The present study assessed the effect of different levels of n-3 fatty acids in the diet on PPI when animals were fed throughout gestation, lactation, and into adulthood. The influence of the diet on acoustic startle reflex when intervention was limited to distinct developmental stages (i.e., during prenatal exposure, early postnatal exposure, pubertal exposure, adult exposure) was beyond the scope of the study, as well as changes in startle responses in dams. Previous studies have found that altered postnatal maternal care can produce disruptions of PPI in adulthood. For example, PPI deficits have been found in rats and mice subjected to the stress of preweaning repeated maternal separation by some researchers (Ellenbroek & Cools, 2002
; Geyer et al., 1993
) but not by others (Lehmann et al., 2000
; Millstein et al., 2006
). Thus, given the fact that behavioral alterations in the dam might lead to long-term impairment of the offspring, we evaluated dam maternal behavior as evidenced by nest building and pup development and growth. No gross abnormalities were observed in any experimental group; all pups gained weight and developed similarly. These observations make it less likely that the observed PPI deficits in the offspring are related to effects of dam behavior but this possibility cannot be excluded.
Our findings of similar time of appearance of developmental milestones in pups from different dietary groups are in disagreement with the previous study by Haubner et al. (2002)
in which dietary supplementation of rat dams with DHA (3% of total fatty acids) during pregnancy and lactation led to a later appearance of acoustic startle response in their pups and longer auditory brainstem conduction times (ABCTs). Because ABCTs are strongly associated with the degree of myelination of the auditory brainstem, the authors suggested that exposure to high levels of DHA during development may negatively impact myelination of the auditory brainstem. In the present study, however, the timing of appearance of the auditory startle reflex was about the same in all four dietary groups. Moreover, imaging studies of DHA supplementation of children with Zellweger’s disease has led to the suggestion that DHA promoted myelination and can even lead to remyelination (Martinez & Vazquez, 1998
The present results indicate that the depletion of n-3 fatty acids from the diet leads to a pronounced deficit in the prepulse inhibition of the acoustic startle response. Mice fed the n-3 Def and Low LNA diets had lower PPI levels compared to the High LNA and DHA+EPA groups (). The n-3 Def and Low LNA groups behaved similarly in terms of magnitude of the prepulse inhibition and habituation to the startle stimuli, although fatty acid composition of the brain tissues was quite different between these groups. The n-3 Def mice accumulated only half the DHA and significantly more ARA, DPAn-6 and DTA than the Low LNA group (). These results suggest that the amount of DHA in the brain does not solely predict the behavioral outcome; the n-6 fatty acid status also influences the PPI measures. This was confirmed by the differences between the Low LNA and High LNA groups: mice fed the High LNA diet exhibited significantly higher PPI compared to the Low LNA mice, whereas the brain DHA content of these mice was only slightly higher, but levels of all three major n-6 fatty acids (ARA, DPAn-6, and DTA) were much lower in the High LNA mice compared to the mice fed Low LNA diet.
Further, a difference between the High LNA group and mice supplemented with DHA and EPA in their diet was observed: the DHA+EPA group showed higher prepulse inhibition levels but only slightly (about 12%) more DHA in the brain tissues, while the content of ARA and DPAn-6 was the same in these mice. This is the first evidence of the differential effects of High LNA and DHA+EPA diets on behavior. This finding suggests that supplementation with preformed DHA and EPA is beneficial for the sensorimotor gating compare to a diet loaded with a high amount of LNA.
Although these diets were designed principally to manipulate the brain DHA content, these results indicate that this variable does not by itself predict the behavioral outcome. Overall, no correlation between the level of any major long-chain fatty acid (DHA, ARA, DPAn-6 and DTA) and PPI magnitude was observed. Rather the complete fatty acid profile may in some manner determine the PPI response.
There has been a discussion about whether deficits in PPI reflect a sensorimotor gating deficit (leading to compromised processing of prepulse) or an impairment of attention leading to a reduced detectability of the prepulse (summarized in Koch, 1998). It seems likely that both mechanisms play a role, as Swerdlow and co-workers have repeatedly shown that treatments that impair PPI do not affect the reduction in the startle peak latency that occurs concomitant to PPI, indicating that the animals are still able to detect the prepulse under conditions that reduce PPI (Swerdlow et al., 1992
). On the other hand, in humans PPI is enhanced if the subjects attended to the prepulse (Jennings et al., 1996
). Obviously, there are important attentional components involved in PPI, indicating that the PPI mechanism is more than a pure sensorimotor gate that is a prerequisite for attention. Attentional mechanisms affect PPI at a perceptual level, whereas higher levels of stimulus processing (cognitive processes) are protected by the gating mechanism underlying PPI (Koch, 1998). Both attentional and sensorimotor mechanisms are involved in the learning process and may underline spatial learning and memory deficits that have been repeatedly reported in n-3 deficient animals (for review, see Fedorova & Salem, 2006
PPI of the acoustic startle reflex is reduced in a variety of neuropsychiatric disorders, for example in schizophrenia, schizotypal personality disorder, Huntington’s disease, obsessive compulsive disorder, Tourette’s syndrome, bipolar disorder, and attention deficit disorder (summarized in Swerdlow and Geyer, 1998
). The PPI deficit observed in patients with schizophrenia is thought to be a measure of the general reduction of the ability to gate intrusive sensory, motor or cognitive information (Geyer at al., 2001
; Braff et al., 1992
; Kumari et al., 2000
; Parwani et al., 2000
). Some attempts have been made to treat symptoms of schizophrenia with n-6 and n-3 fatty acids. The results of these clinical trials are summarized elsewhere (Fenton et al., 2000
; Peet, 2003; Freeman et al., 2006
; Joy et al., 2006
; Kidd, 2007
; Ross et al., 2007
). Double-blind trials of n-6 fatty acid supplementation of neuroleptic medication have yielded negative results (Joy et al., 2006
). In contrast, most of n-3 fatty acid supplementation trials report positive results. To date, six double-blind randomized clinical trials with DHA/EPA have been conducted, involving 390 patients with schizophrenia or schizoaffective disorder. Four trials documented clinical benefit from 2 g EPA daily for three months (Emsley et al., 2002
; Peet et al., 2001
; Peet & Horrobin, 2002
). However, the effect sizes were small in most trials, and significant improvement occurred only at one dose in a group of patients having a specific background treatment (Ross et al., 2007
). Meta-analyses of treatment studies of schizophrenia found that n-3 PUFA failed to improve schizophrenic symptoms as measured by the PANSS total score (Freeman et al., 2006
; Joy et al., 2006
Habituation of the acoustic startle response as assessed by changes of average startle amplitude in the first and last blocks of the experiment (pulse-only trials) showed differences between the mice fed the n-3 Def and the Low LNA diets, and the ones on the High LNA and DHA+EPA supplemented diets (). The High LNA and the DHA+EPA groups demonstrated diminished startle responses at the end of the experiment compare to the beginning, suggesting an adequate habituation process. Whereas the n-3 Def and the Low LNA groups, on the contrary, did not change their responses during the test, indicating a deficit in short-term habituation. Within-session, or short-term habituation, is the decline of the acoustic startle reflex magnitude following repeated presentation of startling stimuli within a single test session. We have previously reported that n-3 deficient mice showed impairment in the habituation to a novel environment as they did not decrease their locomotor and exploratory activity in the open field test with time to the same extent as n-3 adequate animals (Fedorova and Salem, 2006
; Fedorova et al., 2009
). In the present study, we observed a habituation deficit in a different paradigm not requiring locomotion or exploration, suggesting that the impaired habituation phenomenon is attributable to the n-3 fatty acid deficiency. Interestingly, schizophrenic patients are also found to be impaired in the habituation of acoustic startle response (Braff et al., 1992
; Parwani et al., 2000
The results of the present study reveal that n-3 fatty acid deficiency induces profound behavioral changes in mice. For the first time, a deficit in sensorimotor gating as assessed by prepulse inhibition of the acoustic startle response was observed in mice fed the n-3 fatty acid deficient and a Low LNA diet. There is a large body of evidence on the importance of n-3 status for learning and memory, but PPI reflects an automatic, involuntary inhibitory process that functions to protect the initial processing of the information. Further, these are the first data that differentiate in rodents the effects of the High LNA diet from one with added EPA/DHA. This technique is objective, readily used in human studies and clearly sensitive to small changes in nervous system DHA content such as may be induced by variations in dietary DHA intake.