**4.4 Social behaviour**

Capsicum

**4. Discussion**

**4.2 Motor coordination**

**4.3 Pain**

Following the consumption of pepper and capsaicin diets, the pepper and capsaicin groups had lower open arm entry frequency and duration in the elevated plus maze test. Since fearful mice tend to avoid open areas (especially when they are brightly lit), favouring darker and more enclosed spaces [25], these results imply that chilli pepper and capsaicin caused increase in anxiety in the mice [26].

Increase in risk assessment behaviours such as head dipping and stretch attend posture indicate increased anxiety levels [27]. The results showed that though not significant, the head dips and stretch attend postures of the pepper group were slightly higher than control. On the other hand, the head dips and stretch attend postures of the capsaicin group was significantly higher than the control. These results showed that both pepper and capsaicin increased anxiety in the mice, but

These behaviours confirm the anxiogenic tendencies of long term administration of pepper and capsaicin. However, its mechanism of action has not been ascertained. This is in agreement with the study of Choi et al. [28]. Hakimizadeh et al. [29] also reported that direct injection of capsaicin in the hippocampus induces anxiety-like behaviours, but the report of Santos et al. [30] was on the contrary.

Capsaicin and pepper-diet fed mice had significantly reduced foot slips and falls compared to control and these typify improvement in their motor coordination because the lower the frequency of foot slips and number of falls, the better coordinated the animal was [26]. Capsaicin, as the most abundant and commonly occurring capsaicinoid might have achieved this feat (improvement of motor coordination) by aiding in the integration of proprioceptive information with neural processes (TRPV1) in the spinal cord and in the brain (specifically cerebellum) [31].

Tail-flick response of a mouse to thermal stimuli is believed to be a spinally mediated reflex behaviour [32]. From the results of our study, the latencies of tail flick of both the pepper and capsaicin-diet fed mice were significantly longer than those of control in the immersion test. Also, the tail-flick latency of the capsaicin group was longer than that of the pepper group. These results imply that pepper or capsaicin diet increased the pain threshold of the mice leading to decreased pain sensitivity. These results point to a more effective analgesic potential in capsaicin than pepper [33]. In the hot plate test, when an animal perceives pain, it attempts to jump away from the object that elicited the painful stimuli. This supra-spinally controlled behaviour (latency of jump) models an escape behaviour. The latencies of jump of both pepper and capsaicin groups were significantly longer than the control. Also, the latency of jump of the capsaicin group was significantly longer than that of pepper group. These results depicted that both capsaicin and pepper exhibited anti-nociceptive tendencies because it took a long time for mice fed such diets to experience pain compared to control. These results further confirm the analgesic

Capsaicin may have acted by initially activating heat sensitive TRPV1 receptors which induce pain. Repeated and prolonged exposure to capsaicin might have resulted in the reduction of responsiveness of the receptors and ion channels, thus

the anxiogenic effects of capsaicin were greater [26].

**4.1 Anxiety**

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potentials of pepper and capsaicin [33].

The nesting score is an assessment of social behaviour. This nesting behaviour is a reflection of the social behaviour in mice. Nest building in mice correlates to organised behaviour in humans and is very distinct from the findings reported in a research by Alleva et al. [38], where they reported aggressive behaviour in mice treated with capsaicin. A poor performance in the nesting task may indicate impairment in social relationship in mice and likelihood of the presence of autistic behaviour. The nesting score of the capsaicin group was significantly higher compared to that of control. The value for pepper group appeared higher but was not significantly higher than the control. This result indicates enhanced organised social behaviour in mice fed with capsaicin diet. However, its mechanism of action has not been ascertained [33].

### **4.5 Learning and memory**

The hidden-platform task of the Morris water maze is a test of visuospatial learning and memory in the mice and is also hippocampus dependent [39]. The use of extra-maze cues was employed in this task. On the other hand, visible-platform (cued) task of the Morris water maze is a non-hippocampal task and dependent on the dorsal striatum (caudate nucleus and putamen) of the basal ganglia [39]. The visible (cued) platform used a unique intramaze visual cue placed at the location of the escape platform.

The shorter the swim latency, the better the training process. Mice with learning disabilities or impairments were not able to quickly figure out the spatial location/ position of the hidden platform, i.e., it took them a long time. Also, the steeper the gradient of swim latencies within the 3 day acquisition or reversal trainings, the better the learning curve, hence faster learning. Following the consumption of pepper and capsaicin diets, the swim latencies of the pepper and capsaicin groups were significantly longer than control in the first 3 days (acquisition training). This shows that pepper and capsaicin delayed learning process during the acquisition training [40].

During reversal training, the swim latencies of the test groups were also significantly longer on day 1 of the 3 day reversal training task, while on days 2 and 3, the values did not differ from control. This means that on days 2 and 3, the three groups learned equally while the control learned better on day 1. Visuospatial memory was also assessed during the probe trial (exploration without hidden platform). During this trial, it was expected that mice with good memory of the spatial location/position of the hidden platform would spend more time exploring the quadrant which had the platform during reversal training (North-East quadrant), but this was not observed in mice treated with pepper and capsaicin diets. They spent less time in the North-East quadrant. This means that they had memory impairment [40]. This is in contrast to the work by Kong et al. [41] which reported that capsaicin did not significantly alter the learning and memory performance in young adult mice but reduced the number of newly generated cells in the hippocampus. However, this is in line with the work by Kooshki et al. [42]. It is possible that the nociceptive effects of Capsaicin might have also affected learning and memory in the mice.

Learning and memory which are complex cognitive functions of the higher nervous centres encompass a variety of subcomponents with many interactions and overlaps [43]. Memories are stored in the brain by changing the basic sensitivity of synaptic transmission between neurons as a result of previous neural activity. The effects observed might have been due to the presence of the alkaloid called capsaicin. Since capsaicin is neurotoxic [9], it is likely that it impaired synaptic transmission between neurons by interfering with the basic sensitivity of the transmission in the hippocampus leading to impairment of learning and memory of the mice.
