Article Figures & Data
Figures
- Figure 1.
Schematic representation of experimental design. I, Ethical approval for animal experimentation was taken from IAEC, followed by procurement of adult male Wistar rats (180–220 g) from CAHF. Rats were randomly allocated to three experimental groups and maintained at standard housing conditions. All animals were handled for three to four times for one week before starting the experiment for the purpose of familiarization. II, Experimental conditions such as dim light, silence, cleanliness were maintained on all behavioral experiment days. All experiments were performed during the daylight cycle of animals between 9 A.M. to 5 P.M. NOR apparatus was cleaned with 70% ethanol before and after each animal trial to remove the odor cues. A, Habituation. Animals were habituated with empty NOR apparatus for 5 min on day 1. B, Training. Two similar shaped objects were placed diagonally in the NOR apparatus and animals were given weak training (single trial) for 5 min. C, BT(OF) group rats were further exposed to 5-min open field exploration after 15 min of NOR training. D, BT(EE) group rats were given 5-min enriched environment exposure after 15 min of NOR training. The enriched environment comprised of an extralarge fun tunnel for rats, a hut, nesting material such as sizzle pet and dumbbell. E, Test. One object was replaced with a novel object with different shape and color in the NOR apparatus. Animals were tested for LTM 24 h after training session. F, NOR behavioral data were analyzed for percentage of novel object exploration, object exploration time and number of entries in the novel zone. III, After test session, animal brain was excised and hippocampus was snap frozen for analyzing protein expression changes through western blotting.
- Figure 2.
Percentage of novel object exploration was calculated for all experimental groups to study the neurobehavioral effects of different types of novelties, namely, OF and EE, on LTM consolidation through BT phenomenon. A, Statistically significant differences in the percentage of novel object exploration were seen after OF and EE exposure when compared with control group (***p < 0.001 and *p < 0.05, one way ANOVA, n = 11, data represented as mean ± SEM) when tested 24 h after training session. B, Exploratory track plots of control, BT(OF), and BT(EE) group during 5-min NOR test session generated from ANY-maze software. C, Exploratory heat maps of control, BT(OF), and BT(EE) group during test session generated from ANY-maze software. The data are suggestive of EE modulated BT with OF as a positive control. Different type of novelty produces similar extent of LTM consolidation via BT phenomenon when compared with control group in which no novelty was provided.
- Figure 3.
Intragroup comparison of object exploration time for control, BT(OF), and BT(EE) groups. A–C, Object exploration time in training versus test session represented as mean ± SEM. No significant difference was observed (p > 0.05, Mann–Whitney U test, n = 11) in any experimental set, when compared within the groups. The object which was replaced with a novel object on test day was considered as the object of interest for calculating the exploration time for training session, while exploration time of novel object was used for the test session. D–F, Familiar versus novel object exploration time during test session represented as mean ± SEM. Significant differences were observed in novel object exploration time for BT(OF) and BT(EE) groups when intragroup comparison was done with familiar object exploration time (**p < 0.01, Mann–Whitney U test, n = 11) during 5-min NOR test session. The data suggest increased novel object exploration after OF and EE exposure, indicative of LTM formation.
- Figure 4.
Results depict intragroup comparison of number of entries for control, BT(OF), and BT(EE) groups. A–C, No significant difference was observed (p > 0.05, Mann–Whitney U test, n = 11, data represented as mean ± SEM) in any experimental set for number of entries in training versus test session. The zone in which novel object was replaced from familiar object was used as the zone of interest for training session, while the novel object zone was considered for test session. D–F, Number of entries in familiar and novel zone during test session represented as mean ± SEM. Significant differences were observed in novel zone entries for BT(OF) and BT(EE) groups when compared with familiar zone entries (**p < 0.01 and ***p < 0.001, Mann–Whitney U test, n = 11) during 5-min NOR test session. The data are again indicative of LTM consolidation process with increased entries to novel zone after novelty exposure when compared within the group as well.
- Figure 5.
Plasticity related molecular markers induced by different forms of novelty through BT phenomenon was observed. Expression of PKMζ after OF and EE exploration was analyzed using western blotting in hippocampal tissue. GAPDH was used as the normalizing factor for PKMζ relative intensity fold change. Significantly enhanced levels of PKMζ protein in hippocampus were observed in BT(EE) group (***p < 0.001, one way ANOVA) when compared with control. However, no significant difference was found in the PKMζ expression for BT(OF) group when compared with control (ns, non-significant; one-way ANOVA). All data represented as mean ± SEM (n = 4). The data suggest strong induction of PKMζ synthesis in hippocampus of rat brain after EE exposure, indicating the differences in molecular alterations by different form of novelties. The synthesis of PKMζ after EE exploration supports the neurobehavioral findings and establishes EE as the better form of novelty as compared with OF for hippocampus dependent learnings. For full blot image of PKMζ, see Extended Data Figure 5-1; for full blot image of GAPDH, see Extended Data Figure 5-2.
- Figure 6.
Expression of BDNF in hippocampus after OF and EE as different novelty explorations was observed. GAPDH was used as the normalizing factor for BDNF relative intensity fold change. No significant differences were found in the expression of BDNF in hippocampus region after OF and EE exploration when compared with control group (ns, non-significant; one-way ANOVA). All data represented as mean ± SEM (n = 4). For full blot image of BDNF, see Extended Data Figure 6-1.
- Figure 7.
Schematic representation of molecular cascade activated in the hippocampus of rat brain after EE exposure as a process of LTM consolidation through BT phenomenon. Exposure to EE as a novelty after weak training stimulates the release of stored glutamate into the synaptic cleft from presynaptic terminal. The released glutamate is then captured by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on the post-synaptic terminal, resulting in the opening of AMPA receptors and increased influx of Na+ ions in the dendritic space. The increased levels of influxed Na+ ions further help in activating the NMDA receptors by forcing the Mg2+ into the synaptic space. NMDA receptor activation ensures higher influx of Ca2+ into the dendritic space, which binds to the calcium/calmodulin kinase II receptors (CaMKII), thus activating the several kinases such as MAPK inside the cell. In addition, CaMKII mediates the exocytosis of AMPA receptors on the postsynaptic surface, thus further enhancing the synaptic strength and increased activation of synapse for neuronal signaling. Moreover, PKMζ synthesis and CREB phosphorylation are initiated with stronger stimulation of CaMKII by virtue of novelty exposure. Phosphorylated CREB further upregulates the transcription of BDNF protein which in turn enhances the AMPA receptor trafficking as well as activation of its receptor TrkB. The cascade thus facilitates LTM consolidation process after stimulation from EE exposure as the novelty establishing BT as a molecular phenomenon for memory formation.
Tables
Control (n = 11) BT(OF) (n = 11) BT(EE) (n = 11) Day 1: habituation Rats were placed in an empty NOR apparatus and were allowed to freely explore the arena for 5 min. Day 2: training Two similar shaped objects were placed in NOR apparatus and rats were allowed to explore the objects
for 5 min. Single trial training was provided to all groups.No novelty exposure was provided 15 min after training, rats
were subjected to 5-min
open field task as novelty15 min after training, rats were
subjected to 5-min environment
enrichment as noveltyDay 3: test One object was replaced with a novel object in NOR apparatus and rats were tested for 5 min for NOR
long-term memoryExperimental protocol for studying the effect of different types of novelty exposure on memory consolidation process. Animals were randomly divided into three experimental groups: control, BT(OF), and BT(EE) with n = 11 for each group. All groups were subjected to NOR as the learning task, while OF and EE were two different types of novelties used to induce the BT phenomenon. Control group was not given novelty exposure. Complete neurobehavioral experiment consisted of three days protocol following 5 min of habituation, single trial training (followed by novelty for two groups) and testing session.
Extended Data Figure 5-1
Full blot image of PKMζ. Download Figure 5-1, TIF file.
Extended Data Figure 5-2
Full blot image of GAPDH. Download Figure 5-2, TIF file.
Extended Data Figure 6-1
Full blot image of BDNF. Download Figure 6-1, TIF file.
In this issue
