DNA methylation epi-signature is associated with two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid syndrome is characterized by a range of neurodevelopmental phenotypes with incomplete penetrance and variable expressivity. It is caused by a variable size and breakpoint microdeletions in the distal long arm of chromosome 22, referred to as 22q13.3 deletion syndrome, including the SHANK3 gene. Genetic defects in a growing number of neurodevelopmental genes have been shown to cause genome-wide disruptions in epigenomic profiles referred to as epi-signatures in affected individuals. RESULTS: In this study we assessed genome-wide DNA methylation profiles in a cohort of 22 individuals with Phelan-McDermid syndrome, including 11 individuals with large (2 to 5.8 Mb) 22q13.3 deletions, 10 with small deletions (< 1 Mb) or intragenic variants in SHANK3 and one mosaic case. We describe a novel genome-wide DNA methylation epi-signature in a subset of individuals with Phelan-McDermid syndrome. CONCLUSION: We identified the critical region including the BRD1 gene as responsible for the Phelan-McDermid syndrome epi-signature. Metabolomic profiles of individuals with the DNA methylation epi-signature showed significantly different metabolomic profiles indicating evidence of two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

Disruption of MBD5 contributes to a spectrum of psychopathology and neurodevelopmental abnormalities.

Microdeletions of chromosomal region 2q23.1 that disrupt MBD5 (methyl-CpG-binding domain protein 5) contribute to a spectrum of neurodevelopmental phenotypes; however, the impact of this locus on human psychopathology has not been fully explored. To characterize the structural variation landscape of MBD5 disruptions and the associated human psychopathology, 22 individuals with genomic disruption of MBD5 (translocation, point mutation and deletion) were identified through whole-genome sequencing or cytogenomic microarray at 11 molecular diagnostic centers. The genomic impact ranged from a single base pair to 5.4 Mb. Parents were available for 11 cases, all of which confirmed that the rearrangement arose de novo. Phenotypes were largely indistinguishable between patients with full-segment 2q23.1 deletions and those with intragenic MBD5 rearrangements, including alterations confined entirely to the 5'-untranslated region, confirming the critical impact of non-coding sequence at this locus. We identified heterogeneous, multisystem pathogenic effects of MBD5 disruption and characterized the associated spectrum of psychopathology, including the novel finding of anxiety and bipolar disorder in multiple patients. Importantly, one of the unique features of the oldest known patient was behavioral regression. Analyses also revealed phenotypes that distinguish MBD5 disruptions from seven well-established syndromes with significant diagnostic overlap. This study demonstrates that haploinsufficiency of MBD5 causes diverse phenotypes, yields insight into the spectrum of resulting neurodevelopmental and behavioral psychopathology and provides clinical context for interpretation of MBD5 structural variations. Empirical evidence also indicates that disruption of non-coding MBD5 regulatory regions is sufficient for clinical manifestation, highlighting the limitations of exon-focused assessments. These results suggest an ongoing perturbation of neurological function throughout the lifespan, including risks for neurobehavioral regression.

Short-term memory deficits in carrier females with KDM5C mutations.

We present the cognitive abilities of females from five families who carry a mutation in a gene (KDM5C, formerly JARIDIC or SMCX) in Xp 11.2 that encodes a transcriptional regulator with histone demethylase activity that is specific for dimethylated and trimethylated H3K4. In this report, the cognitive abilities of females who carry KDMSC mutations are compared to females who carry mutations in other genes known to cause X-linked intellectual and developmental disability (XLIDD) conditions. The KDM5C mutation carriers had higher mean scores on the abstract/visual and quantitative sections of the Stanford-Binet Intelligence Scale: Fourth Edition and lower mean short term memory scores. Implications for counseling are presented.

Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome.

BRAF, the protein product of BRAF, is a serine/threonine protein kinase and one of the direct downstream effectors of Ras. Somatic mutations in BRAF occur in numerous human cancers, whereas germline BRAF mutations cause cardio-facio-cutaneous (CFC) syndrome. One recurrent somatic mutation, p.V600E, is frequently found in several tumor types, such as melanoma, papillary thyroid carcinoma, colon cancer, and ovarian cancer. However, a germline mutation affecting codon 600 has never been described. Here, we present a patient with CFC syndrome and a de novo germline mutation involving codon 600 of BRAF, thus providing the first evidence that a pathogenic germline mutation involving this critical codon is not only compatible with development but can also cause the CFC phenotype. In vitro functional analysis shows that this mutation, which replaces a valine with a glycine at codon 600 (p.V600G), leads to increased ERK and ELK phosphorylation compared to wild-type BRAF but is less strongly activating than the cancer-associated p.V600E mutation.

Escape of the yolk sac: a hypothesis to explain the embryogenesis of gastroschisis.

Gastroschisis is a significant birth defect that in many countries has shown an increased prevalence in recent decades, and the change has affected primarily younger mothers. Despite numerous epidemiological studies no other consistent associated risk factor has been identified. In this paper we review the five main theories related to the pathogenesis of this malformation and outline the reasons why we think none fully explains the embryogenesis of gastroschisis. We briefly present some clinical observations we have made that we consider germane to the pathogenesis and outline a hypothesis that we think can account for the origins of this malformation. Our proposal is that the determining defect in gastroschisis is failure of the yolk sac and related vitelline structures to be incorporated into the umbilical stalk. Otherwise, ventral closure of the lateral abdominal walls occurs normally, thus orphaning the vitelline duct and yolk sac outside both the main body stalk and the abdominal wall. Thus, in addition to the umbilicus, the abdominal wall has a separate perforation through which the midpoint of the gut is attached to the exteriorized vitelline structures. This connection through the ventral wall prevents normal egress of the gut into the umbilical cord during the second month of development and acts as the egress point for the gut resulting in gastroschisis.

Clinical experience in the evaluation of 30 patients with a prior diagnosis of FG syndrome.

BACKGROUND: FG syndrome (FGS) is an X-linked disorder characterised by mental retardation, hypotonia, particular dysmorphic facial features, broad thumbs and halluces, anal anomalies, constipation, and abnormalities of the corpus callosum. A behavioural phenotype of hyperactivity, affability, and excessive talkativeness is very frequent. The spectrum of clinical findings attributed to FGS has widened considerably since the initial description of the syndrome by Opitz and Kaveggia in 1974 and has resulted in clinical variability and genetic heterogeneity. In 2007, a recurrent R961W mutation in the MED12 gene at Xq13 was found to cause FGS in six families, including the original family described by Opitz and Kaveggia. The phenotype was highly consistent in all the R961W positive patients. METHODS: In order to determine the prevalence of MED12 mutations in patients clinically diagnosed with FGS and to clarify the phenotypic spectrum of FGS, 30 individuals diagnosed previously with FGS were evaluated clinically and by MED12 sequencing. RESULTS: The R961W mutation was identified in the only patient who had the typical phenotype previously associated with this mutation. The remaining 29 patients displayed a wide variety of features and were shown to be negative for mutations in the entire MED12 gene. A definite or possible alternative diagnosis was identified in 10 of these patients. CONCLUSION: This report illustrates the difficulty in making a clinical diagnosis of FGS given the broad spectrum of signs and symptoms that have been attributed to the syndrome. Individuals with a phenotype consistent with FGS require a thorough genetic evaluation including MED12 mutation analysis. Further genetic testing should be considered in those who test negative for a MED12 mutation to search for an alternative diagnosis.

Mutations in JARID1C are associated with X-linked mental retardation, short stature and hyperreflexia.

BACKGROUND: Mutations in the JARID1C (Jumonji AT-rich interactive domain 1C) gene were recently associated with X-linked mental retardation (XLMR). Mutations in this gene are reported to be one of the relatively more common causes of XLMR with a frequency of approximately 3% in males with proven or probable XLMR. The JARID1C protein functions as a histone 3 lysine 4 (H3K4) demethylase and is involved in the demethylation of H3K4me3 and H3K4me2. METHODS: Mutation analysis of the JARID1C gene was conducted in the following cohorts: probands from 23 XLMR families linked to Xp11.2, 92 males with mental retardation and short stature, and 172 probands from small XLMR families with no linkage information. RESULTS: Four novel mutations consisting of two missense mutations, p.A77T and p.V504M, and two frame shift mutations, p.E468fsX2 and p.R1481fsX9, were identified in males with mental retardation. Two of the mutations, p.V504M and p.E468fsX2, are located in the JmjC domain of the JARID1C gene where no previous mutations have been reported. Additional studies showed that the missense mutation, p.V504M, was a de novo event on the grandpaternal X chromosome of the family. Clinical findings of the nine affected males from the four different families included mental retardation (100%), short stature (55%), hyperreflexia (78%), seizures (33%) and aggressive behaviour (44%). The degree of mental retardation consisted of mild (25%), moderate (12%) and severe (63%). CONCLUSION: Based on the clinical observations, male patients with mental retardation, short stature and hyperreflexia should be considered candidates for mutations in the JARID1C gene.

Cholecystokinin octapeptide increases spontaneous glutamatergic synaptic transmission to neurons of the nucleus tractus solitarius centralis.

Cholecystokinin (CCK) is released from enteroendocrine cells after ingestion of nutrients and induces multiple effects along the gastrointestinal tract, including gastric relaxation and short-term satiety. We used whole cell patch-clamp and immunohistochemical techniques in rat brain stem slices to characterize the effects of CCK. In 45% of the neurons of nucleus tractus solitarius subnucleus centralis (cNTS), perfusion with the sulfated form of CCK (CCK-8s) increased the frequency of spontaneous excitatory currents (sEPSCs) in a concentration-dependent manner (1-300 nM). The threshold for the CCK-8s excitatory effect was 1 nM, the EC(50) was 20 nM, and E(max) was 100 nM. The excitatory effects of CCK-8s were still present when the slices were preincubated with tetrodotoxin or bicuculline or when the recordings were conducted with Cs(+) electrodes. Pretreatment with the CCK-A receptor antagonist, lorglumide (1 microM), antagonized the effects of CCK-8s, whereas perfusion with the CCK-B preferring agonist CCK-8 nonsulfated (CCK-ns, 1 microM) did not affect the frequency of sEPSCs. Similarly, pretreatment with the CCK-B receptor antagonist, triglumide (1 microM), did not prevent the actions of CCK-8s. Although the majority (i.e., 76%) of CCK-8s unresponsive cNTS neurons had a bipolar somata shape and were TH-IR negative, no differences were found in either the morphological or the neurochemical phenotype of cNTS neurons responsive to CCK-8s. Our results suggest that the excitatory effects of CCK-8s on terminals impinging on a subpopulation of cNTS neurons are mediated by CCK-A receptors; these responsive neurons, however, do not have morphological or neurochemical characteristics that automatically distinguish them from nonresponsive neurons.

Characterization of neurons of the nucleus tractus solitarius pars centralis.

Esophageal sensory afferent inputs terminate principally in the central subnucleus of the tractus solitarius (cNTS). Neurons of the cNTS comprise two major neurochemical subpopulations. One contains neurons that are nitric oxide synthase (NOS) immunoreactive (-IR) while the other comprises neurons that are tyrosine hydroxylase (TH)-IR. We have shown recently that TH-IR neurons are involved in esophageal-distention induced gastric relaxation. We used whole cell patch clamp techniques in rat brainstem slices combined with immunohistochemical and morphological reconstructions to characterize cNTS neurons. Postrecording reconstruction of cNTS neurons revealed two morphological neuronal subtypes; one group of cells (41 out of 131 neurons, i.e., 31%) had a multipolar soma, while the other group (87 out of 131 neurons, i.e., 66%) had a bipolar soma. Of the 43 cells in which we conducted a neurochemical examination, 15 displayed TH-IR (9 with bipolar morphology, 6 with multipolar morphology) while the remaining 28 neurons did not display TH-IR (18 with bipolar morphology, 10 with multipolar morphology). Even though the range of electrophysiological properties varied significantly, morphological or neurochemical distinctions did not reveal characteristics peculiar to the subgroups. Spontaneous excitatory postsynaptic currents (sEPSC) recorded in cNTS neurons had a frequency of 1.5 +/- 0.15 events s(-1) and an amplitude of 27 +/- 1.2 pA (Vh = -50 mV) and were abolished by pretreatment with 30 muM AP-5 and 10 muM CNQX, indicating the involvement of both NMDA and non-NMDA receptors. Some cNTS neurons also received a GABAergic input that was abolished by perfusion with 30-50 muM bicuculline. In conclusion, our data show that despite the heterogeneity of morphological and neurochemical membrane properties, the electrophysiological characteristics of cNTS neurons are not a distinguishing feature.

TNF(alpha) modulation of visceral and spinal sensory processing.

The cytokine tumor necrosis factor(alpha) (TNF(alpha)) is associated with a constellation of physiological and behavioral characteristics that follow in response to infection such as fever, fatigue, listlessness, loss of appetite, malaise, and tactile hypersensitivity. These responses are examples of central nervous system (CNS) functions modified by the activated immune system. Our studies have focused on the involvement of TNF(alpha) in CNS control of gastrointestinal function and "visceral malaise". We have demonstrated that TNF(alpha) can elicit gastric stasis in a dose-dependent fashion via its interaction with vago-vagal neurocircuitry in the brainstem. Sensory elements of the vago-vagal reflex circuit (i.e., neurons of the solitary tract [NST] and area postrema [AP]) are activated by exposure to TNF(alpha), while the efferent elements (i.e., dorsal motor neurons of the vagus [DMN]) cause gastroinhibition. Transient exposure to low doses of TNF(alpha) cause potentiated (exaggerated) NST responses to stimulation. Subsequent studies suggest that TNF(alpha) presynaptically modulates the release of glutamate from primary afferents to the NST. Using immunohistochemical studies, we have observed the constitutive expression of the TNFR1 receptor on central vagal afferents and spinal trigeminal afferents in the medulla, as well as on cells and afferent fibers within the dorsal root ganglia and within laminae I and II of the dorsal horn throughout the spinal cord. The constitutive presence of these receptors on these afferents may explain why inflammatory or infectious processes that generate TNF(alpha) can disrupt gastrointestinal functions and cause tactile hypersensitivity. These receptors may also play a critical role in the chronic allodynia and hyper-reflexia observed after spinal cord injury or peripheral nerve damage.

Noradrenergic neurons in the rat solitary nucleus participate in the esophageal-gastric relaxation reflex.

Activation of esophageal mechanosensors excites neurons in and near the central nucleus of the solitary tract (NSTc). In turn, NSTc neurons coordinate the relaxation of the stomach [i.e., the receptive relaxation reflex (RRR)] by modulating the output of vagal efferent neurons of the dorsal motor nucleus of the vagus (DMN). The NSTc area contains neurons with diverse neurochemical phenotypes, including a large population of catecholaminergic and nitrergic neurons. The aim of the present study was to determine whether either one of these prominent neuronal phenotypes was involved in the RRR. Immunohistochemical techniques revealed that repetitive esophageal distension caused 53% of tyrosine hydroxylase-immunoreactive (TH-ir) neurons to colocalize c-Fos in the NSTc. No nitric oxide synthase (NOS)-ir neurons in the NSTc colocalized c-Fos in either distension or control conditions. Local brain stem application (2 ng) of alpha-adrenoreceptor antagonists (i.e., alpha1-prazosin or alpha2-yohimbine) significantly reduced the magnitude of the esophageal distension-induced gastric relaxation to approximately 55% of control conditions. The combination of yohimbine and prazosin reduced the magnitude of the reflex to approximately 27% of control. In contrast, pretreatment with either the NOS-inhibitor NG-nitro-l-arginine methyl ester or the beta-adrenoceptor antagonist propranolol did not interfere with esophageal distension-induced gastric relaxation. Unilateral microinjections of the agonist norepinephrine (0.3 ng) directed at the DMN were sufficient to mimic the transient esophageal-gastric reflex. Our data suggest that noradrenergic, but not nitrergic, neurons of the NSTc play a prominent role in the modulation of the RRR through action on alpha1- and alpha2-adrenoreceptors. The finding that esophageal afferent stimulation alone is not sufficient to activate NOS-positive neurons in the NSTc suggests that these neurons may be strongly gated by other central nervous system inputs, perhaps related to the coordination of swallowing or emesis with respiration.

Dissociation of the effects of nucleus raphe obscurus or rostral ventrolateral medulla lesions on eliminatory and sexual reflexes.

Rat preparations were used to investigate long-term changes in external anal sphincter (EAS) contractions and reflexive penile erection following electrolytic lesions of the nucleus raphe obscurus (nRO) or the rostral ventrolateral medulla. EAS contractions were measured electromyographically (EAS EMG) following distention of the EAS with a 5-mm probe. Penile erections were measured using a standard ex copula reflex testing paradigm. At 48 h postlesion, 100% of nRO-lesioned animals displayed reflexive erections and the magnitude of EAS EMG was significantly greater in lesioned animals than in sham controls. These results suggested EAS hyperreflexia following destruction of the nRO. By 14 days postlesion, EAS responsiveness in nRO-lesioned animals had returned to levels comparable to nonlesioned animals. No measures of penile erection were affected by nRO lesions. In animals with nucleus gigantocellularis (Gi) and lateral nucleus paragigantocellularis (Gi-lPGi) lesions, no significant changes to EAS reflexes were observed at any time point. At 48 h postoperative, Gi-lPGi lesions significantly reduced the latency to first erection and increased the number of erections elicited relative to controls. Similar facilitation of erection latency was observed at 14 days postlesion, while erection number and flip total were no longer significantly different from controls. These and previous studies suggest that the nRO regulates defecatory reflexes in the rat. These data further suggest that the comingled EAS and bulbospongiosus (BS) motoneurons are controlled by discrete and separate brainstem circuits and that increases in EAS and penile reflexes after spinal cord lesions are mediated by loss of different descending inputs.

TNF-alpha-induced c-Fos generation in the nucleus of the solitary tract is blocked by NBQX and MK-801.

Previous studies have shown that identified neurons of the nucleus of the solitary tract (NST) are excited by the cytokine tumor necrosis factor-alpha (TNF-alpha). Vagal afferent connections with the NST are predominantly glutaminergic. Therefore, we hypothesized that TNF-alpha effects on NST neurons may be via modulation of glutamate neurotransmission. The present study used activation of the immediate early gene product c-Fos as a marker for neuronal activation in the NST. c-Fos expression was evaluated after microinjections of TNF-alpha in the presence or absence of either the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) or the N-methyl-D- aspartate (NMDA) antagonist MK-801. To assess the specificity of the interaction between TNF-alpha and glutamate, c-Fos expression was also evaluated after injection of oxytocin (OT) (which has a direct excitatory effect in this area of the brain stem) in the presence and absence of NBQX or MK-801. c-Fos labeling was significantly increased in the NST after TNF-alpha exposure. Coinjection of either NBQX or MK-801 with TNF-alpha prevented significant c-Fos induction in the NST. Microinjections of OT also induced significant NST c-Fos elevation, but this expression was unaffected by coinjection of either antagonist with OT. These data lead us to conclude that TNF-alpha activation of NST neurons depends on glutamate and such an interaction is not generalized to all agonists that act on the NST.

Tumor necrosis factor-alpha induces cFOS and strongly potentiates glutamate-mediated cell death in the rat spinal cord.

Excitotoxic cell death due to glutamate release is important in the secondary injury following CNS trauma or ischemia. Proinflammatory cytokines also play a role. Both glutamate and tumor necrosis factor-alpha (TNF(alpha)) are released immediately after spinal cord injury. Neurophysiological studies show that TNF(alpha) can potentiate the effects of glutamatergic afferent input to produce hyperactivation of brain-stem sensory neurons. Therefore, we hypothesized that TNF(alpha) might act cooperatively with glutamate to affect cell death in the spinal cord as well. Nanoinjections of either TNF(alpha) (60 pg) or kainate (KA; 32 ng) alone into the thoracic gray resulted in almost no tissue damage or cell death 90 min after injection. However, the combination of TNF(alpha) plus KA at these same doses produced a large area of tissue necrosis and neuronal cell death, an effect which was blocked by the AMPA receptor antagonist CNQX (17 ng). These results suggest that secondary injury may involve potentiation of AMPA receptor-mediated excitatory cell death by TNF(alpha).

Receptors and transmission in the brain-gut axis: potential for novel therapies. V. Fast and slow extrinsic modulation of dorsal vagal complex circuits.

Vago-vagal reflex circuits in the medulla are responsible for the smooth coordination of the digestive processes carried out from the oral cavity to the transverse colon. In this themes article, we concentrate mostly on electrophysiological studies concerning the extrinsic modulation of these vago-vagal reflex circuits, with a particular emphasis on two types of modulation, i.e., by "fast" classic neurotransmitters and by "slow" neuromodulators. These examples review two of the most potent modulatory processes at work within the dorsal vagal complex, which have dramatic effects on gastrointestinal function. The reader should be mindful of the fact that many more different inputs from other central nervous system (CNS) loci or circulating humoral factors add to this complex mix of modulatory inputs. It is likely that similar long-term modulations of synaptic transmission occur with other neurotransmitters and may represent an important mechanism for the integration and regulation of neuronal behavior. Of course, this fact strongly militates against the success of any single drug or approach in the treatment of motility disorders having a CNS component.

22q13 deletion syndrome.

We have recently collected clinical information on 37 individuals with deletion of 22q13 and compared the features of these individuals with 24 previously reported cases. The features most frequently associated with this deletion are global developmental delay, generalized hypotonia, absent or severely delayed speech, and normal to advanced growth. Minor anomalies include dolicocephaly, abnormal ears, ptosis, dysplastic toenails, and relatively large hands. As with many terminal deletions involving pale G-band regions, the deletion can be extremely subtle and can go undetected on routine cytogenetic analysis. In fact, 32% of the individuals in our study had previous chromosome analyses that failed to detect the deletion. Eight of 37 individuals had deletion of 22q13 secondary to an unbalanced chromosome translocation. In the newborn, this deletion should be considered in cases of hypotonia for which other common causes have been excluded. In the older child, this syndrome should be suspected in individuals with normal growth, profound developmental delay, absent or delayed speech, and minor dysmorphic features. We recommend high-resolution chromosome analysis and fluorescence in situ hybridization studies, or molecular analysis to exclude this diagnosis.

Catecholaminergic neurons in rat dorsal motor nucleus of vagus project selectively to gastric corpus.

Nitric oxide synthase-immunoreactive (NOS-IR) neurons in the rat caudal dorsal motor nucleus of the vagus (DMV) project selectively to the gastric fundus and may be involved in vagal reflexes controlling gastric distension. This study aimed to identify the gastric projections of tyrosine hydroxylase-immunoreactive (TH-IR) DMV neurons, whether such neurons colocalize NOS-IR, and if they are activated after esophageal distension. Gastric-projecting neurons were identified after injection of retrograde tracers into the muscle wall of the gastric fundus, corpus, or antrum/pylorus before removal and processing of the brain stems for TH- and NOS-IR. A significantly higher proportion of corpus- compared with fundus- and antrum/pylorus-projecting neurons were TH-IR (14% compared with 4% and 2%, respectively, P < 0.05). Colocalization of NOS- and TH-IR was never observed in gastric-projecting neurons. In rats tested for c-Fos activation after intermittent esophageal balloon distension, no colocalization with TH-IR was observed in DMV neurons. These findings suggest that TH-IR neurons in the caudal DMV project mainly to the gastric corpus, constitute a subpopulation distinct from that of nitrergic vagal neurons, and are not activated on esophageal distension.

c-Fos generation in the dorsal vagal complex after systemic endotoxin is not dependent on the vagus nerve.

The present study used activation of the c-Fos oncogene protein within neurons in the dorsal vagal complex (DVC) as a marker of neuronal excitation in response to systemic endotoxin challenge [i.e. , lipopolysaccharide (LPS)]. Specifically, we investigated whether vagal connections with the brain stem are necessary for LPS cytokine- induced activation of DVC neurons. Systemic exposure to LPS elicited a significant activation of c-Fos in neurons in the nucleus of the solitary tract (NST) and area postrema of all thiobutabarbital-anesthetized rats examined, regardless of the integrity of their vagal nerves. That is, rats with both vagi cervically transected were still able to respond with c-Fos activation of neurons in the DVC. Unilateral cervical vagotomy produced a consistent but small reduction in c-Fos activation in the ipsilateral NST of all animals within this experimental group. Given that afferent input to the NST is exclusively excitatory, it is not surprising that unilateral elimination of all vagal afferents would diminish NST responsiveness (on the vagotomized side). These data lead us to conclude that the NST itself is a primary central nervous system detector of cytokines.