Blunted diurnal firing in lateral habenula projections to dorsal raphe nucleus and delayed photoentrainment in stress-susceptible mice.

Daily rhythms are disrupted in patients with mood disorders. The lateral habenula (LHb) and dorsal raphe nucleus (DRN) contribute to circadian timekeeping and regulate mood. Thus, pathophysiology in these nuclei may be responsible for aberrations in daily rhythms during mood disorders. Using the 15-day chronic social defeat stress (CSDS) paradigm and in vitro slice electrophysiology, we measured the effects of stress on diurnal rhythms in firing of LHb cells projecting to the DRN (cellsLHb→DRN) and unlabeled DRN cells. We also performed optogenetic experiments to investigate if increased firing in cellsLHb→DRN during exposure to a weak 7-day social defeat stress (SDS) paradigm induces stress-susceptibility. Last, we investigated whether exposure to CSDS affected the ability of mice to photoentrain to a new light-dark (LD) cycle. The cellsLHb→DRN and unlabeled DRN cells of stress-susceptible mice express greater blunted diurnal firing compared to stress-näive (control) and stress-resilient mice. Daytime optogenetic activation of cellsLHb→DRN during SDS induces stress-susceptibility which shows the direct correlation between increased activity in this circuit and putative mood disorders. Finally, we found that stress-susceptible mice are slower, while stress-resilient mice are faster, at photoentraining to a new LD cycle. Our findings suggest that exposure to strong stressors induces blunted daily rhythms in firing in cellsLHb→DRN, DRN cells and decreases the initial rate of photoentrainment in susceptible-mice. In contrast, resilient-mice may undergo homeostatic adaptations that maintain daily rhythms in firing in cellsLHb→DRN and also show rapid photoentrainment to a new LD cycle.

Increased immediate early gene activation in the basolateral amygdala following persistent peripheral inflammation.

Chronic pain results in a variety of neural adaptations, many of which are maladaptive and result in hypersensitivity to pain. In humans, this hypersensitivity can be debilitating and treatment options are limited. Fortunately, there are numerous animal models that mimic clinical populations and have the potential to aid in the evaluation of underlying mechanisms and ultimately the development of better treatments. One of these is the complete Freund's adjuvant (CFA)-model of chronic inflammatory pain. In rodents, this model requires the injection of CFA into the hindpaw, muscle, or joint, which induces inflammation similar to what might be found in individuals with rheumatoid arthritis or tendonitis. While the mechanistic effects CFA on the spinal cord are well established, less is known about the effects of CFA on the brain. Thus, in this study, neuronal activation, as measured by c-Fos immunocytochemistry, in brain regions important to control of pain was evaluated. Animals that received CFA treatment, and tested 3 days later for mechanical allodynia and edema, had an increase in the number of c-Fos immunopositive cells in the basolateral amygdala, but not in any of the other brain regions that were evaluated. Given that the basolateral amygdala is known to be important for pain-related emotional responses, these data suggest that the CFA-model may provide an opportunity to further explore how pain affects this brain region at a mechanistic level, which in turn may shed light on what may be occurring in clinical populations.

Regulation of Locomotor activity in fed, fasted, and food-restricted mice lacking tissue-type plasminogen activator.

BACKGROUND: Circadian rhythms of physiology and behavior are driven by a circadian clock located in the suprachiasmatic nucleus of the hypothalamus. This clock is synchronized to environmental day/night cycles by photic input, which is dependent on the presence of mature brain-derived neurotrophic factor (BDNF) in the SCN. Mature BDNF is produced by the enzyme plasmin, which is converted from plasminogen by the enzyme tissue-type plasminogen activator (tPA). In this study, we evaluate circadian function in mice lacking functional tPA. RESULTS: tPA-/- mice have normal circadian periods, but show decreased nocturnal wheel-running activity. This difference is eliminated or reversed on the second day of a 48-h fast. Similarly, when placed on daily cycles of restricted food availability the genotypic difference in total wheel-running activity disappears, and tPA-/- mice show equivalent amounts of food anticipatory activity to wild type mice. CONCLUSIONS: These data suggest that tPA regulates nocturnal wheel-running activity, and that tPA differentially affects SCN-driven nocturnal activity rhythms and activity driven by fasting or temporal food restriction.

Neural correlates of food anticipatory activity in mice subjected to once- or twice-daily feeding periods.

In rodents, restricted food access to a limited period each day at a predictable time results in the appearance of food anticipatory activity (FAA). Two shorter periods of food access each day can result in two FAA bouts. In this study, we examine FAA under 12:12 and 18:6 photoperiods in mice (Mus musculus) with one or two food access periods per day and measure the activation of the suprachiasmatic, dorsomedial and arcuate nuclei by assaying Fos protein expression, while making use of tissue-type plasminogen activator knockout mice to assess the role of neural plasticity in adaptation to restricted feeding cycles. Long days were utilised to allow for temporal separation of two restricted feeding periods during the light phase. Mice fed twice per day generally divided FAA into two distinct bouts, with mice lacking tissue-type plasminogen activator showing reduced FAA. Increases in Fos expression in response to one restricted feeding period per day were seen in the dorsomedial and arcuate nuclei in both 12:12 and 18:6 conditions, with an increase seen in the SCN in only the 12:12 condition. These increases were eliminated or reduced in the two feeding time conditions (done in 18:6 only). Both activity patterns and Fos expression differed for single restricted feeding times between 18:6 and 12:12 photoperiods. Fos activation was lower during RF in 18:6 than 12:12 across all three brain regions, a pattern not reflective of changes in FAA. These data suggest that involvement of these regions in FAA may be influenced by photoperiodic context.

Urokinase-type plasminogen activator modulates mammalian circadian clock phase regulation in tissue-type plasminogen activator knockout mice.

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue-type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA-/- ; B6.129S2-Plattm1Mlg /J) mice, and identify urokinase-type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA-/- mice entrain to a light-dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA-/- mice take significantly longer to entrain than C57BL/6J wild-type (WT) mice. SCN brain slices from tPA-/- mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose-dependency. Pre-treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), inhibits glutamate-induced phase delays in tPA-/- slices. Selective inhibition of uPA with UK122 prevents glutamate-induced phase resetting in tPA-/- but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA-/- slices. Casein-plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA-/- SCN. Finally, tPA-/- SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.

Changes in brain peptides associated with reproduction and energy homeostasis in photosensitive and photorefractory migratory redheaded buntings.

Present study examined the expression of brain peptides associated with the reproduction and energy homeostasis (GnRH/GnIH, NPY/VIP), and assessed their possible functional association in the photosensitive (non-breeding, pre-breeding), photostimulated (breeding) and photorefractory (post-breeding) migratory redheaded buntings (Emberiza bruniceps), using double-labeled immunohistochemistry. Particularly, we measured immunoreactive (-ir) cell numbers, per cent cell area and cell optical density (OD) in the preoptic area (GnRH-I), midbrain (GnRH-II), paraventricular nucleus (GnIH), dorsomedial hypothalamus, DMH and infundibular complex, INc (NPY and VIP), and lateral septal organ (VIP) of buntings kept under natural photoperiods at the wintering latitude (26°55'N). There was a significant seasonal difference in GnRH-I, not GnRH-II, with reduced -ir cells in the photosensitive and photorefractory buntings, and notably with increased cell OD between the refractory and non-breeding states with no increase in testis size. Also, increased cell OD of GnIH neurons in non-breeding state indicated its role in the maintenance of small testes during the post-refractory period. Overall, seasonal changes in GnRH-I and GnIH were found consistent with their suggested roles in reproductive regulation of absolute photorefractory birds. Further, there was a significant seasonal change in cell OD of NPY neurons in DMH, not the INc. In contrast, VIP immunoreactivity was seasonally altered, with a significantly higher VIP-ir cells in breeding than the pre-breeding state. Finally, close proximity between perikarya with fibres suggested functional interactions between the GnRH and GnIH, and NPY and VIP. Thus, seasonal plasticity of brain peptides is perhaps the part of neural regulation of seasonal reproduction and associated energy homeostasis in migratory songbirds.

Seasonal neuronal plasticity in song-control and auditory forebrain areas in subtropical nonmigratory and palearctic-indian migratory male songbirds.

This study examines whether differences in annual life-history states (LHSs) among the inhabitants of two latitudes would have an impact on the neuronal plasticity of the song-control system in songbirds. At the times of equinoxes and solstices during the year (n = 4 per year) corresponding to different LHSs, we measured the volumetric changes and expression of doublecortin (DCX; an endogenous marker of the neuronal recruitment) in the song-control nuclei and higher order auditory forebrain regions of the subtropical resident Indian weaverbirds (Ploceus philippinus) and Palearctic-Indian migratory redheaded buntings (Emberiza bruniceps). Area X in basal ganglia, lateral magnocellular nucleus of the anterior nidopallium (LMAN), HVC (proper name), and robust nucleus of the arcopallium (RA) were enlarged during the breeding LHS. Both round and fusiform DCX-immunoreactive (DCX-ir) cells were found in area X and HVC but not in LMAN or RA, with a significant seasonal difference. Also, as shown by increase in volume and by dense, round DCX-ir cells, the neuronal incorporation was increased in HVC alone during the breeding LHS. This suggests differences in the response of song-control nuclei to photoperiod-induced changes in LHSs. Furthermore, DCX immunoreactivity indicated participation of the cortical caudomedial nidopallium and caudomedial mesopallium in the song-control system, albeit with differences between the weaverbirds and the buntings. Overall, these results show seasonal neuronal plasticity in the song-control system closely associated with annual reproductive LHS in both of the songbirds. Differences between species probably account for the differences in the photoperiod-response system between the relative refractory weaverbirds and absolute refractory redheaded buntings. J. Comp. Neurol. 524:2914-2929, 2016. © 2016 Wiley Periodicals, Inc.

Annual life-history dependent seasonal differences in neural activity of the olfactory system between non-migratory and migratory songbirds.

Present study investigated seasonal plasticity in neural activity of the olfactory system, and assessed whether this was influenced by differences in seasonal life-history states (LHSs) between the non-migratory and migratory birds. Brains of non-migratory Indian weaver birds and migratory redheaded buntings were processed for ZENK immunohistochemistry, a marker of neuronal activation, at the times of equinoxes (March, September) and solstices (June, December), which correspond with the periods of different seasonal LHSs during the year. Immunoreactivity was quantified in brain regions comprising the olfactory system viz. olfactory bulb (OB), anterior olfactory nucleus (AON), prepiriform cortex (CPP), lateral olfactory tract (LOT) and olfactory cortex (piriform cortex, CPI; lateral olfactory cortex, LOC). In weaver birds, ZENK-like immunoreactive (ZENK-lir) cells were significantly higher in all the brain areas during post-breeding season (September) than during the other seasons; OBs had higher neuronal activity in the breeding season (June), however. A similar neural activity pattern but at enhanced levels was found in migratory buntings almost all the year. These results for the first time show LHS-associated differences in the seasonal plasticity of a sensory system between the non-migratory and migratory songbirds.

Neural correlates of migration: activation of hypothalamic clock(s) in and out of migratory state in the blackheaded bunting (Emberiza melanocephala).

BACKGROUND: Many vertebrates distinguish between short and long day lengths using suprachiasmatic nuclei (SCN). In birds particular, the mediobasal hypothalamus (MBH) is suggested to be involved in the timing of seasonal reproduction. This study investigated the response of SCN and MBH to a single long day, and the role of MBH in induction of the migratory phenotype in night-migratory blackheaded buntings. METHODOLOGY/PRINCIPAL FINDINGS: Experiment 1 immunocytochemically measured c-fos in the SCN, and c-fos, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) in the MBH of buntings exposed to a 20 h light period. Long light period induced significantly stronger c-fos expression, measured as number of c-fos-like immunoreactive (c-fos-lir) cells, in MBH, but not in the SCN. Within the MBH, c-fos-lir cells were significantly denser in the inferior hypothalamic nucleus (IH) and infundibular nucleus (IN), but not in the dorsomedial hypothalamus (DMH). IH and IN also had significantly increased number of VIP and NPY labeled cells. DMH had significantly increased number of VIP labeled cells only. Experiment 2 assayed c-fos, VIP and NPY immunoreactivities in the middle of day and night in the MBH of buntings, after seven long days (day active, non-migratory state) and after seven days of Zugunruhe (night active, migratory state) in long days. In the migratory state, the number of c-fos-lir cells was significantly greater only in DMH; VIP-lir cells were denser in all three MBH regions suggesting enhanced light sensitivity at night. The denser NPY-lir cells only in IN in the non-migratory state were probably due to premigratory hyperphagia. CONCLUSIONS/SIGNIFICANCE: In buntings, SCN may not be involved in the photoperiod-induced seasonal responses. MBH contains the seasonal clock sensitive to day length. VIP and NPY are parts of the neuroendocrine mechanism(s) involved, respectively, in sensing and translating the photoperiodic message in a seasonal response.

Neuropeptide Y mRNA and peptide in the night-migratory redheaded bunting brain.

This study investigated the distribution of neuropeptide Y (NPY) in the brain of the night-migratory redheaded bunting (Emberiza bruniceps). We first cloned the 275-bp NPY gene in buntings, with ≥95% homology with known sequences from other birds. The deduced peptide sequence contained all conserved 36 amino acids chain of the mature NPY peptide, but lacked 6 amino acids that form the NPY signal peptide. Using digosigenin-labeled riboprobe prepared from the cloned sequence, the brain cells that synthesize NPY were identified by in-situ hybridization. The NPY peptide containing cell bodies and terminals (fibers) were localized by immunocytochemistry. NPY mRNA and peptide were widespread throughout the bunting brain. This included predominant pallial and sub-pallial areas (cortex piriformis, cortex prepiriformis, hyperpallium apicale, hippocampus, globus pallidus) and thalamic and hypothalamic nuclei (organum vasculosum laminae terminalis, nucleus (n.) dorsolateralis anterior thalami, n. rotundus, n. infundibularis) including the median eminence and hind brain (n. pretectalis, n. opticus basalis, n. reticularis pontis caudalis pars gigantocellularis). The important structures with only NPY-immunoreactive fibers included the olfactory bulb, medial and lateral septal areas, medial preoptic nucleus, medial suprachiasmatic nucleus, paraventricular nucleus, ventromedial hypothalamic nucleus, optic tectum, and ventro-lateral geniculate nucleus. These results demonstrate that NPY is possibly involved in the regulation of several physiological functions (e.g. daily timing feeding, and reproduction) in the migratory bunting.

Association of rash with outcomes in a randomized phase II trial evaluating cetuximab in combination with mitoxantrone plus prednisone after docetaxel for metastatic castration-resistant prostate cancer.

PURPOSE: Cetuximab (C), a chimeric monoclonal antibody that binds epidermal growth factor receptor (EGFR), is active against androgen-independent prostate cancer cell lines and might enhance the activity of chemotherapy. The efficacy of combining cetuximab with mitoxantrone (M) plus prednisone (MP) was evaluated in progressive metastatic castrate-resistant prostate cancer (CRPC) after receiving docetaxel. MATERIALS AND METHODS: Patients with progression after receiving docetaxel were eligible and randomized 2:1 to CMP or MP. Therapy was mitoxantrone 12 mg/m(2) intravenously (I.V.) on day 1, oral prednisone 10 mg daily in both arms, and cetuximab 250 mg/m(2) I.V. (400 mg/m(2) day 1, cycle 1) on days 1, 8, and 15 in the CMP arm. Cycles were repeated every 21 days. Radiologic assessments of disease and PSA (prostate-specific antigen) occurred every 4 cycles. The primary endpoint was time to progression (TTP). RESULTS: A total of 115 patients were enrolled, 75 in the CMP and 40 in the MP arm: the median TTP was 4.9 and 6.6 months, respectively; the measurable disease response rate was 2% and 4%, the PSA response rate 7.7% and 17.6%, and median survival 11.9 and 15.7 months, respectively. Key grade 3-4 toxicities were neutropenia 44% and 25.6%, anemia 6.7% and 7.7%, thrombocytopenia 6.7% and 2.6%, and fatigue 8% in both arms. In an unplanned exploratory analysis, median TTP with (n = 24) and without rash (n = 51) in the CMP arm was 10.3 months vs. 2.8 months (P = .004). On multivariable analysis,rash was significantly associated with TTP (hazard ratio [HR] = 0.43; P = .01). CONCLUSIONS: The treatment with CMP is not recommended in unselected men with docetaxel-treated CRPC, although rash might help develop tailored therapy.

Food availability affects circadian clock-controlled activity and Zugunruhe in the night migratory male blackheaded bunting (Emberiza melanocephala).

This study investigated the functional linkage between food availability and activity behavior in the Palaearctic Indian night migratory blackheaded bunting (Emberiza melanocephala) subjected to artificial light-dark (LD) cycles. Two experiments were performed on photosensitive birds. In the first one, birds were exposed to short days (LD 10/14; Experiment 1A), long days (LD 13/11; Experiment 1B), or increasing daylengths (8 to 13 h light/d; Experiment 1C) and presented with food either for the whole or a restricted duration of the light period. In Experiments 1A and 1B, illumination of the light and dark periods or of the dark period, alone, was changed to assess the influence of the light environment on direct and circadian responses to food cycles. In the second experiment, birds were exposed to LD 12/12 or LD 8/16 with food availability overlapping with the light (light and food presence in phase) or dark period (light and food presence in antiphase). Also, birds were subjected to constant dim light (LL(dim)) to examine the phase of the activity rhythms under synchronizing influence of the food cycles. Similarly, the presentation of food ad libitum (free food; FF) during an experiment examined the effects of the food-restriction regimes on activity rhythms. A continuous measurement of the activity-rest pattern was done to examine both the circadian and direct effects of the food and LD cycles. Measurement of activity at night enabled assessment of the migratory phenotype, premigratory restlessness, or Zugunruhe. The results show that (i) light masked the food effects if they were present together; (ii) birds had a higher anticipatory activity and food intake during restricted feeding conditions; and (iii) food at night alone reduced both the duration and amount of Zugunruhe as compared to food during the day alone. This suggests that food affects both the daily activity and seasonal Zugunruhe, and food cycles act as a synchronizer of circadian rhythms in the absence of dominant natural environmental synchronizers, such as the light-dark cycle.

Phase inversion of neural activity in the olfactory and visual systems of a night-migratory bird during migration.

Olfactory and visual sensory mechanisms seem to play a critical role in migratory orientation and navigation. How these two mechanisms are functionally linked with other migratory processes is unknown. We investigated this, in relation to the profound behavioural shift that occurs during migration in the night-migratory blackheaded bunting (Emberiza melanocephala). Photosensitive unstimulated birds singly housed in activity cages were subjected to long days (LD 16/8). The activity of each bird was continuously monitored. Daily activity pattern defined the nonmigratory phase (no nocturnal activity) and migratory phase (intense nocturnal activity, Zugunruhe). Body mass and testis size were measured at the beginning and end of the experiment. Long days induced the migratory phenotype (body fattening and Zugunruhe) and testis maturation. The c-fos (Fos) immunoreactivity, as marker of the neural activity of the olfactory and visual subsystems, was measured at midday (8 h after lights-on) and midnight (4 h after lights-off) after the first seven long days (nonmigratory phase) and after seven nights of the Zugunruhe (migratory phase). In the nonmigratory phase, Fos-like immunoreactive (Fos-lir) cells in the olfactory and visual subsystems were high in the day and low at night. In the migratory phase, this was reversed; Fos-lir cells were high at night and low in the day. The phase inversion of neural activity in the olfactory and visual systems in parallel with the behavioral shift suggests a functional coupling between the systems governing migratory flight (expressed as Zugunruhe) and migratory orientation and navigation.