Number of surviving neurons in hippocampal area CA3.
Previous studies have shown that environmental enrichment increases neurogenesis and reverses learning and memory deficits in rats with kainate-induced seizures. We tested the hypothesis that exploring a wooden pyramid for 3h/d augments neurogenesis and attenuates the learning and memory deficits following chemical lesioning of the hippocampus and motor cortex with kainic acid (KA). A pyramid exploration intervention (PEI) was created by subjecting rats to residing in a pyramidal wooden structure of 3 h/d for 30 d. We also compared the effects on neurogenesis for PEI to those for aerobic (swimming) exercise (EX) and environmental enrichment via exploration of a rectangular-shaped wooden cage. Following KA seizures, the PEI increased brain neurogenesis. Differences in measures of neurogenesis were not significantly different than those for EX and EE. Aerobic (swimming) exercise and novel environment exposures appear to increase neural plasticity and may be considered a complementary treatment for epilepsy.
- neural plasticity
- motor cortex
- complementary and alternative medicine
Previous studies from our laboratory have shown that regimens of short periods of daily swimming exercise and environmental enrichment increases brain neurogenesis, learning and memory in the kainate rat model of temporal lobe epilepsy [1, 2, 3]. Temporal lobe epilepsy (TLE) is associated with oxidative stress, a putative mechanism for neuronal apoptosis  and decrements in neural plasticity that impairs the brain’s cognitive functions . Oxidative stress is known to reduce neuronal antioxidant and anti-apoptotic activity which damages the brain’s learning and memory networks . Epilepsy is often resistant to treatment with antiepileptic drugs that are also known to produce oxidative stress and to promote neuronal apoptosis . Manipulation of an animal’s environment appears to have the potential for producing physiological, therapeutic effects and are purported to function as environmental enrichment schemes for improving responsiveness to drug treatments. We have reported increases in neurogenesis and improvements in performance of rats on learning and memory tasks associated with environmental enrichment by allowing rats to explore a novel cage with different objects in different configurations for 3 h/d, for 30 d. Another such environmental enrichment intervention involves subjecting rats to stress restraint in a pyramidal wooden structure. This environmental intervention is shown to reduce the physiological and oxidative stresses associated with immobilization and restraint .
Nikola Tesla was a mathematician-physicist and the inventor of many of today’s most advanced electromagnetic technologies . Among Tesla’s hypotheses more, than a hundred years ago, is the prediction that the architectural design of the great pyramid of Egypt produces electromagnetic effects within its chambers . Pyramids are reported to augment local electric fields via a lightning-rod effect  and function as electromagnetic conductors ; although, the mechanisms remain unclear. It may, therefore, not be an accident of nature that pyramidal-shaped neurons evolved to generate and focus electromagnetic energy in the brain. Pyramidal-shaped neurons comprise 12 billion of the 16 billion neurons in the human cerebral cortex . A psychiatrist-inventor, Hans Berger, in the 1920s, was the first person to demonstrate electroencephalographically-measured brain waves (EEG) . It is now known that each brain wave represents the synchronized action potentials of millions of pyramidal cells in the cerebral cortex . In view of these interesting aspects of pyramids and pyramidal cells in the brain, the present study is designed to test the hypothesis that a pyramid exploration intervention (PEI) augments brain neurogenesis and improves the brain’s learning and memory functions in an animal model of TLE.
These studies were approved by the institutional animal care and use review board of Manipal University, Bangalore India, in partnership with the American University of Antigua College of Medicine.
The animal subjects were 4-month-old male Wistar rats maintained under conditions of 12-hour light-dark diurnal cycles in Manipal University’s Animal Research Facility. The animals were fed
2.2 Experimental design
Figure 1 depicts the study design intended to determine the effects of a pyramid exploration intervention (PEI) on neurogenesis, learning and memory functions in rats. The animals were grouped as follows: 1. Normal control rats (NC); 2. Normal control rats with exposure to the PEI (NC + PEI); 3. Sham control rats with exposure to the PEI (SC + PEI); 4. Kainic acid-lesioned rats; and 5. Kainic acid lesioned rats with exposure to the PEI (KA + PEI). NC animals were undisturbed in their home cages. NC + PEI animals were subjected to the PEI for 3 h/d. SC + PEI animals were subjected to sham surgery followed by exposure to the PEI. The sham surgery involved fixing the rats in a stereotaxic apparatus under general anesthesia after which burr holes were drilled in the skull using stereotaxic coordinates for the lateral ventricles taken from a standard rat brain atlas. A Hamilton syringe was inserted into the lateral ventricles bilaterally and removed without administration of any fluids or drugs. Following suturing of the scalp wounds, the rats were placed back in their home cages and were subjected to the PEI 3 h/d for 30 d. KA animals were administered kainic acid (KA) bilaterally into the lateral ventricles using a Hamilton syringe. Animals in the KA lesioned + PEI group were administered KA and then underwent PEI for 3 h/d for 30 d. The PEI was initiated 1-d after grouping in the NC animals, 1-d after surgery SC + PEI animals and starting on the first postictal day in the KA + PEI group.
2.3 Surgical and related experimental procedures
SC and KA animals were anesthetized with a mixture of ketamine (50 mg/mL), xylazine (4.5 mg/mL) and acepromazine (0.4 mg/mL) at a dose of 0.70 mL/kg body weight and were fixed in the stereotaxic apparatus with the incisor bar situated 3.7 mm below the inter-aural line. The skull was exposed and a burr hole was drilled guided by the stereotaxic coordinates as follows: 3.7 mm from bregma, 4.1 mm lateral from the midline . KA lesioning was accomplished using a Hamilton syringe needle filled with 0.5 μg/μL KA, 1.0 μl of which was administered slowly over 20 min. The KA-filled syringe was lowered from the stereotaxic syringe holder 4.5 mm to reach the lateral ventricles. After the needle was withdrawn, the skin was sutured and the animals were returned to their home cages.
2.4 Pyramid exploration intervention (PEI)
Exposure of animals to PEI was accomplished in a wooden cage of larger dimension than the steel home cage (Figure 2). Rats were allowed to explore the PEI environment for 3 h every d for 30 d, beginning immediately following either grouping (normal control group), sham operation (sham-operated control group) or KA + lesioning (kainate experimental group) for 3 h/d for 30 d beginning 1-d the grouping (normal controls), sham operation (sham controls) or KA lesioning.
2.5 Morphological procedures
2.6 Behavioral testing
Behavioral tests were administered on the 42nd day following grouping for the NC group and on the 42nd day following surgery or KA-induced seizures for the other groups. All testing was done at approximately 7:00 PM to control for the diurnal variation of night-time activity in rodents.
2.7 Data analysis
Analysis of variance (ANOVA) with Bonferroni’s post-hoc test was performed to estimate the statistical significance of differences between groups (GraphPad Prism, version 5). Across the groups, correlations between the most relevant morphometric and behavioral measurements were evaluated by Pearson’s product moment correlation coefficient (r). Neurogenesis parameters were compared for similar experiments performed under conditions of PEI, aerobic (swimming) exercise and another form of environmental enrichment [1, 2, 3]. Statistical significances were guaranteed at P ≤ 0.05.
3.1 Morphometric measurements in hippocampus, amygdala and motor cortex
Figure 3 shows the effects of the PEI, with and without KA lesioning, on the CA3 area of hippocampus by light microscopy. The PEI was associated with significant increases in the numbers of surviving neurons in the normal and sham-operated control groups and after KA lesioning in the experimental group (data not shown). The PEI also produced significant increases in the numbers of surviving neurons in hippocampal area CA1, dentate gyrus, basolateral amygdala and motor cortex in the controls and in the KA-lesioned animals (data not shown).
Figure 4 depicts the effects of the PEI on the dendritic branching of the surviving neurons in hippocampal area CA3 by light microscopic camera lucida tracings.
Figure 5 shows the morphometric measurements of the effects of the PEI on the counts of branch points and intersections in hippocampal area CA3. These data demonstrate that exposure to the PEI produced significant increases in the branch points and intersections both in the presence and in the absence of KA lesioning. Similar increases were found in dentate gyrus, amygdala and motor cortex and in animals subjected to delayed exposure to the PEI after a delay of 60 d, postictal.
3.2 Behavioral measurements
The PEI was associated with significant increases in the percent bias, percentage of correct responses and the number of alternations on the T-maze learning task. Percentage of correct responses for the animals subjected to the PEI for 30 d starting at the first postictal day was positively correlated with the number of surviving neurons in all the brain areas studied, across the five study groups (r = 0.92-0.97, P < 0.001). Percentage of correct responses for these animals was also positively correlated with the number of apical and basal dendritic branch points and intersections in the same brain areas, across the five study groups (r = 0.88-0.94, P < 0.01).
Figure 6 presents the effects of the PEI on the exploration and retention phases of passive-avoidance testing. The PEI was associated with significant decrements in the time spent within a small compartment where an aversive stimulus was previously administered and in the number of crossings. The same pattern of behavioral responses was observed in animals subjected to the PEI for 30 d immediately, starting 1-d postictal, as well as, after a 60-d delay postictal. Similar to the morphometric and T-maze data, these changes were observed in the animals subjected to the PEI. Time spent in the small compartment avoiding the aversive stimulus was negatively correlated with the number of surviving neurons in all the brain areas studied, across the five study groups (r = −0.93 to −0.99, P < 0.001). Time spent in the small compartment was also negatively correlated with the number of apical and basal dendritic branch points and intersections in the same brain areas, across the five study groups (r = −0.88 to −0.95, P < 0.01).
Table 1 summarizes comparisons of the effects of the pyramid exploration intervention (PEI), environmental enrichment in the absence of pyramid exploration (EE) and aerobic (swimming) exercise (EX) on the numbers of surviving neurons in hippocampal area CA3. There were no significant differences in neurogenesis between these three interventions (P > 0.1). Similar results were found for the dendritic branch points and intersections in area CA3 (data not shown).
|PEI||104.3 ± 4.457||EE||108.8 ± 3.601||EX||105.7 ± 4.412|
|SC||103.3 ± 8.359||SC||104.3 ± 4.457||SC||98.83 ± 7.055|
|KA||60.33 ± 1.966||KA||68.50 ± 3.146||KA||64.83 ± 4.491|
The main findings of this study are that morphometrically-measured rat brain neurogenesis and performance-measured learning and memory behavioral tasks are augmented by a pyramidal exploration intervention (PEI). The PEI permitted rats to explore a wooden pyramid for 3 h/d. The PEI was associated with increments in the numbers of surviving neurons, apical and basal dendritic branch points and intersections at hippocampal areas CA1 and CA3, amygdala and motor cortex suggestive of increased neurogenesis and improved learning and memory under control and postictal conditions. This evidence of increased neurogenesis is bolstered by highly significant correlations, across the five study groups. We found significant positive correlations between the animals’ percentage of correct responses on a T-maze (learning) task, the numbers of surviving neurons and the numbers of dendritic branch points (r = 0.88-0.94). We found similar significant, but negative, correlations between the time the animals spent in a small compartment avoiding an aversive stimulus (memory task) and the numbers of surviving neurons, dendritic branch points and intersections (r = −0.88 to −0.99). These findings demonstrate that, whether a control or an experimental animal, rats with more neurons and more dendritic intersections (synapses) in the four brain areas studied had greater learning of a T-maze strategy, as well as, greater memory for avoiding an aversive stimulus.
We have previously shown increases in rat brain neurogenesis and improvements in performance on the same learning and memory tasks as employed in the present study associated with an environmental enrichment intervention involving exploration of a wooden square structure containing novel objects in novel configurations for 3 h/d . We designed the present study to expand the previous studies to a different type of environmental intervention, the exploration of a pyramid-shaped wooden house for 3 h/d. We used a pyramid-shaped structure to emulate prior studies demonstrating that housing in pyramid while exposed to restraint stress counteracts the parameters of oxidative stress associated with the animal restraint . Oxidative stress causes accumulation of superoxide and other free radicals—highly reactive oxygen and nitrogen species such as superoxide and hydroxyl radical anions, nitric oxide, nitrogen dioxide and peroxynitrite . These highly reactive oxygen and nitrogen species (ROS/RNS) do not accumulate under normal conditions because they are neutralized/scavenged by antioxidant enzymes such as superoxide dismutase, catalase and glutathione reductase synthesized by, and stored in, each normal healthy cell . Oxidative stress occurs when the balance between production and neutralizing/scavenging favors accumulation of ROS/RNS . Such accumulation of ROS/RNS causes peroxidation of lipids in the cell’s plasma and organelle membranes . ROS/RNS-dependent enzymes are known to function normally under conditions of very low, physiological levels of ROS/RNS .
The aforementioned pyramid exposure appears to protect against the restraint stress-induced atrophy of the hippocampus . Oxidative stress is shown to be a pathophysiologic feature of KA-induced seizures . The PEI in the present study is, like the aforementioned pyramid study involving stress restraint, associated with physiologically-significant effects, evidenced by significant correlations between morphometric and behavioral measurements across the five study groups. Pyramid exposure is also shown to increase the amplitude of electroencephalographically-measured alpha waves , and positive mood responses associated with meditation [20, 21]. Electroencephalography and monitoring of emotional functions were beyond the scope of the present study. However, hippocampus is an important source of emotional responsiveness and theta waves . Theta and alpha waves propagate together in the neocortex when cognitive tasks are performed . It is, therefore, plausible that the increases in neurogenesis and improvements in behavior observed in the present study are likely to have been reflected in brain waves and emotional responses.
KA-induced seizures are also a stimulus for neurogenesis, associated with increased numbers of neural progenitor (stem) cells within the subgranular zone of hippocampus, amygdala sensorimotor cortex  but motor cortex neurogenesis has not been extensively studied. It was beyond the scope of the present study to identify neural stem cells and to measure the immediate effect of the KA-induced seizures but we assume that our morphometric counts of surviving neurons, dendritic branch points and intersections after a 30-d period is a fair reflection of the seizure-induced neuronal death followed by neural stem cell proliferation known to occur in the KA rat model of TLE.
The results of this study demonstrate that regular exposure of rats to a wooden, pyramidal-shaped environment for their exploration 3 h/d increases neurogenesis learning and memory, under normal control conditions, after a (sham) surgical intervention and following KA-induced seizures. These findings should be interpreted cautiously because previous studies from our laboratory have shown that the same benefits accrue from an environmental enrichment by exposing animals to exploration of a rectangular wooden cage with novel objects in novel configurations for 3 h/d and aerobic (swimming) exercise [1, 2, 3]. It is, therefore, likely that pyramid effects are nonspecific and are the result of exposing animals to any novel environment. We, therefore, conclude that pyramid exposure is probably not different than other modes of environmental enrichment for augmenting brain neurogenesis and improving learning and memory functions. Future studies should determine whether exposure to novel environments are effective as complementary or alternative treatments for the wide variety of neurological diseases wherein full recovery of learning and memory functions may be limited by ineffective neurogenesis.
This work was funded by a grant from Manipal University.
Conflict of interest statement
The authors report no conflicts-of-interest.
The data supporting this research is available upon request to the corresponding author.