Shrew's venom quickly causes circulation disorder, analgesia and hypokinesia.

Multiple representatives of eulipotyphlan mammals such as shrews have oral venom systems. Venom facilitates shrews to hunt and/or hoard preys. However, little is known about their venom composition, and especially the mechanism to hoard prey in comatose states for meeting their extremely high metabolic rates. A toxin (BQTX) was identified from venomous submaxillary glands of the shrew Blarinella quadraticauda. BQTX is specifically distributed and highly concentrated (~ 1% total protein) in the organs. BQTX shares structural and functional similarities to toxins from snakes, wasps and snails, suggesting an evolutional relevancy of venoms from mammalians and non-mammalians. By potentiating thrombin and factor-XIIa and inhibiting plasmin, BQTX induces acute hypertension, blood coagulation and hypokinesia. It also shows strong analgesic function by inhibiting elastase. Notably, the toxin keeps high plasma stability with a 16-h half-life in-vivo, which likely extends intoxication to paralyze or immobilize prey hoarded fresh for later consumption and maximize foraging profit.

Biodistribution of Viscumin after Subcutaneous Injection to Mice and In Vitro Modeling of Endoplasmic Reticulum Stress.

Viscumin (mistletoe lectin I, MLI) in concentrations of 10-11-10-7 M causes endoplasmic reticulum stress and triggers unfolded protein response, a modulator of antitumor immunity, in target cells.

Protein-bound uremic toxins induce tissue remodeling by targeting the EGF receptor.

Indoxyl sulfate and p-cresol sulfate have been suggested to induce kidney tissue remodeling. This study aimed to clarify the molecular mechanisms underlying this tissue remodeling using cultured human proximal renal tubular cells and half-nephrectomized mice treated with indoxyl sulfate or p-cresol sulfate as study models. Molecular docking results suggested that indoxyl sulfate and p-cresol sulfate dock on a putative interdomain pocket of the extracellular EGF receptor. In vitro spectrophotometric analysis revealed that the presence of a synthetic EGF receptor peptide significantly decreased the spectrophotometric absorption of indoxyl sulfate and p-cresol sulfate. In cultured cells, indoxyl sulfate and p-cresol sulfate activated the EGF receptor and downstream signaling by enhancing receptor dimerization, and increased expression of matrix metalloproteinases 2 and 9 in an EGF receptor-dependent manner. Treatment of mice with indoxyl sulfate or p-cresol sulfate significantly activated the renal EGF receptor and increased the tubulointerstitial expression of matrix metalloproteinases 2 and 9. In conclusion, indoxyl sulfate and p-cresol sulfate may induce kidney tissue remodeling through direct binding and activation of the renal EGF receptor.

CD8+ T lymphocyte response against extrahepatic biliary epithelium is activated by epitopes within NSP4 in experimental biliary atresia.

Interferon (IFN)-γ-driven and CD8+ T cell-dependent inflammatory injury to extrahepatic biliary epithelium (EHBE) is likely to be involved in the development of biliary atresia (BA). We previously showed that viral protein NSP4 is the pathogenic immunogen that causes biliary injury in BA. In this study, NSP4 or four synthetic NSP4 (NSP4(157-170), NSP4(144-152), NSP4(93-110), NSP4(24-32)) identified by computer analysis as candidate CD8+ T cell epitopes were injected into neonatal mice. The pathogenic NSP4 epitopes were confirmed by studying extrahepatic bile duct injury, IFN-γ release and CD8+ T cell response against EHBE. The results revealed, at 7 days postinjection, inoculation of glutathione S-transferase (GST)-NSP4 caused EHBE injury and BA in neonatal mice. At 7 or 14 days postinject, inoculation of GST-NSP4, NSP4(144-152), or NSP4(157-170) increased IFN-γ release by CD8+ T cells, elevated the population of hepatic memory CD8+ T cells, and augmented cytotoxicity of CD8+ T cells to rhesus rotavirus (RRV)-infected or naive EHBE cells. Furthermore, depletion of CD8+ T cells in mice abrogated the elevation of GST-NSP4-induced serum IFN-γ. Lastly, parenteral immunization of mouse dams with GST-NSP4, NSP4(144-152), or NSP4(157-170) decreased the incidence of RRV-induced BA in their offspring. Overall, this study reports the CD8+ T cell response against EHBE is activated by epitopes within rotavirus NSP4 in experimental BA. Neonatal passive immunization by maternal vaccination against NSP4(144-152) or NSP4(157-170) is effective in protecting neonates from developing RRV-related BA.

Organic anion transporting polypeptides 1B1 and 1B3 play an important role in uremic toxin handling and drug-uremic toxin interactions in the liver.

PURPOSE: Organic anion-transporting polypeptide (OATP) 1B1 and OATP1B3 contribute to hepatic uptake of numerous drugs. Thus, reduced OATP1B1 and OATP1B3 activity in chronic kidney disease (CKD) may have a major impact on the hepatic clearance of drugs. The effect of drug-uremic toxin interactions on OATP1B1 and OATP1B3 has not been well studied. In the present study, we examine the inhibitory effects of uremic toxins on OATP1B1 and OATP1B3 transport activity to evaluate the interactions between drugs and uremic toxins in patients with chronic kidney disease. METHODS. [3H]Estron-3-sulfate, [3H]taurocholate uptake and [3H]methotrexate by OATP1B1 and OATP1B3 expressing HEK293 cells were performed to evaluate the inhibitory effect of uremic toxins. To clarify whether the uremic toxins that interact with OATP1B1 and/or OATP1B3 were substrates for these transporters, we performed uptake studies. RESULTS. Four uremic toxins, kynurenic acid, indole-3-acetic acid, indoxyl sulfate, and p-cresol, inhibited OATP1B1- and OATP1B3-mediated transport in a concentration-dependent manner, with IC50 values of 180, 770, 2700, and 4600 µM, respectively, for OATP1B1 and 180, 1100, 1300, and 1700 µM, respectively, for OATP1B3. [3H]Methotrexate uptake by OATPs was also inhibited by the four uremic toxins in a dose-dependent manner. Uptake studies revealed that kynurenic acid is a substrate for both the OATP1B1 and OATP1B3. Moreover, OATP1B3 was involved in the transport of indoxyl sulfate. Indole-3-acetic acid and p-cresol were not significantly transported by OATP1B1 and OATP1B3. CONCLUSIONS. We showed that some uremic toxins inhibit OATP-mediated uptake in a concentration-dependent manner, and clarified OATPs contribution to uremic toxin handling in the liver. Thus, we provided basic information to estimate the inhibitory effects of uremic toxins on OATPs in CKD patients. These data suggest that the dose of drugs excreted via renal and non-renal pathways should be carefully adjusted in CKD patients.

Perturbation of bile acid homeostasis is an early pathogenesis event of drug induced liver injury in rats.

Drug-induced liver injury (DILI) is a significant consideration for drug development. Current preclinical DILI assessment relying on histopathology and clinical chemistry has limitations in sensitivity and discordance with human. To gain insights on DILI pathogenesis and identify potential biomarkers for improved DILI detection, we performed untargeted metabolomic analyses on rats treated with thirteen known hepatotoxins causing various types of DILI: necrosis (acetaminophen, bendazac, cyclosporine A, carbon tetrachloride, ethionine), cholestasis (methapyrilene and naphthylisothiocyanate), steatosis (tetracycline and ticlopidine), and idiosyncratic (carbamazepine, chlorzoxasone, flutamide, and nimesulide) at two doses and two time points. Statistical analysis and pathway mapping of the nearly 1900 metabolites profiled in the plasma, urine, and liver revealed diverse time and dose dependent metabolic cascades leading to DILI by the hepatotoxins. The most consistent change induced by the hepatotoxins, detectable even at the early time point/low dose, was the significant elevations of a panel of bile acids in the plasma and urine, suggesting that DILI impaired hepatic bile acid uptake from the circulation. Furthermore, bile acid amidation in the hepatocytes was altered depending on the severity of the hepatotoxin-induced oxidative stress. The alteration of the bile acids was most evident by the necrosis and cholestasis hepatotoxins, with more subtle effects by the steatosis and idiosyncratic hepatotoxins. Taking together, our data suggest that the perturbation of bile acid homeostasis is an early event of DILI. Upon further validation, selected bile acids in the circulation could be potentially used as sensitive and early DILI preclinical biomarkers.

Dose-finding design using mixed-effect proportional odds model for longitudinal graded toxicity data in phase I oncology clinical trials.

Phase I oncology clinical trials are designed to identify the optimal dose that will be recommended for phase II trials. This dose is typically defined as the dose associated with a certain probability of severe toxicity during the first cycle of treatment, although toxicity is repeatedly measured over cycles on an ordinal scale. We propose a new adaptive dose-finding design using longitudinal measurements of ordinal toxic adverse events, with proportional odds mixed-effect models. Likelihood-based inference is implemented. The optimal dose is then the dose producing a target rate of severe toxicity per cycle. This model can also be used to identify cumulative or late toxicities. The performances of this approach were compared with those of the continual reassessment method in a simulation study. Operating characteristics were evaluated in terms of correct identification of the target dose, distribution of the doses allocated and power to detect trends in the risk of toxicities over time. This approach was also used to reanalyse data from a phase I oncology trial. Use of a proportional odds mixed-effect model appears to be feasible in phase I dose-finding trials, increases the ability of selecting the correct dose and provides a tool to detect cumulative effects.

Does indoxyl sulfate, a uraemic toxin, have direct effects on cardiac fibroblasts and myocytes?

AIMS: Indoxyl sulfate (IS) is a uraemic toxin found at high concentration in patients with chronic kidney disease (CKD) co-morbid with chronic heart failure (CHF). The aim of this study was to determine direct effects of IS on cardiac cells as well as the pro-inflammatory effect of IS. METHODS AND RESULTS: Indoxyl sulfate significantly increased neonatal rat cardiac fibroblast collagen synthesis (by 145.7% vs. control, P < 0.05) and myocyte hypertrophy (by 134.5% vs. control, P < 0.001) as determined by (3)H-proline or (3)H-leucine incorporation, respectively. Indoxyl sulfate stimulated tumour necrosis factor-alpha, interleukin-6 (IL-6), and IL-1beta mRNA expression in THP-1 cells as quantified by RT-PCR. Both p38 (RWJ-67657) and MEK1/2 (U0126) inhibitors suppressed all these effects by IS. Furthermore, western blot analysis showed that IS activated mitogen-activated protein kinase (MAPK) (p38, p42/44) and nuclear factor-kappa B (NFkappaB) pathways. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that IS exerted its effects without affecting cell viability. CONCLUSION: This study has, for the first time, demonstrated that IS has pro-fibrotic, pro-hypertrophic, and pro-inflammatory effects, indicating that IS might play an important role in adverse cardiac remodelling mediated via activation of the p38 MAPK, p42/44 MAPK, and NFkappaB pathways. Targeting reduction of IS and/or the pathways it activates may represent a novel therapeutic approach to the management of CHF with concomitant CKD.

A common neuronal mechanism of hypertension and sleep disturbances in spontaneously hypertensive rats: Role of orexinergic neurons.

Epidemiologic studies have shown that sleep disorders are associated with the development of hypertension. The present study investigated dynamic changes in sleep patterns during the development of hypertension across the lifespan in spontaneously hypertensive rats (SHRs) and the neural mechanism that underlies these comorbidities, with a focus on the orexinergic system. Blood pressure in rats was measured using a noninvasive blood pressure tail cuff. Sleep was monitored by electroencephalographic and electromyographic recordings. Immunohistochemistry was used to detect the density and activity of orexinergic neurons in the perifornical nucleus. Hcrt2-SAP (400 or 800 ng) was microinjected in the lateral hypothalamus to lesion orexinergic neurons. Compared with Wistar-Kyoto rats, SHRs exhibited various patterns of sleep disturbances. In SHRs, dynamic changes in hypersomnia in the rats' active phase was not synchronized with the development of hypertension, but hyperarousal in the inactive phase and difficulties in falling asleep were observed concurrently with the development of hypertension. Furthermore, the density and activity of orexinergic neurons in the perifornical nucleus were significantly higher in SHRs than in age-matched Wistar-Kyoto rats. The reduction of orexinergic neurons in the lateral hypothalamus partially ameliorated the development of hypertension and prevented difficulties in falling asleep in SHRs. These results indicate that although the correlation between sleep disturbances and hypertension is very complex, common mechanisms may underlie these comorbidities in SHRs. Overactivity of the orexin system may be one such common mechanism.

Adenine nucleotide-induced contraction on the inner mitochondrial membrane. II. Effect of bongkrekic acid.

In bovine heart mitochondria bongkrekic acid at concentrations as low as about 4 nmol/mg protein (a) completely inhibits phosphorylation of exogenous adenosine diphosphate (ADP) and dephosphorylation of exogenous adenosine triphosphate (ATP), (b) completely reverses atractyloside inhibition of inner membrane contraction induced by exogenous adenine nucleotides, and (c) decreases the amount of adenine nucleotide required to elicit maximal exogenous adenine nucleotide-induced inner membrane contraction to a level which appears to correspond closely with the concentration of contractile, exogenous adenine nucleotide binding sites Bongkrekic acid at concentrations greater than 4 nmol/mg protein induces inner membrane contraction which seems to depend on the presence of endogenous ADP and/or ATP. The findings appear to be consistent with the interpretations (a) that the inner mitochondrial membrane contains two types of contractile, adenine nucleotide binding sites, (b) that the two sites differ markedly with regard to adenine nucleotide affinity, (c) that the high affinity site is identical with the adenine nucleotide exchange carrier, (d) that the low affinity site is accessible exclusively to endogenous adenine nucleotides and is largely unoccupied in the absence of bongkrekic acid, and (e) that bongkrekic acid increases the affinity of both sites in proportion to the amount of the antibiotic bound to the inner membrane.

Recombinant toxins for cancer treatment.

Recombinant toxins target cell surface receptors and antigens on tumor cells. They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem. Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies. They can be mass-produced cheaply in bacteria as homogeneous proteins. Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target.

Thermosensitive liposomes for triggered release of cytotoxic proteins.

Lysolipid-containing thermosensitive liposomes (LTSL) are clinically-relevant drug nanocarriers which have been used to deliver small molecule cytostatics to tumors in combination with local hyperthermia (42 °C) to trigger local drug release. The objective of this study was to investigate the feasibility of LTSL for encapsulation and triggered release of macromolecular drugs such as plant-derived cytotoxins. As therapeutic protein we used Mistletoe lectin-1 (ML1) - a ribosome-inactivating protein with potent cytotoxic activity in tumor cells. Model macromolecules (dextrans, albumin) and ML1 were encapsulated in small unilamellar LTSL with varying lipid compositions by the thin film hydration method and extrusion. LTSLs showed molecular weight dependent heat-triggered release of the loaded cargo. The most promising composition, ML1 formulated in LTSL composed of 86:10:4 %mol DPPC:MSPC:DSPE-PEG2000, was further studied for bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging showed uptake of released ML1 after mild hyperthermia at 42 °C, subsequently leading to potent cytotoxicity by LTSL-ML1. Our study shows that LTSL in combination with localized hyperthermia hold promise as local tumor delivery strategy for macromolecular cytotoxins.

Immunization of Mice by Rotavirus NSP4-VP6 Fusion Protein Elicited Stronger Responses Compared to VP6 Alone.

Due to the limitations and safety issues of the two currently approved live attenuated rotavirus (RV) vaccines "RotaTeq and Rotarix," studies on nonreplicating sources of RV vaccines and search for proper RV antigens are actively carried out. The adjuvant activity of NSP4 and highly immunogenic properties of RV VP6 protein prompted us to consider the construction of a NSP4112-175-VP6 fusion protein and to assess the anti-VP6 IgG, IgA, and IgG subclass responses induced by Escherichia coli-derived NSP4-VP6 fusion protein compared to that of VP6 protein with/without formulation in Montanide ISA 50V2 (M50) in BALB/c mice. Results indicated to the proper expression of the fused NSP4-VP6 and VP6 proteins in E. coli. Intraperitoneal immunization by M50 formulated NSP4-VP6 fusion protein (M5+NSP4-VP6) induced the highest titration of VP6-specific IgG and IgA responses compared to the other groups. Indeed, the presence of NSP4 resulted to the induction of stronger humoral immune responses against the fused protein compared to that elicited by administration of VP6 protein alone (with/without M50 formulation), implying the adjuvant properties of NSP4 for the fused protein. Moreover, the "M50+NSP4-VP6" formulation induced higher serum IgG2a titers than IgG1 and increased Interferon-γ levels, despite unchanged interleukin-4 amounts compared to other groups, indicating Th1-oriented responses with a possible role of NSP4. In conclusion, this study further highlights the potentiality of NSP4-VP6 fusion protein as an efficient and cost-effective immunogen in the field of RV vaccine development.

Effect of delivered dosage of cigarette smoke toxins on the levels of urinary biomarkers of exposure.

Urinary metabolites of tobacco smoke toxins are often used as biomarkers for the evaluation of active and passive exposure to cigarette smoke toxins. In a study of healthy smokers, we investigated concentrations of urinary biomarkers in relation to concentrations of selected toxins in mainstream cigarette smoke as determined by machine smoking of cigarettes in a manner that mimics an individual's smoking behavior (topography). Concentrations of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and benzo(a)pyrene, in mainstream smoke determined under human smoking conditions, and their urinary metabolites cotinine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 1-hydroxypyrene were established for 257 individuals who smoked low-yield (0.1-0.8 mg Federal Trade Commission nicotine/cigarette; mean, 0.66; n = 87), medium-yield (0.9-1.2 mg nicotine/cigarette; mean, 1.1; n = 109), and high-yield cigarettes (nicotine, >1.3 mg nicotine/cigarette; mean, 1.41; n = 61). Levels of urinary metabolites expressed per unit of delivered parent compounds decreased with increased smoke emissions. In smokers of low-, medium-, and high-yield cigarettes, the respective cotinine (ng/mg creatinine)-to-nicotine (mg/d) ratios were 89.4, 77.8, and 57.1 (low versus high; P = 0.06); the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (pmol/mg creatinine)-to-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (ng/d) ratios were 0.81, 0.55, and 0.57 (low versus high; P = 0.05); and the 1-hydroxypyrene (pg/mg creatinine)-to-benzo(a)pyrene (ng/d) ratios were 1.55, 1.13, and 0.97 (low versus high; P = 0.008). Similarly, means of cotinine per unit of delivered nicotine in smokers who consumed <20 cigarettes per day was 3.5-fold higher than in those who smoked >20 cigarettes per day. Likewise, a negative correlation was observed between cotinine-to-nicotine ratios and delivered doses of nicotine in subgroups of smokers who used the identical brand of cigarette, namely a filter tip-vented Marlboro (r = -0.59), which is a popular brand among Euro-Americans, and Newport (r = -0.37), a menthol-flavored cigarette without filter tip vents that is preferred by African-Americans. Thus, the intensity of the exposures significantly affects the levels of urinary biomarkers of exposure and should be taken into account in the evaluation of human exposure to cigarette smoke toxins.

Suppression by intrathecal BmK IT2 on rat spontaneous pain behaviors and spinal c-Fos expression induced by formalin.

The central anti-nociception of BmK IT2, a sodium channel modulator from scorpion Buthus martensi Karsh (BmK) was investigated in this study. It was found that the formalin-induced rat spontaneous flinches and spinal c-Fos expression could be significantly suppressed by intrathecal BmK IT2 pre- or post-formalin injection in a dose-dependent manner. The time course of inhibitory effect exerted by intrathecal BmK IT2 on spontaneous flinches was longer in the pre-treatment group than in post-treatment group. This was consistent with the stronger suppression on spinal c-Fos expression exerted by intrathecal BmK IT2 pre-treatment. In addition, the suppression by intrathecal BmK IT2 on formalin-induced c-Fos expression in superficial laminae was more significant than that in deeper laminae. These results indicate that BmK IT2 can induce central anti-nociceptive response and might thus be a valuable molecular tool for the understanding of pain mechanisms.

The proteasome inhibitor bortezomib augments anti-proliferative effects of mistletoe lectin-I and the PPAR-gamma agonist rosiglitazone in human melanoma cells.

BACKGROUND: The NFkappaB signalling pathway plays an important role in chemoresistance and decreased apoptosis. One indirect way to inhibit the NFkappaB pathway is to slow down the proteasomal degradation of its inhibitor IkappaB, thus preventing NFkappaB from translocation into the nucleus. Hence, the effect of the proteasome inhibitor bortezomib (Velcade) on the cell proliferation of the MV3, FemX-1 and G361 human melanoma cell lines and its action in combination with the PPAR-gamma agonist rosiglitazone or the mistletoe lectin ML-I, both having anti-proliferative effects on melanoma cells in single agent use, was investigated. MATERIALS AND METHODS: Proliferation of melanoma cells under the different treatment regimes over a broad concentration range (0.0001-100 microg/ml) was assessed by means of the XTT cell proliferation assay. RESULTS: At a concentration of 0.1 microg/ml bortezomib significantly reduced the proliferation rate of all melanoma cells to 1-13% of the control, which was mediated through increased apoptosis and inhibition of NFkappaB expression. Furthermore, the combination of bortezomib and rosiglitazone was the most potent and increased the effectiveness against melanoma cell growth by 63-71% (compared to single use of rosiglitazone) and by 27-39% (compared to single use of bortezomib), respectively. CONCLUSION: This combination strategy might be a promising approach for future melanoma therapy.

Antimicrobial peptides in frog poisons constitute a molecular toxin delivery system against predators.

Animals using toxic peptides and proteins for predation or defense typically depend on specialized morphological structures, like fangs, spines, or a stinger, for effective intoxication. Here we show that amphibian poisons instead incorporate their own molecular system for toxin delivery to attacking predators. Skin-secreted peptides, generally considered part of the amphibian immune system, permeabilize oral epithelial tissue and enable fast access of cosecreted toxins to the predator's bloodstream and organs. This absorption-enhancing system exists in at least three distantly related frog lineages and is likely to be a widespread adaptation, determining the outcome of predator-prey encounters in hundreds of species.

Homeopathic mistletoe adverse reaction mimics nodal involvement in 18F-FDG PET/CT performed for evaluation of response to chemotherapy in lymphoma.

Some patients use complementary medicine. We present a patient with Hodgkin's lymphoma, scanned with 18F-FDG PET/CT for evaluation of response after chemotherapy, who was self-administering mistletoe as a homeopathic medicine product. The careful review of the images of the entire scan and patient collaboration in anamnesis were crucial to avoid a false positive result. A review of the published scientific data on the effects of mistletoe is also presented.

Using the continual reassessment method: lessons learned from an EORTC phase I dose finding study.

Many clinicians often do not feel comfortable with the Continual Reassessment Method (CRM). This article reviews its implementation, showing the characteristics, advantages and limitations of this method in Phase I studies as an alternative to the classical 'Fibonacci' escalation schema. A two center, dose escalation phase I study of rViscumin was carried out. Thirty-seven patients were included at 14 different dose-levels (10 to 6400 ng/kg). The complete clinical results are presented elsewhere. A 2-step CRM design enables one to speed-up the study and most importantly to obtain an accurate estimate of the maximum tolerated dose (MTD). Different management issues related to a multicenter study are illustrated and we show how the method can go wrong when severe toxicity, or dose limiting toxicity (DLT), is not considered by the clinician as being sufficient to limit dose escalation (here a grade 3 asthenia related to the drug). This would have affected any dose finding methods. We believe that CRM is a good alternative to the standard method from both a statistical and a practical point of view but further methodological research is necessary to address the issues related to the composite nature of the endpoint.

Insomnia following hypocretin2-saporin lesions of the substantia nigra.

The neuropeptide hypocretin, also known as orexin, has been implicated in waking since its deletion leads to the sleep disorder narcolepsy. Hypocretin neurons project to major arousal areas, and in an effort to determine which region is responsible for the changes in sleep-wake architecture we have developed the neurotoxin hypocretin2-saporin, which lesions hypocretin receptor bearing neurons. Here, in rats, we investigate the effects of hypocretin2-saporin lesions of the substantia nigra and ventral tegmental area in the regulation of sleep and wakefulness. Bilateral injection of hypocretin2-sap into both the ventral tegmental area and substantia nigra (92 and 184 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra) or into the substantia nigra alone (184 ng/microl, 0.5 microl) produced insomnia. The insomnia seemed to be associated with a large increase in locomotion on days 4 and 6 postinjection, as hyperactivity and stereotypic movements were consistently observed on the video recordings in all lesioned rats. In these rats, a nearly complete loss of both tyrosine hydroxylase and neuron-specific nuclear protein (neuronal nuclei) immunoreactive cells in the substantia nigra as well as diminution of tyrosine hydroxylase-immunoreactive fibers in the caudate putamen was found. Following bilateral injection of hypocretin2-sap at a lower concentration (46 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra), very little reduction in the number of tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons and only a temporary increase in wakefulness (17.4% increase during light-off period on day 6 postinjection) were observed. Ventral tegmental area lesions (184 ng/mul of hypocretin2-sap, 0.25 microl, bilateral injections) did not produce significant changes in sleep, although most of the tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons in the ventral tegmental area were destroyed. Insomnia following hypocretin2-sap lesions of the substantia nigra could be secondary to increased motor activity resulting from reduction of tonic inhibitory control by the substantia nigra.