The Effect of Environmental Enrichment on the Number of NADPH-d Positive in the Dentate Gyrus of the Rat Dorsal Zuhayr Khan and Dr. Glenn H Kageyama Abstract

Exposure to an enriched environment has been shown to be beneficial to structure and cognition by preserving neuronal integrity and strengthening the functioning and plasticity of neural circuits [1]. These benefits derive from the added spatial, social, and sensory complexities in an enriched environment [2]. It has recently been established that physical and enriched environments stimulate adult [3] and differentiation in dentate granule cells (DGCs) [4] in the dentate gyrus of the hippocampus. However, possible changes in other cell types, particularly interneurons, has remained elusive. Interneurons are an integral regulator in neurotransmission in the hippocampus. Damage to hippocampal interneurons have serious implications and lead to a decline in cognitive abilities [5]. Our study sought to elucidate possible changes in a subclass of interneurons affected by exposure to an enriched environment (EE) and enriched changing (EC) environment. The topographic arrangement of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) positive interneurons was studied in the dentate gyrus of the rat dorsal hippocampus. We observed an increase in the number of NADPH-d positive neurons found in both enrichment groups. The elevated level of NADPH-d activity was represented uniformly across all six layers of the dentate gyrus and was most significant in the expected granular cell layer (GRCL) and infragranular zone (infraGRZ). The overall results suggest that the brain has the ability to adapt to increased amounts of sensory stimulation. These changes highlight a new mechanism of physiological homeostasis; with an increased demand for energy from new-born DGCs and increased input, the brain will regulate its energy expenditure via an increase in either the activity of existing interneurons, or the actual number of interneurons. Further research will involve observing the differences to the ventral hippocampus, CA1, and CA3 regions, and the effects on other specific subpopulations of interneurons. Introduction

The hippocampus is the primary region of the brain that is often associated with memory consolidation and decision making. The hippocampus is divided into two distinct regions: dorsal and ventral. The dorsal hippocampus performs primarily cognitive functions and is involved with spatial memory [6]; and the other is the ventral hippocampus, which relates to stress, emotion, and affect [7].

The hippocampus is one of the few regions of the brain that has continual addition of new neurons, particularly newborn dentate granule cells (DGCs) through the phenomenon of [8]. Substantial amounts of literature have indicated that these neurons integrate themselves into the circuitry of the hippocampus and are involved in learning and memory [9].

Novel environmental exploration experiences have been shown to increase the addition of adult generated neurons by having DGCs increase their Ca2+ event frequency preferentially [10]. A large portion of these new-born DGCs do not survive an initial developmental phase, termed the

“critical survival period” [11]. In addition to their generation, an enriched environment also contributes to the survival of newborn DGCs. As the number of cells increase in the hippocampus, so does the risk of hyperexcitation [12]. Previous literature reports that vulnerability of GABAergic interneurons to excitotoxic damage was recorded in the hippocampal

CA1 region of pilocarpine models of chronic limbic seizures [13]. GABAergic interneurons have highly specialized functions and different types of hippocampal inhibitory interneurons control spike initiation (e.g., axo-axonic and basket cells) and synaptic integration (e.g. bistratified and oriens-lacunosum moleculare interneurons) within pyramidal neurons and synchronize local network activity, providing a means for functional segregation of neuronal ensembles and proper routing of hippocampal information [14]. Indeed, if inhibitory interneurons are key regulators of hippocampal activity, then it is imperative to determine what mechanisms control the activity of interneurons themselves, as the preservation of these interneurons is vital for hippocampal function. Here, we will highlight a new mechanism of activity regulation via a change in NADPH-d activity of interneurons in the dentate gyrus.

NADPH-d Positive Interneurons

Neuronal nitric oxide synthase (nNOS) activity is determined by NADPH-diaphorase staining. nNOS is primarily expressed by subpopulations of GABAergic interneurons in the mature hippocampus and neocortex, and is responsible for the generation of nitric oxide (NO). As a gaseous very weakly polar molecule without net electric charge and due to its small size, NO can diffuse readily across cell membranes in anterograde and retrograde directions to act presynaptically, postsynaptically, or within the cell that has produced it, functioning as a paracrine neurotransmitter, and also a local vasodilator to increase localized blood flow to active brain areas. Two types of GABAergic nNOS+ neurons have been distinguished histochemically, strongly stained nNOS-Type 1 and weakly stained nNOS-Type 2. Our study focuses only on nNOS-Type 1 interneurons. Type 1 neurons express high levels of nNOS and NADPH-d activity.

These interneurons exhibit a fast spiking firing profile, meaning that they are able to generate a train of action potentials at high frequency [15]. Their axons preferentially target the perisomatic region of granule cells, making them ideally suited to rapidly regulate dentate gyrus output [16].

Materials and Methods

Animals and housing conditions

In order to stimulate the rat dorsal hippocampus, we used varying forms of an enriched environment. Twelve male Sprague-Dawley rats were divided into three groups. The first group was a control (N), and was housed in a 1000g/rat sized caged (Figure 1) and received basic necessities. The cages were changed once a week. The second group was classified as the enriched environment (EE) group. The EE group was housed in a 2000g/rat sized cage (Figure 2) and received basic necessities. The EE group also received toys and square pipes to interact with. At the time of weekly cage changing, the interactive objects were replaced with replicas and were placed in the same location, as found in the former cage, in the new cage.

The third group was classified as the enriched changing (EC) group. The EC group received the same housing conditions as the EE group. However, the location of interactive objects were rearranged twice a week. Furthermore, when undergoing their weekly cage change, the new cage was filled with a set of toys that the rats had not been introduced to before in a random arrangement. Prior to the cage rearrangement and cage changing, the rats were moved into a

40-gallon tank play arena (Figure 3) that was filled with more forms of enrichment for four hours.

Tissue collection, processing, and statistical analyses

After 30 days of housing, rats were anesthetized and perfusion fixed. The were removed and frozen sectioned at 50µm with a sliding microtome. Tissue sections were stained using

NADPH-d histochemistry. The topographical arrangement of NADPH-d positive neurons in the dentate gyrus of the hippocampus, as seen in Figure 4 & 5, from sections was recorded by counting individual neurons (Figure 6) in each of the seven layers: hilum, CA4, infragranular zone (infraGRZ), granule cell layer (GRCL), inner molecular layer (IML), middle molecular layer

(MML), and outer molecular layer (OML). The counts were standardized and expressed as number of stained neurons per millimeter (Figure 7). A standard deviation of counts was determined for each layer examined (Figure 6).

Results & Discussion

Our results in Figure 6 show an increase in the number of NADPH-d neurons across all seven layers⎼⎼ associated with both enrichment groups compared to controls. With the developed standard measurement in Figure 7, we saw that the control group (n=7) had 2.32, the EE group

(n=7) with 3.45, and the EC group (n=10) with 3.52 stained neurons per millimeter. The overall results suggest that the brain has the ability to adapt to increased sensory input from an enriched environment. These changes suggest a new mechanism of physiological homeostasis; with an increased demand for energy from new-born DGCs and increased input, the brain will regulate its energy expenditure via an increase in either the activity of existing interneurons, or the actual number of interneurons. Which one it is, is still yet to be determined. This is very exciting because an increase in interneuron activity has very rarely been shown before in works of literature. Many papers focus on restoration numbers of interneurons after lesions or induced injury, however, very few have shown that there is an increase in the activity of original numbers of interneurons. Although this alternation in neuron number is potentially compelling, we recognize that our samples are relatively small, and should be recognized as preliminary.

Future Work

A new protocol was submitted to run a restudy on NADPH-d activity of interneurons. The CA1 and CA3 regions will be studied in conjunction with the dentate gyrus. Additionally, we will study the effects of social isolation on the ventral hippocampus with the hypothesis that NADPH-d activity will be reduced due to a decrease in stimuli from social isolation. Subsequently, in both dorsal and ventral hippocampus, we will be looking at the effects of enriched environment and social isolation on specific subpopulations of calcium-binding interneurons. These include parvalbumin (PV), calbindin (CB), calretnin (CR), somatostatin (SOM) and a few others as these interneurons have been associated with high energy expenditure and their critical roles in transmission regulation [17]. Double-labeling with a neurogenesis marker, doublecortin (DCX), is also planned to determine if any of the increase in NADPH-d activity was due to the adult neurogenesis of interneurons, a phenomenon observed only in the olfactory system.

Appendix

Figure 1

Fiigure ,4 Figure 5

N umber o f NADPHd + ln t e r neurons A c r oss t he D ent a t e Gyrus 50 45 40 35 30 25 20 15 10 5 0 O ML D N 7 • EE 8.36 • EC 6 . 13 6.2 Contr-o l n=7. EE n=7. EC n=to

D N • EE • EC Figure 6

Number of NADPHd+ l nterneurons Per M illimeterof H i ppocampus

3 . 4 5

L5 2 2.5 3 3.5 Control n=7, EE n=7, EC n=lO Figure 7 References

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Acknowledgements

Special Thanks to Vivianne Mitri, Hosne Afrin, Amrin Vajifdar, Julie Truong, Toni Sykes, Rosa Guerra, Samuel Payan, Sandie Macias, Brennan Kidder, Leeland Liu, Austin Pyles, Solomon Washington, Scott Weber, Jacob Eagleton, Aayushi Mardia, Jordan Wong, Romualda Aquino, Girisha Bharadwaj, Colin Campell, Alexis Colon, Eric Huynh, Summer Kim, Breana Navarro, Aleena Newsum, Anh Nguyen, Jon Provens, Breanna Robles, Kenneth Rangel.

This study could not have been conducted without the Teacher Scholar Grant 2018-2019.

And of course, Dr. Glenn H Kageyama.