De novo truncating variants in the intronless IRF2BPL are responsible for developmental epileptic encephalopathy.

PURPOSE: Developmental and epileptic encephalopathies (DEEs) are severe clinical conditions characterized by stagnation or decline of cognitive and behavioral abilities preceded, accompanied or followed by seizures. Because DEEs are clinically and genetically heterogeneous, next-generation sequencing, especially exome sequencing (ES), is becoming a first-tier strategy to identify the molecular etiologies of these disorders. METHODS: We combined ES analysis and international data sharing. RESULTS: We identified 11 unrelated individuals with DEE and de novo heterozygous truncating variants in the interferon regulatory factor 2-binding protein-like gene (IRF2BPL). The 11 individuals allowed for delineation of a consistent neurodevelopmental disorder characterized by mostly normal initial psychomotor development followed by severe global neurological regression and epilepsy with nonspecific electroencephalogram (EEG) abnormalities and variable central nervous system (CNS) anomalies. IRF2BPL, also known as enhanced at puberty protein 1 (EAP1), encodes a transcriptional regulator containing a C-terminal RING-finger domain common to E3 ubiquitin ligases. This domain is required for its repressive and transactivating transcriptional properties. The variants identified are expected to encode a protein lacking the C-terminal RING-finger domain. CONCLUSIONS: These data support the causative role of truncating IRF2BPL variants in pediatric neurodegeneration and expand the spectrum of transcriptional regulators identified as molecular factors implicated in genetic developmental and epileptic encephalopathies.

Impaired hypercarbic and hypoxic responses from developmental loss of cerebellar Purkinje neurons: implications for sudden infant death syndrome.

Impaired responsivity to hypercapnia or hypoxia is commonly considered a mechanism of failure in sudden infant death syndrome (SIDS). The search for deficient brain structures mediating flawed chemosensitivity typically focuses on medullary regions; however, a network that includes Purkinje cells of the cerebellar cortex and its associated cerebellar nuclei also helps mediate responses to carbon dioxide (CO2) and oxygen (O2) challenges and assists integration of cardiovascular and respiratory interactions. Although cerebellar nuclei contributions to chemoreceptor challenges in adult models are well described, Purkinje cell roles in developing models are unclear. We used a model of developmental cerebellar Purkinje cell loss to determine if such loss influenced compensatory ventilatory responses to hypercapnic and hypoxic challenges. Twenty-four Lurcher mutant mice and wild-type controls were sequentially exposed to 2% increases in CO2 (0-8%) or 2% reductions in O2 (21-13%) over 4 min, with return to room air (21% O2/79% N2/0% CO2) between each exposure. Whole body plethysmography was used to continuously monitor tidal volume (TV) and breath frequency (f). Increased f to hypercapnia was significantly lower in mutants, slower to initiate, and markedly lower in compensatory periods, except for very high (8%) CO2 levels. The magnitude of TV changes to increasing CO2 appeared smaller in mutants but only approached significance. Smaller but significant differences emerged in response to hypoxia, with mutants showing smaller TV when initially exposed to reduced O2 and lower f following exposure to 17% O2. Since cerebellar neuropathology appears in SIDS victims, developmental cerebellar neuropathology may contribute to SIDS vulnerability.

Research challenges in central nervous system manifestations of inborn errors of metabolism.

The Research Challenges in CNS Manifestations of Inborn Errors of Metabolism workshop was designed to address challenges in translating potential therapies for these rare disorders, and to highlight novel therapeutic strategies and innovative approaches to CNS delivery, assessment of effects and directions for the future in the treatment of these diseases. Therapies for the brain in inborn errors represent some of the greatest challenges to translational research due to the special properties of the brain, and of inborn errors themselves. This review covers the proceedings of this workshop as submitted by participants. Scientific, ethical and regulatory issues are discussed, along with ways to measure outcomes and the conduct of clinical trials. Participants included regulatory and funding agencies, clinicians, scientists, industry and advocacy groups.

Rediscovering Tocophersolan: A Renaissance for Nano-Based Drug Delivery and Nanotheranostic Applications.

A unique and pleiotropic polymer, d-alpha-tocopheryl polyethylene glycol succinate (Tocophersolan), is a polymeric, synthetic version of vitamin E. Tocophersolan has attracted enormous attention as a versatile excipient in different biomedical applications including drug delivery systems and nutraceuticals. The multiple inherent properties of Tocophersolan allow it to play flexible roles in drug delivery system design, including excipients with outstanding biocompatibility, solubilizer with the ability to promote drug dissolution, drug permeation enhancer, P-glycoprotein inhibitor, and anticancer compound. For these reasons, Tocophersolan has been widely used for improving the bioavailability of numerous pharmaceutical active ingredients. Tocophersolan has been approved by stringent regulatory authorities (such as the US FDA, EMA, and PMDA) as a safe pharmaceutical excipient. In this review, the current advances in nano-based delivery systems consisting of Tocophersolan, with possibilities for futuristic applications in drug delivery, gene therapy, and nanotheranostics, were systematically curated.

A Community-Based Lifestyle-Integrated Physical Activity Intervention to Enhance Physical Activity, Positive Family Communication, and Perceived Health in Deprived Families: A Cluster Randomized Controlled Trial.

Background: Zero-time exercise (ZTEx) is an approach integrating simple strength- and stamina-enhancing physical activity into daily life. The study evaluated the effectiveness of a community-based lifestyle-integrated physical activity intervention using ZTEx to enhance participants' physical activity, family communication, perceived health and happiness, and family harmony. Methods: A parallel group, cluster randomized controlled trial was conducted in a sample of 673 participants from eight Integrated Family Service Centers in Hong Kong. The experimental group (n = 316) received a physical activity intervention. The control group (n = 357) received information on healthy eating. Both groups received three face-to-face intervention sessions (totalling 6 h and 30 min) and 16 text messages. The primary outcome was the change in days spent engaged in ZTEx. Secondary outcomes included changes in sitting time, days engaged in moderate or vigorous physical activities, family communication (encouraging and engaging family members in ZTEx), dietary habits, perceived health and happiness, and family harmony. Self-administered questionnaires were used at baseline and at 3, 6, and 12 months. Mixed effects models with intention-to-treat analysis was used. Results: Compared with the control group at 3 months, the experimental group showed significantly greater increases of 1.3 days spent doing ZTEx (Cohen's d: 0.60), 0.3 days spent doing moderate physical activity (Cohen's d: 0.08), 0.3 days encouraging family members to do ZTEx (Cohen's d: 0.16), and 0.7 days doing ZTEx with family members (Cohen's d: 0.39) during the 7 days prior. At 3 months, the experimental group also showed a significantly greater improvement in perceived health, by a score of 0.2 (Cohen's d: 0.14). The effect sizes ranged from small to medium, with similar intervention effects at the 6-month and 1-year assessments. Compared with the experimental group, the control group showed a significantly greater reduction of 0.4 days on which sweetened beverages were consumed (95% CI: 0.01, 0.9, p < 0.05, Cohen's d: 0.28). The qualitative results supported the quantitative findings. Conclusions: Our findings show that a community-based lifestyle-integrated physical activity (PA) intervention can enhance physical activity, family communication, and perceived health in deprived families in Hong Kong. Trial registration: The research protocol was retrospectively registered at the National Institutes of Health (identifier number: NCT02601534) on November 10, 2015.

Continuous infusion of enzyme replacement therapy is inferior to weekly infusions in MPS I dogs.

Intravenous enzyme replacement therapy with recombinant human α-L-iduronidase (rhIDU) is used weekly to treat mucopolysaccharidosis (MPS) I. We tested continuous administration of rhIDU at two dosing levels (0.58 mg/kg per week and 2 mg/kg per week) in MPS I dogs, and compared the efficacy of continuous infusion with the clinically used 0.58 mg/kg weekly three-hour infusion. Peak plasma concentrations of rhIDU were much higher in weekly-treated dogs (mean 256 units/ml) than steady-state concentrations in dogs treated with continuous infusion (mean 1.97 units/ml at 0.58 mg/kg per week; 8.44 units/ml at 2 mg/kg per week). Dogs receiving continuous IV rhIDU, even at a higher (2 mg/kg per week) dose, had consistently lower iduronidase levels in tissues than dogs receiving a weekly (0.58 mg/kg per week) dose. GAG storage was also less improved by continuous intravenous infusion. Adverse events were similar in all dosing groups. We found that continuous administration of 2 mg/kg per week rhIDU to MPS I dogs was insufficient to achieve GAG storage reduction comparable to 0.58 mg/kg weekly dosing.

Novel treatments and future perspectives: outcomes of intrathecal drug delivery.

Intrathecal enzyme replacement therapy (ERT) holds promise as a treatment for the central nervous system manifestations of lysosomal storage diseases. Treatment via the cerebrospinal fluid represents a potential method of delivering recombinant enzyme across the blood-brain barrier. Experiments in animal models of mucopolysaccharidosis (MPS) Type I, MPS II and MPS IIIA have shown that ERT delivered via the intrathecal route distributes throughout the central nervous system and penetrates brain tissue, where it promotes clearance of lysosomal storage material. Studies are underway to investigate the safety and efficacy of intrathecal ERT in patients with MPS I.

A review of the mechanism by which exploding bridge-wire detonators function.

An introduction to exploding bridge-wire (EBW) detonators is given followed by an extensive critical review of open source literature pertaining to these devices. The aim is to better establish the mechanism of operation. Some authors state that the key mechanism is shock-to-detonation while others maintain it is more thermal in nature, or a complex combination of both. In addition to EBW detonators, arc detonators and direct optical initiation detonators are also reviewed, and it is demonstrated that in this manner the usually coupled effects from both shock and deflagration can be somewhat decoupled. As a result, it is hypothesized that the mechanism of operation in all three detonators is in fact the same: the formation of a hot plasma with a power of ≈1 MW and emission in the ultraviolet drives a thermal explosion process.

Multi-type child maltreatment: prevalence and its relationship with self-esteem among secondary school students in Tanzania.

BACKGROUND: Child maltreatment is becoming predominantly multi-type in nature. Studies report that multi-type child maltreatment is associated with low self-esteem in adolescence and adulthood. There is a lack of published studies in Tanzania regarding multi-type child maltreatment and its relationship with self-esteem in adolescence. This study investigates the prevalence of multi-type child maltreatment and its relationship with self-esteem among secondary school students in Tanzania. METHODS: A cross-sectional, community-based study of secondary school students was conducted in randomly selected secondary schools in Tanzania. A multistage cluster sampling technique was employed to obtain the required number of study participants. The Rosenberg Self-Esteem Scale and the Adverse Childhood Experiences (ACE) questionnaire were used to measure the variables under investigation in the study. A total of 1000 participants (M: F ratio = 1.2:1) were studied. The mean age at presentation was 16.24 ± 7.36 years. The modal age group was 16-18 years (54.2%). RESULTS: The prevalence of multi-type child maltreatment was 97.6%. The prevalence of physical abuse, physical neglect, emotional neglect emotional abuse and sexual abuse was 82.1, 26.2, 51.9, 21.8 and 24.7%, respectively. Females reported a higher prevalence of physical abuse (84.3%), physical neglect (28.0%) and sexual abuse (26.2%) than their male counterparts. Emotional abuse (53.3%) was reported more often by males. In terms of ACE, participants were classified as having zero (2.4%), one (22.4%), two (20.3%), three (18.2%), four (14.7%), five (12.8%) and over five (9.2%) types of maltreatment. With regard to multi-type child maltreatment, emotional abuse (X2 = 2.925, p = 0.001), emotional neglect (X2 = 2.329, p = 0.032), physical neglect (X2 = 22.508, p < 0.001) and physical abuse (X2 = 6.722, p = 0.036) were significantly associated with low self-esteem. CONCLUSION: The current study demonstrates that multi-type child maltreatment exists in Tanzania and has adversely affected self-esteem among secondary school students. We believe that this study has significantly added to the body of literature on child maltreatment by investigating exposure to 10 types of ACEs as opposed to single types, as the majority of previous studies have investigated.

ETHE1 mutations are specific to ethylmalonic encephalopathy.

Mutations in ETHE1, a gene located at chromosome 19q13, have recently been identified in patients affected by ethylmalonic encephalopathy (EE). EE is a devastating infantile metabolic disorder, characterised by widespread lesions in the brain, hyperlactic acidaemia, petechiae, orthostatic acrocyanosis, and high levels of ethylmalonic acid in body fluids. To investigate to what extent ETHE1 is responsible for EE, we analysed this gene in 29 patients with typical EE and in 11 patients presenting with early onset progressive encephalopathy with ethylmalonic aciduria (non-EE EMA). Frameshift, stop, splice site, and missense mutations of ETHE1 were detected in all the typical EE patients analysed. Western blot analysis of the ETHE1 protein indicated that some of the missense mutations are associated with the presence of the protein, suggesting that the corresponding wild type amino acid residues have a catalytic function. No ETHE1 mutations were identified in non-EE EMA patients. Experiments based on two dimensional blue native electrophoresis indicated that ETHE1 protein works as a supramolecular, presumably homodimeric, complex, and a three dimensional model of the protein suggests that it is likely to be a mitochondrial matrix thioesterase acting on a still unknown substrate. Finally, the 625G-->A single nucleotide polymorphism in the gene encoding the short chain acyl-coenzyme A dehydrogenase (SCAD) was previously proposed as a co-factor in the aetiology of EE and other EMA syndromes. SNP analysis in our patients ruled out a pathogenic role of SCAD variants in EE, but did show a highly significant prevalence of the 625A alleles in non-EE EMA patients.

Cerebellar contribution to higher and lower order rule learning and cognitive flexibility in mice.

Cognitive flexibility has traditionally been considered a frontal lobe function. However, converging evidence suggests involvement of a larger brain circuit which includes the cerebellum. Reciprocal pathways connecting the cerebellum to the prefrontal cortex provide a biological substrate through which the cerebellum may modulate higher cognitive functions, and it has been observed that cognitive inflexibility and cerebellar pathology co-occur in psychiatric disorders (e.g., autism, schizophrenia, addiction). However, the degree to which the cerebellum contributes to distinct forms of cognitive flexibility and rule learning is unknown. We tested lurcher↔wildtype aggregation chimeras which lose 0-100% of cerebellar Purkinje cells during development on a touchscreen-mediated attentional set-shifting task to assess the contribution of the cerebellum to higher and lower order rule learning and cognitive flexibility. Purkinje cells, the sole output of the cerebellar cortex, ranged from 0 to 108,390 in tested mice. Reversal learning and extradimensional set-shifting were impaired in mice with⩾95% Purkinje cell loss. Cognitive deficits were unrelated to motor deficits in ataxic mice. Acquisition of a simple visual discrimination and an attentional-set were unrelated to Purkinje cells. A positive relationship was observed between Purkinje cells and errors when exemplars from a novel, non-relevant dimension were introduced. Collectively, these data suggest that the cerebellum contributes to higher order cognitive flexibility, lower order cognitive flexibility, and attention to novel stimuli, but not the acquisition of higher and lower order rules. These data indicate that the cerebellar pathology observed in psychiatric disorders may underlie deficits involving cognitive flexibility and attention to novel stimuli.

Management and outcomes following pancreaticoduodenectomy for ampullary adenocarcinoma.

INTRODUCTION: The purpose of this study was to evaluate outcomes following pancreaticoduodenectomy(PD) for ampullary adenocarcinoma(AAC). METHODS: We evaluated patients having undergone PD for AAC and the impact of clinical/histopathologic factors and adjuvant therapy(AT) on survival. RESULTS: 52 patients underwent potentially curative PD. Perineural and lymphovascular invasion were associated with decreased survival. There was no difference in survival between patients treated with PD vs. PD+AT, however, AT was more often administered to patients with N1 vs. N0 and stage II/III vs. I disease. Among patients receiving AT, we observed a trend towards improved survival when radiation was included. Recurrence occurred in 7/18(39%) stage I patients, only 2(7%) of which received AT. CONCLUSION: AT did not improve survival, however was more commonly administered in advanced disease. Stage I patients had high recurrence rates but rarely received AT. Prospective evaluation of appropriate AT regimens and use in early stage patients should be considered.

Antitumor efficacy of AAV-mediated systemic delivery of interferon-beta.

Type I interferons (alpha/beta) have significant antitumor activity although their short half-life and systemic side effects have limited their clinical utility. An alternative dosing schedule of continuous, low-level delivery, as is achieved by gene therapy, rather than intermittent, high concentration pulsed-dosing, might avoid the toxicity of interferon while maintaining its antitumor efficacy. We have tested a gene therapy approach in murine tumor models to treat malignancies that have shown responsiveness to interferon in clinical trials. The tumor cell lines used were moderately sensitive to the direct effects of human interferon-beta (hIFN-beta) in vitro. For in vivo testing, systemic delivery of hIFN-beta was generated following liver-targeted delivery of adeno-associated virus (AAV) vector carrying the hIFN-beta transgene. This prevented engraftment of subcutaneous human gliomas, and orthotopic, localized (intrarenal) and disseminated (primarily pulmonary) human renal cell carcinomas; and caused regression of established tumors at these sites. In a syngeneic, immunocompetent model of melanoma, AAV IFN-beta treatment limited subcutaneous tumor growth and prevented disseminated disease. A significant decrease in mean intratumoral vessel density was demonstrated in hIFN-beta-treated tumors, suggesting that in addition to a direct tumoricidal effect, the antitumor efficacy of AAV IFN-beta in this study was due to its ability to inhibit angiogenesis.

Data from subjects receiving intrathecal laronidase for cervical spinal stenosis due to mucopolysaccharidosis type I.

Five subjects with mucopolysaccharidosis type I and symptomatic cervical spinal stenosis received intrathecal laronidase in a 4-month pilot study and/or a 12-month extension study [1]. Clinical descriptions of study subjects, nonserious adverse events, individual data tables, and scoring system methods are provided. There were ten nonserious adverse events that occurred in more than one study subject. Somatosensory evoked potentials were absent in two subjects and normal in two subjects, limiting their utility as an endpoint. There were no significant changes in magnetic resonance imaging of cervical spinal cord or brain, pulmonary function tests, or cerebrospinal fluid opening pressure. These data are presented along with the scoring methods used in evaluation of the study subjects.

Distinct miRNA expression in dorsal striatal subregions is associated with risk for addiction in rats.

Recently, we published data using an animal model that allowed us to characterize animals into two groups, addiction vulnerable and addiction resilient, where we identified that addiction/relapse vulnerability was associated with deficits in synaptic plasticity-associated gene expression in the dorsal striatum (DS). Notable was the strong reduction in expression for activity-regulated cytoskeleton-associated protein (Arc) considered a master regulator of synaptic plasticity. In the present study, we confirmed that Arc messenger RNA was significantly decreased in the DS, but importantly, we identified that this reduction was restricted to the dorsomedial (DMS) and not dorsolateral striatum (DLS). There is recent evidence of microRNA (miRNA)-associated posttranscriptional suppression of Arc and animal models of addiction have identified a key role for miRNA in the regulation of addiction-relevant genes. In further support of this link, we identified several differentially expressed miRNA with the potential to influence addiction-relevant plasticity genes, including Arc. A key study recently reported that miR-212 expression is protective against compulsive cocaine-seeking. Supporting this hypothesis, we found that miR-212 expression was significantly reduced in the DMS but not DLS of addiction-vulnerable animals. Together, our data provide strong evidence that miRNA promote ongoing plasticity deficits in the DS of addiction-vulnerable animals.

Oral stereotypy induced by amphetamine microinjection into striatum: an anatomical mapping study.

The ventrolateral striatum has been shown to specifically contribute to expression of psychostimulant-induced orofacial stereotypies. Adult male Sprague-Dawley rats were implanted with bilateral cannulae directed at one of eight striatal subareas, and were injected with saline or amphetamine (20 micrograms/0.5 microliter/side) in a counterbalanced order. Behaviors were observed and scored using a time-sampling procedure. In the middle ventrolateral striatum, amphetamine injections produced intense stereotypy, primarily consisting of bar biting, non-injurious self-biting and repetitive paw-to-mouth movements, while having no effect on locomotion or rearing. Amphetamine injections 2 mm medial or 1 mm dorsal produced no oral stereotypy, while injections 1 mm rostral or caudal to the effective site produced only low levels of stereotypy. Injections into the surrounding sites, particularly in the ventromedial region, also had stimulatory effects on locomotion and rearing. In a separate experiment, animals were given either unilateral or bilateral injection of amphetamine into the ventrolateral striatum. The unilateral injection produced stereotypy half as intense as that observed with bilateral injections. These results suggest that the ventrolateral striatum is a discrete neuroanatomical region which is primarily responsible for expression of psychostimulant-induced orofacial stereotypies, and which is likely to be the striatal region controlling normal oral motor function. Further investigation of this area may provide valuable insights concerning the etiology and treatment of orofacial dyskinesias associated with basal ganglia dysfunction.

Synthesis of transthyretin (pre-albumin) mRNA in choroid plexus epithelial cells, localized by in situ hybridization in rat brain.

The sites of synthesis of transthyretin in the brain were investigated using in situ hybridization with [35S]-labeled recombinant cDNA probes specific for transthyretin mRNA. Autoradiography of hybridized coronal sections of rat brain revealed specific cellular localization of transthyretin mRNA in choroid plexus epithelial cells of the lateral, third, and fourth ventricles. Transferrin mRNA was also investigated and, in contrast to transthyretin mRNA, was localized mainly in the lateral ventricles. Our results indicate that substantial synthesis of transthyretin and transferrin mRNA may occur in the choroid plexus.

The acute phase response in the rodent.

In the rodent, the general response to acute inflammation and tissue damage is characterized by a complex rearrangement in the pattern of concentrations of proteins in the plasma leading to an increase in the sedimentation rate of erythrocytes, an increase in leukocyte concentration in the bloodstream, and a decrease in the hematocrit. Body temperature changes only slightly or not at all. The reasons for the change in plasma concentrations of proteins are changes in their rates of synthesis in the liver. Degradation of plasma proteins is not affected. The details of the acute phase response evolved in the interaction of species with their environment. Therefore, it is not surprising to find differences in the details of the acute phase response among species. For example, alpha 2-macroglobulin is a strongly positive acute phase reactant in the rat, but not in the mouse; C-reactive protein is a strongly positive acute phase protein in the mouse, but is not found in the rat. An inducible acute phase cysteine proteinase inhibitor system, which has evolved from a primordial kininogen gene, has been observed so far only in the rat. The changes in the synthesis rates of acute phase proteins during inflammation are closely reflected by corresponding changes in intracellular mRNA levels. In the liver, the capacity to induce the acute phase pattern of synthesis and secretion of plasma proteins probably develops around birth. Changes in mRNA levels are brought about by changes in transcription rates or by changes in mRNA stability. Kinetics of mRNA changes during the acute phase response differ for individual proteins. The main signal compound for eliciting the acute phase response in liver seems to be interleukin-6/interferon-beta 2/hepatocyte stimulating factor, whereas interleukin-1 leads to typical acute phase changes in mRNA levels only for alpha 1-acid glycoprotein, albumin, and transthyretin. Plasma protein genes are expressed in various extrahepatic tissues, such as the choroid plexus, the yolk sac, the placenta, the seminal vesicles, and other sites. All these tissues are involved in maintaining protein homeostasis in associated extracellular compartments by synthesis and secretion of proteins. Synthesis and secretion of plasma proteins in paracompartmental organs other than the liver is not influenced by the acute phase stimuli.

GRF-induced feeding: evidence for protein selectivity and opiate involvement.

Growth hormone-releasing factor (GRF) is a hypothalamic peptide named for its ability to induce release of growth hormone from the anterior pituitary. GRF also acts as a neurotransmitter in the suprachiasmatic nucleus/medial preoptic area (SCN/MPOA) to stimulate food intake. The purpose of this series of experiments was to explore the nature of GRF-induced feeding, with a particular emphasis on macronutrient selectivity, and to examine the role of opiate activity in the paraventricular nucleus of the hypothalamus (PVN). Chow intake stimulated by GRF microinjection (1 pmol/0.5 microliters) into the SCN/MPOA was blocked by injection of methyl-naltrexone (3 micrograms/0.5 microliters) into the PVN. In animals habituated to macronutrient diets (Teklad, WI), GRF preferentially stimulated intake of protein at 2 and 4 h postinjection, whereas it had no effect on carbohydrate intake. Further, this effect was blocked by injection of naloxone (40 nmol/0.5 microliters) into the PVN. Microinjection of morphine (0, 1, 10, and 17 micrograms/0.5 microliter) into the PVN also specifically stimulated protein intake at 2 and 4 h postinjection. These results suggest that feeding derived from GRF actions in the SCN/MPOA is macronutrient selective, and is dependent on PVN opiate activity for expression.

Blockade of endogenous GRF at dark onset selectively suppresses protein intake.

This study examined whether endogenous central GRF activity contributes to the increase in macronutrient intake shown by rats at dark onset. Animals were habituated to two diets: carbohydrate-fat and protein-fat. Antiserum raised against GRF (aGRF; 1% and 10% solutions) was microinjected into the suprachiasmatic nucleus/medial preoptic area (SCN/MPOA) at dark onset, and macronutrient intake was determined at 1, 2, and 4 h postinjection. aGRF blocked the increase in protein intake normally seen at dark onset, but had no effect on carbohydrate intake. These findings suggest that endogenous GRF activity in the SCN/MPOA region of the brain contributes to the circadian and nutritional organization of food intake.