Psychological Handling

Abstract

Psychological handling is the physical manipulation of animals and humans to induce a behavioral or other psychological reaction, and MeSH classifies it under motivation. This article covers the neonatal handling paradigm in rats and its effects on the pituitary-adrenal response to stress, the debate over whether those effects are mediated by maternal care, the handling of laboratory mice and rats as a source of stress and of welfare refinement, and tactile stimulation of preterm human infants. The central question is what handling changes: an early theory held that stimulation in infancy reduces emotional reactivity, later work pointed to the behavior of the mother, and current work on laboratory animals turns on the method by which an animal is picked up.

Keywords: handling, neonatal handling, maternal care, stress, laboratory animal welfare

As one of the first rodent models designed to investigate the effects of early-life experiences, the neonatal handling paradigm has helped show how subtle changes in the infant environment can drive neurodevelopment of the immature brain in typical or atypical trajectories (Raineki et al., 2014). Specific experimental manipulations of the rat postnatal environment have been demonstrated to exert robust and marked effects on neurobiological, physiological and behavioral phenotypes in adulthood (Pryce & Feldon, 2003).

What Psychological Handling Is

MeSH defines the descriptor Handling, Psychological as physical manipulation of animals and humans to induce a behavioral or other psychological reaction, notes that in experimental psychology the animal is handled to induce a stress situation or to study the effects of gentling or mothering, and lists Handling (Psychology) and Handling, Psychology as entry terms. MeSH files the descriptor under motivation and lists no narrower descriptors beneath it.

The word covers two experimental practices. In the developmental literature it usually names a brief daily separation of pups from the mother: in one protocol, rat pups 2 to 14 days of age were exposed daily to handling (15 min of separation from mother and home cage), to maternal separation (180 min of comparable separation), or were left entirely undisturbed (Plotsky & Meaney, 1993). In laboratory husbandry it names the routine picking up of animals, and handling can stimulate stress and anxiety in laboratory animals that negatively impacts welfare and introduces a confounding factor in many areas of research (Gouveia & Hurst, 2019).

Infantile Handling and Stress

Two 1957 reports by Levine and colleagues addressed infantile experience and resistance to physiological stress (Levine, 1957) and infantile experience and the maturation of the pituitary-adrenal axis (Levine et al., 1957). Weininger studied the effects of early experience on behavior and growth characteristics (Weininger, 1956).

Denenberg offered a general theory of these effects. He contested the critical period hypothesis, that there are certain limited time periods in infancy during which a particular class of stimuli will have profound effects upon subsequent behavior, arguing that experiments using rats and mice do not support it and that, where findings are consistent with the hypothesis, the critical period is a complex function of the amount of infantile stimulation (Denenberg, 1964). His central hypothesis was that the amount of stimulus input in infancy acts to reduce emotional reactivity in a monotonic fashion; it follows that an inverted U function should be obtained between amount of infantile stimulation and adult performance for tasks involving some noxious element and of moderate difficulty, whereas for easy or difficult tasks the relationships should be monotonic, though opposite in slope (Denenberg, 1964). A 2020 editorial on early adversity returns to that literature, noting that too little and too much stimulation were associated with poorer outcomes and that the idea has been rediscovered in later theorizing (Gunnar, 2020).

The receptor-level account came later. Rats handled during infancy had a permanent increase in concentrations of receptors for glucocorticoids in the hippocampus, a critical region in the negative-feedback inhibition of adrenocortical activity, and at all ages tested rats that were not handled secreted more glucocorticoids in response to stress than did handled rats (Meaney et al., 1988). Hippocampal cell loss and pronounced spatial memory deficits emerged with age in the nonhandled rats but were almost absent in the handled rats, and the authors concluded that a subtle manipulation early in life can retard the emergence of a degenerative cascade of aging (Meaney et al., 1988).

Handling also altered the hypothalamic stress system. As adults, rats that had undergone maternal separation showed increased hypothalamic corticotropin-releasing factor (CRF) mRNA levels compared with nonhandled rats, while CRF mRNA levels in handled rats were significantly lower than in either group, and restraint stress produced significantly higher increases in plasma corticosterone in maternally separated and nonhandled animals than in handled animals (Plotsky & Meaney, 1993).

Figure 1

Daily Time Away From the Mother in Three Early-Life Conditions

Duration matters. In the rodent, brief periods of separation result in an attenuated adrenal response to stress, whereas longer periods of separation result in an exaggerated response and several behavioral anomalies, such as increased alcohol consumption and an increased startle response (Levine, 2005). The long-separation side of that contrast is less consistent than the summary suggests: an earlier review found contrary findings for almost all parameters investigated, and suggested that maternal separation has become a collective term for a variety of extremely different experimental manipulations (Lehmann & Feldon, 2000). In primates, disruptions of the mother-infant relationship reveal a pattern of behavioral disturbance but little influence on the endocrine response, although brief maternal separations result in a blunted cortisol response in juvenile squirrel monkeys (Levine, 2005). A review of more than 50 years of data challenges the classical view of neonatal handling as an animal model that results only in beneficial outcomes: both beneficial and negative outcomes may occur, depending on the parameters of testing, the sex of the subject and the neurobehavioral system analyzed (Raineki et al., 2014). Sex differences appeared early: on a hole-board, nonhandled rats decreased head-dipping over days whereas handled rats maintained a high rate, and males and females showed completely opposite patterns of exploration depending on handling treatment and on level of stimulus variation (Weinberg et al., 1978).

Maternal Mediation

One account locates the handling effect in the mother. Levine had proposed that handling of pups modifies the mother-pup interaction, thereby causing a change in maternal behavior, so that handling effects in infancy are mediated via the mother (Denenberg, 1999).

Natural variation in maternal care produced a similar profile. As adults, the offspring of mothers that exhibited more licking and grooming of pups during the first 10 days of life showed reduced plasma adrenocorticotropic hormone and corticosterone responses to acute stress, increased hippocampal glucocorticoid receptor mRNA expression, enhanced glucocorticoid feedback sensitivity and decreased hypothalamic corticotropin-releasing hormone mRNA, and each measure was significantly correlated with the frequency of maternal licking and grooming (Liu et al., 1997). The authors suggested that maternal behavior serves to program hypothalamic-pituitary-adrenal responses to stress in the offspring (Liu et al., 1997), and, as Denenberg summarized their comparison of handled with nonhandled litters, Liu and colleagues found that mothers of handled pups licked and groomed their young significantly more often than did mothers of control litters (Denenberg, 1999).

The maternal behavior itself has been measured closely. Licking and grooming is normally distributed across dams, peaks in the first few days postpartum and gradually declines, is transmitted to female offspring, and is a stable, individual characteristic of rat dams (Champagne et al., 2003). Increased pup licking and grooming and arched-back nursing altered the offspring epigenome at a glucocorticoid receptor gene promoter in the hippocampus; the differences emerged over the first week of life, were reversed with cross-fostering and persisted into adulthood (Weaver et al., 2004). A review added that patterns of maternal care that increase stress reactivity in offspring are enhanced by stressors imposed on the mother, and took this as evidence for the importance of parental care as a mediator of the effects of environmental adversity on neural development (Meaney, 2001).

The mediation account is widely held but contested. A 2020 editorial describes it as largely accepted, though sometimes controversial, that stimuli as disparate as handling and shock produce similar outcomes because they both result in heightened maternal care (Gunnar, 2020). Denenberg summarized data obtained with mice and rabbits that in part support and in part challenge the conclusions of Liu and colleagues (Denenberg, 1999). In Lister hooded rats, both early handling and maternal separation induced elevated levels of maternal care compared with nonhandled litters, yet only early handling reduced the adult offspring's HPA and fear responses, which the authors took to indicate that maternal care cannot be the sole mediator of these effects (Macrì et al., 2004). A critical review of the maternal mediation hypothesis argued that developmental plasticity of HPA and fear responses in rats cannot be explained by a linear single-factor model based on environment-dependent variation in active maternal care (Macrì & Würbel, 2006). In its two-factor model, active maternal care and environmental stress exert independent, yet opposing, effects on HPA reactivity and fearfulness in the offspring, which accounts for the finding that completely safe and stable, as well as highly stressful, maternal environments result in high HPA reactivity and fearfulness compared with moderately challenging maternal environments (Macrì & Würbel, 2006). The same review offered an alternative in which the effects are a function of maternal HPA activation, which raises the possibility that variation in active maternal care is an epiphenomenon rather than a causal factor (Macrì & Würbel, 2006). A later review led by Macrì revised the account: it proposed that neonatal circulating levels of corticosteroids may constitute a potential mediator connecting early and adult defensive systems, and that the link between early and adult stress follows a U-shaped curve, with low levels down-regulating individual reactivity to external stressors and high levels exerting opposite effects (Macrì et al., 2011).

Handling Laboratory Animals

Balcombe and colleagues reviewed laboratory routines as a cause of animal stress (Balcombe et al., 2004). Routine laboratory animal handling has profound effects on anxiety and stress responses, and when the method was compared, picking up mice by the tail induced aversion and high anxiety, whereas use of tunnels or the open hand led to voluntary approach, low anxiety and acceptance of physical restraint (Hurst & West, 2010). The standard method used to pick up laboratory mice, tail handling, involves the capture, elevation and restraint of mice via their tails (Henderson et al., 2020), whereas handling stress can be reduced substantially by using a handling tunnel, or by cupping mice without restraint on the open hand (Gouveia & Hurst, 2017).

The method also changes data. In a habituation-dishabituation paradigm in which mice must discriminate between two urine stimuli, tail-handled mice showed little willingness to explore and investigate test stimuli, leading to poor test performance, whereas those handled by tunnel explored readily and showed robust responses, though responses were more variable for cup handling (Gouveia & Hurst, 2017). In ICR mice, voluntary interaction with the experimenter was much greater in mice handled by a tunnel than in those picked up by the tail, and tunnel-handled mice showed less anxiety in the open field test but not in the elevated plus maze (Nakamura & Suzuki, 2018).

Familiarization is cheap. Very brief handling (2 s) was sufficient to familiarize mice with tunnel handling, even when experienced only during cage cleaning, whereas brief but more frequent handling was needed for familiarization with cup handling, and pick-up by the tail induced strong aversion even when handling was brief and infrequent (Gouveia & Hurst, 2019). After repeated restraint, tunnel-handled mice showed increased willingness to interact with a handler and reduced anxiety in standard behavioral tests compared with tail-handled mice (Henderson et al., 2020). Handling influenced affective behavior and stress hormone release in a sex-dependent manner, though the handling technique did not influence fecal corticosterone metabolite concentrations in either sex: frequent tail handling led to a reduction of wellbeing-associated burrowing and increased despair-like behavior in male mice, whereas females displayed a stress response to a low handling frequency, and both sexes preferred to interact with the tunnel (Sensini et al., 2020).

Rats are handled differently. Rats initially fear humans, which can increase stress and impact study results, and rat tickling is a promising habituation technique that can also be used to model and measure positive affect (LaFollette et al., 2017). Cloutier and colleagues studied playful handling of laboratory rats by caretakers (Cloutier et al., 2012). A systematic review identified 32 articles (56 experiments); the most common method cycled through 15 seconds of tickling and 15 seconds of rest for 2 minutes for 3 to 5 days, and experiments with a control showed that tickling increases positive vocalization and approach behavior, decreases anxiety measures, improves handling, and in some cases decreases stress hormones (LaFollette et al., 2017). A later study set out to determine an efficient and effective dosage of tickling (LaFollette et al., 2018).

Handling in Human Infants

MeSH extends handling to humans, and the human work concerns touch. Tactile/kinesthetic stimulation, consisting of body stroking and passive movements of the limbs for three 15-minute periods per day for 10 days, was given to 20 preterm neonates with a mean gestational age of 31 weeks and a mean birth weight of 1,280 g, and compared with 20 control neonates the stimulated neonates averaged a 47% greater weight gain per day (25 g versus 17 g), were more active and alert, and showed more mature habituation, orientation, motor and range-of-state behavior on the Brazelton scale (Field et al., 1986). Their hospital stay was 6 days shorter, and the authors suggested that such stimulation may be a cost-effective way of facilitating growth and behavioral organization even in very small preterm neonates (Field et al., 1986). Pooled trials temper the single result. A Cochrane review found that massage interventions improved daily weight gain by 5.1 g and appeared to reduce length of stay by 4.5 days, though there are methodological concerns about the blinding of this outcome, and it concluded that serious concerns about the methodological quality of the included studies weaken credibility in these findings and that evidence of benefit for developmental outcomes is weak and does not warrant wider use of preterm infant massage (Vickers et al., 2004). A later meta-analysis of 17 studies found that massage improved daily weight gain by 5.32 g and reduced length of stay by 4.41 days, found no significant differences on the Neonatal Behavioral Assessment Scale, and concluded that the association between massage and neurobehavioral development is still elusive (Wang et al., 2013). Field later reviewed touch for socioemotional and physical well-being (Field, 2010).

The human evidence is harder to read than the rodent evidence. The long-term effects of early experiences in humans are very difficult to interpret, because it is not possible to determine the length and severity of the experiences or when in development they were imposed, although there is a general consensus that adverse early experiences contribute to adult psychopathology (Levine, 2005).

Worked Example

The preterm study gives the arithmetic of a handling effect. The stimulated neonates gained a mean of 25 g a day against 17 g for controls (Field et al., 1986). The difference is 8 g a day, and 8 / 17 = 0.47, which is the reported 47% greater weight gain per day. The rodent protocols scale differently: maternal separation lasted 180 min a day against 15 min for handling (Plotsky & Meaney, 1993), a twelvefold difference in daily time away (180 / 15 = 12), and it was the brief separation, not the long one, that was followed by the lowest adult CRF mRNA.

Discussion

Three conclusions follow for psychology. First, handling is not one manipulation. In rats, brief separation attenuated the adrenal response to stress while longer separation did not (Macrì et al., 2004) and in some reports exaggerated it (Levine, 2005), and both beneficial and negative outcomes may follow neonatal handling depending on the parameters of testing, sex and the system analyzed (Raineki et al., 2014).

Second, the mechanism is debated. Maternal licking and grooming predicted adult stress responses in the offspring (Liu et al., 1997), but a single-factor model based on active maternal care could not explain the effects of postnatal manipulations, and maternal care may be an epiphenomenon rather than a causal factor (Macrì & Würbel, 2006).

Third, handling is itself an experimental variable. Tail handling impaired mouse performance in behavioral tests compared with tunnel handling (Gouveia & Hurst, 2017), so the way animals are picked up is part of what a study measures.

Current Directions

Three lines of work are active. Refinement: replacing tail with tunnel handling during routine cage cleaning and procedures has been described as a major refinement with little if any cost for familiarization (Gouveia & Hurst, 2019), and Henderson and colleagues' findings suggest that its welfare benefits are not diminished by other putatively more invasive procedures (Henderson et al., 2020). Sex differences: the impact of handling may differ between the sexes (Sensini et al., 2020). Standards: methods to reduce bias were insufficiently reported in the tickling literature, and the establishment of tickling best practices has been called essential because its outcomes can be moderated by several factors (LaFollette et al., 2017).

Common Misconceptions

“Handling a young animal always makes it calmer.”

A review of neonatal handling concludes that both beneficial and negative outcomes may occur, depending on the parameters of testing, the sex of the subject and the system analyzed (Raineki et al., 2014).

“Any time away from the mother has the same effect.”

In rodents, brief separations attenuated the adrenal response to stress, whereas longer separations did not (Macrì et al., 2004) and in some reports exaggerated it (Levine, 2005).

“How a laboratory mouse is picked up makes no difference.”

Picking mice up by the tail induced aversion and high anxiety, whereas tunnels or the open hand led to voluntary approach and low anxiety (Hurst & West, 2010).

Glossary

Adrenocorticotropic hormone.

A hormone whose plasma response to acute stress was reduced in adult offspring of mothers that licked and groomed their pups more.

Arched-back nursing.

A rat nursing posture that, with licking and grooming, altered the offspring epigenome at a hippocampal glucocorticoid receptor gene promoter.

Corticosterone.

A rodent adrenal hormone whose rise under restraint stress was smaller in rats handled in infancy.

Corticotropin-releasing factor.

A hypothalamic factor whose mRNA was lower in adult rats handled in infancy than in maternally separated or nonhandled rats.

Critical period hypothesis.

The hypothesis that there are limited time periods in infancy during which a particular class of stimuli has profound effects on later behavior.

Cup handling.

Cupping a mouse without restraint on the open hand.

Glucocorticoid receptor.

A receptor whose hippocampal concentration was permanently increased in rats handled during infancy.

Hypothalamic-pituitary-adrenal axis.

The stress-hormone system whose responses maternal behavior has been suggested to program in the offspring.

Licking and grooming.

A rat maternal behavior that is normally distributed across dams and a stable, individual characteristic of each dam.

Maternal mediation hypothesis.

The proposal that developmental plasticity of HPA and fear responses is mediated by environment-dependent variation in active maternal care.

Maternal separation.

A longer daily separation of pups from the mother, such as 180 min, in contrast with the 15 min of handling.

Neonatal handling.

One of the first rodent models of early-life experience, in which pups are briefly separated each day from mother and home cage.

Non-aversive handling.

Picking up mice in a tunnel or cupped on the open hand rather than by the tail.

Tail handling.

The standard method of capturing, elevating and restraining laboratory mice by their tails.

Tickling.

A handling method for rats, used as a habituation technique and to model and measure positive affect.

Tunnel handling.

Picking up a mouse with a handling tunnel instead of by the tail.

Two-factor model.

An account in which active maternal care and environmental stress exert independent, opposing effects on HPA reactivity and fearfulness in the offspring.

Key Researchers

Frances A. Champagne

Professor of Psychology at the University of Texas at Austin; co-authored the analysis of licking and grooming across cohorts of rat dams and the study of epigenetic programming by maternal behavior. ORCID - Wikipedia - Wikidata - Faculty Page

Victor H. Denenberg

(1925-2008). Developmental psychobiologist at the University of Connecticut; proposed that stimulation in infancy reduces emotional reactivity and questioned the critical period hypothesis. Wikipedia - Wikidata

Jane L. Hurst

Professor at the University of Liverpool; co-authored the comparison of tail, tunnel and open-hand handling of laboratory mice. Wikipedia - Wikidata - Faculty Page

Seymour Levine

(1925-2007). Psychoneuroendocrinologist at Stanford University; reported on infantile experience and resistance to physiological stress. Obituary

Michael J. Meaney

Neuroscientist at the Douglas Research Centre, McGill University; co-authored the studies of neonatal handling and the hippocampus and of maternal care and stress responses. ORCID - Wikipedia - Wikidata - Faculty Page

Joanne Weinberg

Professor Emerita of Cellular and Physiological Sciences at the University of British Columbia; co-authored a study of how handling differentially affects exploration in male and female rats, and a review of the positive and negative effects of neonatal handling. Faculty Page - Google Scholar

Hanno Würbel

Professor of Animal Welfare at the University of Bern; co-authored a study dissociating maternal care from offspring stress responses and the critical review of the maternal mediation hypothesis. Faculty Page

Frequently Asked Questions

What is psychological handling?

In one widely cited rat protocol it meant a brief daily separation of pups from the mother and home cage, 15 minutes a day between 2 and 14 days of age (Plotsky & Meaney, 1993).

What is the neonatal handling paradigm?

It is one of the first rodent models designed to investigate the effects of early-life experiences (Raineki et al., 2014).

How does handling in infancy affect the stress response?

Rats handled during infancy had more hippocampal glucocorticoid receptors and secreted fewer glucocorticoids in response to stress than nonhandled rats at all ages tested (Meaney et al., 1988).

Does handling work through the mother?

The view is widely held but disputed: a 2020 editorial describes it as largely accepted, though sometimes controversial, that stimuli as disparate as handling and shock produce similar outcomes because they both result in heightened maternal care (Gunnar, 2020).

Is neonatal handling always beneficial?

No. Both beneficial and negative outcomes may occur, depending on the parameters of testing, the sex of the subject and the system analyzed (Raineki et al., 2014).

What is the least stressful way to pick up a laboratory mouse?

Using a tunnel or the open hand led to voluntary approach and low anxiety, whereas picking mice up by the tail induced aversion and high anxiety (Hurst & West, 2010).

What is rat tickling?

It is a handling method used as a habituation technique and to model and measure positive affect in rats (LaFollette et al., 2017).

Does touch help premature babies?

A Cochrane review found that massage improved daily weight gain by 5.1 g, but judged that serious concerns about the methodological quality of the included studies weaken credibility in its findings (Vickers et al., 2004).

References