Neuroscientists have at their disposal numerous model systems with which to carry out their research. A key decision in designing an experiment is choosing the best model with which to tackle the question at hand. In simpler model systems such as cell lines or in those that more closely resemble neural systems, such as embryonic or postnatal neuronal cultures, the focus of the study can be reductionist, allowing consideration of aspects of complex systems in narrower terms. Such models are extremely useful for the study of cellular and molecular mechanisms, and the limitations of these simplified systems are recognized by most readers and authors. For example, those studying the biophysical properties of ion channels in cell lines generally recognize that they are not modelling the intact nervous system.
Over the last decade there has been a noticeable shift from a ‘reductionist’ to a ‘systems’ perspective and with this has come a substantial growth in the use of ex vivo brain slices - in which neuronal architecture is better preserved - for the study of synaptic and cellular biology. This trend has been facilitated by technological advances. For example, techniques such as patch-clamp electrophysiology and cellular imaging, once only possible in isolated cells, are now routinely practiced in tissue slices. However, such studies are performed using animals of various ages and it is seldom acknowledged that they are often limited to very young animals. This is important because findings in young animals do not necessarily extrapolate to adults. Although this limitation is not confined to slice physiology, it is here that the use of young animals is most evident.
To assess the extent to which animals of different ages are being used in neuroscience research, we performed a systematic review of the literature in which we tabulated the ages at which rodents were tested. We examined (i) studies on hippocampal long-term potentiation (LTP) in rodents and (ii) studies using the Morris water maze test of spatial memory (see supporting information
for methodology and references). In rodents, hippocampal LTP has been extensively investigated as a mechanism for spatial memory (Morris et al., 2003
In our review on hippocampal LTP, we first examined studies that used ex vivo
brain tissue preparations (see supporting information
). shows that only a minority of studies (34%) used adult animals, while the majority (59%) used animals that had not yet reached adulthood (see next section for age definitions). The remaining 7% of studies did not specify an age or weight. Overall, the studies used a wide range of ages, from postnatal day 1 (P1) to P915 (i.e. 30 months). Furthermore, the age ranges at which animals were tested did not just vary between studies, but also within. Approximately 30% of experiments pooled animals whose ages varied by 3–4 weeks, often spanning critical age periods, such as puberty. We also found that 42% of studies defined animals as “adults”, although the methods section or supplementary online material
clearly indicated that the average age of the animals was not within an adult range. When we repeated this analysis using the lowest age in each study (see supporting information
for detailed explanation) we found that 60% of studies incorrectly defined young animals as “adults”. To highlight the problems in controlling for the developmental stage of rodents we looked at the various definitions of “adult” (or mature) in this cohort of papers and found that this varied across studies from a low of P24-25 to a high of P549 (18 months) (Larson et al., 2005
; Fukata et al., 2006
; Sim et al., 2006
). The definition of “young adult” varied from a low of P21-22 to a high of P92 (3 months) (Xu et al., 2000
; Meredith et al., 2003
; Miyamoto et al., 2005
; Sim et al., 2006
; Lauterborn et al., 2007
Figure 1 Results of a systematic review examining age distribution of rodents used in neuroscience experiments. A, Age distribution of subjects used in studies on hippocampal LTP using ex vivo (solid line, n=336) and in vivo (dashed line, n=112) preparations. (more ...)
When we confined our analysis of ex vivo studies to experiments using voltage- or current-clamp slice electrophysiology (patch clamp or sharp electrodes) we found that 75% of studies were using young animals and only 20% examined adult animals; the rest (5%) did not specify the age of the experimental subjects ().
Studies on hippocampal LTP and Morris water maze (Morris WM) analysed with respect to experimental approaches and ages at which animals were tested.
Possible reasons for this bias towards young animals include improved cell visibility and identification in young tissue, primarily due to less advanced myelination. Although there are newer methods available to conduct such studies in older animals, they are more challenging. Interestingly though, the scarce use of adult animals was not just limited to voltage- and current-clamp studies; only 38% of experiments using field recordings in the tissue slice, and 45% of those using other techniques (e.g. western blots, immunohistochemistry, electron microscopy, etc.) examined adult animals (). Such techniques do not rely on good individuation of cells, and could thus be performed easily in adult animals. Although a few of these ex vivo studies may have been specifically examining development and could therefore justify this choice of age, this is unlikely to be the case for the majority. Thus, studies of the same phenomenon (hippocampal LTP) in the live animal show quite a different age profile. As our next analysis shows ( and ), studies of hippocampal LTP using in vivo electrophysiology and/or behavioural tests, focused mostly on adult animals (65%); only 26% used young animals, and the rest (9%) did not report age. Likewise, studies using the Morris water maze to test spatial memory (, ) were performed mainly on adult subjects (77%); only 17% of the studies were performed in young animals and in 5%, age was not specified. The lack of significant overlap between these different techniques makes it difficult to extrapolate between them and means that information gleaned from ex vivo work often does not directly complement studies of in vivo physiology and behaviour.