Brain-derived neurotrophic factor regulation of N-methyl-D-aspartate receptor-mediated synaptic currents in suprachiasmatic nucleus neurons

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells. Previous work raises the possibility that brain-derived neurotrophic factor (BDNF) and its high-affinity receptor TrkB may be important as modulators of this excitatory input into the SCN. To test this possibility, we used whole-cell patch-clamp methods to measure excitatory currents in rat SCN neurons. These currents were evoked by electrical stimulation of the optic nerve. We found that the amplitude of the N-methyl-D-aspartate (NMDA) component of the evoked excitatory postsynaptic currents (NMDA-EPSC) was increased by application of BDNF. The neurotrophin also increased the magnitude of NMDA-evoked currents in SCN neurons. The BDNF enhancement of the NMDA-EPSC was blocked by treatment with the neurotrophin receptor antagonist K252a as well as treatment with the soluble form of the TrkB receptor engineered as an immunoadhesin (TrkB IgG). Finally, the BDNF enhancement was lost in brain slices treated with the NR2B antagonist ifenprodil. The results demonstrate that BDNF and TrkB receptors are important regulators of retinal glutamatergic synaptic transmission within the SCN.     

Mechanism of bilateral communication in the suprachiasmatic nucleus

The central circadian pacemaker of the suprachiasmatic nuclei (SCN) is a bilaterally symmetrical structure. Little is known about the physiological mechanisms underlying communication between the left and right SCN and yet the degree of synchronization between SCN neurons can have a critical impact on the properties of the circadian system. In this study, we used electrophysiological tools and calcium (Ca²⁺) imaging to examine the mechanisms underlying bilateral signaling in mouse SCN. Electrical stimulation of one SCN produced responses in the contralateral SCN with a short delay (approximately 5 ms) and Ca²⁺‐dependence that are consistent with action potential‐mediated chemical synaptic transmission. Patch‐clamp recordings of stimulated cells revealed excitatory postsynaptic inward‐currents (EPSCs), which were sufficient in magnitude to elicit action potentials. Electrical stimulation evoked tetrodotoxin‐dependent Ca²⁺ transients in about 30% of all contralateral SCN neurons recorded. The responding neurons were widely distributed within the SCN with a highest density in the posterior SCN. EPSCs and Ca²⁺ responses were significantly reduced after application of a glutamate receptor antagonist. Application of antagonists for receptors of other candidate transmitters inhibited the Ca²⁺ responses in some of the cells but overall the impact of these antagonists was variable. In a functional assay, electrical stimulation of the SCN produced phase shifts in the circadian rhythm in the frequency of multiunit activity rhythm in the contralateral SCN. These phase shifts were blocked by a glutamate receptor antagonist. Taken together, these results implicate glutamate as a transmitter required for communication between the left and right SCN.     

Role for the NR2B subunit of the N-methyl-D-aspartate receptor in mediating light input to the circadian system

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells that utilize glutamate as a neurotransmitter. A variety of evidence suggests that the release of glutamate then activates N -methyl- d -aspartate (NMDA) receptors within the SCN and triggers a signaling cascade that ultimately leads to phase shifts in the circadian system. In this study, we first sought to explore the role of the NR2B subunit in mediating the effects of light on the circadian system of hamsters and mice. We found that localized microinjection of the NR2B subunit antagonist ifenprodil into the SCN region reduces the magnitude of light-induced phase shifts of the circadian rhythm in wheel-running activity. Next, we found that the NR2B message and levels of phospho-NR2B vary with time of day in SCN tissue using semiquantitative real-time polymerase chain reaction and western blot analysis, respectively. Functionally, we found that blocking the NR2B subunit with ifenprodil significantly reduced the magnitude of NMDA currents recorded in SCN neurons. Ifenprodil also significantly reduced the magnitude of NMDA-induced Ca²⁺ changes in SCN cells. Together, these results demonstrate that the NR2B subunit is an important component of NMDA receptor-mediated responses within SCN neurons and that this subunit contributes to light-induced phase shifts of the mammalian circadian system.     

Brain-derived neurotrophic factor upregulates and maintains AMPA receptor currents in neocortical GABAergic neurons

The regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors is implicated in synaptic plasticity. Although we have found that brain-derived neurotrophic factor (BDNF) triggers surface translocation of AMPA receptor proteins, the physiological significance of the BDNF effect remained to be determined. The present immunohistochemical studies revealed that cortical GABAergic neurons exhibited the most striking response to BDNF. Accordingly, we monitored AMPA-triggered currents through GABAergic neurons: Chronic BDNF treatment increased the AMPA-triggered currents but not NMDA-triggered currents in culture. In parallel, the amplitude, but not frequency, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) was elevated in GABAergic neurons. In agreement, BDNF enhanced GABA release triggered by AMPA compared to the amount triggered by high potassium. Conversely, there was a significant decrease in the mEPSC amplitude of GABAergic neurons in heterozygous BDNF-knockout mice. These findings indicate that the neurotrophin enhances the input sensitivity of GABAergic neurons to facilitate their inhibitory function in the neocortex.     

Differential regulation of neurotrophin expression in basal forebrain astrocytes by neuronal signals

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3) promote the function and/or survival of basal forebrain (BF) cholinergic neurons in vivo and in culture. The neurotrophin source is commonly thought to be targets of cholinergic neurons and the possibility that local glial sources support cholinergic neurons has not been well examined. These sources, however, may be critical to BF neurons before or even after they reach their targets. We investigated neurotrophin expression in BF astrocytes and its regulation by neural signals. Solution hybridization and immunocytochemical assays revealed that NGF, BDNF, and NT(3) mRNA and proteins were expressed in cultured BF astrocytes. To investigate roles of neuronal signals in neurotrophin regulation, effects of K(+), glutamate, and the cholinergic agonist carbachol were examined. These stimuli affected neurotrophin expression differentially. KCl increased BDNF mRNA but did not alter NGF or NT(3) mRNA. The effect was blocked by nifedipine, suggesting that it was mediated by L-type voltage-dependent calcium currents. Carbachol also increased BDNF mRNA levels without changing NGF or NT(3). Effects were blocked by the muscarinic antagonist, atropine. In contrast, glutamate increased both NGF and BDNF mRNA. NT(3) mRNA again was unaffected. The metabotropic agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) reproduced glutamate effects, whereas kainate or N-methyl-D-aspartate (NMDA) plus glycine did not. Lack of antagonism by ionotropic antagonists and blockade of glutamate effects by metabotropic antagonists confirmed metabotropic mediation. We suggest that BF astrocytes are local sources of neurotrophins for BF cholinergic neurons during development and are regulated differentially by specific neuronal signals. Critical neuronal-glial interactions may underlie basal forebrain function.     

Involvement of vasoactive intestinal polypeptide in NMDA-induced phase delay of firing activity rhythm in the suprachiasmatic nucleus in vitro

Glutamate has been reported to be involved in the transmission of photic information from the retina to the suprachiasmatic nucleus (SCN). Therefore, we investigated whether the application of (NMDA), a glutamate receptor agonist could, reset the circadian rhythm of SCN firing activity in vitro. Treatment with NMDA for 1 h between projected zeitgeber time (ZT) 13–14 produced a phase delay in a concentration-dependent manner. The NMDA-induced phase delay was antagonized by an NMDA-receptor antagonist, MK-801 (100 μM). The retinohypothalamic tract has been reported to make terminals on neurons possessing vasoactive intestinal polypeptide (VIP). Therefore, we investigated the effects of NMDA on VIP release from the SCN and on VIP immunoreactivity in the SCN. Application of NMDA for 15 min between ZT 13–15 increased release of VIP from the SCN. In contrast to release, the content of VIP in the SCN tissue was reduced by application of NMDA. Immunohistochemical analysis revealed that application of NMDA for 4 h or 1 h reduced VIP immunoreactivity in the SCN. To investigate the possibility that VIP released by NMDA could reset SCN neuronal activity, we examined the effects of VIP on the SCN neuronal activity rhythm. Cotreatment with VIP (1 μM) and gastrin-releasing peptide (1 μM) for 1 h between ZT 13–14 caused a phase-delay of SCN activity rhythm. These findings suggest that activation of NMDA receptors during early subjective night causes a phase delay of the SCN neuronal activity via facilitation of VIP release in this nucleus.     

BDNF enhancement of postsynaptic NMDA receptors is blocked by ethanol

The neurotrophin brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission. Physiological and biochemical evidence implicates the NMDA glutamate receptor as one of the targets for BDNF modulation. In the present studies, murine brain slices containing hippocampus and neocortex were used to study the effects of BDNF on excitatory neurotransmission. Acute exposure to BDNF rapidly and reversibly enhanced the magnitude of NMDA-mediated, but not AMPA receptor-mediated, synaptic currents, specifically enhancing the activity of NMDA receptors containing the NR2B subunit. This effect of BDNF was dependent on activation of trkB neurotrophin receptors because similar effects were not seen with the related neurotrophins NT-3 or NGF. Furthermore, activation of trkB receptors in the postsynaptic neuron was required, as BDNF-induced potentiation was blocked by postsynaptic injection of a trk tyrosine kinase inhibitor. Interestingly, the effect of BDNF was also completely blocked by pretreatment with ethanol, even at concentrations of ethanol that had minimal direct effects on NMDA-mediated responses. These results provide a potential mechanism for the proposed role for BDNF in activity-dependent synaptic plasticity and, potentially, learning and memory processes.     

Developmental changes in the BDNF-induced modulation of inhibitory synaptic transmission in the Kölliker-Fuse nucleus of rat

The Kölliker–Fuse nucleus (KF), part of the pontine respiratory group, is involved in the control of respiratory phase duration, and receives both excitatory and inhibitory afferent input from various other brain regions. There is evidence for developmental changes in the modulation of excitatory inputs to the KF by the neurotrophin brain-derived neurotrophic factor (BDNF). In the present study we investigated if BDNF exerts developmental effects on inhibitory synaptic transmission in the KF.
Recordings of inhibitory postsynaptic currents (IPSCs) in KF neurons in a pontine slice preparation revealed general developmental changes. Recording of spontaneous and evoked IPSCs (sIPSCs, eIPSCS) revealed that neonatally the γ-aminobutyric acid (GABA)ergic fraction of IPSCs was predominant, while in later developmental stages glycinergic neurotransmission significantly increased. Bath-application of BDNF significantly reduced sIPSC frequency in all developmental stages, while BDNF-mediated modulation on eIPSCs showed developmental differences. The eIPSCs mean amplitude was uniformly and significantly reduced following BDNF application only in neurons from rats younger than postnatal day 10. At later postnatal stages the response pattern became heterogeneous, and both augmentations and reductions of eIPSC amplitudes occurred. All BDNF effects on eIPSCs and sIPSCs were reversed with the tyrosine kinase receptor-B inhibitor K252a.
We conclude that developmental changes in inhibitory neurotransmission, including the BDNF-mediated modulation of eIPSCs, relate to the postnatal maturation of the KF. The changes in BDNF-mediated modulation of IPSCs in the KF may have strong implications for developmental changes in synaptic plasticity and the adaptation of the breathing pattern to afferent inputs.     

Involvement of vasoactive intestinal polypeptide in NMDA-induced phase delay of firing activity rhythm in the suprachiasmatic nucleus in vitro

Glutamate has been reported to be involved in the transmission of photic information from the retina to the suprachiasmatic nucleus (SCN). Therefore, we investigated whether the application of N-methyl-d-aspartate (NMDA), a glutamate receptor agonist could, reset the circadian rhythm of SCN firing activity in vitro. Treatment with NMDA for 1 h between projected zeitgeber time (ZT) 13–14 produced a phase delay in a concentration-dependent manner. The NMDA-induced phase delay was antagonized by an NMDA-receptor antagonist, MK-801 (100 μM). The retinohypothalamic tract has been reported to make terminals on neurons possessing vasoactive intestinal polypeptide (VIP). Therefore, we investigated the effects of NMDA on VIP release from the SCN and on VIP immunoreactivity in the SCN. Application of NMDA for 15 min between ZT 13–15 increased release of VIP from the SCN. In contrast to release, the content of VIP in the SCN tissue was reduced by application of NMDA. Immunohistochemical analysis revealed that application of NMDA for 4 h or 1 h reduced VIP immunoreactivity in the SCN. To investigate the possibility that VIP released by NMDA could reset SCN neuronal activity, we examined the effects of VIP on the SCN neuronal activity rhythm. Cotreatment with VIP (1 μM) and gastrin-releasing peptide (1 μM) for 1 h between ZT 13–14 caused a phase-delay of SCN activity rhythm. These findings suggest that activation of NMDA receptors during early subjective night causes a phase delay of the SCN neuronal activity via facilitation of VIP release in this nucleus.     

Involvement of glutamate release in substance P-induced phase delays of suprachiasmatic neuron activity rhythm in vitro

The suprachiasmatic nucleus (SCN) has been identified as a mammalian circadian rhythm clock. Treatment with substance P (SP) at zeitgeber time 13-14 produced phase delays of circadian rhythm in spontaneous neural activity in SCN neurons in vitro. SP-induced phase delays are blocked by treatment with not only SP receptor antagonist, spantide, but N-methyl-D-aspartate receptor antagonist, MK-801. In the biochemical experiment, we demonstrated that SP-induced glutamate release from the SCN slices was observed by the high-performance liquid chromatography assay. The present results suggest that glutamate release may be involved in SP-induced phase delays.     

Transforming growth factor alpha attenuates the functional expression of AMPA receptors in cortical GABAergic neurons

In the developing neocortex, brain-derived neurotrophic factor (BDNF) exerts a trophic activity to increase the expression and channel activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits. Here, we demonstrate that the epidermal growth factor (EGF) receptor (ErbB1) ligands exert the opposite biological activity in cultured neocortical neurons. Subchronic stimulation of ErbB1 with transforming growth factor alpha (TGFalpha), EGF, or heparin-binding EGF (HB-EGF) down-regulated protein expression of the GluR1 AMPA receptor subunit in cultured neocortical neurons. In agreement, TGFalpha treatment decreased the Bmax of [3H] AMPA binding and GluR1 mRNA levels. Immunocytochemistry revealed that the decrease in GluR1 was most pronounced in multipolar GABAergic neurons. To examine the physiological consequences, we recorded AMPA-evoked currents as well as miniature excitatory postsynaptic currents in morphologically identified putative GABAergic neurons in culture. Subchronic TGFalpha treatment decreased AMPA-triggered currents as well as the amplitude and frequency of miniature excitatory postsynaptic currents. An ErbB1 tyrosine kinase inhibitor, PD153035, inhibited the TGFalpha effect. Moreover, TGFalpha counteracted the neurotrophic activity of BDNF on AMPA receptor expression. Co-application of TGFalpha with BDNF blocked the BDNF-triggered up-regulation of AMPA receptor expression and currents. These observations reveal a negative regulatory activity of the ErbB1 ligand, TGFalpha, which reduces the input sensitivity of cortical GABAergic neurons to attenuate their inhibitory function.     

Brain-derived neurotrophic factor facilitates glutamate and inhibits GABA release from hippocampal synaptosomes through different mechanisms

Brain-derived neurotrophic factor (BDNF) has an acute excitatory effect on rat hippocampal synaptic transmission. To compare the action of BDNF upon the release of excitatory and inhibitory neurotransmitters in the hippocampus, we studied the effect of acutely applied BDNF on the K+-evoked glutamate and on the K+-evoked gamma-aminobutyric acid (GABA) release from rat hippocampal nerve terminals (synaptosomes). The acute application of BDNF (30-100 ng/ml) enhanced the K+-evoked [3H]glutamate release. This effect involved tyrosine-kinase B (TrkB) receptor phosphorylation and Ca2+ entry into synaptosomes through voltage-sensitive calcium channels, since it was abolished by K252a (200 nM), which prevents TrkB-mediated phosphorylation, and by CdCl2 (0.2 mM), a blocker of voltage-sensitive calcium channels. In contrast, BDNF (3-100 ng/ml) inhibited K+-evoked [3H]GABA release from hippocampal synaptosomes. This action was also mediated by phosphorylation of the TrkB receptor, but was independent of Ca2+ entry into synaptosomes through voltage-sensitive calcium channels. Blockade of transport of GABA with SKF 89976a (20 microM) prevented the inhibitory action of BDNF upon GABA release, indicating that BDNF influences the activity of GABA transporters. It is concluded that BDNF influences in an opposite way, through distinct mechanisms, the release of glutamate and the release of GABA from hippocampal synaptosomes.     

Role of membrane conductances and protein synthesis in subjective day phase advances of the hamster circadian clock by neuropeptide Y

Neurons of the mammalian circadian pacemaker in the hypothalamic suprachiasmatic nuclei exhibit a rhythm in firing rate that can be reset by neuropeptide Y. We recorded the effects of neuropeptide Y on Na+ and K+ conductances of hamster suprachiasmatic nuclei neurons using whole-cell, perforated-patch and cell-attached patch-clamp recordings, both in dissociated and brain slice preparations. While neuropeptide Y had no effect on voltage-gated Na+ currents, neuropeptide Y activated a leak K+ current. Neuropeptide Y phase advances in the suprachiasmatic nuclei brain slice preparation were blocked by a number of K+ channel blockers (tetraethylammonium chloride, dendrotoxin-I, glybenclamide). However, a K+ ionophore, valinomycin, did not shift the rhythm. The inhibition by tetraethylammonium chloride did not persist in the presence of glutamatergic receptor blockers. We have previously shown that glutamate can oppose neuropeptide Y phase-shifting actions, suggesting that K+ channel inhibition acts by inducing glutamate release. Protein synthesis inhibitors had no effect on clock phase when applied during the subjective day, and had no influence on neuropeptide Y-induced phase shifts. On the other hand, glutamate's ability to inhibit neuropeptide Y shifts was abolished by protein synthesis inhibition. Thus, while neuropeptide Y phase shifts do not require protein synthesis, glutamate blocks neuropeptide Y shifts via increased gene expression during the subjective day, at a time when it does not reset the clock. These results indicate that neuropeptide Y phase shifts via a mechanism that does not involve changes in membrane conductance or protein synthesis.     

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.     

NMDA-evoked calcium transients and currents in the suprachiasmatic nucleus: gating by the circadian system

A variety of evidence suggests that the effects of light on the mammalian circadian system are mediated by glutamatergic mechanisms and that the N-methyl- D-aspartate (NMDA) receptor plays an important role in this regulation. One of the fundamental features of circadian oscillators is that their response to environmental stimulation varies depending on the phase of the daily cycle when the stimuli are applied. For example, the same light treatment, which can produce phase shifts of the oscillator when applied during subjective night, has no effect when applied during the subjective day in animals held in constant darkness (DD). We examined the hypothesis that the effects of NMDA on neurons in the suprachiasmatic nucleus (SCN) also vary from day to night. Optical techniques were utilized to estimate NMDA-induced calcium (Ca2+) changes in SCN cells. The resulting data indicate that there was a daily rhythm in the magnitude and duration of NMDA-induced Ca2+ transients. The phase of this rhythm was determined by the light-dark cycle to which the rats were exposed with the Ca2+ transients peaking during the night. This rhythm continued when animals were held in DD. gamma-Aminobutyric acid (GABA)ergic mechanisms modulated the NMDA response but were not responsible for the rhythm. Finally, there was a rhythm in NMDA-evoked currents in SCN neurons that also peaked during the night. This study provides the first evidence for a circadian oscillation in NMDA-evoked Ca2+ transients in SCN cells. This rhythm may play an important role in determining the periodic sensitivity of the circadian systems response to light.     

Brain-derived neurotrophic factor enhances fast excitatory synaptic transmission in human epileptic dentate gyrus

Brain-derived neurotrophic factor (BDNF) has trophic effects and modulates synaptic transmission in the hippocampal formation in animal studies. It is also upregulated in acute and chronic epilepsy models and in human temporal lobe epilepsy. This study was undertaken to examine the effects of BDNF on fast synaptic transmission in the human epileptic dentate gyrus. Hippocampal specimens were acquired from patients with temporal lobe epilepsy during surgical removal of the anterior temporal lobe intended to treat the epileptic condition. Whole-cell patch-clamp recordings were obtained from dentate granule cells in transverse hippocampal slices in vitro. Application of BDNF increased the amplitude and frequency of spontaneous excitatory postsynaptic currents and increased the amplitude of evoked excitatory postsynaptic currents. BDNF had no effect on spontaneous inhibitory postsynaptic currents but produced a decrease in amplitude of evoked inhibitory postsynaptic currents. BDNF's effects were abolished by coapplication of the tyrosine kinase inhibitor K252a. Therefore, BDNF enhances fast excitatory transmission in the epileptic human dentate gyrus and may play an important role in epileptogenesis in temporal lobe epilepsy. This raises the possibility of designing therapies for this disorder that may be both anticonvulsant and antiepileptogenic.     

Voltage-gated calcium channels play crucial roles in the glutamate-induced phase shifts of the rat suprachiasmatic circadian clock

The resetting of the circadian clock based on photic cues delivered by the glutamatergic retinohypothalamic tract is an important process helping mammals to function adaptively to the daily light-dark cycle. To see if the photic resetting relies on voltage-gated Ca(2+) channels (VGCCs), we examined the effects of VGCC blockers on the glutamate-induced phase shifts of circadian firing activity rhythms of suprachiasmatic nucleus (SCN) neurons in hypothalamic slices. First, we found that a cocktail of amiloride, nimodipine and omega-conotoxin MVIIC (T-, L- and NPQ-type VGCC antagonists, respectively) completely blocked both phase delays and advances, which were, respectively, induced by glutamate application in early and late night. Next, we discovered that: (i) amiloride and another T-type VGCC antagonist, mibefradil, completely obstructed the delays without affecting the advances; (ii) nimodipine completely blocked the advances while having less impact on delays; and (iii) omega-conotoxin MVIIC blocked largely, if not entirely, both delays and advances. Subsequent whole-cell recordings revealed that T-type Ca(2+) currents in neurons in the ventrolateral, not dorsomedial, region of the SCN were larger during early than late night, whereas L-type Ca(2+) currents did not differ from early to late night in both regions. These results indicate that VGCCs play important roles in glutamate-induced phase shifts, T-type being more important for phase delays and L-type being so for phase advances. Moreover, the results point to the possibility that a nocturnal modulation of T-type Ca(2+) current in retinorecipient neurons is related to the differential involvement of T-type VGCC in phase delays and advances.     

In vitro entrainment of the circadian rhythm of vasopressin-releasing cells in suprachiasmatic nucleus by vasoactive intestinal polypeptide

Mammalian circadian pacemaker is located in suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is entrained by light-dark cycle; the photic information is transmitted primarily via the retino-hypothalamic tract (RHT). The main neurotransmitter of the tract is glutamate. RHT fibers end on the ventrolateral part of the nucleus, where vasoactive intestinal peptide (VIP)-immunopositive neurons are localized. They send their axons into dorsomedial SCN, where most of the vasopressinergic (AVP) neurones are located. The AVP neurons retain the clock-like properties in vitro. Vasopressin release from the cultured neurons shows circadian rhythm peaking in the middle of subjective day. VIP induces phase-shifts of the rhythm, magnitude and direction of the shift depending on timing of the application. VIP applied 6-12 h before the peak of vasopressin rhythm induces advances, application 4-8 h after the peak induces delays. The lowest concentration required to induce the phase-shift is 30 nM, further increase of the concentration does not affect the magnitude of the shift. In contrast, glutamate has no effect on the phase of vasopressin rhythm, although in high concentrations it transiently stimulates vasopressin release. The data indicate that the vasopressinergic cells in the SCN contain circadian oscillators, whose rhythms run mutually synchronized in our cultures. VIP acts directly on the vasopressinergic cells to shift the phase of their pacemakers; glutamate has no such effect presumably because in vivo it acts through the VIP-ergic cells but the neuronal network is altered after the dissociation of the cells.