Babri, Ghotbi, Razavi, and Bavil:

Effect of Intrahippocampal Injection of Dihydrotestosterone on Spatial Memory in Kindled Male Rats

Shirin Babria, Roshanak Ghotbib, Samira Razavib, Fariba Mirzaei Bavilb

aNeurosciences Research Centre (NSRC), Tabriz University of Medical Sciences, Tabriz, Iran
bDrug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Correspondence
Fariba Mirzaei Bavil, Department of Physiology, Tabriz University of Medical Sciences, Tabriz, Iran. Tel/Fax: +98-41-33364664, Email: bavil2000@yahoo.com

Received: 2016-05-21
Accepted: 2017-01-25

DOI: 10.13183/jecns.v4i1.49

Abstract


Objective: Dihydrotestosterone (DHT) is an endogenous hormone, derived from testosterone in the enzymatic reaction mediated by 5-alpha reductase. The high density of androgen receptors and 5-alpha reductase enzyme has been found in the hippocampus. According to the recent studies, androgens may have an improving effect on spatial memory. On the other hand, kindling can result impairment in learning and memory of rats. In this study, we investigated the effect of intrahippocampal DHT on spatial memory in male kindled rats.

Materials and Methods: 160 male rats were randomly divided into 20 groups (n=8). Control, shame, saline (0.5 µl intrahippocampal), DMSO (0.5 µl intrahippocampal), kindled, DHT (5, 10, 20 μg 30 min or 24 h before test), kindled + DHT (5, 10, 20 μg: 30 min before or after stimulation and then test was done), kindled + DHT (5, 10, 20 μg injection 30 min before the test). Two canules were implanted in CA1 region of animals in all groups except control group. DHT was solved in 0.5 µl DMSO acquisition and retrievals were assessed by Morris Water Maze.

Results: Our findings showed that DHT in doses of 10 and 20 μg can improve acquisition and retrieval in kindled male rats. DHT doesn’t have any effect on seizure parameters.

Conclusion: Intrahippocampal DHT can improve acquisition and retrieval in kindled rats but it doesn’t have any effect on seizure parameters.

Keywords: Dihydrotestosterone, Hippocampus, Seizure, Acquisition, Retrieval, Kindling

© 2017 Swedish Science Pioneers, All rights reserved.


Introduction

Frequent stimuli causing convulsions (kindling), manifest as steady increase in sensitivity to stimuli. Increasing evidences show that kindling may cause permanent changes in some kinds of behavior [1-3]. It has been shown in several studies that spatial cognition is one of the disturbed behaviors in kindling. Kindling disrupts spatial performance both in Morris Water Maze (MWM) and redial arm maze [3-5].

It has been shown that there is high density of androgen receptors in regions related with memory and learning such as hippocampus, and androgens are related with different aspects of cognition in the brain [3,6]. It has been shown that intra-hippocampal injection of testosterone and its reduced metabolite Dihydrotestosterone (DHT) and 3-a-DL, increase learning in avoidance tasks, compared with gonadectomized rats [7].

Seizure activity has profound effects on hippocampus and interestingly, epileptic activity originating from hippocampal focus can impair hypothalamic-pituitary axis, causing gonadal dysfunction [8,9].

Because learning tasks activity mediated by hippocampus disturbed following seizure, proper function of verbal memory is depends on hippocampal function [10].

In rodents, hippocampal-mediated function of spatial memory is impaired following seizure [9,11,12].

Androgens have neuroprotective effects on hippocampus. In-vitro experiments show that androgens can ameliorate b-amyloid toxicity and cell death induced by oxidative stress [13]. Neuroprotective effects of androgens, had also been proved in in-vivo experiments. Androgens can reduce hippocampal injury resulting from adrenalectomy and administration of kainic acid [14].

In fact, current data indicate that protective effectsof androgens can be attributed to their reduced metabolites. Testosterone, DHT and 3-a-DL can prevent hippocampal cell death induced by adrenalectomy [15].

Studies showed that hippocampus is susceptible to damage induced by seizure, and further research on protective effectsof androgens on memory, is mandatory.

This study is tailored to evaluate the effects of DHT on spatial memory in male rats, elucidating potential effects of DHT on spatial memory in kindled rats.

Materials and Methods

Animals

All experiments were performed under guidelines of Tabriz University of Medical Sciences for care and use of laboratory animals. One hundred-sixty male adult Wistar rats (250-300gr) were housed in room temperature and light-controlled environment. Rats were handled during experiment, except first 7 days after surgery.

Surgery

Rats were anesthetized with ketamine (100 mg/kg) and placed in the stereotaxic apparatus. A three-polar electrode with a cannula was placed in right and other cannula was placed in left CA1 region. Stereotaxic apparatus was set according to Paxinos-Watson method (AP=3.5; ML=2.6; DV=3.5).

Injection

Intracranial injection was done by 21-gauge canula and 27-gauge needle, attached to Hamilton syringe by a polyethylene tube. Injections were 30 minutes before and 30 minutes after stimulation, and 30 minutes before MWM (0.5 ml DMSO; 5, 10 & 20 mg DHT dissolved in 0.5 ml DMSO).

Animals stimulation

Kindling was started 7 days after surgery. Stimulation threshold was established in first day of kindling. On the first day, basic EEG was performed. 10 mA electrical current was applied on CA1 region and stimulation threshold was determined.

Current intensity was considered satisfactory in the case that recorded discharge flow was at least 5 seconds. Otherwise, flow rate was increased to reach thethreshold. Rats were stimulated every 24 hours until animals achieved complete kindling that meant appearance of stage-fiveseizure three times a day for three consecutive days [16].

Behavioral assessment

Apparatus

Morris Water Maze (MWM) is a water-full tank with a diameter of 130cm and aheight of 130 cm. Maze composed four quadrants: Northeast, northwest, Southeast, and Southwest. Platform was placed in one of the squares, 1 cm under water. The camera was located at top of tank for recording rat swimming; Trackingsystem was used to measure the distance traveled by rat to reach the podium, thetime spent to reach the platform (escape latency), speed of swimming (movement), and ratio of distance traveled and time spent in each quarter [17].

Tests

All tests were begun at 8.00 a.m. Each rate was tested on two successivedays, andin each test, 2 blocks consist of 4 trials, were tested. Between successive blocks, 5 minutes break was allowed for each rat.

The animals were allowed to swim and find hidden platform during 60 seconds ineach trial at a fixed position (northeast). If the rat found platform within 60 seconds, he was allowed to rest 30 seconds on the podium. Otherwise, rat was ledto the platform and allowed to rest for 30 seconds.

On the second day, the animal was assessed after platform removed. Next, distance and elapsed time were measured in the target quadrant (test probe); in the second blocks, platform was transferred to another quadrant and after coveringby a white cover, visible platform test was performed [7].

Effects of kindling on memory

In this study, 16 rats were divided into two groups. In the first group, rats werestimulated every day until demonstrating three successive seizures. In the second group, rats were undergone surgery without receiving any stimulation.

Effect of DHT on spatial memory in kindled rats

Injection 30 minutes before test MWM

Thirty-two kindled rats were divided into 4 groups; the first group received 5.0 mL DMSO, and second, third and fourth groups received 5, 10 and 20 mg DHT dissolved in 5.0 mL DMSO, respectively.

Injection 30 minutes after stimulation

Groups of rats were assigned in similar way with previous test. However, injections were performed 24 hours before MWM and 30 minutes after stimulation.

Histology

After performing tests, rats were killed with high dose of chloroform. Brains wereplaced and fixed in formalin solution, followed by obtaining 100 mm thicknesssections from CA1 region. Then, slices were placed on slides and sections werestained with methylene blue dye solution.

Data analysis

The data were analyzed using SPSS (version 16.0). At first step, statistical assessment was done based on Kolmogorov-Smirnovtest. If three was normal distribution, ANOVA test followed by Tukey test wereperformed. Differences were considered as significant if p<0.05. In statestatistical software, was used for calculations and analyses.

Results

Based on achieved results, there was no significant difference between three groups (control, DMSO and saline groups) in terms of distance traveled by ratsfor reaching platform, as well as probe test results and visible platform test results (Figure 1).

thumblarge

Figure 1. (a) Traveled distance, (b) percentage of present in target quadrant and (c) traveled distance to find the visible platform in visible platform test in control, DMSO and saline groups. Mean±SEM (n=8).

Effects of kindling on spatial memory

In the animals of kindled group, distance traveled to reach the platform showed significant increase compared to control group (p<0.05). There was significant difference between kindled and control groups in terms of percentage of time spent in the target quadrant (probe test) (p<0.01). However, no significant differences were seen in the visible platform test (Figure 2).

thumblarge

Figure 2. (a) Traveled distance and (b) escape latencies in target quadrant in probe test in control and kindle groups. *p<0.05 and **p<0.01 indicates the difference vs control group. Mean±SEM (n = 8).

Effects of DHT on spatial memory in kindled rats Thirty minutes before the test MWM

The distance traveled by rats to reach platform in groups receiving 10 and 20 mg DHT, decreased in 10 and 20 mg groups significantly (p<0.01 and p<0.05 respectively).

The results of probe test showed significant differences in the percentage of elapsed time in target quadrant, between group receiving 10 mg DHT and solventsolution (Figure 3).

thumblarge

Figure 3. Effects of different doses of DHT thirty minutes before the test MWM in kindled rat on (a) traveled distance and (b) escape latencies in target quadrant in probe test. *p<0.05 and **p<0.01 indicates the difference between each group and DMSO group. Mean±SEM (n = 8).

Thirty minutes after stimulation

Distance traveled by rats to reach platform, as well as results of the probe test, did not demonstrate any significant difference between groups (Figure 4).

thumblarge

Figure 4. Effects of different doses of DHT thirty minutes after stimulation in kindled rats on (a) traveled distance and (b) escape latencies in target quadrant in probe test. The test was done after 24 hours. Mean±SEM (n = 8).

Thirty minutes before stimulation

There was no significant difference in terms of traveled distance and elapsed time for reaching platform, as well as results of probe test, between study groups (Figure 5).

thumblarge

Figure 5. Effects of different doses of DHT, thirty minutes before stimulation in kindled rats on (a) traveled distance and (b) escape latencies in target quadrant in probe test. The test was done after 24 hours. Mean±SEM (n = 8).

Discussion

Based on these results, there were no significant differences between saline and DMSO group. Distance traveled for reaching platform, increased significantly in kindled group, and DHT injection 30 minutes before MWM, increased distance traveled andelapsed time in the quadrant. However, DHT injection before and after stimulation, had no significant effect on spatial memory.

These results showed that kindling could disrupt learning abilities. Special profile produced by kindling, is consistent with disrupting hippocampus functional mechanisms.

On the other words, kindling may impair hippocampal function both directly and exclusively [2]. It has been shown that kindling can cause changes in NMDA receptors by deleting NMDA receptor inhibitory mechanisms [1]. Kindling can cause changes in the inhibitory properties of GABA (A) receptors.

It can be expected that physiologic properties of plasticity forms such as long-term potentials, are changed as well [1]. These changes can alter effectiveness of plasticity associated with learning abilities. Thus, kindling is a type of metaplasia, leads to shift from the optimal situation of plasticity in hippocampus [7].

Studies have shown that there are many androgen receptors in hippocampus, associating with memory and learning. Also, it has been shown that there are correlations between various aspects of cognition and androgens. Steroid hormones probably act via neurotransmitters such as acetylcholine [18], dopamine [19], noradrenaline, glutamate [20] and serotonin. Steroid hormones can increase affinity of neurotransmitters by changing ionic permeability in the brain structures such as hippocampus. For example, sex hormones effect on brain cholinergic function and memory [18]. Improved spatial cognitive function in kindled rats receiving intra-hippocampal DHT is a newly found phenomenon that may be due to DHT effects on kindling and properties of this hormone on mitigating the effectsof seizure. On the other hand, this study showed that DHT has no effect on epilepsy. Effect of DHT on spatial memory in the kindled rats, presumably is mediated by another ways.

Seizure activity has a dramatic effect on hippocampal functions. Interestingly, seizures originating from hippocampal focus can impair pituitary hormones regulation by hypothalamus and cause impairment of gonadal function.

Generalized and focal seizures disrupt physiologic structure and circulating levels of androgens in rats [21]. Thus, epileptic activity in the hippocampus could effect on secretion of androgens.

In healthy middle-aged rats with hippocampal seizures, androgen levels were changed compared with middle-aged rats without seizures [22]. There was no difference between concentrations of hippocampal androgens in gonadectomized GDX rats with epilepsy compared to normal GDX rats [20].

These data indicate that intact hypothalamic-pituitary axis is mandatory for gonadal response. These in formations, are consistent with clinical findings obtained from men with epilepsy. In this case, age-related decline in androgens levels occurs sooner. 5-a-reductase levels that considered essential for the metabolism of testosterone to DHT and 3-a-DL, are also changes. Studies have shown that the amount of this enzyme reduced significantly after chemical CA1 hippocampal kindling, compared to administrating carrier solution alone [22].

Although it is unknown whether this effect is due to reducing the amount of enzyme or reducing cell number, functional outcomes of these changes are clear: although in small quantities, the amount of androgens is changes. In fact, testosterone levels in rats suffering from seizures are higher than normal rats and the amount of DHT and 3-a-DL in hippocampus of epileptic rats is slightly lower than normal rats; this observation can be justified by reduction 5-a-reductase levels and inability of testosterone to convert to DHT and 3-a-DL, testosterone levels are increased [22].

Because spatial memory is improved with administration of DHT gel in hypo gonadal men and with administrating DHT in kindled rats, and because responsiveness of hypothalamic-pituitary-gonadal axis is reduced and decline of androgen levels is accelerated in patients with epilepsy, it may be possible that intrahippocampal DHT can normalized intrahippocampal environment and reversed factors disturbing learning activities and induction of optimal plasticity.

It is important that NMDA receptors play an important role in spatial learning and DHT increases N-methyl-D-aspartate affinity for NMDA receptors in the CA1 region [23]. Kindling not only disrupt NMDA and GABA (A) receptors, but also alter hypothalamic-pituitary-gonadal axis. In fact, kindling impairs spatialmemory in two dimensions that previously are not taken into consideration.

Considering results of this trial, we can conclude that the effects of androgens on memory are not due to their neuroprotective properties and these effects depend on other factors.

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Notes

Conflict of interest

The authors have declared that there is no conflict of interest.

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