L Amino acids and D-glucose bind stereospecifically to a colloidal clay.

L-Leucine, L-aspartate, and D-glucose bind in a stereospecific manner to a colloidal clay, bentonite. This binding has high-affinity, saturable characteristics. The biologically uncommon enantiomers, D-leucine, D-asparatate, and L-glucose, do not exhibit any selective absorption on bentonite. It is suggested that this difference between stereoisomers could account for the evolution of life forms possessing a great preponderance of L amino acids and D-glucose.

Brain blood flow: alteration by prior exposure to a learned task.

Chicks with the eyelids of one eye sutured were trained to discriminate between grains and pebbles. The learned experience was completely recognizable by the naive eye that had been occluded during training. When both eyes were opened after monocular training, the velocity of blood flow through paired left and right brain regions was identical. However, when chicks were reexposed to the discrimination situation, blood flow through the cerebral hemisphere associated with the naive eye was greater than that through the hemisphere associated with the trained eye.

c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms.

Circadian rhythms are endogenous cycles with periods close to, but not exactly equal to, 24 h. In mammals, circadian rhythms are generated in the suprachiasmatic nucleus (SCN) of the hypothalamus as well as several peripheral cell types, such as fibroblasts. Protein kinases are key regulators of the circadian molecular machinery. We investigated the role of the c-Jun N-terminal kinases (JNK), which belong to the mitogen-activated protein kinases family, in the regulation of circadian rhythms. In rat-1 fibroblasts, the p46 kDa, but not the p54 kDa, isoforms of JNK expressed circadian rhythms in phosphorylation. The JNK-inhibitor SP600125 dose-dependently extended the period of Period1-luciferase rhythms in rat-1 fibroblasts from 24.23+/-0.17-31.48+/-0.07 h. This treatment also dose-dependently delayed the onset of the bioluminescence rhythms. The effects of SP600125 on explant cultures from Period1-luciferase transgenic mice and Period2(Luciferase) knockin mice appeared tissue-specific. SP600125 lengthened the period in SCN, pineal gland, and lung explants in Period1-luciferase and Period2(Luciferase) mice. However, in the kidneys circadian rhythms were abolished in Period1-luciferase, while circadian rhythms were not affected by SP600125 treatment in Period2(Luciferase) mice. Valproic acid, already known to affect period length, enhanced JNK phosphorylation and, as predicted, shortened the period of the Period1-bioluminescence rhythms in rat-1 fibroblasts. In conclusion, our results showed that SP600125 treatment, as well as valproic acid, alters JNK phosphorylation levels, and modulates the period length in various tissues. We conclude that JNK phosphorylation levels may help to set the period length of mammalian circadian rhythms.

Prediction of the hip joint centre in adults, children, and patients with cerebral palsy based on magnetic resonance imaging.

The location of the hip joint centre (HJC) is required for calculations of hip moments, the location and orientation of the femur, and muscle lengths and lever arms. In clinical gait analysis, the HJC is normally estimated using regression equations based on normative data obtained from adult populations. There is limited relevant anthropometric data available for children, despite the fact that clinical gait analysis is predominantly used for the assessment of children with cerebral palsy. In this study, pelvic MRI scans were taken of eight adults (ages 23-40), 14 healthy children (ages 5-13) and 10 children with spastic diplegic cerebral palsy (ages 6-13). Relevant anatomical landmarks were located in the scans, and the HJC location in pelvic coordinates was found by fitting a sphere to points identified on the femoral head. The predictions of three common regression equations for HJC location were compared to those found directly from MRI. Maximum absolute errors of 31 mm were found in adults, 26 mm in children, and 31 mm in the cerebral palsy group. Results from regression analysis and leave-one-out cross-validation techniques on the MRI data suggested that the best predictors of HJC location were: pelvic depth for the antero-posterior direction; pelvic width and leg length for the supero-inferior direction; and pelvic depth and pelvic width for the medio-lateral direction. For single-variable regression, the exclusion of leg length and pelvic depth from the latter two regression equations is proposed. Regression equations could be generalised across adults, children and the cerebral palsy group.

Potentiation of the resetting effects of light on circadian rhythms of hamsters using serotonin and neuropeptide Y receptor antagonists.

Circadian rhythms are entrained by light/dark cycles. In hamsters, the effects of light on circadian rhythms can be modulated by serotonergic input to the suprachiasmatic nucleus from the raphe nuclei and by neuropeptide Y containing afferents to the suprachiasmatic nucleus from the intergeniculate leaflet in the thalamus. In this study we measured effects of compounds acting on serotonergic 1A and neuropeptide Y Y5 receptors to determine if combined serotonergic-neuropeptide Y inhibition could synergistically potentiate effects of light on rhythms. We used mixed serotonergic agonist/antagonists BMY 7378 or NAN-190 as well as a neuropeptide Y Y5 antagonist CP-760,542. Both BMY 7378 and NAN-190 are thought to block serotonin release via acting as agonists at the 5-hydroxytryptamine 1A (5-HT1A) autoreceptors on cells in the raphe, and also block response of target cells by acting as antagonists at post-synaptic 5-HT1A receptors, for example, in the suprachiasmatic nuclei or the intergeniculate leaflet. Replicating prior work, we found that pretreatment with either drug alone increased the phase shift to light at circadian time 19. The combined effect of BMY 7378 and CP-760,542 given prior to light at circadian time 19 was to further potentiate the subsequent phase shift in wheel-running rhythms (the phase shift was 317% of controls; light alone: 1.35 h phase shift vs. BMY 7378, CP-760,542, and light: 4.27 h phase shift). Combined treatment with NAN-190 and CP-760,542 produced a light-induced phase shift 576% of controls (phase shift to light alone: 1.23 h vs. NAN-190, CP-760,542, and light: 7.1 h phase shift). These results suggest that the resetting effects of light on circadian rhythms can be greatly potentiated in hamsters by using pharmacological treatments that block both serotonergic and neuropeptide Y afferents to the suprachiasmatic nuclei.

Let there be "more" light: enhancement of light actions on the circadian system through non-photic pathways.

Circadian rhythms are internally generated circa 24 h rhythms. The phase of the circadian pacemaker in mammals can be adjusted by external stimuli such as the daily cycle of light, as well as by internal stimuli such as information related to the physiological and behavioral status of the organism, collectively called "non-photic stimuli". We review a large number of studies regarding photic-non-photic interactions on the circadian system, with special focus on two widely described neurotransmitters associated with non-photic input pathways: neuropeptide Y (NPY) and serotonin 5-HT. Both neurotransmitters are capable of phase advancing the master pacemaker oscillation when applied during the subjective day, as do several behavioral manipulations. Also, both are capable of inhibiting light-induced phase shifts during the subjective night, suggesting a dynamic interaction between photic and non-photic stimuli in the fine-tuning of the pacemaker function. Suppression of the NPYergic and/or serotonergic non-photic input pathways can in turn potentiate the phase-shifting effects of light. These findings pose new questions about the possibility of a physiological role for the dynamic interaction between photic and non-photic inputs. This might be particularly important in the case of circadian system adjustments under certain conditions, such as depression, shift work or jet lag.

The neuropeptide Y Y5 receptor mediates the blockade of "photic-like" NMDA-induced phase shifts in the golden hamster.

Circadian or daily rhythms generated from the mammalian suprachiasmatic nuclei (SCN) of the hypothalamus can be synchronized by light and nonphotic stimuli. Whereas glutamate mediates photic information, nonphotic information can in some cases be mediated by neuropeptide Y (NPY) or serotonin. NPY or serotonin can reduce the phase-resetting effect of light or glutamate; however, the mechanisms and level of interaction of these two kinds of stimuli are unknown. Here we investigate the effect of NPY on the NMDA-induced phase shift of the hamster SCN circadian neural activity rhythm by means of single-unit recording techniques. NMDA (10-100 microm) applied in the early subjective night induced phase delays in the time of peak firing, whereas doses in the millimolar range disrupted firing patterns. The NMDA-induced phase delay was blocked by coapplication of NPY (0.02-200 microm). NPY Y1/Y5 and Y5 receptor agonists, but not the Y2 receptor agonist, blocked the NMDA-induced phase delay in a similar manner as NPY. The coapplication of a Y5 but not Y1 receptor antagonist eliminated NPY blockade of NMDA-induced phase delays, suggesting that the Y5 receptor is capable of mediating the inhibitory effect of NPY on photic responses. These results indicate that nonphotic and photic stimuli may interact at a level at or beyond NMDA receptor response and indicate that the Y5 receptor is involved in this interaction. Alteration of Y5 receptor function may therefore be expected to alter synchronization of circadian rhythms to light.

Assessment of sub-division of plantar pressure measurement in children.

Methods for the measurement of plantar pressure are poorly defined particularly when describing sub-sections of the plantar surface of the foot in the presence of deformity. The aim of this study was to assess foot pressure measurement in healthy children, using an automatic technique of sub-area definition that has the potential for objective evaluation of treatment of foot deformity. Twelve healthy children were examined on three occasions. Plantar pressure data were collected and time synchronised with force plate and stereophotogrammetric data. The footprint was divided into five sub-sections by using the position of the markers on the foot at mid-stance projected onto the pressure footprint. Repeatability for peak pressure and peak force was assessed. Automatic sub-area definition based on marker placement was found to be reliable in healthy children. A comparison of results revealed that peak vertical force was a more consistent measure than peak pressure for each of the five sub-areas. This suggests that force may be a more appropriate measurement for outcome studies.

Lesions of the thalamic intergeniculate leaflet alter hamster circadian rhythms.

We have investigated the effects of destruction of the geniculo-hypothalamic tract (GHT) on the circadian system of golden hamsters. In the first experiment, intact hamsters were housed in constant darkness, and phase shifts in running-wheel activity rhythms were assessed following 15-min light pulses administered at circadian time (CT) 12 (defined as the beginning of activity), CT 14, CT 18, and CT 20. Responses to light pulses at the same CTs were then reassessed after GHT lesions. Hamsters with complete lesions showed decreases in phase advances caused by light pulses at CT 18 and CT 20. Phase delays elicited by light at CT 12 and CT 14 were not altered. In a second study, intact and GHT-ablated hamsters housed in constant light received 6-hr dark pulses at various CTs. Hamsters with complete GHT ablation showed smaller advances than controls to dark pulses centered on CT 8-10. After 110 days in constant light, 7 of 10 intact hamsters showed splitting of their activity rhythms into two components, while only 1 of the 8 similarly treated ablated hamsters displayed dissociated activity components. Ablated hamsters had significantly shorter free-running periods during the first 35 days of exposure to constant light than did the intact hamsters. These results demonstrate that destruction of the GHT in the hamster alters phase shifting in response to periods of light or dark, and they indicate a role for the GHT in mediating several photic effects on the circadian system.

Histamine phase shifts the hamster circadian pacemaker via an NMDA dependent mechanism.

The SCN acts as the central pacemaker for circadian rhythms in mammals. Histamine has been shown to affect circadian rhythms both in vivo and in vitro. We investigated the mechanism by which histamine phase shifts circadian rhythms in vitro. Hypothalamic slices containing the SCN were prepared from golden hamsters, and spontaneous firing rates of individual cells were recorded on the second day in vitro. Application of histamine (1 microM-10 mM) at the extrapolated time of 2 h after lights off (ZT 14) on day 1 in vitro delayed the time of peak firing in a dose-dependent manner. Pre-exposure to the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)-2-amino-5-phosphonopentanoic acid (AP-5; 100 microM-1 mM) 5 min before histamine (1 microM) was applied to the slice blocked the phase-delaying effects of histamine. Application of the H1 blocker mepryamine (100 nM) or the H2 blocker cimetidine (10 microM) followed by histamine had no effect on the phase delay induced by histamine. In whole cell recordings from acutely dissociated neurons of hamster SCN, histamine (50 microM) was shown to potentiate NMDA-evoked currents by 52 +/- 12%. These experiments demonstrate that histamine phase shifts of the circadian clock are dependent on NMDA receptor activation and that histamine can directly potentiate NMDA currents in SCN neurons. Histamine may alter circadian clock function by acting directly on NMDA receptors, possibly via binding to the polyamine site.

Suprachiasmatic nucleus neurons are glucose sensitive.

The suprachiasmatic nucleus (SCN) in the hypothalamus serves as the pacemaker for mammalian circadian rhythms. In a hamster brain slice preparation, the authors were able to record spontaneous activity from SCN cells for up to 4 days in vitro and verify a self-sustained rhythm in firing. The phase of this rhythm was altered by the concentration of glucose in the bathing medium, with time of peak firing advanced for a 20 mM glucose condition and slightly delayed for a 5 mM glucose condition, relative to 10 mM. The advancing effect of 20 mM glucose and the delaying effect of 5 mM glucose were not maintained during a 2nd day in vitro after changing the bathing medium back to 10 mM glucose, thus indicating the effect was not a permanent phase shift of the underlying oscillation. In experiments recording from cell-attached membrane patches on acutely dissociated hamster SCN neurons, exchanging the bathing medium from high (20 mM) to zero glucose increased potassium (K+)-selective channel activity. With inside-out membrane patches, the authors revealed the presence of a glybenclamide-sensitive K+ channel (190 pS) and a larger conductance (260 pS) Ca(2+)-dependent K+ channel that were both reversibly inhibited by ATP at the cytoplasmic surface. Furthermore, 1 mM tetraethylammonium chloride was demonstrated to advance peak firing time in the brain slice in a similar manner to a high concentration of glucose (20 mM). The authors interpret the result to imply that SCNs are sensitive to glucose, most probably via ATP modulation of K+ channel activity in these neurons. Tonic modulation of K+ channel activity appears to alter output of the pacemaker but does not reset the phase.

The ventral lateral geniculate nucleus and the intergeniculate leaflet: interrelated structures in the visual and circadian systems.

The ventral lateral geniculate nucleus (vLGN) and the intergeniculate leaflet (IGL) are retinorecipient subcortical nuclei. This paper attempts a comprehensive summary of research on these thalamic areas, drawing on anatomical, electrophysiological, and behavioral studies. From the current perspective, the vLGN and IGL appear closely linked, in that they share many neurochemicals, projections, and physiological properties. Neurochemicals commonly reported in the vLGN and IGL are neuropeptide Y, GABA, enkephalin, and nitric oxide synthase (localized in cells) and serotonin, acetylcholine, histamine, dopamine and noradrenalin (localized in fibers). Afferent and efferent connections are also similar, with both areas commonly receiving input from the retina, locus coreuleus, and raphe, having reciprocal connections with superior colliculus, pretectum and hypothalamus, and also showing connections to zona incerta, accessory optic system, pons, the contralateral vLGN/IGL, and other thalamic nuclei. Physiological studies indicate species differences, with spectral-sensitive responses common in some species, and varying populations of motion-sensitive units or units linked to optokinetic stimulation. A high percentage of IGL neurons show light intensity-coding responses. Behavioral studies suggest that the vLGN and IGL play a major role in mediating non-photic phase shifts of circadian rhythms, largely via neuropeptide Y, but may also play a role in photic phase shifts and in photoperiodic responses. The vLGN and IGL may participate in two major functional systems, those controlling visuomotor responses and those controlling circadian rhythms. Future research should be directed toward further integration of these diverse findings.

Uptake and release of putative neurotransmitters. Measurements in regions of the normal and Newcastle disease virus-infected mouse brain.

The capacity of various regions of the mouse brain to accumulate a series of putative neurotransmitter compounds has been studied in cerebral homogenates. This uptake is selective, sodium dependent, energy dependent, and exhibits characteristics of high affinity transport. Calcium-stimulated release under depolarizing conditions of accumulated radioactive compounds was also examined. Large regional variations of uptake and release capacity existed. No clear relation between intensity of uptake and releasability of transported compounds was seen. The effect of infection of mice with Newcastle disease virus on these processes was investigated. No significant differences were seen in infected mice despite their depressed metabolic rate.

Is novel wheel inhibition of per1 and per2 expression linked to phase shift occurrence?

We studied whether access to a novel running wheel in vivo could reset the suprachiasmatic nuclei (SCN) in vitro. Golden hamsters were transferred to dim red light at Zeitgeber time (ZT) 4, given their first exposure to a running wheel for 3 h, and killed at either ZT7 or ZT9. Using a brain slice preparation, the SCN firing rate rhythm in vitro was advanced relative to controls only in the slices prepared at ZT9 (phase shift: 2.36+/-0.06 h, n=4) but not ZT7 (-0.26+/-0.16 h, n=4). Transitions to dim red light or brain slice preparation at ZT7 or ZT9 alone do not shift the rhythm. Hamsters with wheels had significantly lower levels of SCN per1 mRNA compared with controls at ZT7, and lower per2 mRNA when examined at ZT9. We conclude that 3 h of novel wheel access appears to require some extended time in vivo in order for the SCN to be reset, even beyond the time when per1 mRNA levels have been altered.

Neuropeptide Y and glutamate block each other's phase shifts in the suprachiasmatic nucleus in vitro.

The suprachiasmatic nuclei contain a circadian clock whose activity can be recorded in vitro for several days. Photic information is conveyed to the nuclei primarily via a direct projection from the retina, the retinohypothalamic tract, utilizing an excitatory amino acid neurotransmitter. Photic phase shifts may be mimicked by application of glutamate in vitro. A second, indirect pathway to the suprachiasmatic nuclei via the geniculohypothalamic tract utilizes neuropeptide Y as a transmitter. Phase shifts to neuropeptide Y in vitro are similar to those seen to non-photic stimuli in vivo. We have used the hypothalamic slice preparation to examine the interactions of photic and non-photic stimuli in the suprachiasmatic nuclei. Coronal hypothalamic slices containing the suprachiasmatic nuclei were prepared from Syrian hamsters and 3 min recordings of the firing rate of individual cells were performed throughout a 12 h period. Control slices receiving either no application or application of artificial cerebrospinal fluid to the suprachiasmatic nucleus showed a consistent daily peak in their rhythms. Glutamate produces phase shifts of the circadian clock in the hamster hypothalamic slice preparation during the subjective night but not during the subjective day. These phase shifts were similar in timing and direction to the photic phase response curve in vivo confirming previous work with the rat slice preparation. Neuropeptide Y produces phase shifts of the circadian clock during the subjective day but not during the subjective night. The phase shifts are similar in timing and direction to the non-photic phase response curve in vivo, confirming previous in vitro work. We then examined the interaction of these neurochemicals with each other at various times during the circadian cycle. We found that both advances and delays to glutamate in the slice are blocked by application of neuropeptide Y. We also found that phase shifts to neuropeptide Y in the slice are blocked by application of glutamate. These results indicate that photic and non-photic associated neurochemicals can block each others phase shifting effects within the suprachiasmatic nucleus in vitro. These experiments demonstrate the ability of photic and non-photic associated neurochemicals to interact at the level of the suprachiasmatic nucleus. It is clear that neuropeptide Y antagonizes the effect of glutamate during the subjective night, and that glutamate antagonizes the effect of neuropeptide Y during the subjective day. Great care must be taken when devising treatments where photic and non-photic signals may interact.

NPY opposes PACAP phase shifts via receptors different from those involved in NPY phase shifts.

The suprachiasmatic nuclei (SCN) of the hamster can be maintained for several days in vitro, allowing electrophysiological investigation of the mammalian circadian clock. Application of pituitary adenylate cyclase activating peptide (PACAP) at Zeitgeber time (ZT) 6 on the first day in vitro can phase shift the rhythm of firing rate expressed by SCN neurons on a subsequent day in vitro. Here we report that co-application of neuropeptide Y (NPY) will block the phase-shifting action of PACAP. This blocking action is mimicked by [Leu31,Pro34]NPY and [D-Trp32]NPY but not by NPY(22-36) or avian pancreatic polypeptide. The results indicate that NPY has actions in the SCN via receptors distinct from the Y2 receptor, which mediates the phase-shifting action of NPY.

Neuropeptide Y applied in vitro can block the phase shifts induced by light in vivo.

The mammalian suprachiasmatic nuclei (SCN) can be synchronized by light, with direct glutamatergic input from the retina. Input to the SCN from the intergeniculate leaflet contains neuropeptide Y (NPY) and can modulate photic responses. NPY can reduce the phase-resetting effect of light or glutamate. We investigated the effect of NPY applied in vitro on light-induced phase shifts of the SCN neural activity rhythm. Light pulses delivered in vivo induced phase shifts in brain slice preparations similar to those as measured by behavioral activity rhythms. NPY applied after the light pulse blocked the phase shifts during both the early and late subjective night. NPY applied 30 min after the light pulse could block the phase delay induced by light. Our results show that NPY can inhibit photic resetting of the clock during the subjective night. The time course of this inhibitory effect suggests a mechanism downstream of the glutamate receptor.

Neuropeptide Y phase shifts the circadian clock in vitro via a Y2 receptor.

The suprachiasmatic nuclei (SCN) contain a circadian clock whose activity can be recorded in vitro for several days. This clock can be reset by the application of neuropeptide Y. In this study, we focused on determination of the receptor responsible for neuropeptide Y phase shifts of the hamster circadian clock in vitro. Coronal hypothalamic slices containing the SCN were prepared from Syrian hamsters housed under a 14 h:10 h light:dark cycle. Tissue was bathed in artificial cerebrospinal fluid (ACSF), and the firing rates of individual cells were sampled throughout a 12 h period. Control slices received either no application or application of 200 nl ACSF to the SCN at zeitgeber time 6 (ZT6; ZT12 was defined as the time of lights off). Application of 200 ng/200 nl of neuropeptide Y at ZT6 resulted in a phase advance of 3.4 h. Application of the Y2 receptor agonist, neuropeptide Y (3-36), induced a similar phase advance in the rhythm, while the Y1 receptor agonist, [Leu31, Pro34]-neuropeptide Y had no effect. Pancreatic polypeptide (rat or avian) also had no measurable phase-shifting effect. Neuropeptide Y applied at ZT20 or 22 had no detectable phase-shifting effect. These results suggest that the phase-shifting effects of neuropeptide Y are mediated through a Y2 receptor, similar to results found in vivo.

Circadian modulation of the ryanodine receptor type 2 in the SCN of rodents.

We examined the temporal modulation of intracellular calcium release channels in the suprachiasmatic nucleus (SCN). We found a circadian rhythm in [3H]ryanodine binding that was specific to the SCN. The peak in the rhythm occurred at CT 7 and was due to an increase in Bmax, which correlated well with immunoblots showing an increase in RyR-2 expression in the SCN. Double immunohistochemical studies showed that RyR-2 was expressed exclusively in neurons. Ryanodine and caffeine applied around CT 7-9 advanced the clock phase in a hamster brain slice preparation. No rhythm of IP3R was seen in any of the brain areas studied. Our results indicate that RyR-2 exhibits an endogenous rhythm, which influences the intracellular calcium dynamics and thus modulates SCN activity.

Neuropeptide Y in the mammalian circadian system: effects on light-induced circadian responses.

The present review examine the role of neuropeptide (NPY) in the circadian system, focusing on the interactions between light and NPY, especially during the subjective night. NPY has two different effects on the circadian system of mammals. On one hand, NPY, similar to behavioral stimulation, can change the phase of the clock by itself during the subjective day. On the other hand, NPY, again similar to behavioral stimulation, can inhibit the phase-shifting effect of light during the night. These effects of NPY may occur through different receptor subtypes, the Y2 receptor mediating day-time effects and the Y5 receptor mediating night-time effects of NPY. Our results also indicate that there are differences between in vivo and in vitro studies: NPY inhibition of in vivo light-induced phase shifts was observed only late in the subjective night; however, NPY applied in vitro could block light-induced phase shifts early in the subjective night as well. Contrasting these in vivo and in vitro results led us to suggest that the time of day of maximal effect of NPY in the intact animal may be a time when exogenous administration of NPY has little effect, due to saturation of the system. This situation could be an example of how the measurable output of the clock can be affected by the behavioral state in a different way at different time points, depending not only on the clock itself but also on behavior. If verified in human beings, the ability of NPY to modulate the circadian-clock responses to light may be of clinical importance.