Neuronal dysfunctions and cognitive deficits in a multi-hit rat model following cumulative effects of early life stressors

Perinatal protein malnourishment is a leading cause for mental and physical retardation in children with poor socioeconomic conditions. Such malnourished children are vulnerable to additional stressors, that may synergistically act to cause neurological disorders at adulthood. In this study, the above mentioned condition is mimicked via a multi-hit rat model in which pups born to protein malnourished mothers (LP) were co-injected with polyinosinic:polycytidylic acid (Poly I:C; viral mimetic) at Postnatal day (PND) 3 and lipopolysaccharide (LPS; bacterial mimetic) at PND 9. Individual exposure of Poly I:C and LPS was also given to LP pups to correlate chronicity of stress. Similar treatments were also given to control pups. Hippocampal cellular apoptosis, β III tubulin catastrophe, altered neuronal profiling and spatial memory impairments were assessed at PND 180, using specific immunohistochemical markers (active caspase 3, β III tubulin, doublecortin), Golgi studies and cognitive mazes (Morris Water Maze and T maze). Increase in cellular apoptosis, loss of dendritic arborization and spatial memory impairments were higher in multi-hit group, than the single-hit groups. Such impairments observed due to multi-hit stress, mimic conditions similar to many neurological disorders and hence it is hypothesized that later life neurological disorders might be an outcome of multiple early life hits. Summary Statement This study is first of its kind which practically studies the combined effects of major early life stressors like protein malnourishment, viral and bacterial infections on the nervous system.


Introduction
Approximately, 4 million neonatal deaths occur every year due to social crisis and 99% of such neonates belong to underdeveloped countries (Malqvist 2011). A child that survives poverty and social crisis might be at a higher risk for developing later life neurological disorders and hence, the concept of perinatal multi-stress emerges according to which, the children that encounter stress during perinatal period in the form of malnourishment, viral and bacterial infection, parental separation and abuse are vulnerable to development of neuropsychiatric disorders (Lai and Huang, 2011;Hoeijmakers et al., 2015;Syed and Nemeroff, 2017;Malinovskaya et al., 2018;Sarkar et al., 2019). The UNICEF data 2018, projects that 50% of death among neonates is due to undernutrition and additionally, nutritional inadequacy is also attributable to severity and death caused by common infections by delaying the recovery process of body. Again, according to world hunger index among different type of malnourishment, protein malnourishment is majorly responsible for retardation, delayed physical and mental growth and child death.
Furthermore, viral and bacterial infectionsare another common issue in already malnourished children belonging to the poorer community because of the unhygienic surroundings in which the children grow (Rytter et al., 2014;Walson and Berkley, 2018). All these stressed conditions can together modulate the CNS and may be responsible for causing later life brain adversities.
Protein malnourishment, Poly I:C (double stranded RNA used to create a viral mimic animal model) and LPS, (bacterial endotoxin, widely used as a bacterial infection agent) are reported to change the neuronal architecture of fetal brain by destabilizing the synaptic connectivity leading to weakening the process of memory formation, retention and consolidation capacity in rats (Nishi et al., 2010;Yirmiya et al., 2011;Alamy and Bengelloun, 2012;Naik et al., 2015;Sinha et al., 2018). Both malnourishment and immune inflammation during early age play deleterious role via interacting with cellular integrity of the brain, hence multi stress together might increase the chances of brain deterioration by many folds. Connectivity failure in neuronal circuitry is one of the consequences of early life stress exposure, caused due to deformities of residential hippocampal neurons that further lead to memory wreckage in stressed individuals (Aas et al., 2012) which is very much common in case of neurological disorders like Alzheimer's, Schizophrenia, autism etc. (Taylor et al., 2014;Dalle and Mabandla, 2018;Justice, 2018).
Decline in the number of viable neurons by caspase mediated apoptotic pathways are common in stress induced models (Moleur et al., 1998;Watanabe et al., 2002). Caspases are main executer cysteine proteases, that when activated, nucleophilically attack targeted proteins resulting in cleavage and cellular apoptosis. Neuronal degeneration could be due to stress mediated apoptotic pathways during which activated caspases cleaves cytoskeletal and integral cellular proteins, fragmenting and decreasing the overall cell number in brain. Cytoskeletal proteins are the building blocks of cellular extensions including neuronal arborization and are crucial for stable synapse formation, proper translation, transport and alignment of cytoskeletal subunits (Koleske, 2013).
β III tubulin is a subclass of tubulin family encoded by TUBB3 gene in humans, specific to neurons and can be used as a marker for mature neuronal profiling. Neuronal damage can be identified as fragmentation and disruption of β III tubulin 4 subunits leading to loss of dendritic arborization and connectivity (Poirier et al., 2010;Verstraelen et al., 2017), further heading an affected individual towards neurological disorders like Alzheimer's, Autism, ADHD and Schizophrenia (Bishop et al., 2010). Among neuronal sub types, pyramidal neurons are associated with memory functioning (Kasai et al., 2010) and during memory impairment pyramidal neurons of CA1 (CornuAmmonis) and CA3 regions of hippocampus are largely found to be affected (McEwen et al., 2016). The foremost effects of stress on neurons was identified as alterations in the classical morphology of the neurons that appeared either stubbed, confined or extra-long types. In the stress induced brains, there is also an overall increase in dendritic fragmentations and neuronal death. Decrease in the arborization of neurons due to cell death and fragmentation causes connectivity failure interrupting synapse formation which is a common cause for memory impairment and neurological abnormalities (Kulkarni and Firestein, 2012).
Loss of mature neurons also calls for formation of new neurons (Kuhn et al., 2015;Quadrato et al., 2014).
New neurons during adult neurogenesis are formed in the Dentate Gyrus (DG) in all mammalian species including humans (Synder et al., 2011). The newly formed immature neurons subsequently migrate and integrate into the circuitry on demand i.e., when and where necessary. Naive neurons are initially high in excitability and low in inhibition but, after maturation and integration in the functional layers, they become stable and start extending their processes, forming new arborizations and connections with the residential local neurons (Hastings and Gould, 1999;Dieni et al., 2016). Doublecortin (DCX), a neuronal migratory microtubule protein encoded by DCX gene is specific for naive and migratory neurons (Brown et al., 2003). Stress induced changes in adult neurogenesis have been reported by many researchers but relation between neuronal disintegration, adult neurogenesis, neuronal migration and memory impairment has not been well established.
The overall cellular malfunctioning in hippocampus due to various early life stressors may finally lead to memory impairments, which can be considered as onset of neuropsychiatric disorders. Hippocampus is associated with spatial memory impairments (Broadbent et al., 2004;Shrager et al., 2007) which includes both spatial reference and spatial working memory (Niewoehner et al., 2007;Bizon et al., 2012). Spatial reference memory can be differentiated from spatial working memory as the former allows left right discrimination whereas the latter is used to keep an account of recent activities like completing a task or understanding a sentence (Cowan, 2014).
Hippocampus was initially found to be responsible only for declarative or long-term memory but recent studies have supported the involvement of hippocampus with working memory retention (Jeneson et al., 2011;Leszczynski, 2011;Yee et al., 2014). Such is the need for cognition in normal living conditions and the impairment of cognitive abilities can directly be associated with neuropsychiatric disorders (Lajud and Torner, 2015).
Most stress-oriented studies deal with a single type of stressor. Early developmental windows are prone to every minute changes occurring in the environment and hence the developing period is susceptible to multiple stressors which cumulatively may act on the developing brain causing later life abnormalities. Moreover, the synergistic and coordinated action of multiple early life stressors on the developing brain is highly challenging and require utmost attention. In this study, maternal malnourishment is used to create a stressed uterine environment and the F 1 pups born from both healthy and malnourished mothers were further subjected to viral and bacterial infections 5 with either polyinosinic:polycytidylic acid (Poly I:C) or Lipopolysaccharide (LPS) or both simultaneously. As malnourished subjects might be susceptible to immune infection, such multi-hit models depict the scenario relatable to most of the underdeveloped countries. Altered morphology and degeneration of neurons, in the rat hippocampus, was studied in context to memory impairment, using specific immunohistochemical markers and memory oriented cognitive mazes. and LPS were administered simultaneously to LP animals (LP+Poly I:C+LPS group), a vigorous increase in active caspase 3 positivity was recorded (Fig. 2H). Similar treatments were also administered to the HP animals (Figs. 2B, C, D) but the extent of cellular damage in HPgroupswere much less as compared totheir respective LP groups.

Results
The changes vide supra were further confirmed through the quantitative data ( Fig. 2I)   Additionally, from the low magnified images of hippocampus, it was visible that on chronic stress exposure i.e., in LP+Poly I:C+LPS animals, the CA layers became improvident due to increased density and overcrowding of damaged dendrites (Figs. 4H a).
Sholl analysis on camera lucida traced neuronal images was performed to assess morphometric variation including dendritic arborization and length among treated and control groups. Total number of Sholl circles at an interval of 20µm gave length of dendrites whereas dendritic arborization was calculated from the mean total number of intersections made by dendrites on the circles.Specifically, pyramidal neurons were chosen for analysis and hence as per the shape of such neurons, numbers of intersecting points in the Sholl circles were maximum at 40µm, which further decreased with increase in the length of the neurons. The histogram was plotted with values of the distance from the soma by X axis and mean number of intersections between dendrites and Sholl circles by Y axis, which gave an idea of the morphometric differences between pyramidal neurons of HP control and other treated groups  Table 1, given under supplementary data).

Stress triggered spotting of immature neurons, identified by anti-DCX immunolabelling of naive neurons
Damage induced increase in DCX positive neurons (arrows), was observed in treated groups. The spotting of DCX positive neurons in CA layer were directly proportional to the amount of damage that occurred to residential neurons. Chronic stress due to multiple-hitin LP animals i.e., LP+Poly I:C+LPS (Figs. 6H, 7H) led to a vigorous increase in DCXpositive cells in and around CA layers and DG. Additionally, the DCX positive cells were not discrete and appeared to be clumped in DG may be because of chronic stress related damage to naive DG neurons.
Comparatively The morphometric analysis of DCX immunoreactivity in CA1, CA3 and DG (Fig. 7I)

Impaired spatial memory during MWM and T maze task, on exposure to various early life stressors MWM
Animals were trained to use spatial clues for navigation and localization of escape platform during MWM task. From the latency and mean path efficiency graph (Fig. 8I, J), it was noted that due to severe chronicity, the latency was highest and path efficiency was lowest in multi-hit, i.e., LP+Poly I:C+LPS animals when compared with HP control (F (7,88) =16.17, P≤0.001, F (7,88) =11.4, P≤0.001) and LP alone group (F (7,88) =13.59, P≤0.001), which meant that they took more time and followed complex path while reaching the escape platform. LP alone animals 9 were also found to have an increasedlatency and decreased path efficiency depicting impaired spatial memory when compared to HP control (F (1,66) =9.54; P≤0.001, impact of LP diet) which on further immune activation with either Poly I:C or LPS was heightened as the latency increased and path efficiency decreased in LP+Poly I:C and LP+LPS animals when compared to HP control (F (7,88) =5.6; P≤0.001; F (7,88) =5.8; P≤0.001) and similarly treated HP groups i.e., HP+Poly I:C (F (1,66) =5.9; P≤0.001) group. Significant difference in latency were also found between LP, LP+Poly I:C (F (3,44) =10.4, P≤0.001) and LP, LP+LPS groups (F (3,44) =12.6, P≤0.001). Also, when percent time spent in target quadrant data was analyzed (Fig. 8K), it was seen that HP control group had maximum time spent in the target quadrant to which the platform belongs, avoiding the other zones. However, such effects of multi-hit stress were much less severe in HP animals may be because of their ability to resist or rectify.
From the track records of MWM task, indirect and complex trajectory depicting impaired spatial memory was seen in LP ( Fig. 8E) alone, LP+Poly I:C (Fig. 8F), and LP+LPS (Fig. 8G) groups. The HP control animals (Fig.   8A) followed a direct path from the start point to the escape platform which the treated animals failed to do. The complexity of tracks and impairment of memory increased in with increase in chronicity of stress and hence, the LP+Poly I:C+LPS animals as they took indirect and haphazard path and reached the escape platform after doing multiple errors when compared with other groups (Fig. 8H). Single or combo exposure of Poly I:C or LPS to HP animals also showed some impairment in path efficiency (Figs. 8B, C, D), but was comparatively lesser than the respective LP group animals.

T Maze
Alternate baited arm protocol was followed in T maze task to check spatial working memory among all the groups. The mean path efficiency data was plotted in graph (Fig. 9I), from which, it was interpreted that spatial working memory was impaired following stress. Such spatial memory impairment was seen to be highest in multihit group i.e., LP+Poly I:C+LPS as the animals were not able to distinguish between left and right arms, they committed more error while choosing the correct arm to obtain reward (F ( The track records revealed distorted and repeated track in treated animals showing errors while choosing the right baited arm. It was evident from the complex and haphazard track of LP+Poly I:C+LPS animals that they had maximum impairment in their spatial working memory when compared to other treated groups (Fig. 9H).
Lastly, with in the HP groups also, there was impairment in memory upon treatment with Poly I:C and LPS either singularly or in combination.

Discussion
The role of perinatal stress as causative factor for neurological disorders like Schizophrenia, Alzheimer's, Parkinson's etc. has been well reported (Huang, 2014, Hoeijmakers et al., 2015, Hohmann et al., 2017. However, the mechanism of action of stressors and the influence of one type of stressor on another still remains unclear. Some of the studies have focused the dual-hit hypothesis according to which, first hit at genetic level during critical developmental periods increases the risk of second-hit, which is generally an environmental insult like infection and nutritional deficiency. Dual-hit exposure may act synergistically and give rise to Schizophrenic symptoms in adult individuals (Maynard et al., 2001;Feigenson et al., 2015). However, due to a wide range of stressors and their diverse mode of action, dual-hit hypothesis alone is not sufficient to provide a link between stressors and neurological disorders. This study is the first one to address and streamline the events that lead to symptoms seen during CNS malfunctioning in a multi-hit model.
In this study, perinatally multi-hit animals were found to have highest active caspase 3 expressions in hippocampus when compared with other groups and additionally, dendrites of such chronically stressed animals also showed active caspase 3 expressions depicting their degeneration and neuronal dysfunction. Such upregulation of active caspase 3 could be a reason for low β III expression in multi-hit groups. The multi-hit animals also subsequently exhibited damaged neuronal profile and decreased dendritic arborization, may be because of vigorous active caspase 3 triggered β III tubulin damage. Simultaneous upregulation of DCX protein was also found in DG and CA layers of multi-hit animals with chronic stress leading to scattering of DCX positive cells in CA layers. Such altered cytoarchitecture of neurons in hippocampus was finally reflected as the impaired spatial memory in stressed animals which was directly proportional to the chronicity of stress. Lastly, it was also seen that LP animals were prone to infection as they reacted more vigorously to a subsequent stress exposure when compared to HP animals suggesting thereby that the stressors are interdependent and act synergistically which has been variously hypothesized (Schaible and Kaufmann, 2007;Katona and Apte,2008).
Active caspase 3 is an apoptotic marker used to study the extent of cellular damage during stress exposure (Mcllwain et al., 2015). Chronic stress in multi-hit (LP+Poly I:C+LPS) animals was found to be responsible for increase in caspase 3 protein in hippocampus, which was higher than the single-hit groups. Such chronic stress dependent increase in active caspase 3 protein was also reported in a depressed rat model by Bachis and co-workers (2008). Restrained stress when combined with forced swim stress was also found to increase the caspase 3 level in 1 1 prefrontal cortex of Wistar rats (Zhang et al., 2017). Moreover, Toll like receptors (TLR) activated by viral and bacterial pathogens along with oxidative stress are also found to be responsible for activating caspases and inducing apoptosis in cells leading to neurodegeneration (Snigdha et al., 2012;Loeches et al., 2014;Sharma et al., 2016).
Active caspase 3 dependent neuronal death in case of specific virus-oriented encephalitis, viz., West Nile virus encephalitis and Japanese encephalitis are also being reported (Samuel et al., 2007;Mishra and Basu, 2008). Thus, all the single stress-oriented studies (vide supra) indicate that caspase 3 mediated cell death is a major contributor to neurodegeneration and hence tally with the active caspase 3 data presented in this study. Moreover, the combined exposure of multiple stressors (multi-hit) is responsible for vigorous cell death in hippocampus, further accelerating the occurrence of neurodegeneration and related disorders.
Beside cell death, activated caspase 3 also causes microtubule distortion leading to dendritic loss and stunting in neurons, prominent in Alzheimer's disease (Troy and Jean, 2015). Stress dependent damage of mitochondrial membrane also leads to activation of caspase 3 in dendrites, causing fragmentation (Mattson et al., 1998;Amelio et al., 2011). Such activecaspase 3 labeled dendrites were also seen in chronically stressed multi-hit group in the present study confirming their fragmentation. Moreover, caspase 3 has been reported to cleave the tubulin protein causing both axonal and dendritic degeneration (Sokolowski et al., 2014). Thus, the caspase 3 activation during various stress conditions may be a crucial factor leading to β III tubulin degradation.
Multi-hit stress in our study led to an extensive β III tubulin degeneration in neurons, which could be linked . Thus, from our β III tubulin data it can be interpreted that multi-hit stress can make an individual prone to neurological disorders as cytoskeletal changes may lead to neurodegeneration leading to behavioral and cognitive deficits through compromised neuronal arbors.
Stunted neurons due to fragmented and drooping dendrites were common in chronically stressed multi-hit group. Although no multi-hit studies have been reported so far, our result can still be supported by the single-hit studies which reported that following viral or bacterial infections, the CA1 neurons in rats becomes architecturally damaged with reduced dendritic length and arborization further leading to cognitive impairment (Vyas et al., 2002;Radley et al., 2004;Murmu et al., 2006;Baharnoori et al., 2009;Jurgens et al., 2012). Thus, multi-hit stress could be a major factor that accelerates cellular damage further causing memory impairment leading to neurodegenerative disorders.
Alongside cellular damage, stressors are also reported to enhance adult neurogenesis in order to compensate for regional damage. In the present study as well, the DCX positive cells were spotted around CA layers and DG of stressed animals. DCX is reported to be strongly expressed by migrating immature neurons (Kaindl et al.,

2
2006; Klempin et al., 2011;Madhyastha et al., 2013) and in our study, mainly after the multi-hit exposure, DCX expression was upregulated, suggesting an increase in migration of newly generated neurons. Some studies have reported the difference in extent of neurogenesis between sub ventricular (SVZ) and sub granular (SGZ) zone, stating that SVZ regions contains more neural stem cells and with additional amplifying signals, the extent of neurogenesis in SVZ is somewhat higher than SGZ region (Zhu et al., 2014;Ghosh et al., 2019). However, some studies have also reported a decrease in DCX positive cells in hippocampus of rats on chronic stress exposure (Dagyte et al., 2009). This could be because that DCX positive immature neurons were unable to be incorporated in to the circuitry due to chronicity of stress and hence were prone to degeneration suggesting that increase in chronicity decreases the chance of maturation and survival of neurons born as a result of compensation mechanism (Nacher et al., 2004, Lemaire et al., 2005Naninck et al., 2014). Hence in our study even with an increase in adult neurogenesis, the cognitive abilities of stressed animals remain impaired with the multi-hit animals showing maximum memory impairment in MWM and T maze task which can be further linked with occurrence of neurodegeneration related disorders in adult animals.
Memory impairment is a hallmark of neurodegenerative disorders which occurs due to stress dependent altered neuronal circuitry (Esch et al., 2002;Akers et al., 2006;Wen et al., 2017). Early life exposure to stressors like Poly I:C, LPS and perinatal protein malnourishment individually are well reported to cause memory impairment in rats (Naik et al., 2015;Zakaria et al., 2017;Baghel et al., 2018). However, in the present study all these early life stressors in combination were found to heighten later life memory impairments. Such multi stress exposure during early life was found to cause chronic damage to spatial memory in LP+Poly I:C+LPS animals via stress dependent caspase 3 activation and β III tubulin catastrophe leading to loss of neuronal connectivity and subsequent neurodegeneration in adult rats.

Animal husbandry and early life stress induction:
Wistar rats were maintained in the animal house facility under controlled physical environment (temperature=25±1 o C, humidity=65±2%, light and dark cycle=12 hr). Before shifting to the experimental diet, all F 0 animals were given ad libitum access to clean drinkable water (reverse osmosis water) and rat pellet feed. 32 Virgin females (3 months old, body weight; b.wt., 120-140gms) were selected and shifted to control i.e., high protein (HP; 20 % protein, n=16) and low protein (LP; 8% protein, n=16) diets, 15 days prior to mating and then maintained on their respective diets throughout gestation and lactation periods (isocaloric rat feed, both 8% and 20% protein diet were obtained from National Institute of Nutrition, Hyderabad, India). The day of parturition was noted as postnatal day 0 (PND 0). The F 1 pups from both HP and LP dams were used for creating the following groups. Litter size adjusted to 8 to avoid variation.
Control: HP F 1 pups (n=32, from four different dams) without any treatment were considered as controls. They were maintained with their respective dams and used for various studies at the age of PND 180 (b.wt. 230±10gms).
1 3 Low protein model (LP, single stress): F 1 pups from LP dams (n=32, from four different dams) were used as LP alone group. The animals were maintained in LP diet and used for various studies at the age of PND 180 (b.wt. 180±10gms).  Golgi Technique: From each group, animals (n=6, belonging to different dams/group) were decapitated at PND 180, followed by dissection and immediate immersion of brain tissues in Golgi fixative solution (consists of sodium dichromate, chloral hydrate, formaldehyde, glutaraldehyde and DMSO). The tissues were immersed fixed for 72 hrs and then treated with 0.75% silver nitrate (Qualigens) solution for minimum 48 hrs. After complete impregnation, the sections (100µm thickness) were cut using a Leica Vibratome (VT 1000s). The sections were dehydrated, mounted in DPX and stored for neuronal analysis (Kumar et al., 2013).

Morphological analysis of neurons:
Neurons belonging to different animals from each group were traced using camera Lucida, attached to Leica DME microscope (n=36 neurons from 6 different slides/group). Sholl analysis for dendritic length and arborization was performed for each neuron, using ImageJ freeware software. The number of intersections in each Sholl circle was analyzed for complexity and length of neurites (Baharnoori et al., 2008).

Cognitive experiment for memory analysis at PND 180 (n=12/group from different dams):
Morris Water Maze (MWM): Spatial memory was assessed using MWM (Columbus Instruments) as per the procedure opted by Naik et al., (2015). Initially, the animals were acclimatized and taught to navigate the hidden/escape platform using spatial clues for three consecutive days (4 trial each animal for 120 second). 60 min of gap was given between the trials. During the learning period, if the animals failed to reach the platform, they were manually guided to the escape platform following the shortest and direct path from the start point to the escape platform. On the fourth day i.e., after 24 hrs, the animals were subjected to MWM test to locate the escape platform within 120 sec. Platform was removed on the fourth day to avoid visual error (probe test). Data was recorded for three trials using a vertical camera connected to an Any Maze software version 4.82. Parameters like the mean path efficiency i.e., the shortest route taken by the animal to reach the escape platform, latency i.e., the time taken to reach the escape platform and the percent time spent in the target quadrant (north-west) was analyzed for each and every group.
T Maze: For spatial working memory assessment, T maze from Columbus instrument was used and the protocol was designed according to Nagayach et al., (2014). It is a reward-based test, in which animals were trained and taught to detect the baited arm by remembering previous visited arm. It is a T shaped maze with one start and two reward arms. The animals were also made to learn left right discrimination in this test. Initially, the animals were acclimatized by placing in the maze and allowing them to explore the whole maze. During training, the animals were placed in the start arm and allowed to arbitrarily choose an arm for 30s. The animals were then removed and 1 6 again placed on the start arm, expecting to choose the unexplored arm. The reward was placed at the end of either left or right arm and the position of the reward was changed after every trial. The animals were taught to obtain the reward making minimum effort while choosing the correct arm. 4 trials each of 120s were given to each animal with a gap of 60 min between trials for 3 consecutive days. On the 4 th day, 3 trials for each animal were performed and the mean path efficiency to reach the reward/baited arm was recorded using a vertical camera attached to an Any Maze software version 4.82.
Statistical Analysis: Data were analyzed using One Way ANOVA (for group wise comparison) and Two-Way ANOVA (for comparison between groups belonging to two independent variables i.e., diet and treatment) followed by post hoc Holm-Sidak test using Sigma Plot 12. Significance level was preset at P≤0.05.
The animals were maintained and experimental plan was designed with prior permission from Institutional Animal Ethics Committee of Jiwaji University, Gwalior (M.P), India.
1    control (A) and other treated groups (B, C, D, E, F, G). The LP animals on treatment with either Poly I:C or LPS (F, G) also performed poorly than the HP control (A) and corresponding HP treated group (B, C), (n=12/group).

Figure legends
The graphical analysis of mean latency, path efficiency and percent time spent in target quadrant (I, J, K) depicts memory impairment in HP and LP animals on Poly I:C and LPS administration as the animals belonging to treated groupstook longer time to reach the escape platform, followed complicated trajectory and spent less time in the target quadrant. Maximum deficit was shown by LP+Poly I:C+LPS animals, when compared to rest of the groups. Within the group significant difference was found between HP+Poly I:C and LP+Poly I:C animals, showing that on administration of Poly I:C or LPS, LP animals suffered more damage when compared to HP animals. HP and LP treated Single-hit groups showed spatial memory impairment with decrease in path efficiency as seen from the T maze (I) when compared to HP control but damage was chronic in case of multi-hit group when Poly I:C and LPS was administered sequentially in LP animals i.e., LP+Poly I:C+LPS group. (n=12/group).Valuesof One-and Two-Way ANOVA are expressed as mean±SEM; **P≤0.005, ***P≤0.001 with respect to controls; α α P≤0.005, with respect to HP+Poly I:C and LP+Poly I:C