J Comp Physiol A (2005) 191: 23–30 DOI 10.1007/s00359-004-0569-5

O R I GI N A L P A P E R

Xiaodong Li Æ Jenifer Gilbert Æ Fred C. Davis

Disruption of masking by hypothalamic lesions in Syrian hamsters

Received: 29 May 2004 / Revised: 28 July 2004 / Accepted: 12 August 2004 / Published online: 24 September 2004
Springer-Verlag 2004

Abstract Negative masking of locomotor activity by light in nocturnal rodents is mediated by a non-image-forming irradiance-detection system in the retina. Structures receiving input from this system potentially contribute to the masking response. The suprachiasmatic nucleus (SCN) regulates locomotor activity and receives dense innervation from the irradiance-detection system via the retinohypothalamic tract, but its role in masking is unclear. We studied masking in adult Syrian hamsters (Mesocricetus auratus) with electrolytic lesions directed at the SCN. Hamsters were exposed to a 3.5:3.5 ultradian light/dark cycle and their wheel-running activity was monitored. Intact hamsters showed robust masking, expressing less than 20% of their activity in the light even though light and dark occurred equally during their active times. In contrast, hamsters with lesions showed, on average, as much activity in the light as in the dark. Tracing of retinal projections using cholera toxin b subunit showed that the lesions damaged retinal projections to the SCN and to the adjacent subparaventricular zone. Retinal innervation outside the hypothalamus was not obviously affected by the lesions. Our results indicate that retinohypothalamic projections, and the targets of these projections, to the SCN and/or adjacent hypothalamic areas play an important role in masking. Keywords Circadian Æ Retinohypothalamicx Æ Suprachiasmatic Æ Melanopsin Æ Subparaventricular Abbreviations CTB: Cholera toxin b subunit Æ CTB-ir: CTB immunoreactivity Æ DD: Constant darkness Æ DLG: Dorsal lateral geniculate Æ IGL: Intergeniculate leaflet Æ LD: Light/dark Æ RHT: Retinohypothalamic tract Æ SCN: Suprachiasmatic

X. Li Æ J. Gilbert Æ F. C. Davis (&) Department of Biology, Northeastern University, Boston, MA 02115, USA E-mail: [email protected] Tel.: +1-617-3734039 Fax: +1-617-3733724

nucleus Æ SPZ: Subparaventricular zone Æ VLG: Ventral lateral geniculate

Introduction The mammalian circadian system is traditionally divided into three parts: input pathways for external temporal information (light), the central pacemaker, and output effector systems for behavior and physiology (Reppert and Weaver 2002). The anatomical correlates of these elements are the retina, the suprachiasmatic nucleus (SCN) and the neural targets under SCN control. In constant darkness (DD), the interaction between the pacemaker and downstream processes shapes the temporal profiles of overt rhythms. These profiles are also subject to influences by external cues. For example, light, the principal Zeitgeber, can affect locomotor activity through both entrainment of the central pacemaker and positive and negative masking [enhancement and inhibition of wheel-running activity by light, respectively (Mrosovsky 1999)]. There is evidence that the negative (inhibitory) masking effect of light on a nocturnal animal’s wheel-running activity is mediated by the retinal nonimage-forming irradiance-detection system that entrains circadian rhythms (Mrosovsky et al. 1999, 2001). Mouse genetic studies of the recently identified melanopsin gene demonstrate a key role for the protein in non-visual functions such as entrainment, negative masking and pupillary constriction (Hattar et al. 2003; Lucas et al. 2003; Panda et al. 2003). Retinal ganglion cells expressing melanopsin are directly light sensitive and give direct projection to targets within the brain (Berson et al. 2002; Hattar et al. 2002). These ganglion cells may also receive indirect photic input from rods and cones (Belenky et al. 2003). Evidence to date indicates that both the classic photoreceptors (rods and cones) and the non-visual melanopsin-expressing ganglion cells contribute to the entrainment of circadian rhythms as

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well as to other non-visual functions, with their relative contributions influenced by the ambient light intensity (Lucas et al. 2003; Mrosovsky and Hattar 2003). The anatomical sites mediating some non-visual functions have been identified. For example, photoentrainment is mediated by the SCN, and pupillary constriction is mediated by the olivary pretectal nucleus (Clarke and Ikeda 1985). In contrast, little is known about the anatomical sites that contribute to the masking response. The SCN regulates wheel-running activity and receives dense innervation from retinal melanopsin cells, suggesting its possible role in masking (Ralph et al. 1990; Hattar et al. 2002; Gooley et al. 2003; Morin et al. 2003). In support of this are reports of abnormal responses to light in SCN-lesioned animals (Rusak 1977; Schwartz and Zimmerman 1991). Due to the close proximity of the SCN to the optic chiasm and tracts, it is a concern that a lesion of the SCN may also affect retinal projections to other areas that could mediate masking. In addition, a direct study of the role of the SCN in masking concluded that electrolytic SCN lesions do not affect negative masking in hamsters (Redlin and Mrosovsky 1999b). Thus the role of the SCN in negative masking is presently unclear. To clarify the role of the SCN and to ultimately link the masking response with a neural substrate, we studied masking in hamsters with lesions directed at their SCN. To complement the study by Redlin and Mrosovsky (1999b), we examined masking in hamsters kept on a 3.5:3.5 ultradian light/dark (LD) cycle. This protocol solved problems using light pulses or 24-h LD cycles in studies of SCN-lesioned hamsters. The former is impractical because the timing of activity expressed by SCN-lesioned hamsters is unpredictable, and on 24-h cycles, the timing of activity in intact, and possibly in SCN-lesioned hamsters as well, is largely determined by entrainment rather than by masking. On an ultradian LD cycle with a period of 7 h, intervals of light scan all circadian phases of intact hamsters, and in SCN-lesioned hamsters the intervals of light and dark occur equally at many different times when activity may or may not occur. In addition to the analysis of masking, we examined retinal projections in the intact and SCN-lesioned hamsters in order to identify the areas likely to have been deprived of retinal input as a result of the lesions. In the long term, the identification of the neural substrates that mediate masking may contribute to the broader problem of understanding non-visual effects of light on human physiology and behavior (e.g., Cajochen et al. 2000).

Materials and methods Animals Adult male and female hamsters (Mesocricetus auratus, LVG, Charles River Laboratories, MA, USA) were kept in individual running-wheel cages with food and water continuously available. The cages were kept in

light-tight chambers (five or six cages per chamber) with forced ventilation. Light (150 lux at the cage-top level) was provided by a single 40-in. fluorescent tube partially wrapped in black tape about 50 cm above the cage top. LD cycles were controlled by a ChronTrol clock (model XT, ChronTrol). Surgeries Hamsters were anesthetized with sodium pentobarbital (100 mg/kg body weight) and placed in a stereotaxic instrument (Kopf model 900). The skull was exposed around bregma and a small burr hole was drilled just anterior to bregma. An electrode was lowered midline through the hole. Coordinates for lesions were 0.6 mm anterior to bregma and 8.4 mm below the skull surface with the tooth bar set at 2. After the desired coordinates were reached, a 4-mA constant current was passed for 10 s. The electrode was then slowly withdrawn, the burr hole filled with gelatin foam, and the skin closed with wound clips. Experiments with males or females were conducted independently at different times. The assessment of masking on the 3.5:3.5 LD cycle began 15 weeks after the lesions for males and 11 weeks after the lesions for females. Males were kept on the ultradian cycle for 9 days and the females for 25. During the 9 days on the ultradian cycle, any particular circadian time would be exposed to 4–5 light and dark intervals. Before the ultradian cycle, both groups were kept on 24-h LD cycles most of the time with some days in DD or constant dim red light (<10 lux). The time between the ultradian cycle and sacrifice for histological analysis was 2 weeks for males and 10 weeks for females. Before sacrifice, hamsters received intra-vitreous injections of cholera toxin b subunit (CTB). Hamsters were anesthetized with sodium pentobarbital (100 mg/kg body mass) and 2 ll of the reconstituted CTB (List Biological Laboratories) solution (1%, pH 7.4) was slowly injected into the vitreous humor compartment of each eye using a 10-ll Hamilton syringe. After injection, the needle was left in the eye for 10 s before being slowly withdrawn. The hamsters were sacrificed for immunohistochemical analysis after 48–72 h. Wheel-running activity recording and analysis Wheel turns were registered by a microswitch on each cage and recorded in 1-min bins using the ClockLab system (Actimetrics). Data files were analyzed using the ClockLab data analysis software (Actimetrics) to generate actograms and average waveforms. The average activity during the light or dark intervals was divided by the combined average. The resulting percentages were transformed by the arc-sine transformation (Sokal and Rolf 1995) and statistically analyzed using the Student’s t-test.

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Histology Hamsters were anesthetized with sodium pentobarbital (100 mg/kg body weight) and perfused transcardially with 50–100 ml of saline followed by 100 ml of 4% paraformaldehyde buffered in PBS (0.01 M, pH 7.4). Brains were taken from the skull and fixed in the same fixative for an additional 1–2 h before being immersed in 20% (w/v) sucrose in PBS overnight at 4C. Brains were then frozen on dry ice and four series of 40-lm sections were cut on a freezing microtome. Sections were collected into PBS and used for Nissl staining and immunostaining for CTB. The free-floating immunostaining procedure was adapted from that used in Lu et al. (1999). Briefly, sections were rinsed in PBS (3·5 min) followed by 30 min in 0.25% Triton-X 100 (in 0.01 M PBS) solution (PBST). Primary antibody against CTB (goat anti-CTB, List Biological Laboratories) was used at 1:100,000 dilution in PBST. Sections were incubated overnight at room temperature and rinsed in PBST for 3·5 min before being incubated in the secondary antibody solution (donkey antigoat, Jackson Immuno Research, used at 1:1,000) for 1 h before being incubated in the avidin/biotin complex solution (ABC kit from Vector) and subsequently stained using DAB as the chromogen. All reagents from the ABC kit were used at half of the concentration recommended by the manufacturer. Sections were then mounted onto gelatin-coated slides, air-dried and coverslipped using Permount (Fisher).

Results SCN-lesioned hamsters showed similar amounts of wheel-running activity in the light as in the dark during the 3.5:3.5 ultradian LD cycle Figures 1 and 2 show examples of wheel-running records during exposure to the 3.5:3.5 LD cycle. The Fig. 1a–f Examples of wheelrunning activity records plotted with a 24-h folding period. Records from one intact male (a) and one intact female (d) and two SCN-lesioned males (b and c) and two SCN-lesioned females (e and f) are shown. For each animal, the wheel-running record over 7 days during the 3.5:3.5-h LD cycle is shown. The LD cycle (gray shading represents light periods) was directly recorded on the females’ records (d, e, and f) via photosensors near the cages. The cycle is not shown on the male records because a direct record of the light was not available

activity records are folded at both 24 h (Fig. 1) and at 7 h (Fig. 2) to facilitate viewing of the activity distribution relative to the ultradian LD cycle. As expected, bouts of wheel-running activity in SCN-lesioned hamsters were more scattered relative to intact hamsters when viewed at the 24-h folding period (Fig. 1). When the records are folded at 7 h, it can be seen that intact males and females showed most of their activity in the dark even though their circadian activity time was equally likely to occur during dark or light intervals (Fig. 2a, d). In contrast, SCN-lesioned hamsters regularly expressed wheel-running activity in the light (Fig. 2 b, c, e and f). This difference between intact and SCN-lesioned hamsters is also apparent when activity counts are averaged to produce activity profiles folded at 7 h (Fig. 3). The percentage of activity in the light is summarized for all animals in Fig. 4. Overall, the lesioned animals, both males and females, showed equal amounts of wheel-running activity in the light as in the dark, while intact hamsters showed a preference for the dark. SCN lesions disrupted retinal projections to the subparaventricular zone The degree of lesion damage was estimated using both Nissl-stained sections and CTB tracing. The pattern of CTB-immunoreactive (CTB-ir) projections within the intact hypothalamus was similar to that previously described for hamsters (Johnson et al. 1988b). The RHT projection includes dense innervation of the SCN and much less innervation of the retrochiasmatic area, the subparaventricular zone (SPZ), and adjacent anterior hypothalamic (AH) areas. Fibers to areas immediately adjacent to the SCN appear to course through the SCN (Fig. 5 a, d). In all but one of the lesioned hamsters, CTB-ir fibers in the subparaventricular zone (SPZ) were greatly diminished (for males, see Fig. 6a, b, d and e; for

26 Fig. 2a–f Examples of wheelrunning activity records plotted with a 7-h folding period. Records for the same animals (a–f) and the same days as in Fig. 1 are displayed using a 7-h folding period. The left half of each record is the dark interval (D dark, L light)

Fig. 3a–f Examples of the average waveform of wheelrunning activity at the 7-h folding period. Waveforms for the same animals (a–f) as in Figs. 1 and 2 are displayed for a 7-h folding period. The waveform is constructed from the average number of counts per minute across the LD cycle (D dark, L light). The average waveforms are scaled to be the same size even though absolute levels of activity were different. The peak values for each panel are: a 12, b 18, c 3.5, d 12, e 2.5, f5

Fig. 4 Summary data for percentage of wheel-running activity expressed in the light in intact and SCN-lesioned hamsters. Intact males (n=9) showed 17.8±3.4% (mean±SE) of their activity in the light during the 3.5:3.5-h ultradian cycle, while SCN-lesioned males (n=8) showed 51.1±5.5% (P<0.005). Intact females (n=10) showed 18.2±4.9% of their activity in the light while SCN-lesioned females (n=10) showed 47.0±6.9% (P<0.005)

females, see Fig. 7a, b, d and e). In two of the lesioned hamsters, small bundles of CTB-ir fibers remained in the SCN region (Figs. 6d and 7a) indicating that some of the SCN and its retinal innervation were spared. Even in those cases, projections to the SPZ were greatly diminished (e.g., Figs. 6e and 7b). Compared to intact hamsters, these hamsters also showed disrupted negative masking (e.g., Fig. 2c, e). Examples of retinal projections to the dorsal lateral geniculate (DLG) region in SCN-lesioned hamsters are shown in Figs. 6 and 7. The similarity between intact and lesioned hamsters is consistent with results of a previous study in which the RHT projection to the SCN was cut while leaving projections to the thalamus intact (Johnson et al. 1988a). In the present study, retinal projections to targets other than the hypothalamus, including the intergeniculate leaflet (IGL), ventral lateral geniculate (VLG), pretectum, and tectum were observed in all SCN-lesioned hamsters. Furthermore, hamsters that were unlikely to have any damage to the optic chiasm or optic tracts (e.g., Figs. 6a, d and 7a, d), still

27 Fig. 5a–f Examples of retinal projections as revealed by CTB tracing in intact male (a, b, and c) and female (d, e, and f) hamsters. SCN (a and d), SPZ (b and e) and DLG (c and f) levels are shown. All panels are at the same magnification. The scale bar in panel f represents 500 lm

Fig. 6a–f Examples of retinal projections as revealed by CTB tracing in male SCN-lesioned hamsters. SCN (a and d), SPZ (b and e) and DLG (c and f) levels are shown. Tissue for the sections shown in (a), (b) and (c) was from the hamster whose wheel-running record is shown in Fig. 2b. Tissue for the sections shown in (d), (e) and (f) was from the hamster whose wheel-running record is shown in Fig. 2c. All panels are at the same magnification. The scale bar in panel f represents 500 lm

Fig. 7a–f Examples of retinal projections as revealed by CTB tracing in female SCN-lesioned hamsters. SCN (a and d), SPZ (b and e) and DLG (c and f) levels are shown. Tissue for the sections shown in (a), (b) and (c) was from the hamster whose wheel-running record is shown in Fig. 2e. Tissue for the sections shown in (d), (e) and (f) was from the hamster whose wheel-running record is shown in Fig. 2f. All panels are at the same magnification. The scale bar in panel f represents 500 lm

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showed a disruption of masking. In two lesioned hamsters, however, the pretectal area appeared to have less CTB-ir than in intact hamsters but this was not correlated in any obvious way with their behavior.

Discussion Under a 3.5:3.5-h LD cycle (200 lux), SCN-lesioned male and female hamsters showed impaired negative masking compared to intact hamsters. This impairment was associated with ablation of the SCN as well as with a loss of retinal innervation to the subparaventricular zone (SPZ), a region previously suggested to contribute to the masking response in intact hamsters (Kramer et al. 2001). The impairment in masking was not associated with disruption of retinal innervation to other targets outside of the hypothalamus. A 3.5:3.5 LD cycle was previously used to study the masking response in mice and hamsters (Redlin and Mrosovsky 1999a, 1999b; Hattar et al. 2003; Mrosovsky and Hattar 2003). This paradigm is especially useful for the study of SCN-lesioned animals whose activity pattern is unpredictable. It should be noted that although we observed significant impairment of masking in general among the SCN-lesioned animals, the distribution of their wheel-running activity across the ultradian LD cycle showed variation. Some animals showed activity scattered across the LD phases (e.g., Fig. 2c), while others showed a relatively stable phase relationship with the LD transition (e.g., Fig. 2b). Others seemed to prefer the light (e.g., Fig. 2e). Thus, with regard to the timing of wheel-running activity, we cannot conclude that SCN-lesioned hamsters are entirely blind to the light. The present results contrast with a previous study that used the same ultradian LD cycle. In that study, SCNlesioned hamsters showed masking as strong as control hamsters and masking similar to that shown by intact hamsters in the present study (Redlin and Mrosovsky 1999b). Although there is no obvious explanation for the different results of these studies, one possible explanation is the way lesions were created. Redlin and Mrosovsky created two lesions in each animal (one for each of the bilateral SCN) while the present study used a single midline lesion. Perhaps RHT projections to hypothalamic areas other than the SCN were affected differently by the two methods (for example, differential sparing of projections to the SPZ). The status of retinal projections in the Redlin and Mrosovsky study is not known since retinal tracing was not done. Other differences between the studies that could be relevant include the source of the hamsters used, the overall level of activity expressed by hamsters, and a difference in the time between creating the lesions and testing for masking. The retinal tracing results of the present study showed that electrolytic SCN-lesions diminished retinal projections to the SPZ. The SPZ is thought to be the primary relay for the SCN output regulation of locomotor activity (Watts and Swanson 1987; Lu et al. 2001; Moore

and Danchenko 2002). Since retinal projections to both the SCN and the SPZ were disrupted, it is not possible to ascribe an exclusive role in masking to either. It was previously shown that the SPZ is innervated by SCN cells that are directly activated by light (de la Iglesia and Schwartz 2002), suggesting that SPZ cells are capable of indirectly responding to light information from the retina through the relay of the SCN. On the other hand, retinal projections to the SPZ might directly influence the functional state of cells in this area, thus affecting behavior downstream of the SCN. It is possible that both direct (retinal input) and indirect (via the SCN) routes convey light information to the SPZ to influence behavior. It should also be noted that the RHT has a broader distribution than the SCN and SPZ (as defined by SCN projections) and extends into the adjacent AH area. Further lesion studies in the SPZ and AH area will help to delineate the roles of those structures in masking. Retinal target areas outside of the hypothalamus could contribute to the acute effects of light on locomotor activity. Previous studies suggested that activation of the classical visual system promotes wheel-running activity, an effect termed ‘‘positive masking.’’ For example, visual cortex or DLG lesions, as well as deficits in outer retina photoreceptor cells, impair a positive masking response in mice (Mrosovsky et al. 1999; Edelstein and Mrosovsky 2001; Redlin et al. 2003). Positive masking is particularly evident under lower light intensity. At higher intensity, the negative masking effect is more prominent (Mrosovsky et al. 1999). In all lesioned animals of the present study, we observed retinal projections to the lateral geniculate, including the DLG and IGL. It is possible that projections to the DLG promoted wheel-running activity when our SCN-lesioned hamsters were exposed to light, while negative masking mediated by the RHT was diminished. This change in relative strength between positive and negative masking may have helped to shape the distribution of wheel-running activity across the 3.5:3.5 cycle and cause some animals to show an apparent preference for the light. Preference for the light might be even more evident if lower intensities are used. Retinal projections to the pretectum area have been implicated in direct behavior-state control (Miller et al. 1998, 1999). These studies measured triggering of REM sleep in response to a light-to-dark transition, but it is unclear how this effect relates to wheel-running activity. Pretectal lesions have been created in hamsters and were found to have no effect on the free-running-period lengthening effect of constant light (Morin and Pace 2002), an effect of light that is affected by IGL lesions (Pickard et al. 1987). Masking was not examined in hamsters with pretectal lesions. The IGL does not appear to be necessary for negative masking since lesions of the IGL result in enhanced negative masking in hamsters (Redlin et al. 1999). This study is complicated, however, by possible damage to the adjacent DLG, which would be expected to cause the observed effects (Edelstein and Mrosovsky 2001).

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In summary, we observed impairment of negative masking in SCN-lesioned hamsters relative to that in intact hamsters. We also observed disruption of retinal projections, not only to the SCN, but also to the SPZ, a structure previously implicated in SCN output control. Signals that mediate the circadian regulation of wheelrunning activity and signals that mediate the masking effects of light on wheel-running activity must converge at some point within the neural circuits that control this behavior. It is reasonable to expect that these influences on wheel-running activity are closely integrated in shaping the daily profile of activity during exposure to LD cycles. The present results suggest that circadian control and masking converge on the regulation of wheel-running activity within the SCN or within an immediate downstream target of the SCN such as the SPZ. Future studies should use fiber-sparing excitotoxic lesions and/or microinjection of retinal ganglion cell neurotransmitters/neuromodulators in discrete retinal projection targets to better delineate the anatomical site(s) that mediate masking. Acknowledgements Special thanks to Dr. Jun Lu for advice on the use of CTB and to Christina Giuliano for technical help. Supported by NIH grants HD18686 and MH068796 to FCD. The experiments reported here comply with the ‘‘Principles of Animal Care’’, publication no. 86–23, revised 1985 of the National Institute of Health, and with Northeastern University’s Institutional Animal Care and Use Committee.

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