Slow Physical Growth, Delayed Reflex Ontogeny, and Permanent Behavioral as Well as Cognitive Impairments in Rats Following Intra-generational Protein Malnutrition

Abstract

Environmental stressors including protein malnutrition (PMN) during pre-, neo- and post-natal age have been documented to affect cognitive development and cause increased susceptibility to neuropsychiatric disorders. Most studies have addressed either of the three windows and that does not emulate the clinical conditions of intra-uterine growth restriction (IUGR). Such data fail to provide a complete picture of the behavioral alterations in the F1 generation. The present study thus addresses the larger window from gestation to F1 generation, a new model of intra-generational PMN. Naive Sprague Dawley (SD) dams pre-gestationally switched to LP (8% protein) or HP (20% protein) diets for 45 days were bred and maintained throughout gestation on same diets. Pups born (HP/LP dams) were maintained on the respective diets post-weaningly. The present study aimed to show the sex specific differences in the neurobehavioral evolution and behavioral phenotype of the HP/LP F1 generation pups. A battery of neurodevelopmental reflex tests, behavioral (Open field and forelimb gripstrength test), and cognitive [Elevated plus maze (EPM) and Morris water maze (MWM)] assays were performed. A decelerated growth curve with significantly restricted body and brain weight, delays in apparition of neuro-reflexes and poor performance in the LP group rats was recorded. Intra-generational PMN induced poor habituation-with-time in novel environment exploration, low anxiety and hyperactive like profile in open field test in young and adult rats. The study revealed poor forelimb neuromuscular strength in LP F1 pups till adulthood. Group occupancy plots in MWM test revealed hyperactivity with poor learning, impaired memory retention and integration, thus modeling the signs of early onset Alzehemier phenotype. In addition, a gender specific effect of LP diet with severity in males and favoring female sex was also noticed.

Keywords: Morris water maze; anxiety; cliff avoidance; elevated plus maze; intra-generational protein malnutrition; open field test.

Figure 1

Maternal PMN permanently compromises the body weight gain in later life: file photo of HP and LP group pups at the age of PND 21 (A) and 3 months (B) showing physical slowing and reduced body weight gain in LP F1 generation rats. Loss of hair was a prominent feature at 3 months of age in LP rats.

Figure 2

Lactational but not gestational protein malnutrition results in decreased body and brain weight gain in F1 generation: A daily based pre-weaning body weight gain trend in HP and LP group pups with advancing postnatal age revealed a decelerated growth in the LP F1 pups. Surprisingly, no significant difference was noticed at birth (PND 0). However, the body weight difference was highly significant from PND 3 to 30 (A) and even at maturity (B). Graph (C) shows the gross brain weight of LP and HP group animals on some representative days. By PND 5 onwards, the gain in brain weight was consistently and significantly lower in LP animals and continued till the last study time-point. Data is presented as Mean body/brain weight in grams ± SE. ^***P < 0.001.

Figure 3

Maternal PMN delays neurodevelopment and performance in F1 generation rats: Graphs showing the Surface righting reflex in HP and LP group males (A) and Females (B) as Mean score value against postnatal day. A complete surface righting reflex with a score of 2 was attained by PND 6 in both HP and LP Pups with no significant sex related difference. Inclined Plane test results in HP and LP F1 males (C) and Females (D) as mean score value against postnatal day for 100% attainment of the reflex reveal a significant effect of pre-gestational protein malnutrition on the reflex maturation, with HP F1 pups presenting the negative geotaxis response significantly earlier and better in terms of scoring. Data is presented as Mean Score Value ± SE. ^**P < 0.01, ^***P < 0.001.

Figure 4

Maternal PMN significantly delays cliff avoidance reflex in F1 generation pups: Graphs showing the Cliff avoidance reflex data as mean score value in HP and LP group males (A) and Females (B) at representative postnatal days. HP pups attained this reflex by PND 7 with no sex dependent difference in performance on score levels and reflex completion. A statistically significant delay of 5 day was recorded in LP group pups as the reflex maturation was attained by PND 12. Data is presented as Mean Score Value ± SE. ^**P < 0.01, ^***P < 0.001.

Figure 5

Hyperactivity and anxiety like behavior displayed by LP F1 generation animals: Bar graphs representing the distance traveled (A,B) and resting time (C,D) in Open field test of HP and LP group male and female rats at 2, 3, and 6 months of postnatal age. LP F1 animals showed hyperactivity as evident by significantly increased distance traveled and less resting time as compared to HP group animals. Data is presented as Mean ± SE. ^**P < 0.01, ^***P < 0.001.

Figure 6

Maternal protein malnutrition induces hyperactivity and low basal anxiety in F1 generation pups: Graphical representation of horizontal counts (A,B) and time spent in center zone (C,D) of the Open field test in HP and LP F1 generation rats at 2, 3, and 6 months of age as a measure of the anxiety. Horizontal counts in open field estimate the basal activity of the animal with increased horizontal counts as a measure of the hyperactive behavior. LP males at the age of 2 and 6 months displayed increased horizontal activity. The behavioral bias in response at 3 months can be correlated to the sexual maturity and associated HPA hormonal axis. Time in square analysis revealed low basal anxiety levels and reduced habituation in LP group animals at 2 and 6 months of postnatal age irrespective of sex as compared to the HP controls. Data is presented as Number of counts and Time spent in seconds (Mean ± SE). ^**P < 0.01, ^***P < 0.001.

Figure 7

Track reports of open field test: Representative open field activity tracks of 2, 3, and 6 month old rats of HPM (HP F1 males), LPM (LP F1 males), HPF (HP F1 females), and LPF (LP F1 females) groups. The time in square analysis reveal low basal anxiety and reduced habituation in LP group animals irrespective of sex with significantly more time spent in center zone as compared to HP age matched controls.

Figure 8

Percent time spent in open arms of Elevated Plus maze as a measure of anxiety level in rodents: Bar graphs representing the percent time spent in open arms at 2, 3, and 6 months of postnatal age. A significant increase in both percent time in open arms and open arm entries in LP group animals at 2, 3, and 6 month postnatal age is an indicative of low anxiety and low fear behavior further validating the results from open field test. Data is presented as % time spent (Mean ± SE) (males, A; females, B) and open arm entries (males, C; females, D). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 9

Maternal PMN induces poor neuromuscular strength in F1 generation: Bar graph showing Forelimb gripstrength in HP and LP group males (A) and females (B) subjected to gripstrength test at 3 and 6 months of postnatal age. A significantly poor neuromuscular strength was recorded in LP group animals irrespective of sex at 3 and 6 months of postnatal age. Data is presented as Mean ± SE. ^***P < 0.001.

Figure 10

Morris water maze (MWM) tracks indicate poor spatial learning and memory: Representative swim tracks of HP and LP F1 animals at 3 and 6 months of postnatal age. The swim tracks represent poor spatial learning and memory in LP group animals with severe effects manifested in LP males. HP group animals swam less, presented good memory with a high path efficiency.

Figure 11

Group occupancy plots in MWM test reveal hyperactivity and learning and memory impairments in LP animals. ANYMaze Software generated group occupancy plots for activity in MWM test reveals efficient learning in HP males and females at 3 months whereas LP males and Females show more swim activity reflecting poor spatial memory. Spatial learning and memory deficits with hyperactivity in MWM task in LP group animals is evident in 6 month age group.

Figure 12

Maternal PMN leads to impaired spatial learning and memory in later life: Bar graphs representing the latency to locate the hidden platform (A,B) and path efficiency (C,D) in MWM test at 2, 3, and 6 months of postnatal age in HP and LP group animals. Data from the MWM test reveals a significantly high latency in the LP group animals to locate the hidden platform. A significant better path efficiency was noticed in HP group animals with males displaying high efficiency as compared to females well correlating with the natural tendency of females loving to swim and float. Data is presented as Mean ± SE. ^**P < 0.01, ^***P < 0.001.