Methodological aspects

In rodent models, repeated separations of the pups from the mother (or care-giving female) during the first two to three postnatal weeks are frequently used to examine the consequences of different early-life experiences. These models are commonly denominated maternal separation (MS). However, MS is not one defined model but comprises a variety of experimental paradigms, see Table 1 and Fig. 1.

Table 1

A summary of experimental conditions that commonly are used in maternal separation studies. Note that there are some inconsistencies in the literature regarding the nomenclature of the procedures (for details, see the text). In the table, examples of the most common denominations are listed

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Fig. 1

Common experimental groups in rodent maternal separation (MS) paradigms. Animals in the horizontal groups are subjected to the same handling by the experimenter and exposed to the same housing conditions. The duration of MS differs and, consequently, the effect of being separated from the dam different length of time can be assessed. The vertical groups represent control groups commonly used to examine the outcome of short or prolonged periods of MS. These groups differ in a number of aspects and care must be taken in choosing the proper control depending on the question asked. Modified from (Nylander and Roman 2012). NH, non-handling; AFR, animal facility rearing; MS0, brief handling of the pup with less than 1 min loss of contact with the dam; MS15 and MS180-360, maternal separation for 15 min or 180–360 min, common protocols utilize 180, 240 or 360 min separations

Early studies showed that handling, i.e. gently holding rat pups for 10 min every day for 3 weeks post-weaning, resulted in positive physiological and behavioural effects in adulthood (Weininger 1954). That early handling was related to long-term effects in the offspring when comparing with non-handled (NH) rats was further supported in studies where it was shown that separating mother and pups daily for only 3 min during the first 3 weeks of life reduced the physiological responses to stress (Levine 1957; Levine and Lewis 1959). Over the years, MS paradigms have been developed and used in a number of studies to investigate the long-term effects of early-life events on the offspring. These studies also include prolonged MS and they have not only provided valuable knowledge about neurobiological and behavioural consequences of early-life experiences but also about experimental factors that influence these effects (e.g. reviews by Kuhn and Schanberg 1998; Lehmann and Feldon 2000; Levine 2002; Macri and Würbel 2006; Nylander and Roman 2012; Pryce and Feldon 2003).

In MS studies, the duration of the separation is used to simulate different environmental settings. When the aim is to simulate a beneficial environment, i.e. conditions that relate to positive behavioural effects and protection against negative influences, the pups are subjected to shorter, that is minutes rather than hours, separations from the dam. For example, repeated short separations for 15 min (MS15) per day are used to simulate naturalistic conditions based on knowledge from the wildlife rearing conditions where the lactating dam leaves the nest regularly, often around 10 min and not longer than 1 h depending on the age of the offspring (Grota and Ader 1969). When the aim is to simulate a risk environment, i.e. adverse conditions relating to risk for negative consequences such as excessive ethanol intake or symptoms reminiscent of pathological conditions, longer periods of maternal absence are used. The newborn rat is dependent on the mother for a normal development and repeated separations for longer periods of time, commonly 180–360 min per day, are used to disrupt the early social mother–pup interactions that are vital for normal neuronal and behavioural development. Many studies support the notion that prolonged MS is regarded a risk environment that is associated with early-life stress and negative consequences later in life (Holmes et al. 2005; Ladd et al. 2000; Levine 2002; Pryce and Feldon 2003). Maternal deprivation (MD) is also seen in the literature but is, however, not used consistently; MD can refer to occasional 24 h MS but also be used interchangeably with MS to describe 180–360 min separations from the dam (Hall et al. 1999; Ogawa et al. 1994; Vazquez et al. 2005; Viveros et al. 2009). Furthermore, early deprivation has been used to describe separation of the pup from the dam and littermates (see below, individual MS) to distinguish from separation of the litter from the dam (Ruedi-Bettschen et al. 2005). In the various MS protocols, the separations are done repeatedly, either daily or occasionally.

The separation conditions differ between MS studies. During the separation from the dam, the rat pups can be either placed individually or kept together in litters, either in the maternity cage or in a separation cage. In the litter-wise paradigm, the pups experience loss of contact with the dam, i.e. they cannot see, communicate with, smell or feel her presence, whereas the contact with the littermates is undisturbed. In individual MS paradigms, the rat pups are individually placed during the separations. They are, for example, placed separately in small compartments in a cage so that they can communicate with and smell the other pups but they are deprived the tactile contact with the littermates. In all MS paradigms, the ambient temperature should be controlled in order to avoid hypothermia (Ruedi-Bettschen et al. 2004; Zimmerberg and Shartrand 1992) and it is of course of particular importance to keep control over temperature when the rat pups are isolated from both dam and littermates. Another factor that can differ is the use of cross-fostering procedures versus the use of biological littermates. Often, pups are cross-fostered on the day of birth and arranged in litters to contain an equal number of male and female pups. However, there is no standardized procedure for cross-fostering and how this is done varies between studies.

Another experimental design used in the literature to investigate the effects of disturbed social interactions is social isolation. The animals are single housed, usually for longer periods of time, and the effects of deprivation of social contacts at different ages can be assessed. Recent comprehensive reviews discuss the behavioural effects seen after isolation rearing immediately after weaning, including effects on ethanol intake (Becker et al. 2011; Neisewander et al. 2012). The compiled studies show that social stress post-weaning facilitates drug abuse-related behaviours and that animal models can be used to further study the consequences of social isolation during adolescence. However, in these social isolation studies, the isolation occurs after weaning and thus does not include disturbance of the early-life environment with the dam. Thus, these conditions are different from the MS conditions and the models should be considered to be different models since they study different aspects of social stress. Hall et al. discuss the different and the common effects, respectively, elicited by either isolation in weanling rats or MS and suggest a common role for mesolimbic dopamine in consequences seen after social stress, but still, they could observe differences in how dopamine function was affected by these two procedures (Hall et al. 1999). Taken together, since social behaviour and social interactions differ before and after weaning and since the social stress induced by the isolation occurs at different developmental time windows, it is important to distinguish between effects elicited by MS from those seen after social isolation later in life.

The choice of control groups in the experimental design of a MS study is of vital importance in the evaluation of results from a MS experiment. As mentioned above, short periods of MS can be used to examine the basis for environmental protective factors per se, but they can also be used as a control condition in the comparison to effects induced by the prolonged separations, i.e. the risk environment. Common experimental groups that are used as controls include MS15 and MS0 or brief (early) handling that usually refer to shorter separations for up to 5 min per day (Jaworski et al. 2005; Lehmann and Feldon 2000; Pryce and Feldon 2003; Roman and Nylander 2005). Comparisons between short/brief and prolonged separations enable analysis of the effects of the duration of maternal absence with all other experimental conditions, such as handling by the experimenter and general housing conditions, kept the same (Fig. 1). Other controls are listed in Table 1 and they include the non-handling (NH) paradigm and animal facility rearing (AFR). NH relates to an unnatural, understimulated environment that in itself may induce effects in the offspring (Macri and Würbel 2006; Pryce and Feldon 2003), which indicates that it is not a suitable control. AFR refers to conventional animal facility housing and conditions with experimenter contact only when the cages are changed. However, although AFR is similar between laboratories, there are still several possible confounding factors due to different laboratory environments and handling procedures (Chesler et al. 2002; Crabbe et al. 1999; Wahlsten et al. 2003).

Methodological aspects

A number of experimental paradigms can be used to assess effects on ethanol consumption in rodents (Becker 2013; Crabbe et al. 2011; Sanchis-Segura and Spanagel 2006). The animal can have access to ethanol in one or several bottles in the home cage and thereby be given a free choice to drink from the preferred bottle/s. A common protocol includes a two-bottle free choice between ethanol and water. In such voluntary drinking paradigms, the animals can have either continuous or intermittent access to ethanol. A drawback with continuous access to ethanol is that the delay between consumption and onset of effects often results in overall low voluntary ethanol consumption (Meisch and Lemaire 1993). Rats establish a higher voluntary ethanol intake when they are provided intermittent access to ethanol (Wayner and Greenberg 1972; Wise 1973). Recent studies using an intermittent access protocol with free choice (20 % ethanol and water on Mondays, Wednesdays and Fridays) reported higher intake and preference as compared to continuous access (Simms et al. 2008). However, in the continuous as well as the intermittent paradigms, ethanol is available for 24 h and during this time period the animals drink in sporadic bouts. Of relevance for the achieved blood ethanol concentration is the number of drinking bouts that occur in a unit of time and the amount of ethanol consumed on each occasion. Despite similar intake during 24 h, high volume and short duration bouts that occur frequently may result in different blood ethanol patterns when compared to lower volume, longer duration bouts occurring less frequently (Samson and Czachowski 2003). There are also limited access paradigms in which ethanol is available only a limited period over a 24-h period. In limited access paradigms, it has been stressed that the animals drink more when access to ethanol is given during their active phase, i.e. during the dark period, also known as “drinking in the dark” (Rhodes et al. 2005; Sprow and Thiele 2012). Other protocols use sweetened ethanol, for example by addition of sucrose, to increase voluntary intake but that requires extra controls for the rewarding effects of the sweet solution. In forced drinking paradigms, the animals have no water available and thus no choice but to drink ethanol. Forced paradigms are less suitable whenever the animal’s propensity to drink ethanol is of interest.

Most rats like to drink ethanol in low concentrations such as 2–6 % (Meisch and Lemaire 1993; Richter and Campbell 1940) possibly because low concentrations have a mild-sweet taste (Sanchis-Segura and Spanagel 2006). In continuous access paradigms, it is therefore common to use schedules with a gradually increasing ethanol concentration for example from 2 to 10 % ethanol over a couple of weeks. Voluntary drinking paradigms also include free choice protocols with more than one ethanol bottle in addition to water, for example, three- or four-bottle choice paradigms. With these protocols, it is possible to examine the individual preference for lower or higher concentrations of ethanol, for example in animals that have been exposed to different early-life environmental conditions.

Operant techniques can also be used for self-administration of ethanol. The animals learn to work for a reward and the self-administration of a drug and motivation to take the drug can be assessed. Operant techniques are used primarily for heroin and central stimulants and less for ethanol but can be used for oral delivery of ethanol, or intravenous or intragastric self-administration (see Crabbe et al. 2011; Sanchis-Segura and Spanagel 2006).

Influence of separation conditions

In contrast to the consistent findings in litter-wise paradigms, it was recently shown that there were no differences in adult ethanol consumption between repeated MS15 and MS360 when the rat pups were placed individually during the separation (Oreland et al. 2011; Fig. 2 and Table 2). The rats that were subjected to individual MS15 had higher ethanol consumption than usually seen after litter-wise MS15 and not different from the intake seen in rats subjected to MS360 or AFR. However, it is worth noting that even though the ethanol intake was similar, the ethanol-induced effects differed in MS15 and MS360 rats (Oreland et al. 2011). Thus, the rearing conditions were of importance for later ethanol response. In the study by Hilakivi-Clarke et al., the rat pups were separated in litters postnatal day (PND) 5–10 and then individually from PND 11 to 20. With this paradigm, there was a difference between prolonged (60 min) separations and MS15 (Hilakivi-Clarke et al. 1991) indicating that it is most important to keep the litters intact during the first ten postnatal days.

The findings of no differences in ethanol consumption after individual MS are in agreement with previous results showing different neurobiological outcome after individual and litter-wise separations (Gustafsson et al. 2008; Oreland et al. 2010). The individual and litter-wise separations in those reports were performed in one and the same MS study to exclude other confounding factors and show that the tactile contact during separations from the dam is of vital importance for the outcome of MS. Deprivation of littermate contact seem to be stressful, even though it is just for 15 min, which can explain why the loss of this sensory input also results in loss of the protective influences from short periods of separations from the dam such as the litter-wise MS15 condition.

In contrast, a recent study reported increased ethanol consumption in adult Long-Evans rats subjected to prolonged MS (Romano-Lopez et al. 2012). In this study, the rats were individually separated from the dam twice daily for 180 min or remained with the dam and handled like AFR rats. Notably, another ethanol intake paradigm was used to assess consumption. The animals were introduced to ethanol in a fading saccharin schedule and then first exposed to forced consumption followed by voluntary drinking of 10 % ethanol. These results indicate that although no differences can be seen using voluntary drinking protocols, differences in ethanol consumption may emerge when using forced consumption paradigms.