Chidambaram Ramanathan

Combined Application of BDNF to the Eye and Brain Enhances Ganglion Cell Survival and Function in the Cat after Optic Nerve Injury

PURPOSE To determine whether application of BDNF to the eye and brain provides a greater level of neuroprotection after optic nerve injury than treatment of the eye alone. METHODS Retinal ganglion cell survival and pattern electroretinographic responses were compared in normal cat eyes and in eyes that received (1) a mild nerve crush and no treatment, (2) a single intravitreal injection of BDNF at the time of the nerve injury, or (3) intravitreal treatment combined with 1 to 2 weeks of continuous delivery of BDNF to the visual cortex, bilaterally. RESULTS Relative to no treatment, administration of BDNF to the eye alone resulted in a significant increase in ganglion cell survival at both 1 and 2 weeks after nerve crush (1 week, 79% vs. 55%; 2 weeks, 60% vs. 31%). Combined treatment of the eye and visual cortex resulted in a modest additional increase (17%) in ganglion cell survival in the 1-week eyes, a further significant increase (55%) in the 2-week eyes, and ganglion cell survival levels for both that were comparable to normal (92%-93% survival). Pattern ERG responses for all the treated eyes were comparable to normal at 1 week after injury; however, at 2 weeks, only the responses of eyes receiving the combined BDNF treatment remained so. CONCLUSIONS Although treatment of the eye alone with BDNF has a significant impact on ganglion cell survival after optic nerve injury, combined treatment of the eye and brain may represent an even more effective approach and should be considered in the development of future optic neuropathy-related neuroprotection strategies.

Temporal and spatial distribution of immunoreactive PER1 and PER2 proteins in the suprachiasmatic nucleus and peri-suprachiasmatic region of the diurnal grass rat (Arvicanthis niloticus)

The suprachiasmatic nucleus (SCN) of the hypothalamus contains the primary circadian pacemaker in both diurnal and nocturnal mammals. The lower subparaventricular zone (LSPV) immediately dorsal to the SCN may also play an important role in the regulation of circadian rhythms. The SCN contains a multitude of oscillator cells that generate circadian rhythms through transcriptional/translational feedback loops involving a set of clock genes including per1 and per2. Little is known about the temporal and spatial features of the proteins encoded by these genes in day-active mammals. The first objective of this study was to characterize the expression of PER1 and PER2 in the SCN of a diurnal rodent, the unstriped Nile grass rat (Arvicanthis niloticus). The second objective was to evaluate the hypothesis that a molecular clock could exist in the LSPV, where endogenous rhythms in Fos expression are seen in grass rats but not in laboratory rats. Animals were kept on a 12:12 light/dark cycle and perfused at 4-h intervals, and their brains were processed for immunohistochemical detection of PER1 and PER2. Both proteins were seen in the SCN where they peaked early in the dark phase, providing further evidence that the differences between diurnal and nocturnal patterns of behavior emerge from mechanisms lying downstream from the pacemaker within the SCN. Rhythmic expression of PER1 and PER2 was also seen in the LSPV providing support for the hypothesis that this region might participate in circadian time keeping in the diurnal grass rat. In addition, rhythms were seen lateral to the LSPV and the SCN. Results of this study are discussed in light of similarities and differences in the circadian time-keeping systems of day- and night-active animals.

Phase preference for the display of activity is associated with the phase of extra-suprachiasmatic nucleus oscillators within and between species.

Many features of the suprachiasmatic nucleus (SCN) are the same in diurnal and nocturnal animals, suggesting that differences in phase preference are determined by mechanisms downstream from the SCN. Here, we examined this hypothesis by characterizing rhythmic expression of Period 1 (PER1) and Period 2 (PER2) in several extra-SCN areas in the brains of a diurnal murid rodent, Arvicanthis niloticus (grass rats). In the shell of the nucleus accumbens, dorsal striatum, piriform cortex, and CA1 of the hippocampus, both PER1 and PER2 were rhythmic, with peak expression occurring at ZT10. PER1 in the dentate gyrus also peaked at ZT10, but PER2 was arrhythmic in this region. In general, these patterns are 180 degrees out of phase with those reported for nocturnal species. In a second study, we examined inter-individual differences in the multioscillator system of grass rats. Here, we housed grass rats in cages with running wheels, under which conditions some individuals spontaneously adopt a day active (DA) and others a night active (NA) phase preference. In the majority of the extra-SCN regions sampled, the patterns of PER1 and PER2 expression of NA grass rats resembled those of nocturnal species, while those of DA grass rats were similar to the ones seen in grass without access to running wheels. In contrast, the rhythmic expression of both PER proteins was identical in the SCN and ventral subparaventricular zone (vSPZ) of DA and NA animals. Differences in the phase of oscillators downstream from the SCN, and perhaps the vSPZ, appear to determine the phase preference of particular species, as well as that of members of a diurnal species that show voluntary phase reversals. The latter observation has important implications for the understanding of health problems associated with human shift work.

Responses of brain and behavior to changing day-length in the diurnal grass rat (Arvicanthis niloticus)

Seasonal affective disorder (SAD) is a major depressive disorder that recurs in the fall and winter when day-length gets short. It is well accepted that day-length is encoded by the principal circadian clock located in the suprachiasmatic nucleus (SCN), but very little is known about day-length encoding in diurnal mammals. The present study utilized the grass rat, Arvicanthis niloticus, to investigate how the circadian system responds to photoperiodic changes in a diurnal mammal that shows day-length-dependent mood changes. The animals were initially housed in equatorial day-length (12h, EP) followed by either long (16h, LP) or short (8h, SP) photoperiods. The LP animals showed an expansion of the peak phase of the PER1 and PER2 rhythm in the SCN as well as an extended behavioral active phase. In contrast, the SP animals did not show any compression of their active phase nor a change in the peak duration of PER1 or PER2 expression, compared to those in EP. The results suggest that the circadian system in the diurnal grass rats is less responsive when day-length gets short compared to when it gets longer. The depression-like behaviors were assessed using sweet solution preference (SSP) and forced swimming test (FST). Animals in the SP group showed decreased SSP and increased immobility time in FST as compared to the EP group, suggesting a depressive phenotype. The present study serves as the first step toward exploring the role that the circadian system plays in SAD using a diurnal rodent model.

Daily rhythms and sex differences in vasoactive intestinal polypeptide, VIPR2 receptor and arginine vasopressin mRNA in the suprachiasmatic nucleus of a diurnal rodent, Arvicanthis niloticus

Diurnal and nocturnal animals differ with respect to the time of day at which the ovulatory surge in luteinizing hormone occurs. In some species this is regulated by the suprachiasmatic nucleus (SCN), the primary circadian clock, via cells that contain vasoactive intestinal polypeptide (VIP) and vasopressin (AVP). Here, we evaluated the hypothesis that chronotype differences in the timing of the luteinizing hormone surge are associated with rhythms in expression of the genes that encode these neuropeptides. Diurnal grass rats (Arvicanthis niloticus) were housed in a 12/12‐h light–dark cycle and killed at one of six times of day (Zeitgeber time 1, 5, 9, 13, 17, 21; ZT 0 = lights‐on). In‐situ hybridization was used to compare levels of vip, avp and VIP receptor mRNA (vipr2) in the SCN of intact females, ovariectomized females, ovariectomized females given estradiol and intact males. We found a sex difference in vip rhythms with a peak occurring at ZT 13 in males and ZT 5 in intact females. In all groups avp mRNA rhythms peaked during the day, from ZT 5 to ZT 9, and had a trough in the dark at ZT 21. There was a modest rhythm and sex difference in the pattern of vipr2. Most importantly, the patterns of each of these SCN rhythms relative to the light–dark cycle resembled those seen in nocturnal rodents. Chronotype differences in timing of neuroendocrine events associated with ovulation are thus likely to be generated downstream of the SCN.

The Substructure of the Suprachiasmatic Nucleus: Similarities between Nocturnal and Diurnal Spiny Mice

Evolutionary transitions between nocturnal and diurnal patterns of adaptation to the day-night cycle must have involved fundamental changes in the neural mechanisms that coordinate the daily patterning of activity, but little is known about how these mechanisms differ. One reason is that information on these systems in very closely related diurnal and nocturnal species is lacking. In this study, we characterize the suprachiasmatic nucleus (SCN), the primary brain structure involved in the generation and coordination of circadian rhythms, in two members of the genus Acomys with very different activity patterns, Acomys russatus (the golden spiny mouse, diurnal) and Acomys cahirinus (the common spiny mouse, nocturnal). Immunohistochemical techniques were used to label cell bodies containing vasoactive intestinal polypeptide (VIP), vasopressin (VP), gastrin-releasing peptide (GRP) and calbindin (CalB) in the SCN, as well as two sets of inputs to it, those containing serotonin (5-HT) and neuropeptide Y (NPY), respectively. All were present in the SCN of both species and no differences between them were seen. On the basis of neuronal phenotype, the SCN was organized into three basic regions that contained VIP-immunoreactive (-ir), CalB-ir and VP-ir cells, in the ventral, middle and dorsal SCN, respectively. In the rostral SCN, GRP-ir cells were in both the VIP and the CalB cell regions, and in the caudal area they were distributed across a portion of each of the other three regions. Fibers containing NPY-ir and serotonin (5-HT)-ir were most concentrated in the areas containing VIP-ir and CalB-ir cells, respectively. The details of the spatial relationships among the labeled cells and fibers seen here are discussed in relation to different approaches investigators have taken to characterize the SCN more generally.

Daily rhythms in PER1 within and beyond the suprachiasmatic nucleus of female grass rats (Arvicanthis niloticus).

Although circadian rhythms of males and females are different in a variety of ways in many species, their mechanisms have been primarily studied in males. Furthermore, rhythms are dramatically different in diurnal and nocturnal animals but have been studied predominantly in nocturnal ones. In the present study, we examined rhythms in one element of the circadian oscillator, the PER1 protein, in a variety of cell populations in brains of diurnal female grass rats. Every 4 h five adult female grass rats kept on a 12-h light/dark (LD) cycle were perfused and their brains were processed for immunohistochemical detection of PER1. Numbers of PER1-labeled cells were rhythmic not only within the suprachiasmatic nucleus (SCN), the locus of the primary circadian clock in mammals, but also in the peri-suprachiasmatic region, the oval nucleus of the bed nucleus of the stria terminalis, the central amygdala, and the nucleus accumbens. In addition, rhythms were detected within populations of neuroendocrine cells that contain tyrosine hydroxylase. The phase of the rhythm within the SCN was advanced compared with that seen previously in male grass rats. Rhythms beyond the SCN were varied and different from those seen in most nocturnal species, suggesting that signals originating in the SCN are modified by its direct and/or indirect targets in different ways in nocturnal and diurnal species.

Compartmentalized expression of light-induced clock genes in the suprachiasmatic nucleus of the diurnal grass rat (Arvicanthis niloticus)

Photic responses of the circadian system are mediated through light-induced clock gene expression in the suprachiasmatic nucleus (SCN). In nocturnal rodents, depending on the timing of light exposure, Per1 and Per2 gene expression shows distinct compartmentalized patterns that correspond to the behavioral responses. Whether the gene- and region-specific induction patterns are unique to nocturnal animals, or are also present in diurnal species is unknown. We explored this question by examining the light-induced Per1 and Per2 gene expression in functionally distinct SCN subregions, using diurnal grass rats Arvicanthis niloticus. Light exposure during nighttime induced Per1 and Per2 expression in the SCN, showing unique spatiotemporal profiles depending on the phase of the light exposure. After a phase delaying light pulse (LP) in the early night, strong Per1 induction was observed in the retinorecipient core region of the SCN, while strong Per2 induction was observed throughout the entire SCN. After a phase advancing LP in the late night, Per1 was first induced in the core and then extended into the whole SCN, accompanied by a weak Per2 induction. This compartmentalized expression pattern is very similar to that observed in nocturnal rodents, suggesting that the same molecular and intercellular pathways underlying acute photic responses are present in both diurnal and nocturnal species. However, after an LP in early subjective day, which induces phase advances in diurnal grass rats, but not in nocturnal rodents, we did not observe any Per1 or Per2 induction in the SCN. This result suggests that in spite of remarkable similarities in the SCN of diurnal and nocturnal rodents, unique mechanisms are involved in mediating the phase shifts of diurnal animals during the subjective day.

Tyrosine hydroxylase positive neurons and their contacts with vasoactive intestinal polypeptide-containing fibers in the hypothalamus of the diurnal murid rodent, Arvicanthis niloticus

Diurnal and nocturnal animals differ with respect to the timing of a host of behavioral and physiological events including those associated with neuroendocrine functions, but the neural bases of these differences are poorly understood. In nocturnal species, rhythms in tyrosine hydroxylase-containing (TH+) neurons in the hypothalamus appear to be responsible for rhythms in prolactin secretion. Here we investigated TH+ cells in a diurnal rodent (Arvicanthis niloticus, the unstriped Nile grass rat), and comparing them with those of a nocturnal rodent (Rattus norvegicus, Sprague-Dawley rat). We also examined relationships between TH+ cells and fibers containing vasoactive intestinal polypeptide (VIP) that are thought to originate from cells in the suprachiasmatic nucleus (SCN), the site of the primary circadian clock in mammals. The distribution of TH+ neurons was very similar in the two species except for a population of cells in the basal forebrain that was only present in grass rats. Fibers containing VIP appeared to contact neuroendocrine TH+ cells in both species. These data suggest that, though there may be subtle species differences, temporal information is likely to be carried along the same direct pathways from the SCN to the TH+ neurons in day- and night-active species.

PER2 rhythms in the amygdala and bed nucleus of the stria terminalis of the diurnal grass rat (Arvicanthis niloticus)

The suprachiasmatic nucleus (SCN) of the hypothalamus is the central pacemaker that controls circadian rhythms in mammals. In diurnal grass rats (Arvicanthis niloticus), many functional aspects of the SCN are similar to those of nocturnal rodents, making it likely that the difference in the circadian system of diurnal and nocturnal animals lies downstream from the SCN. Rhythms in clock genes expression occur in several brain regions outside the SCN that may function as extra-SCN oscillators. In male grass rats PER1 is expressed in the oval nucleus of the bed nucleus of the stria terminalis (BNST-ov) and in the central and basolateral amygdala (CEA and BLA, respectively); several features of PER1 expression in these regions of the grass rat brain differ substantially from those of nocturnal species. Here we describe PER2 rhythms in the same three brain regions of the grass rat. In the BNST-ov and CEA PER2 expression peaked early in the light period Zeitgeber time (ZT) 2 and was low during the early night, which is the reverse of the pattern of nocturnal rodents. In the BLA, PER2 expression was relatively low for most of the 24-h cycle, but showed an acute elevation late in the light period (ZT10). This pattern is also different from that of nocturnal rodents that show elevated PER2 expression in the mid to late night and into the early day. These results are consistent with the hypothesis that diurnal behavior is associated with a phase change between the SCN and extra-SCN oscillators.