- Email: [email protected]
Polysialylated neural cell adhesion molecule modulates photic signaling in the mouse suprachiasmatic nucleus
Neuroscience Letters 280 (2000) 207±210 www.elsevier.com/locate/neulet
Polysialylated neural cell adhesion molecule modulates photic signaling in the mouse suprachiasmatic nucleus J. David Glass a,*, Huaming Shen a, Lenka Fedorkova a, Lei Chen a, Henry Tomasiewicz b, Michiko Watanabe c a
Department of Biological Sciences, Kent State University, Kent, OH 44242, USA Department of Anatomy and Cell Biology, Emory University, Atlanta, GA 30322, USA c Department of Pediatrics, Division of Pediatric Cardiology, Rainbow Babies and Children's Hospital, Case Western Reserve University, School of Medicine, Cleveland, OH 44106, USA b
Received 9 December 1999; accepted 28 December 1999
Abstract Polysialic acid (PSA), a sialic acid polymer that regulates plasticity and cell-cell interactions in neural tissues, is expressed in the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). In vivo enzymatic removal of PSA from the mouse SCN signi®cantly impaired both the photic induction of Fos protein in SCN cells and lightinduced phase-resetting of the circadian locomotor activity rhythm. Genetic deletion of PSA and it's neural cell adhesion molecule (NCAM) carrier correspondingly attenuated light-induced circadian phase-shifting. Comparison of PSA levels between young and old mice revealed a large aging-related reduction in SCN PSA content that accompanies the diminished capacity for circadian photic response reported in old rodents. Collectively these data support the contention that PSA modulates photic signaling in the SCN, and that normal reductions in the cell surface molecule contribute to aging-related de®cits in SCN circadian clock function. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Suprachiasmatic nucleus; Aging; Polysialic acid; Neural cell adhesion molecule
The hypothalamic suprachiasmatic nuclei (SCN) are the principal site for the generation and entrainment of mammalian circadian rhythms [7]. Such rhythms are synchronized to the daily light±dark cycle via a direct monosynaptic projection from the retina to the SCN, the retinohypothalamic tract (RHT) [8], and an indirect pathway from the intergeniculate lea¯et, the geniculohypothalamic tract [2]. Photic entrainment occurs through daily adjustments of SCN circadian clock phase induced by photic information primarily supplied by the RHT. Photic stimulation elicits a cascade of events in the SCN initiated by the release of glutamate from RHT terminals. This in turn stimulates the production of immediate±early gene products, including Fos protein, in different populations of SCN cells, and ultimately, changes in the phase of pacemaker activity. There is evidence that RHT mediated photic signaling in the SCN is a gated response, involving inhibitory serotonergic input [4], and possibly the production of neurotrophic factors [6]. * Corresponding author. Tel.: 11-330-672-2934; fax: 11-330672-3713. E-mail address: [email protected] (J.D. Glass)
The SCN express a highly polysialylated form of neural cell adhesion molecule (PSA-NCAM) [15] that promotes plasticity and regulates synaptic ef®cacy in select regions of the adult brain [9,17]. This unique form of NCAM, characterized by long a2,8-linked sialic acid polymers (PSA), promotes changes in tissue architecture. Removal of PSA by a speci®c endoneuraminidase (endo N) or by genetic deletion dramatically disrupts functions characteristic of adult brain regions that express the PSA-NCAM. For example, endo N treatment of hippocampal slices abolishes long-term potentiation (LTP) and long-term depression (LTD) [9]. Also, application of endo N to the supraoptic nuclei inhibit hypothalamo-neurohypophysial response to physiological stimuli [17], and intra-SCN injection of endo N alters the free-running period of circadian locomotor activity under constant darkness (DD; 16). Mice whose PSA-NCAM has been genetically deleted exhibit defects in learning [3], LTP [9] and striking abnormalities in circadian rhythm period and duration [16]. Further implicating PSA-NCAM in the regulation of adult neural activity are reports that dramatic state-dependent changes in the expression of this molecule are associated with learning [14] and physiological stimula-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 78 6- 2
208
J.D. Glass et al. / Neuroscience Letters 280 (2000) 207±210
tion [11]. There is also evidence that PSA-NCAM modulates neuronal response to brain-derived neurotrophic factor (BDNF) [19]. In view of the compelling evidence for involvement of PSA-NCAM in cell-cell communication and synaptic physiology, together with its robust expression in the SCN, the aim of the present study was to assess a possible modulatory role of PSA-NCAM in photic signaling the SCN circadian clock. Although the phenomenon of photic circadian phase-resetting implies involvement of plastic neural reorganization, this idea has not yet been tested. Alterations in the photic resetting response of the SCN by manipulations of PSA expression like those undertaken in the present study would support this implication. Male C57BL/6J mice obtained from the Jackson Laboratory (Bar Harbor, ME), mutant Ncam tm1Cwr mice generated using homologous recombination in embryonic stem cells after deletion of the 180-speci®c exon 18 and matched wildtype litter mates (Tomasiewicz et al. [18]) aged 8±10 weeks were used in this study. General circadian locomotor activity of the mice was measured using an infrared motion sensor placed above the cage. Output from the sensors was integrated with an IBM-compatible computer running Datacol data acquisition software (Minimitter). Analyses of rhythm characteristics and graphical output (actograms) were undertaken using Tau software (Minimitter). The free-running period of the locomotor activity rhythm under constant darkness (DD) was obtained from the x 2 periodogram averaged over a period of at least 7 days. Under DD the onset of activity, designated as circadian time (CT) 12, was used as the phase reference point for the onset of the subjective night. Activity onset was de®ned as the ®rst 20 min bout of activity that exceeded 30% of maximal activity rate for the day, was preceded by a period of at least 4 h of inactivity and was followed by a period of at least 30 min of sustained activity. Phase-shifts were calculated as the difference between projected times of activity onset on the day after photic stimulation as determined by back-extrapolation of the least-squares regression line through activity onsets on days 2±7 after treatment and the least-squares line through activity onsets during 7 days prior to treatment. The photic stimulation consisted of a 30 min pulse of incandescent light (40 lux). The relationship between PSA expression and photic signaling in the SCN was assessed in three separate experiments. In experiment 1, light induced circadian phase-shifting and SCN Fos protein expression were examined C57BL/ 6J mice whose PSA was removed for up to 18 days by intraSCN injection of endoneuraminidase (endo N) that speci®cally cleaves sialic acid polymers in chains of seven to nine or greater [5]. Mice under DD for 2 weeks received a 1.5 ml injection of heat-inactivated endo N (control) or active endo N, and were then exposed to a 30 min light pulse from CT 14.5±15.0 9 days later. Mice were killed 1 h after the pulse to examine SCN fos expression or were left undisturbed for 9 days after the light pulse to evaluate behavioral phaseshifting. Selected sections containing the SCN from both
groups of mice were stained for PSA. In experiment 2, light induced circadian phase-shifting was examined in Ncam tm1Cwr mice whose PSA was eliminated by genetic deletion of the NCAM-180 carrier of PSA and in wildtype litter mates with normal PSA expression. After 10 days under DD a light pulse was administered as described above, and phase-shifting response was assessed over the subsequent 9-day period. In experiment 3, measurements of SCN PSA content were undertaken to determine if an agingrelated reduction in SCN PSA accompanies declining circadian photic response (20). The PSA-NCAM content in the SCN of young (2-month-old) and old (25-month-old) wildtype mice killed at ZT 3.5 (zeitgeber time; ZT 12 designated as time of lights-off) were assessed by immunoblot analysis. For immunohistological analyses, mice were deeply anesthetized with Nembutal, the brains removed, blocked and ®xed in buffered paraformaldehyde overnight at 48C. Coronal vibratome sections (50 mm thick) were processed for the demonstration of Fos protein using a rabbit polyclonal antibody (c-fos-ab-2; Oncogene Science, Unionville, NY) or PSA using ascites ¯uid containing a mouse monoclonal antibody, 5A5 (IgM [1]), against a-2,8-linked PSA of NCAM. Treatment effects on Fos protein expression in the SCN were quanti®ed by counting immunostained nuclei in consecutive sections of the mid region of the SCN that contained the largest number of immunostained nuclei [4]. Aging effects on PSA expression in the SCN were assessed by immunoblot analysis. Intra-SCN microinjection of endo N puri®ed from bacteriophage K1F was carried out under Nembutal anesthesia using a unilateral stereotaxicallyguided 28 gauge cannula aimed at the SCN with the following coordinates: anteroposterior 12.2 mm from bregma; lateral 1.3 mm from midline at 58 from vertical, and horizontal 4.5 mm from dura, with head level. Data from the photic behavioral phase-shifting and Fos protein induction experiments, as well as from the immunoblot analyses were analyzed using a one-way repeated-measures analysis of variance (ANOVA) followed by the Student Newman±Keuls post-hoc mean comparison test. The level of statistical signi®cance was set at P , 0:05. Results from the present study con®rm previous immunohistochemical analyses of PSA depletion by intra-SCN injection of endo N, where PSA expression in the SCN region was suppressed for up to 18 days after endo N treatment [16] (Fig. 1). Controls treated with heat-inactivated endo N (n 6) exhibited a robust expression of Fos protein throughout the rostrocaudal axis of the SCN induced by a 30 min light pulse from CT 14.5±15.0. The majority of cells that expressed Fos immunoreactivity were located in the mid-region of this axis (Fig. 2). In animals that received intra-SCN injection of active endo N (n 6), the number of light-induced Fos immunoreactive cells was signi®cantly reduced compared to that of the control animals (77.6 ^ 4.2% of control; P , 0:05; Figs. 2 and 3). The inhibitory effect of this treatment was most evident in the ventrolateral region of the SCN. Correspondingly, the extent of light-
J.D. Glass et al. / Neuroscience Letters 280 (2000) 207±210
Fig. 1. Bright ®eld photographs of immunostaining for PSA in coronal hemisections of the SCN with 3,3-diaminobenzidine used as chromogen. The left panel reveals strong immunostaining in a control animal that received an intra-SCN injection of inactivated endo N. The right panel reveals an absence of reaction product in the SCN of an animal that received active endo N 18 days prior to staining. OC, optic chiasma; SCN, suprachiasmatic nucleus; 3V, third ventricle.
induced behavioral phase-shifting in control animals was signi®cantly larger than that in animals that received active endo N (2.3 ^ 0.2 vs. 1.6 ^ 0.1 h phase-delays, respectively; P , 0:05; Figs. 2 and 3; both groups, n 6). Also, the extent of light-induced phase-shifting in wild-type Ncam tm1Cwr mice was signi®cantly greater compared to the mice homozygous for the Ncam tm1Cwr mutation that lack PSA (1.08 ^ 0.06 vs. 0.68 ^ 0.06 h phase-delays, respec-
Fig. 2. Upper panels: bright ®eld photographs of immunostaining for light-induced Fos in the midregion of the SCN of (A) a control animal treated with inactive endo N, and (B) an animal treated with active endo N 9 days prior to staining. The abbreviations are same as in Fig. 1. Lower panels: representative pro®les of the effects of endo N on light-induced phase-resetting of the free-running circadian activity rhythm under DD. (A) A control animal treated with inactive endo N, and (B) an animal treated with active endo N 9 days prior to the light pulse. The actograms are double-plotted such that each horizontal trace represents 48 h, and the record of each day is presented to the right and beneath that of the preceding day. Arrows represent the time of intra-SCN injection. The light ¯ash is represented by an asterisk.
209
Fig. 3. Graphical presentation of the effects of intra-SCN injection of endo N on light-induced Fos protein in the SCN and the effects of this injection and genetic deletion of PSA-NCAM on light-induced circadian phase-resetting. *Treatment effect signi®cantly different from respective control, P , 0:05.
tively; P , 0:05; Fig. 3; both groups, n 11). Immunoblot analysis of the PSA in SCN extracts from young (8 weeks of age; n 3) and old (2 years of age; n 3) mice revealed a signi®cant aging-related loss of PSA, with the old mice having only 59 ^ 14% of the PSA content of the young mice (P , 0:05). The mechanism whereby PSA-NCAM could modulate photic signaling in the SCN is speculative, however, a number of possibilities exist based on work from various neural systems. The ®rst possibility is that PSA-NCAM expression in or near synaptic complexes could regulate synaptic ef®cacy. Immunoreactive PSA has been demonstrated at the synapse by electron microcopy in the hippocampus (9) and SCN (15). The PSA, by way of its large relative size or charge repulsion, could physically affect neurotransmitter release and/or diffusion across the synaptic cleft. Observations that removal of PSA using endo N blocks LTP and LTD, and that the expression of PSANCAM at the synapse is modulated by neuronal activity [9] suggest that this expression may be critical for synaptic plasticity and information processing in the hippocampus. It is reasonable that PSA-NCAM could also have a related role in processing photic information in the SCN, as LTD- and LTP-related forms of cellular memory have been reported in the SCN [10,12]. As these processes could be functionally relevant to phase-resetting of the circadian clock, their suppression by removal of PSA by endo N in the present study could have resulted in the attenuated circadian-related response to light. A related consideration is the possible interaction between PSA-NCAM and brain-derived neurotrophic factor (BDNF). Removal of PSA from cortical neurons with endo N has been shown to block BDNF-induced responses, including Fos protein induction [19]. Signi®cantly, BDNF, which is rhythmically expressed by SCN cells, is thought to enhance synaptic transmission associated with photic signaling in the SCN, and could help regulate the phase of sensitivity to the phase-resetting action of light [6]. Both
210
J.D. Glass et al. / Neuroscience Letters 280 (2000) 207±210
PSA-NCAM and BDNF are expressed in the retinorecipient area of the SCN, suggesting that they could have a common target in the photic signaling cascade. Changes in the degree of polysialylation of the 180 kDa NCAM carrier of PSA in the SCN could also dramatically in¯uence intracellular signal transduction in the SCN. The NCAM 180 isoform interacts with the cytoskeleton-protein linker, spectrin (which promotes the docking of synaptic vesicles to the presynaptic plasma membrane) and thus changes in PSANCAM expression/turnover could regulate neurotransmitter release through interaction with spectrin [13]. In the context of photic signaling in the SCN, this PSA-NCAM cytoskeletal interaction could serve to modulate glutamate release from RHT terminals, in turn in¯uencing Fos induction and circadian clock resetting. Removal of the PSA from the NCAM 180 by endo N treatment could impair this interaction, resulting in a suboptimal capacity for photically-stimulated glutamate release. It will be important in subsequent experiments to determine if presynaptic RHT terminals express PSA. The present results represent the ®rst demonstration of a signi®cant aging-related reduction in PSA content in the SCN. Experiments in hamsters have revealed that aging is associated with severe de®cits in circadian activities, including photic signaling in the SCN [20]. Although many factors are involved in aging-related changes of the circadian system, the causes for these changes remain unknown. In view of the role of PSA-NCAM in the regulation of synaptic ef®cacy and plasticity, and the deleterious effects of PSA removal on SCN function, we speculate that age-related reductions in PSA could contribute to the decline in the circadian timing structure of old animals. From the present results, this could be particularly relevant to the reduced sensitivity of the aged circadian clock system to the phase-resetting effects of light. Supported by National Institutes of Health grant MH57034 to J.D.G. The authors are indebted to Dr. Urs Rutishauser for providing endo N. [1] Acheson, A., Sunshine, J. and Rutishauser, U., NCAM polysialic acid can regulate both cell±cell and cell±substrate interactions. J. Cell Biol., 114 (1991) 143±153. [2] Card, J.P. and Moore, R.Y., Organization of lateral geniculate±hypothalamic connections in the rat. J. Comp. Neurol., 284 (1989) 135±147. [3] Cremer, H., Lange, K., Christoph, A., Plomann, M., Vopper, G., Roes, J., Brown, R., Baldwin, S., Kraemer, P. and Scheff, S., Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and de®cits in spacial learning. Nature, 367 (1994) 455±457. [4] Glass, J.D., Selim, M. and Rea, M.A., Modulation of lightinduced C-fos expression in the suprachiasmatic nuclei by 5-HT1A receptor agonists. Brain Res., 638 (1994) 235±242. [5] Hallenbeck, P.C., Vimr, E.R., Yu, F., Bassler, B. and Troy, F.A., Puri®cation and properties of a bacteriophage-
[6] [7] [8] [9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
induced endo-N-acetylneuraminidase speci®c for polyalpha-2-8-sialosyl carbohydrate units. J. Biol. Chem., 262 (1987) 3553±3561. Liang, F.-Q., Walline, R. and Earnest, D.J., Circadian rhythm of brain-derived neurotrophic factor in the rat suprachiasmatic nucleus. Neurosci. Lett., 242 (1998) 89±92. Moore, R.Y., Organization and function of a central nervous system oscillator: The suprachiasmatic nucleus. Fed. Proc., 42 (1983) 2783±2789. Moore, R.Y. and Lenn, N.J., A retinohypothalamic projection in the rat. J. Comp. Neurol., 146 (1971) 1±14. MuÈller, D., Wang, C., Skibo, G., Toni, N., Cremer, H., Calaora, V., Rougon, G. and Kiss, J.Z., PSA-NCAM is required for activity-induced synaptic plasticity. Neuron, 17 (1996) 413±422. Nishikawa, Y., Shibata, S. and Watanabe, S., Circadian changes in long-term potentiation of rat suprachiasmatic ®eld potentials elicited by optic nerve stimulation in vitro. Brain Res., 695 (1995) 158±162. Nothias, F., Vernier, P., von Boxberg, Y., Mirman, S. and Vincent, J.-D., Modulation of NCAM polysialylation is associated with morpho-functional modi®cations in the hypothalamoneurohypophysial system during lactation. Eur. J. Neurosci., 9 (1997) 1553±1565. Obrietan, K. and van den Pol, A.N., Neuropeptide Y depresses GABA-mediated calcium transients in developing suprachiasmatic nucleus neurons: a novel form of calcium long-term depression. J. Neurosci., 16 (1996) 3521±3533. Pollerberg, G., Burridge, K., Krebs, K., Goodman, S. and Schachner, M., The 180-kDa component of the neural cell adhesion molecule NCAM is involved in cell±cell contacts and cytoskeleton-membrane interactions. Cell Tiss. Res., 250 (1987) 227±236. Regan, C.M. and Fox, G.B., Polysialylation as a regulator of neural plasticity in rodent learning and aging. Neurochem. Res., 20 (1995) 593±598. Shen, H., Glass, J.D., Seki, T. and Watanabe, M., Ultrastructural analysis of polysialylated neural cell adhesion molecule in the suprachiasmatic nuclei of the adult mouse. Anat. Rec., 256 (1999) 448±457. Shen, H., Watanabe, M., Tomasiewicz, H., Rutishauser, U., Magnuson, T. and Glass, J.D., Role of neural cell adhesion molecule and polysialic acid in mouse circadian clock function. J. Neurosci., 17 (1997) 5221±5229. Theodosis, D.T., El Majdoubi. M., Pierre, K. and Poulain, D.A., Factors governing activity-dependent structural plasticity of the hypothalamoneurohypophysial system. Cell. Mol. Neurobiol., 18 (1998) 285±298. Tomasiewicz, H., Ono, K., Yee, D., Thompson, C., Gordis, C., Rutishauser, U. and Magnuson, T., Genetic deletion of a neural cell adhesion molecule variant (N-CAM 180) produces distinct de®cits in the central nervous system. Neuron, 11 (1993) 1163±1174. Vutskits, L., Paccaud, J.-P., Muller, D., Djebbara-Hannas, Z., Durbec, P., Rougon, G. and Kiss, J.Z., PSA-NCAM modulates the responsiveness of cortical neurons to BDNF, Proc. Polysialic acid: Biochemistry, Cell biology and Diseases, ile des Embiez, France, 1998, pp. 53. Zhang, Y., Kornhauser, J.M., Zee, P.C., Mayo, K.E., Takahashi, J.S. and Turek, F.W., Effects of aging on light-induced phase-shifting of circadian behavioral rhythms, fos expression and CREB phosphorylation in the hamster suprachiasmatic nucleus. Neuroscience, 70 (1996) 951±961.
Polysialylated neural cell adhesion molecule modulates photic signaling in the mouse suprachiasmatic nucleus J. David Glass a,*, Huaming Shen a, Lenka Fedorkova a, Lei Chen a, Henry Tomasiewicz b, Michiko Watanabe c a
Department of Biological Sciences, Kent State University, Kent, OH 44242, USA Department of Anatomy and Cell Biology, Emory University, Atlanta, GA 30322, USA c Department of Pediatrics, Division of Pediatric Cardiology, Rainbow Babies and Children's Hospital, Case Western Reserve University, School of Medicine, Cleveland, OH 44106, USA b
Received 9 December 1999; accepted 28 December 1999
Abstract Polysialic acid (PSA), a sialic acid polymer that regulates plasticity and cell-cell interactions in neural tissues, is expressed in the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). In vivo enzymatic removal of PSA from the mouse SCN signi®cantly impaired both the photic induction of Fos protein in SCN cells and lightinduced phase-resetting of the circadian locomotor activity rhythm. Genetic deletion of PSA and it's neural cell adhesion molecule (NCAM) carrier correspondingly attenuated light-induced circadian phase-shifting. Comparison of PSA levels between young and old mice revealed a large aging-related reduction in SCN PSA content that accompanies the diminished capacity for circadian photic response reported in old rodents. Collectively these data support the contention that PSA modulates photic signaling in the SCN, and that normal reductions in the cell surface molecule contribute to aging-related de®cits in SCN circadian clock function. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Suprachiasmatic nucleus; Aging; Polysialic acid; Neural cell adhesion molecule
The hypothalamic suprachiasmatic nuclei (SCN) are the principal site for the generation and entrainment of mammalian circadian rhythms [7]. Such rhythms are synchronized to the daily light±dark cycle via a direct monosynaptic projection from the retina to the SCN, the retinohypothalamic tract (RHT) [8], and an indirect pathway from the intergeniculate lea¯et, the geniculohypothalamic tract [2]. Photic entrainment occurs through daily adjustments of SCN circadian clock phase induced by photic information primarily supplied by the RHT. Photic stimulation elicits a cascade of events in the SCN initiated by the release of glutamate from RHT terminals. This in turn stimulates the production of immediate±early gene products, including Fos protein, in different populations of SCN cells, and ultimately, changes in the phase of pacemaker activity. There is evidence that RHT mediated photic signaling in the SCN is a gated response, involving inhibitory serotonergic input [4], and possibly the production of neurotrophic factors [6]. * Corresponding author. Tel.: 11-330-672-2934; fax: 11-330672-3713. E-mail address: [email protected] (J.D. Glass)
The SCN express a highly polysialylated form of neural cell adhesion molecule (PSA-NCAM) [15] that promotes plasticity and regulates synaptic ef®cacy in select regions of the adult brain [9,17]. This unique form of NCAM, characterized by long a2,8-linked sialic acid polymers (PSA), promotes changes in tissue architecture. Removal of PSA by a speci®c endoneuraminidase (endo N) or by genetic deletion dramatically disrupts functions characteristic of adult brain regions that express the PSA-NCAM. For example, endo N treatment of hippocampal slices abolishes long-term potentiation (LTP) and long-term depression (LTD) [9]. Also, application of endo N to the supraoptic nuclei inhibit hypothalamo-neurohypophysial response to physiological stimuli [17], and intra-SCN injection of endo N alters the free-running period of circadian locomotor activity under constant darkness (DD; 16). Mice whose PSA-NCAM has been genetically deleted exhibit defects in learning [3], LTP [9] and striking abnormalities in circadian rhythm period and duration [16]. Further implicating PSA-NCAM in the regulation of adult neural activity are reports that dramatic state-dependent changes in the expression of this molecule are associated with learning [14] and physiological stimula-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 78 6- 2
208
J.D. Glass et al. / Neuroscience Letters 280 (2000) 207±210
tion [11]. There is also evidence that PSA-NCAM modulates neuronal response to brain-derived neurotrophic factor (BDNF) [19]. In view of the compelling evidence for involvement of PSA-NCAM in cell-cell communication and synaptic physiology, together with its robust expression in the SCN, the aim of the present study was to assess a possible modulatory role of PSA-NCAM in photic signaling the SCN circadian clock. Although the phenomenon of photic circadian phase-resetting implies involvement of plastic neural reorganization, this idea has not yet been tested. Alterations in the photic resetting response of the SCN by manipulations of PSA expression like those undertaken in the present study would support this implication. Male C57BL/6J mice obtained from the Jackson Laboratory (Bar Harbor, ME), mutant Ncam tm1Cwr mice generated using homologous recombination in embryonic stem cells after deletion of the 180-speci®c exon 18 and matched wildtype litter mates (Tomasiewicz et al. [18]) aged 8±10 weeks were used in this study. General circadian locomotor activity of the mice was measured using an infrared motion sensor placed above the cage. Output from the sensors was integrated with an IBM-compatible computer running Datacol data acquisition software (Minimitter). Analyses of rhythm characteristics and graphical output (actograms) were undertaken using Tau software (Minimitter). The free-running period of the locomotor activity rhythm under constant darkness (DD) was obtained from the x 2 periodogram averaged over a period of at least 7 days. Under DD the onset of activity, designated as circadian time (CT) 12, was used as the phase reference point for the onset of the subjective night. Activity onset was de®ned as the ®rst 20 min bout of activity that exceeded 30% of maximal activity rate for the day, was preceded by a period of at least 4 h of inactivity and was followed by a period of at least 30 min of sustained activity. Phase-shifts were calculated as the difference between projected times of activity onset on the day after photic stimulation as determined by back-extrapolation of the least-squares regression line through activity onsets on days 2±7 after treatment and the least-squares line through activity onsets during 7 days prior to treatment. The photic stimulation consisted of a 30 min pulse of incandescent light (40 lux). The relationship between PSA expression and photic signaling in the SCN was assessed in three separate experiments. In experiment 1, light induced circadian phase-shifting and SCN Fos protein expression were examined C57BL/ 6J mice whose PSA was removed for up to 18 days by intraSCN injection of endoneuraminidase (endo N) that speci®cally cleaves sialic acid polymers in chains of seven to nine or greater [5]. Mice under DD for 2 weeks received a 1.5 ml injection of heat-inactivated endo N (control) or active endo N, and were then exposed to a 30 min light pulse from CT 14.5±15.0 9 days later. Mice were killed 1 h after the pulse to examine SCN fos expression or were left undisturbed for 9 days after the light pulse to evaluate behavioral phaseshifting. Selected sections containing the SCN from both
groups of mice were stained for PSA. In experiment 2, light induced circadian phase-shifting was examined in Ncam tm1Cwr mice whose PSA was eliminated by genetic deletion of the NCAM-180 carrier of PSA and in wildtype litter mates with normal PSA expression. After 10 days under DD a light pulse was administered as described above, and phase-shifting response was assessed over the subsequent 9-day period. In experiment 3, measurements of SCN PSA content were undertaken to determine if an agingrelated reduction in SCN PSA accompanies declining circadian photic response (20). The PSA-NCAM content in the SCN of young (2-month-old) and old (25-month-old) wildtype mice killed at ZT 3.5 (zeitgeber time; ZT 12 designated as time of lights-off) were assessed by immunoblot analysis. For immunohistological analyses, mice were deeply anesthetized with Nembutal, the brains removed, blocked and ®xed in buffered paraformaldehyde overnight at 48C. Coronal vibratome sections (50 mm thick) were processed for the demonstration of Fos protein using a rabbit polyclonal antibody (c-fos-ab-2; Oncogene Science, Unionville, NY) or PSA using ascites ¯uid containing a mouse monoclonal antibody, 5A5 (IgM [1]), against a-2,8-linked PSA of NCAM. Treatment effects on Fos protein expression in the SCN were quanti®ed by counting immunostained nuclei in consecutive sections of the mid region of the SCN that contained the largest number of immunostained nuclei [4]. Aging effects on PSA expression in the SCN were assessed by immunoblot analysis. Intra-SCN microinjection of endo N puri®ed from bacteriophage K1F was carried out under Nembutal anesthesia using a unilateral stereotaxicallyguided 28 gauge cannula aimed at the SCN with the following coordinates: anteroposterior 12.2 mm from bregma; lateral 1.3 mm from midline at 58 from vertical, and horizontal 4.5 mm from dura, with head level. Data from the photic behavioral phase-shifting and Fos protein induction experiments, as well as from the immunoblot analyses were analyzed using a one-way repeated-measures analysis of variance (ANOVA) followed by the Student Newman±Keuls post-hoc mean comparison test. The level of statistical signi®cance was set at P , 0:05. Results from the present study con®rm previous immunohistochemical analyses of PSA depletion by intra-SCN injection of endo N, where PSA expression in the SCN region was suppressed for up to 18 days after endo N treatment [16] (Fig. 1). Controls treated with heat-inactivated endo N (n 6) exhibited a robust expression of Fos protein throughout the rostrocaudal axis of the SCN induced by a 30 min light pulse from CT 14.5±15.0. The majority of cells that expressed Fos immunoreactivity were located in the mid-region of this axis (Fig. 2). In animals that received intra-SCN injection of active endo N (n 6), the number of light-induced Fos immunoreactive cells was signi®cantly reduced compared to that of the control animals (77.6 ^ 4.2% of control; P , 0:05; Figs. 2 and 3). The inhibitory effect of this treatment was most evident in the ventrolateral region of the SCN. Correspondingly, the extent of light-
J.D. Glass et al. / Neuroscience Letters 280 (2000) 207±210
Fig. 1. Bright ®eld photographs of immunostaining for PSA in coronal hemisections of the SCN with 3,3-diaminobenzidine used as chromogen. The left panel reveals strong immunostaining in a control animal that received an intra-SCN injection of inactivated endo N. The right panel reveals an absence of reaction product in the SCN of an animal that received active endo N 18 days prior to staining. OC, optic chiasma; SCN, suprachiasmatic nucleus; 3V, third ventricle.
induced behavioral phase-shifting in control animals was signi®cantly larger than that in animals that received active endo N (2.3 ^ 0.2 vs. 1.6 ^ 0.1 h phase-delays, respectively; P , 0:05; Figs. 2 and 3; both groups, n 6). Also, the extent of light-induced phase-shifting in wild-type Ncam tm1Cwr mice was signi®cantly greater compared to the mice homozygous for the Ncam tm1Cwr mutation that lack PSA (1.08 ^ 0.06 vs. 0.68 ^ 0.06 h phase-delays, respec-
Fig. 2. Upper panels: bright ®eld photographs of immunostaining for light-induced Fos in the midregion of the SCN of (A) a control animal treated with inactive endo N, and (B) an animal treated with active endo N 9 days prior to staining. The abbreviations are same as in Fig. 1. Lower panels: representative pro®les of the effects of endo N on light-induced phase-resetting of the free-running circadian activity rhythm under DD. (A) A control animal treated with inactive endo N, and (B) an animal treated with active endo N 9 days prior to the light pulse. The actograms are double-plotted such that each horizontal trace represents 48 h, and the record of each day is presented to the right and beneath that of the preceding day. Arrows represent the time of intra-SCN injection. The light ¯ash is represented by an asterisk.
209
Fig. 3. Graphical presentation of the effects of intra-SCN injection of endo N on light-induced Fos protein in the SCN and the effects of this injection and genetic deletion of PSA-NCAM on light-induced circadian phase-resetting. *Treatment effect signi®cantly different from respective control, P , 0:05.
tively; P , 0:05; Fig. 3; both groups, n 11). Immunoblot analysis of the PSA in SCN extracts from young (8 weeks of age; n 3) and old (2 years of age; n 3) mice revealed a signi®cant aging-related loss of PSA, with the old mice having only 59 ^ 14% of the PSA content of the young mice (P , 0:05). The mechanism whereby PSA-NCAM could modulate photic signaling in the SCN is speculative, however, a number of possibilities exist based on work from various neural systems. The ®rst possibility is that PSA-NCAM expression in or near synaptic complexes could regulate synaptic ef®cacy. Immunoreactive PSA has been demonstrated at the synapse by electron microcopy in the hippocampus (9) and SCN (15). The PSA, by way of its large relative size or charge repulsion, could physically affect neurotransmitter release and/or diffusion across the synaptic cleft. Observations that removal of PSA using endo N blocks LTP and LTD, and that the expression of PSANCAM at the synapse is modulated by neuronal activity [9] suggest that this expression may be critical for synaptic plasticity and information processing in the hippocampus. It is reasonable that PSA-NCAM could also have a related role in processing photic information in the SCN, as LTD- and LTP-related forms of cellular memory have been reported in the SCN [10,12]. As these processes could be functionally relevant to phase-resetting of the circadian clock, their suppression by removal of PSA by endo N in the present study could have resulted in the attenuated circadian-related response to light. A related consideration is the possible interaction between PSA-NCAM and brain-derived neurotrophic factor (BDNF). Removal of PSA from cortical neurons with endo N has been shown to block BDNF-induced responses, including Fos protein induction [19]. Signi®cantly, BDNF, which is rhythmically expressed by SCN cells, is thought to enhance synaptic transmission associated with photic signaling in the SCN, and could help regulate the phase of sensitivity to the phase-resetting action of light [6]. Both
210
J.D. Glass et al. / Neuroscience Letters 280 (2000) 207±210
PSA-NCAM and BDNF are expressed in the retinorecipient area of the SCN, suggesting that they could have a common target in the photic signaling cascade. Changes in the degree of polysialylation of the 180 kDa NCAM carrier of PSA in the SCN could also dramatically in¯uence intracellular signal transduction in the SCN. The NCAM 180 isoform interacts with the cytoskeleton-protein linker, spectrin (which promotes the docking of synaptic vesicles to the presynaptic plasma membrane) and thus changes in PSANCAM expression/turnover could regulate neurotransmitter release through interaction with spectrin [13]. In the context of photic signaling in the SCN, this PSA-NCAM cytoskeletal interaction could serve to modulate glutamate release from RHT terminals, in turn in¯uencing Fos induction and circadian clock resetting. Removal of the PSA from the NCAM 180 by endo N treatment could impair this interaction, resulting in a suboptimal capacity for photically-stimulated glutamate release. It will be important in subsequent experiments to determine if presynaptic RHT terminals express PSA. The present results represent the ®rst demonstration of a signi®cant aging-related reduction in PSA content in the SCN. Experiments in hamsters have revealed that aging is associated with severe de®cits in circadian activities, including photic signaling in the SCN [20]. Although many factors are involved in aging-related changes of the circadian system, the causes for these changes remain unknown. In view of the role of PSA-NCAM in the regulation of synaptic ef®cacy and plasticity, and the deleterious effects of PSA removal on SCN function, we speculate that age-related reductions in PSA could contribute to the decline in the circadian timing structure of old animals. From the present results, this could be particularly relevant to the reduced sensitivity of the aged circadian clock system to the phase-resetting effects of light. Supported by National Institutes of Health grant MH57034 to J.D.G. The authors are indebted to Dr. Urs Rutishauser for providing endo N. [1] Acheson, A., Sunshine, J. and Rutishauser, U., NCAM polysialic acid can regulate both cell±cell and cell±substrate interactions. J. Cell Biol., 114 (1991) 143±153. [2] Card, J.P. and Moore, R.Y., Organization of lateral geniculate±hypothalamic connections in the rat. J. Comp. Neurol., 284 (1989) 135±147. [3] Cremer, H., Lange, K., Christoph, A., Plomann, M., Vopper, G., Roes, J., Brown, R., Baldwin, S., Kraemer, P. and Scheff, S., Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and de®cits in spacial learning. Nature, 367 (1994) 455±457. [4] Glass, J.D., Selim, M. and Rea, M.A., Modulation of lightinduced C-fos expression in the suprachiasmatic nuclei by 5-HT1A receptor agonists. Brain Res., 638 (1994) 235±242. [5] Hallenbeck, P.C., Vimr, E.R., Yu, F., Bassler, B. and Troy, F.A., Puri®cation and properties of a bacteriophage-
[6] [7] [8] [9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
induced endo-N-acetylneuraminidase speci®c for polyalpha-2-8-sialosyl carbohydrate units. J. Biol. Chem., 262 (1987) 3553±3561. Liang, F.-Q., Walline, R. and Earnest, D.J., Circadian rhythm of brain-derived neurotrophic factor in the rat suprachiasmatic nucleus. Neurosci. Lett., 242 (1998) 89±92. Moore, R.Y., Organization and function of a central nervous system oscillator: The suprachiasmatic nucleus. Fed. Proc., 42 (1983) 2783±2789. Moore, R.Y. and Lenn, N.J., A retinohypothalamic projection in the rat. J. Comp. Neurol., 146 (1971) 1±14. MuÈller, D., Wang, C., Skibo, G., Toni, N., Cremer, H., Calaora, V., Rougon, G. and Kiss, J.Z., PSA-NCAM is required for activity-induced synaptic plasticity. Neuron, 17 (1996) 413±422. Nishikawa, Y., Shibata, S. and Watanabe, S., Circadian changes in long-term potentiation of rat suprachiasmatic ®eld potentials elicited by optic nerve stimulation in vitro. Brain Res., 695 (1995) 158±162. Nothias, F., Vernier, P., von Boxberg, Y., Mirman, S. and Vincent, J.-D., Modulation of NCAM polysialylation is associated with morpho-functional modi®cations in the hypothalamoneurohypophysial system during lactation. Eur. J. Neurosci., 9 (1997) 1553±1565. Obrietan, K. and van den Pol, A.N., Neuropeptide Y depresses GABA-mediated calcium transients in developing suprachiasmatic nucleus neurons: a novel form of calcium long-term depression. J. Neurosci., 16 (1996) 3521±3533. Pollerberg, G., Burridge, K., Krebs, K., Goodman, S. and Schachner, M., The 180-kDa component of the neural cell adhesion molecule NCAM is involved in cell±cell contacts and cytoskeleton-membrane interactions. Cell Tiss. Res., 250 (1987) 227±236. Regan, C.M. and Fox, G.B., Polysialylation as a regulator of neural plasticity in rodent learning and aging. Neurochem. Res., 20 (1995) 593±598. Shen, H., Glass, J.D., Seki, T. and Watanabe, M., Ultrastructural analysis of polysialylated neural cell adhesion molecule in the suprachiasmatic nuclei of the adult mouse. Anat. Rec., 256 (1999) 448±457. Shen, H., Watanabe, M., Tomasiewicz, H., Rutishauser, U., Magnuson, T. and Glass, J.D., Role of neural cell adhesion molecule and polysialic acid in mouse circadian clock function. J. Neurosci., 17 (1997) 5221±5229. Theodosis, D.T., El Majdoubi. M., Pierre, K. and Poulain, D.A., Factors governing activity-dependent structural plasticity of the hypothalamoneurohypophysial system. Cell. Mol. Neurobiol., 18 (1998) 285±298. Tomasiewicz, H., Ono, K., Yee, D., Thompson, C., Gordis, C., Rutishauser, U. and Magnuson, T., Genetic deletion of a neural cell adhesion molecule variant (N-CAM 180) produces distinct de®cits in the central nervous system. Neuron, 11 (1993) 1163±1174. Vutskits, L., Paccaud, J.-P., Muller, D., Djebbara-Hannas, Z., Durbec, P., Rougon, G. and Kiss, J.Z., PSA-NCAM modulates the responsiveness of cortical neurons to BDNF, Proc. Polysialic acid: Biochemistry, Cell biology and Diseases, ile des Embiez, France, 1998, pp. 53. Zhang, Y., Kornhauser, J.M., Zee, P.C., Mayo, K.E., Takahashi, J.S. and Turek, F.W., Effects of aging on light-induced phase-shifting of circadian behavioral rhythms, fos expression and CREB phosphorylation in the hamster suprachiasmatic nucleus. Neuroscience, 70 (1996) 951±961.