In Vivo Bio-Activation of JWH-175 to JWH-018: Pharmacodynamic and Pharmacokinetic Studies in Mice.

3-(1-Naphthalenylmethyl)-1-pentyl-1H-indole (JWH-175) is a synthetic cannabinoid illegally marketed for its psychoactive cannabis-like effects. This study aimed to investigate and compare in vitro and in vivo pharmacodynamic activity of JWH-175 with that of 1-naphthalenyl (1-pentyl-1H-indol-3-yl)-methanone (JWH-018), as well as evaluate the in vitro (human liver microsomes) and in vivo (urine and plasma of CD-1 male mice) metabolic profile of JWH-175. In vitro binding studies showed that JWH-175 is a cannabinoid receptor agonist less potent than JWH-018 on mouse and human CB1 and CB2 receptors. In agreement with in vitro data, JWH-175 reduced the fESPS in brain hippocampal slices of mice less effectively than JWH-018. Similarly, in vivo behavioral studies showed that JWH-175 impaired sensorimotor responses, reduced breath rate and motor activity, and increased pain threshold to mechanical stimuli less potently than JWH-018. Metabolic studies demonstrated that JWH-175 is rapidly bioactivated to JWH-018 in mice blood, suggesting that in vivo effects of JWH-175 are also due to JWH-018 formation. The pharmaco-toxicological profile of JWH-175 was characterized for the first time, proving its in vivo bio-activation to the more potent agonist JWH-018. Thus, it highlighted the great importance of investigating the in vivo metabolism of synthetic cannabinoids for both clinical toxicology and forensic purposes.

Long-Term, Targeted Delivery of GDNF from Encapsulated Cells Is Neuroprotective and Reduces Seizures in the Pilocarpine Model of Epilepsy.

Neurotrophic factors are candidates for treating epilepsy, but their development has been hampered by difficulties in achieving stable and targeted delivery of efficacious concentrations within the desired brain region. We have developed an encapsulated cell technology that overcomes these obstacles by providing a targeted, continuous, de novo synthesized source of high levels of neurotrophic molecules from human clonal ARPE-19 cells encapsulated into hollow fiber membranes. Here we illustrate the potential of this approach for delivering glial cell line-derived neurotrophic factor (GDNF) directly to the hippocampus of epileptic rats. In vivo studies demonstrated that bilateral intrahippocampal implants continued to secrete GDNF that produced high hippocampal GDNF tissue levels in a long-term manner. Identical implants robustly reduced seizure frequency in the pilocarpine model. Seizures were reduced rapidly, and this effect increased in magnitude over 3 months, ultimately leading to a reduction of seizures by 93%. This effect persisted even after device removal, suggesting potential disease-modifying benefits. Importantly, seizure reduction was associated with normalized changes in anxiety and improved cognitive performance. Immunohistochemical analyses revealed that the neurological benefits of GDNF were associated with the normalization of anatomical alterations accompanying chronic epilepsy, including hippocampal atrophy, cell degeneration, loss of parvalbumin-positive interneurons, and abnormal neurogenesis. These effects were associated with the activation of GDNF receptors. All in all, these results support the concept that the implantation of encapsulated GDNF-secreting cells can deliver GDNF in a sustained, targeted, and efficacious manner, paving the way for continuing preclinical evaluation and eventual clinical translation of this approach for epilepsy.SIGNIFICANCE STATEMENT Epilepsy is one of the most common neurological conditions, affecting millions of individuals of all ages. These patients experience debilitating seizures that frequently increase over time and can associate with significant cognitive decline and psychiatric disorders that are generally poorly controlled by pharmacotherapy. We have developed a clinically validated, implantable cell encapsulation system that delivers high and consistent levels of GDNF directly to the brain. In epileptic animals, this system produced a progressive and permanent reduction (>90%) in seizure frequency. These benefits were accompanied by improvements in cognitive and anxiolytic behavior and the normalization of changes in CNS anatomy that underlie chronic epilepsy. Together, these data suggest a novel means of tackling the frequently intractable neurological consequences of this devastating disorder.

Tropospheric ozone effect on olfactory perception and olfactory bulb dopaminergic interneuron excitability.

Ozone (O3) forms in the Earth's atmosphere, both naturally and by reactions of man-made air pollutants. Deleterious effects of O3 have been found in the respiratory system. Here, we examine whether O3 alters olfactory behavior and cellular properties in the olfactory system. For this purpose, mice were exposed to O3 at a concentration found in highly polluted city air [0.8 ppm], and the behavior elicited by social and non-social odors in habituation/dishabituation tests was assessed. In addition, the electrical responses of dopaminergic olfactory bulb (OB) neurons were also evaluated. O3 differentially compromises olfactory perception to odors: it reduces responses to social and non-social odors in Swiss Webster mice, while this effect was observed in C57BL/6 J mice only for some non-social odors. Additionally, O3 reduced the rate of spontaneous spike firing in periglomerular dopaminergic cells (PG-DA) of the OB. Because this effect could reflect changes in excitability and/or synaptic inputs, the ability of O3 to alter PG-DA spontaneous activity was also tested together with cell membrane resistance, membrane potential, rheobase and chronaxie. Taken together, our data suggest the ability of O3 to affect olfactory perception.

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats during EEG Recordings.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Cognitive dysfunction and impaired neuroplasticity following repeated exposure to the synthetic cannabinoid JWH-018 in male mice.

BACKGROUND AND PURPOSE: Psychotic disorders have been reported in long-term users of synthetic cannabinoids. This study aims at investigating the long-lasting effects of repeated JWH-018 exposure. EXPERIMENTAL APPROACH: Male CD-1 mice were injected with vehicle, JWH-018 (6 mg·kg-1 ), the CB1 -antagonist NESS-0327 (1 mg·kg-1 ) or co-administration of NESS-0327 and JWH-018, every day for 7 days. After 15 or 16 days washout, we investigated the effects of JWH-018 on motor function, memory, social dominance and prepulse inhibition (PPI). We also evaluated glutamate levels in dialysates from dorsal striatum, striatal dopamine content and striatal/hippocampal neuroplasticity focusing on the NMDA receptor complex and the neurotrophin BDNF. These measurements were accompanied by in vitro electrophysiological evaluations in hippocampal preparations. Finally, we investigated the density of CB1 receptors and levels of the endocannabinoid anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and their main synthetic and degrading enzymes in the striatum and hippocampus. KEY RESULTS: The repeated treatment with JWH-018 induced psychomotor agitation while reducing social dominance, recognition memory and PPI in mice. JWH-018 disrupted hippocampal LTP and decreased BDNF expression, reduced the synaptic levels of NMDA receptor subunits and decreased the expression of PSD95. Repeated exposure to JWH-018, reduced hippocampal CB1 receptor density and induced a long-term alteration in AEA and 2-AG levels and their degrading enzymes, FAAH and MAGL, in the striatum. CONCLUSION AND IMPLICATIONS: Our findings suggest that repeated administration of a high dose of JWH-018 leads to the manifestation of psychotic-like symptoms accompanied by alterations in neuroplasticity and change in the endocannabinoid system.

Synthetic cannabinoid JWH-073 alters both acute behavior and in vivo/vitro electrophysiological responses in mice.

JWH-073 is a synthetic cannabinoid (SCB) that is illegally marketed within an "herbal blend", causing psychoactive effects more intense than those produced by Cannabis. Users report that JWH-073 causes less harmful effects than other SCBs, misrepresenting it as a "safe JWH-018 alternative", which in turn prompts its recreational use. The present study is aimed to investigate the in vivo pharmacological activity on physiological and neurobehavioral parameters in male CD-1 mice after acute 1 mg/kg JWH-073 administration. To this aim we investigate its effect on sensorimotor (visual, acoustic, and tactile), motor (spontaneous motor activity and catalepsy), and memory functions (novel object recognition; NOR) in mice coupling behavioral and EEG data. Moreover, to clarify how memory function is affected by JWH-073, we performed in vitro electrophysiological studies in hippocampal preparations using a Long-Term Potentiation (LTP) stimulation paradigm. We demonstrated that acute administration of JWH-073 transiently decreased motor activity for up to 25 min and visual sensorimotor responses for up to 105 min, with the highest effects at 25 min (~48 and ~38%, respectively), while the memory function was altered up to 24 h (~33%) in treated-mice as compared to the vehicle. EEG in the somatosensory cortex showed a maximal decrease of α (~23%) and γ (~26%) bands at 15 min, ß (~26%) band at 25 min, a maximal increase of θ (~14%) band at 25 min and δ (~35%) band at 2 h, and a significant decrease of θ (~18%), α (~26%), and ß (~10%) bands during 24 h. On the other hand, EEG in the hippocampus showed a significant decrease of all bands from 10 min to 2 h, with the maximal effect at 30 min for θ (~34%) and γ (~26%) bands and 2 h for α (~36%), ß (~29%), and δ (~15%) bands. Notably, the δ band significant increase both at 5 min (~12%) and 24 h (~19%). Moreover, in vitro results support cognitive function impairment (~60% of decrease) by interfering with hippocampal synaptic transmission and LTP generation. Our results suggest that JWH-073 deeply alters brain electrical responsiveness with minor behavioral symptoms. Thus, it poses a subtle threat to consumers who mistakenly consider it safer than other SCBs.

Low-dose 7,8-Dihydroxyflavone Administration After Status Epilepticus Prevents Epilepsy Development.

Temporal lobe epilepsy often manifests months or even years after an initial epileptogenic insult (e.g., stroke, trauma, status epilepticus) and, therefore, may be preventable. However, no such preventive treatment is currently available. Aim of this study was to test an antioxidant agent, 7,8-dihydroxyflavone (7,8-DHF), that is well tolerated and effective in preclinical models of many neurological disorders, as an anti-epileptogenic drug. However, 7,8-DHF also acts as a TrkB receptor agonist and, based on the literature, this effect may imply an anti- or a pro-epileptogenic effect. We found that low- (5 mg/kg), but not high-dose 7,8-DHF (10 mg/kg) can exert strong anti-epileptogenic effects in the lithium-pilocarpine model (i.e., highly significant reduction in the frequency of spontaneous seizures and in the time to first seizure after status epilepticus). The mechanism of these different dose-related effects remains to be elucidated. Nonetheless, considering its excellent safety profile and antioxidant properties, as well as its putative effects on TrkB receptors, 7,8-DHF represents an interesting template for the development of effective and well-tolerated anti-epileptogenic drugs.

Improvement of HSV-1 based amplicon vectors for a safe and long-lasting gene therapy in non-replicating cells.

A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics in vivo using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.

Metals detected by ICP/MS in wound tissue of war injuries without fragments in Gaza.

BACKGROUND: The amount and identity of metals incorporated into "weapons without fragments" remain undisclosed to health personnel. This poses a long-term risk of assumption and contributes to additional hazards for victims because of increased difficulties with clinical management. We assessed if there was evidence that metals are embedded in "wounds without fragments" of victims of the Israeli military operations in Gaza in 2006 and 2009. METHODS: Biopsies of "wounds without fragments" from clinically classified injuries, amputation (A), charred (C), burns (B), multiple piercing wounds by White Phosphorus (WP) (M), were analyzed by ICP/MS for content in 32 metals. RESULTS: Toxic and carcinogenic metals were detected in folds over control tissues in wound tissues from all injuries: in A and C wounds (Al, Ti, Cu, Sr, Ba, Co, Hg, V, Cs and Sn), in M wounds (Al, Ti, Cu, Sr, Ba, Co and Hg) and in B wounds (Co, Hg, Cs, and Sn); Pb and U in wounds of all classes; B, As, Mn, Rb, Cd, Cr, Zn in wounds of all classes, but M; Ni was in wounds of class A. Kind and amounts of metals correlate with clinical classification of injuries, exposing a specific metal signature, similar for 2006 and 2009 samples. CONCLUSIONS: The presence of toxic and carcinogenic metals in wound tissue is indicative of the presence in weapon inducing the injury. Metal contamination of wounds carries unknown long term risks for survivors, and can imply effects on populations from environmental contamination. We discuss remediation strategies, and believe that these data suggest the need for epidemiological and environmental surveys.

Fgf-2 overexpression increases excitability and seizure susceptibility but decreases seizure-induced cell loss.

Fibroblast growth factor 2 (FGF-2) has multiple, pleiotropic effects on the nervous system that include neurogenesis, neuroprotection and neuroplasticity. Thus, alteration in FGF-2 expression patterns may have a profound impact in brain function, both in normal physiology and in pathology. Here, we used FGF-2 transgenic mice (TgFGF2) to study the effects of endogenous FGF-2 overexpression on susceptibility to seizures and to the pathological consequences of seizures. TgFGF2 mice display increased FGF-2 expression in hippocampal pyramidal neurons and dentate granule cells. Increased density of glutamatergic synaptic vesicles was observed in the hippocampus of TgFGF2 mice, and electrophysiological data (input/output curves and patch-clamp recordings in CA1) confirmed an increase in excitatory inputs in CA1, suggesting the presence of a latent hyperexcitability. Indeed, TgFGF2 mice displayed increased susceptibility to kainate-induced seizures compared with wild-type (WT) littermates, in that latency to generalized seizure onset was reduced, whereas behavioral seizure scores and lethality were increased. Finally, WT and TgFGF2 mice with similar seizure scores were used for examining seizure-induced cellular consequences. Neurogenesis and mossy fiber sprouting were not significantly different between the two groups. In contrast, cell damage (assessed with Fluoro-Jade B, silver impregnation and anti-caspase 3 immunohistochemistry) was significantly lower in TgFGF2 mice, especially in the areas of overexpression (CA1 and CA3), indicating reduction of seizure-induced necrosis and apoptosis. These data suggest that FGF-2 may be implicated in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring ictogenesis but reducing seizure-induced cell death.

Novel Synthetic Opioids: The Pathologist's Point of View.

BACKGROUND: New Psychoactive Substances (NPS) constitute a broad range of hundreds of natural and synthetic drugs, including synthetic opioids, synthetic cannabinoids, synthetic cathinones, and other NPS classes, which were not controlled from 1961 to 1971 by the United Nations drug control conventions. Among these, synthetic opioids represent a major threat to public health. METHODS: A literature search was carried out using public databases (such as PubMed, Google Scholar, and Scopus) to survey fentanyl-, fentanyl analogs-, and other synthetic opioid-related deaths. Keywords including "fentanyl", "fentanyl analogs", "death", "overdose", "intoxication", "synthetic opioids", "Novel Psychoactive Substances", "MT-45", "AH-7921", and "U-47700" were used for the inquiry. RESULTS: From our literature examination, we inferred the frequent implication of fentanyls and synthetic opioids in side effects, which primarily affected the central nervous system and the cardiovascular and pulmonary systems. The data showed a great variety of substances and lethal concentrations. Multidrug-related deaths appeared very common, in most reported cases. CONCLUSIONS: The investigation of the contribution of novel synthetic opioid intoxication to death should be based on a multidisciplinary approach aimed at framing each case and directing the investigation towards targeted toxicological analyses.

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Personalized Needles for Microinjections in the Rodent Brain.

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

Cellular resilience: 5-HT neurons in Tph2(-/-) mice retain normal firing behavior despite the lack of brain 5-HT.

Considerable evidence links dysfunction of serotonin (5-hydroxytryptamine, 5-HT) transmission to neurodevelopmental and psychiatric disorders characterized by compromised "social" cognition and emotion regulation. It is well established that the brain 5-HT system is under autoregulatory control by its principal transmitter 5-HT via its effects on activity and expression of 5-HT system-related proteins. To examine whether 5-HT itself also has a crucial role in the acquisition and maintenance of characteristic rhythmic firing of 5-HT neurons, we compared their intrinsic electrophysiological properties in mice lacking brain 5-HT, i.e. tryptophan hydroxylase-2 null mice (Tph2(-/-)) and their littermates, Tph2(+/-) and Tph2(+/+), by using whole-cell patch-clamp recordings in a brainstem slice preparation and single unit recording in anesthetized animals. We report that the active properties of dorsal raphe nucleus (DRN) 5-HT neurons in vivo (firing rate magnitude and variability; the presence of spike doublets) and in vitro (firing in response to depolarizing current pulses; action potential shape) as well as the resting membrane potential remained essentially unchanged across Tph2 genotypes. However, there were subtle differences in subthreshold properties, most notably, an approximately 25% higher input conductance in Tph2(-/-) mice compared with Tph2(+/-) and Tph2(+/+) littermates (p<0.0001). This difference may at least in part be a consequence of slightly bigger size of the DRN 5-HT neurons in Tph2(-/-) mice (approximately 10%, p<0.0001). Taken together, these findings show that 5-HT neurons acquire and maintain their signature firing properties independently of the presence of their principal neurotransmitter 5-HT, displaying an unexpected functional resilience to complete brain 5-HT deficiency.

Mechanisms of action of CHF3381 in the forebrain.

(1) Aim of this study was to gain insight into the mechanism of action of CHF3381, a novel putative antiepileptic and neuroprotective drug. (2) CHF3381 blocked NMDA currents in primary cultures of cortical neurons: maximal effect was nearly -80% of the NMDA-evoked current, with EC(50) of approximately 5 micro M. This effect was selective, reversible, use-dependent and elicited at the concentrations reached in the rodent brain after peripheral administration of therapeutic doses. (3) CHF3381 also inhibited voltage-gated Na(+) currents in an apparently voltage-dependent manner. However, this effect could be obtained only at relatively high concentrations (100 micro M). (4) Consistent with the mild effects on voltage-gated Na(+) channels, CHF3381 (100 micro M) failed to affect electrical stimulation-evoked glutamate overflow in hippocampal slices. In contrast, the anti-convulsant agent and Na(+) channel blocker lamotrigine (100 micro M) inhibited stimulation-evoked glutamate overflow by approximately 50%. (5) CHF3381 reduced kindled seizure-induced c-fos mRNA levels within the same brain regions, and to a similar level, as the selective NMDA receptor antagonist MK801, providing circumstantial evidence to the idea that CHF3381 blocks NMDA receptors in vivo. (6) The present mechanistic studies suggest that the primary mechanism of action of CHF3381 in the forebrain is blockade of NMDA receptors. On this basis, this compound may have a potential use in other diseases caused by or associated with a pathologically high level of NMDA receptor activation.

Bystander effect on brain tissue of mesoangioblasts producing neurotrophins.

Neurotrophic factors (NTFs) are involved in the regulation of neuronal survival and function and, thus, may be used to treat neurological diseases associated with neuronal death. A major hurdle for their clinical application is the delivery mode. We describe here a new strategy based on the use of progenitor cells called mesoangioblasts (MABs). MABs can be isolated from postnatal mesoderm tissues and, because of a high adhesin-dependent migratory capacity, can reach perivascular targets especially in damaged areas. We generated genetically modified MABs producing nerve growth factor (MABs-NGF) or brain-derived neurotrophic factor (MABs-BDNF) and assessed their bystander effects in vitro using PC12 cells, primary cultures, and organotypic cultures of adult hippocampal slices. MABs-NGF-conditioned medium induced differentiation of PC12 cells, while MABs-BDNF-conditioned medium increased viability of cultured neurons and slices. Slices cultured with MABs-BDNF medium also better retained their morphology and functional connections, and all these effects were abolished by the TrkB kinase blocker K252a or the BDNF scavenger TrkB-IgG. Interestingly, the amount of BDNF released by MABs-BDNF produced greater effects than an identical amount of recombinant BDNF, suggesting that other NTFs produced by MABs synergize with BDNF. Thus, MABs can be an effective vehicle for NTF delivery, promoting differentiation, survival, and functionality of neurons. In summary, MABs hold distinct advantages over other currently evaluated approaches for NTF delivery in the CNS, including synergy of MAB-produced NTF with the neurotrophins. Since MABs may be capable of homing into damaged brain areas, they represent a conceptually novel, promising therapeutic approach to treat neurodegenerative diseases.

Swim stress enhances nociceptin/orphanin FQ-induced inhibition of rat dorsal raphe nucleus activity in vivo and in vitro: role of corticotropin releasing factor.

The effects of nociceptin/orphanin FQ on putative serotonin (5HT) neurons of the dorsal raphe nucleus (DRN), known to modulate the behavioral responses to stress, were investigated in vivo and in vitro. In DRN slices from unstressed rats, nociceptin/orphanin FQ concentration-dependently inhibited the firing rate of putative 5HT neurons (EC(50) = 21.6 +/- 1.21 nM) and the selective NOP receptor antagonist UFP-101 shifted the concentration-response curve to the right (estimated pA(2) 6.86). Nociceptin/orphanin FQ potency was enhanced in slices prepared from rats previously subjected to a 15 min swim stress (EC(50) = 1.98 +/- 0.11 nM). Swim stress did not change the number or affinity of NOP receptors in DRN. Stress-elicited potentiation involved corticotropin-releasing factor (CRF)(1) receptors, GABA signaling and protein synthesis, being attenuated by pre-treatment with antalarmin (20 mg/kg, i.p.), diazepam (2.4 mg/kg, i.p.) and cycloheximide (2.5 mg/kg, i.p.), respectively. In anesthetized unstressed rats, locally applied nociceptin/orphanin FQ (0.03 and 0.1 ng/30 nl) inhibited the firing rate of DRN neurons (to 80 +/- 7 and 54 +/- 10% of baseline, respectively). Nociceptin/orphanin FQ inhibition was potentiated both 24 h after swim stress and 1 h after CRF (30 ng/30 nl intra-DRN). Stress-induced potentiation was prevented by the selective CRF(1) receptor antagonist, NBI 30755 (20 mg/kg, i.p.). In contrast, the inhibitory response of DRN neurons to the 5HT(1A) agonist, 8OH-DPAT (1 microg/1 microl, intra-DRN) was not potentiated by swim stress, ruling out a non-specific enhanced permeability of GIRK channel. Together, these findings suggest that CRF and the nociceptin/orphanin FQ/NOP system interact in the DRN during stress to control 5HT transmission; this may play a role in stress-related neuropsychopathologies.

Inhibition of serotonin outflow by nociceptin/orphaninFQ in dorsal raphe nucleus slices from normal and stressed rats: Role of corticotropin releasing factor.

In the dorsal raphe nucleus (DRN) many inputs converge and interact to modulate serotonergic neuronal activity and the behavioral responses to stress. The effects exerted by two stress-related neuropeptides, corticotropin releasing factor (CRF) and nociceptin/orphaninFQ (N/OFQ), on the outflow of [(3)H]5- hydroxytryptamine were investigated in superfused rat dorsal raphe nucleus slices. Electrical stimulation (100 mA, 1 ms for 2 min) evoked a frequency-dependent peak of [(3)H]5- hydroxytryptamine outflow, which was sodium and calcium-dependent. Corticotropin releasing factor (1-100 nM), concentration-dependently inhibited the stimulation (3 Hz)-evoked [(3)H]5-hydroxytryptamine outflow; the inhibition by 30 nM corticotropin releasing factor (to 68 +/- 5.7%) was prevented both by the non selective CRF receptor antagonist alpha-helicalCRF(9-41) (alpha-HEL) (300 nM) and by the CRF(1) receptor antagonist antalarmin (ANT) (100 nM). The CRF(2) agonist urocortin II (10 nM) did not modify [(3)H]5- hydroxytryptamine outflow, ruling out the involvement of CRF(2) receptors. Bicuculline (BIC), a GABAA antagonist (10 microM), prevented the inhibitory effect of corticotropin releasing factor (30 nM), supporting the hypothesis that the inhibition was mediated by increased gamma-aminobutyric acid (GABA) release. Nociceptin/ orphaninFQ (1 nM-1 microM) exerted an antalarmin- and bicuculline-insensitive inhibition on [(3)H]5- hydroxytryptamine outflow, with the maximum at 100 nM (to 63+/- 4.2%), antagonized by the NOP receptor antagonist UFP-101 (1 microM). Dorsal raphe nucleus slices prepared from rats exposed to 15 min of forced swim stress displayed a reduced [(3)H]5-hydroxytryptamine outflow, in part reversed by antalarmin and further inhibited by nociceptin/orphaninFQ. These findings indicate that (i) both corticotropin releasing factor and nociceptin/orphaninFQ exert an inhibitory control on dorsal raphe nucleus serotonergic neurons; (ii) the inhibition by corticotropin releasing factor involves gamma-aminobutyric acid neurons; (iii) nociceptin/ orphaninFQ inhibits dorsal raphe nucleus serotonin system in a corticotropin releasing factor- and gamma-aminobutyric acid-independent manner; (iv) nociceptin/orphaninFQ modulation is still operant in slices prepared from stressed rats. The nociceptin/orphaninFQ-NOP receptor system could represent a new target for drugs effective in stress-related disorders.

Alterations in seizure susceptibility and in seizure-induced plasticity after pharmacologic and genetic manipulation of the fibroblast growth factor-2 system.

PURPOSE: The adult brain undergoes activity-dependent plastic modifications during pathologic processes that are reminiscent of those observed during development. For example, seizures induce neuronal loss, neurogenesis, axonal and dendritic sprouting, gliosis, and circuit remodeling. Neurotrophic factors and fibroblast growth factor-2 (FGF-2), in particular, are well-known mediators in each of these cellular events. The aim of this minireview is to summarize and discuss the data supporting the idea that FGF-2 may be involved in seizure generation and in their sequelae. METHODS: We used epilepsy models of kainate and kindling, with FGF-2 knockout mice and FGF-2 overexpressing mice. RESULTS: Seizures increase FGF-2 mRNA and protein levels in specific brain areas and upregulate the expression of its receptor FGFR-1. Short-term intrahippocampal injection of FGF-2 cause seizures, whereas long-term i.c.v. infusion of low-dose FGF-2 does not affect kainate seizures but promotes behavioral recovery and reduces hippocampal damage. Kainate seizure severity is not altered in FGF-2 knockout mice, but is increased in FGF-2 overexpressing mice. CONCLUSIONS: FGF-2 is implicated in seizure susceptibility and in seizure-induced plasticity.