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Context: Prognostic prediction models (PPMs) can help clinicians predict outcomes., Objective: To critically examine peer-reviewed PPMs predicting delayed recovery among pediatric patients with concussion., Data Sources: Ovid Medline, Embase, Ovid PsycInfo, Web of Science Core Collection, Cumulative Index to Nursing and Allied Health Literature, Cochrane Library, Google Scholar., Study Selection: The study had to report a PPM for pediatric patients to be used within 28 days of injury to estimate risk of delayed recovery at 28 days to 1 year postinjury. Studies had to have at least 30 participants., Data Extraction: The Critical Appraisal and Data Extraction for Systematic Reviews of Prediction Modeling Studies checklist was completed., Results: Six studies of 13 PPMs were included. These studies primarily reflected male patients in late childhood or early adolescence presenting to an emergency department meeting the Concussion in Sport Group concussion criteria. No study authors used the same outcome definition nor evaluated the clinical utility of a model. All studies demonstrated high risk of bias. Quality of evidence was best for the Predicting and Preventing Postconcussive Problems in Pediatrics (5P) clinical risk score., Limitations: No formal PPM Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) process exists., Conclusions: The 5P clinical risk score may be considered for clinical use. Rigorous external validations, particularly in other settings, are needed. The remaining PPMs require external validation. Lack of consensus regarding delayed recovery criteria limits these PPMs., (Copyright © 2024 by the American Academy of Pediatrics.)

Comparing self-reported symptom scores across time requires longitudinal measurement invariance (LMI), a psychometric property that means the measure is functioning identically across all time points. Despite its prominence as a measure of depression symptom severity in both research and health care, LMI has yet to be firmly established for the Patient Health Questionnaire-9 depression module (PHQ-9), particularly over the course of antidepressant pharmacotherapy. Accordingly, the objective of this study was to assess for LMI of the PHQ-9 during pharmacotherapy for major depressive disorder. This was a secondary analysis of data collected during a randomized controlled trial. A total of 1,944 veterans began antidepressant monotherapy and completed the PHQ-9 six times over 24 weeks of treatment. LMI was assessed using a series of four confirmatory factor analysis models that included all six time points, with estimated parameters increasingly constrained across models to test for different aspects of invariance. Root-mean-square error of approximation of the chi-square difference test values below 0.06 indicated the presence of LMI. Exploratory LMI analyses were also performed for separate sex, age, and race subgroups. Root-mean-square error of approximation of the chi-square difference test showed minimal change in model fits during invariance testing (≤ 0.06 for all steps), supporting full LMI for the PHQ-9. LMI was also supported for all tested veteran subgroups. As such, PHQ-9 sum scores can be compared across extended pharmacotherapy treatment durations. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Background: Individuals with a history of moderate-severe traumatic brain injury (TBI) experience a significantly higher prevalence of insomnia compared to the general population. While individuals living with TBI have been shown to benefit from traditional insomnia interventions (e.g., face-to-face [F2F]), such as Cognitive Behavioral Therapy for Insomnia (CBTI), many barriers exist that limit access to F2F evidence-based treatments. Although computerized CBT-I (CCBT-I) is efficacious in terms of reducing insomnia symptoms, individuals with moderate-severe TBI may require support to engage in such treatment. Here we describe the rationale, design, and methods of a randomized controlled trial (RCT) assessing the efficacy of a guided CCBT-I program for reducing insomnia symptoms for participants with a history of moderate-severe TBI., Methods: This is an RCT of a guided CCBT-I intervention for individuals with a history of moderate-severe TBI and insomnia. The primary outcome is self-reported insomnia severity, pre- to post-intervention. Exploratory outcomes include changes in sleep misperception following CCBT-I and describing the nature of guidance needed by the Study Clinician during the intervention., Conclusion: This study represents an innovative approach to facilitating broader engagement with an evidence-based online treatment for insomnia among those with a history of moderate-severe TBI. Findings will provide evidence for the level and nature of support needed to implement guided CCBT-I. Should findings be positive, this study would provide support for a strategy by which to deliver guided CCBT-I to individuals with a history of moderate-severe TBI., Competing Interests: Declaration of competing interest Dr. Brenner reports grants from the VA, DOD, NIH, and the State of Colorado, editorial renumeration from Wolters Kluwer, and royalties from the American Psychological Association and Oxford University Press. In addition, she consults with sports leagues via her university affiliation. Dr. Forster reports grants from the VA, DOD and NIH., (Copyright © 2023. Published by Elsevier Inc.)

Background: Bright light therapy (BLT) is efficacious for seasonal and non-seasonal depression. However, the current state of BLT use in practice is unknown, impeding efforts to identify and address utilization gaps. This study's objective was to investigate BLT delivery in a nationwide U.S. healthcare system., Methods: This was a retrospective observational study of electronic medical records from all veterans who received outpatient mood disorder-related care in the Veterans Health Administration (VHA) from October 2008 through September 2020. BLT delivery was measured through the placement of light box consults., Results: Of the 3,442,826 veterans who received outpatient mood disorder care, only 57,908 (1.68 %) received a light box consult. Consults increased by 548.44 % (99.9 % credible interval: 467.36 %, 638.74 %) over the timeframe and displayed a robust yearly cycle that peaked on either December 21st or December 22nd. Past mental health treatment for a mood disorder was associated with a higher probability of a consult (relative risk = 4.79, 99.9 % CI: 4.21, 5.60). There was low representation related to veteran age, gender, race, and ethnicity., Limitations: No information on patients who declined light boxes or actual light box use following consult placement., Conclusions: Outpatient BLT delivery for mood disorders in the VHA remains low, despite significant growth over the past decade. It also displays a strong seasonal rhythm that peaks on the winter solstice, suggesting a limited focus on seasonal depression and a suboptimal reactive approach to changing sunlight. Overall, there exists ample opportunity for novel implementation efforts aimed at increasing utilization of BLT., Competing Interests: Declaration of competing interest The authors have no known conflicts of interest to disclose., (Published by Elsevier B.V.)

Background: Increasing evidence suggests that conditions with decreased morning and increased evening light exposure, including shift work, daylight-saving time, and eveningness, are associated with elevated mortality and suicide risk. Given that the alignment between the astronomical, biological, and social time varies across a time zone, with later-shifted daylight exposure in the western partition, we hypothesized that western time zone partitions would have higher suicide rates than eastern partitions., Methods: United States (U.S.) county-level suicide and demographic data, from 2010 to 2018, were obtained from a Centers for Disease Control database. Using longitude and latitude, counties were sorted into the western, middle, or eastern partition of their respective time zones, as well as the northern and southern halves of the U.S. Linear regressions were used to estimate the associations between suicide rates and time zone partitions, adjusting for gender, race, ethnicity, age group, and unemployment rates., Results: Data were available for 2872 counties. Across the U.S., western partitions had statistically significantly higher rates of suicide compared to eastern partitions and averaged up to two additional yearly deaths per 100,000 people (p < .001)., Limitations: Ecological design and limited adjustment for socioeconomic factors., Conclusions: To our knowledge, this is the first study of the relationship between longitude-based time zone partitions and suicide. The results were consistent with the hypothesized elevated suicide rates in the western partitions, and concordant with previous reports on cancer mortality and transportation fatalities. The next step is to retest the hypothesis with individual-level data, accounting for latitude, photoperiodic changes, daylight-saving time, geoclimatic variables, physical and mental health indicators, as well as socioeconomic adversity and protection., Competing Interests: Declaration of competing interest The authors have no known conflicts of interest to disclose., (Published by Elsevier B.V.)

Bright light therapy (BLT) is a promising non-pharmacological treatment for a range of psychiatric conditions. The goal of this review was to provide a comprehensive overview of the efficacy of BLT across mental and behavioral illnesses. Using systematic umbrella review methodology, we searched Ovid MEDLINE, Embase, PsycInfo, Web of Science, and Google Scholar for systematic reviews of randomized controlled trials (RCTs) evaluating BLT for any mental or behavioral illness from the date of inception until March 2021. Review quality was assessed using the AMSTAR 2 tool and summary efficacy data were extracted from recent reviews. Of 792 unique records, 67 systematic reviews were included which targeted a range of disorders related to mood, neurocognition, sleep, and eating. Recent meta-analyses targeting seasonal or non-seasonal depression found that BLT outperformed light-related control conditions. Reviews of other disorders identified few RCTs and generally did not support the efficacy of BLT for various outcomes. Overall, the extant literature supports the efficacy of BLT for seasonal and non-seasonal depression, although higher quality systematic reviews are needed to increase confidence in these findings. There was no specific funding for this review, and it was preregistered on Prospero (ID: CRD42021240751).

Objective: Bayesian meta-analyses offer several advantages over traditional approaches, including improved accuracy when using a small number of studies and enhanced estimation of heterogeneity. However, psychological trauma research has yet to see widespread adoption of these statistical methods, potentially due to researchers' unfamiliarity with the processes involved. The purpose of this article is to provide a practical tutorial for conducting random-effects Bayesian meta-analyses., Method: Explanations and recommendations are provided for completing the primary steps of a Bayesian meta-analysis, ranging from model specification to interpretation of results. Furthermore, an illustrative example is used to demonstrate the application of each step. In the example, results are synthesized from six studies included in a previously published systematic review (Holliday et al., 2020), with a combined sample size of 21,244,109, examining the association between posttraumatic stress disorder and risk of suicide in veterans and military personnel., Results: The posterior distributions for each model estimate, such as the pooled effect size and the heterogeneity parameter, are discussed and interpreted with regard to the probability of increased suicide risk., Conclusions: Our hope is that this tutorial, along with the provided data and code, facilitate the use of Bayesian meta-analyses in the study of psychological trauma. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Objective: Kibler et al. (2009) reported that hypertension was related to PTSD independent of depression. These two conditions have significant diagnostic overlap. The present study sought to conceptually replicate this work with a veteran sample, using Bayesian estimation to directly update past results, as well as examine symptom severity scores in relation to hypertension., Method: This was a secondary analysis of data obtained from the United States-Veteran Microbiome Project. Lifetime diagnoses of PTSD and major depressive disorder (MDD) were obtained from a structured clinical interview and hypertension diagnoses were extracted from electronic medical records. PTSD and depressive symptom severity were obtained from self-report measures. Logistic regressions with Bayesian estimation were used to estimate the associations between hypertension and (a) psychiatric diagnostic history and (b) symptom severity scores., Results: Compared with veterans without lifetime diagnoses of either disorder, the PTSD-only group was estimated to have a 29% increase in hypertension risk, and the PTSD + MDD group was estimated to have a 66% increase in hypertension risk. Additionally, higher levels of PTSD symptom severity were associated with a higher risk of hypertension., Conclusion: PTSD diagnosis and symptom severity are uniquely associated with hypertension, independent of MDD or depressive symptom severity. These results support previous findings that PTSD might be a modifiable risk factor for the prevention and treatment of hypertension. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Background: Physical exercise offers benefits for treating psychological disorders, particularly depression. Exercise is associated with reduction of posttraumatic stress disorder (PTSD) symptoms in civilians. Given the comorbidities and unique trauma experiences of the veteran population, the current work aims to estimate the effect of exercise on PTSD symptoms in veteran samples., Observations: A systematic review identified 6 single-arm studies and 3 randomized controlled trials (RCTs) using exercise as an interventional treatment among veteran samples with full or subsyndromal PTSD. Most single-arm studies used yoga-based interventions, whereas RCTs showed more variety and included yoga, aerobic activity, and resistance exercises. Data synthesis of study results revealed a medium standardized mean difference for the single-arm trials (Hedges g , -0.60, P = .03) and a small-to-medium standardized mean difference for the RCTs (Hedges g , -0.40, P = .06). Single-arm studies were all rated at serious risk of bias. Only 1 RCT was rated at high risk of bias, although the remaining RCTs showed some concern of elevated bias., Conclusions: There is preliminary evidence that exercise may be a useful treatment option for PTSD symptom reduction in veterans. Our review also highlights the need for additional high-quality randomized trials to confirm the benefits of exercise for PTSD symptom reduction in veterans., Competing Interests: Author disclosures The authors report no actual or potential conflicts of interest or outside sources of funding with regard to this article., (Copyright © 2022 Frontline Medical Communications Inc., Parsippany, NJ, USA.)

Importance: Understanding the extent to which population-level suicide risk screening facilities follow-up and engagement in mental health treatment is important as engaging at-risk individuals in treatment is critical to reducing suicidal behaviors., Objective: To evaluate mental health follow-up and treatment engagement in the Veterans Health Administration (VHA) following administration of the Columbia-Suicide Severity Rating Scale (C-SSRS) screen, a component of the VHA's universal suicide risk screening program., Design: This cross-sectional study used data from VA's Corporate Data Warehouse., Settings: 140 VHA Medical Centers., Participants: Patients who completed the C-SSRS screen in ambulatory care between October 1, 2018-September 30, 2020., Exposure: Standardized suicide risk screening., Main Outcomes and Measures: Mental health follow-up (one or more visits within 30 days of C-SSRS screening) and treatment engagement (two or more visits within 90 days of C-SSRS screening) were examined., Results: 97,224 Veterans in Fiscal Year 2019 (FY19) (mean age 51.4 years; 86.8% male; 64.8% white, 22.4% African-American) and 58,693 Veterans in FY20 (mean age 49.6 years; 85.5% male; 63.4% white, 21.9% African-American) received the C-SSRS screen. Across FYs, a positive C-SSRS screen was associated with increased probability of mental health follow-up and treatment engagement. Patients who were not seen in mental health in the year prior to screening had the greatest increase in probability of mental health follow-up and engagement following a positive screen (P<0.001). For FY19, a positive C-SSRS screen in non-mental health connected patients was associated with an increased probability of follow-up from 49.8% to 79.5% (relative risk = 1.60) and engagement from 39.5% to 63.6% (relative risk = 1.61). For mental health-connected patients, a positive C-SSRS screen was associated with a smaller increase in probability of follow-up from 75.8% to 87.6% (relative risk = 1.16) and engagement from 63.3% to 76.4% (relative risk = 1.21). Results for FY20 were similar., Conclusions and Relevance: Identification of suicide risk through population-level screening was associated with increased mental health follow-up and engagement, particularly for non-mental health connected patients. Findings support the use of a standardized, comprehensive suicide risk screening program for managing elevated suicide risk in a large healthcare system., Competing Interests: The authors have declared that no competing interests exist.

The Beck Anxiety Inventory (BAI) is widely used within the Veterans Health Administration (VHA), both as an assessment tool and as a part of measurement-based care practices. However, there is preliminary evidence that the BAI may perform uniquely in veteran samples, emphasizing the need for a comprehensive investigation of the BAI in this population. The present study compared the normative data reported by Beck and Steer (1993) to secondary data generated by a nationwide sample of U.S. military veterans receiving treatment through the VHA. Secondary data, including initial BAI scores, demographic characteristics, treatment location, and diagnoses originally recorded during the course of usual VHA care over a 5-year period for 57,088 individual veterans, were extracted through the VA Informatics and Computing Infrastructure. BAI scores were compared across samples and various veteran subgroups. Exploratory and confirmatory factor analyses were also conducted. Results revealed that the BAI performed similarly across veteran and normative samples. Male and older veterans were found to have lower BAI scores than their respective counterparts. Factor analyses indicated that a three-factor model best fit the veteran data. Additionally, a cut score of 18 best differentiated between veterans with and without anxiety and related disorders. This study helps support the use of the BAI as a reliable and valid instrument for assessing anxiety symptoms in veterans. Additional research is recommended to better guide BAI interpretation across age groups and sexes/genders. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

The Beck Depression Inventory-II (BDI-II) is used within the Veterans Health Administration (VHA) to measure depression symptom severity. This naturalistic study aimed to examine VHA-specific BDI-II use and establish normative data and psychometric properties. Initial BDI-II data for 152,260 individual veterans were extracted from preexisting medical records using the VA Informatics and Computing Infrastructure. BDI-II scores were compared against Beck, Steer, and Brown (1996)'s original sample, as well as across veteran subgroups. Exploratory and confirmatory factor analyses were also conducted. Similar to Beck et al.'s (1996) sample, the BDI-II was most frequently administered in outpatient psychiatric VHA settings, although it was also used in inpatient and medical settings. Veterans scored significantly higher on the BDI-II than the original comparison groups. This was true across diagnostic categories. The largest discrepancy was seen between nondepressed veterans and corresponding patients from the original sample (Cohen's d = 1.34). Older veterans endorsed less severe levels of depression symptomatology. Additionally, a 2-factor model similar to Beck et al.'s (1996) original solution provided the best fit to the data. Veterans reported higher levels of somatic-affective symptoms than cognitive symptoms. Although potentially useful, the BDI-II requires further investigation in veterans. Standard cut scores are not recommended for use in this population when evaluating severity of depression. A cut score of 27 or higher best differentiated between veterans with and without mood disorders in the current sample. Treatment providers should also consider using BDI-II factor scores, rather than the total score, to measure depressive symptom change. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Added sugars are ubiquitous in contemporary Western diets. Although excessive sugar consumption is now robustly associated with an array of adverse health consequences, comparatively little research has thus far addressed its impact on the risk of mental illness. But ample evidence suggests that high-dose sugar intake can perturb numerous metabolic, inflammatory, and neurobiological processes. Many such effects are of particular relevance to the onset and maintenance of depressive illness, among them: systemic inflammation, gut microbiota disruption, perturbed dopaminergic reward signaling, insulin resistance, oxidative stress, and the generation of toxic advanced glycation end-products (AGEs). Accordingly, we hypothesize that added dietary sugars carry the potential to increase vulnerability to major depressive disorder, particularly at high levels of consumption. The present paper: (a) summarizes the existing experimental and epidemiological research regarding sugar consumption and depression vulnerability; (b) examines the impact of sugar ingestion on known depressogenic physiological processes; and (c) outlines the clinical and theoretical implications of the apparent sugar-depression link. We conclude that the extant literature supports the hypothesized depressogenic impact of added dietary sugars, and propose that an improved understanding of the effects of sugar on body and mind may aid in the development of novel therapeutic and preventative measures for depression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Evidence from recent animal studies suggest that minocycline, a broad-spectrum antibiotic capable of regulating immune processes, may possess antidepressant properties. These studies, however, have yet to be comprehensively reviewed. Accordingly, this systematic review and meta-analysis summarizes the extant literature examining the effect of minocycline on depressive-like behavior in rodent models. PubMed, PsycINFO, and Web of Science databases were systematically searched for articles that met prespecified inclusion and exclusion criteria, and standardized mean differences (SMDs) were calculated for each continuous measure of depressive-like behavior. The overall effect of minocycline on depressive-like behavior was estimated using robust variance estimation meta-analysis. Separate subgroup analyses were conducted on diseased vs healthy animal models, different rodent species, and immobility-based vs anhedonia-based measures of depressive-like behavior. A total of 22 preclinical studies (816 animals) were included. Overall, minocycline reduced depressive-like behavior in rodents (SMD = -1.07, 95% CI -1.41--0.74, p < 0.001). Subgroup analyses revealed that minocycline reduced depressive-like behavior in diseased, but not healthy, animal models. Finally, minocycline was found to reduce both immobility-based and anhedonia-based outcomes. These findings suggest that minocycline may be an effective treatment of core depressive symptoms, and that further investigation of minocycline treatment for clinically relevant depression in humans is warranted.

Background: Probiotics have generated intensive research interest in recent years as a novel mode of treatment for physical and mental illness. Nevertheless, the anxiolytic potential of probiotics remains unclear. The present systematic review and meta-analysis aimed to evaluate the clinical and preclinical (animal model) evidence regarding the effect of probiotic administration on anxiety., Methods: The PubMed, PsycINFO, and Web of Science databases were reviewed for preclinical and clinical studies that met the defined inclusion and exclusion criteria. The effects of probiotics on anxiety-like behavior and symptoms of anxiety were analyzed by meta-analyses. Separate subgroup analyses were conducted on diseased versus healthy animals, specific preclinical probiotic species, and clinical versus healthy human samples., Results: Data were extracted from 22 preclinical studies (743 animals) and 14 clinical studies (1527 individuals). Overall, probiotics reduced anxiety-like behavior in animals (Hedges' g = -0.47, 95% CI -0.77 --0.16, p = 0.004). Subgroup analyses revealed a significant reduction only among diseased animals. Probiotic species-level analyses identified only Lactobacillus (L.) rhamnosus as an anxiolytic species, but these analyses were broadly under-powered. Probiotics did not significantly reduce symptoms of anxiety in humans (Hedges' g = -0.12, 95% CI -0.29-0.05, p = 0.151), and did not differentially affect clinical and healthy human samples., Conclusions: While preclinical (animal) studies suggest that probiotics may help reduce anxiety, such findings have not yet translated to clinical research in humans, perhaps due to the dearth of extant research with clinically anxious populations. Further investigation of probiotic treatment for clinically relevant anxiety is warranted, particularly with respect to the probiotic species L. rhamnosus., Competing Interests: The authors have declared that no competing interests exist.

Psychotherapists risk making 2 types of errors with clients who struggle with addictive behaviors: failure to diagnose addictive behaviors and failure to effectively treat addictive behaviors. Given the high prevalence of addictive behaviors in clinical populations, therapists are in a unique position to assist individuals with these problems. It is assumed that therapists possess general diagnostic and treatment skills and yet many do not diagnose or do not treat addictive behaviors. Reasons for making these errors include prohibitive beliefs and limited knowledge about addictive behaviors. We offer specific recommendations to reduce these psychotherapy errors. These include: (a) more deliberate screening and diagnosis of addictive behaviors, (b) increased application of empirically supported addiction treatments, (c) required education and training in addictive behaviors, (d) modification of prohibitive attitudes about addressing addictive behaviors, and (e) increased attention paid to the addictive behaviors by professional psychotherapy organizations., (PsycINFO Database Record (c) 2016 APA, all rights reserved)

Background: The dynorphin (DYN)/kappa-opioid receptor (KOR) system undergoes neuroadaptations following chronic alcohol exposure that promote excessive operant self-administration and negative affective-like states; however, the exact mechanisms are unknown. The present studies tested the hypothesis that an upregulated DYN/KOR system mediates excessive alcohol self-administration that occurs during withdrawal in alcohol-dependent rats by assessing DYN A peptide expression and KOR function, in combination with site-specific pharmacologic manipulations., Methods: Male Wistar rats were trained to self-administer alcohol using operant behavioral strategies and subjected to intermittent alcohol vapor or air exposure. Changes in self-administration were assessed by pharmacologic challenges during acute withdrawal. In addition, 22-kHz ultrasonic vocalizations were utilized to measure negative affective-like states. Immunohistochemical techniques assessed DYN A peptide expression and [(35)S]GTPγS coupling assays were performed to assess KOR function., Results: Alcohol-dependent rats displayed increased alcohol self-administration, negative affective-like behavior, DYN A-like immunoreactivity, and KOR signaling in the amygdala compared with nondependent control rats. Site-specific infusions of a KOR antagonist selectively attenuated self-administration in dependent rats, whereas a mu-opioid receptor/delta-opioid receptor antagonist cocktail selectively reduced self-administration in nondependent rats. A mu-opioid receptor antagonist/partial KOR agonist attenuated self-administration in both cohorts., Conclusions: Increased DYN A and increased KOR signaling could set the stage for a one-two punch during withdrawal that drives excessive alcohol consumption in alcohol dependence. Importantly, intracentral nucleus of the amygdala pharmacologic challenges functionally confirmed a DYN/KOR system involvement in the escalated alcohol self-administration. Together, the DYN/KOR system is heavily dysregulated in alcohol dependence and contributes to the excessive alcohol consumption during withdrawal., (Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Rationale: Once dependent on alcohol or opioids, negative affect may accompany withdrawal. Dependent individuals are hypothesized to "self-medicate" in order to cope with withdrawal, which promotes escalated alcohol and drug use., Objectives: The current study aimed to develop a reliable animal model to assess symptoms that occur during spontaneous alcohol and opioid withdrawal., Methods: Dependence was induced using intermittent alcohol exposure or pulsatile heroin delivery and assessed for the presence of withdrawal symptoms during acute withdrawal by measuring somatic signs, behavior in the forced swim test (FST), and air-puff-induced 22-kHz ultrasonic vocalizations (USVs). Additional animals subjected to 8 weeks of alcohol vapor exposure were evaluated for altered somatic signs, operant alcohol self-administration, and 22-kHz USV production, as well as performance in the elevated plus maze (EPM)., Results: During spontaneous withdrawal from pulsatile heroin or intermittent alcohol vapor, animals displayed increased somatic withdrawal signs, FST immobility, and 22-kHz USV production but did not show any behavioral change in the EPM unless the duration of alcohol exposure was extended to 4 weeks. Following 8 weeks of alcohol vapor exposure, animals displayed somatic withdrawal signs, escalated alcohol self-administration, and increased 22-kHz USVs., Conclusions: These paradigms provide consistent methods to evaluate the behavioral ramifications, and neurobiological substrates, of alcohol and opioid dependence during spontaneous withdrawal. As immobility in the FST and percent open-arm time in the EPM were dissociable, with 22-kHz USVs paralleling immobility in the FST, assessment of air-puff-induced 22-kHz USVs could provide an ethologically valid alternative to the FST.

Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis after transection of the medial forebrain bundle. We have assessed the temporal and sequential activities of microglia in these events by examining the complement-3 (OX-42), major histocompatibility complex class II antigen presentation (OX-6) and phagocytic activity (ED1), and correlating these indicators with dopaminergic neuronal loss. Microglia in the ipsilateral substantia nigra pars reticulata evinced activation morphology at 12 h postaxotomy. Phagocytic microglia apposed dying dopaminergic neurons in the pars compacta starting at 3 days postlesion; their number increased through 14 days and slowly decreased. Nuclear chromatin condensation and significant loss of tyrosine hydroxylase-positive dopaminergic neurons occurred around 7 days postlesion. In contrast to microglial expression of interleukin-1beta and inducible nitric oxide synthase at the axotomy site, nigral microglia were interleukin-1beta and inducible nitric oxide synthase-negative. Consistently, RNase protection assays showed that interleukin-1beta and inducible nitric oxide synthase transcripts in nigra were equivocal. The present data support the idea that phagocytosis of axotomized neurons by activated microglia is not limited to dead neurons but includes dying neurons probably without cytotoxic effects of inflammatory substances, such as interleukin-1beta or nitric oxide.

Agmatine, decarboxylated from arginine by arginine decarboxylase, is particularly prominent in the hypothalamus. The present study utilized the rat hypothalamo-neurohypophysial system to determine expression and "pre-synaptic" modulation of agmatine in the central nervous system (CNS). Under confocal-laser scanning, agmatine-like immunoreactivity (Agm-LI) was found enriched in arginine-vasopressin (AVP)-containing magnocellular neurons of the supraoptic nuclei (SON) and paraventricular nuclei (PVN). In addition, using electron microscopy, Agm-LI was found closely associated with large neurosecretory-like vesicles in neurohypophysial nerve terminals of posterior pituitary gland. Radioimmunoassay revealed that 10 and 30 microM agmatine concentration-dependently inhibited the depolarization-evoked AVP release from isolated neurohypophysial terminals. Using perforated patch-clamp, effects of agmatine on whole-terminal voltage-gated ion currents in the isolated neurohypophysial nerve terminals were examined. While it did not significantly affect either tetrodotoxin (TTX)-sensitive Na(+) or sustained Ca(2+)-activated K(+) channel currents, agmatine (1-40 microM) inhibited Ca(2+) channel currents in approximately 53% of the total nerve terminals investigated. The onset of inhibitory effect was immediate, and the inhibition was reversible and concentration-dependent with an IC(50)=4.6 microM. In the remaining (approximately 47%) neurohypophysial nerve terminals, only a higher (120 microM) concentration of agmatine could moderately inhibit Ca(2+) channel currents. The results suggest that: (1) endogenous agmatine is co-expressed in AVP-containing, hypothalamic magnocellular neurons of the SON/PVN and in neurohypophysial nerve terminals of posterior pituitary gland; (2) agmatine may serve as a physiological neuromodulator by regulating the voltage-gated Ca(2+) channel and, as a result, the release of AVP from neurohypophysial nerve terminals.

Electrical stimulation of the cerebellar fastigial nucleus (FN) elicits a prolonged ( approximately 10 days) and substantial (50-80%) protection against ischemic and excitotoxic injuries. The mechanism(s) of protection are unknown. We investigated whether FN stimulation directly protects brain cells against apoptotic cell death in an in vitro rat brain slice culture model. Rats were electrically stimulated in FN or, as control, the cerebellar dentate nucleus (DN). Coronal slices through the forebrain were explanted, exposed to staurosporine, harvested, and analyzed for caspase-3 activity by a fluorescence assay. FN, but not DN, stimulation significantly reduced staurosporine-induced caspase-3 activity by 39 +/- 7% at 3 h, 31 +/- 3% at 6 h and 26 +/- 4% at 10 h of incubation. Immunocytochemistry revealed FN-specific reductions in activated caspase-3 mainly in glial-like cells throughout the forebrain. FN stimulation also results in a 56.5% reduction in cytochrome c release upon staurosporine incubation. We conclude that neuroprotection elicited from FN stimulation can directly modify the sensitivity of brain cells to apoptotic stimuli and thereby suppress staurosporine induced apoptosis in adult rat brain slices. This model indicates that neuroprotection can be studied in vitro and provides new insight into the potential role of glial cells in ischemic protection of neurons induced by FN stimulation.

Agmatine (decarboxylated l-arginine), an endogenous ligand of imidazoline and alpha(2) adrenoreceptors, is particularly enriched in the rat hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. The present study utilized light and electron microscopic immunocytochemical methods to determine the distribution and extent of colocalization of agmatine relative to subpopulations of vasopressin- (VP) and oxytocin- (OT) producing neurons in PVN and SON nuclei. By light microscopy, agmatine-immunoreactive perikarya were found in both the magnocellular and the parvocellular neuronal subdivisions of PVN and SON. Confocal and electron microscopy revealed that agmatine-immunoreactivity (I) within neuronal perikarya was associated with the nuclear membrane as well as mitochondria, Golgi complexes, endoplasmic reticula, and plasmalemma. Additionally, agmatine-I was identified in both axons and axonal terminals, which were enriched in large dense-core vesicles. Dual and triple immunocytochemical labeling experiments also demonstrated that agmatine coexists with VP or OT in most PVN and SON magnocellular neurons. Combinations of iontophoretic injections of Fluorogold into the dorsomedullary complex with immunocytochemical labeling revealed that many retrogradely labeled neurons in the parvocellular region of the PVN contained agmatine-I and either VP or OT. These findings provide evidence that agmatine may function as a modulator of both hypothalamically mediated neuroendocrine and autonomic responses., (Copyright 2001 Academic Press.)

We investigated whether stimulation of the functionally discrete subthalamic region, subthalamic cerebrovasodilator area (SVA), which increases cerebral blood flow (CBF) when excited, would, like stimulation of cerebellar fastigial nucleus (FN), produce central neurogenic neuroprotection. A 1-h electrical stimulation of SVA or FN reduced infarctions triggered by permanent occlusion of middle cerebral artery (MCA) by 48-55% in Sprague-Dawley rats and by 59% in Fisher rats. The salvaging effect of SVA stimulation, similar to FN, was long lasting and reduced the volume of infarctions placed 72 h or 10 days later by 58 and 26%, respectively, in Fisher rats. Bilateral lesioning of FN neurons by the microinjection of ibotenic acid 5 days before SVA stimulation did not affect SVA-evoked neuroprotection. Bilateral lesions of SVA neurons administered 5 days before FN stimulation had no effect on FN-induced neuroprotection but reversed the stimulus-locked increase in CBF accompanying FN stimulation. This study demonstrates that (1) excitation of neurons and/or fibers projecting through the SVA reduces ischemic infarctions as substantially as excitation of FN neurons; (2) the effects are long-lasting and not attributable to increases in cerebral blood flow, changes in blood gases or brain temperature, or rat strain; (3) the neuroprotective effects of SVA and FN stimulation are mutually independent and (4) FN-evoked cerebrovasodilation is mediated by SVA neurons. The SVA and FN are part of a neuronal system in CNS, which is distributed and, when excited, acts to protect the brain from ischemic injury.

The present study examined specific effects of sodium cyanide (CN) on the membrane potential (MP), spontaneous discharge (SD) and voltage-gated ion current of the identified bulbospinal rostral ventrolateral medulla (RVLM) neuron in the rat pup brainstem slice. 125 microM CN rapidly depolarized MP in the RVLM neuron by 11.6 mV as well as enhanced the SD rate by 300%. In contrast, the same dose of CN immediately hyperpolarized unlabeled, non-RVLM neurons by 4.8 mV. 50 microM CN did not significantly affect voltage-gated Ca(++) or A-type K(+) currents. The same concentration of CN, however, rapidly and reversibly suppressed voltage-gated Na(+) currents and sustained outward K(+) currents in the RVLM neuron by 22.5% and 23%, respectively. Tetraethylammonium could mimic the effect of CN on MP, SD and sustained K(+) current in the RVLM neuron. It is concluded that: (1) like that from the adult rat, the rat pup bulbospinal RVLM neuron can be selectively and rapidly excited by CN; (2) the hypoxia-sensitive, sustained outward K(+) channel may play an important role in the acute hypoxia-induced excitation of the RVLM neurons.

Sympathoexcitatory reticulospinal neurons of the rostral ventrolateral medulla (RVLM) are oxygen detectors excited by hypoxia to globally elevate regional cerebral blood flow (rCBF). The projection, which accounts for >50% of hypoxic cerebral vasodilation, relays through the medullary vasodilator area (MCVA). However, there are no direct cortical projections from the RVLM/MCVA, suggesting a relay that diffusely innervates cortex and possibly originates in thalamic nuclei. Systematic mapping by electrical microstimulation of the thalamus and subthalamus revealed that elevations in rCBF were elicited only from a limited area, which encompassed medial pole of zona incerta, Forel's field, and prerubral zone. Stimulation (10 sec train) at an active site increased rCBF by 25 +/- 6%. Excitation of local neurons with kainic acid mimicked effects of electrical stimulation by increasing rCBF. Stimulation of the subthalamic cerebrovasodilator area (SVA) with single pulses (0.5 msec; 80 microA) triggered cortical EEG burst-CBF wave complexes with latency 24 +/- 5 msec, which were similar in shape to complexes evoked from the MCVA. Selective bilateral lesioning of the SVA neurons (ibotenic acid, 2 microg, 200 nl) blocked the vasodilation elicited from the MCVA and attenuated hypoxic cerebrovasodilation by 52 +/- 12% (p < 0.05), whereas hypercarbic vasodilation remained preserved. Lesioning of the vasodilator site in the basal forebrain failed to modify SVA-evoked rCBF increase. We conclude that (1) excitation of intrinsic neurons of functionally restricted region of subthalamus elevates rCBF, (2) these neurons relay signals from the MCVA, which elevate rCBF in response to hypoxia, and (3) the SVA is a functionally important site conveying vasodilator signal from the medulla to the telencephalon.

In anesthetized spinalized rat, electrical stimulation of the nucleus tractus solitarius (NTS) synchronizes the EEG by increasing the power of 4-6-Hz waves (>100%; P<0.01), and elevates cerebral blood flow (rCBF) by 18+/-5% (P<0.05). The coordinated response appears within seconds, is global, reversible, graded, evoked from the commissural sub-nucleus, and replicated by L-glutamate. The responses are markedly reduced by bilateral lesions or muscimol microinjections restricted to a region of ventral medullary reticular formation, the medullary cerebral vasodilator area (MCVA), a region from which stimulation elicits identical responses and mediates the comparable responses to hypoxic/ischemic excitation of sympathoexcitatory neurons of rostral ventrolateral medulla (RVLM). We conclude that: (a) excitation of intrinsic neurons of commissural NTS synchronizes the EEG and coordinately elevates rCBF; (b) the responses are mediated by excitation of neurons in MCVA; (c) the MCVA may be a common final pathway mediating cerebrovascular and EEG responses from multiple areas of CNS; and (d) the NTS-MCVA pathway may be a part of the anatomical substrate for behaviors, including slow-wave sleep and seizure suppression evoked by stimulation of visceral afferents terminating in NTS.

We sought to identify the medullary relay area mediating the elevations of regional cerebral blood flow (rCBF) and synchronization of the electroencephalogram (EEG) in the rat cerebral cortex elicited by hypoxic excitation of reticulospinal sympathoexcitatory neurons of the rostral ventrolateral medulla (RVLM ). In anaesthetized spinalized rats electrical stimulation of RVLM elevated rCBF (laser-Doppler flowmetry) by 31 +/- 6 %, reduced cerebrovascular resistance (CVR) by 26 +/- 8 %, and synchronized the EEG, increasing the power of the 5-6 Hz band by 98 +/- 25 %. Stimulation of a contiguous caudal region, the medullary cerebral vasodilator area (MCVA), had comparable effects which, like responses of RVLM, were replicated by microinjection of L-glutamate (5 nmol, 20 nl). Microinjection of NaCN (300 pmol in 20 nl) elevated rCBF (17 +/- 5 %) and synchronized the EEG from RVLM, but not MCVA, while nicotine (1.2 nmol in 40 nl) increased rCBF by 13 +/- 5 % and synchronized the EEG from MCVA. In intact rats nicotine lowered arterial pressure only from MCVA (101 +/- 3 to 52 +/- 9 mmHg). Bilateral electrolytic lesions of MCVA significantly reduced, by over 59 %, elevations in rCBF and, by 78 %, changes in EEG evoked from RVLM. Bilateral electrolytic lesions of RVLM did not affect responses from MCVA. Anterograde tracing with BDA demonstrated that RVLM and MCVA are interconnected. The MCVA is a nicotine-sensitive region of the medulla that relays signals elicited by excitation of oxygen-sensitive reticulospinal neurons in RVLM to reflexively elevate rCBF and slow the EEG as part of the oxygen-conserving (diving) reflex initiated in these neurons by hypoxia or ischaemia.

Antagonists of glutamate receptors of the N-methyl-d-aspartate subclass (NMDAR) or inhibitors of nitric oxide synthase (NOS) prevent nervous system plasticity. Inflammatory and neuropathic pain rely on plasticity, presenting a clinical opportunity for the use of NMDAR antagonists and NOS inhibitors in chronic pain. Agmatine (AG), an endogenous neuromodulator present in brain and spinal cord, has both NMDAR antagonist and NOS inhibitor activities. We report here that AG, exogenously administered to rodents, decreased hyperalgesia accompanying inflammation, normalized the mechanical hypersensitivity (allodynia/hyperalgesia) produced by chemical or mechanical nerve injury, and reduced autotomy-like behavior and lesion size after excitotoxic spinal cord injury. AG produced these effects in the absence of antinociceptive effects in acute pain tests. Endogenous AG also was detected in rodent lumbosacral spinal cord in concentrations similar to those previously detected in brain. The evidence suggests a unique antiplasticity and neuroprotective role for AG in processes underlying persistent pain and neuronal injury.

We investigated whether the medullary cerebrovasodilator area (MCVA), a region of ventral medulla mediating elevations of regional cerebral blood flow (rCBF) and electroencephalogram (EEG) synchronization elicited in cerebral cortex from stimulation of reticulospinal neurons of rostral ventrolateral medulla (RVLM), also mediates comparable responses from the cerebellar fastigial nucleus (FN). In spinalized rats, electrical stimulation of MCVA, RVLM or FN elevated rCBF and synchronized the EEG. The FN-evoked responses were significantly attenuated or blocked by bilateral lesions of MCVA. The MCVA is a novel region of medullary reticular formation mediating actions of medullary and cerebellar centers on rCBF and EEG to link visceral centers of brainstem and cerebral cortex.

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylomithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.

Recent evidence suggests that agmatine, which is an intermediate in polyamine biosynthesis, might be an important neurotransmitter in mammals. Agmatine is synthesized in the brain, stored in synaptic vesicles in regionally selective neurons, accumulated by uptake, released by depolarization, and inactivated by agmatinase. Agmatine binds to alpha2-adrenoceptors and imidazoline binding sites, and blocks NMDA receptor channels and other ligand-gated cationic channels. Furthermore, agmatine inhibits nitric oxide synthase, and induces the release of some peptide hormones. As a result of its ability to inhibit both hyperalgesia and tolerance to, and withdrawal from, morphine, and its neuroprotective activity, agmatine has potential as a treatment of chronic pain, addictive states and brain injury.

The fundamental neuronal substrates of the arterial baroreceptor reflex have been elucidated by combining anatomical, neurophysiological, and pharmacological approaches. A serial pathway between neurons located in the nuclei of the solitary tract (NTS), the caudal ventrolateral medulla (CVL), and the rostral ventrolateral medulla (RVL) plays a critical role in inhibition of sympathetic outflow following stimulation of baroreceptor afferents. In this paper, we summarize our studies using tract-tracing and electron microscopic immunocytochemistry to define the potential functional sites for synaptic transmission within this circuitry. The results are discussed as they relate to the literature showing: (1) baroreceptor afferents excite second-order neurons in NTS through the release of glutamate; (2) these NTS neurons in turn send excitatory projections to neurons in the CVL; (3) GABAergic CVL neurons directly inhibit RVL sympathoexcitatory neurons; and (4) activation of this NTS-->CVL-->RVL pathway leads to disfacilitation of sympathetic preganglionic neurons by promoting withdrawal of their tonic excitatory drive, which largely arises from neurons in the RVL. Baroreceptor control may also be regulated over direct reticulospinal pathways exemplified by a newly recognized sympathoinhibitory region of the medulla, the gigantocellular depressor area. This important autonomic reflex may also be influenced by parallel, multiple, and redundant networks.

Electrical stimulation of the cerebellar fastigial nucleus (FN) in spontaneously hypertensive (SHR), Wistar-Kyoto (WKY) and Fisher rats reduced, by approximately 50%, the infarctions produced by occlusion of the middle cerebral artery. Blockade of ATP-dependent potassium (K-ATP) channels with glibenclamide (i.c.v.) abolished salvage only in the SHR rat. While blockade of K-ATP channels failed to abolish salvage in WKY and Fisher rats, participation of potassium channels in neurogenic neuroprotection cannot be excluded.

Agmatine, an amine and organic cation, is an endogenous ligand at alpha 2-adrenergic and imidazoline (I-) receptors, to which it binds with high affinity. In addition, agmatine has properties of an endogenous neurotransmitter. Thus, agmatine (a) is locally synthesized in brain by a specific enzyme, arginine decarboxylase; (b) is stored in a large number of neurons with selective distribution in the CNS; (c) is associated with small vesicles in axon terminals that, at least in hippocampus, make synaptic asymmetric (excitatory) synapses on pyramidal cells; (d) is released from synaptosomes in a Ca(2+)-dependent manner; (e) can be enzymatically degraded by agmatinase in synaptosomes; (f) can be inactivated by selective reuptake; (g) blocks the ligand-gated NMDA receptor channel at sites distinct from ligand-binding and polyamine sites; and (h) has systemic actions when administered intraventricularly. Additionally, (i) agmatine is a precursor of brain putrescine and, hence, of higher polyamines, and (j) it competitively inhibits the activity of all isozymes of nitric oxide synthase. Agmatine meets most criteria to establish it as a novel neurotransmitter/neuromodulator in the CNS. However, agmatine differs from forms of clonidine displacing system with respect to distribution, bioactivity, and capacity to interact with antibodies raised to imidazoline-like drugs. Thus, there are multiple endogenous ligands of the imidazoline receptors, one of which is agmatine.

We have shown that cultured vascular smooth muscle cells (VSMC) and brain astroglial cells express I-receptors of the I2 subtype. While imidazoline agents are anti-proliferative in smooth muscle cells, they increase the expression of glial fibrillary acidic protein (GFAP) in astrocytes. Because increases in GFAP suppress the induction of calcium-independent, inducible nitric oxide synthase (NOS-2), we measured whether idazoxan and related imidazolines and agmatine would also suppress the expression of NOS-2. Cultured astrocytes and macrophages, RAW 264.7 cell line, were incubated with lipopolysaccharide (LPS, 1 microgram/ml) or cytokine mixture in the presence of 1-100 microM of idazoxan, agmatine, or other imidazoline agents. Idazoxan potently (IC50 10 microM) decreased the activity of NOS-2 in astrocytes, but was less potent in RAW 264.7 cells. By contrast, agmatine was most potent in RAW 264.7 cells (IC50, 10 microM) but less potent in glial cells and VSMC. Both idazoxan and agmatine decreased the activity of NOS-2 by reducing the levels of enzyme protein as measured by immunoblot and immunocytochemistry. No specific binding of [3H]-idazoxan was observed in RAW 264.7 cell membranes. We conclude that idazoxan, agmatine, and selected imidazoline agents inhibit the expression of NOS-2 and proliferation in primary glial cells and VSMC. While the antiproliferative actions appear mediated by I-receptors of the I2 type, the anti-inflammatory response is probably not mediated by I-receptors but possibly by direct actions on signal transduction enzymes.

Electrical stimulation of the cerebellar fastigial nucleus (FN) elevates regional cerebral blood flow (rCBF) and arterial pressure (AP) and provides long-lasting protection against focal and global ischemic infarctions. We investigated which neuronal element in FN, perikarya or axons, mediates this central neurogenic neuroprotection and whether it also protects against excitotoxicity. In anesthetized rats, the FN was stimulated for 1 hr, and ibotenic acid (IBO) was microinjected unilaterally into the striatum. In unstimulated controls, the excitotoxic lesions averaged approximately 40 mm3. Stimulation of FN, but not dentate nucleus (DN), significantly reduced lesion volumes up to 80% when IBO was injected 15 min, 72 hr, or 10 d, but not 30 d, thereafter. In other rats, intrinsic neurons of FN or DN were destroyed by pretreatment with IBO. Five days later, the FN was stimulated, and 72 hr later, IBO was microinjected into the striatum. Lesions of FN, but not DN, abolished neuroprotection but not the elevations of rCBF and AP elicited from FN stimulation. Excitotoxic lesions of FN, but not DN, also abolished the 37% reduction in focal ischemic infarctions produced by middle cerebral artery occlusion. Excitation of intrinsic FN neurons provides long-lasting, substantial, and reversible protection of central neurons from excitotoxicity, as well as focal ischemia, whereas axons in the nucleus, probably collaterals of ramified brainstem neurons, mediate the elevations in rCBF, which do not contribute to neuroprotection. Long-lived protection against a range of injuries is an unrecognized function of FN neurons transmitted over pathways distinct from those regulating rCBF.

Reticulospinal sympathoexcitatory neurons of rostral ventrolateral medulla (RVL) are selectively excited by hypoxia to elevate arterial pressure (AP) and cerebral blood flow (rCBF), that are elements of the oxygen-conserving (diving) reflex. We investigated whether KATP+-channels participate in this. Tolbutamide and glibenclamide, KATP+-channel blockers, microinjected into RVL in anesthetized rats, dose-dependently and site-specifically elevated AP and rCBF and potentiated responses to hypoxemia. KATP+-channels may mediate hypoxic excitation of oxygen-sensing RVL neurons., (Copyright 1999 Elsevier Science B.V.)

In rat, electrical stimulation of the cerebellar fastigial nucleus (FN) for 1 h reduces the volume of focal ischemic infarctions produced by occluding the middle cerebral artery (MCAO), even 10 days later. The mechanism by which this 'central neurogenic neuroprotection' salvages ischemic brain is not known but does not result from changes in cerebral perfusion. MCAO also triggers periodic periinfarction depolarizing waves (PIDs) in the ischemic penumbra, the territory of salvage. These may contribute to neuronal death and promote infarct expansion. Conceivably, FN stimulation, which can otherwise modify cortical excitability, may alter the development of PIDs. We investigated in anesthetized rats whether FN stimulation modifies PIDs expression and, if so, the threshold for evoking cortical spreading depression (CSD), a process sharing characteristics with PIDs and an index of cortical excitability. Stimulation of FN immediately or 72 h before MCAO decreased infarction volumes by approximately 45% (p<0.01), increased PID latency >10-fold, and decreased the number of PIDs by >50% (p<0.001). In normal rats, stimulation of FN increased the threshold current for eliciting CSD by 175% and slowed its propagation velocity by 35% (p<0.01 for each) immediately, but not 72 h, after FN stimulation. We conclude: FN stimulation elicits long-lasting suppression of PIDs in parallel with neuroprotection. However, PIDs suppression over time is unlikely to result from a major increase in cortical tolerance to depolarization and probably is not the principal mechanism of salvage., (Copyright 1999 Elsevier Science B.V.)

We investigated in rat hippocampus neurons whether 4-(aminobutyl)guanidine (agmatine), formed by decarboxylation of L-arginine by arginine decarboxylase and metabolized to urea and putrescine, can modulate the function of N-methyl-D-aspartate (NMDA) receptor channels. In cultured hippocampal neurons studied by whole-cell patch clamp, extracellular-applied agmatine produced a voltage- and concentration-dependent block of NMDA but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid nor kainate currents. Analysis of the voltage dependence of the block suggests that agmatine binds at a site located within the NMDA channel pore with a dissociation constant of 952 microM at 0 mV and an electric distance of 0.62. We also tested effects of several agmatine analogs. Arcaine (1,4-butyldiguanidine) also produced a similar voltage-dependent block of the NMDA current, whereas putrescine (1, 4-butyldiamine) had little effect, suggesting that the guanidine group of agmatine is the active moiety when blocking the NMDA channel. Moreover, spermine (an endogenous polyamine) potentiated the NMDA current even in the presence of blocker agmatine or arcaine, suggesting that the guanidine-containing compounds agmatine and arcaine interact with the NMDA channel at a binding site different from that of spermine. Our results indicate that in hippocampal neurons agmatine selectively modulates the NMDA subclass of glutamate receptor channels mediated by the interaction between the guanidine group and the channel pore. The results support other data that agmatine may function as an endogenous neurotransmitter/neuromodulator in brain.

Binding of idazoxan (IDA) to imidazoline receptors of the I2 subtype in astrocytes influences astroglial gene expression as evidenced by increased expression of glial fibrillary acidic protein and mRNA. To determine whether IDA affected glial inflammatory gene expression, we tested the effects of IDA on astroglial nitric oxide synthase type-2 (NOS-2) expression. NOS-2 was induced in primary rat astrocytes and C6 glioma cells by incubation with 1 microgram/ml lipopolysaccharide (LPS) plus three cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma) or three cytokines alone. Cells were incubated with 1-100 microM IDA, and at 24 h NOS-2 expression assessed. In astrocytes and C6 cells, preincubation with IDA dose-dependently inhibited nitrite accumulation (IC50 approximately 25 microM), accompanied by a reduction in NOS-2 protein levels and L-citrulline synthesis activity in cell lysates. IDA also inhibited nitrite production in LPS stimulated RAW 264.7 macrophages. In astrocytes, but not C6 cells, longer preincubation times with IDA yielded significantly greater suppression, and maximal suppression (>90%) was achieved after a 8 h preincubation in 100 microM IDA. The degree of inhibition was diminished whether IDA was added after LPS plus cytokine mixture. In contrast to NE, continuous incubation with IDA was required to achieve suppression. IDA reduced induction of NOS-2 protein levels, steady state NOS-2 mRNA levels, and activity of a NOS-2 promoter construct stably transfected in C6 cells. These results show that IDA inhibits NOS-2 activity and protein expression in glial cells and macrophages, and suggest that this occurs by decreasing transcription from the NOS-2 promoter.

Electrical stimulation of the cerebellar fastigial nucleus (FN) in rat protects the brain against ischemia. We studied whether FN could reduce the cerebrovascular inflammation as a mechanism of protection. FN or dentate nucleus (sham controls) was electrically stimulated for 1 h, and 72 h later rats were either injected with interleukin (IL)-1beta into the striata or processed to analyze inflammatory responses in isolated brain microvessels. In striata, IL-1beta induced a recruitment of leukocytes that was reduced by 50% by FN stimulation. In isolated microvessels, IL-1beta induced the transient and dose-dependent upregulation of the mRNAs encoding for the inducible nitric oxide synthase (NOS-2), intercellular adhesion molecule 1 (ICAM-1), and inhibitory kappaB-alpha (IkappaB-alpha), an inhibitor of nuclear factor-kappaB. FN stimulation decreased the upregulation of NOS-2 and ICAM-1 mRNAs, whereas it increased IkappaB-alpha mRNA expression. Dentate nucleus stimulation did not mimic the FN actions. These findings suggest that FN stimulation may render brain microvessels refractory to IL-1beta by overproduction of IkappaB-alpha and support the hypothesis that alteration of microvascular inflammation may contribute to the central neurogenic neuroprotection elicited from the FN.

Agmatine, which in other life forms serves as a metabolic intermediate for polyamine biosynthesis, appears to have properties in mammals consistent with its actions as a neurotransmitter/neuromodulator. Thus, agmatine is synthesized unequally in brain by arginine decarboxylase (ADC); is stored in neurons and axon terminals with a heterogeneous distribution; is released from synaptosomes by depolarization; is enzymatically converted by agmatinase to putrescine; interacts not only with alpha2-adrenergic and I-receptors in the CNS, but also may selectively block NMDA receptor channels; and, when administered centrally, has several potent biological actions. Clarification of its role in normal brain function, however, has not yet been fully established, in part because of the absence of agents that selectively affect its biosynthesis or degradation.

We examined the cellular and subcellular localization of agmatine in the hippocampal CA1 region by immunocytochemistry. By light microscopy, agmatine-like immunoreactivity (agmatine-LI) was found primarily in the perikarya and dendritic profiles of pyramidal cells and in punctate processes preponderantly in stratum radiatum. Electron microscopy revealed that agmatine-LI was cytoplasmic and concentrated in 'clusters' associated with mitochondria and tubular vesicles. In stratum radiatum, agmatine-LI was primarily in axons and axon terminals associated with small, synaptic vesicles. The terminals almost exclusively formed asymmetric synapses on the spines of dendrites, many of which originated from pyramidal cells. Some agmatine-LI also was present in shafts and spines of pyramidal cell dendrites and in astrocytic processes. The results demonstrate that agmatine in the hippocampus is found primarily in terminals forming excitatory (asymmetric) synapses on pyramidal cells, some of which contain agmatine-LI. These findings further implicate agmatine as an endogenous neurotransmitter which may be co-stored with L-glutamate and may act in part in the rat hippocampus as a blocker of the N-methyl-D-aspartate receptor and nitric oxide synthase.

A focal infarction produced by occlusion of the middle cerebral artery (MCAO) in spontaneously hypertensive rats induced expression of inducible nitric oxide synthase (iNOS) mRNA, measured by competitive reverse transcription-polymerase chain reaction. The mRNA appeared simultaneously in the ischemic core and penumbra at 8 h, peaked between 14 and 24 h, and disappeared by 48 h. At 24 h, inducible nitric oxide synthase (iNOS)-like immunoreactivity was present in the endothelium of cerebral microvessels and in scattered cells, probably representing leukocytes or activated microglia. Electrical stimulation of the cerebellar fastigial nucleus (FN) for 1 h, 48 h before MCAO, reduced infarct volumes by 45% by decreasing cellular death in the ischemic penumbra. It also reduced by >90% the expression of iNOS mRNA and protein in the penumbra, but not core, and decreased by 44% the iNOS enzyme activity. We conclude that excitation of neuronal networks represented in the cerebellum elicits a conditioned central neurogenic neuroprotection associated with the downregulation of iNOS mRNA and protein. This neuroimmune interaction may, by blocking the expression of iNOS, contribute to neuroprotection.

We investigated whether electrical stimulation of the cerebellar fastigial nucleus (FN) can protect pyramidal neurons of the CA1 zone of dorsal hippocampus from delayed neuronal death caused by global ischemia. Stimulation of the FN for 1 h prior to transient 4-vessel occlusion in anesthetized rats salvaged 57% (p < 0.01) of pyramidal neurons from degeneration. This effect could be preconditioned. Sham simulation of FN or stimulation of the rostral ventrolateral medulla (RVL) were without effect (p > 0.5). Excitation of intrinsic neuronal pathways represented in FN can protect central neurons from global as well as focal ischemic degeneration. The brain contains systems designed to protect it from ischemia by mechanisms of central neurogenic neuroprotection acting independently of actions on cerebral blood flow.

The distribution of agmatine (decarboxylated arginine) was mapped in the central nervous system (CNS) in the rat. Agmatine-like immunoreactivity was identified by light microscopy, exclusively in the cytoplasm of neuronal perikarya. Immunoreactive neurons were present in the cerebral cortex, predominantly within laminae VI and V and, to a lesser extent, III and mainly in retrosplenial, cingulate, primary somatosensory and auditory cortices, and the subiculum. In the lower brainstem, immunoreactivity was selectively localized to visceral relay nuclei: the nucleus tractus solitarii and pontine parabrachial complex, and periventricular areas including the laterodorsal nucleus, locus coeruleus and dorsal raphe. In the midbrain, immunolabeled cells were concentrated in the ventral tegmental area and periaqueductal gray. In the forebrain, subcortical neurons were labeled predominantly in the preoptic area, amygdala, septum, bed nucleus of the stria terminalis, midline thalamus, and the hypothalamus. Ultrastructural analysis of layer V of the somatosensory cortex demonstrated agmatine-immunoreactivity in neurons, primarily in large dense-core vesicles located in the cytoplasm. Agmatine immunoreactivity was also affiliated with endoplasmic reticulum and the plasmalemma. Cortical neurons and the subiculum were labeled in animals not administered the axonal transport inhibitor, colchicine; thus, may normally contain higher concentrations of the amine than other brain regions. The central distribution of agmatine is consistent with the hypothesis that the amine may be a novel neurotransmitter of neurons involved in behavioral and visceral control., (Copyright 1998 Elsevier Science B.V.)

Agmatine is an amine derived from the decarboxylation of arginine by arginine decarboxylase (ADC) and metabolized to putrescine by agmatinase. While prevalent in bacteria and plants, agmatine and its metabolic enzymes have been recently identified in mammalian tissues. In the present study we sought to determine: (a) whether macrophages (cell line RAW 264.7) express ADC and agmatinase, and (b) if the enzymes are regulated by lipopolysaccharide (LPS), and/or by the inhibitory cytokines transforming growth factor-beta (TGF-beta), interleukin-10 (IL-10) and interleukin-4 (IL-4). LPS induced a dose-dependent stimulation of agmatinase, while it decreased ADC, the effect in both cases being maximum at 20 h. As expected, LPS dose-dependently stimulated the inducible nitric oxide synthase activity (iNOS). A strong correlation was observed between the effects of LPS on the agmatine-related enzymes and iNOS. By contrast, exposure to IL-10 and TGF-beta caused a reduction in ADC and agmatinase, whereas IL-4 was ineffective on ADC, but reverted the LPS-induced increase of agmatinase. We conclude that the agmatine pathway may be an alternative metabolic route for arginine in macrophages, suggesting a regulatory role of agmatine during inflammation.

Imidazoline receptors (I-receptors) are considered as potential therapeutic targets for a spectrum of stress-induced illnesses. Yet, I-receptors remain poorly defined at the molecular level. In this study, candidate imidazoline receptor proteins were compared using two imidazoline receptor-selective antisera of diverse origins. One antiserum was derived from affinity-purified imidazoline-binding protein. The second antiserum was produced as an anti-idiotypic antiserum, from purified IgG selective for imidazolines. Despite such diverse origins, both antisera co-identified an 85 kDa band on western blots from a variety of tissues. The integrity of the 85 kDa band was dependent on protection by eight different protease inhibitors. Other proteolytic breakdown products (obtained after homogenization with only one protease inhibitor) were comparable in size to previously reported smaller immunoreactive bands. The full-size 85 kDa band was also enriched in plasma membrane fractions and abundant in rat PC12 cells and brain regions known to be abundant in I1 binding sites. Furthermore, the immunodensity of the 85 kDa band, against anti-idiotypic antiserum, was linearly correlated with reported I1 site radioligand Bmax values (r2 = 0.8736, P = 0.0002) across nine rat tissues. Therefore, a possible candidate for the full-length imidazoline receptor(s) appears to be an 85 kDa protein.