DOI 10.1515/revneuro-2012-0074   Rev. Neurosci. 2012; 23(5-6): 707–722

Antonia Manduca, Patrizia Campolongo and Viviana Trezza*

Cannabinoid modulation of mother-infant interaction: is it just about milk? Abstract: Mother-infant interactions are essential for

proper neurobehavioral development of the offspring,

and disruptions in those relationships may result in

neuroendocrine, neurochemical and behavioral altera-

tions at adulthood. The neural circuitries involved in

mother-infant interactions have not been completely

elucidated yet. The brain endocannabinoid system plays

an essential role in prenatal and postnatal neurobehav-

ioral development. Here, we will summarize and discuss

the available findings about the role of endocannabi-

noids in three key aspects of mother-infant interactions

in rodents: suckling, maternal behavior and separation-

induced ultrasonic vocalizations (USVs). The studies

reviewed here show that endocannabinoids are not

only involved in suckling initiation and, therefore, in

the feeding and growth of the offspring, but also regu-

late the emotional reactivity of rodent pups, as meas-

ured by the rate of isolation-induced USVs. Conversely,

less information is available about endocannabinoid

modulation of maternal behavior, and therefore more

research in this direction is warranted. Indeed, since

Cannabis sativa preparations are widely used by young

people, including pregnant and lactating women, it is

important to understand whether developmental expo-

sure to cannabinoids interferes with mother-infant bond

formation, potentially leading to neurodevelopmental

alterations and increased vulnerability to psychopathol-

ogy later in life.

Keywords: endocannabinoids; maternal behavior;

mother-infant interaction; suckling; ultrasonic vocaliza-

tions.

*Corresponding author: Viviana Trezza, Department of Biology,

University “ Roma Tre ” , I-00146 Rome, Italy,

e-mail: viviana.trezza@uniroma3.it

Antonia Manduca: Department of Biology, University “ Roma Tre ” ,

I-00146 Rome, Italy

Patrizia Campolongo: Department of Physiology and Pharmacology,

Sapienza University of Rome, I-00185 Rome, Italy

Introduction

Cannabis sativa is one of the most widely used psycho-

active drugs with a documented history of consumption

going back thousands of years (Touwn, 1981; Russo, 2005,

2007; Di Marzo, 2006; Di Marzo and Petrocellis, 2006). The

main psychoactive principle, Δ 9-tetrahydrocannabinol

(THC), a highly lipophilic molecule, was identified in 1964

(Gaoni and Mechoulam, 1964) and led to the identifica-

tion of the endogenous cannabinoid system that includes

cannabinoid receptors (CB1 and CB2), endogenous lipid

ligands [endocannabinoids (ECs), such as anandamide

(AEA) and 2-arachidonoyl-glycerol (2-AG)] and enzymes

involved in EC synthesis, transport and degradation

(Devane et al., 1992; Freund et al., 2003; Piomelli, 2003;

Kogan and Mechoulam, 2006).

The CB1 cannabinoid receptor, cloned in 1990 from

the mammalian brain (Matsuda et al., 1990), is expressed

almost ubiquitously throughout the brain (Tsou et al.,

1998; Moldrich and Wenger, 2000) and exhibits some

expression patterns in peripheral tissues, such as immune

cells, vascular tissue and adipocytes (Parolaro, 1999;

Hillard, 2000; Cota et al., 2003). Three years after the dis-

covery of the CB1 cannabinoid receptor, a second cannabi-

noid receptor, termed the CB2 cannabinoid receptor, was

identified in the marginal zone of the spleen (Munro et al.,

1993), and its localization was postulated to be predomi-

nantly in peripheral immune cells and organs (Benito

et al., 2008). However, recent evidence shows that CB2

cannabinoid receptors are expressed not only by micro-

glial cells in injured, infected or inflamed central nervous

system (CNS) tissues (Benito et al., 2008), but also under

normal physiological conditions (Van Sickle et al., 2005;

Gong et al., 2006; Palazuelos et al., 2006).

ECs are synthesized ‘ on demand ’ at postsynaptic sites

of neurons after an increase in neural activity and calcium

ion influx and are then released into the synaptic cleft,

where they act as retrograde neurotransmitters to acti-

vate presynaptically located cannabinoid receptors, thus

leading to the inhibition of the release of neurotransmit-

ters, including glutamate, GABA, acetylcholine, seroto-

nin and norepinephrine (Schlicker and Kathmann, 2001;

Wilson et al., 2001; Piomelli, 2003).

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708   A. Manduca et al.: Endocannabinoids and mother-infant interaction

Over the years, it has become evident that the endo-

cannabinoid system plays an essential role in multiple

aspects of brain function, including an influence on the

hypothalamic-pituitary-adrenal (HPA) axis and conse-

quent regulation of the stress response (Hill et al., 2009;

Riebe and Wotjak, 2011), modulation of emotional states

(Viveros et al., 2007; Moreira and Lutz, 2008; Lutz, 2009;

Taber and Hurley, 2009; Marco and Laviola, 2012) and

cognitive processes (Wotjak, 2005; Rubino and Parolaro,

2008; Campolongo et al., 2009a,b, 2012; Akirav, 2011;

Marco et al., 2011; Terzian et al., 2011; Zanettini et al., 2011;

Atsak et al., 2012) along the lifespan.

Mother-infant interaction is essential for offspring

survival, and it has been shown that early-life adversi-

ties might have lasting neuroendocrine consequences,

alter cognitive functioning and increase the vulnerabi lity

to developing psychopathologies, such as depression,

anxiety and schizophrenia later in life (Breier et al., 1988;

Agid et al., 1999; Pechtel and Pizzagalli, 2011). Survival

of infants depends on attachment to the caregiver, and

elaborate mother-infant relationships have been identi-

fied across most mammalian species, including rodents

(Smotherman, 1983; Moore et al., 1997). The establish-

ment of proper mother-infant interactions requires

infants to identify, learn, remember and approach their

attachment figure and vice versa, as it is also necessary

that the mother develops attaching behaviors toward the

offspring.

The clinical literature suggests that the interactions of

infants with their caregivers are important for child brain

development and in shaping adult behavior and that

individuals that experience early abuse or neglect have

a greater probability of developing psychopathological

disorders later in life (Glaser, 2000; Hildyard and Wolfe,

2002; Shaffer et al., 2009). These clinical data have their

counterpart in preclinical findings (Berman, 1990; Francis

et al., 1999; Meaney, 2001; Champagne et al., 2003; Cirulli

et al., 2003).

The endocannabinoid system is actively present

and functional from the earliest stages of ontogenetic

development until early and late postnatal life and plays

an essential role in prenatal and postnatal neurobeha-

vioral development (Rodriguez de Fonseca et al., 1993;

Berrendero et al., 1998, 1999; Fernandez-Ruiz et al.,

2000; Mato et al., 2003; Fride, 2004b; Harkany et al.,

2007; Schneider, 2009).

In the present review, we will summarize and discuss

the available findings about the effects of developmental

cannabinoid exposure on three key aspects of mother-

infant interaction in rodents, namely suckling, maternal

behavior and isolation-induced ultrasonic vocalizations

(USVs). Studying cannabinoid modulation of mother-

infant interactions in laboratory animals is important

for several reasons. First, as the molecular basis and the

neural circuitries involved in the human attachment pro-

cesses are still unclear, studies in rodents might provide

a greater understanding of the neurobiology of mother-

infant bonding. Second, investigating the neural sub-

strates of the bond between mother and infants, which

represents a critical experience for both the newborn and

the mother, might help to understand the neural under-

pinnings of neuropsychiatric disorders that may be asso-

ciated with altered mother/child relationships. Addition-

ally, given the frequent abuse of Cannabis preparations

among young people, including pregnant women, it is

important to elucidate the possible residual behavio-

ral effects of cannabinoid administration during early

postnatal developmental stages. Last, the endocannabi-

noid system is now emerging as a potential therapeutic

target for the treatment of some neuropsychiatric disor-

ders (Gobbi et al., 2005; Bortolato et al., 2006; Vinod and

Hungund, 2006; Marco et al., 2011); it is therefore essen-

tial to investigate the neurobehavioral effects induced by

early exposure to cannabinoid drugs.

Cannabinoid modulation of suckling and milk intake Breastfeeding in humans (Kavanaugh et al., 1997) and

pup suckling in animals (Ferris et al., 2005) represent

rewarding social stimuli that encourage maternal behav-

ior and bonding. Functional magnetic resonance imaging

studies have shown that suckling stimulation in lactat-

ing dams activates the brain reward system (Febo et al.,

2005; Ferris et al., 2005). Interestingly, pups seem to be

more salient for the dams than other rewarding stimuli,

such as cocaine (Ferris et al., 2005). Indeed, pup suckling

in both lactating dams and virgin females activated the

dorsal and ventral striatum and prefrontal cortex (Ferris

et al., 2005). However, lactating dams exposed to cocaine

instead of pups showed a suppression of activity in those

brain regions (Ferris et al., 2005). Thus, the ability of early

postpartum rats to choose against competing hedonic

stimuli may have evolved to ensure the engagement in

interaction with pups at the expense of other rewarding

activities.

In female rats, suckling induces prolactin secretion,

and this involves stimulation by a prolactin-releasing

hormone, possibly oxytocin (Freeman et al., 2000). Of

particular importance in the past was the question of

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A. Manduca et al.: Endocannabinoids and mother-infant interaction   709

whether the surge of these two hormones, induced by

each suckling episode and necessary for the continuation

of adequate lactation, might be sensitive to the actions

of the main cannabinoid compound THC. To this aim,

postpartum lactating rats were treated with THC, and

their blood samples were collected; compared to vehicle-

injected controls, THC reduced plasma pro lactin levels,

which inhibited suckling-induced release in postpartum

rats (Bromley et al., 1978). Furthermore, THC influenced

the release of oxytocin in response to suckling, causing

a transient suspension of milk ejections, although oxy-

tocin injections 10 or 30 min after THC treatment evoked

increases in intramammary pressure, indicating con-

tinued responsiveness of the mammary gland to the

hormone stimulation (Tyrey and Murphy, 1988).

Later in time, a series of studies performed in neona-

tal mice has shown that CB1 cannabinoid receptor activa-

tion is critically important for the initiation of the suckling

response (Fride et al., 2001, 2003, 2009; Fride, 2008). A

single injection of the CB1 cannabinoid receptor antago-

nist/inverse agonist SR141716A within 24 h after birth to

newborn mice of three different strains (Sabra, C57BL/6

and ICR) completely inhibited milk ingestion and subse-

quent growth in most pups, causing death within 4 – 8 days

(Fride et al., 2001, 2003). Administration of SR141716A on

postnatal day (PND) 2 resulted in half death rate, whereas

administration on PND 5 had no effect at all on pup growth

and survival (Fride et al., 2003). The effects induced by a

blockade of CB1 cannabinoid receptors in mouse neonates

were dose-dependent and specifically mediated by can-

nabinoid receptors, as co-administration of THC almost

fully reversed the effect of SR141716A (Fride et al., 2001).

To determine whether the proximity to birth, rather than

the developmental stage of the pups, was critical for the

impaired suckling induced by SR141716A, the drug was

injected into newborn precocial mice that were born with

open eyes and the ability to walk, run and ingest solid

food. Again, SR141716A significantly delayed development

in those pups (Fride, 2008). SR141716A is not only a CB1

cannabinoid receptor antagonist, it is, more precisely, an

antagonist/inverse agonist able to elicit behavioral and

neural response even if there is little or no endogenous CB1

cannabinoid receptor tone (Meschler et al., 2000). There-

fore, it was not clear from these experiments whether the

developmental deficiencies were induced by a neutral CB1

cannabinoid receptor blockade, thereby inhibiting endog-

enous cannabinoid tone, or by inverse agonist reduction

of constitutive CB1 cannabinoid receptors. To better under-

stand the mechanisms underlying cannabinoid modula-

tion of suckling and milk intake, Fride et al. (2007) inves-

tigated the effects of a neutral CB1 cannabinoid receptor

antagonist, VCHSR1, that binds with a 14-fold lower affinity

to the CB1 cannabinoid receptor compared to SR141716A

and that has been hypothesized to only cause a neutral

receptor blockade (Hurst et al., 2002). VCHSR1 impaired

suckling and had growth-arresting effects similar to those

induced by SR141716A. In fact, VCHSR1, administered on

PND 1 to mouse pups, dose-dependently reduced weight

gain, gastric milk content (expressed as milkbands, which

represent the milk in the stomach that is visible through the

transparent newborn skin and thus can be conveniently

scored), axillary temperature and survival between PND

1 and 10 (Fride et al., 2007). Despite the CB1 cannabinoid

receptor antagonist/inverse agonist SR141716A showing

more dramatic effects than the CB1 cannabinoid receptor

neutral antagonist VCHSR1, including higher mortality

rates in view of the much lower binding affinity of VCHSR1

to the CB1 cannabinoid receptor, the two compounds had

very similar effects on neonatal feeding and growth (Fride

et al., 2001, 2003). Collectively, these data show that the

growth failure induced by SR141716A is generalized to other

CB1 cannabinoid receptor antagonists and that activation

of cannabinoid receptors by ECs is essential for normal milk

ingestion and development in mice. As the administration

of the CB1 cannabinoid receptor antagonist/inverse agonist

SR141716A on PND 1 to newborn mice completely inhibited

milk ingestion and subsequent growth in most pups and

caused death within a few days after administration (Fride

et al., 2001), the existence and viability of mice completely

lacking CB1 cannabinoid receptors seems somehow para-

doxical. To solve this apparent paradox, experiments in CB1

cannabinoid-receptor-deficient mice have been performed.

Interestingly, CB1 knockout pups (CB1 -/- ) did not nurse on

day 1 of life, displaying deficient milk suckling; however,

they developed normal suckling behavior by day 2 of life

(Fride et al., 2003; Fride, 2004a). Thus, a possible expla-

nation for these findings is that CB1 -/- neonates possess a

compensatory mechanism that helps them to overcome

the lack of CB1 cannabinoid receptors. This compensatory

mechanism might involve other neurotransmitter systems,

such as the endogenous opioid system, that closely inter-

act with the endocannabinoid system (Maldonado and

Rodriguez de Fonseca, 2002; Trezza and Vanderschuren,

2008b, 2009; Fattore et al., 2010; Parolaro et al., 2010;

Trezza et al., 2010) and play a regulatory role in milk suck-

ling (Petrov et al., 1998). Therefore, in view of the interac-

tion between the two systems, it is tempting to speculate

that the opioid system takes over some of the functions

normally regulated by ECs in CB1 -/- mice, including suck-

ling behavior (Fride et al., 2003). Interestingly, however,

despite the genetic ablation of CB1 cannabinoid receptors,

SR141716A significantly inhibited the survival rate and the

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710   A. Manduca et al.: Endocannabinoids and mother-infant interaction

initiation of the suckling response in CB1 -/- , although to a

lesser extent than in wild-type animals (Fride et al., 2003).

These data support the hypothesis of an unknown can-

nabinoid receptor with partial control over milk ingestion

in newborns. Recently, the orphan receptor GPR55 has

attracted much attention as another member of the cannabi-

noid family, potentially explaining physiological effects

that are non-CB1/CB2-mediated (Sharir and Abood, 2010;

Anavi-Goffer et al., 2012). Although this putative additional

cannabinoid receptor needs still to be fully characterized,

it is tempting to speculate that it might be involved in the

partial control of suckling and milk ingestion in newborns.

To understand the physiological and behavioral

mediators by which blockade of CB1 cannabinoid recep-

tors prevents normal suckling behavior, PND 2 – PND 11

mouse pups were injected with SR141716A on day 1 of life

and were exposed to anesthetized nursing dams (Fride,

2004a). Whereas vehicle-injected pups located the nipples

and nursed from the dam on every testing day, SR141716A-

injected pups approached the nipples but could not

suckle, leading to the hypothesis that the drug affects

the oral-motor strength necessary for the pups to ingest

milk through the nipple (Fride, 2004b). The hypothesis of

a severe oral-motor impairment induced by the CB1 can-

nabinoid receptor antagonist/inverse agonist SR141716A

was confirmed by the observation that, when exposed to

a dish with a milk ⁄ cream mixture, which can be ingested

by licking without the need for sucking, SR141716A-treated

pups were able to ingest the same amount of milk as

controls (Fride, 2004b, 2008). This dramatic oral-motor

impairment found explanation in the fact that the two

major ECs, AEA and 2-AG, play a fundamental role in axon

guidance and synaptogenesis (Berghuis et al., 2007) and

axonal growth (Williams et al., 2003), respectively. More-

over, CB1 cannabinoid receptor activation modulates gly-

cinergic synaptic currents in hypoglossal motoneurones of

postnatal rats (Mukhtarov et al., 2005) that are essential

for rat pup milk suckling (Fujita et al., 2006). Therefore,

it has been hypothesized (Fride, 2008) that when pups

are treated with SR141716A at birth, incomplete synap-

togenesis of the hypoglossal nerve may fail to adequately

activate tongue movements (Fujita et al., 2006) that are

critical for sucking (Mukhtarov et al., 2005). Thus, a CB1

receptor blockade does not seem to interfere with the moti-

vation to suckle milk, but with the efficiency of the oral

musculature (Fride, 2004b). Human infants with decel-

erated or arrested physical growth (height and weight

measurements below the fifth percentile or a downward

change in growth across two major growth percentiles)

associated with poor developmental and emotional func-

tioning without any organic cause and with an oral-motor

weakness observed are classified as suffering from non-

organic failure to thrive (NOFTT) (Ramsay et al., 2002).

On the basis of the data obtained in neonatal mice, Fride

et al. (2007) hypothesized that endocannabinoid defi-

ciency and CB1 cannabinoid receptor dysfunction might

represent the uncharacterized biologic vulnerability that

underlies NOFTT and proposed that infant mice treated

with CB1 cannabinoid receptor antagonists might repre-

sent the first animal model for NOFTT in newborns (Fride

et al., 2007).

ECs can be found in bovine as well as human and

rodent milk, with 2-AG being present in at least 100 – 1000-

fold higher concentrations than AEA (Di Marzo et al., 1998;

Fride et al., 2001, 2005a). Furthermore, high levels of CB1

cannabinoid receptor mRNA and 2-AG have been observed

on the first day of life in brain structures involved in feeding

and in the suckling response, such as the hypothalamic

ventromedial nucleus. On the basis of these observations,

Fride (2008) proposed a model to explain cannabinoid

modulation of pup suckling behavior. According to this

model, more than AEA, pup-derived 2-AG released at birth

enables the first milk sucking session (via CB1 receptor

activation). In normal conditions, pup-derived 2-AG will

be supplemented with milk-derived 2-AG, thus enabling

CB1 cannabinoid receptor activation during the next

nursing session (Fride, 2008). A blockade of CB1 cannabi-

noid receptors immediately after birth prevents the activa-

tion of the pups sucking apparatus by brain-derived 2-AG,

and therefore milk is not ingested and brain-derived 2-AG

levels remain too low to activate enough of the CB1 can-

nabinoid receptors required for sucking during the next

nursing session. As a result, the neonate does not ingest

sufficient milk for growth and survival (Fride, 2008).

To summarize, it is evident from preclinical studies

that CB1 cannabinoid receptor antagonists administered

to newborn rodent pups dramatically interfere with their

ability to ingest milk because of an oral-motor weakness.

These effects are dose-dependent and mediated by CB1 can-

nabinoid receptors (Fride et al., 2007), although other not

yet identified cannabinoid receptors might also be involved.

Cannabinoid modulation of maternal behavior The term ‘ maternal behavior ’ , when referring to non-

human mammals, includes a wide range of behaviors

exhibited in preparation for the arrival of newborns and

in the care, protection and subsequent weaning of the

newly arrived neonates, and represents a complex pattern

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A. Manduca et al.: Endocannabinoids and mother-infant interaction   711

of behavioral interactions between the mother and the

offspring, some of which are common to most mammals,

whereas others are species-specific and depend primar-

ily on the maturity of the newborns at birth (Cirulli et al.,

2003; Ogren and Lombroso, 2008; Champagne and Curley,

2009; Kristal, 2009; Levy and Keller, 2009).

Maternal care represents the first experience of social

interaction for the newborns and is essential for proper

development and establishment of social competence

throughout life (Champagne and Curley, 2009; Levy and

Keller, 2009; Schechter et al., 2012). In humans, mother-

infant interactions influence the future life of the off-

spring (Bowlby, 1978; Goldberg, 1991; Carter et al., 2004),

and subsequently, child abuse or childhood neglect cor-

relate with severe, deleterious long-term effects on a

child ’ s cognitive, socio-emotional and behavioral devel-

opment (Hildyard and Wolfe, 2002). Similarly, impair-

ment of mother-infant bonding strongly influences off-

spring sociality in non-human primates (Agid et al., 1999;

Heim and Nemeroff, 2001) and rodents (Broad et al., 2006;

Kaffman and Meaney, 2007; Bosch and Neumann, 2012).

For example, as human infants raised in socially deprived

environments show decreased social interaction as chil-

dren (Rutter et al., 1999), rhesus monkeys reared in a

nursery away from their mother manifest deficits in social

and emotional behavior (Suomi et al., 1971; Sackett, 1984;

Champoux et al., 1991; Winslow et al., 2003).

In rodents, maternal behavior is a complex set of

activities, including nest repair, pup sniffing, licking and

exploration, pup retrieval, grooming and various forms

of nursing (arched-back nursing, prone nursing, blanket

nursing) (Pryce et al., 2001; Numan and Insel, 2003; Capone

et al., 2005). Mother-infant bond formation is reinforced

by various social stimuli, such as tactile stimuli and USVs

from the pups to the mother and feeding and tactile stimu-

lation from the mother to the pups (Fleming et al., 1994;

Magnusson and Fleming, 1995; Branchi et al., 2001; Trezza

et al., 2011). The reciprocal exhibition of maternal behav-

iors and newborn signalling promotes physiological and

immunological resilience, physical maturation and spe-

cies-typical social, emotional and cognitive development

of the pups (Fleming et al., 1999; Francis and Meaney,

1999; Caspi and Moffitt, 2006; R ä s ä nen and Kruuk, 2007;

Mogi et al., 2011). Ultimately, reciprocal mother-infant

behaviors increase the probability that the newborns will

survive and, once having reached sexual maturity, that

they will mate and successfully rear their own offspring.

The brain regions crucial for the regulation of pup-

directed maternal behavior substantially overlap with

those underlying other forms of social bonding. In par-

ticular, both rodent (Numan, 1994; Numan and Sheehan,

1997; Tsou et al., 1998; Leckman and Herman, 2002; Fride,

2008) and human (Swain et al., 2007) studies point to a

network of highly conserved hypothalamic-midbrain –

limbic-paralimbic-cortical circuits that interact to support

specific aspects of the maternal response to infants. Thus,

infant stimuli activate basal forebrain regions, which in

turn regulate brain circuits that handle specific nurtur-

ing and caregiving responses and activate more general

neural circuits for handling emotions, motivation and

attention, all of which are crucial for effective parent-

ing (Numan and Sheehan, 1997; Swain et al., 2007). The

maternal brain undergoes remarkable physiological and

behavioral changes in the peripartum period that are

aimed at the care of the offspring, and several hormones,

neuropeptides and neurotransmitters that include mainly

estrogen, progesterone, prolactin, oxytocin, vasopressin,

endogenous opioids and dopamine play important roles

in the induction and regulation of maternal behavior in

both animals and humans (Panksepp et al., 1980; Numan

and Sheehan, 1997; Nelson and Panksepp, 1998; Fleming

et al., 1999; Moles et al., 2004; Nemsadze and Silagava,

2010; Numan and Woodside, 2010).

Although there is abundant information about the

influence of most hormones and neurotransmitters on

maternal behavior in rodents, less is known about its modu-

lation by ECs. The effects of Cannabis extract on maternal

behavior date back to the late 1970s and early 1980s, when

it was first reported that acute and sub-chronic adminis-

tration of THC dose dependently suppressed retrieval of

nesting material in mice (Moschovakis et al., 1978; Sieber

et al., 1980); conversely, cannabidiol, a non-psychotropic

cannabinoid compound of the plant Cannabis sativa

(Leweke et  al., 2012; Rock et al., 2012), had no effect by

itself on maternal behavior and did not modify the effects

of THC on nest-building behavior (Lazaratou et al., 1980).

Similar effects have been reported in postpartum lactat-

ing rats, in which intravenously injected THC disrupted

all components of maternal behavior, inhibiting prolac-

tine release (Bromley et al., 1978). A clear impairment of

nest-building behavior was also found after multiple and

chronic oral administration of an extract of hashish to

pairs of mice, although no general sedation was observed

because a tolerance to the sedative effects of hashish

developed very rapidly (Frischknecht et al., 1982). Subse-

quent studies confirmed that acute administration of THC

depressed nursing and pup-retrieving behavior (Navarro

et al., 1995). Interestingly, adult mice treated from parturi-

tion to weaning with a hashish extract showed an early

impairment of maternal behavior, whereas a partial tole-

rance occurred later in the perinatal period with normal

care for the offspring by the drugged dams (Frischknecht

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712   A. Manduca et al.: Endocannabinoids and mother-infant interaction

et al., 1980). Furthermore, pups that were born to drug-

naive dams showed impairments in later life when they

were fostered by dams that had been treated with Can-nabis extract during the perinatal period (Abel et al.,

1979), which indicates that being raised by cannabinoid-

treated dams markedly affects the future behavior of the

offspring. As correctly pointed out by Schneider (2009),

a potential problem of these early studies is that rather

high doses of cannabinoid compounds were often used.

Less information is available so far on the consequences

of lower cannabinoid doses on maternal behavior. More

recently, it has been shown that mouse dams treated with

the CB1 cannabinoid receptor antagonist/inverse agonist

SR141716A during postpartum days 1 – 8 tended overall to

retrieve their pups with a longer delay than vehicle-treated

dams and sniffed and licked their pups less compared to

control animals, which suggests an impairment in mater-

nal care (Schechter et al., 2012). As maternal behavior is

a key factor in shaping adult social skills (Hildyard and

Wolfe, 2002; Champagne et al., 2003; Kikusui et al., 2005;

Levy and Keller, 2009; Schechter et al., 2012), any change

in maternal behavior induced by cannabinoid exposure

might be related to altered neurobehavioral development

in the offspring. In line with this hypothesis, blocking CB1

cannabinoid receptors in lactating mouse dams caused

changes in the offspring ’ s behavior during early and late

developmental stages (Schechter et al., 2012). Indeed, at

adulthood, animals reared by SR141716A-treated dams

showed higher levels of active social interaction and

rearing, hypoactivity in the open field, less immobility

in the catalepsy test and more analgesia in the hot-plate

test (Schechter et al., 2012). These findings show that

the endocannabinoid system is critical for the establish-

ment of maternal behavior and support the impact of

maternal ECs on future sensorimotor and social behav-

ior of offspring. It should be noted, however, that other

authors failed to detect changes in maternal care in rhesus

monkeys exposed to THC during pregnancy and lactation

(Golub et al., 1981). Therefore, due to the scarce number

of studies performed and to the sometimes contradictory

results obtained, more well-controlled studies in labora-

tory animals should be performed to better elucidate the

role of the endocannabinoid system in maternal behavior.

Cannabinoid modulation of ultrasonic vocalizations (USVs) Like many other vertebrates, rodents emit USVs across a

broad range of frequencies. USVs are emitted in different

social situations and serve to communicate information

regarding individual and group identity, status or mood

(e.g., dominance, submission, fear or aggression), antici-

patory behavior (e.g., approach, play, groom or mount)

and environmental conditions (e.g., presence of preda-

tors, location of food or separation from mother and sib-

lings) (Cuomo et al., 1988; D ’ Amato, 1991; Panksepp and

Burgdorf, 2000; Panksepp et al., 2007; Portfors, 2007;

Scattoni et al., 2009, 2011; Lahvis et al., 2011). The trans-

mission of different types of information through vocali-

zations depends on the specific frequency and temporal

properties of the acoustic signals (Portfors, 2007).

Although rodent USVs are generally inaudible to humans,

they can be studied in laboratory settings using special-

ized equipment. Data on USVs in rodents have indeed been

reported since the 1950s, when it was observed that adult

laboratory rats emit calls at frequencies around 23 – 28 kHz

when socially isolated (Anderson, 1954). Two years later,

Zippelius and Schleidt published their important discov-

ery that infant mice produce USVs when separated from

their mother and siblings (Zippelius and Schleidt, 1956).

Since then, hundreds of studies have been performed

showing that measuring the USVs emitted by laboratory

animals under particular physical, environmental or

social conditions can provide essential information about

their affective state and new insights into specific aspects

of social interaction, such as mating, nursing, aggres-

sion and defence (Cuomo et al., 1988; D ’ Amato, 1991;

Panksepp and Burgdorf, 2000; Panksepp et al., 2007;

Portfors, 2007; Scattoni et al., 2009, 2011; Lahvis et al.,

2011). In particular, the USVs emitted by rodent pups in

response to separation from the mother and the nest are

whistle-like sounds with frequencies between 30 and 90

kHz (Insel et al., 1986; Cuomo et al., 1987; Branchi et al.,

2001; Scattoni et al., 2009; Lahvis et al., 2011) and play an

essential communicative role in mother – offspring inter-

action (Oswalt and Meier, 1975; Hofer and Shair, 1991).

They are, indeed, a potent stimulus for maternal retrieval

and elicit caregiving behaviors in the dam (Insel et al.,

1986; Cuomo et al., 1987; Branchi et al., 2001; Farrell and

Alberts, 2002; Scattoni et al., 2009; Lahvis et al., 2011;

Trezza et al., 2011; Schechter et al., 2012). USV emis-

sion in rodent pups is modulated by both physical and

social parameters (Branchi et al., 1998, 2001). Concern-

ing physical parameters, a decrease in body temperature

increases the rate of USVs emitted by the pups (Oswalt

and Meier, 1975; Blumberg et al., 1992), as an adaptive

response to induce maternal retrieval into the nest; olfac-

tory and tactile cues also strongly modulate the number

of USVs (Branchi et al., 1998; Branchi et al., 2001). As for

social parameters, it has been reported that the presence

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A. Manduca et al.: Endocannabinoids and mother-infant interaction   713

of the anaesthetised mother or even of a single anesthe-

tized littermate was enough to reduce the number of USVs

emitted by the pups more than 70% (Hofer and Shair,

1978). Conversely, USV emission was increased when the

pup was allowed a brief period of contact with its anesthe-

tized dam during isolation, a phenomenon that has been

named ‘ maternal potentiation ’ (Hofer et al., 1998).

Pup retrieval is a complex type of social interaction

involving both the mother and pup (Hahn and Schanz,

1996); thus, the rate of calling or call characteristics pro-

duced by the pup may alter the behavior of the mother

(Brudzynski, 2005). Likewise, individual differences in

the mother ’ s behavior, such as more or less exploration to

find a pup, may alter the behavior of the pup (Hahn and

Lavooy, 2005).

The emotional state of the pup is a crucial determi-

nant of USV production; for this reason, measuring iso-

lation-induced USVs in rodent pups has been extensively

validated and widely used to investigate the ontogeny of

emotionality and the potential anxiolytic or anxiogenic

effects induced by pharmacological or genetic manipu-

lations (Insel et al., 1986; Cuomo et al., 1987; Branchi

et al., 2001, 2004a,b). High rates of USVs are generally

indicative of an anxiety-like state in pups; conversely, a

reduced frequency of USVs emitted by pups in response to

separation from the mother and siblings usually indicates

a less anxious phenotype and can be the consequence of

anxiolytic drug administration (Insel et al., 1986; Cuomo

et al., 1987; Branchi et al., 2001, 2004b).

Cannabis sativa is one of the most prevalent mood-

altering drugs used by humans and, considering the impor-

tant role of the endocannabinoid system in the regulation

of emotional states (Wotjak, 2005; Moreira and Lutz, 2008;

Trezza et al., 2008b, 2012; Rubino and Parolaro, 2011) and

the functionality of endocannabinoid neurotransmission

from early developmental stages (Fried, 2002; Harkany

et al., 2007; Lee and Gorzalka, 2012; Marco and Laviola,

2012), it is reasonable to hypothesize that cannabinoid

drugs modulate the emission of pup isolation-induced

USVs ( Table 1 ). In line with this hypothesis, it has been

shown that the acute administration of the potent CB1

cannabinoid receptor agonist CP55,940 produced a dose-

dependent reduction in USVs in Long-Evans hooded rats

isolated from their mother and siblings (McGregor et al.,

1996); this reduction in the frequency of USVs occurred

in the presence of a substantial drug-induced hypother-

mia (McGregor et al., 1996). The CB1 cannabinoid recep-

tor antagonist SR141716A reversed the reduction of USVs

induced by the synthetic cannabinoid agonist CP55,940,

thus indicating that the observed effects were mediated

by the activation of CB1 cannabinoid receptors (McGregor Drug

Mec

hani

sm o

f act

ion

Effe

ctAn

tago

nism

Expo

sure

Stra

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fere

nces

CP

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0 (

0.1

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mg

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; i.

p.)

CB

1 c

an

na

bin

oid

re

cep

tor

ag

on

ist

↓S

R1

41

71

6A

(2

0 m

g/k

g;

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.)P

os

tna

tal

(to

pu

ps

at

PN

D 1

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Lon

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van

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oo

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d

rat

pu

ps

McG

reg

or

et

al.

, 1

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6

SR

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A (

20

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CB

1 c

an

na

bin

oid

re

cep

tor

an

tag

on

ist/

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rse

ag

on

ist

↑P

os

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tal

(to

pu

ps

at

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D 1

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g-E

van

s h

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de

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pu

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reg

or

et

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, 1

99

6

SR

14

17

16

A (

10

mg

/kg

; i.

p.)

CB

1 c

an

na

bin

oid

re

cep

tor

an

tag

on

ist/

inve

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ag

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↓P

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(to

mo

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r

fro

m P

ND

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o P

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Sa

bra

ou

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ps

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17

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A (

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os

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tal

(to

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ps

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D 1

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Wis

tar

rat

pu

ps

Ka

thu

ria

et

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, 2

00

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; i.

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Po

stn

ata

l (t

o p

up

s a

t

PN

D 1

0)

Wis

tar

rat

pu

ps

Bo

rto

lato

et

al.

, 2

00

6

THC

(5

mg

/kg

; o

r.)

CB

1 c

an

na

bin

oid

re

cep

tor

ag

on

ist

↑P

eri

na

tal

(to

mo

the

r

fro

m G

D 1

5 t

o P

ND

9)

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(P

ND

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zza

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00

8a

,b

WIN

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2–

2 (

0.5

mg

/kg

;

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.)

CB

1 c

an

na

bin

oid

re

cep

tor

ag

on

ist

↓P

ren

ata

l (t

o m

oth

er

fro

m G

D 5

to

GD

20

)

Wis

tar

rat

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ps

(P

ND

10

)

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ton

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t a

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es

tati

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al

da

y.

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714   A. Manduca et al.: Endocannabinoids and mother-infant interaction

et al., 1996). Interestingly, the number of USVs emitted

by pups administered both CP55,940 and SR141716A was

increased compared to animals given the agonist alone,

which suggests a possible intrinsic effect of SR141716A.

Therefore, it is possible that ECs inhibit pup USV emis-

sion under conditions of isolation, and that SR141716A

blocks this inhibitory effect to produce a disinhibition

of USVs. There are close functional interactions between

the endocannabinoid and opioid systems in the regula-

tion of reward processes, including drug, food and social

rewards (Fattore et al., 2005; Solinas and Goldberg, 2005;

Maldonado et al., 2006; Trezza et al., 2008b; Trezza and

Vanderschuren, 2008b, 2009). The neuronal mechanisms

underlying functional cannabinoid-opioid interactions in

the regulation of social rewards are already present in ado-

lescent animals (Spano et al., 2010; Trezza et al., 2010). In

the study by McGregor et al. (1996), however, the opioid

receptor antagonist naloxone was not able to antagonize

the reduction of USVs induced by the cannabinoid recep-

tor agonist CP55,940. Interactions between cannabinoids

and brain D1 dopamine (Rodriguez de Fonseca et al., 1994)

and benzodiazepine (Onaivi et al., 1990) receptors have

also been described. However, the dopamine D1 receptor

antagonist SCH 23390 and the benzodiazepine receptor

antagonist flumazenil did not antagonize the reduction

in USV emissions in pups treated with the cannabinoid

receptor agonist CP55,940 (McGregor et al., 1996). On

the basis of these data, it is possible to speculate that

cannabinoid-opioid-dopamine interactions, although

already functional in adolescent rats engaged in positive

social interactions (Trezza and Vanderschuren, 2008a,

2009), are not yet mature in pre-weaning pups. In lactat-

ing dams, daily SR141716A injections during postpartum

days 1 – 8 induced fewer vocalizations in pups at PND 6

and PND 8, together with reduced maternal behaviors

from the mother towards the pups (Schechter et al., 2012).

It is accepted that the number of calls emitted by mouse

pups can reflect maternal responsiveness (Cohen-Salmon,

1987), and when there is no response or there is a delay

to respond to the needs of pups, pups lower this mode of

connection with the caregiver (D ’ Amato et al., 2005). Thus,

low levels of maternal care in SR141716A-treated dams

induced a decrease in the rate of pup USVs. The reason

for lower body weight and hypothermia in pups exposed

to SR141716A during lactation is likely a lack of nursing

and crouching which, together with pup retrieval, are

considered fundamental maternal behaviors (Schechter

et al., 2012). As USVs are typically interpreted as express-

ing ‘ separation distress ’ (Brunelli and Hofer, 2007; Braw

et al., 2008), a modification in their rate might indicate

an abnormal development of the brain circuitry involved

in the regulation of emotionality (Panksepp, 1982), a cir-

cuitry that includes a variety of neuropeptides strategi-

cally distributed throughout the mammalian system that

serve to mediate the expression of attachment, separa-

tion distress, social comfort and a variety of other social

behaviors (Panksepp, 1982; Francis et al., 2000). Other

preclinical evidence of endocannabinoid modulation of

USVs in pups has been provided by the use of the AEA

hydrolysis inhibitor URB597, which inhibits the enzyme

fatty acid amide hydrolase (FAAH) and leads to prolonged

ongoing AEA signaling, and the endocannabinoid trans-

port inhibitor AM404, which inhibits the still controver-

sial high-affinity transport system that removes ECs from

the synaptic space in neural and non-neural cells (Fowler,

2012). URB597 reduced the number of stress-induced USVs

emitted by rat pups removed from their nest at doses that

had no effect on pup motor activity; these anxiolytic-like

responses were blocked by the CB1 antagonist/inverse

agonist SR141716A (Kathuria et al., 2003). Similarly, the

endocannabinoid uptake inhibitor AM404 reduced USVs

in 10-day-old rat pups removed from the nest without

alteration of axillary temperature or locomotor activ-

ity; again, these effects were mediated by activation of

CB1 cannabinoid receptors, as they were antagonized by

preventive administration of SR141716A (Bortolato et al.,

2006). The anxiolytic-like effects induced by URB597 and

AM404 were confirmed in adult animals (Kathuria et al.,

2003; Bortolato et al., 2006). On the basis of these data,

it was proposed that drugs that interfere with endocan-

nabinoid deactivation, thus prolonging local endocan-

nabinoid activity, might represent new therapeutic tools

to treat anxiety-related disorders (Moreira et al., 2008;

Scherma et al., 2008; Ruehle et al., 2012).

Exposure to cannabinoid drugs during pregnancy

and/or lactation has also been related to changes in USV

emissions in rodent pups. For example, 12-day-old pups

exposed to THC during the perinatal period displayed an

increased rate of USVs when separated from their mother

and siblings compared to the control group (Trezza et al.,

2008a,b); however, a reduction of separation-induced

USVs in rat pups prenatally exposed to the synthetic can-

nabinoid agonist WIN55,212 – 2 has also been reported

(Antonelli et al., 2005). Differences in the type and dose

of cannabinoid agonist used (i.e., direct or indirect can-

nabinoid agonist or antagonist) and the time window

of exposure could account for the apparent discrepan-

cies between these preclinical findings, as bidirectional

responses of cannabinoids on emotional reactivity have

been extensively demonstrated (Viveros et al., 2005;

Moreira and Lutz, 2008). Furthermore, it would be inter-

esting to determine whether the altered isolation-induced

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A. Manduca et al.: Endocannabinoids and mother-infant interaction   715

USV pattern found in rats exposed to cannabinoid drugs

during pregnancy and/or lactation could be due to a

direct effect of the drugs on brain areas involved in emo-

tional behavior and/or to an indirect effect of the drug on

maternal behavior.

To investigate the consequences of CB1 cannabinoid

receptor deletion on the ontogeny of emotionality, Fride

et al. (2005b) evaluated the emission of USVs induced

by maternal separation in wild-type and CB1 -/- pups. As

expected (Bell et al., 1972; Branchi et al., 2001), wild-type

mouse pups reached the highest frequency of USV emis-

sion between days 3 and 6 of age until day 10; in contrast,

CB1 - /- pups showed very low levels of USVs throughout

development (Fride et al., 2005b).

Altogether, the results outlined here suggest that ECs

regulate USV production in rodent pups, plausibly via

activation of brain CB1 cannabinoid receptors. As isola-

tion-induced USVs are an index of the affective and emo-

tional state of the animal, the available literature indicates

that endocannabinoid modulation of emotional reactivity

appears at early developmental ages.

Conclusions Mother-infant interactions are essential for both the

physical and neurobehavioral development of the young,

and disruptions in such relationships result in neuroen-

docrine, neurochemical and behavioral changes in the

adult organism (Cirulli et al., 2003; Swain et al., 2007;

Champagne and Curley, 2009; Mogi et al., 2011). However,

the basic mechanisms underlying such changes have not

been completely elucidated yet.

The endocannabinoid system is actively present and

functional from the earliest stages of ontogenetic devel-

opment, from fertilization and pre-implantation until

prenatal and postnatal life (Fernandez-Ruiz et al., 2000;

Harkany et al., 2007; Trezza et al., 2008a,b, 2011, 2012;

Trezza and Vanderschuren, 2008a,b; Fride et al., 2009;

Schneider, 2009). In the present review, we discuss pre-

clinical data showing that, immediately after birth, ECs

play an essential role in the initiation of suckling, which

is necessary for successful postnatal growth, development

and thriving. However, the range of functions subserved

by ECs in the newborn goes well beyond the control of milk

intake. Indeed, there is ample preclinical evidence that

ECs are essential in the modulation of isolation-induced

USVs, which play a crucial role in mother-infant interac-

tion. Conversely, less information is available on endocan-

nabinoid modulation of maternal behavior, and the few

available studies provide sometimes contradictory results.

Further preclinical and clinical studies on the role of

the endocannabinoid system in mother-infant bonding are

essential for three main reasons. First, it is important to

fully elucidate the physiological role played by ECs in all

the facets of this crucial form of social interaction. Second,

as exogenously administered cannabinoids can be trans-

ferred from the mother to the offspring through the placen-

tal blood during gestation and through the maternal milk

during lactation (Jakubovic and McGeer, 1977; Hutchings

et al., 1989; Fernandez-Ruiz et al., 2004), it is important to

determine whether, by interfering with the mother-infant

bond formation, developmental exposure to cannabinoid

compounds could lead to the onset of neurodevelopmen-

tal alterations and lead to increased vulnerability to psy-

chopathology later in life. Third, in recent years the endo-

cannabinoid system has emerged as a novel therapeutic

target for the treatment of some neuropsychiatric disorders

(Vinod and Hungund, 2006; Marco et al., 2011); however,

the potential therapeutic use of cannabinoid drugs in

young populations requires a profound investigation of

possible adverse effects of such compounds, particularly

on the CNS of immature individuals. For these reasons, as

social experiences early in life epigenetically shape brain

development and adult behavior (Champagne and Curley,

2005), it is particularly important to evaluate whether can-

nabinoid drugs may affect social interaction and social

motivation from early developmental stages.

Acknowledgements : This research was supported by

Netherlands Organization for Scientific Research (NWO)

Veni grant 91611052 (V.T.), Marie Curie Career Reintegra-

tion Grant PCIG09-GA-2011 – 293589 (V.T.) and by MIUR,

FIRB Futuro in Ricerca (P.C. and V.T.).

Received July 10, 2012; accepted September 16, 2012; previously

published online October 25, 2012

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722   A. Manduca et al.: Endocannabinoids and mother-infant interaction

Antonia Manduca is a PhD student in Biology Applied to Human

Health at the Department of Biology, Roma Tre University, Rome,

Italy. Under the supervision of Dr. Viviana Trezza, she is involved in

psychopharmacological research on the role of the endocannabi-

noid system in the modulation of emotions and cognition.

Dr. Patrizia Campolongo is Assistant Professor at the Department

of Physiology and Pharmacology of the University of Rome “ La

Sapienza ” . The thrust of her research is to investigate the role of

the endocannabinoid system in the control of cognition and

emotions and to unveil the neural underpinnings of emotional

memory processing. She has won several prestigious awards,

among them the European Brain and Behavioral Society (EBBS)

young investigator award and the “ Sapienza Ricerca 2010 ” young

investigator prize, for the six most promising scientists performing

fore-front research within the whole University. She is a member of

the US Society for Neuroscience (Sfn), the International Behavioral

Neuroscience Society (IBNS), the EBBS, the European Behavioural

Pharmacology Society (EBPS), the International Cannabinoid Re-

search Society (ICRS) and the Italian Society of Pharmacology (SIF).

Dr. Viviana Trezza is Assistant Professor at the Department of

Biology of Roma Tre University. Her research focuses on the

following topics: 1. the role of endocannabinoid neurotransmis-

sion in animal models of cognitive and emotional disorders; 2.

neurobiology of social behavior in rats; 3. the developmental

neurotoxicity of drugs of abuse and environmental chemicals. Dr.

Trezza ’ s research activity is funded by the EU (Marie Curie Career

Reintegration Grant, Seventh Framework Programme People), NWO

(Nederlandse Organisatie voor Wetenschappelijk Onderzoek) and

MIUR (Italian Ministry of Education, University and Research).

Viviana Trezza is a member of the US Society for Neuroscience

(SfN), the International Behavioral Neuroscience Society (IBNS),

the European Brain and Behavioral Society (EBBS), the European

Behavioural Pharmacology Society (EBPS), the International

Cannabinoid Research Society (ICRS) and the Italian Society of

Pharmacology (SIF).

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