Induction Of C-fos mRNA Research Articles

Cell-type expression and activation by light of neuropsins in the developing and mature Xenopus retina.

Photosensitive opsins detect light and perform image- or nonimage-forming tasks. Opsins such as the "classical" visual opsins and melanopsin are well studied. However, the retinal expression and functions of a novel family of neuropsins are poorly understood. We explored the developmental time-course and cell-type specificity of neuropsin (opn5, 6a, 6b, and 8) expression in Xenopus laevis by in situ hybridization and immunohistochemistry. We compared the Xenopus results with publicly available single cell RNA sequencing (scRNA-seq) data from zebrafish, chicken, and mouse. Additionally, we analyzed light-activation of neuropsin-expressing cells through induction of c-fos mRNA. opn5 and opn8 expression begins at stage 37/38 when the retinal circuits begin to be activated. Once retinal circuits connect to the brain, opn5 mRNA is distributed across multiple retinal cell types, including bipolar (~70%-75%), amacrine (~10%), and retinal ganglion (~20%) cells, with opn8 present in amacrine (~70%) and retinal ganglion (~30%) cells. opn6a and opn6b mRNAs emerge in newborn-photoreceptors (stage 35), and are colocalized in rods and cones by stage 37/38. Interestingly, in the mature larval retina (stage 43/44), opn6a and opn6b mRNAs become preferentially localized to rods and cones, respectively, while newborn photoreceptors bordering the proliferative ciliary marginal zone express both genes. In zebrafish, opn6a and opn6b are also expressed in photoreceptors, while Müller glia and amacrine cells express opn8c. Most neuropsin-expressing retinal ganglion cells display c-fos expression in response to light, as do over half of the neuropsin-expressing interneurons. This study gave a better understanding of retinal neuropsin-expressing cells, their developmental onset, and light activation.

The power of innate: Behavioural attachment and neural activity in responses to natural and artificial objects in filial imprinting in chicks.

Newly hatched domestic chicks are known to orient preferentially toward naturalistic stimuli, resembling a conspecific. Here, we examined to what extent this behavioral preference can be transcended by an artificial imprinting stimulus in both short-term and long-term tests. We also compared the expression maps of the plasticity-associated c-fos gene in the brains of chicks imprinted to naturalistic (rotating stuffed jungle fowl) and artificial (rotating illuminated red box) stimuli. During training, the approach activity of chicks to a naturalistic object was always higher than that to an artificial object. However, the induction of c-fos mRNA was significantly higher in chicks imprinted to a box than to a fowl, especially in the intermediate medial mesopallium, hyperpallium apicale, arcopallium, and hippocampus. Initially, in the short-term test (10min after the end of training), chicks had a higher preference for a red box than for a stuffed fowl. However, in the long-term test (24h after imprinting), the response to an artificial object decreased to the level of preference for a naturalistic object. Our results thus show that despite the artificial object causing a stronger c-fos novelty response and higher behavioral attachment in the short term, this preference was less stable and fades away, being overtaken by a more stable innate predisposition to the naturalistic social object.

Somatosensory cortex hyperconnectivity and impaired whisker-dependent responses in Cntnap2−/− mice

Sensory abnormalities are a common feature in autism spectrum disorders (ASDs). Tactile responsiveness is altered in autistic individuals, with hypo-responsiveness being associated with the severity of ASD core symptoms. Similarly, sensory abnormalities have been described in mice lacking ASD-associated genes. Loss-of-function mutations in CNTNAP2 result in cortical dysplasia-focal epilepsy syndrome (CDFE) and autism. Likewise, Cntnap2−/− mice show epilepsy and deficits relevant with core symptoms of human ASDs, and are considered a reliable model to study ASDs. Altered synaptic transmission and synchronicity found in the cerebral cortex of Cntnap2−/− mice would suggest a network dysfunction. Here, we investigated the neural substrates of whisker-dependent responses in Cntnap2+/+ and Cntnap2−/− adult mice. When compared to controls, Cntnap2−/− mice showed focal hyper-connectivity within the primary somatosensory cortex (S1), in the absence of altered connectivity between S1 and other somatosensory areas. This data suggests the presence of impaired somatosensory processing in these mutants. Accordingly, Cntnap2−/− mice displayed impaired whisker-dependent discrimination in the textured novel object recognition test (tNORT) and increased c-fos mRNA induction within S1 following whisker stimulation. S1 functional hyperconnectivity might underlie the aberrant whisker-dependent responses observed in Cntnap2−/− mice, indicating that Cntnap2 mice are a reliable model to investigate sensory abnormalities that characterize ASDs.

Abnormal Whisker-Dependent Behaviors and Altered Cortico-Hippocampal Connectivity in Shank3b-/- Mice.

Abnormal tactile response is an integral feature of Autism Spectrum Disorders (ASDs), and hypo-responsiveness to tactile stimuli is often associated with the severity of ASDs core symptoms. Patients with Phelan-McDermid syndrome (PMS), caused by mutations in the SHANK3 gene, show ASD-like symptoms associated with aberrant tactile responses. The neural underpinnings of these abnormalities are still poorly understood. Here we investigated, in Shank3b−/− adult mice, the neural substrates of whisker-guided behaviors, a key component of rodents’ interaction with the surrounding environment. We assessed whisker-dependent behaviors in Shank3b−/− adult mice and age-matched controls, using the textured novel object recognition (tNORT) and whisker nuisance (WN) test. Shank3b−/− mice showed deficits in whisker-dependent texture discrimination in tNORT and behavioral hypo-responsiveness to repetitive whisker stimulation in WN. Sensory hypo-responsiveness was accompanied by a significantly reduced activation of the primary somatosensory cortex (S1) and hippocampus, as measured by c-fos mRNA induction, a proxy of neuronal activity following whisker stimulation. Moreover, resting-state fMRI showed a significantly reduced S1-hippocampal connectivity in Shank3b mutants, in the absence of altered connectivity between S1 and other somatosensory areas. Impaired crosstalk between hippocampus and S1 might underlie Shank3b−/− hypo-reactivity to whisker-dependent cues, highlighting a potentially generalizable somatosensory dysfunction in ASD.

Remote CB1 receptor antagonist administration reveals multiple sites of tonic and phasic endocannabinoid neuroendocrine regulation

Endogenous cannabinoids (endocannabinoids, eCB) are expressed throughout the body and contribute to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and general stress reactivity. This study assessed the contributions of CB1 receptors (CB1R) in the modulation of basal and stress-induced neural and HPA axis activities. Catheterized adult male rats were placed in chambers to acclimate overnight, with their catheters connected and exteriorized from the chambers for relatively stress-free remote injections. The next morning, the CB1R antagonist AM251 (1 or 2 mg/kg) or vehicle was administered, and 30 min later, rats were exposed to loud noise stress (30 min) or no noise (basal condition). Blood, brains, pituitary and adrenal glands were collected immediately after the procedures for analysis of c-fos and CB1R mRNAs, corticosterone (CORT) and adrenocorticotropin hormone (ACTH) plasma levels. Basally, CB1R antagonism induced c-fos mRNA in the basolateral amygdala (BLA) and auditory cortex (AUD) and elevated plasma CORT, indicating disruption of eCB-mediated constitutive inhibition of activity. CB1R blockade also potentiated stress-induced hormone levels and c-fos mRNA in several regions such as the bed nucleus of the stria terminalis (BST), lateral septum (LS), and basolateral amygdala (BLA) and the paraventricular nucleus of the hypothalamus (PVN). CB1R mRNA was detected in all central tissues investigated, and the adrenal cortex, but at very low levels in the anterior pituitary gland. Interestingly, CB1R mRNA was rapidly and bidirectionally regulated in response to stress and/or antagonist treatment in some regions. eCBs therefore modulate the HPA axis by regulating both constitutive and activity-dependent inhibition at multiple levels.

Driving Neurogenesis in Neural Stem Cells with High Sensitivity Optogenetics.

Optogenetic stimulation of neural stem cells (NSCs) enables their activity-dependent photo-modulation. This provides a spatio-temporal tool for studying activity-dependent neurogenesis and for regulating the differentiation of the transplanted NSCs. Currently, this is mainly driven by viral transfection of channelrhodopsin-2 (ChR2) gene, which requires high irradiance and complex in vivo/vitro stimulation systems. Additionally, despite the extensive application of optogenetics in neuroscience, the transcriptome-level changes induced by optogenetic stimulation of NSCs have not been elucidated yet. Here, we made transformed NSCs (SFO-NSCs) stably expressing one of the step-function opsin (SFO)-variants of chimeric channelrhodopsins, ChRFR(C167A), which is more sensitive to blue light than native ChR2, via a non-viral transfection system using piggyBac transposon. We set up a simple low-irradiance optical stimulation (OS)-incubation system that induced c-fos mRNA expression, which is activity-dependent, in differentiating SFO-NSCs. More neuron-like SFO-NCSs, which had more elongated axons, were differentiated with daily OS than control cells without OS. This was accompanied by positive/negative changes in the transcriptome involved in axonal remodeling, synaptic plasticity, and microenvironment modulation with the up-regulation of several genes involved in the Ca2+-related functions. Our approach could be applied for stem cell transplantation studies in tissue with two strengths: lower carcinogenicity and less irradiance needed for tissue penetration.

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.

Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus.

Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus—derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A—positive cells in a tamoxifen-dependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c-Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability.

Activation of AP-1 By JAK2V617F through Induction and Stabilization of c-FOS

Abstract JAK2V617F, a gain-of-function mutation in the tyrosine kinase JAK2, has been detected in most patients with Philadelphia chromosome (Ph)-negative classical myeloproliferative neoplasms (MPNs). The identification of the JAK2 mutation in MPNs led to the development of JAK2 inhibitors, which suppress the symptoms of this disease. However, treatment with these inhibitors rarely leads to molecular remission and is associated with side effects caused by blockade of function of wild-type JAK2. In addition, resistance has been reported; therefore, a new therapeutic strategy based on understanding of MPN development is required. Here, we found that JAK2V617F dramatically upregulates cytomegalovirus (CMV) promoter activity in HEK293T cells. By performing a reporter assay using luciferase, we found that a non-canonical AP-1 binding motif is responsible for this activation of the CMV promoter. By performing a series of biochemical analyses, including an electrophoresis mobility shift assay and a supershift assay with antibodies, we found that c-FOS, a member of the AP-1 family of proteins, bound to oligo duplex DNA containing the identified motif only when JAK2V617F was expressed. A reporter construct harboring the identified sequence alone showed a robust increase in reporter assay when JAK2V617F was induced, while mutation of the AP-1 binding motif abolished the response to JAK2V617F. The results showed that c-FOS overexpression alone is sufficient for activation of the promoter containing the non-canonical AP-1 binding site, demonstrating that c-FOS is a downstream target of JAK2V617F in CMV promoter activation. The activation of this reporter construct was also observed in leukemia cell lines UT-7/EPO and UT-7/GM when JAK2V617F is expressed, which implies that this activation is not limited to HEK293T cells. We found that the activation of AP-1 by JAK2V617F was partly due to robust induction of c-FOS mRNA (41 fold) followed by increase in c-FOS protein levels. In addition, we found that, the ubiquitin-proteasome protein degradation pathway responsible for c-FOS degradation was perturbed in HEK293T cells expressing JAK2V617F, which at least partly contributed to c-FOS protein accumulation and subsequent AP-1 activation. Multiple inhibitors of known downstream pathways of JAK2, such as the mitogen activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways, were analyzed using the reporter assay. The results showed that none of them suppressed JAK2V617F-dependent reporter activation. Furthermore, inhibition of the STAT pathway by expression of the dominant-negative form of STAT1, STAT3, or STAT5 failed to suppress AP-1 activation by JAK2V617F. Consistent with this observation, expression of the active form of STAT1, STAT3, and STAT5 did not affect AP-1 activity. Taken together, we conclude that AP-1 activation by JAK2V617F through c-FOS induction involves an unidentified molecular mechanism. Although our observation is limited to an in vitro system, our finding suggests that deregulation of AP-1 plays a role in MPN development. The AP-1 reporter assay system that we have established should be useful not only for screening novel compounds for MPN treatment but also in studying the novel pathway regulated by JAK2V617F, which would lead to the development of a novel therapeutic strategy against MPNs. Disclosures No relevant conflicts of interest to declare.

P.1.g.102 Modulation of the conditioned emotional response by dorsal hippocampus TRPV1 receptor

We have shown previously that unconditioned stressors inhibit neurons of the lateral/capsular division of the central nucleus of the amygdala (CEAl/c) and oval division of the bed nucleus of the stria terminalis (BSTov), which form part of the central extended amygdala. The current study investigated whether conditioned fear inhibits c-fos mRNA expression in these regions. Male rats were trained either to associate a visual stimulus (light) with footshock or were exposed to the light alone. After training, animals were replaced in the apparatus, and 2 h later injected remotely, via a catheter, with amphetamine (2 mg/kg i.p.), to induce c-fos mRNA and allow inhibition of expression to be measured. The rats were then presented with 15 visual stimuli over a 30 minute period. As expected, fear conditioned animals that were not injected with amphetamine, had extremely low levels of c-fos mRNA in the central extended amygdala. In contrast, animals that were trained with the light alone (no fear conditioning) and were injected with amphetamine had high levels of c-fos mRNA in the CEAl/c and BSTov. Animals that underwent fear conditioning, and were re-exposed to the conditioned stimulus after amphetamine injection had significantly reduced levels of c-fos mRNA in both the BSTov and CEAl/c, compared to the non-conditioned animals. These data suggest that conditioned fear can inhibit neurons of the central extended amygdala. Because these neurons are GABAergic, and project to the medial CEA (an amygdaloid output region), this may be a novel mechanism whereby conditioned fear potentiates amygdaloid output.

Wheel running alters patterns of uncontrollable stress-induced cfos mRNA expression in rat dorsal striatum direct and indirect pathways: A possible role for plasticity in adenosine receptors

Emerging evidence indicates that adenosine is a major regulator of striatum activity, in part, through the antagonistic modulation of dopaminergic function. Exercise can influence adenosine and dopamine activity, which may subsequently promote plasticity in striatum adenosine and dopamine systems. Such changes could alter activity of medium spiny neurons and impact striatum function. The purpose of this study was twofold. The first was to characterize the effect of long-term wheel running on adenosine 1 (A1R), adenosine 2A (A2AR), dopamine 1 (D1R), and dopamine 2 (D2R) receptor mRNA expression in adult rat dorsal and ventral striatum structures using in situ hybridization. The second was to determine if changes to adenosine and dopamine receptor mRNA from running are associated with altered cfos mRNA induction in dynorphin- (direct pathway) and enkephalin- (indirect pathway) expressing neurons of the dorsal striatum following stress exposure. We report that chronic running, as well as acute uncontrollable stress, reduced A1R and A2AR mRNA levels in the dorsal and ventral striatum. Running also modestly elevated D2R mRNA levels in striatum regions. Finally, stress-induced cfos was potentiated in dynorphin and attenuated in enkephalin expressing neurons of running rats. These data suggest striatum adenosine and dopamine systems are targets for neuroplasticity from exercise, which may contribute to changes in direct and indirect pathway activity. These findings may have implications for striatum mediated motor and cognitive processes, as well as exercise facilitated stress-resistance.

Increased dopaminergic innervation in the brain of conditional mutant mice overexpressing Otx2: Effects on locomotor behavior and seizure susceptibility

The homeobox-containing transcription factor Otx2 controls the identity, fate and proliferation of mesencephalic dopaminergic (mesDA) neurons. Transgenic mice, in which Otx2 was conditionally overexpressed by a Cre recombinase expressed under the transcriptional control of the Engrailed1 gene (En1Cre/+; tOtx2ov/+), show an increased number of mesDA neurons during development. In adult mice, Otx2 is expressed in a subset of neurons in the ventral tegmental area (VTA) and its overexpression renders mesDA more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Here we further investigated the neurological consequences of the increased number of mesDA neurons in En1Cre/+; tOtx2ov/+ adult mice. Immunohistochemistry for the active, glycosylated form of the dopamine transporter (glyco-Dat) showed that En1Cre/+; tOtx2ov/+ adult mice display an increased density of mesocortical DAergic fibers, as compared to control animals. Increased glyco-Dat staining was accompanied by a marked hypolocomotion in En1Cre/+; tOtx2ov/+ mice, as detected in the open field test. Since conditional knockout mice lacking Otx2 in mesDA precursors (En1Cre/+; Otx2floxv/flox mice) show a marked resistance to kainic acid (KA)-induced seizures, we investigated the behavioral response to KA in En1Cre/+; tOtx2ov/+ and control mice. No difference was observed between mutant and control mice, but En1Cre/+; tOtx2ov/+ mice showed a markedly different c-fos mRNA induction profile in the cerebral cortex and hippocampus after KA seizures, as compared to controls. Accordingly, an increased density of parvalbumin (PV)-positive inhibitory interneurons was detected in the deep layers of the frontal cortex of naïve En1Cre/+; tOtx2ov/+ mice, as compared to controls. These data indicate that Otx2 overexpression results in increased DAergic innervation and PV cell density in the fronto-parietal cortex, with important consequences on spontaneous locomotor activity and seizure-induced gene expression. Our results strengthen the notion that Otx2 mutant mouse models are a powerful genetic tool to unravel the molecular and behavioral consequences of altered development of the DAergic system.

D-serine deficiency attenuates the behavioral and cellular effects induced by the hallucinogenic 5-HT2A receptor agonist DOI

Both the serotonin and glutamate systems have been implicated in the pathophysiology of schizophrenia, as well as in the mechanism of action of antipsychotic drugs. Psychedelic drugs act through the serotonin 2A receptor (5-HT2AR), and elicit a head-twitch response (HTR) in mice, which directly correlates to 5-HT2AR activation and is absent in 5-HT2AR knockout mice. The precise mechanism of this response remains unclear, but both an intrinsic cortico-cortical pathway and a thalamo-cortical pathway involving glutamate release have been proposed. Here, we used a genetic model of NMDAR hypofunction, the serine racemase knockout (SRKO) mouse, to explore the role of glutamatergic transmission in regulating 5-HT2AR-mediated cellular and behavioral responses. SRKO mice treated with the 5-HT2AR agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) showed a clearly diminished HTR and lower induction of c-fos mRNA. These altered functional responses in SRKO mice were not associated with changes in cortical or hippocampal 5-HT levels or in 5-HT2AR and metabotropic glutamate-2 receptor (mGluR2) mRNA and protein expression. Together, these findings suggest that D-serine-dependent NMDAR activity is involved in mediating the cellular and behavioral effects of 5-HT2AR activation.

Regulation of Primary Response Genes in B Cells

Deregulated gene expression in B cells often results in various lymphoid malignancies and immune deficiencies. Therefore, understanding signal-induced gene regulatory pathways involved during B cell activation is important to tackle pathologies associated with altered B cell function. Primary response genes (PRGs) are rapidly induced upon signaling in B cells and other cell types and often encode oncogenic transcription factors, which are associated with various malignancies. However, an important issue that remains unclear is whether the fundamental mechanism of activation of these genes is essentially the same under such diverse conditions. c-fos is a PRG that is induced rapidly upon activation of B cells in response to a wide variety of stimuli. Using the c-fos gene as a candidate PRG, we addressed here how it is regulated in response to tumor-promoting and antigen-mimicking signals. Our results show that although the mRNA was induced and extinguished within minutes in response to both signals, surprisingly, apparently full-length unspliced pre-mRNA persisted for several hours in both cases. However, although the mitogenic signal resulted in a more sustained mRNA response that persisted for 4 h, antigenic signaling resulted in a more robust but very transient response that lasted for <1 h. Moreover, the pre-mRNA profile exhibited significant differences between the two signals. Additionally, the splicing regulation was also observed with egr-2, but not with c-myc. Together, these results suggest a previously underappreciated regulatory step in PRG expression in B cells.

Su2115 Altered Activation of Cardiac Sympathetic Discharge and Covariated Brain Region During Colorectal Distention With Corticotropin-Releasing Hormone in Irritable Bowel Syndrome

G A A b st ra ct s plasma ghrelin levels at 2 h after ICV UCN. UCN induces c-fos mRNA and Fos protein expression in the supraoptic nucleus, paraventricular nucleus of hypothalamus (PVN), locus coeruleus, solitary tract nucleus (NTS), and ventrolateral nucleus of medulla oblongata (VLM), known to influence sympathetic outflow. Fos expression in NTS was markedly suppressed by coadministration of RKT (control 84 ± 28, UCN 352 ± 130, UCN + RKT 48 ± 15; P , 0.05). UCN-induced Fos expression in VLM was also suppressed by RKT (control 35 ± 10, UCN 289 ± 94, UCN + RKT 133 ± 27), but not significant (P=0.08). RKT also suppressed UCN-induced c-fos expression in PVN. In contrast, the dorsal motor nucleus involved in vagal outflow did not show any changes in Fos and fos expression by RKT. Conclusions: These data indicate that a) RKT inhibits the activity of the neuron of NTS suggesting that RKT inhibits vagal input from the periphery to central nerves. b) As the result of inhibiton of vagal input, activity of hypothalamic satiety center, PVN is suppressed resulting in the inhibition of the sympathetic outflow from the central nerve to the periphery. RKT may restore disturbed appetite and ghrelin secretion through the restoration of balance of central autonomic nervous system.

Su2116 Regional Neuroplastic Brain Changes in Patients With Inflammatory Bowel Diseases (IBD) and Irritable Bowel Syndrome (IBS)

G A A b st ra ct s plasma ghrelin levels at 2 h after ICV UCN. UCN induces c-fos mRNA and Fos protein expression in the supraoptic nucleus, paraventricular nucleus of hypothalamus (PVN), locus coeruleus, solitary tract nucleus (NTS), and ventrolateral nucleus of medulla oblongata (VLM), known to influence sympathetic outflow. Fos expression in NTS was markedly suppressed by coadministration of RKT (control 84 ± 28, UCN 352 ± 130, UCN + RKT 48 ± 15; P , 0.05). UCN-induced Fos expression in VLM was also suppressed by RKT (control 35 ± 10, UCN 289 ± 94, UCN + RKT 133 ± 27), but not significant (P=0.08). RKT also suppressed UCN-induced c-fos expression in PVN. In contrast, the dorsal motor nucleus involved in vagal outflow did not show any changes in Fos and fos expression by RKT. Conclusions: These data indicate that a) RKT inhibits the activity of the neuron of NTS suggesting that RKT inhibits vagal input from the periphery to central nerves. b) As the result of inhibiton of vagal input, activity of hypothalamic satiety center, PVN is suppressed resulting in the inhibition of the sympathetic outflow from the central nerve to the periphery. RKT may restore disturbed appetite and ghrelin secretion through the restoration of balance of central autonomic nervous system.

Cholinergic denervation attenuates phencyclidine-induced c-fos responses in rat cortical neurons

The cortical cholinergic innervation, which is important for memory and cognition, has been implicated in schizophrenia. To experimentally analyze such a possible role of the cholinergic system, we have used the dissociative drug phencyclidine (PCP), known to produce schizophrenia-like psychosis in humans, to model aspects of schizophrenia in rats. We previously showed that induced cortical cholinergic hypofunction leads to enhanced PCP-induced locomotor activity and attenuated social interaction. After PCP, rats lacking cortical cholinergic innervation also show impaired declarative memory. To directly study the role of the basalo-cortical cholinergic projections for PCP-induced neural activation in different cortical areas, we have now monitored the rapid (30 and 60min) effects of low doses of PCP (2 and 3mg/kg) on neural activation as reflected by transcriptional activation of c-fos in cortical areas, using quantitative in situ hybridization. We find an almost pan-cortical neural induction of c-fos mRNA with doses of PCP low enough not to alter levels of either BDNF or Nogo receptor mRNA levels. Specific unilateral lesioning of the uncrossed cholinergic projections to the cortical mantle by 192-IgG-saporin immunotoxin delivery to nc basalis (NBM) caused a striking ipsilateral decrease of the PCP-induced cortical c-fos mRNA induction, restricted to areas which had become effectively denervated. Because PCP at low doses is unlikely to directly influence cortical neurons, we suggest that it acts by activation of the cholinergic input, which in turn leads to cortical c-fos mRNA increases. Our results are compatible with a role for the cholinergic system in symptoms of schizophrenia, by showing that the basalo-cortical cholinergic projections are needed in order for PCP to have full activating effects on cortical neurons.

Cannabinoid receptor type 1 antagonism significantly modulates basal and loud noise induced neural and hypothalamic-pituitary-adrenal axis responses in male Sprague–Dawley rats

Altered regulation of the hypothalamic-pituitary-adrenal (HPA) axis is associated with stress-induced changes in cognitive, emotional, and physical health. Recent evidence indicates that the endogenous cannabinoid (eCB) system may modulate HPA-axis function both directly and more centrally, via regulation of limbic brain systems that control HPA-axis activity. The current study examines the contribution of cannabinoid type 1 (CB1) receptor modulation throughout the neuraxis on control and stress-induced HPA-axis activity. Adult male Sprague–Dawley rats were given intraperitoneal injections of either CB1 receptor antagonist (AM251, 2 mg/kg) or vehicle 30 min prior to a session of loud white noise stress (95 dBA for 30 min) or placement in a familiar sound-proof chamber. Immediately following stress and control treatments, rats were killed, the brains and pituitary glands were excised for subsequent immediate early gene (c-fos mRNA) measurement, and trunk blood was collected for subsequent determination of corticosterone (CORT) and adrenocorticotropic (ACTH) hormone levels. AM251 treatment resulted in a potentiated plasma ACTH response to loud noise stress. AM251 treatment also increased stress-induced plasma CORT levels, but that increase may be due to an increase in basal plasma CORT levels, as was evident in control rats. AM251 treatment produced three distinctive c-fos mRNA response patterns across the various brain regions examined. In cortical (prelimbic, infralimbic, somatosensory, and auditory) and some subcortical structures (basolateral amygdala and paraventricular nucleus of the hypothalamus), AM251 treatment produced a substantial increase in c-fos mRNA that was comparable with the elevated c-fos mRNA levels present in those brain regions of both vehicle and AM251-treated stressed rats. In some other subcortical structures (bed nucleus of the stria terminalis and medial preoptic area) and the anterior pituitary, AM251 treatment produced a c-fos mRNA response pattern that was similar to the response pattern of ACTH hormone levels, that is, no effect on no noise control levels, but an augmentation of stress-induced levels. Conversely, in the medial geniculate and ventral posterior thalamus, AM251 treatment inhibited stress-induced c-fos mRNA induction. These data indicate that disruption of eCB signaling through CB1 receptors results in potentiated neural and endocrine responses to loud noise stress, but also substantial increases in activity in various brain regions and the adrenal gland.This article is part of a Special Issue entitled: Stress, Emotional Behavior and the Endocannabinoid System.

The role of endogenous neurotensin in psychostimulant-induced disruption of prepulse inhibition and locomotion

The neuropeptide neurotensin (NT) is closely associated with dopaminergic and glutamatergic systems in the rat brain. Central injection of NT into the nucleus accumbens (NAcc) or peripheral administration of NT receptor agonists, reduces many of the behavioral effects of psychostimulants. However, the role of endogenous NT in the behavioral effects of psychostimulants (e.g. DA agonists and NMDA receptor antagonists) remains unclear. Using a NTR antagonist, SR142948A, the current studies were designed to examine the role of endogenous NT in DA receptor agonist- and NMDA receptor antagonist-induced disruption of prepulse inhibition of the acoustic startle response (PPI), locomotor hyperactivity and brain-region specific c-fos mRNA expression. Adult male rats received a single i.p. injection of SR142948A or vehicle followed by d-amphetamine, apomorphine or dizocilpine challenge. SR142948A had no effect on baseline PPI, but dose-dependently attenuated d-amphetamine- and dizocilpine-induced PPI disruption and enhanced apomorphine-induced PPI disruption. SR142948A did not significantly affect either baseline locomotor activity or stimulant-induced hyperlocomotion. Systemic SR142948A administration prevented c-fos mRNA induction in mesolimbic terminal fields (prefrontal cortex, lateral septum, NAcc, ventral subiculum) induced by all three psychostimulants implicating the VTA as the site for NT modulation of stimulant-induced PPI disruption. Further characterization of the NT system may be valuable to find clinical useful compounds for schizophrenia and drug addiction.

A food predictive cue must be attributed with incentive salience for it to induce c-fos mRNA expression in cortico-striatal-thalamic brain regions.

Cues associated with rewards acquire the ability to engage the same brain systems as rewards themselves. However, reward cues have multiple properties. For example, they not only act as predictors of reward capable of evoking conditional responses (CRs), but they may also acquire incentive motivational properties. As incentive stimuli they can evoke complex emotional and motivational states. Here we sought to determine whether the predictive value of a reward cue is sufficient to engage brain reward systems, or whether the cue must also be attributed with incentive salience. We took advantage of the fact that there are large individual differences in the extent to which reward cues are attributed with incentive salience. When a cue (conditional stimulus, CS) is paired with delivery of food (unconditional stimulus, US), the cue acquires the ability to evoke a CR in all rats; that is, it is equally predictive and supports learning the CS-US association in all. However, only in a subset of rats is the cue attributed with incentive salience, becoming an attractive and desirable incentive stimulus. We used in situ hybridization histochemistry to quantify the ability of a food cue to induce c-fos mRNA expression in rats that varied in the extent to which they attributed incentive salience to the cue. We found that a food cue induced c-fos mRNA in the orbitofrontal cortex, striatum (caudate and nucleus accumbens), thalamus (paraventricular, intermediodorsal and central medial nuclei), and lateral habenula, only in rats that attributed incentive salience to the cue. Furthermore, patterns of "connectivity" between these brain regions differed markedly between rats that did or did not attribute incentive salience to the food cue. These data suggest that the predictive value of a reward cue is not sufficient to engage brain reward systems-the cue must also be attributed with incentive salience.