Effect of Bacopa monniera on amygdaloid neurons Vollala VR et al. the superficial or cortical-like group; and 3) the centrome- dial group.14 The amygdala is associated with a range of cognitive functions, including emotion, learning, memory, attention and perception.15 Most current views of amygdala function emphasize its role in negative emotions, such as fear, and in the linking of negative emotions with other aspects of cognition, such as learning and memory.16 However, recent studies of neuronal activity in the monkey amygdala have shown that, contrary to this widely held view, the amygdala is just as important for the processing of positive reward and reinforcement.17-19 In addition to its role in associative learning, the amygdala modulates memory processes that occur elsewhere, such as the hippocampus, the striatum18,21 and, perhaps, the pre- frontal cortex. Many forms of emotionally driven memory tasks that are dependent on the amygdala and are impaired by pre-training amygdaloid lesions are spared by post- training amygdaloid lesions,22,23 which suggests that the amygdala is involved in only part of the learning process (i.e., adaptation). The role of the amygdala may not be limited to events that occur during training. Some studies have reported an increase or impairment of retention information when the amygdala is manipulated shortly after training, which suggests that the effects on memory are partly mediated by post-training neuronal activity.24,25 The hippocampus and amygdala interact with each other and also operate independently of each other, as shown by research on monkeys. In these monkeys, lesions of the hippocampus alone caused an impairment of the memory for object–place associations, but lesions of the amygdala alone caused an impairment of the memory for object- reward associations.26 The amygdala modulates both the encoding and the storage of hippocampal-dependent memories25 and is concerned with the programming of memories, especially those memories that are related to emotions.16-18,28-33 Although there are reports that show that BM improves learning and memory in rats, there is no direct experimental evidence to show a correlation of the action of this plant extract on behavior, especially learning and memory, with histological changes in the brain. Our earlier study using BM extract treatment in adult rats showed improvements in learning and memory, including spatial learning and passive avoidance learning.34 Advanced neuroscience research has shown that the learning process is associated with alterations in the dendritic morphology of the hippocampal33-38 and amygdaloid neurons.33,39 The ques- tion of whether BM extract affects the neurons of the amygdala remains unanswered. Therefore, we attempted to answer whether this plant extract induced changes in amygdaloid neurons. METHODS Animals and experimental groups Wistar albino rats of random sex, approximately 2K months old and weighing approximately 150 to 200 g were used for these studies. The animals were bred in the central animal house of Manipal University, Manipal, India. The rats were fed Amrut rat and mice pellets manufactured by Pranav Agro Industries Ltd, E/5-6, M.I.D.C., Kupwad Block, SANGLI – 416436 (Maharashtra), India. Four rats were housed in each polypropylene cage. All rats were maintained under a 12:12 hour cycle of darkness and light. The 664 CLINICS 2011;66(4):663-671 experimental protocol was approved by the Institutional Animal Ethical Committee for Experimental Clearance of Manipal University IAEC/KMC/02/2005-2006. Rats were assigned to 2-, 4- and 6-week treatment groups. Rats in each of these groups were divided into 20 mg/kg, 40 mg/kg and 80 mg/kg dose groups (n = 8 for each dose). Each rat in a given dose group was fed the designated amount of standardized extract of BM daily for 2, 4 or 6 weeks. In addition to these experimental groups, an untreated normal control group (NC) and a gum acacia vehicle control group (GAC) (n = 8 in both groups) were also maintained. Extraction and administration of BM Standardized plant extract of BM was supplied by the herbal manufacturer, M/s. Natural Remedies Private Limited, Bangalore, India. The shelf life of this extract is 2 years. The first step was the extraction of the botanically identified plant material with alcohol. The alcoholic extract was re-extracted with water, and the water-soluble matter was retained. The final re-extraction was concentrated and dried to make a powder. Phytochemical analysis using high- performance liquid chromatography (HPLC) and high- performance thin layer chromatography (HPTLC) revealed that the final extract contained approximately 10% w/w (10% of the total mass of the extract) of the active ingredients (bacosides A and B). The plant extract was administered orally with 5% gum acacia using an oral feeding needle attached to a syringe. Behavioral tests Following BM treatment, all groups (NC, GAC and BM) were subjected to behavioral tests during the 12-hour dark period (which started at 7 PM). The behavioral tests consisted of a spatial learning (T-maze) test and a passive avoidance test and were performed as previously detailed.34 Rapid Golgi staining procedure At the end of the treatment period (2, 4 or 6 weeks), the rats were anesthetized with ether and killed by cervical dislocation. The brains were removed and fixed in rapid Golgi fixative. Tissue was processed for rapid Golgi staining as previously detailed.36 Briefly, tissues were fixed for 5 days in Golgi fixative and impregnated with a 1.5% aqueous silver nitrate solution for 48 hours. Sledge microtome sections of 120-mm thickness were excised, dehydrated, cleared and mounted with Distrin plasticizer xylene mounting media. Camera lucida tracing Eight to 10 basolateral amygdaloid neurons from each rat were traced using camera lucida. The basolateral amygdala exhibits pyramidal-like neurons. These cells have pyriform or pyramidal somata that vary in size, depending on the nucleus. The dendrites arise from the cell body. Normally, the morphology of all of these cells is the same, and there is no variation between cells. Dendritic branching points (a measure of dendritic arborization), dendritic intersections (a measure of dendritic length) and dendritic processes that arose from the somata of the amygdaloid neurons were counted (Fig. 1). Neurons with a minimal overlap of dendrites, heavily impregnated with silver nitrate and without truncated dendrites were selected for tracing.