Cholinergic and noradrenergic axonal activity contains a behavioral-state signal that is coordinated across the dorsal cortex.

Fluctuations in brain and behavioral state are supported by broadly projecting neuromodulatory systems. In this study, we use mesoscale two-photon calcium imaging to examine spontaneous activity of cholinergic and noradrenergic axons in awake mice in order to determine the interaction between arousal/movement state transitions and neuromodulatory activity across the dorsal cortex at distances separated by up to 4 mm. We confirm that GCaMP6s activity within axonal projections of both basal forebrain cholinergic and locus coeruleus noradrenergic neurons track arousal, indexed as pupil diameter, and changes in behavioral engagement, as reflected by bouts of whisker movement and/or locomotion. The broad coordination in activity between even distant axonal segments indicates that both of these systems can communicate, in part, through a global signal, especially in relation to changes in behavioral state. In addition to this broadly coordinated activity, we also find evidence that a subpopulation of both cholinergic and noradrenergic axons may exhibit heterogeneity in activity that appears to be independent of our measures of behavioral state. By monitoring the activity of cholinergic interneurons in the cortex, we found that a subpopulation of these cells also exhibit state-dependent (arousal/movement) activity. These results demonstrate that cholinergic and noradrenergic systems provide a prominent and broadly synchronized signal related to behavioral state, and therefore may contribute to state-dependent cortical activity and excitability.

Vagus nerve stimulation induces widespread cortical and behavioral activation.

Vagus nerve stimulation (VNS) is used for management of a variety of neurological conditions, although the therapeutic mechanisms are not fully understood. Accumulating evidence suggests that VNS may modulate cortical state and plasticity through activation of broadly projecting neuromodulatory systems. Using a mouse model, we compared arousal-linked behaviors with dorsal cortical activity obtained with widefield and two-photon GCaMP6s calcium imaging and electrophysiological recordings. We observed robust and reliable cortical and behavioral dose-dependent activation in waking mice to VNS, including pupil dilation and, frequently, whisker movements and locomotion. Widefield calcium imaging and multiunit recording during VNS revealed that this observed increase in arousal state is coupled with a rapid and widespread increase in excitatory activity, including, but not limited to, activation of somatosensory, visual, motor, retrosplenial, and auditory cortical regions. Two-photon GCaMP6s calcium imaging of cholinergic and noradrenergic cortical axons revealed that VNS strongly activates these neuromodulatory systems. Importantly, VNS-evoked activation of neuromodulatory axons and excitatory neurons in the cortex persisted in mice under light anesthesia, in the absence of overt movement. Arousal state changes were abolished by vagus nerve transection, confirming that observed VNS effects were specific to nerve stimulation and triggered widespread activity above that which can be explained by motor activity. Taken together, our results support a model of VNS in which activation of subcortical structures leads to widespread activation of cortex and an increase in arousal state, at least partially due to the activation of cholinergic and noradrenergic modulatory pathways.

Zinc chloride inhibits lysine decarboxylase production from Eikenella corrodens in vitro and its therapeutic implications.

OBJECTIVES: Dentifrices containing zinc reduce gingival inflammation and bleeding better than control dentifrices (no zinc). How zinc might work is not understood. We have shown that lysine decarboxylase (LdcE), an enzyme from Eikenella corrodens, converts lysine to cadaverine in dental biofilms. The lack of lysine impairs the dentally attached cell barrier to biofilm, causing biofilm products to leak into junctional epithelium and stimulate inflammation. In year-old beagle dogs, immunization with LdcE, induces antibodies that inhibit LdcE activity and retard gingivitis development. We therefore examined whether a zinc-mediated loss of LdcE activity could explain the beneficial effect of zinc dentifrices. METHODS: We grew E. corrodens in modified tryptic soy broth with or without zinc chloride, and extracted LdcE from the cell surface using a Potter Elvehjem homogenizer. RESULTS: Up to 0.96 mM zinc chloride in the bacterial growth medium did not change cell yield, but reduced the extracted protein content by 41% (R2 = 0.27, p < 0.05) and LdcE activity/mg extracted protein by 85% (R2 = 0.90, p < 0.001). In extracts from cells grown without zinc, 78 times this zinc chloride concentration (73 mM) was required to reduce LdcE activity by 75%. CONCLUSIONS: Zinc ions inhibit the production of protein with LdcE activity at E. corrodens cell surfaces. The zinc ions may attach to cysteine residues that are unique to the N-terminal region of LdcE by interfering with the non-covalent polypeptide assembly that produces enzyme activity. CLINICAL SIGNIFICANCE: Zinc ion-mediated inhibition of LdcE assembly may provide a rationale for the improved control of gingival inflammation by zinc dentifrices.

Experimental Demyelination of the Lateral Olfactory Tract and Anterior Commissure.

Demyelination significantly affects brain function. Several experimental methods, each inducing varying levels of myelin and neuronal damage, have been developed to understand the process of myelin loss and to find new therapies to promote remyelination. The present work investigates the effect of one such method, lysolecithin administration, on the white matter tracts in the olfactory system. The olfactory forebrain contains two distinct tracts with differing developmental histories, axonal composition, and function: the lateral olfactory tract (LOT), which carries ipsilateral olfactory information from the olfactory bulb to olfactory cortex, and the anterior commissure (AC), which interconnects olfactory regions across hemispheres. The effects of lysolecithin injections were assessed in two ways: (1) the expression of myelin basic protein, a component of compacted myelin sheaths, was quantified using immunohistochemistry and (2) electron microscopy was used to obtain measurements of myelin thickness of individual axons as well as qualitative descriptions of the extent of damage to myelin and surrounding tissue. Data were collected at 7, 14, 21, and 30 days post-injection (dpi). While both the LOT and AC exhibited significant demyelination at 7 dpi and had returned to control levels by 30 dpi, the process differed between the two tracts. Remyelination occurred more rapidly in the LOT: substantial recovery was observed in the LOT by 14 dpi, but not in the AC until 21 dpi. The findings indicate that (a) the LOT and AC are indeed suitable tracts for studying lysolecithin-induced de- and remyelination and (b) experimental demyelination proceeds differently between the two tracts.

The mouse olfactory peduncle 4: Development of synapses, perineuronal nets, and capillaries.

Olfaction is critical for survival in neonatal mammals. However, little is known about the neural substrate for this ability as few studies of synaptic development in several olfactory processing regions have been reported. Odor information detected in the nasal cavity is first processed by the olfactory bulb and then sent via the lateral olfactory tract to a series of olfactory cortical areas. The first of these, the anterior olfactory nucleus pars principalis (AONpP), is a simple, two layered cortex with an outer plexiform and inner cell zone (Layers 1 and 2, respectively). Five sets of studies examined age-related changes in the AONpP. First, immunocytochemistry for glutamatergic (VGlut1 and VGlut2) and GABAergic (VGAT) synapses demonstrated that overall synaptic patterns remained uniform with age. The second set quantified synaptic development with electron microscopy and found different developmental patterns between Layers 1 and 2. As many of the interhemispheric connections in the olfactory system arise from AONpP, the third set examined the development of crossed projections using anterograde tracers and electron microscopy to explore the maturation of this pathway. A fourth study examined ontogenetic changes in immunostaining for the proteoglycans aggrecan and brevican, markers of mesh-like extracellular structures known as perineuronal nets whose maturation is associated with the end of early critical periods of synaptogenesis. A final study found no age-related changes in the density of vasculature in the peduncle from P5 to P30. This work is among the first to examine early postnatal changes in this initial cortical region of the olfactory system.

Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation.

BACKGROUND: Lysine, a nutritionally essential amino acid, enters the oral cavity in gingival crevicular fluid (GCF). During oral hygiene restriction (OHR), lysine decarboxylase (LDC) in dento-gingival biofilms converts lysine to cadaverine. Lysine depletion impairs the dental epithelial barrier to bacterial proinflammatory products. Antibodies to LDC from Eikenella corrodens (Ecor-LDC) inhibit LDC activity and retard gingival inflammation in beagle dogs. Whether E. corrodens is the major source of LDC in dental biofilms and whether the lysine analog tranexamic acid (TA) inhibits LDC activity, biofilm accumulation, and GCF exudation in a human gingivitis model were examined. METHODS: Antibodies raised in goats to LDC-rich extracts from E. corrodens cell surfaces were used to inhibit Ecor-LDC and detect it in biofilm extracts using Western blots. Ecor-LDC activity was measured at pH 4.0 to 11.0 and its TA dissociation constant (Ki) at pH 7.0. Young adults used a 5% or 10% TA mouthwash three times daily during OHR for 1 week. RESULTS: Ecor-LDC antibodies and TA inhibited biofilm LDC. Ki of TA for Ecor-LDC was 940 µM. TA reduced plaque index (PI) by downshifting the PI correlation with biofilm lysine content after OHR without TA. GCF was correspondingly suppressed. However, greater TA retention in saliva partially relieved GCF suppression but not biofilm lysine depletion. CONCLUSIONS: TA slightly inhibits LDC but strongly reduces biofilm by inhibiting bacterial lysine uptake. Unfortunately, TA may impair dental epithelial attachments by also inhibiting lysine transporter uptake. Ecor-LDC inhibitors other than lysine analogs may maintain sufficient lysine levels and attachment integrity to prevent periodontal inflammation.

The mouse olfactory peduncle. 3. Development of neurons, glia, and centrifugal afferents.

The present series of studies was designed to provide a general overview of the development of the region connecting the olfactory bulb to the forebrain. The olfactory peduncle (OP) contains several structures involved in processing odor information with the anterior olfactory nucleus (cortex) being the largest and most studied. Results indicate that considerable growth occurs in the peduncle from postnatal day (P)10-P20, with reduced expansion from P20 to P30. No evidence was found for the addition of new projection or interneurons during the postnatal period. GABAergic cells decreased in both number and density after P10. Glial populations exhibited different patterns of development, with astrocytes declining in density from P10 to P30, and both oligodendrocytes and microglia increasing through the interval. Myelination in the anterior commissure emerged between P11 and P14. Dense cholinergic innervation was observed at P10 and remained relatively stable through P30, while considerable maturation of serotonergic innervation occurred through the period. Unilateral naris occlusion from P1 to P30 resulted in about a 30% reduction in the size of the ipsilateral peduncle but few changes were observed on the contralateral side. The ipsilateral peduncle also exhibited higher densities of GAD67-containing interneurons and cholinergic fibers suggesting a delay in normal developmental pruning. Lower densities of interneurons expressing CCK, somatostatin, and NPY and in myelin basic protein staining were also observed. Understanding variations in developmental trajectories within the OP may be an important tool for unraveling the functions of the region.

Attention to endogenous and exogenous cues affects auditory localization.

Three experiments examine attentional differences in auditory localization using either endogenous or exogenous visual cues. Participants were presented with visual cues on a computer screen and asked to localize auditory targets presented through headphones. In conditions in which the auditory and visual stimuli traveled in the same direction, participants showed illusory directional hearing (Hari in Neurosci Lett 189:29-30, 1995) in that the targets were perceived to travel through the head. In conditions in which the directions of the auditory and visual stimuli were conflicting, participants localized the auditory targets as traveling in the direction of the visual cues. These data suggest that visual capture plays a predominate role in the processes of auditory localization that occurs within the head. Additionally, endogenous response times were significantly greater than exogenous response times. We propose this is the result of additional time required to shift one's attentional window in the endogenous condition.

Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development.

Periodontal disease, gingival inflammation (gingivitis) and periodontal attachment loss (periodontitis), causes tooth loss and susceptibility to chronic inflammation. Professionally scaling and cleaning the teeth regularly controls the disease, but is expensive in companion animals. Eikenella corrodens is common in canine oral cavities where it is a source of lysine decarboxylase (LDC). In human dental biofilms (plaques), LDC converts lysine to cadaverine and impairs the gingival epithelial barrier to bacteria. LDC vaccination may therefore retard gingivitis development. Year-old beagle dogs provided blood samples, and had weight and clinical measurements (biofilm and gingivitis) recorded. After scaling and cleaning, two dogs were immunized subcutaneously with 0.2mg native LDC from E. corrodens and 2 sets of four dogs with 0.2mg recombinant LDC purified from Escherichia coli. A third set of 4 dogs was immunized intranasally. Rehydragel(®), Emulsigen(®), Polygen™ or Carbigen™ were used as adjuvant. Four additional pairs of dogs were sham-immunized with each adjuvant alone (controls). Immunizations were repeated twice, 3 weeks apart, and clinical measurements were obtained after another 2 weeks, when the teeth were scaled and cleaned again. Tooth brushing was then stopped and the diet was changed from hard to soft chow. Clinical measurements were repeated after 1, 2, 3, 4, 6 and 8 weeks. Compared with sham-immunized dogs, gingivitis was reduced over all 8 weeks of soft diet after subcutaneous immunization with native LDC, or after intranasal immunization with recombinant LDC in Carbigen™, but for only 6 of the 8 weeks after subcutaneous immunization with recombinant LDC in Emulsigen(®) (repeated measures ANOVA). Subcutaneous vaccination induced a strong serum IgG antibody response that decreased during the soft diet period, whereas intranasal immunization induced a weak serum IgA antibody response that did not decrease. Immunization with recombinant LDC may provide protection from gingivitis if procedures are optimized.

Social anhedonia and schizotypy in a community sample: the Maryland longitudinal study of schizotypy.

Social anhedonia has been employed in psychometric high-risk studies to identify putative schizotypes. To date, this research has focused almost exclusively on college samples. The current study sought to examine the validity of social anhedonia as an indicator of risk for schizophrenia-spectrum disorders within a community sample. Furthermore, we evaluated the role of other individual difference variables in accounting for variable clinical severity within the social anhedonia group including trait affectivity, social support, and family environment. Following the mailed questionnaire screening of 2434 eighteen-year olds, laboratory assessments were conducted with individuals identified as being high in social anhedonia (n=86) and a comparison sample (n=89). Compared with the control group, individuals in the social anhedonia group were found to have higher rates of mood disorders, elevated schizophrenia-spectrum personality disorder characteristics, greater negative symptom characteristics, and lower global functioning. Individuals within the social anhedonia group also reported greater trait negative affectivity, lower positive affectivity, less social support, and more family conflict. Low social support and problematic family environment were found to be related to elevations in spectrum personality disorder characteristics and poorer functioning within the social anhedonia group. These cross-sectional findings from a community sample provide further support for social anhedonia as a possible indicator of schizotypy.

Pathologic role of stressed-induced glucocorticoids in drug-induced liver injury in mice.

UNLABELLED: We previously reported that acetaminophen (APAP)-induced liver injury (AILI) in mice is associated with a rise in serum levels of the glucocorticoid (GC), corticosterone. In the current study, we provide evidence that endogenous GC play a pathologic role in AILI. Specifically, pretreatment of mice with the GC receptor (GCR) inhibitor, RU486 (mifepristrone), protected normal but not adrenalectomized mice from AILI, while pretreatment with dexamethasone, a synthetic GC, exacerbated AILI. RU486 did not affect the depletion of whole liver reduced GSH or the formation of APAP-protein adducts. It also had no effects on the formation of reactive oxygen species or the depletion of mitochondrial GSH or ATP. While RU486 pretreatment also protected against halothane-induced liver injury, it exacerbated concanavalin A (ConA)- and carbon tetrachloride (CCl(4))-induced liver injury, demonstrating the complexity of GC effects in different types of liver injury. CONCLUSION: These results suggest that under certain conditions, elevated levels of GC might represent a previously unappreciated risk factor for liver injury caused by APAP and other drugs through the diverse biological processes regulated by GCR.

The role of cytokines in the mechanism of adverse drug reactions.

Cytokines are thought to play a role in acute and/or immune-mediated adverse drug reactions (ADRs) due to their ability to regulate the innate and adaptive immune systems. This role is highly complex owing to the pluripotent nature of cytokines, which enables the same cytokine to play multiple roles depending on target organ(s) involved. As a result, the discussion of cytokine involvement in ADRs is organized according to target organ(s); specifically, ADRs targeting skin and liver, as well as ADRs targeting multiple organs, such as drug-induced autoimmunity and infusion-related reactions. In addition to discussing the mechanism(s) by which cytokines contribute to the initiation, propagation, and resolution of ADRs, we also discuss the usefulness and limitations of current methodologies available to conduct such mechanistic studies. While animal models appear to hold the most promise for uncovering additional mechanisms, this field is plagued by a lack of good animal models and, as a result, the mechanism of cytokine involvement in ADRs is often studied using less informative in vitro studies. The recent formation of the Drug-Induced Liver Injury Network, whose goal is collect thousands of samples from drug-induced liver injury patients, has enormous potential to advance knowledge in this field, by enabling large-scale cytokine polymorphism studies. In conclusion, we discuss how further advances in this field could be of significant benefit to patients in terms of preventing, predicting, and treating ADRs.

Nuclear trafficking of the human cytomegalovirus pp71 (ppUL82) tegument protein.

The human cytomegalovirus tegument protein pp71 localizes to the nucleus immediately upon infection, and functions to initiate viral gene expression. Analysis of a series of random insertion mutations revealed that sequences within the mid region (MR) of pp71 are important for localization to the nucleus. Fusion of MR sequences with eGFP revealed that amino acids 94 to 300 were sufficient to target proteins to the nucleus. Random substitution mutagenesis within this domain resulted in two double substitution mutants, pp71P203T/T223M and pp71T228M/L275Q, with a predominantly cytoplasmic localization. Disruption of nuclear targeting resulted in relocalization of the fusion proteins to a distinct perinuclear region. Using tandem mass spectrometry, we determined that threonine 223 can be phosphorylated. Mutation of this residue to a phosphomimetic amino acid resulted in abrogation of nuclear targeting. These results strongly suggest that the intracellular trafficking of pp71 is regulated by phosphorylation.

Behavioral signs of schizoidia and schizotypy in social anhedonics.

Social anhedonia appears to be a promising indicator of Meehl's construct of schizotypy. While findings from diagnostic, cognitive, and psychophysiological studies have supported the validity of social anhedonia as an indicator of schizotypy, the behavioral characteristics of these putative schizotypes are not yet fully understood. This study utilized a rating system for behavioral signs of schizoidia and schizotypy to determine whether atypical interpersonal behaviors were observable in social anhedonics and to examine if these behavioral signs provide unique information, beyond traditional symptom ratings, in the identification of putative schizotypes. A community sample of 170 18-19-year-olds (85 social anhedonics, 85 controls) received diagnostic evaluations which were videotaped and subsequently rated for behavioral signs of schizoidia and schizotypy. Compared to controls, the social anhedonia group displayed significantly more behavioral signs characteristic of schizoid and schizotypal personality disorders. Behavioral signs of schizoidia accounted for a significant amount of group variance even after controlling for clinical symptom ratings. These results indicate that social anhedonics display interpersonal behaviors consistent with risk for schizophrenia-spectrum disorders and that these behavioral signs convey information about group status that is not accounted for by traditional clinical interview ratings of symptomatology.

Examining the latent structure of negative symptoms: is there a distinct subtype of negative symptom schizophrenia?

Negative symptoms have emerged as a replicable factor of symptomatology within schizophrenia. Although rating scales provide assessments along dimensions of severity, categorization into a negative symptom subtype is typically conducted. A categorical view of negative symptoms is best reflected in the proposal that enduring, primary negative symptoms, or deficit symptoms, reflect a distinct subtype of schizophrenia . Despite an accumulation of findings that support a categorical conceptualization, the data are also consistent with a dimensional-only model where negative symptom subtypologies simply reflect an extreme on a continuum of severity. Using taxometric statistical methods , the present study examined whether a taxonic, or latent class, model best describes negative symptoms in a sample of 238 schizophrenia patients. In order to obtain more stable estimates of symptoms, ratings on the Scale for the Assessment of Negative Symptoms [Andreasen, N.C., 1982. Negative symptoms in schizophrenia: Definition and reliability. Arch. Gen. Psychiatry 39, 784-788.] were averaged across two assessments over a 6-month period. Two taxometric methods, maximum covariance analysis (MAXCOV) and mean above minus below a cut (MAMBAC) identified a latent class or taxon with a base rate of approximately 28-36%. Members of the negative symptom taxon differed from the nontaxon class in that taxon members were more likely to be male and demonstrated poorer social functioning. Taxon and nontaxon schizophrenia patients did not differ in psychotic or affective symptoms. The findings converge to provide support for a categorical view of negative symptoms. Further research is required to replicate the present taxonic findings and to examine characteristics (including possible etiological factors) associated with this negative symptom taxon.

Affectivity in the problem-solving interactions of schizophrenia patients and their family members.

This study sought to examine the relationship between symptomatology and the affect expressed between individuals with schizophrenia and their family members. It was hypothesized that, because of their impact on patient social behavior and potential burden on relatives, greater negative symptoms would be associated with less emotional expression in patients but would be related to the greater expression of negative emotions in their relatives within a problem-solving discussion. Informed by research on the structure of emotion, a broad assessment of affect, including Negativity, Positivity, and Disengagement, was utilized to examine affect expressed by patients with schizophrenic disorders (N=91) and their family members during videotaped problem-solving discussions. Although individuals with schizophrenia were comparable to their family members in displays of Negativity, patients displayed less Positivity and greater Disengagement. Greater negative symptoms (in particular blunted or flat affect) were related to a general diminution of affective expression in the schizophrenia group. However, negative symptoms were unrelated to the emotional expression of family members. Other symptoms such as thought disorder and mood symptoms of anxiety, depression, and hostility were not related to displays of affect by either patients or their family members. The findings indicate the importance of examining domains of affect other than negativity and demonstrate that negative symptoms are related to interpersonal displays of affect in schizophrenia. Additionally, these results suggest that schizophrenic symptoms, by themselves, may contribute little to the conflict between patients and their family members.