Developmental patterns of NMDAR expression within the song system do not recur during adult vocal plasticity in zebra finches

All songbirds learn to sing during postnatal development but then display species differences in the capacity to learn song in adulthood. While the mechanisms that regulate avian vocal plasticity are not well characterized, one contributing factor may be the composition of N-methyl-D-aspartate receptors (NMDAR). Previous studies of an anterior forebrain pathway implicated in vocal plasticity revealed significant regulation of NMDAR subunit expression during the developmental sensitive period for song learning. Much less is known about the developmental regulation of NMDAR subunit expression in regions that participate more directly in motor aspects of song behavior. We show here that an increase in NR2A subunit mRNA and a decrease in NR2B subunit mRNA within the vocal motor pathway accompany song learning in zebra finches; however, manipulations that can alter the timing of song learning did not alter the course of these developmental changes. We also tested whether adult deafening, a treatment that provokes vocal change in songbirds that normally sing a stable song throughout adulthood, would render NMDAR subunit expression more similar to that observed developmentally. We report that NR2A and NR2B mRNA levels did not change within the anterior forebrain or vocal motor pathways after adult deafening, even after substantial changes in song structure. These results indicate that vocal plasticity does not require "juvenile patterns" of NMDAR gene expression in the avian song system.     

Serotonergic Modulation of Photic Entrainment in the Syrian Hamster

In this review, we describe six lines of evidence that reveal a modulatory role for serotonin (5-HT) in the regulation of the response of suprachiasmatic nucleus (SCN) neurons to retinal illumination in the Syrian hamster. Electrical stimulation of the median raphe nucleus, sufficient to elicit the release of 5-HT in the SCN, inhibits light-induced phase shifts of the hamster circadian activity rhythm. Two 5-HT receptors capable of mediating the effects of 5-HT on photic responses, the 5-HT₇ receptor and the 5-HT_1B receptor, are present in the hamster SCN. Light-induced phase shifts are attenuated by systemic and local administration of two 5-HT receptor agonists, 8-OH-DPAT, and TFMPP, and these agents attenuate photic phase shifts by acting on pharmacologically distinct receptors. Furthermore, both compounds also attenuate light-induced Fos expression and photic suppression of pineal melatonin content, indicating that serotonergic modulation of photic signal transduction in the SCN is not limited to the regulation of circadian phase. Finally, both 8-OH-DPAT and TFMPP inhibit RHT neurotransmission in the hypothalamic slice preparation. Further, TFMPP fails to attenuate responses to exogenous glutamate on retinorecipient SCN neurons, consistent with a presynaptic site of action for the drug. Based on these data, we propose that 5-HT modulates RHT neurotransmission in the SCN through at least two distinct mechanisms: (1) via activation of 5-HT₇ receptors probably located on retinorecipient neurons; and (2) via activation of presynaptic 5-HT_1B receptors leading to reduced release of glutamate from RHT terminals in the SCN.     

Cloning and Localization of Exon 5-Containing Isoforms of the NMDAR1 Subunit in Human and Rat Brains

Abstract: Nine isoforms of the rat NMDAR1 receptor subunit have been previously identified, of which several have an alternatively spliced N-terminal insert believed to be important in proton sensitivity of the receptor. The cloning of the human homologues of NMDAR1-3b (hNMDA1-1) and NMDAR1-4b (hNMDA1-2), both bearing the insert, is reported here. A monoclonal antibody generated against the N-terminal region of these isoforms showed reactivity with at least two distinct human brain proteins of ∼115 kDa. This antibody was further characterized by using a series of truncated fusion proteins and splice variants of NMDAR1 demonstrating its specific recognition of an epitope within the 21-amino acid N-terminal insert, encoded by exon 5. Western blot and immunocytochemical studies were performed to examine the expression of the exon 5-containing isoforms of the NMDAR1 subunit in both rat and human brain.     

Light pulses do not induce c-fos or per1 in the SCN of hamsters that fail to reentrain to the photocycle

Circadian activity rhythms of most Siberian hamsters (Phodopus sungorus sungorus) fail to reentrain to a 5-h phase shift of the light-dark (LD) cycle. Instead, their rhythms free-run at periods close to 25 h despite the continued presence of the LD cycle. This lack of behavioral reentrainment necessarily means that molecular oscillators in the master circadian pacemaker, the SCN, were unable to reentrain as well. The authors tested the hypothesis that a phase shift of the LD cycle rendered the SCN incapable of responding to photic input. Animals were exposed to a 5-h phase delay of the photocycle, and activity rhythms were monitored until a lack of reentrainment was confirmed. Hamsters were then housed in constant darkness for 24 h and administered a 30-min light pulse 2 circadian hours after activity onset. Brains were then removed, and tissue sections containing the SCN were processed for in situ hybridization. Sections were probed with Siberian hamster c-fos and per1 mRNA probes because light rapidly induces these 2 genes in the SCN during subjective night but not at other circadian phases. Light pulses induced robust expression of both genes in all animals that reentrained to the LD cycle, but no expression was observed in any animal that failed to reentrain. None of the animals exhibited an intermediate response. This finding is the first report of acute shift in a photocycle eliminating photosensitivity in the SCN and suggests that a specific pattern of light exposure may desensitize the SCN to subsequent photic input.     

Serotonergic Modulation of Photic Entrainment in the Syrian Hamster

In this review, we describe six lines of evidence that reveal a modulatory role for serotonin (5-HT) in the regulation of the response of suprachiasmatic nucleus (SCN) neurons to retinal illumination in the Syrian hamster. Electrical stimulation of the median raphe nucleus, sufficient to elicit the release of 5-HT in the SCN, inhibits light-induced phase shifts of the hamster circadian activity rhythm. Two 5-HT receptors capable of mediating the effects of 5-HT on photic responses, the 5-HT₇ receptor and the 5-HT_1B receptor, are present in the hamster SCN. Light-induced phase shifts are attenuated by systemic and local administration of two 5-HT receptor agonists, 8-OH-DPAT, and TFMPP, and these agents attenuate photic phase shifts by acting on pharmacologically distinct receptors. Furthermore, both compounds also attenuate light-induced Fos expression and photic suppression of pineal melatonin content, indicating that serotonergic modulation of photic signal transduction in the SCN is not limited to the regulation of circadian phase. Finally, both 8-OH-DPAT and TFMPP inhibit RHT neurotransmission in the hypothalamic slice preparation. Further, TFMPP fails to attenuate responses to exogenous glutamate on retinorecipient SCN neurons, consistent with a presynaptic site of action for the drug. Based on these data, we propose that 5-HT modulates RHT neurotransmission in the SCN through at least two distinct mechanisms: (1) via activation of 5-HT₇ receptors probably located on retinorecipient neurons; and (2) via activation of presynaptic 5-HT_1B receptors leading to reduced release of glutamate from RHT terminals in the SCN.     

NMDA and PACAP Receptor Signaling Interact to Mediate Retinal-Induced SCN Cellular Rhythmicity in the Absence of Light

The “core” region of the suprachiasmatic nucleus (SCN), a central clock responsible for coordinating circadian rhythms, shows a daily rhythm in phosphorylation of extracellular regulated kinase (pERK). This cellular rhythm persists under constant darkness and, despite the absence of light, is dependent upon inputs from the eye. The neural signals driving this rhythmicity remain unknown and here the roles of glutamate and PACAP are examined. First, rhythmic phosphorylation of the NR1 NMDA receptor subunit (pNR1, a marker for receptor activation) was shown to coincide with SCN core pERK, with a peak at circadian time (CT) 16. Enucleation and intraocular TTX administration attenuated the peak in the pERK and pNR1 rhythms, demonstrating that activation of the NMDA receptor and ERK in the SCN core at CT16 are dependent on retinal inputs. In contrast, ERK and NR1 phosphorylation in the SCN shell region were unaffected by these treatments. Intraventricular administration of the NMDA receptor antagonist MK-801 also attenuated the peak in SCN core pERK, indicating that ERK phosphorylation in this region requires NMDA receptor activation. As PACAP is implicated in photic entrainment and is known to modulate glutamate signaling, the effects of a PAC₁ receptor antagonist (PACAP _6-38) on SCN core pERK and pNR1 also were examined. PACAP _6-38 administration attenuated SCN core pERK and pNR1, suggesting that PACAP induces pERK directly, and indirectly via a modulation of NMDA receptor signaling. Together, these data indicate that, in the absence of light, retinal-mediated NMDA and PAC₁ receptor activation interact to induce cellular rhythms in the SCN core. These results highlight a novel function for glutamate and PACAP release in the hamster SCN apart from their well-known roles in the induction of photic circadian clock resetting.     

Age and Visual Experience-dependent Expression of NMDAR1 Splice Variants in Rat Retina

The N-methyl-D-aspartate receptor (NMDAR) is a key molecule mediating brain plasticity related processes. Knowing that alternative splicing of the NMDAR1 (NR1) subunit offers molecular diversity to NMDAR, controls the forward trafficking of the NR1 protein and is important for placing NMDA receptors at synapses, we investigated herein the postnatal developmental expression and the influence of visual deprivation on NR1 subunit splice variants in rat retina. Real-time PCR was performed using oligonucleotide primers specific for N- terminal (NR1a, NR1b) and C-terminal splice variants (NR1-1, NR1-2, NR1-3, NR1-4). The developmental profiles of mRNA expression levels of all NR1 isoforms peaked at the end of the third week. Dark rearing led to reductions in both N- and C-terminal NR1 variants in several developmental ages and a significant interaction between age and visual experience was observed for NR1a, NR1-2 and NR1-4 expression. Our results have demonstrated a developmental and visual experience-dependent regulation of NR1 splicing in rat retina.     

Acute light exposure suppresses circadian rhythms in clock gene expression

Light can induce arrhythmia in circadian systems by several weeks of constant light or by a brief light stimulus given at the transition point of the phase response curve. In the present study, a novel light treatment consisting of phase advance and phase delay photic stimuli given on 2 successive nights was used to induce circadian arrhythmia in the Siberian hamster ( Phodopus sungorus). We therefore investigated whether loss of rhythms in behavior was due to arrhythmia within the suprachiasmatic nucleus (SCN). SCN tissue samples were obtained at 6 time points across 24 h in constant darkness from entrained and arrhythmic hamsters, and per1, per2 , bmal1, and cry1 mRNA were measured by quantitative RT-PCR. The light treatment eliminated circadian expression of clock genes within the SCN, and the overall expression of these genes was reduced by 18% to 40% of entrained values. Arrhythmia in per1, per2, and bmal1 was due to reductions in the amplitudes of their oscillations. We suggest that these data are compatible with an amplitude suppression model in which light induces singularity in the molecular circadian pacemaker.     

Kinetic expression of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) during embryonic stem cell differentiation

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is widely used as a marker during vasculogenesis and angiogenesis from embryonic stem (ES) cells. However, the expression of PECAM-1 isoforms in ES cells has not been determined. The present study was designed to determine the role of PECAM-1 isoforms during in vitro endothelial differentiation of ES cells. It was found that undifferentiated ES cells expressed high level of PECAM-1, which primarily located at cell-cell junction, but the expression of PECAM-1 was sharply down-regulated during early ES cell differentiation. In addition, undifferentiated ES cells were found the expressed all eight known alternatively spliced PECAM-1 isoforms, among them the expression of PECAM-1 isoforms lacking exon 15 or 14&15 was predominant. Quantitative analysis revealed a significant increase in the expression of PECAM-1 isoform lacking exon 12&14&15 as vascular development of ES cells. These results indicate a constitutive expression of PECAM-1 in undifferentiated murine ES cells and suggest a developmental role of PECAM-1 isoform changes during vasculogenesis and angiogenesis.     

Developmental regulation of NMDA receptor 2B subunit mRNA and ifenprodil binding in the zebra finch anterior forebrain

In passerine songbirds, song learning often is restricted to an early sensitive period and requires the participation of several discrete regions within the anterior forebrain. Activation of N-methyl-D-aspartate (NMDA) receptors is implicated in song learning and in one forebrain song region, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), NMDA receptors decrease in density, their affinity for the antagonist MK-801 increases, and their currents decay more quickly as young male zebra finches lose the ability to imitate new song elements. These developmental changes in NMDA receptor pharmacology and physiology suggest that the subunit composition of NMDA receptors changes developmentally. Here, we have used in situ hybridization and [3H]ifenprodil receptor autoradiography to study the developmental regulation of the NMDA receptor 2B subunit (NR2B) within the anterior forebrain of male zebra finches. NR2B mRNA expression within the IMAN was twice as great in 30-day-old males (early in the sensitive period for song learning) as in adult males, and this developmental decrease in NR2B mRNA expression was mirrored by a decrease in high-affinity (NR2B-associated) [3H]ifenprodil binding within this song region. In another anterior forebrain song region, Area X, NR2B mRNA also declined significantly after 30 days posthatch, but this decline was not accompanied by a significant decrease in [3H]ifenprodil binding. The results are consistent with the hypothesis that developmental changes in NMDA receptor function mediated by regulation of subunit composition contribute to the sensitive period for vocal learning in birds.     

Rhythmicity of the cGMP-related signal transduction pathway in the mammalian circadian system

Entrainment of mammalian circadian rhythms requires the activation of specific signal transduction pathways in the suprachiasmatic nuclei (SCN). Pharmacological inhibition of kinases such as cGMP-dependent kinase (PKG) or Ca2+/calmodulin-dependent kinase, but not cAMP-dependent kinase, blocks the circadian responses to light in vivo. Here we show a diurnal and circadian rhythm of cGMP levels and PKG activity in the hamster SCN, with maximal values during the day or subjective day. This rhythm depends on phosphodiesterase but not on guanylyl cyclase activity. Five-minute light pulses increased cGMP levels at the end of the subjective night [circadian time 18 (CT18)], but not at CT13.5. Western blot analysis indicated that the PKG II isoform is the one present in the SCN. Inhibition of PKG or guanylyl cyclase in vivo significantly attenuated light-induced phase shifts at CT18 (after 5-min light pulses) but did not affect c-Fos expression in the SCN. These results suggest that cGMP and PKG are related to SCN responses to light and undergo diurnal and circadian changes.