CT22, and a repression at CT16. However, DOI significantly increased Per1 expression when applied at CT16, but had no effect at either CT22 or CT6. This response was similar to that previously observed in vivo. This provides support for the hypothesis that the actions of 5-HT 2C receptor agonists are on cells located within the SCN.

Morphometric analysis of hypothalamic cells showing c-Fos proteins after movement restriction and EHF-irradiation

A morphometric analysis of hypothalamic cells containing c-Fos-like protein detected by an indirect immunoperoxidase method was carried out to clarify the responses to movement restriction and skin electromagnetic high frequency (EHF) irradiation (7.1 mm, 42.2 GHz, 20 mW output power) simultaneously to three acupuncture projection areas. The morphometry of c-Fos-protein positive neurons by their number and type was analyzed. Movement restriction (40 min) induced c-Fos protein expression primarily in cells with 10–50 μm 2 sizes (associative type neurons) only in anterior hypothalamic nucleus and lateral hypothalamic area; while additional EHF-irradiation of acupuncture projection areas (under movement restriction) induced c-Fos expression in all hypothalamic structures and mostly in cells with 70–150 μm 2 sizes (relay type neurons), i.e. changed the pattern of activated cell type distribution. In conclusion, the findings show that modest stress under which experimental animals often are exposed increase c-Fos protein expression in hypothalamic centers and skin EHF-irradiation of acupuncture projection areas seem to increase that.